Sculpting the bicyclo[3.1.0]hexane template of carbocyclic nucleosides to improve recognition by herpes thymidine kinase

The replacement of the furanose ring by a cyclopentane in nucleosides generates a group of analogues known generically as carbocyclic nucleosides. These compounds have increased chemical and enzymatic stability due to the absence of a true glycosyl bond that characterizes conventional nucleosides. The additional fusion of a cyclopropane ring to the cyclopentane produces a bicyclo[3.1.0]hexane system that depending on its location relative to the nucleobase is able to lock the embedded cyclopentane ring into conformations that mimic the typical north and south conformations of the furanose ring in conventional nucleosides. These bicyclo[3.1.0]hexane templates have already provided important clues to differentiate the contrasting conformational preferences between kinases and polymerases. Herein, we describe the design, synthesis, and phosphorylation pattern of a new bicyclo[3.1.0]hexane thymidine analogue that seems to possess an ideal spatial distribution of pharmacophores for an optimal interaction with herpes simplex 1 thymidine kinase. The bicyclo[3.1.0]hexane template represents a privileged rigid template for sculpting other carbocyclic nucleosides to meet the demands of specific receptors.     

Synthesis of nucleoside analogues bearing the five naturally occurring nucleic acid bases built on a 2-oxabicylo[3.1.0]hexane scaffold

Hitherto unknown nucleoside analogues incorporating the five naturally occurring nucleic acid bases built on a 2-oxabicyclo[3.1.0]hexane template were synthesized. The synthesis of these new conformationally restricted nucleoside analogues involved the preparation of a suitable sugar precursor bearing the 2-oxabicyclo[3.1.0]hexane scaffold. This sugar was readily obtained from [(3aS,6aS)-2,2-dimethyl-3a,6a-dihydrofuro[2,3-d][1,3]dioxol-5-yl]methyl benzyl ether (4) following a Simons-Smith-type cyclopropanation reaction. Finally, glycosylation reactions and deprotection provided the nucleoside analogues. Using nucleoside 14 bearing thymine base as a model, we found that the conformation of such nucleoside analogue was restricted toward a (0)T(1) conformation.     

Synthesis of conformationally locked carbocyclic nucleosides built on an oxabicyclo[3.1.0]hexane system

The rigid 6-oxobicyclo[3.1.0]hexane scaffold, characteristic of the natural antibiotic neplanocin C (3), was used to build prototypes of conformationally locked deoxynucleosides in the North hemisphere of the pseudorotational cycle. The purine analogues 6 and 7 are conformationally equivalent to carbocyclic nucleosides built with the bicyclo[3.1.0]hexane template. The pyrimidine nucleosides were unstable and underwent a facile intramolecular epoxide ring-opening reaction leading to heterocycle 22. Only the deoxyguanosine analogue 7 showed antiviral activity against EBV.     

Construction of the bicyclo[3.1.0]hexane template of a conformationally locked carbocyclic adenosine via an olefin keto-carbene cycloaddition

An intramolecular olefin keto-carbene cycloaddition reaction created the bicyclo[3.1.0]hexane template 10 that was necessary for the synthesis of carbocyclic amine 15. This amine is a direct precursor to a family of rigid nucleosides that are conformationally locked in the Southern hemisphere of the pseudorotational cycle. The synthesis of the conformationally locked adenosine analogue is reported herein as an illustrative example of the methodology. The racemic (South)-methanocarba adenosine analogue (+/-)-4 is the first example of a conformationally locked ribonucleoside version in the Southern hemisphere.     

Enantioselective synthesis of bicyclo[3.1.0]hexane carbocyclic nucleosides via a lipase-catalyzed asymmetric acetylation. Characterization of an unusual acetal byproduct

The bicyclo[3.1.0]hexane scaffold can lock the conformation of a carbocyclic nucleoside into one of the two antipodal (north or south) conformations typical of conventional nucleosides that normally exist in a rapid, two-state equilibrium in solution. In a recent brief communication, we reported a practical method to access the requisite bicyclo[3.1.0]hexane pseudosugar for the north antipode via an intramolecular olefin-ketocarbene cycloaddition. The most attractive features of this synthesis was that a relatively complex synthon was obtained from simple and inexpensive starting materials and that the resulting racemic mixtures of purine nucleosides could be successfully resolved by adenosine deaminase (ADA) hydrolysis. In this work, we describe the development of a more general, lipase-catalyzed double-acetylation reaction, which could successfully resolve an earlier precursor, 4-(tert-butyldiphenylsilamethoxy)-1-(hydroxymethyl)bicyclo[3.1.0]hexan-2-ol [(+/-)-7], into enantiomerically pure (+)-diacetate 8 and (-)-monoacetate 9. The former diacetate was converted to the conformationally locked (north)-carbocyclic guanosine (+)-17 identical to the one obtained previously by ADA resolution. The present method represents a more general and efficient process applicable to the synthesis of all classes of (north) bicyclo[3.1.0]hexane nucleosides, including pyrimidine analogues. During the lipase-catalyzed resolution, we were able to demonstrate the presence of an unusual acetal-forming reaction that consumed small amounts of the unreactive monoacetate (-)-9. This side reaction was also enzyme-catalyzed and was triggered by the byproduct acetaldehyde generated during the reaction.     

Understanding how the herpes thymidine kinase orchestrates optimal sugar and nucleobase conformations to accommodate its substrate at the active site: a chemical approach

The herpes virus thymidine kinase (HSV-tk) is a critical enzyme for the activation of anti-HSV nucleosides. However, a successful therapeutic outcome depends not only on the activity of this enzyme but also on the ability of the compound(s) to interact effectively with cellular kinases and with the target viral or cellular DNA polymerases. Herein, we describe the synthesis and study of two nucleoside analogues built on a conformationally locked bicyclo[3.1.0]hexane template designed to investigate the conformational preferences of HSV-tk for the 2'-deoxyribose ring. Intimately associated with the conformation of the 2'-deoxyribose ring is the value of the C-N torsion angle chi, which positions the nucleobase into two different domains (syn or anti). The often-conflicting sugar and nucleobase conformational parameters were studied using North and South methanocarbadeoxythymidine analogues (6 and 7), which forced HSV-tk to make a clear choice in the conformation of the substrate. The results provide new insights into the mechanism of action of this enzyme, which cannot be gleaned from a static X-ray crystal structure.     

An efficient synthesis of endo,exo-furofuranone derivatives

The ring openings of 1-acetyl-4-phenyl-3-oxabicyclo[3.1.0]hexane afforded alpha-acetyl-gamma-butyrolactone that underwent a novel diazo-transfer reaction, followed by C-H insertion, to provide a series of endo,exo-furofuranone analogues.     

Lipase-catalyzed synthesis of a tri-substituted cyclopropyl chiral synthon: a practical method for preparation of chiral 1-alkoxycarbonyl-2-oxo-3-oxabicyclo[3.1.0]hexane

An efficient method for the preparation of optically active enantiomers of 1-ethoxycarbonyl-2-oxo-3-oxabicyclo[3.1.0]hexane 1 has been developed. Treatment of 1 with lipase Amano PS gave (1S,5R)-1-carboxy-2-oxo-3-oxabicyclo[3.1.0]hexane 4a which was converted to (1S,5R)-1-ethoxycarbonyl-2-oxo-3-oxabicyclo[3.1.0]hexane 1a with high enantiomeric purity (98.0% ee, 75% yield), while the (1R,5S)-lactone ester 1b remained intact. A simple procedure for the recovery of 4a from the reaction mixture was also established.     

First synthesis of 2′-oxabicyclo[3.1.0]hexyl nucleosides with a north conformation

The first synthesis of 2′-oxabicyclo[3.1.0]hexyl nucleosides, a novel class of bicyclonucleosides, with a north conformation was successfully accomplished starting from (S)-epichlorohydrin via a tandem alkylation-lactonization, a less steric hindrance-dependent silylation in equilibrium and a coupling reaction with nucleobases under Vorbruggen conditions. Addition of acetic acid prevented a benzoyl group from migrating during desilylation with TBAF. ¹H NMR and X-ray crystallographic analysis indicated that the anomeric effect worked on the β-2′-oxabicyclo[3.1.0]hexyl nucleosides.     

Preparation and reactions of some 2,2-difunctional 1,1-dibromocyclopropanes

The synthesis of 2,2-dibromocyclopropane-1,1-dicarboxylic acids is described. Reaction of substituted 1,1-dibromo-2-acyloxymethylcyclopropanes with methyl lithium at low temperature leads to a bromine-lithium exchange and then either formal protonation to give the corresponding monobromocyclopropanes or intramolecular cyclisation to give a substituted 3-oxabicyclo[3.1.0]hexane. Oxidative ring opening of these compounds leads stereoselectively to 1,1,2,2-tetrasubstituted cyclopropanes with four functionalities on the ring.     

Reaction of nitrenes with strained olefins. Preparation and reactions of some 6-azabicyclo[3.1.0]hexanes

A number of examples of the 6-azabicycIo[3.1.0]hexane ring system have been prepared by the oxidation of N-aminophthalimide or 3-amino-2-methyl-4-quinazoIone with lead tetraacetate in the presence of variously substituted cyclopentenes. Thus, 6-phthalimidyl-6-azabicyclo[3.1.0]hexane, dimethy 1–6-phthalimidyl-6-azabicyclo[3.1.0]-hexane-1,5-dicarboxylate, 2,3-benzo-6-phthalimidyl-6-azabicycIo[3.1.0]hexane and N-3-(2-methyl-4-quinazolyl)-6-azabicyclo [3.1.0]hexane were prepared for the first time. All of the new compounds were found to be stable in refluxing carbon tetrachloride and chlorobenzene. Refluxing 6-phthalimidyl-6-azabieyclo[3,1.0]hexane in acetic acid for 24 hours resulted in quantitative rearrangement to a phthalohydrazide, 8 .     

Enantioselective synthesis of bicyclo[6.1.0]nonane-9-carboxylic acids via Me2AlOTf-promoted intramolecular Friedel-Crafts alkylation of arenes with the gamma-lactone moiety of 3-oxabicyclo[3.1.0]hexan-2-ones

A strategy to rapidly assemble enantiomerically pure bicyclo[6.1.0]nonane-9-carboxylic acids via Me2AlOTf-promoted intramolecular Friedel-Crafts alkylation of tethered pi-nucleophiles with the gamma-lactone moiety of 3-oxabicyclo[3.1.0]hexan-2-ones is described. The approach begins with the enantioselective synthesis of 3-oxabicyclo[3.1.0]hexan-2-ones bearing a tethered pi-nucleophile at the 6-position by intramolecular Rh(II)-catalyzed cyclopropanation of allylic diazoacetates, prepared from the corresponding (Z)-allylic alcohols. Me2AlOTf-induced intramolecular Friedel-Crafts cyclization provides medium-sized carbocycles and heterocycles in high yields without requiring high-dilution or slow substrate addition techniques. The scope and limitations of this synthetic methodology are presented.     

Synthesis of cyclopropane-fused α-chlorolactones utilizing the nucleophilicity of cyclopropylmagnesium carbenoids

A novel method for the synthesis of cyclopropane-fused α-chloro-γ-lactones was developed utilizing the nucleophilicity of cyclopropylmagnesium carbenoids. Cyclopropylmagnesium carbenoids were generated from i-PrMgCl and 1-chlorocyclopropyl p-tolyl sulfoxides with a [(phenoxycarbonyl)oxy]methyl group at the 2-position of the cyclopropane ring. The resulting cyclopropylmagnesium carbenoids reacted with an intramolecular carbonate unit to give 1-chloro-3-oxabicyclo[3.1.0]hexan-2-ones in moderate to good yields. The asymmetric synthesis of 1-chloro-3-oxabicyclo[3.1.0]hexan-2-one was achieved using optically active dichloromethyl p-tolyl sulfoxide as a starting material.     

Synthesis of bicyclo[3.1.0]hexane derivatives as conformationally restricted analogues of beta-arabinofuranosyl and alpha-galactofuranosyl rings

A route for the synthesis of bicyclo[3.1.0]hexane-derived conformationally restricted analogues of beta-arabinofuranosyl and alpha-galactofuranosyl rings is described. Advantage is taken of the pseudo-enantiomeric relationship between the two ring systems to develop a route that provides both targets from a single precursor. Key steps include a base-promoted ring contraction of an epoxy ketone obtained from cyclohexane-1,4-dione to give the bicyclo[3.1.0]hexane ring system and a late stage resolution involving esterification with O-acetyl-(S)-mandelic acid.     

Synthesis of conformationally constrained lysine analogues

The synthesis of two conformationally constrained lysine analogues is reported. The synthesis of the novel analogue 1 based on the 3-aza-bicyclo[3.1.0]hexane system is accomplished from the known tricycle 3 in eight steps. The synthesis of the analogue 2 is accomplished in eight steps from 4-hydroxy proline. Both analogues are synthesized appropriately protected for Fmoc/Boc solid-phase peptide synthesis.     

Synthesis and structure of novel conformationally constrained 1',2'-azetidine-fused bicyclic pyrimidine nucleosides: their incorporation into oligo-DNAs and thermal stability of the heteroduplexes

[structures: see text] The synthesis of novel 1',2'-aminomethylene bridged (6-aza-2-oxabicyclo[3.2.0]heptane) "azetidine" pyrimidine nucleosides and their transformations to the corresponding phosphoramidite building blocks (20, 39, and 42) for automated solid-phase oligonucleotide synthesis is reported. The novel bicyclonucleoside "azetidine" monomers were synthesized by two different strategies starting from the known sugar intermediate 6-O-benzyl-1,2:3,4-bis-O-isopropylidene-D-psicofuranose. Conformational analysis performed by molecular modeling (ab initio and MD simulations) and NMR showed that the azetidine-fused furanose sugar is locked in a North-East conformation with pseudorotational phase angle (P) in the range of 44.5-53.8 degrees and sugar puckering amplitude (phi(m)) of 29.3-32.6 degrees for the azetidine-modified T, U, C, and 5-Me-C nucleosides. Thermal denaturation studies of azetidine-modified oligo-DNA/RNA heteroduplexes show that the azetidine-fused nucleosides display improved binding affinities when compared to that of previously synthesized North-East sugar constrained oxetane fused analogues.     

Intramolecular Radical Aziridination of Allylic Sulfamoyl Azides by Cobalt(II)‐Based Metalloradical Catalysis: Effective Construction of Strained Heterobicyclic Structures

Cobalt(II)‐based metalloradical catalysis (MRC) has been successfully applied for effective construction of the highly strained 2‐sulfonyl‐1,3‐diazabicyclo[3.1.0]hexane structures in high yields through intramolecular radical aziridination of allylic sulfamoyl azides. The resulting [3.1.0] bicyclic aziridines prove to be versatile synthons for the preparation of a diverse range of 1,2‐ and 1,3‐diamine derivatives by selective ring‐opening reactions. As a demonstration of its application for target synthesis, the metalloradical intramolecular aziridination reaction has been incorporated as a key step for efficient synthesis of a potent neurokinin 1 (NK₁) antagonist in 60 % overall yield.     

Using conformationally locked nucleosides to calibrate the anomeric effect: Implications for glycosyl bond stability

Steric and electronic parameters, such as the anomeric effect (AE) and gauche effect play significant roles in steering the North⇆South equilibrium of nucleosides in solution.Two isomeric oxa-bicyclo[3.1.0]hexane nucleosides that are conformationally locked in either the North or the South conformation of the pseudorotational cycle were designed to study the consequences of having the AE operational or not, independent of other parameters. The rigidity of the system allowed the orientation of the orbitals involved to be set in ‘fixed’ relationships, either antiperiplanar where the AE is permanently ‘on’, or gauche where the AE is impaired. The consequences of these two alternatives were subjected to high-level calculations and measured experimentally by X-ray crystallography, hydrolytic stability of the glycosyl bond, and pK_a values.     

Development of Bicyclo[3.1.0]hexane-Based A3 Receptor Ligands: Closing the Gaps in the Structure–Affinity Relationships

The adenosine A₃ receptor is a promising target for treating and diagnosing inflammation and cancer. In this paper, a series of bicyclo[3.1.0]hexane-based nucleosides was synthesized and evaluated for their P1 receptor affinities in radioligand binding studies. The study focused on modifications at 1-, 2-, and 6-positions of the purine ring and variations of the 5′-position at the bicyclo[3.1.0]hexane moiety, closing existing gaps in the structure–affinity relationships. The most potent derivative 30 displayed moderate A₃AR affinity (Ki of 0.38 μM) and high A₃R selectivity. A subset of compounds varied at 5′-position was further evaluated in functional P2Y₁R assays, displaying no off-target activity.     

1H-NMR-spektroskopische Untersuchungen an 1-Ethynyl-6-oxabicyclo[3.1.0]hexanen

The¹H-NMR-spectra of a number of substituted 1-ethynyl-6-oxabicyclo[3.1.0]hexanes are discussed with regard to the influence of the ring size of the carbocyclic ring and the substituents at the triple bond on the chemical shift and coupling constant of the epoxidic proton with the vicinal methylene protons.