Synthesis of 3′-O2-(Azaheterocycle)-Thymidines

Abstract

The synthesis of 3′-O ²-(azaheterocycle)-thymidines is presented from 1-thia-3-aza-1,3-butadiene precursors (N-thioacylamidines). A variety of heterocycles is accessible using the dienic, the electrophilic or the nucleophilic reactivity of these thia-azabutadiene systems. 3′-O ²-(azaheterocycle)-thymidine analogues are regarded as potential substrates to interfere with the DNA-polymerization process.     

Single diastereomers of unsymmetrical tris-spirocyclic cyclotriphosphazenes based on 1,1'-bi-2-naphthol--synthesis and structures

Diastereoselective synthesis and characterization of chiral unsymmetrical tris-spirocyclic cyclotriphosphazenes based on chiral 1,1'-bi-2-naphthol (BINOL) are reported. Specifically, the chiral compounds (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)Cl(2) [(-)-4] and (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](OCH(2)CH(2)NMe)(2) [(-)-5] are prepared by starting with the chiral mono-spiro compound (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)]Cl(4) [(-)-3]. Synthesis of four other chiral spirocyclics, N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](OCH(2)CH(2) NMe)(O-2,2'C(6)H(4)-C(6)H(4)O)[(-)-6 and (+)-6], N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](NMe(2))(4) [(-)-7], N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)(NMeCH(2)CH(2)OH)(2) [(-)-8 and (+)-8], and N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)[NHCH(2)CH(2)CH(2)Si(OEt)(3)](2) (9) is also reported herein. Compounds 4-6 are obtained in the solid state diastereoselectively and their X-ray structures have been determined and discussed. The diastereoselectivity is also shown by structural characterization of two distinct isomers in the case of 6 [(-)-6 and (+)-6, respectively] by starting with precursor of 3 having (R) or (S)-BINOL residue. The (1)H NMR spectra of 7 and 8 exhibit doublets with virtual coupling for the methyl protons, consistent with the chiral nature of the binaphthoxy residue. The potential of 9, which hydrolyzes readily in CDCl(3) solution, as a useful precursor for chiral polymer applications is highlighted.     

Synthesis and properties of 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) as effective antisense oligonucleotides

Novel bicyclo nucleosides, 2'-O,4'-C-ethylene nucleosides and 2'-O,4'-C-propylene nucleosides, were synthesized as building blocks for antisense oligonucleotides to further optimize the 2'-O,4'-C-methylene-linkage of bridged nucleic acids (2',4'-BNA) or locked nucleic acids (LNA). Both the 2'-O,4'-C-ethylene- and propylene-linkage within these nucleosides restrict the sugar puckering to the N-conformation of RNA as do 2',4'-BNA/LNA. Furthermore, ethylene-bridged nucleic acids (ENA) having 2'-O,4'-C-ethylene nucleosides had considerably increased the affinity to complementary RNA, and were as high as that of 2',4'-BNA/LNA (DeltaT(m)=+3 approximately 5 degrees C per modification). On the other hand, addition of 2'-O,4'-C-propylene modifications in oligonucleotides led to a decrease in the affinity to complementary RNA. As for the stability against nucleases, incorporation of one 2'-O,4'-C-ethylene or one 2'-O,4'-C-propylene nucleoside into oligonucleotides considerably increased their resistance against exonucleases to an extent greater than 2',4'-BNA/LNA. These results indicate that ENA is more suitable as an antisense oligonucleotide and is expected to have better antisense activity than 2',4'-BNA/LNA.     

Conformational properties of branched RNA fragments in aqueous solution

The conformational properties of branched trinucleoside diphosphates ACC, ACG, AGC, AGG, AUU, AGU, AUG, ATT, GUU, and aAUU [XYZ = X(2'p5'Y)3'p5'Z] have been studied in aqueous solution by nuclear magnetic resonance (1H, 13C), ultraviolet absorption, and circular dichroism. It is concluded from these studies that the purine ring of the central residue (X; e.g., adenosine) forms a base-base stack exclusively with the purine or pyrimidine ring of the 2'-nucleotidyl unit (Y; 2'-residue). The residue attached to the central nucleoside via the 3'-5'-linkage (Z; 3'-residue) is "free" from the influence of the other two heterocyclic rings. The ribose rings of the central nucleoside and the 2'- and 3'-residues exist as equilibrium mixtures of C2'-endo (2E)-C3'-endo (3E) conformers. The furanose ring of the central nucleoside (e.g., A) when linked to a pyrimidine nucleoside via the 2'-5'-linkage shows a higher preference for the 2E pucker conformation (e.g., AUG, AUU, ACG, ca. 80%) than those linked to a guanosine nucleoside through the same type of bond (AGU, AGG, AGC, ca. 70%). This indicates some correlation between nucleotide sequence and ribose conformational equilibrium. The 2E-3E equilibrium of 2'-pyrimidines (Y) shows significant, sometimes exclusive, preference (70-100%) for the 3E conformation; 3'-pyrimidines and 2'-guanosines have nearly equal 2E and 3E rotamer populations; and the ribose conformational equilibrium of 3'-guanosines shows a preference (60-65%) for the 2E pucker. Conformational properties were quantitatively evaluated for most of the bonds (C4'-C5', C5'-O5', C2'-O2', and C3'-O3') in the branched "trinucleotides" AUU and AGG by analysis of 1H-1H, 1H-31P, and 13C-31P coupling constants. The C4'-C5' bond of the adenosine units shows a significant preference for the gamma + conformation. The dominant conformation about C4'-C5' and C5'-O5' for the 2'-and 3'-nucleotidyl units is gamma + and beta t, respectively, with larger gamma + and beta t rotamer populations for the 2'-unit. The increased conformational purity in the 2'-residue, compared to the 3'-residue, is ascribed to the presence of an ordered (adenine----2'-residue) stacked state. The favored rotamers about C3'-O3' and C2'-O2' are epsilon- and epsilon'-, respectively. The conformational features of AUU and AGG were compared to those of their constitutive dimers A3'p5'G, A2'p5'G, A3'p5'U, and A2'p5'U and monomers 5'pG and 5'pU.     

Carbon-13 NMR in conformational analysis of nucleic acid fragments. 3. The magnitude of torsional angle epsilon in d(TpA) from CCOP and HCOP NMR coupling constants.

Carbon-13 NMR spectra of the deoxyribonucleotide d(TpA), 3',5'-cyclic AMP and 3',5'-cyclic dAMP were measured. It is shown that the different substitution of C2' in deoxyribonucleotides versus ribonucleotides does not affect the vicinal C2'-C3'-O3'-P coupling to a measurable extent. Therefore, the same set of Karplus parameters may be used for the C2'-C3-O3'-P couplings in ribonucleotides and in deoxyribonucleotides. Vicinal carbon-phosphorus and proton-phosphorus coupling constants are used to calculate the magnitude of the torsion angle epsilon (C4'-C3'-O3'-P), which amounts to 195(0) in the trans conformer and to 261(0) in the gauche(-) conformer.     

Preparation of oligodeoxyribonucleoside phosphorodithioates by a triester method.

A method to prepare thymidine phosphorodithioate dimers (ref. 1) has been extended to allow the preparation of oligo-2'-deoxyribonucleotide phosphorodithioates containing all four bases. The method is suitable for large-scale synthesis and gives phosphorodithioates without phosphorothioate impurities (31P nmr, detection limit 0.5 to 1%). Oligonucleotides up to octamers which contain -0-(PS2-)-0- linkages at all positions have been prepared by block synthesis in solution. The phosphorodithioate linkage is introduced by the reaction of a 5'-O, N-protected nucleoside (or oligonucleotide) with a dithiophosphorylating agent RSP(S)(ODhbt)2, R = 2,4-dichlorobenzyl, Dhbt = 3,4-dihydro-4-oxo-benzotriazin-3-yl, followed by coupling of the product to a 3'-O,N-protected nucleoside (or oligonucleotide). This method gives pure protected oligodeoxyribonucleoside phosphorodithioates, and phosphorothioate linkages are only introduced if contact with conc. aqueous ammonia during or after deblocking is unduly prolonged.     

The effect of (2''-5'') and (3''-5'') phosphodiester linkages on conformational and stacking properties of cytidylyl-cytidine in aqueous solution.

Conformational properties of (2'-5') and (3'-5') CpC have been determined by proton magnetic resonance spectroscopy at 220 MHz. The ribose ring structures are predominantly 3E with the exception of the ring from the 2'-phosphate fragment of C(2'-5')pC which exhibits an 2E pucker. Bases are oriented anti with respect to the ribose and the conformations about C4'-C5', C5'-O5', C3'-O3' (C2'-O2') are gg, g'g', and g+ in equilibrium g-, respectively. The dimers exist as mixtures of stacked (g+g+ and g-g- about the P-O(C) bonds) and unstacked species at 20 degrees C. Stacking is estimated to be 35% in both dimers.     

Differential inhibition of mRNA degradation pathways by novel cap analogs

mRNA degradation predominantly proceeds through two alternative routes: the 5'-->3' pathway, which requires deadenylation followed by decapping and 5'-->3' hydrolysis; and the 3'-->5' pathway, which involves deadenylation followed by 3'-->5' hydrolysis and finally decapping. The mechanisms and relative contributions of each pathway are not fully understood. We investigated the effects of different cap structure (Gp(3)G, m(7)Gp(3)G, or m(2)(7,3'-O) Gp(3)G) and 3' termini (A(31),A(60), or G(16)) on both translation and mRNA degradation in mammalian cells. The results indicated that cap structures that bind eIF4E with higher affinity stabilize mRNA to degradation in vivo. mRNA stability depends on the ability of the 5' terminus to bind eIF4E, not merely the presence of a blocking group at the 5'-end. Introducing a stem-loop in the 5'-UTR that dramatically reduces translation, but keeping the cap structure the same, does not alter the rate of mRNA degradation. To test the relative contributions of the 5'-->3' versus 3'-->5' pathways, we designed and synthesized two new cap analogs, in which a methylene group was substituted between the alpha- and beta-phosphate moieties, m(2)(7,3'-O)Gpp(CH2)pG and m(2)(7,3'-O)Gp(CH2)ppG, that are predicted to be resistant to cleavage by Dcp1/Dcp2 and DcpS, respectively. These cap analogs were recognized by eIF4E and conferred cap-dependent translation to mRNA both in vitro and in vivo. Oligonucleotides capped with m(2)(7,3'-O)Gpp(CH2)pG were resistant to hydrolysis by recombinant human Dcp2 in vitro. mRNAs capped with m(2)(7,3'-O)Gpp(CH2)pG, but not m(2)(7,3'-O)Gp(CH2)ppG, were more stable in vivo, indicating that the 5'-->3' pathway makes a major contribution to overall degradation. Luciferase mRNA containing a 5'-terminal m(2)(7,3'-O)Gpp(CH2)pG and 3'-terminal poly(G) had the greatest stability of all mRNAs tested.     

Methoxylation of 3',4'-aromatic side chains improves P-glycoprotein inhibitory and multidrug resistance reversal activities of 7,8-pyranocoumarin against cancer cells

The overexpression of P-glycoprotein (Pgp), an ATP-driven membrane exporter of hydrophobic xenobiotics, is one of the major causes of multidrug resistance (MDR) in cancer cells. Through extensive screening we have found that the extracts of Peucedanum praeruptorum Dunn. and one of the major components (+/-)-praeruptorin A (PA) may reverse Pgp-mediated multidrug resistance. Studies on novel PA derivatives have shown that (+/-)-3'-O,4'-O-dicinnamoyl-cis-khellactone (DCK) is more active than PA or verapamil and is a non-competitive inhibitor of Pgp. Here, we report that methoxylation of the cinnamoyl groups on DCK may further enhance its bioactivity. The structure-activity relationship is demonstrated by comparing two new pyranocoumarins (+/-)-3'-O,4'-O-bis(3,4-dimethoxycinnamoyl)-cis-khellactone (DMDCK) and (+/-)-3'-O,4'-O-bis(4-methoxycinnamoyl)-cis-khellactone (MMDCK). While the co-existence of 3- and 4-methoxy groups on cinnamoyl remarkably enhanced the Pgp-inhibitory activity, the lone existence of the 4-methoxy group on cinnamoyl reduced the activity. Contrary to DCK, DMDCK promoted the binding of UIC2 antibody to Pgp which signifies a conformational change of Pgp similar to that induced by transport substrates. While DCK moderately stimulated the basal Pgp-ATPase activity, DMDCK inhibited the activity. A pharmacophore search with verapamil-based template revealed that four functional groups of DMDCK could be simultaneously involved in interaction with Pgp whereas for DCK or MMDCK only three groups were involved. It is speculated that the additional 3-methoxy group on cinnamoyl allows DMDCK to interact more efficiently with Pgp substrate site(s). If DMDCK was tightly bind to Pgp substrate site(s) the complexes could be inactive with regard to transportation and ATP hydrolysis could also be inhibited.     

Remarkable substrate-inhibitor properties of nicotine enantiomers towards a guinea pig lung aromatic azaheterocycle N-methyltransferase

The kinetics of nicotine methylation by guinea pig lung homogenates has been investigated. An interesting stereospecificity has been observed for nicotine enantiomers. R-(+)-Nicotine is a substrate Km = 1.42 X 10(-5)M for an SAM-dependent guinea pig lung aromatic azaheterocycle N-methyltransferase, whereas S-(-)-nicotine acts as a competitive inhibitor (Ki = 6.25 X 10(-5)M) of the N-methylation of its antipode.     

Recognition of T.A interruption by 2',4'-BNAs bearing heteroaromatic nucleobases through parallel motif triplex formation

2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) monomers bearing novel unnatural nucleobases, 4-(3-benzamidophenyl)-2-pyridone and 2-(N-methylbenzamido)thiazole, were synthesized and successfully incorporated into oligonucleotides. UV melting experiments showed that the corresponding oligonucleotide derivatives formed stable triplexes with dsDNA targets even in the presence of a T.A interruption.     

Advanced nuclear magnetic resonance lanthanide probe analyses of short-range conformational interrelations controlling ribonucleic acid structures

An advanced method was developed for lanthanide-probe analyses of the conformations of flexible biomolecules such as nucleotides. The new method is to determine structure parameters (such as internal-rotation angles) and population parameters for local conformational equilibria of flexible sites, together with standard deviations of these parameters. As the prominent advantage of this method, the interrelations among local conformations of flexible sites may be quantitatively elucidated from the experimental data of lanthanide-induced shifts and relaxations and vicinal coupling constants. As a structural unit of ribonucleic acids, the molecular conformations and conformational equilibria of uridine 3'-monophosphate in aqueous solution were analyzed. The stable local conformers about the C3'-O3' bond are the G+ (phi' = 281 +/- 11 degrees) and G- (phi' = 211 +/- 8 degrees) forms. The internal rotation about the C3'-O3' bond and the ribose-ring puckering are interrelated; 97 +/- 5% of the C3'-endo ribose ring is associated with the G- form while 70 +/- 22% o the C2'-endo ribose ring is associated with the G+ form. An interdependency also exists between the internal rotation about the C4'-C5' bond and the ribose-ring puckering. These short-range conformational interrelations are probably important in controlling the dynamic aspects of ribonucleic acid structures.     

Synthesis of 5-azacytidine nucleosides with rigid sugar moiety as potential antitumor agents

The bicyclic 3'-O,5'-C-methylene-linked and 2'-O,5'-C-methylene-linked 5-azacytidine derivatives were readily synthesized from 1,2;5,6-di-O-isopropylidene-D-glucose and evaluated against several cancer cell lines.     

Novel pyridinyl and pyrimidinylcarbazole sulfonamides as antiproliferative agents

A series of azaheterocyclic carbazole sulfonamides was synthesized and evaluated for antiproliferative activity. The most potent compounds N-(2,6-dimethoxypyridine-3-yl)-9-ethyl and 9-methylcarbazole-3-sulfonamide (13 and 14) gave significant cytotoxicity (IC50 = 122 and 101 nM). Compound 13 displayed submicromolar activities against seven human tumor cell lines. The SARs of this series of sulfonamides which includes the influence of azaheterocycle rings, sulfonamide linkage, and the carbazole ring are described.     

DFT studies of the disaccharide, alpha-maltose: relaxed isopotential maps

The disaccharide, alpha-maltose, forms the molecular basis for the analysis of the structure of starch, and determining the conformational energy landscape as the molecule oscillates around the glycosidic bonds is of importance. Thus, it is of interest to determine, using density functionals and a medium size basis set, a relaxed isopotential contour map plotted as a function of the phi(H) and psi(H) dihedral angles. The technical aspects include the method of choosing the starting conformations, the choice of scanning step size, the method of constraining the specific dihedral angles, and the fitting of data to obtain well defined contour maps. Maps were calculated at the B3LYP/6-31+G( *) level of theory in 5 degrees intervals around the (phi(H),psi(H))=(0 degrees ,0 degrees ) position, out to approximately +/-30 degrees or greater, for gg-gg'-c, gg-gg'-r, gt-gt'-c, gt-gt'-r, tg-tg'-c, and tg-tg'-r conformers, as well as one-split gg(c)-gg'(r) conformer. The results show that the preferred conformation of alpha-maltose in vacuo depends strongly upon the hydroxyl group orientations ('c'/'r'), but the energy landscape moving away from the minimum-energy position is generally shallow and transitions between conformational positions can occur without the addition of significant energy. Mapped deviations of selected parameters such as the dipole moment; the C1-O1-C4', H1-C1-O1, and H4'-C4'-O1 bond angles; and deviations in hydroxymethyl rotamers, O5-C5-C6-O6, O5'-C5'-C6'-O6', C5-C6-O6-H, and C5'-C6'-O6'-H', are presented. These allow visualization of the structural and energetic changes that occur upon rotation about the glycosidic bonds. Interactions across the bridge are visualized by deviations in H(O2)...O3', H(O3')...O2, and H1...H4' distances and the H(O2)-O2-C2-C1 and H'(O3')-O3'-C3'-C4' hydroxyl dihedral angles.     

Conversion of A3 adenosine receptor agonists into selective antagonists by modification of the 5'-ribofuran-uronamide moiety

The highly selective agonists of the A(3) adenosine receptor (AR), Cl-IB-MECA (2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine), and its 4'-thio analogue, were successfully converted into selective antagonists simply by appending a second N-methyl group on the 5'-uronamide position. The 2-chloro-5'-(N,N-dimethyl)uronamido analogues bound to, but did not activate, the human A(3)AR, with K(i) values of 29 nM (4'-O) and 15 nM (4'-S), showing >100-fold selectivity over A(1), A(2A), and A(2B)ARs. Competitive antagonism was demonstrated by Schild analysis. The 2-(dimethylamino)-5'-(N,N-dimethyl)uronamido substitution also retained A(3)AR selectivity but lowered affinity.     

Preparation and properties of 2',5'-linked oligonucleotide analogues containing 3'-O,4'-C-methyleneribonucleosides

Bicyclic nucleoside analogues, 3'-O,4'-C-methyleneuridine and -5-methyluridine, were successfully incorporated into oligonucleotides via connection with 2',5'-phosphodiester linkage, and hybridization behavior and nuclease stability of the modified oligonucleotides were investigated.     

Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity

During mRNA synthesis, the polymerase of vesicular stomatitis virus (VSV) copies the genomic RNA to produce five capped and polyadenylated mRNAs with the 5'-terminal structure 7mGpppA(m)pApCpApGpNpNpApUpCp. The 5' mRNA processing events are poorly understood but presumably require triphosphatase, guanylyltransferase, [guanine-N-7]- and [ribose-2'-O]-methyltransferase (MTase) activities. Consistent with a role in mRNA methylation, conserved domain VI of the 241-kDa large (L) polymerase protein shares sequence homology with a bacterial [ribose-2'-O]-MTase, FtsJ/RrmJ. In this report, we generated six L gene mutations to test this homology. Individual substitutions to the predicted MTase active-site residues K1651, D1762, K1795, and E1833 yielded viruses with pinpoint plaque morphologies and 10- to 1,000-fold replication defects in single-step growth assays. Consistent with these defects, viral RNA and protein synthesis was diminished. In contrast, alteration of residue G1674 predicted to bind the methyl donor S-adenosylmethionine did not significantly perturb viral growth and gene expression. Analysis of the mRNA cap structure revealed that alterations to the predicted active site residues decreased [guanine-N-7]- and [ribose-2'-O]-MTase activity below the limit of detection of our assay. In contrast, the alanine substitution at G1674 had no apparent consequence. These data show that the predicted MTase active-site residues K1651, D1762, K1795, and E1833 within domain VI of the VSV L protein are essential for mRNA cap methylation. A model of mRNA processing consistent with these data is presented.