Synthesis of 2,6‐Dihalogenated Purine Nucleosides by Thermostable Nucleoside Phosphorylases

The enzymatic transglycosylation of 2,6‐dichloropurine (26DCP) and 6‐chloro‐2‐fluoropurine (6C2FP) with uridine, thymidine and 1‐(β‐D ‐arabinofuranosyl)‐uracil as the pentofuranose donors and recombinant thermostable nucleoside phosphorylases from G. thermoglucosidasius or T. thermophilus as biocatalysts was studied. Selection of 26DCP and 6C2FP as substrates is determined by their higher solubility in aqueous buffer solutions compared to most natural and modified purines and, furthermore, synthesized nucleosides are valuable precursors for the preparation of a large number of biologically important nucleosides. The substrate activity of 26DCP and 6C2FP in the synthesis of their ribo‐ and 2′‐deoxyribo‐nucleosides was closely similar to that of related 2‐amino‐ (DAP), 2‐chloro‐ and 2‐fluoroadenines; the efficiency of the synthesis of β‐D ‐arabinofuranosides of 26DCP and 6C2FP was lower vs. that of DAP under similar reaction conditions. For a convenient and easier recovery of the biocatalysts, the thermostable enzymes were immobilized on MagReSyn^® epoxide beads and the biocatalyst showed high catalytic efficiency in a number of reactions. As an example, 6‐chloro‐2‐fluoro‐(β‐D ‐ribofuranosyl)‐purine ( 9 ), a precursor of various antiviral and antitumour drugs, was synthesized by the immobilized enzymes at 60 °C under high substrate concentrations (uridine:purine ratio of 2:1, mol). The synthesis was successfully scaled‐up [uridine (2.5 mmol), base (1.25 mmol); reaction mixture 50 mL] to afford 9 in 60% yield. The reaction reveals the great practical potential of this enzymatic method for the efficient production of modified purine nucleosides of pharmaceutical interest.

     

Discovery of a Novel Inhibitor of Human Purine Nucleoside Phosphorylase by a Simple Hydrophilic Interaction Liquid Chromatography Enzymatic Assay

Human purine nucleoside phosphorylase (HsPNP) belongs to the purine salvage pathway of nucleic acids. Genetic deficiency of this enzyme triggers apoptosis of activated T-cells due to the accumulation of deoxyguanosine triphosphate (dGTP). Therefore, potential chemotherapeutic applications of human PNP inhibitors include the treatment of T-cell leukemia, autoimmune diseases and transplant tissue rejection. In this report, we present the discovery of novel HsPNP inhibitors by coupling experimental and computational tools. A simple, inexpensive, direct and non-radioactive enzymatic assay coupled to hydrophilic interaction liquid chromatography and UV detection (LC-UV using HILIC as elution mode) was developed for screening HsPNP inhibitors. Enzymatic activity was assessed by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx) by LC-UV. A small library of 6- and 8-substituted nucleosides was synthesized and screened. The inhibition potency of the most promising compound, 8-aminoinosine ( 4 ), was quantified through K_i and IC₅₀ determinations. The effect of HsPNP inhibition was also evaluated in vitro through the study of cytotoxicity on human T-cell leukemia cells (CCRF-CEM). Docking studies were also carried out for the most potent compound, allowing further insights into the inhibitor interaction at the HsPNP active site. This study provides both new tools and a new lead for developing novel HsPNP inhibitors.     

Synthesis and Antiviral Evaluation of TriPPPro‐AbacavirTP,TriPPPro‐CarbovirTP,and Their 1′,2′‐cis‐Disubstituted Analogues

Herein we describe the synthesis of lipophilic triphosphate prodrugs of abacavir, carbovir, and their 1′,2′‐cis‐substituted carbocyclic analogues. The 1′,2′‐cis‐carbocyclic nucleosides were prepared by starting from enantiomerically pure (1R,2S)‐2‐((benzyloxy)methyl)cyclopent‐3‐en‐1‐ol by a microwave‐assisted Mitsunobu‐type reaction with 2‐amino‐6‐chloropurine. All four nucleoside analogues were prepared from their 2‐amino‐6‐chloropurine precursors. The nucleosides were converted into their corresponding nucleoside triphosphate prodrugs (TriPPPro approach) by application of the H‐phosphonate route. The TriPPPro compounds were hydrolyzed in different media, in which the formation of nucleoside triphosphates was proven. While the TriPPPro compounds of abacavir and carbovir showed increased antiviral activity over their parent nucleoside, the TriPPPro compounds of the 1′,2′‐cis‐substituted analogues as well as their parent nucleosides proved to be inactive against HIV.     

Flow‐Synthesis of Nucleosides Catalyzed by an Immobilized Purine Nucleoside Phosphorylase from Aeromonas hydrophila: Integrated Systems of Reaction Control and Product Purification

A purine nucleoside phosphorylase from Aeromonas hydrophyla (AhPNP) was covalently immobilized in a pre‐packed stainless steel column containing aminopropylsilica particles via Schiff base chemistry upon glutaraldehyde activation. The resulting AhPNP‐IMER (Immobilized Enzyme Reactor, immobilization yield ≈50%) was coupled on‐line through a 6‐way switching valve to an HPLC apparatus containing an analytical or a semi‐preparative chromatographic column. The synthesis of five 6‐modified purine ribonucleosides was carried out by continuously pumping the reaction mixture through the AhPNP‐IMER until the highest conversion was reached, and then directing the reaction mixture to chromatographic separation. The conditions of the AhPNP‐catalyzed transglycosylations (2:1 ratio sugar donor:base acceptor; 10 mM phosphate buffer; pH 7.5; temperature 37 °C, flow rate 0.5 mL min⁻¹) were optimized by a fractional factorial experimental design. Coupling the bioconversion step with the product purification in such an integrated platform resulted in a fast and efficient synthetic process (yield=52–89%; <10 mg) where sample handling was minimized. To date, AhPNP‐IMER has retained completely its activity upon 50 reactions in 10 months.

     

Ruthenium‐Catalyzed Oxidative Annulation of 6‐Anilinopurines with Alkynes via C?H Activation: Synthesis of Indole‐Substituted Purines/Purine Nucleosides

Indole‐substituted purine nucleobases have been synthesized by Ru‐catalyzed oxidative annulation of 6‐anilinopurines with internal alkynes that involves C H activation. Unsymmetrical aryl(alkyl)alkynes led to high regioselectivity. The reaction was also successful with nucleosides by delivering unprotected indole‐substituted nucleosides. In the presence of [RuCl₂(p‐cymene)]₂ and copper(II) acetate hydrate [Cu(OAc)₂⋅H₂O], in some cases, we have observed two‐fold C H activation products that exhibit fluorescence. A ruthenacycle intermediate was characterized by crystallography, which suggests that the N‐1 nitrogen atom of the purine acts as a directing group for the present transformation.

     

Synthesis of 2′‐Deoxynucleosides by Transglycosylation with New Immobilized and Stabilized Uridine Phosphorylase and Purine Nucleoside Phosphorylase

Multimeric uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) of Bacillus subtilis have been expressed from genes cloned in Escherichia coli, purified, characterized, immobilized and stabilized on solid support. A new immobilization strategy has been developed for UP onto Sepabeads coated with polyethyleneamine followed by cross‐linking with aldehyde‐dextran. PNP has been immobilized onto glyoxyl‐agarose. At pH 10 and 45 °C these derivatives catalyzed the transglycosylation of 2′‐deoxyuridine to 2′‐deoxyguanosine in high yield (92%). Under the same conditions the not immobilized enzymes were promptly inactivated.     

A Conformational Mimetic Approach for the Synthesis of Carbocyclic Nucleosides as Anti‐HCV Leads

Computer‐aided approaches coupled with medicinal chemistry were used to explore novel carbocyclic nucleosides as potential anti‐hepatitis C virus (HCV) agents. Conformational analyses were carried out on 6‐amino‐1H‐pyrazolo[3,4‐d]pyrimidine (6‐APP)‐based carbocyclic nucleoside analogues, which were considered as nucleoside mimetics to act as HCV RNA‐dependent RNA polymerase (RdRp) inhibitors. Structural insight gained from the modeling studies revealed the molecular basis behind these nucleoside mimetics. The rationally chosen 6‐APP analogues were prepared and evaluated for anti‐HCV activity. RdRp SiteMap analysis revealed the presence of a hydrophobic cavity near C7 of the nucleosides; introduction of bulkier substituents at this position enhanced their activity. Herein we report the identification of an iodinated compound with an EC₅₀ value of 6.6 μM as a preliminary anti‐HCV lead.     

North‐ and South‐Bicyclo[3.1.0]Hexene Nucleosides: The Effect of Ring Planarity on Anti‐HIV Activity

The syntheses of new conformationally locked North‐ and South‐bicyclo[3.1.0]hexene nucleosides is reported. The North analogues were synthesized by a convergent approach from the known (1S,2R,5R)‐5‐[(tert‐butyldiphenylsilyloxy)methyl]bicyclo[3.1.0]hex‐3‐en‐2‐ol by Mitsunobu coupling with the nucleobases. The South analogues were synthesized from their bicyclo[3.1.0]hexane nucleoside precursors by the selective protection of the primary hydroxy group, conversion of the secondary alcohol into a good leaving group, and base‐catalyzed elimination to generate the olefin. The transformation of a bicyclo[3.1.0]hexane nucleoside into a bicyclo[3.1.0]hexene nucleoside flattens the five‐membered ring of the bicyclic system and rescues anti‐HIV activity for North‐D4T, North‐D4A, and South‐D4C. The relationship between planarity and the anti/syn disposition of the nucleobase that is favored by a particular pseudosugar platform are proposed as key parameters in controlling biological activity.     

An Expedient Synthesis of Flexible Nucleosides through Enzymatic Glycosylation of Proximal and Distal Fleximer Bases

The structurally unique “fleximer” nucleosides were originally designed to investigate how flexibility in a nucleobase could potentially affect receptor–ligand recognition and function. Recently they have been shown to have low-to-sub-micromolar levels of activity against a number of viruses, including coronaviruses, filoviruses, and flaviviruses. However, the synthesis of distal fleximers in particular has thus far been quite tedious and low yielding. As a potential solution to this issue, a series of proximal fleximer bases (flex-bases) has been successfully coupled to both ribose and 2′-deoxyribose sugars by using the N-deoxyribosyltransferase II of Lactobacillus leichmannii (LlNDT) and Escherichia coli purine nucleoside phosphorylase (PNP). To explore the range of this facile approach, transglycosylation experiments on a thieno-expanded tricyclic heterocyclic base, as well as several distal and proximal flex-bases were performed to determine whether the corresponding fleximer nucleosides could be obtained in this fashion, thus potentially significantly shortening the route to these biologically significant compounds. The results of those studies are reported herein.     

Different Binding Modes of Small and Large Binders of GAT1

Well‐known inhibitors of the γ‐aminobutyric acid (GABA) transporter GAT1 share a common scaffold of a small cyclic amino acid linked by an alkyl chain to a moiety with two aromatic rings. Tiagabine, the only FDA‐approved GAT1 inhibitor, is a typical example. Some small amino acids such as (R)‐nipecotic acid are medium‐to‐strong binders of GAT1, but similar compounds, such as proline, are very weak binders. When substituted with 4,4‐diphenylbut‐3‐en‐1‐yl (DPB) or 4,4‐bis(3‐methylthiophen‐2‐yl)but‐3‐en‐1‐yl (BTB) groups, the resulting compounds have similar pK_i and pIC₅₀ values, even though the pure amino acids have very different values. To investigate if small amino acids and their substituted counterparts share a similar binding mode, we synthesized butyl‐, DPB‐, and BTB‐substituted derivatives of small amino acids. Supported by the results of docking studies, we propose different binding modes not only for unsubstituted und substituted, but also for strong‐ and weak‐binding amino acids. These data lead to the conclusion that following a fragment‐based approach, not pure but N‐butyl‐substituted amino acids should be used as starting points, giving a better estimate of the activity when a BTB or DPB substituent is added.     

Synthese von Hetaryl‐pyridiniumsalzen und kondensierten 3‐Amino‐pyrid‐2‐onen

1‐(3‐Coumaryl)‐pyridinium salts 3 and 1‐(3‐coumaryl)‐tetrahydrothiophenium salts 5 were synthesized from 2‐acylphenyl chloro‐ or bromoacetates 2 . 2‐Chloro‐N¹‐(3,4‐dimethoxyphenyl)‐acetamide and substituted 2‐chloro‐N¹‐(2‐thienyl)‐acetamides 8 react with acetyl chloride and pyridine to yield the quinolinyl‐ and (thieno[2,3‐b]pyridin‐5‐yl)‐pyridinium salts 10 . Fused thieno[2,3‐b]pyridin‐ones 19 were formed from N‐chloroacetyl‐2‐aminothiophen‐3‐carbonitriles 16 with pyridine via Thorpe‐Ziegler cyclization and followed by cyclodehydrogenation. In presence of pyridine alkyl 2‐chloro‐acetylaminobenzoates 21 yield 3‐(1‐pyridinio)‐quinoline‐4‐olates 23 . Zincke‐cleavage of 10 and 23 with hydrazinium hydroxide leads to fused 3‐amino‐pyridine‐2‐ones 11 and 3‐amino‐4‐hydroxy‐quinoline‐2‐ones 24 , respectively. Oxazoloquinolines 25 were synthesized from 24 with acetic anhydride.     

The Regioisomeric Triphenylaminoethanols –Comparison of their Efficiency in Enantioselective Catalysis

Both enantiomers of the novel amino alcohol (R)‐ and (S)‐ 2 are prepared from the corresponding enantiomer of the mandelic acid‐derived ethanediol 3 . The regioisomeric amino alcohols 1 and 2 are converted into the imines 7 and 8 , respectively. Titanium complexes 9 and 10 derived therefrom are used as catalysts for the addition of diethylzinc to benzaldehyde and yield the alcohol 11 in up to 92% ee. On the other hand, the chloro‐substituted titanium complexes 14 and 15 are able to mediate the Torgov cyclization reaction of the diketone 16 to give the estrone derivative 17 . In both reactions titanium complexes 10 and 15 derived of the novel amino alcohol 2 give higher enantioselectivities than the complexes 9 and 14 that are based on the regioisomeric amino alcohol 1 .     

Potent,Metabolically Stable 2‐Alkyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐adenines as Adenosine A2A Receptor Ligands

Inhibition of adenosine A_2A receptors has been shown to elicit a therapeutic response in preclinical animal models of Parkinson’s disease (PD). We previously identified the triazolo‐9H‐purine, ST1535, as a potent A_2AR antagonist. Studies revealed that ST1535 is extensively hydroxylated at the ω‐1 position of the butyl side chain. Here, we describe the synthesis and evaluation of derivatives in which the ω‐1 position has been substituted (F, Me, OH) in order to block metabolism. The stability of the compounds was evaluated in human liver microsomes (HLM), and the affinity for A_2AR was determined. Two compounds, (2‐(3,3‐dimethylbutyl)‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐6‐amine ( 3 b ) and 4‐(6‐amino‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐2‐yl)‐2‐methylbutan‐2‐ol ( 3 c ), exhibited good affinity against A_2AR (K_i=0.4 nM and 2 nM , respectively) and high in vitro metabolic stability (89.5 % and 95.3 % recovery, respectively, after incubation with HLM for two hours).     

Design and synthesis of novel cyclooxygenase-1 inhibitors as analgesics: 5-amino-2-ethoxy-N-(substituted-phenyl)benzamides

We previously found that N‐(4‐aminophenyl)‐4‐trifluoromethylbenzamide (TFAP), a COX‐1 inhibitor, exhibits an analgesic effect without causing gastric damage. Unfortunately, TFAP causes reddish purple coloration of urine, and its analgesic effect is less potent than that of indomethacin. Herein we describe our study focusing on the development of 4‐ and 5‐amino‐2‐alkoxy‐N‐phenylbenzamide scaffolds, designed on the basis of the structures of TFAP and parsalmide, another known COX‐1 inhibitory analgesic agent. 5‐Amino‐2‐ethoxy‐N‐(2‐ or 3‐substituted phenyl)benzamide derivatives exhibited analgesic activity in a murine acetic acid induced writhing test. Among these compounds, 5‐amino‐2‐ethoxy‐N‐(2‐methoxyphenyl)benzamide ( 9 v ) possesses potent COX‐1 inhibitory and analgesic activities, similar to those of indomethacin. In addition, 5‐amino‐2‐ethoxy‐N‐(3‐trifluoromethylphenyl)benzamide ( 9 g ) showed a more potent analgesic effect than indomethacin or 9 v without causing apparent gastric damage or coloration of urine, although its COX‐1 inhibitory activity was weaker than that of indomethacin or 9 v . Thus, 9 g and 9 v appear to be promising candidates for analgesic agents and are attractive lead compounds for further development of COX‐1 inhibitors.     

Intermolecular Hydrogen Abstraction Reaction between Nitrogen Radicals in Purine Rings and Alkyl Ethers: A Highly Selective Method for the Synthesis of N‐9 Alkylated Purine Nucleoside Derivatives

A highly selective method for the synthesis of N‐9 alkylated purine nucleoside derivatives via an intermolecular hydrogen abstraction reaction between nitrogen radicals in purine rings and alkyl ethers was developed. Novel purine nucleoside derivatives were obtained with good to high yields in the presence of (diacetoxyiodo)benzene (DIB) and iodine in one‐step reaction.     

Improvement of SNAr Reaction Rate by an Electron‐Withdrawing Group in the Crosslinking of DNA Cytosine‐5 Methyltransferase by a Covalent Oligodeoxyribonucleotide Inhibitor

DNA cytosine 5‐methyltransferase (DNMT) catalyzes methylation at the C5 position of the cytosine residues in the CpG sequence. Aberrant DNA methylation patterns are found in cancer cells. Therefore, inhibition of human DNMT is an effective strategy for treating various cancers. The inhibitors of DNMT have an electron‐deficient nucleobase because this group facilitates attack by the catalytic Cys residue in DNMTs. Recently, we reported the synthesis and properties of mechanism‐based modified nucleosides, 2‐amino‐4‐halopyridine‐C‐nucleosides (d^XP), as inhibitors of DNMT. To develop a more efficient inhibitor of DNMT for oligonucleotide therapeutics, oligodeoxyribonucleotides (ODNs) containing other nucleoside analogues, which react more quickly with DNMT, are needed. Herein, we describe the design, synthesis, and evaluation of the properties of 2‐amino‐3‐cyano‐4‐halopyridine‐C‐nucleosides (d^XP^CN) and ODNs containing d^XP^CN, as more reactive inhibitors of DNMTs. Nucleophilic aromatic substitution (S_NAr) of the designed nucleosides, d^XP^CN, was faster than that of d^XP, and the ODN containing d^XP^CN effectively formed a complex with DNMTs. This study suggests that the incorporation of an electron‐withdrawing group would be an effective method to increase reactivity toward the nucleophile of the DNMTs, while maintaining high specificity.     

Structure-Guided Tuning of a Selectivity Switch towards Ribonucleosides in Trypanosoma brucei Purine Nucleoside 2′-Deoxyribosyltransferase

The use of nucleoside 2′-deoxyribosyltransferases (NDTs) as biocatalysts for the industrial synthesis of nucleoside analogues is often hindered by their strict preference for 2′-deoxyribonucleosides. It is shown herein that a highly versatile purine NDT from Trypanosoma brucei (TbPDT) can also accept ribonucleosides as substrates; this is most likely because of the distinct role played by Asn53 at a position that is usually occupied by Asp in other NDTs. Moreover, this unusual activity was improved about threefold by introducing a single amino acid replacement at position 5, following a structure-guided approach. Biophysical and biochemical characterization revealed that the TbPDT_Y5F variant is a homodimer that displays maximum activity at 50 °C and pH 6.5 and shows a remarkably high melting temperature of 69 °C. Substrate specificity studies demonstrate that 6-oxopurine ribonucleosides are the best donors (inosine>guanosine≫adenosine), whereas no significant preferences exist between 6-aminopurines and 6-oxopurines as base acceptors. In contrast, no transferase activity could be detected on xanthine and 7-deazapurines. TbPDT_Y5F was successfully employed in the synthesis of a wide range of modified ribonucleosides containing different purine analogues.     

Palladium‐Catalyzed Aryl Amination Reactions of 6‐Bromo‐ and 6‐Chloropurine Nucleosides

Palladium‐catalyzed C N bond forming reactions of 6‐bromo‐ as well as 6‐chloropurine ribonucleosides and the 2′‐deoxy analogues with arylamines are described. Efficient conversions were observed with palladium(II) acetate/Xantphos/cesium carbonate, in toluene at 100 °C. Reactions of the bromonucleoside derivatives could be conducted at a lowered catalytic loading [5 mol% Pd(OAc)₂/7.5 mol% Xantphos], whereas good product yields were obtained with a higher catalyst load [10 mol% Pd(OAc)₂/15 mol% Xantphos] when the chloro analogue was employed. Among the examples evaluated, silyl protection for the hydroxy groups appears better as compared to acetyl. The methodology has been evaluated via reactions with a variety of arylamines and by synthesis of biologically relevant deoxyadenosine and adenosine dimers. This is the first detailed analysis of aryl amination reactions of 6‐chloropurine nucleosides, and comparison of the two halogenated nucleoside substrates.     

Synthesis of Chiral Cyclopropyl Carbocyclic Purine Nucleosides via Asymmetric Intramolecular Cyclopropanations Catalyzed by a Chiral Ruthenium(II) Complex

The synthesis of chiral cyclopropyl carbocyclic purine nucleoside analogues via the highly enantioselective intramolecular cyclopropanation reactions has been reported. With a chiral ruthenium(II)‐phenyloxazoline complex as the catalyst, cyclopropyl carbocyclic purine nucleoside analogues containing three contiguous stereocenters were obtained with up to 99% yield and 99% ee. Furthermore, a chiral cyclopropyl carbocyclic adenosine nucleoside having anti‐BLV activity could be synthesized in a concise manner using this strategy.

     

A One‐Pot Synthesis of [1,2,4]Triazino[4,3‐b]‐[1,2,4,5]tetrazines

Reactions of hydrazonoyl halides 6 with either 4‐amino‐2,3‐dihydro‐6‐substituted‐3‐thioxo‐[1,2,4]‐triazin‐5(4H)ones 1 ( 2 ) or 4‐amino‐3‐methylthio‐6‐substituted‐[1,2,4]‐triazin‐5(4H)ones 3 ( 4 ) gave [1,2,4]‐triazino‐[4,3‐b][1,2,4,5]tetrazine derivatives 9 ( 10 ), respectively. The mechanism of the reactions studied is discussed.