Fluorescent Biaryl Uracils with C5-Dihydro- and Quinazolinone Heterocyclic Appendages in PNA.

There has been much effort to exploit fluorescence techniques in the detection of nucleic acids. Canonical nucleic acids are essentially nonfluorescent; however, the modification of the nucleobase has proved to be a fruitful way to engender fluorescence. Much of the chemistry used to prepare modified nucleobases relies on expensive transition metal catalysts. In this work, we describe the synthesis of biaryl quinazolinone-uracil nucleobase analogs prepared by the condensation of anthranilamide derivatives and 5-formyluracil using inexpensive copper salts. A selection of modified nucleobases were prepared, and the effect of methoxy- or nitro- group substitution on the photophysical properties was examined. Both the dihydroquinazolinone and quinazolinone modified uracils have much larger molar absorptivity (~4-8×) than natural uracil and produce modest blue fluorescence. The quinazolinone-modified uracils display higher quantum yields than the corresponding dihydroquinazolinones and also show temperature and viscosity dependent emission consistent with molecular rotor behavior. Peptide nucleic acid (PNA) monomers possessing quinazolinone modified uracils were prepared and incorporated into oligomers. In the sequence context examined, the nitro-substituted, methoxy-substituted and unmodified quinazolinone inserts resulted in a stabilization (∆Tm = +4.0/insert; +2.0/insert; +1.0/insert, respectively) relative to control PNA sequence upon hybridization to complementary DNA. All three derivatives responded to hybridization by the "turn-on" of fluorescence intensity by ca. 3-to-4 fold and may find use as probes for complementary DNA sequences.

Synthesis and biological evaluation of 5-nitropyrimidine-2,4-dione analogues as inhibitors of nitric oxide and iNOS activity.

Fifty two compounds based on 5-nitropyrimidine-2,4-dione moiety have been synthesized and evaluated for their inhibitory potency on the production of nitric oxide. Among them, compound 36 inhibited the production of nitric oxide (IC50 : 8.6 µm) on lipopolysaccharide-induced RAW 264.7 cells and inducible nitric oxide synthase activity (IC50 : 6.2 µm), as well as exerted no potential cytotoxicity (IC50 > 80.0 µm). Docking study confirmed that compound 36 was an inducible nitric oxide synthase inhibitor with perfect binding to the active site of inducible nitric oxide synthase. At a dose of 10 mg/kg, oral administration of 36 possessed protective properties in carrageenan-induced paw edema of male ICR mice.

Janus-AT bases: synthesis, self-assembly, and solid state structures.

The high yielding synthesis of heterocycles with defined H-bond accepting and donating capabilities provides for the design of self-assembling structures and specific recognition of biological targets. Herein we report the syntheses and solid-state structures of three self-complementary uracil/thymine derivatives where each presents the standard ADA face inherently complementary to adenine and a synthetically appended DAD face complementary to uracil/thymine. These heterocycles, which have never before been reported or characterized, represent diaminopurine-uracil/thymine hybrids that, in two of the three cases, relate to previously reported heterocyclic hybrids of G and C. All three heterocycles crystallized to afford the first X-ray crystal structures of self-complementary heterocycles capable of ADA-DAD pairing. The potential use in DNA and RNA recognition are briefly discussed.

Structure-activity relationship studies on N3-substituted willardiine derivatives acting as AMPA or kainate receptor antagonists.

N3-substitution of the uracil ring of willardiine with a variety of carboxyalkyl or carboxybenzyl substituents produces AMPA and kainate receptor antagonists. In an attempt to improve the potency and selectivity of these AMPA and kainate receptor antagonists a series of analogues with different terminal acidic groups and interacidic group spacers was synthesized and pharmacologically characterized. (S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxythiophene-3-ylmethyl)pyrimidine-2,4-dione (43, UBP304) demonstrated high potency and selectivity toward native GLU(K5)-containing kainate receptors (K(D) 0.105 +/- 0.007 microM vs kainate on native GLU(K5); K(D) 71.4 +/- 8.3 microM vs (S)-5-fluorowillardiine on native AMPA receptors). On recombinant human GLU(K5), GLU(K5)/GLU(K6), and GLU(K5)/GLU(K2), K(B) values of 0.12 +/- 0.03, 0.12 +/- 0.01, and 0.18 +/- 0.02 microM, respectively, were obtained for 43. However, 43 displayed no activity on homomeric GLU(K6) or GLU(K7) kainate receptors or homomeric GLU(A1-4) AMPA receptors (IC(50) values > 100 microM). Thus, 43 is a potent and selective GLU(K5) receptor antagonist.

Microwave-assisted synthesis and biological evaluation of novel uracil derivatives inhibiting human thymidine phosphorylase.

New 5-chloro-6-substituted-uracil derivatives have been prepared by microwave assisted-synthesis and tested in vitro as thymidine phosphorylase inhibitors. One of these compounds showed potent inhibitory activity, with an IC50 value in the submicromolar range. The biological activity of the new compounds is discussed in terms of structure-activity relationship.

Synthesis and evaluation of new potential HIV-1 non-nucleoside reverse transcriptase inhibitors. New analogues of MKC-442 containing Michael acceptors in the C-6 position.

Analogues of MKC-442 capable of undergoing Michael addition reactions were synthesised in order to investigate the activity against the HIV-1 mutant (Y181C). An improved activity was postulated on the basis of a possible covalent binding to the mercapto group of Cys181. Lithiation of the C-6 position of 1-ethoxymethyl-5-ethyl-1H-pyrimidine-2,4-dione (5) was followed by reaction with alpha,beta-unsaturated aldehydes and oxidation of the alcohols formed to give the alkenoyl analogues 1a-3a. Analogues 1b-3b containing an allyloxymethyl group in the N-1 position instead of the ethoxymethyl group could not be synthesised due to isomerisation of the allylic group during the metallation reaction. The NMR data for compounds 1a-3a showed a hindered rotation, which was more pronounced for the 6-cyclohexenylcarbonyl derivative 3a than for the propenyl derivatives 1a and 2a. Moderate activity against wild type HIV-1 was observed for the alcohol 8 and the ketones 2a-3a. However, no activity was observed against the Y181C mutant.

Synthesis and biological evaluation of CX-659S and its related compounds for their inhibitory effects on the delayed-type hypersensitivity reaction.

In order to find novel nonsteroidal compounds possessing an inhibitory activity against delayed-type hypersensitivity (DTH) reactions, we conducted random screening using a picryl chloride (PC)-induced contact hypersensitivity reaction (CHR) in mice, and found compound 1 as a lead compound. Then we synthesized and evaluated an extensive series of 5-carboxamidouracil derivatives focused on both the uracil and the antioxidative moieties. Among them, we found that the hindered phenol moiety was necessary to exhibit the activities; especially, compounds 28a-28c having the partial structure of vitamin E were found to exert potent activities against the DTH reaction by both oral and topical administration. And compound 28c showed antioxidative activity against lipid peroxidation with an IC50 of 5.9 microM. Compound 28c (CX-659S) was chosen as a candidate drug for the treatment of cutaneous disorders such as atopic dermatitis and allergic contact dermatitis.

The selenium analog of 6-propylthiouracil. Measurement of its inhibitory effect on type I iodothyronine deiodinase and of its antithyroid activity.

6-Propylthiouracil (PTU), a widely used antithyroid drug for the treatment of Graves' disease, is also a potent inhibitor of Type I iodothyronine deiodinase (ID-1). Inhibition of ID-1 was attributed initially to the formation of a mixed disulfide between PTU and a putative cysteine residue at the active site. It has been demonstrated recently that ID-1 is a selenium-containing enzyme, with selenocysteine, rather than cysteine, at the active site. It seemed possible, therefore, that the selenium analog of PTU (PSeU) might be a more potent inhibitor of ID-1 than PTU. To test this possibility, we developed a procedure for the synthesis of PSeU, and we compared PSeU and PTU as inhibitors of ID-1 in a test system containing 125I-rT3, rat liver microsomes, and dithiothreitol. Deiodinase activity was measured by the increase in 125I-iodide. PTU and PSeU were tested at 0.1, 0.3, 1 and 3 microM. Based on results of four separate experiments, the drugs were essentially equipotent as inhibitors of ID-1, although statistical analysis suggested that PSeU may be slightly more potent than PTU. PTU and PSeU were also compared for antithyroid activity in vivo and in vitro. As inhibitors of the catalytic activity of thyroid peroxidase (TPO), the two drugs were essentially equipotent in iodination and guaiacol assays involving measurements made shortly after the addition of H2O2. However, in in vivo experiments with rats, PSeU showed no appreciable inhibition of organic iodine formation in the thyroid, whereas PTU, as expected, was a potent inhibitor. The lack of inhibition of organic iodine formation in vivo by PSeU suggests that, unlike PTU, it is not concentrated by the thyroid gland. In an iodination system in which H2O2 was generated by glucose-glucose oxidase, both PTU and PSeU, when present at 10 microM, acted as reversible inhibitors of iodination. However, when the drug concentration was raised to 50 microM, TPO was inactivated and iodination was irreversibly inhibited. These results suggest that PTU and PSeU inhibit TPO-catalyzed iodination by similar mechanisms. Under the same conditions, the selenium analog of methimazole (another widely used antithyroid drug) does not inactivate TPO. It acts primarily as a reversible inhibitor of TPO-catalyzed iodination.

Phenylselenenyl- and phenylthio-substituted pyrimidines as inhibitors of dihydrouracil dehydrogenase and uridine phosphorylase.

Lithiation of 5-bromo-2,4-bis(benzyloxy)pyrimidine (3) with n-BuLi at -80 degrees C followed by the addition of diphenyl diselenide or diphenyl disulfide as an electrophile furnished the corresponding 5-(phenylhetera)-2,4-bis(benzyloxy)pyrimidine, which on exposure to trimethylsilyl iodide in CH2-Cl2 at room temperature yielded the 5-(phenylhetera)uracils in 70-75% yield. Similarly, the 6-(phenylhetera)uracils were prepared from 6-bromo-2,4-bis(benzyloxy)pyrimidine (10). 1-[(2-Hydroxyethoxy)methyl]-5-(phenylselenenyl)uracil (PSAU, 18) and 1-(ethoxymethyl)-5-(phenylselenenyl)uracil (17) were synthesized by the electrophilic addition of benzeneselenenyl chloride to the acyclic uracils under basic conditions. These compounds were evaluated for their ability to inhibit dihydrouracil dehydrogenase (DHUDase, E.C. 1.3.1.2), orotate phosphoribosyltransferase (OPRTase, E.C. 2.4.2.10), uridine phosphorylase (UrdPase, E.C. 2.4.2.3), and thymidine phosphorylase (dThdPase, E.C. 2.4.2.4). 5-(Phenylselenenyl)uracil (PSU, 6) and 5-(phenylthio)uracil (PTU, 7) inhibited DHUDase with apparent K(i) values of 4.8 and 5.4 microM, respectively. The corresponding 6-analogues, compounds 13 and 14, demonstrated inhibitory activity against OPRTase. PTU as well as PSU and its riboside, 2'-deoxyriboside, and acyclonucleosides were inhibitors of UrdPase, with PSAU (18) being the most potent with an apparent K(i) value of 3.8 microM. None of the compounds evaluated had any effect on dThdPase. Interestingly, most of the compounds showed modest selective anti-human-immunodeficiency-virus activity in acutely infected primary human lymphocytes.

Synthesis of 5-selenium-substituted uracil derivatives. Inhibition of thymidylate synthetase by 5-hydroseleno-2'-deoxyuridylate.

5-Selenium-substituted derivatives (diselenides) or uracil, 2'-deoxyuridine, and 2'-deoxyuridylic acid were synthesized via the addition of methyl hypobromite to the 5,6 double bond, followed by reaction of the adducts with sodium diselenide. The physical and chemical properties of these compounds (including their facile reduction by dithiothreitol and rapid reoxidation) were similar to those of the corresponding 5-sulfur analogues. 5-Hydroseleno-2'-deoxyuridylic acid was as potent as 5-mercapto-2'-deoxyuridylate in inhibiting thymidylate synthetase from L. casei (ki approximately 6 X 10(-8) M) but the nucleoside III was considerably less active than 5-mercapto-2'-deoxyuridine in the inhibition of growth of the leukemia L1210 cell in culture.