Molecular mechanisms of transitions induced by cytosine analogue: comparative quantum-chemical study

Using the simplest molecular models at the MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) level of the theory it has been shown for the first time that in addition to traditional incorporational errors caused by facilitated (compared with the canonical DNA bases cytosine (Cyt)) tautomerization of 6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one (DCyt), this mutagen causes the replication errors, increasing one million times the population of mispair Gua.DCyt* (asterisk marked mutagenic tautomer) as compared with mispair Gua.Cyt*. It is also proved that DCyt in addition to traditional incorporational errors also induces similar errors by an additional mechanism - due to a facilitated tautomerization of the wobble base pair Ade.DCyt (compared to the same pair Ade.Cyt) to a mispair Ade.DCyt* which is quasirisomorphic Watson-Crick base pair. Moreover, the obtained results allowed interpreting non-inconsistently the existing experimental NMR data.     

Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo

We have studied mutagenic specificities of DNA lesions in vivo in yeast CYC1 oligonucleotide transformation assay. We introduced two lesions into oligonucleotides. One was a nucleoside analog, 3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one 2'-deoxyriboside (dP), which is highly mutagenic to bacteria. It is supposed to be a miscoding, but otherwise good template for DNA polymerases. The other lesion was the TT pyrimidine(6-4)pyrimidone photoproduct, one of the typical UV lesions, which blocks DNA replication. These oligonucleotides were used to transform yeast cyc1 mutants with ochre nonsense mutation to Cyc1+. As expected from its templating properties in vitro, the transforming activity of dP-containing oligonucleotides was similar to those of unmodified oligonucleotides. Results indicated that dP may direct incorporation of guanine and adenine at a ratio of 1:20 or more in vivo. An oligonucleotide containing the photoproduct showed the transforming activity of as low as 3-5% of that of the corresponding unmodified oligonucleotide. This bypass absolutely required REV1 gene. The sequence analysis of the transformants has shown that the lesion was read as TT and TC at a ratio of 3:7, indicating its high mutagenic potential.     

Selection of novel ligands from a whole-molecule randomly mutated C5a library

Novel antagonists of the proinflammatory leukocyte chemoattractant C5a have been produced from a phage display library of whole-molecule random mutants. The cDNA for the inflammatory polypeptide C5adR(74) was used as template in a PCR reaction doped with the mutagenic nucleoside triphosphates dPTP [dP: 6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-c][1,2]oxazin-7-one] and 8-oxodGTP (8-oxodG: 8-oxo-2'-deoxyguanosine) to allow the introduction of mutations in a highly controlled manner throughout the cDNA. The resultant library of mutants was displayed on bacteriophage M13 using a jun/fos linker sequence. Functional polypeptides were isolated by several rounds of selection against the receptor for C5a expressed on the surface of CHO cells. From this selection procedure, a limited number of variants of C5adR(74) were obtained. When expressed as free polypeptide, the binding affinities of the selected C5adR(74) sequences were increased 5-fold relative to wild-type protein. Site-directed mutagenesis of the C-terminus of these variants resulted in the production of antagonists of C5adR(74) activity.     

Synthesis and duplex stability of oligonucleotides containing cytosine-thymine analogues.

The synthesis of the deoxynucleoside derived from the base P, 6H,8H-3,4-dihydro-pyrimido[4,5-c] [4,5-c] [1,2]oxazin-7-one, 2, and its introduction by established phosphoramidite and H-phosphonate chemistry into oligonucleotides is described. The melting transition temperatures (Tm) of a range of heptadecamer duplexes containing P/A and P/G base-pairs are compared with corresponding ones having N4-methoxycytosine (M) 1 and mismatched normal bases. P/A and P/G pairs allow closely similar duplex stabilities and have the potential to reduce the multiplicity of probes and primers based on amino acid sequences by removing the T/C degeneracy.     

Synthesis of Bicyclic N4-Oxycytidine Derivatives

Abstract

5-B-D-Ribofuranosyl-3H,5H,7H-pyrimido[4,5-c][1,2]oxazol-6-one, 13, a fixed anti-conformer of N⁴-alkoxycytidines was synthesised to investigate its hydrogen-bonding potential.     

Synthesis of oligodeoxyribonucleotides containing degenerate bases and their use as primers in the polymerase chain reaction.

Heptadecaoligodeoxyribonucleotides containing one or more of the bases, 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P), 2-amino-6-methoxyaminopurine (K), and hypoxanthine (I) and combinations of P with K and I have been synthesised on a DNA synthesiser. The stability of duplexes containing these basemodified oligomers with P/A, P/G, K/C and K/T; P/A, P/G, I/C, I/T and I/A, I/G, I/C, I/T base pairs were compared by measuring their melting transition (Tm) values. Oligomers containing both P and K and P and I were more stable than those with I alone or with mismatches. These oligomers together with one with a P base at the 3'-end were used as primers in polymerase chain reaction (PCR) experiments. They were all effective primers except one with I alone and a triple mismatch. Thus the use of the degenerate bases P and K in primer design is established.     

The mechanism of mutation induction by a hydrogen bond ambivalent, bicyclic N4-oxy-2'-deoxycytidine in Escherichia coli.

The triphosphate of the nucleoside deoxyribosyl dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is known to be incorporated into DNA efficiently by Taq polymerase and is a useful tool for polymerase-mediated in vitro mutagenesis. It is shown here that dP is a potent mutagen in Escherichia coli and Salmonella typhimurium . In E.coli , this deoxycytidine analog induces both GC-->AT and AT-->GC transitions. No induced transversions are observed. It is highly mutagenic in wild-type E.coli, but this is much reduced in a strain lacking thymidine kinase. Mutagenesis induced by dP is efficiently inhibited by the addition of thymidine. Partially purified thymidine kinase from E.coli catalyzes phosphorylation of dP to its 5'-monophosphate. When E.coli was grown in the presence of dP, the nucleoside analog was incorporated into its DNA. The content of dP in DNA was dependent on the concentration of dP added to the medium. The incorporation characteristics of the 5'-triphosphate of dP (dPTP) were also studied using E.coli DNA polymerase I large fragment. The results confirm that this triphosphate can be incorporated opposite A and G in the template with similar efficiencies. This indicates that dP is metabolized as a thymidine analog and that the resulting triphosphate induces a high rate of mutagenesis through replicational errors.     

Identification of antitumour activity of novel derivatives of 8-aryl-2,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazine-3,4-dione and 8-aryl-4-imino-2,3,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3(6H)-one

The series of 8-aryl-2,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazine-3,4-diones (11-20) and 8-aryl-4-imino-2,3,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3(6H)-ones (21-25) were designed and their in vitro cytotoxic activities against human LS180, HeLa, T47D, A549 and RPMI 8226 carcinoma cells are presented. In the crystalline state molecule 12 exists as the predominant tautomeric 3-oxo form, whereas the second possible 3-hydroxy tautomer is not observed. Compound 19 revealed a strong affection to LS180 cancer cells at lower tested concentration (37.9 microM) and simultaneously was found to be non-toxic towards the normal cell line investigated--GMK cells. Furthermore, this compound was proved to possess the efficiency for DNA strand breakage of the examined cancer cell lines. However, imidazotriazin-3,4-dione 20 was able to cause significant viability decreases in human RPMI 8226 peripheral blood myeloma cells. Compound 22 has exhibited remarkable inhibitory effects against LS180 and A549 carcinoma cells, whereas 24 revealed the highest growth inhibition against A549 cell line. Simultaneously, at lower tested concentration these compounds were proved to be completely non-toxic for GMK cells. Moreover, cytotoxic and antibacterial properties of starting, tautomeric 1-aryl-2-hydrazonoimidazolidines (1-6 and 8-9) are presented. Six of them (1-2, 4-6 and 9) proved active as antimicrobials. All these compounds revealed MIC values in the range of 15.0-78.6 microM. Their activities were compared to those of ampicillin and chloramphenicol.     

RO4383596, an orally active KDR, FGFR, and PDGFR inhibitor: synthesis and biological evaluation

(+/-)-1-(anti-3-Hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido[4,5-d]pyrimidin-2-one (RO4383596) is a potent and selective inhibitor of the pro-angiogenic receptor tyrosine kinases KDR, FGFR, and PDGFR. This agent has an excellent pharmacokinetic profile and is highly efficacious in rodent models of angiogenesis upon oral administration.     

Synthesis, antioxidant and toxicological study of novel pyrimido quinoline derivatives from 4-hydroxy-3-acyl quinolin-2-one

A series of novel pyrimido and other fused quinoline derivatives like 4-methyl pyrimido [5,4-c]quinoline-2,5(1H,6H)-dione (4a), 4-methyl-2-thioxo-1,2-dihydropyrimido [5,4-c]quinoline-5(6H)-one (4b), 2-amino-4-methyl-1,2-dihydropyrimido [5,4-c]quinolin-5(6H)-one (4c), 3-methylisoxazolo [4,5-c]quinolin-4(5H)-one (4d), 3-methyl-1H-pyrazolo [4,3-c]quinoline-4(5H)-one (5e), 5-methyl-1H-[1,2,4] triazepino [6,5-c]quinoline-2,6(3H,7H)-dione (5f), 5-methyl-2-thioxo-2,3-dihydro-1H-[1,2,4]triazepino [6,5-c]quinolin-6(7H)-one (5 g) were synthesized regioselectively from 4-hydroxy-3-acyl quinolin-2-one 3. They were screened for their in vitro antioxidant activities against radical scavenging capacity using DPPH(), Trolox equivalent antioxidant capacity (TEAC), total antioxidant activity by FRAP, superoxide radical (O(2)(°-)) scavenging activity, metal chelating activity and nitric oxide scavenging activity. Among the compounds screened, 4c and 5 g exhibited significant antioxidant activities.     

Mechanism of hydroxylamine mutagenesis: tautomeric shifts and proton exchange between the promutagen N6-methoxyadenosine and cytidine

Whereas the amino, but not imino, tautomer of the promutagen N6-methoxyadenosine (OMe6A) forms planar associates (base pairs) with the potentially complementary uridine [Stolarski, R., Kierdaszuk, B., Hagberg, C.-E., & Shugar, D. (1984) Biochemistry 23, 2906-2913], it has now been found, with the aid of 1H NMR spectroscopic techniques, that only the imino tautomer of OMe6A base pairs with the potentially complementary cytidine. The association constant for such heteroassociates is more than an order of magnitude higher than that for autoassociates of OMe6A. The formation of heteroassociates is accompanied by a marked shift in tautomeric equilibrium of OMe6A, with an increase in the population of the amino form from 18% to as high as 44% and a corresponding decrease in the population of the imino species. Furthermore, the presence of cytidine in a solution of OMe6A appreciably enhances the rate of tautomeric exchange between the two tautomeric forms. Formation of planar heteroassociates between cytidine and the imino form of OMe6A is also accompanied by proton exchange between the cytidine NH2 and the N6-H of the amino form of OMe6A. The rate constants for this exchange and for tautomeric exchange, determined by the saturation transfer technique, have been measured at various concentrations and temperatures. A model is advanced for proton exchange that takes into account the interdependence of tautomeric exchange and proton exchange, as well as the role of auto- and heteroassociates. The relevance of these results to the molecular basis of hydroxylamine and methoxyamine mutagenesis and to the phenomenon of proton exchange in other systems is briefly discussed.     

Saturation of DNA mismatch repair and error catastrophe by a base analogue in Escherichia coli

Deoxyribosyl-dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is a potent mutagenic deoxycytidine-derived base analogue capable of pairing with both A and G, thereby causing G. C --> A. T and A. T --> G. C transition mutations. We have found that the Escherichia coli DNA mismatch-repair system can protect cells against this mutagenic action. At a low dose, dP is much more mutagenic in mismatch-repair-defective mutH, mutL, and mutS strains than in a wild-type strain. At higher doses, the difference between the wild-type and the mutator strains becomes small, indicative of saturation of mismatch repair. Introduction of a plasmid containing the E. coli mutL(+) gene significantly reduces dP-induced mutagenesis. Together, the results indicate that the mismatch-repair system can remove dP-induced replication errors, but that its capacity to remove dP-containing mismatches can readily be saturated. When cells are cultured at high dP concentration, mutant frequencies reach exceptionally high levels and viable cell counts are reduced. The observations are consistent with a hypothesis in which dP-induced cell killing and growth impairment result from excess mutations (error catastrophe), as previously observed spontaneously in proofreading-deficient mutD (dnaQ) strains.     

The use of natural product scaffolds as leads in the search for trypanothione reductase inhibitors

Twenty-three heterocyclic compounds were evaluated for their potential as trypanothione reductase inhibitors. As a result, the harmaline, 10-thiaisoalloxazine, and aspidospermine frameworks were identified as the basis of inhibitors of Trypanosoma cruzi trypanothione reductase. Two new compounds showed moderately strong, linear competitive inhibition, namely N,N-dimethyl-N-[3-(7-methoxy-1-methyl-3,4-dihydro-9H-beta-carbolin-9-yl)propyl]amine (15) and 1,3-bis[3-(dimethylamino)propyl]-1,5-dihydro-2H-pyrimido[4,5-b][1,4]benzothiazine-2,4(3H)-dione (21), with K(i) values of 35.1+/-3.5microM and 26.9+/-1.9microM, respectively. Aspidospermine (25) inhibited T. cruzi TryR with a K(i) of 64.6+/-6.2microM. None of the compounds inhibited glutathione reductase. Their toxicity toward promastigotes of Leishmania amazonensis was assessed.     

Synthesis and anti-inflammatory activities of N4,N5-disubstituted-3-methyl- H-pyrazolo[3,4-c]pyridazines

The synthesis and anti-inflammatory activity of 4,5-dihydroxy-3-methyl-1H-pyrazolo[3,4-c]pyridazine (4), 4,5-dichloro-3-methyl-1H-pyrazolo[3,4-c]pyridazine (5), 4,-benzoyloxy-3-methyl-1-benzoyl-1H-pyrazolo[3,4-c]pyridazin-5yl benzoate (6), 3-methyl-N4,N5-bis(4-methylphenyl)-1H-pyrazolo[3,4-c]pyridazine-4,5-diamine (7), 4[[5-(4-carboxyanilino)-3-methyl-1H-pyrazolo[3,4-c]pyridazin-4yl]amino]benzoic acid (8), N-[5-(benzoylamino)-3-methyl-1H-pyrazolo[3,4-c]pyridazin-4-yl]benzamide (9) and 3-methyl-N4,N5-bis[4-(1H-benzimidazol-2yl)phenyl]-1H-pyrazolo[3,4-c]pyridazine-4,5-diamine (10) are being reported.     

Synthesis and in vitro antitumor activity of thiophene analogues of 5-chloro-5,8-dideazafolic acid and 2-methyl-2-desamino-5-chloro-5,8-dideazafolic acid

N-[5-[N-(2-Amino-5-chloro-3,4-dihydro-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (6) and N-[5-[N-(5-chloro-3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-L-glutamic acid (7), the first reported thiophene analogues of 5-chloro-5,8-dideazafolic acid, were synthesized and tested as inhibitors of tumor cell growth in culture. 4-Chloro-5-methylisatin (10) was converted stepwise to methyl 2-amino-5-methyl-6-chlorobenzoate (22) and 2-amino-5-chloro-3,4-dihydro-6-methyl-4-oxoquinazoline (19). Pivaloylation of the 2-amino group, followed by NBS bromination, condensation with di-tert-butyl N-(5-amino-2-thenoyl)-L-glutamate (28), and stepwise cleavage of the protecting groups with ammonia and TFA yielded. Treatment of 9 with acetic anhydride afforded 2,6-dimethyl-5-chlorobenz[1,3-d]oxazin-4-one (31), which on reaction with ammonia, NaOH was converted to 2,6-dimethyl-5-chloro-3,4-dihydroquinazolin-4-one (33). Bromination of, followed by condensation with and ester cleavage with TFA, yielded. The IC(50) of and against CCRF-CEM human leukemic lymphoblasts was 1.8+/-0.1 and 2.1+/-0.8 microM, respectively.     

Synthesis of polyhydroxyindolizidines from 5,6-dihydro-2H-pyran-2-one.

(1aR,5aR,5bS,6S,7S)-6,7-Di-tert-butoxy-5-oxo-pyrrolidino[1,2-b]isoxazolidino[4,5-c]tetrahydropyran (8) prepared by (1,3)-dipolar cycloaddition of the cyclic nitrone 6 derived from tartaric acid to 5,6-dihydro-2H-pyran-2-one (7) was transformed into indolizidine 11 via a sequence of reactions involving methanolysis of the lactone ring, intramolecular alkylation of the nitrogen atom promoted by a carbontetrabromide-triphenylphosphine mixture and hydrogenolysis of the N single bond O bond. Decarboxylation of 11 provided known 7-hydroxylentiginosine derivative 14, whereas oxidative decarboxylation gave indolizidine 15 structurally related to castanospermine.