A practical synthesis of xylo- and arabinofuranoside precursors by diastereoselective reduction using Corey-Bakshi-Shibata catalyst.

The Corey-Bakshi-Shibata (CBS) catalyst provides an efficient mechanism to reduce ketones and achieve desired enantiopure alcohols. Herein, the diastereoselective reduction of C-2' and C-3'-keto ribofuranoside derivatives to the corresponding arabino- and xylofuranosides in greater than 95% diastereomeric excess is reported. The stereo-directed substitution with an azido group as well as the synthesis of prodrugs cytarabine and vidarabine are also described. The reported strategy offers superior diastereoselectivity, shorter reaction times, and obviates cooling required with comparable protocols involving achiral reductants.

Double-headed nucleotides with arabino configuration: synthesis and hybridization properties.

The formation of new nucleic acid motifs by using double-headed nucleotides is reported. Modified phosphoramidites carrying additional thymine or adenine attached to the 2'-position of arabinouridine through a methylene linker are conveniently prepared and incorporated into oligonucleotides to obtain the modified nucleotide monomers (a)U(T) and (a)U(A), respectively. The extension of a DNA double helix by one or two additional A:T base pairs is achieved by placing these modified monomers in the opposite strands in a so-called (+1)-zipper arrangement. Hence, 12 basepairs can be presented in an 11-mer or even a 10-mer duplex. The modified nucleotide monomers also behave as dinucleotides when base-paired with two complementary nucleotides from the opposite strand. A new nucleic acid motif is introduced when two (a)U(A) monomers recognize each other in the center of a duplex.

The readout of base-pair information in adenine-thymine α-D-Arabinonucleosides.

Structurally modified nucleosides are central players in the field of nucleic acid chemistry. Adenine-thymine (AT) pyrimido[4,5-d]pyrimidine furanosyl and pyranosyl arabinonucleosides have been synthesized for the first time. Single-crystal X-ray diffraction analysis reveals novel base pairs that, in synergy with the sugar residues, direct the emergence of distinct networks containing channels and cavities. The microscopic noncovalent connections can be translated into macroscopic levels in which robust organogels are formed by the furanoside but not the pyranoside. The influences of the sugars are also displayed by the different shaped superstructures of the free nucleosides in solution. The readout of the information in the base moiety is therefore tailored by the sugar configuration, and the interplays exert subtle effects on the structures, from solid to gel and to the solution state. The potential for forming these appealing base pairs and higher structures enables these intriguing nucleosides to serve as unique building blocks in various areas or to construct innovative nucleic acid structures.

Synthesis and biological properties of caffeic acid-PNA dimers containing guanine.

Caffeic acid (CA; 3,4-dihydroxycinnamic acid) is endowed with high antioxidant activity. CA derivatives (such as amides) have gained a lot of attention due to their antioxidative, antitumor and antimicrobial properties as well as stable characteristics. Caffeoyl-peptide derivatives showed different antioxidant activity depending on the type and the sequence of amino acid used. For these reasons, we decided to combine CA with Peptide Nucleic Acid (PNA) to test whether the new PNA-CA amide derivatives would result in an improvement or gain of CA's biological (i.e., antioxidant, cytotoxic, cytoprotective) properties. We performed the synthesis and characterization of seven dimer conjugates with various combinations of nucleic acid bases and focused NMR studies on the model compound ga-CA dimer. We demonstrate that PNA dimers containing guanine conjugated to CA exhibited different biological activities depending on composition and sequence of the nucleobases. The dimer ag-CA protected HepG2, SK-B-NE(2), and C6 cells from a cytotoxic dose of hydrogen peroxide (H2O2).

Synthesis and in vitro cytostatic activity of 1,2- and 1,3-diacylglycerophosphates of clofarabine.

The conjugates of anticancer nucleoside clofarabine [2-chloro-9-(2-deoxy-2-fluoro-ß-d-arabinofuranosyl)adenine] with 1,2- and 1,3-diacylglycerophosphates have been prepared by the phosphoramidite method using a combination of 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl protecting group for the sugar moiety of the nucleoside and 2-cyanoethyl protection for the phosphate fragment. Some of the synthesized conjugates exhibited cytostatic activity against HL-60, A-549, MCF-7, and HeLa tumor cell lines.

A stereoselective approach to ß-L-arabino nucleoside analogues: synthesis and cyclization of acyclic 1',2'-syn N,O-acetals.

Reported herein is a novel and versatile strategy for the stereoselective synthesis of unnatural ß-L-arabinofuranosyl nucleoside analogues from acyclic N,OTMS-acetals bearing pyrimidine and purine bases. These unusual acetals undergo a C1' to C4' cyclization where the OTMS of the acetal serves as the nucleophile to generate 2'-oxynucleosides with complete retention of configuration at the C1' acetal center. N,OTMS-acetals are obtained diastereoselectively from additions of silylated nucleobases onto acyclic polyalkoxyaldehydes in the presence of MgBr(2)·OEt(2). The strategy reported is addressing important synthetic challenges by providing stereoselective access to unnatural L-nucleosides starting from easily accessible pools of D-sugars and, as importantly, by allowing the formation of the sterically challenging 1',2'-cis nucleosides. A wide variety of nucleoside analogues were synthesized in 7-8 steps from easily accessible D-xylose.

Sugar-modified derivatives of cytostatic 7-(het)aryl-7-deazaadenosines: 2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides.

A series of novel sugar-modified derivatives of cytostatic 7-hetaryl-7-deazaadenosines (2'-C-methylribonucleosides, 2'-deoxy-2'-fluoroarabinonucleosides, arabinonucleosides and 2'-deoxyribonucleosides) was prepared and screened for biological activity. The synthesis consisted of preparation of the corresponding sugar-modified 7-iodo-7-deazaadenine nucleosides and their aqueous-phase Suzuki-Miyaura cross-coupling reactions with (het)arylboronic acids or Stille couplings with hetarylstannanes in DMF. The synthesis of 7-iodo-7-deazaadenine nucleosides was based on a glycosidation of 6-chloro-7-iodo-7-deazapurine with a suitable sugar synthon or on an interconversion of 2'-OH stereocenter (for arabinonucleosides). Several examples of 2'-C-Me-ribonucleosides showed moderate anti-HCV activities in a replicon assay accompanied by cytotoxicity. Several 7-hetaryl-7-deazaadenine fluoroarabino- and arabinonucleosides exerted moderate micromolar cytostatic effects. The most active was 7-ethynyl-7-deazaadenine fluoroarabinonucleoside which showed submicromolar antiproliferative activity. However, all the sugar-modified derivatives were less active than the parent ribonucleosides.

An improved strategy for the synthesis of [¹8F]-labeled arabinofuranosyl nucleosides.

The expression of the herpes simplex virus type-1 thymidine kinase (HSV1-tk) gene can be imaged efficaciously using a variety of 2'-[(18)F]fluoro-2'-deoxy-1-b-D-arabinofuranosyl-uracil derivatives [[(18)F]-FXAU, X=I(iodo), E(ethyl), and M(methyl)]. However, the application of these derivatives in clinical and translational studies has been impeded by their complicated and long syntheses (3-5h). To remedy these issues, in the study at hand we have investigated whether microwave or combined catalysts could facilitate the coupling reaction between sugar and nucleobase and, further, have probed the feasibility of establishing a novel approach for [(18)F]-FXAU synthesis. We have demonstrated that the rate of the trimethylsilyl trifluoromethanesulfonate (TMSOTf)-catalyzed coupling reaction between the 2-deoxy-sugar and uracil derivatives at 90 °C can be significantly accelerated by microwave-driven heating or by the addition of Lewis acid catalyst (SnCl(4)). Further, we have observed that the stability of the α- and ß-anomers of [(18)F]-FXAU derivatives differs during the hydrolysis step. Using the microwave-driven heating approach, overall decay-corrected radiochemical yields of 19%-27% were achieved for [(18)F]-FXAU in 120min at a specific activity of >22MBq/nmol (595Ci/mmol). Ultimately, we believe that these high yielding syntheses of [(18)F]-FIAU, [(18)F]-FMAU and [(18)F]-FEAU will facilitate routine production for clinical applications.

Synthesis of 2'-deoxy-2'-[18F]fluoro-9-ß-D-arabinofuranosylguanine: a novel agent for imaging T-cell activation with PET.

PURPOSE: 9-(ß-D-Arabinofuranosyl)guanine (AraG) is a guanosine analog that has a proven efficacy in the treatment of T-cell lymphoblastic disease. To test the possibility of using a radiofluorinated AraG as an imaging agent, we have synthesized 2'-deoxy-2'-[(18)F]fluoro-9-ß-D-arabinofuranosylguanine ([(18)F]F-AraG) and investigated its uptake in T cells. PROCEDURE: We have synthesized [(18)F]F-AraG via a direct fluorination of 2-N-acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3',5'-di-O-trityl-2'-O-trifyl-ß-D-ribofuranosyl)guanine with [(18)F]KF/K.2.2.2 in DMSO at 85°C for 45 min. [(18)F]F-AraG uptake in both a CCRF-CEM leukemia cell line (unactivated) and activated primary thymocytes was evaluated. RESULTS: We have successfully prepared [(18)F]F-AraG in 7-10% radiochemical yield (decay corrected) with a specific activity of 0.8-1.3 Ci/µmol. Preliminary cell uptake experiments showed that both a CCRF-CEM leukemia cell line and activated primary thymocytes take up the [(18)F]F-AraG. CONCLUSION: For the first time to the best of our knowledge, [(18)F]F-AraG has been successfully synthesized by direct fluorination of an appropriate precursor of a guanosine nucleoside. This approach maybe also useful for the synthesis of other important positron emission tomography (PET) probes such as [(18)F]FEAU, [(18)F]FMAU, and [(18)F]FBAU which are currently synthesized by multiple steps and involve lengthy purification. The cell uptake studies support future studies to investigate the use of [(18)F]F-AraG as a PET imaging agent of T cells.

Efficient syntheses of clofarabine and gemcitabine from 2-deoxyribonolactone.

The development of a new methodology to achieve electrophilic fluorination of triisopropylsilyl-protected 2-deoxyribonolactone has been employed to synthesize clofarabine and gemcitabine with improved synthetic efficiency versus prior synthetic methods. These studies highlight the versatility of this new methodology to obtain medically relevant 2'-fluoronucleosides.

3'-bromo analogues of pyrimidine nucleosides as a new class of potent inhibitors of Mycobacterium tuberculosis.

Tuberculosis (TB) is a major health problem worldwide. We herein report a new class of pyrimidine nucleosides as potent inhibitors of Mycobacterium tuberculosis (M. tuberculosis). Various 2'- or 3'-halogeno derivatives of pyrimidine nucleosides containing uracil, 5-fluorouracil, and thymine bases were synthesized and evaluated for antimycobacterial activities. Among the compounds tested, 3'-bromo-3'-deoxy-arabinofuranosylthymine (33) was the most effective antituberculosis agent in the in vitro assays against wild-type M. tuberculosis strain (H37Ra) (MIC(50) = 1 microg/mL) as well as drug-resistant (H37Rv) (rifampicin-resistant and isoniazid-resistant) strains of M. tuberculosis (MIC(50) = 1-2 microg/mL). Compound 33 also inhibited intracellular M. tuberculosis in a human monocytic cell line infected with H37Ra, demonstrating higher activity against intramacrophagic mycobacteria (80% reduction at 10 microg/mL concentration) than extracellular mycobacteria (75% reduction at 10 microg/mL concentration). In contrast, pyrimidine nucleosides possessing 5-fluorouracil base were weak inhibitors of M. tuberculosis. No cytotoxicity was found up to the highest concentration of compounds tested (CC(50) > 100-200 microg/mL) against a human cell line. Overall, these encouraging results substantiate the potential of this new class of compounds as promising antituberculosis agents.

CD and NMR assignment of the anomeric configuration of 4-(5-deoxy-alpha,beta-l-arabinofuranosyl)-2-phenyl-2H-1,2,3-triazole C-nucleoside analogs.

Acid-catalyzed dehydrative cyclization of 5-deoxy-L-manno-pentitol-1-yl)-2-heptulose bisphenylhydrazone and subsequent reflux with copper sulfate gave an anomeric mixture of 4-(5-deoxy-alpha,beta-L-arabinofuranosyl)-2-phenyl-2H-1,2,3-triazole C-nucleoside analogs. The mixture was separated by chromatography, and the anomeric compositions configurations of the components were determined by CD, NMR, mass spectroscopy, and acylation.

9-(2-C-Cyano-2-deoxy-beta-D-arabino-pentofuranosyl)guanine, a potential antitumor agent against B-lymphoma infected with Kaposi's sarcoma-associated herpesvirus.

Several 9-(2-C-cyano-2-deoxy-l-beta-D-arabino-pentofuranosyl)purine derivatives were tested against Kaposi's sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma (PEL) cells. The guanine derivative (2, CNDAG) as well as the 2-amino-6-substituted-purine derivatives 3, 4 and 5 inhibited KSHV-positive cell growth but showed no cytotoxicity against KSHV-negative cells at >15 muM concentrations. Therefore, it was found that compounds 2, 3, 4 and 5 showed selective cytotoxicity against PEL cells infected with KSHV.

Synthesis of 9-beta-d-arabinofuranosylguanine by combined use of two whole cell biocatalysts.

Unlike the preparation of other purine nucleosides, transglycosylation from a pyrimidine nucleoside and guanine is difficult because of the low solubility of this base. Thus, another strategy, based on the coupled action of two whole cell biocatalyzed reactions, transglycosylation and deamination, was used. Enterobacter gergoviae and Arthrobacter oxydans were employed to synthesize 9-beta-d-arabinofuranosylguanine (AraG), an efficient anti leukemic drug.

Synthesis and hybridization studies of alpha-configured arabino nucleic acids.

Synthesis of alpha-L-arabino- and alpha-D-arabino-configured pentofuranosyl nucleosides of four of the natural bases [thymine (ara-T), adenine (ara-A), cytosine (ara-C) and guanine (ara-G)] is reported together with hybridization properties of oligonucleotides containing alpha-L-ara-T and -A, alpha-D-ara-T and -A, and 2'-amino-alpha-L-ara-T monomers. 2'-O-Acetylated alpha-L-ara-T, -A, -C and -G, alpha-D-ara-T, -A, -C and -G, and N2'-acylated-alpha-L-ara-T phosphoramidite building blocks were synthesized and used together with standard DNA phosphoramidites for solid-phase synthesis of 18-mer oligonucleotides. Thermal denaturation experiments showed that incorporation of three or six of the arabino-configured monomers into DNA-oligonucleotides reduced the binding affinity towards antiparallel DNA/RNA complements. Fully modified alpha-L-ara-oligonucleotides did not hybridize with DNA/RNA complements, whereas hybridization of fully modified alpha-D-ara-oligonucleotides with complementary DNA/RNA in parallel strand orientation was confirmed.

Synthesis and anticancer evaluation of 4'-C-methyl-2'-fluoro arabino nucleosides.

As part of an ongoing program to develop novel antitumor agents over the years, we have synthesized and evaluated a number of 4'-C-substituted nucleosides. A few years ago, we reported the first synthesis of 4'-C-hydroxymethyl-2'-fluoro arabino nucleosides, which did not exhibit any cytotoxicity. In our exploration of related compounds, we synthesized and evaluated the 4'-C-methyl-2'-fluoro arabino nucleosides in both the purine and pyrimidine series. In the pyrimidine series, 1-(4-C-methyl-2-fluoro-beta-D-arabinofuranosyl) cytosine (13) was found to be highly cytotoxic and had significant antitumor activity in mice implanted with human tumor xenografts. The synthesis and anticancer activity of this series of nucleosides are reported.

Synthesis and mycobacterial growth inhibition activities of bivalent and monovalent arabinofuranoside containing alkyl glycosides.

Arabinofuranosides constitute one of the important components of cell wall structures of mycobacteria. With this importance of arabinofuranosides in mind, alkyl glycosides bearing arabinofuranoside trisaccharides were prepared, wherein the sugars were presented either in the monovalent or bivalent forms. Following the synthesis, the monovalent and bivalent alkyl glycosides were tested for their activities in a mycobacterial growth assay. The growth of the mycobacterial strain M. smegmatis was assessed in the presence of the alkyl glycosides and it was realized that the alkyl glycosides acted as inhibitors of the mycobacterial growth. The inhibition of the growth, caused by the above alkyl glycosides, was not observed for the arabinofuranose trisaccharide alone, without the alkyl groups, and for an alkyl glycoside bearing maltose as the sugar component.

9-(2-C-Cyano-2-deoxy-beta-D-arabino-pentofuranosyl)guanine, a potential antitumor agent against B-lymphoma infected with Kaposi's sarcoma-associated herpesvirus.

Several 9-(2-C-cyano-2-deoxy-l-beta-d-arabino-pentofuranosyl)purine derivatives were tested against Kaposi's sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma (PEL) cells. The guanine derivative (3, CNDAG), as well as the 2-amino-6-substituted-purine derivatives 4, 5, and 6, exhibited cell growth inhibitory activity against KSHV-infected cells, but showed no cytotoxicity against KSHV-negative cells at >15 microM concentrations. Therefore, it was found that compounds 3, 4, 5, and 6 showed selective cytotoxicity against PEL cells infected with KSHV.

Approval summary: nelarabine for the treatment of T-cell lymphoblastic leukemia/lymphoma.

PURPOSE: To describe the clinical studies, chemistry manufacturing and controls, and clinical pharmacology and toxicology that led to Food and Drug Administration approval of nelarabine (Arranon) for the treatment of T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma. EXPERIMENTAL DESIGN: Two phase 2 trials, one conducted in pediatric patients and the other in adult patients, were reviewed. The i.v. dose and schedule of nelarabine in the pediatric and adult studies was 650 mg/m2/d daily for 5 days and 1,500 mg/m2 on days 1, 3, and 5, respectively. Treatments were repeated every 21 days. Study end points were the rates of complete response (CR) and CR with incomplete hematologic or bone marrow recovery (CR*). RESULTS: The pediatric efficacy population consisted of 39 patients who had relapsed or had been refractory to two or more induction regimens. CR to nelarabine treatment was observed in 5 (13%) patients and CR+CR* was observed in 9 (23%) patients. The adult efficacy population consisted of 28 patients. CR to nelarabine treatment was observed in 5 (18%) patients and CR+CR* was observed in 6 (21%) patients. Neurologic toxicity was dose limiting for both pediatric and adult patients. Other severe toxicities included laboratory abnormalities in pediatric patients and gastrointestinal and pulmonary toxicities in adults. CONCLUSIONS: On October 28, 2005, the Food and Drug Administration granted accelerated approval for nelarabine for treatment of patients with relapsed or refractory T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma after at least two prior regimens. This use is based on the induction of CRs. The applicant will conduct postmarketing clinical trials to show clinical benefit (e.g., survival prolongation).

Synthesis and properties of trans-3',4'-bridged nucleic acids having typical S-type sugar conformation.

The synthesis of nucleoside analogues with a conformationally restricted sugar moiety is of great interest. The present research describes the synthesis of BNA (bridged nucleic acid) monomers 1 and 2 bearing a 4,7-dioxabicyclo[4.3.0]nonane skeleton and a methoxy group at the C2' position. Conformational analysis showed that the sugar moiety of these monomers is restricted in a typical S-type conformation. It was difficult to synthesize the phosphoramidite derivative of the ribo-type monomer 1, while the phosphoramidite of the arabino-type monomer 2 was successfully prepared and incorporated into oligodeoxynucleotides (ODNs). The hybridization ability of the obtained ODN derivatives containing 2 with complementary strands was evaluated by melting temperature (T(m)) measurements. As a result, the ODN derivatives hybridized with DNA and RNA complements in a sequence-selective manner, though the stability of the duplexes was lower than that of the corresponding natural DNA/DNA or DNA/RNA duplex.