Thymidine Derivatives Research Articles

Charge Dependent Substrate Activity of C3′ and N3 Functionalized, Organometallic Technetium and Rhenium-Labeled Thymidine Derivatives toward Human Thymidine Kinase 1

Human cytosolic thymidine kinase (hTK1) has proven to be a suitable target for the noninvasive imaging of cancer cell proliferation using radiolabeled thymidine analogues such as [(18)F]3'-fluoro-3'-deoxythymidine ([(18)F]FLT). A thymidine analogue for single photon emission computed tomography (SPECT), which incorporates the readily available and inexpensive nuclide technetium-99m, would be of considerable practical interest. hTK1 is known to accommodate modification of the structure of the natural substrate thymidine at the positions N3 and C3' and, to a lesser extent, C5. In this work, we used the copper-catalyzed azide-alkyne cycloaddition to synthesize two series of derivatives in which thymidine is functionalized at either the C3' or N3 position with chelating systems suitable for the M(CO)(3) core (M = (99m)Tc, Re). The click chemistry approach enabled complexes with different structures and overall charges to be synthesized from a common precursor. Using this strategy, the first organometallic hTK1 substrates in which thymidine is modified at the C3' position were identified. Phosphorylation of the organometallic derivatives was measured relative to thymidine. We have shown that the influence of the overall charge of the derivatives is dependent on the position of functionalization. In the case of the C3'-functionalized derivatives, neutral and anionic substrates were most readily phosphorylated (20-28% of the value for the parent ligand thymidine), whereas for the N3-functionalized derivatives, cationic and neutral complexes were apparently better substrates for the enzyme (14-18%) than anionic derivatives (9%).

Human polymerase α inhibitors for skin tumors. Part 2. Modeling, synthesis and influence on normal and transformed keratinocytes of new thymidine and purine derivatives

Recently, the three-dimensional structure of the active site of human DNA polymerase α (pol α) was proposed based on the application of molecular modeling methods and molecular dynamic simulations. The modeled structure of the enzyme was used for docking selective inhibitors (nucleotide analogs and the non-nucleoside inhibitor aphidicolin) in its active site in order to design new drugs for actinic keratosis and squamous cell carcinoma (SCC). The resulting complexes explained the geometrical and physicochemical interactions of the inhibitors with the amino acid residues involved in binding to the catalytic site, and offered insight into the experimentally derived binding data. The proposed structures were synthesized and tested in vitro for their influence on human keratinocytes and relevant tumor cell lines. Effects were compared to aphidicolin which inhibits pol α in a non-competitive manner, as well as to diclofenac and 5-fluorouracil, both approved for therapy of actinic keratosis. Here we describe three new nucleoside analogs inhibiting keratinocyte proliferation by inhibiting DNA synthesis and inducing apoptosis and necrosis. Thus, the combination of modeling studies and in vitro tests should allow the derivation of new drug candidates for the therapy of skin tumors, given that the agents are not relevant substrates of nucleotide transporters expressed by skin cancer cells. Kinases for nucleoside activation were detected, too, corresponding with the observed effects of nucleoside analogs.

A convenient protection for 4-oxopyrimidine moieties in nucleosides by the pivaloyl group

Application of the pivaloyl group as a protection for the N3 position of thymidine and uridine was investigated. Pivaloylation of thymidine is a very rapid reaction proceeding under mild conditions with excellent regioselectivity for sugar or thymine moiety, depending on the amines used. Several pivaloylated thymidine derivatives were obtained by treatment of unprotected thymidine with pivaloyl chloride under various experimental conditions. Stability of the N3-pivaloyl protecting group under basic and acidic conditions was evaluated and the conditions for its selective removal were found.

Synthesis of thymidine derivatives bearing aromatic oligoamides with rigidified backbone

Synthesis of thymidine derivatives bearing aromatic oligoamides with rigidified backbone

A ‘click chemistry’ approach to the efficient synthesis of modified nucleosides and oligonucleotides for PET imaging

Different thymidine derivatives bearing either an iodoaryl moiety at the 5′ position or a dialkylsilyl group at the 3′ position have been efficiently synthesized as precursors for carbon-11 or fluorine-18 labeling, respectively. Furthermore, iodoarylated thymidine derivatives have been incorporated into oligonucleotides giving an original way to label them with carbon-11.

Reaction of the 4'-benzenesulfonyl derivatives of thymidine with organosilicon and organoaluminum reagents

With an aim to develop a new approach to synthesize 4'-substituted nucleosides, reactions of thymidine derivatives having a benzenesulfonyl leaving group at the 4'-position with organosilicon and organoaluminum reagents were investigated. Two substrates 4alpha (alpha-L-isomer) and 4 beta (beta-D-isomer) were prepared for this purpose. Although reaction of 4alpha with organosilicon reagents gave preferentially the 4'-substituted (allyl and N(3)) beta-D-nucleoside, its reaction with AlMe(3) gave the 4'-methyl-alpha-L-thymidine as the major product. On the other hand, the substrate 4beta, upon reacting with AlMe(3), furnished the desired 4'-methylthymidine exclusively in high yield.

Functionalized thymidine derivatives as carriers for the γ-emitter technetium tricarbonyl moiety

Nuclear medicine imaging of cell proliferation has gained broad interest in clinical oncology. A good tracer to image cell proliferation, possibly associated to tumour progression, should rapidly and specifically be incorporated into growing DNA. Following this idea, we have singled out thymidine as a potential biological carrier for delivery of the organometallic fragment [M(CO) 3] + (M = “cold” Re, γ-emitter 99mTc) to DNA. In the present paper we report the two-step synthesis of thymidine N-3 derivatives and their coordination complexes. Thymidine could be alkylated without protecting the sugar alcoholic functions. Methyl-2,2′-diaminodiethylamine thymidine derivative was quickly reacted with rhenium and technetium carbonyls in almost quantitative yield. The resulting 99mTc complex represents a convenient radiopharmaceutical for single photon emission tomography (SPECT).

Organometallic [Re(CO)3]+and [Re(CO)2(NO)]2+Labeled Substrates for Human Thymidine Kinase 1

Thymidine was functionalized at position N3 with a tridentate iminodiacetic acid chelating system and a potentially tetradentate mercaptoethyliminodiacetic acid chelating system. Spacers of different lengths (ethyl and butyl) were introduced between the chelators and thymidine. The derivatives were labeled with the [Re(CO)(2)(NO)](2+) and [Re(CO)(3)](+) cores to give isostructural complexes with different overall charges. All complexes were analyzed by NMR, MS, and IR, and in addition, the X-ray structure of a [Re(CO)(2)(NO)](2+) labeled thymidine derivative functionalized at the N3 position was solved. The ligands incorporating the potentially tetradentate mercaptoethyliminodiacetic acid chelating system coordinated tridentately through iminodiacetic acid to both the [Re(CO)(2)(NO)](2+) core and the [Re(CO)(3)](+) core. This was surprising given that the reaction of [NEt(4)][Re(CO)(2)(NO)Br(3)] with the model ligand ethylmercaptoethyliminodiacetic acid led to dissociation of a carbonyl ligand and formation of a monocarbonyl-mononitrosyl complex, as confirmed by X-ray structure analysis. All of the organometallic thymidine derivatives were substrates for human thymidine kinase 1, a key enzyme in (cancer) cell proliferation. Neutral [Re(CO)(2)(NO)](2+) labeled thymidine derivatives revealed substrate activity ranging from 24 to 40%, and the structurally analogous anionic [Re(CO)(3)](+) labeled thymidine derivatives from 20 to 38% compared with the natural substrate thymidine.

Nucleophilic substitution approach to 4′-substituted thymidines by employing 4′-benzenesulfonyl leaving group

Synthesis of 4′-substituted thymidines was investigated based on nucleophilic substitution using organosilicon and organoaluminum reagents. Two substrates having a benzenesulfonyl leaving group at the 4′-position were prepared for this purpose: 1-[4-benzenesulfonyl-3,5-bis- O-( tert-butyldimethylsilyl)-2-deoxy-α- l- threo-pentofuranosyl]thymine ( 8α) and the 4′-(benzenesulfonyl)thymidine derivative ( 8β). The reaction of 8α with organosilicon reagents (Me 3SiCH 2CH CH 2 and Me 3SiN 3) in combination with SnCl 4 gave preferentially the 4′-substituted β- d-isomer: the 4′-allyl ( 12β) and 4′-azido ( 15β) derivatives, respectively. The reaction of 8α with AlMe 3, however, gave the 4′-methyl-α- l-isomer ( 16α) as the major product, presumably through an ion pair mechanism. By employing the substrate 8β in this reaction, the 4′-methylthymidine derivative ( 16β) was obtained exclusively in high yield. The 4′-ethyl ( 20β) and 4′-cyano ( 24β) derivatives were also synthesized by reacting 8β with the respective organoaluminum reagent.

2-({3-[(2R,4S,5R)-4-Hydr-oxy-5-hydroxy-methyl-2,3,4,5-tetra-hydro-furan-2-yl]-5-methyl-2,6-dioxo-1,2,3,6-tetra-hydro-pyrimidin-1-yl}meth-yl)isoindoline-1,3-dione.

The title compound, C19H19N3O7, is a thymidine derivative and serves as an inter­mediate in the synthesis of a 99mTc radiolabeled nucleoside analog. Inter­molecular O—H⋯O hydrogen bonding is observed between the hydr­oxy functionalities of the ribose unit themselves as well as between a hydr­oxy group and an O atom of the phthalimide group of an adjacent mol­ecule. The mol­ecules are stacked on top of each other in the direction of the a axis. The crystal packing is further stabilized by weak intra- and inter­molecular C—H⋯O hydrogen bonds. The absolute configuration of the compound is known from the synthesis.

Facile Synthesis and Anti-Tumor Cell Activity of Se-Containing Nucleosides

Many organic compounds containing selenium have shown anticancer effects and some have been used in chemoprevention of cancers and other diseases. Though Se-containing amino acids are generally used for these purposes, the natural nucleosides may also be used as Se-carriers for these important applications. Therefore, we describe here the convenient synthesis of the new 3′-MeSe-thymidine nucleoside and the other uridine and thymidine derivatives modified with MeSe at the 2′ and 5′ positions, and report their anti-tumor activity against prostate cancer cell lines. Our work demonstrates for the first time anticancer activity of the methylseleno nucleosides.

Design, synthesis, and evaluation of a novel bridged nucleic acid, 2′,5′-BNA ON, with S-type sugar conformation fixed by N– O linkage

We designed a novel 2′- O,5′- N bridged nucleic acid, 2′,5′-BNA ON, whose sugar puckering was fixed to S-type conformation by an N– O linkage. A dimer unit formed from 2′,5′-BNA ON-U and thymidine was synthesized via a coupling reaction between a protected 2′,5′-BNA ON-U monomer and a thymidine derivative. Introduction of 2′,5′-BNA ON-U into DNA was carried out using conventional phosphoramidite chemistry with a DNA synthesizer. The hybridization abilities of 2′,5′-BNA ON-U-modified oligonucleotides against DNA or RNA complement were evaluated.

Synthesis of 5‐Formyl‐2′‐Deoxyuridine and Its Incorporation into Oligodeoxynucleotides

A straightforward, efficient method for the synthesis of 5-formyl-2'-deoxyuridine (dfU) and solid-phase synthesis of oligodeoxynucleotides containing dfU using a phosphoramidite method are described. The synthesis of dfU is achieved by oxidation of the 5-methyl group in thymidine derivatives. However, incorporation of the dfU 3'-O-phosphoramidite into oligodeoxynucleotides proceeds in low yield, due to instability of the 5-formyl group under conditions used for automated DNA synthesis. Therefore, oligodeoxynucleotides containing a 5-(1,2-dihydroxyethyl)uracil derivative are first prepared and finally oxidized by periodate to give the desired oligodeoxynucleotides containing 5-formyluracil.

Chemical synthesis of oligodeoxyribonucleotides containing N 3- and O4 -carboxymethylthymidine and their formation in DNA

Humans are exposed to N-nitroso compounds from both endogenous and exogenous sources. Many N-nitroso compounds can be metabolically activated to give diazoacetate, which can result in the carboxymethylation of DNA. The remarkable similarity in p53 mutations found in human gastrointestinal tumors and in shuttle vector studies, where the human p53 gene-containing vector was treated with diazoacetate and propagated in yeast cells, suggests that diazoacetate might be an important etiological agent for human gastrointestinal tumors. The O6-carboxymethyl-2′-deoxyguanosine was previously detected in isolated DNA upon exposure to diazoacetate and in blood samples of healthy human subjects. The corresponding modifications of thymidine and 2′-deoxyadenosine have not been assessed, though significant mutations at A:T base pairs were found in the p53 tumor suppressor gene in human gastrointestinal tumors and in shuttle vector studies. To understand the implications of the carboxymethylation chemistry of thymidine in the observed mutations at A:T base pairs, here we synthesized authentic N3-carboxymethylthymidine (N3-CMdT) and O4-carboxymethylthymidine (O4-CMdT), incorporated them into DNA, and demonstrated, for the first time, that they were the major carboxymethylated derivatives of thymidine formed in calf thymus DNA upon exposure to diazoacetate. The demonstration of the formation of N3-CMdT and O4-CMdT in isolated DNA upon treatment with diazoacetate, together with the preparation of authentic oligodeoxyribonucleotide substrates housing these two lesions, laid the foundation for investigating the replication and repair of these lesions and for understanding their implications in the mutations observed in human gastrointestinal tumors.

Synthesis, In Vitro, and In Silico Evaluation of Organometallic Technetium and Rhenium Thymidine Complexes with Retained Substrate Activity toward Human Thymidine Kinase Type 1

Human cytosolic thymidine kinase (hTK1) has proven to be a suitable target for noninvasive imaging of cancer cell proliferation using radiolabeled substrates such as [ (18)F]fluorothymidine ([ (18)F]FLT). However, a thymidine tracer useful for single photon emission tomography (SPECT) based on the inexpensive radionuclide technetium-99m would be of significant interest. In this work, a series of thymidine derivatives labeled with the organometallic [M(CO) 3] (+) core (M = (99m)Tc, Re) were synthesized. Neutral, cationic, and anionic complexes were readily formed in aqueous media, and all were substrates of recombinant hTK1 when incubated with ATP. The neutral complexes were phosphorylated to a greater extent than the charged complexes. The extent of phosphorylation was further improved by increasing the spacer length separating thymidine and the organometallic core. A molecular dynamics simulation study performed with a modified hTK1 structure supported the experimental findings. In vitro cell internalization experiments performed in a human neuroblastoma cell line (SKNMC) showed low uptake of the charged complexes but significant uptake for the neutral, lipophilic complexes with a log P value >1.

BODIPY-modified uridines as potential fluorescent probes for nucleic acids that are recognized by DNA-polymerases

A new type of BODIPY-modified uridines that contain the fluorophore attached to the 5-position of uridine via a short phenylene bridge have been prepared and characterized by methods of the optical spectroscopy and electrochemistry. The spectroscopic and redox properties can be manipulated by substituent and ligand exchange. After synthetic incorporation into DNA via phosphoramidite chemistry the canonical base pairing is maintained due to the rigid phenylene bridge. Moreover, primer extension experiments show that BODIPY-modified uridines are still recognized as thymidine derivatives by DNA-polymerases and adenine is inserted as the correct counter base.

Nucleic acid duplexes with zippers of additional nucleobases and aromatics in minor or major groove

Two series of thymidine derivatives with additional nucleobases/aromatics attached to either the 5'(S)-C- or the 5-position were prepared by epoxide opening and/or "click chemistry" cycloaddition protocols and introduced into DNA duplexes. Interstrand base-base communication in the minor groove and intrastrand stacking interactions in the major groove were detected.

SO2-Extrusion Reactions of Sulfonylated Nucleosides: A Novel Strategy for the Synthesis of Exocyclic Olefinic Thymidines

Analogues of 3′-C-methylidene-2′,3′-dideoxy-5-meth­yluridine have been synthesized from the 3′-deoxy-3′-sulfonylated derivatives of thymidine using sulfur dioxide extrusion reaction.

Theoretical investigation of 3'-substituted-2'-3'-dideoxythymidines related to AZT. SAR, infrared and substituent electronic effect studies

Several substituted 3'-azido-3'-deoxythymidine derivatives have been theoretically investigated by performing the BLYP to obtain approximate minimum energy structures, as well as the DZVP basis set. A quantitative structure–activity relationship (QSAR) of the same series, in regard to their anti HIV, has been studied. The substituents investigated are X= N3, H, Cl, F, CH3, OCH3, COCH3 and COOH as well as the linearity groups such as -NO, -N=CHAr, and CN. The vibrational spectrum was calculated beyond the harmonic approximation. The correlation analysis of νOH values failed to correlate with substituent Hammett's σ parameter. The electrondonating groups at C-3’ of the thymidine derivatives are more suitable than N3 group and would increase the electron density on the C5’-OH group.

Interstrand Cross-Link Formation in Duplex and Triplex DNA by Modified Pyrimidines

DNA interstrand cross-links have important biological consequences and are useful biotechnology tools. Phenylselenyl substituted derivatives of thymidine (1) and 5-methyl-2'-deoxycytidine (5) produce interstrand cross-links in duplex DNA when oxidized by NaIO4. The mechanism involves a [2,3]-sigmatropic rearrangement of the respective selenoxides to the corresponding methide type intermediates, which ultimately produce the interstrand cross-links. Determination of the rate constants for the selenoxide rearrangements indicates that the rate-determining step for cross-linking is after methide formation. Cross-linking by the thymidine derivative in duplex DNA shows a modest kinetic preference when flanked by pyrimidines as opposed to purines. In contrast, the rate constant for cross-link formation from 5 opposite dG in duplex DNA is strongly dependent upon the flanking sequence and, in general, is at least an order of magnitude slower than that for 1 in an otherwise identical sequence. Introduction of mispairs at the base pairs flanking 5 or substitution of the opposing dG by dI significantly increases the rate constant and yield for cross-linking, indicating that stronger hydrogen bonding between the methide derived from it and dG compared to dA and the respective electrophile derived from 1 limits reaction by increasing the barrier to rotation into the required syn-conformation. Incorporation of 1 or 5 in triplex forming oligonucleotides (TFOs) that utilize Hoogsteen base pairing also yields interstrand cross-links. The dC derivative produces ICLs approximately 10x faster than the thymidine derivative when incorporated at the 5'-termini of the TFOs and higher yields when incorporated at internal sites. The slower, less efficient ICL formation emanating from 1 is attributed to reaction at N1-dA, which requires local melting of the duplex. In contrast, 5 produces cross-links by reacting with N7-dG. The cross-linking reactions of 1 and 5 illustrate the versatility and utility of these molecules as mechanistic probes and tools for biotechnology.