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A variety of 4'-aryl-3-(arylmethylidene)-1″-[(cyclic-amino)methylene]-1'-methyl-dispiro[cyclohexane-1,3'-pyrrolidine-2',3″-[3H]indole]-2,2″(1″H)-diones 4a-u were prepared via reaction of 2E,6E-bis(arylidene)-1-cyclohexanones 1a-i with azomethine ylides, generated in situ via a decarboxylative condensation of isatins 2a-c and sarcosine (3). Single crystal X-ray study of 4a, revealed structural and stereochemical features of these derivatives. While most of the synthesized compounds exhibit mild antitumor properties when tested against various human tumor cell lines (HEPG2 "liver", HELA "cervical" and PC3 "prostate" cancers), three of them, 4d and 4p (active against HEPG2), and compound 4g (active against HELA), demonstrated higher activities, that were close or even higher than that of the reference standard Doxorubicin. QSAR studies revealed good predictive and statistically significant 3 descriptor models (r2=0.903-0.812, r2adjusted=0.855-0.672, r2prediction=0.773-0.605)., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Nucleoside H-phosphonates activated with a condensing agent spontaneously formed nucleoside 3',5'-cyclic H-phosphonates. The cyclization was stereoselective and produced one of the P-diastereomers in preponderance (de ca. 80%). Nucleoside 3',5'-cyclic H-phosphonates were stereochemically unstable and underwent epimerization affording the thermodynamically more stable diastereomer as a major product (de ca. 80%). They were susceptible to hydrolysis, transesterification, and oxidation and by changing oxidation protocols nucleoside 3',5'-cyclic phosphate analogues, e.g. phosphodiesters, phosphorothioate diesters, and phosphotriesters, were obtained.

The mechanism and sources of selectivity in the palladium-catalyzed propargylic substitution reaction that involves phosphorus nucleophiles, and which yields predominantly allenylphosphonates and related compounds, have been studied computationally by means of density functional theory. Full free-energy profiles are computed for both H-phosphonate and H-phosphonothioate substrates. The calculations show that the special behavior of H-phosphonates among other heteroatom nucleophiles is indeed reflected in higher energy barriers for the attack on the central carbon atom of the allenyl/propargyl ligand relative to the ligand-exchange pathway, which leads to the experimentally observed products. It is argued that, to explain the preference of allenyl- versus propargyl-phosphonate/phosphonothioate formation in reactions that involve H-phosphonates and H-phosphonothioates, analysis of the complete free-energy surfaces is necessary, because the product ratio is determined by different transition states in the respective branches of the catalytic cycle. In addition, these transition states change in going from a H-phosphonate to a H-phosphonothioate nucleophile., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

1,3-Dipolar cycloaddition reaction of 1-aryl-1H-pyrrole-2,5-diones 1a-e with non-stabilized azomethine ylides, generated in situ via decarboxylative condensation of isatins 2a-c and sarcosine (3) in refluxing ethanol, afforded 4'-aryl-5'a,6'-dihydro-1'-methyl-spiro[3H-indole-3,2'(1'H)-pyrrolo[3,4-c]pyrrole]-2,3',5'(1H,2'aH,4'H)-triones 4a-o in good yields. Compound 4l exhibited high anti-tumor activity against HEPG2 (liver cancer) cell line (IC(50) = 12.16 μM) compared to that of Doxorubicin (IC(50) = 7.36 μM), and the other synthesized compounds revealed moderate anti-tumor properties against HCT116 (colon), MCF7 (breast) and HEPG2 (liver) human tumor cell lines. 3D-Pharmacophore modeling and quantitative structure-activity relationship (QSAR) analysis were combined to explore the structural requirements controlling the observed anti-tumor properties. It was found that the major structural factors affecting potency of these compounds were related to their basic skeleton., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

31P NMR spectroscopy was used to investigate a stereochemical course of a nitrite-promoted conversion of phosphoramidate diesters into the corresponding phosphotriesters. It was found that this reaction occurred with almost complete epimerization at the phosphorus center and at the C1 atom in the amine moiety. On the basis of the 31P NMR data, a plausible mechanism for the reaction was proposed. The density functional theory calculation of the key step of the reaction, i.e., breaking of the P-N bond and formation of the P-O bond, suggested a one-step S(N)2(P) process with retention of configuration at the phosphorus center.

A new, efficient method is developed, based on a palladium(0)-catalyzed reaction of propargylic derivatives with various phosphorus nucleophiles, to produce allenylphosphonates and their analogues with defined stereochemistry in the allenic and the phosphonate moiety.

Efficiency and stereoselectivity of condensations of ribonucleoside 3'-H-phosphonates with ethanol promoted by pivaloyl chloride were investigated as a function of tertiary amines used. Side reactions leading to an increased demand for the condensing agent were identified as derived from an attack of the pivalate anion at carbonyl centers of reactive pivaloyl derivatives. The conditions that secured quantitative yields of H-phosphonate diester condensations were assessed. Several tertiary amines promoted condensations with stereoselectivity higher than that observed for pyridine derivatives. A correlation between diastereoselectivity of the product formation and Brønsted and H-bonding basicities of the amine used was found.

We propose O-silylated C3-halohydrins [1(3)-O-silyl-2-O-acyl-, 1,2(2,3)-O-bis(silyl)-, and 1(3)-O-acyl-2-O-silyl-3(1)-halo-sn-glycerides] as new chirons in the total synthesis of glycerolipid constructs. These are efficiently producible via opening of the oxirane ring of the corresponding glycidyl derivatives and permit (i) displacement of the iodine by a requisite carboxylate in the presence of O-triisopropylsilyl (O-TIPS), O-tert-butyldimethylsilyl (O-TBDMS), and O-acyl substituents; (ii) selective acylation across an appropriate silyloxy system [e.g., O-TBDMS or O-triethylsilyl (O-TES)] of monoesterified haloglycerides; (iii) direct exchange of an O-silyl protection (e.g., O-TBDMS or O-TIPS) for a trichloroacetyl group; (iv) conversion of a terminal TBDMS group into the corresponding trifluoroacetate without affecting O-TIPS-, O-acyl- and iodo functions. The above transformations secure flexible routes to a variety of otherwise difficult-to-access key-intermediates [e.g., 1,2(2,3)-O-bis(acyl)-3(1)-trichloroacetyl-, 1,3-O-bis(acyl)-2-trichloroacetyl-, 1,2(2,3)-O-bis(acyl)-3(1)-O-TBDMS/TIPS-, 1,3-O-bis(acyl)-2-O-TIPS/TBDMS-, 1(3)-O-acyl-2-O-TIPS-, 1,2(2,3)-O-bis(acyl)-3(1)-iodo-sn-glycerols, etc.] and lend themselves to a powerful methodology for the preparation of di- and triacylglycerols as well as glycerol-based cationic lipids. The reactions involved are entirely regio- and stereospecific, avoid acyl migration, and can provide target compounds with a chosen absolute configuration from a single synthetic precursor.

Recently, we have proposed a new D(P)/L(P) stereochemical notation for P-chiral dinucleoside monophosphate analogues based on a structural relationship between compounds. As an extension of this work, we present here applications of the D(P)/L(P) notation for tracking stereochemistry of reaction pathways involving H-phosphonate, phosphoramidite, phosphorotriester, and other intermediates frequently met in the nucleotide chemistry.

A general and efficient method for the microwave-assisted formation of the C-P bond was developed. Using a prevalent palladium catalyst, Pd(PPh3)4, a quantitative cross-coupling of various H-phosphonate diesters with aryl and vinyl halides was achieved in less than 10 min. The reactions occurred with retention of configuration at the phosphorus center and in the vinyl moiety. Using this protocol, several C-phosphonates, including those bearing nucleoside and cholesteryl moieties, were prepared in high yields.

Studies on the reaction of H-phosphonothioate, H-phosphonodithioate, and H-phosphonoselenoate monoesters with iodine in the presence of a base led to identification of a unique oxidation pathway, which consists of the initial oxidation of the sulfur or selenium atom in these compounds, followed by oxidative elimination of hydrogen iodide to generate the corresponding metaphosphate analogues. The intermediacy of the latter species during oxidation of the investigated H-phosphonate monoester derivatives with iodine was supported by various diagnostic experiments. The scope and limitation of these oxidative transformations for the purpose of the synthesis of nucleoside phosphorothioate, nucleoside phosphorodithioate, and nucleoside phosphoroselenoate diesters was also investigated.

A trifluoroacetate-catalyzed opening of the oxirane ring of glycidyl derivatives bearing allylic acyl or alkyl functionalities with trifluoroacetic anhydride (TFAA), provides an efficient entry to configurationally homogeneous 1(3)-acyl- or 1(3)-O-alkyl-sn-glycerols. Selective introduction of tert-butyldimethylsilyl- (TBDMS), or triisopropylsilyl- (TIPS) transient protections at the terminal sites within these key intermediates secures 1(3)-acyl- or 1(3)-O-alkyl-3(1)-O-TBDMS (or TIPS)-sn-glycerols as general bifunctional precursors to 1,2(2,3)-diacyl-, 1(3)-O-alkyl-2-acyl- and 1,3-diacyl-sn-glycerols and hence triester isosters. Incorporation of a requisite acyl residue at the central carbon of the silylated synthons with a subsequent Et(3)N.3HF-promoted, direct trichloroacetylation across the siloxy system by trichloroacetic anhydride (TCAA), followed by cleavage of the trichloroacetyl group, affords the respective 1,2(2,3)-diacyl- or 1(3)-O-alkyl-2-acyl-sn-glycerols. Alternatively, a reaction sequence involving: (i) attachment of a trichloroacetyl fragment at the stereogenic C2-centre of the monosilylated glycerides; (ii) replacement of the silyl moiety by a short- or long-chain carboxylic acid residue by means of the acylating agent: tetra-n-butylammonium bromide (TBABr)-carboxylic acid anhydride (CAA)-trimethylsilyl bromide (TMSBr); and (iii) removal of the trichloroacetyl replacement, provides pure 1,3-diacyl-sn-glycerols. The TBABr-CAA-TMSBr reagent system allows also a one-step conversion of 1,2-diacylglycerol silyl ethers into homochiral triglycerides with predefined asymmetry and degree of unsaturation. These compounds can also be accessed via a two-step one-pot approach where the trichloroacetyl derivatives of 1,2(2,3)- or 1,3-diacyl-sn-glycerols serve as triester building blocks for establishing the third ester bond at preselected C3(1)- or C2-positions within the glycerol skeleton at the very last synthetic stage. In all instances, the target compounds were produced under mild conditions, in high enantiomeric purity, and in practically quantitative yields.

The D(P)/L(P) convention is a stereochemical notation for P-chiral nucleotide analogs and related compounds. In contrast to the absolute R(P)/S(P) notation, the D(P)/L(P) system is based on a geometrical relationship between substituents in a dinucleoside monophosphate skeleton. The D(P)/L(P) notation is a convenient alternative to the R(P)/S(P) notation for stereochemical correlation analysis of physical, chemical, and biological properties of nucleotide and oligonucleotide analogs bearing any type of tri- or tetra-coordinated phosphorus moiety.

Glycidyl esters upon treatment with a mixture of carboxylic acid anhydride (CAA) and trimethylsilyl halide (TMSX) in the presence of tetra-n-butylammonium halide (Bu(4)NX, X=Cl, Br or I) undergo stereospecific and regioselective opening of the oxirane ring to afford mixed-(or mono)-acid 1,2(2,3)-diacyl-3(1)-halo-sn-glycerols in high yields.

A configuration of ligands around a phosphorus atom in P-chiral dinucleoside monophosphate analogues can be described using DP/LP stereochemical notation, which allows immediate correlation between the notation of configuration and the actual spatial arrangement of the phosphorus ligands. The area of applications of this new stereochemical nomenclature covers dinucleoside units bridged by virtually any type of tri-and tetra-coordinated phosphorus moieties, that is, phosphorothioates, phosphoramidates, phosphoramidites, boranephosphates, methanephosphonates, H-phosphonates, and many others.

2-,3-,4-Pyridylphosphonates and their phosphonothioate congeners were analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). It was found that the fragmentation pathways of these compounds were not influenced to any detectable extent by the stereochemistry at the phosphorus centers but were sensitive to the position of a nitrogen atom in the pyridine ring of these compounds. Possible mechanisms for fragmentations of the investigated compounds are discussed in detail.

Recently, we have proposed a new DP/LP stereochemical notation for P-chiral dinucleoside monophosphate analogues that permits simple correlation between spatial arrangement of the substituents and the configuration at the phosphorus center. As an extension of this work, we present here applications of the DP/LP notation to derivatives containing only one nucleoside unit (e.g., alkyl nucleoside phosphodiesters, nucleoside phosphomonoesters, cyclic phosphate derivatives, nucleoside di-, and triphosphates) and to nonnucleosidic phosphorus compounds.

Stereoselectivity in condensation of protected ribonucleoside 3'-H-phosphonates with hydroxylic components was investigated using 31P NMR spectroscopy. The correlation between absolute configuration at the phosphorus center and the chemical shifts of the produced H-phosphonate diesters and the corresponding phosphorothioates, was studied.

9-Fluorenemethyl H-phosphonoselenoate monoester has been used to produce thymidine 3'-O-phosphoroselenoate monoester from which various P(V) derivatives containing multiple modifications at phosphorus were obtained; e.g., thymidine 3'-O-phosphoroselenofluoridate, 3'-O-phosphoroselenothioate, or 3'-O-phosphorodiselenoate monoesters.

It was found that in stereoselective condensations of ribonucleoside 3'-H-phosphonates with alcohols, the major diastereomer of the produced H-phosphonate diesters is formed from the minor diastereomer of the intermediate phosphonic-pivalic anhydride.

Sixteen diribonucleoside (3'-5')-H-phosphonates were synthesized via condensation of the protected ribonucleoside 3'-H-phosphonates with nucleosides, and the influence of a nucleoside sequence on the observed stereoselectivity was analyzed. 31P NMR spectroscopy was used to evaluate a relationship between chemical shift and absolute configuration at the phosphorous center of the H-phosphonate diesters as well as of the corresponding phosphorothioate diesters. Although for the most cases such correlation was found, there was however several exceptions to the rule where the relative positions of resonances arisingfrom Rp and Sp diastereomers were reversed.

A new stereochemical notation for P-chiral nucleotide analogues and related compounds is proposed In this notation, the names of configurations, designated as D(P) and L(P), are derived from a geometrical relationship, rather than from priority rules, of substituents at the phosphorus centre. This new stereochemical description offers clear advantages over the CIP R/S nomenclature, particularly when used for comparing the influence of absolute configuration at the phosphorus centre on physicochemical and biological properties of oligonucleotide analogues or in stereochemical correlation analysis of P-chiral nucleotide derivatives.

In this paper a short account of our recent basic studies aiming toward development of new synthetic methods for the preparation of nucleotide analogues using H-phosphonate chemistry is presented.

In this chapter, a concise account of the synthesis of oligonucleotides using the H-phosphonate methodology is given. It includes various methods for the preparation of the starting material, nucleoside 3'-H-phosphonate monoesters, their conversion into dinucleoside H-phosphonate diesters, oxidative transformations of dinucleoside H-phosphonates into the corresponding phosphate and phosphorothioate derivatives, and protocols for the synthesis of oligonucleotides and their phosphorothio analogs.

An efficient entry to nucleoside 3'-H-phosphonoselenoate monoesters via phosphinate intermediates was developed. It involves a reaction of suitably protected nucleosides with triethylammonium phosphinate in the presence of pivaloyl chloride, followed by selenization of the intermediate nucleoside phosphinates with triphenylphosphine selenide, to produce the corresponding nucleoside H-phosphonoselenoates in 86-92% yields.

Oligonucleotides can be synthesized by condensing a protected nucleoside H-phosphonate monoester with a second nucleoside in the presence of a coupling agent to produce a dinucleoside H-phosphonate diester. This can then be converted to a dinucleoside phosphate or to a backbone-modified analog such as a phosphorothioate or phosphoramidite. This unit discusses four alternative methods for synthesizing nucleoside H-phosphonate monoesters. The methods are efficient and experimentally simple, and use readily available reagents. The unit describes the activation of the monoesters, as well as competing acylation and other potential side reactions.

Stereochemical outcome of oxidative coupling of H-phosphonate diesters with amines, promoted by iodine, can be controlled to obtain the corresponding phosphoramidate diesters with inversion or with retention of configuration at the phosphorus centre.

A new method for stereospecific synthesis of P-chiral phosphorothioates based on intramolecular nucleophile catalysis was developed.

An efficient and stereospecific synthesis of dinucleoside 4'-(2,2':6',2''-terpyridyl)phosphonate 2 and 5-(2,2'-bipyridyl)phosphonate 3 via a palladium(0) cross coupling strategy has been developed.

This paper expands the available methods for preparation of H-phosphonoselenoate using a new reagent, 9-fluorenemethyl H-phosphonoselenoate.

Chemoselectivity and stereospecificity of iodine mediated oxidative couplings using separate diastereomers of dinucleoside H-phosphonate and H-phosphonothioate with various N- and O-binucleophiles were investigated.

In this paper a short account of our recent research concerning the development of new synthetic methods and reagents for the preparation of nucleotides and their analogues, is given.

Most methods for preparing H-phosphonate monoesters suffer from variable yields and are often incompatible with common protecting groups used in oligonucleotide synthesis. This unit describes four procedures that consistently give high yields of the desired products. Taken together, they provide an arsenal of phosphonylation procedures that it compatible with most common protecting groups.

The hydration pattern of thymidyl(3'->5') thymidine 1 and those of Rp and Sp diastereomers of the corresponding methylphosphonate analogue 2, have been studied using Molecular Dynamics (MD) computer simulation. It was found that the methylphosphonate modification leads to significant changes in the coordination of water molecules around the internucleotidic linkage and these, in turn, affect the hydration pattern of other parts of the molecule. The most notable differences between Rp and Sp diastereomers 2a and 2b were found to occur at the deoxyribose moieties of the nucleosid-5'-yl units.

The first method for solid support synthesis of all-Rp-oligo(ribonucleoside phosphorothioate)s is presented as well as attempts to increase the stereoselectivity of the key step in this approach. The synthetic strategy consists of (i) a solid support synthesis procedure, using 5'-O-(4-methoxytriphenylmethyl)-2'-O-tert-butyldimethylsilyl-ri bon ucleoside 3'-H- phosphonates, that due to stereoselectivity in the condensation step, gives oligomers with mostly Sp-H-phosphonate diesters (72-89% under standard conditions), (ii) stereospecific sulfurization with S8 in pyridine to produce oligo(ribonucleoside phosphorothioate)s enriched with internucleosidic linkages of Rp configuration, (iii) treatment of the deprotected oligonucleotides with the enzyme Nuclease P1 from Penicillium citrinum, that specifically catalyses cleavage of Sp-phosphorothioate diester linkages, which leaves a mixture of oligomers having all internucleosidic linkages as Rp-phosphorothioates, and finally (iv) isolation and HPLC purification of the full length all-Rp oligomer. Mixed sequences containing the four common nucleosidic residues up to the chain length of a heptamer were synthesized. Change of N-4-protection on the cytidine building block from propionyl to N-methylpyrrolidin-2-ylidene gave a slightly improved diastereoselectivity in H-phosphonate diester formation. Increased selectivity up to 99+% was obtained with the guanosine building block when the amount of pyridine in the coupling step was reduced.

Formation of H-phosphonothioate diesters via condensation of H-phosphonate monoesters with a hydroxylic component in the presence of various coupling agents and possible side reactions that may accompany this process were studied using (31)P NMR spectroscopy. Optimal reaction conditions, which eliminate or significantly suppress the side reactions, have been designed and assessed in syntheses of dinucleoside H-phosphonothioate diesters.

A new synthetic method for the preparation of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotides (5'-methylenephosphonate analogues) was developed. The method is based on the use of a phosphonate protecting group, 4-methoxy-1-oxido-2-picolyl, enabling intramolecular nucleophilic catalysis which together with the condensing agent, 2,4,6-triisopropylbenzenesulfonyl chloride, secures fast and efficient formation of the 5'-methylenephosphonate internucleosidic bonds. The produced protected oligomers were treated with thiophenol and triethylamine to remove the phosphonate protecting groups, cleaved from the solid support using concentrated aqueous ammonia, and purified by HPLC. Several thymidine oligonucleotide analogues with the chain length of up to 20 nucleotidic units, in which all internal 5'-oxygen atoms have been replaced by methylene groups directly bound to phosphorus, were synthesised using this methodology.

The monomeric and symmetrical dimeric 5'-hydrogenphosphonate derivatives of AZT were prepared and evaluated for their inhibitory properties against HIV-1 in several cell lines. The synthesis of the compounds was achieved by reaction of AZT with in situ prepared phosphorus tris(imidazolide) or with phosphonic acid in the presence of pivaloyl chloride. The two title compounds showed in vitro anti-HIV activity similar to (but not better than) that of AZT in three cell lines which were not deficient in thymidine kinase. On the other hand they were inactive in CEM-TK- cells. Pharmacokinetic studies in several media corroborate the assumption that these compounds must not be considered as 'true antiviral agents', but that they act by releasing their nucleoside entity.

The molecular structure of one diastereomer of the dinucleoside methylphosphonate Tp(Me)sT (1) has been determined by X-ray diffraction methods. The crystal asymmetric unit contains one molecule of 1 and one methanol in an orthorhombic unit cell of dimensions a = 13.241(4), b = 13.844(3), c = 14.944(7) A, space group P2(1)2(1)2(1). Both pyrimidine bases in 1 are oriented anti relative to the 2'-deoxyribose rings, and the sugar conformations are 2E and 2(3)T in the 4-thiothymidine and thymidine moieties, respectively. The deoxyribose-phosphonate backbone has an extended conformation with the bases completely unstacked and almost parallel. The absolute configuration at the phosphorus center in 1 is Sp.

Two model compounds, 1 and 2, have been studied to test the stability of the t-butyldimethylsilyl (t-BDMSi) group towards conditions used during chemical synthesis of RNA fragments by the H-phosphonate approach. When 1 was treated with anhydrous acid for 16 h both the H-phosphonate diester and the t-BDMSi group remained intact. Removal of the t-BDMSi group from 2 with 1.0 M tetrabutylammonium fluoride (TBAF .3H2O) in THF was complete within 4 h and neither concomitant cleavage nor migration of the phosphodiester linkage could be detected even after 24 h. The dimer 2 was not completely stable towards concentrated aqueous ammonia and both loss of the t-BDMSi group and concomitant cleavage of the phosphodiester linkage occurred upon prolonged treatment. These reactions were substantialy suppressed in ethanol containing ammonia solutions, however to alleviate this problem during oligoribonucleotide synthesis, more labile protecting groups for heterocyclic bases would be desired. In conclusion, these studies indicate that 2'-O-t-BDMSi can be considered as a convenient and safe protecting group, which should secure synthesis of oligoribonucleotides with exclusively 3'-5' internucleotidic linkages.

Various pivaloyltetrazoles and chlorophosphates have been investigated as potential condensing reagents for H-phosphonate diester formation. Acylating or phosphorylating properties as well as possible side-reactions triggered by these reagents were checked using TLC and 31P NMR analysis.

Chemical synthesis of nucleoside-phospholipid conjugates based on hydrogenphosphonate chemistry has been achieved via coupling of 1,2-dipalmitoylglycero-3-H-phosphonate with suitable protected nucleosides or via coupling of nucleoside H-phosphonates with 1,2-dipalmitoylglycerol. It was also found that 1,2-dipalmitoylglycero-3-H-phosphonate, which is a stable compound, can serve as a convenient intermediate in the synthesis of various phospholipids.