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Several 6-anilino-2-thiouracils were synthesized and tested for their ability to inhibit the inner-ring iodothyronine deiodinase from human placenta. The p-ethyl and p-n-butyl analogues were strongly inhibitory to the enzyme and were much more effective than the standard deiodinase inhibitor, 6-propyl-2-thiouracil. The degree of inhibition caused by 6-(p-n-butylanilino)-2-thiouracil was, moreover, unaffected by high concentrations of reducing agent in the enzyme assay. Attempts to prepare 3-alkyl derivatives via S-debenzylation of 2-benzylthio intermediates led to rearrangement to, for example, 3-methyl-5-benzyl-6-amino-2-thiouracil. This compound also strongly inhibited the deiodinase reaction. Preliminary results suggest that these compounds are useful to study in vitro and in vivo metabolism of thyroid hormones and may be clinically useful to enhance the availability of active thyroid hormones to certain organs.

Quantitative structure-activity relationships (QSAR) of a series of 6-anilinouracil derivatives were developed for their inhibitory activity against the wild-type DNA polymerase III (pol III) and a mutant enzyme, pol III/azp-12, derived from Bacillus subtilis. Interaction between inhibitors and both enzymes appears to result solely from hydrophobic binding. Comparison of the substituent contributions indicates increased hydrophobic character and a minor change of shape of the inhibitor binding site of the mutant enzyme. Because the two enzymes have identical Km values for substrates, the inhibitor binding site is thought to be distinct from the enzyme active site.

6-(Phenylhydrazino)uracils inhibit the replication-specific enzyme DNA polymerase III of Bacillus subtilis by forming a strong, reversible complex with template-primer DNA and enzyme. The phenyl ring interacts with a hydrophobic enzyme site which, on the basis of structure-activity relationships of substituted analogues, appears to possess the following characteristics: (1) planarity or near-planarity; (2) a finite capacity to accommodate bulky substituents; and (3) location near the domain of the enzyme active site. A mutant DNA polymerase III, derived from a mutant strain of B. subtilis selected for resistance to 6-(p-hydroxyphenylazo)pyrimidines, is resistant only to inhibitors bearing p-hydroxy or amino groups and is hypersensitive to inhibitors containing nonpolar substituents; these results suggest the existence of mutable, secondary regions of the binding site which interact with para substituents and, thus, influence the strength of the primary phenyl-enzyme interaction.

Impairment of DNA synthesis by lack of thymine nucleotides was long considered the critical cytotoxic event in cells exposed to methotrexate (MTX) (1). Recent work has shown, however, that the thymineless state is associated with increases in intracellular dUMP and dUTP with resulting uracil misincorporation into DNA (2-4). Subsequent unrepaired excisions of uracils by uracil-DNA-glycosylase (5,6) lead to DNA fragmentation (7-11).

6-(p-Hydroxyphenylazo) uracil (HPUra, Fig. 8.1), 6-(p-hydroxy phenylazo) 2-amino 4 oxo pyrimidine (HPlsocytosine, Fig. 8.1) and several related 6 (arylazo)pyrimidines were conceived and synthesised by Bernard Langley of Imperial Chemical Industries, Ltd in the early 1960s. Langley and his colleagues, in their initial studies of biological activity of the arylazopyrimidines, observed that they were potent inhibitors of the growth of Gram-positive bacteria and that they possessed little or no toxicity for Gram-negative organisms or for animal cells.

We have identified and characterized a distinct non-linearity in the time course of the reaction of mammalian DNA polymerase beta with synthetic polynucleotides. Nucleotide incorporation is biphasic; an initial burst of activity decays exponentially to a lower steady-state velocity. This slow transition in polymerase activity is not due to substrate depletion, abortive complex formation, or enzyme inactivation. The data are consistent with description of the beta-polymerase as a hysteretic enzyme, a finding which provides a potential explanation for the non-hyperbolic kinetics which have been reported previously for this polymerase. We have also found, in contrast to some previous data, that the nucleotide analogue, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPdGTP), is an inhibitor of the beta-polymerase. When poly(dC).oligo(dG) is used as template.primer, inhibition of the initial velocity is competitive with dGTP with a Ki of 1.25 microM. On activated DNA, however, beta-polymerase displays sensitivity to BuPdGTP which overlaps with that previously reported for DNA polymerase delta; 100 microM BuPdGTP is required to inhibit the initial velocity of a dGTP-deficient, truncated assay. Finally, we demonstrate that, in addition to its inhibition of initial velocity, BuPdGTP also modulates both the rate constant of the slow transition in polymerase activity, and the steady-state velocity of the beta-polymerase.

Substituent effects governing inhibition of DNA polymerase III from Bacillus subtilis were examined in several series of N6-substituted 6-aminopyrimidines. The presence of alkyl groups as large as n-butyl in the 3-position of 6-(5-indanylamino)uracil had no effect on inhibitor-enzyme binding. Substituents in the 4-position of a series of 2-amino-6-(benzylamino)pyrimidines had complex effects: alkoxy and phenoxy derivatives were less active than the parent 4-oxo (isocytosine) compound, but alkylphenoxy and halophenoxy derivatives were more active than the 4-phenoxy compound itself, suggesting that hydrophobic binding can occur between 4-substitutents and the enzyme surface and that space between the pyrimidine ring and pol III may represent the active site of the enzyme. Replacement of 5-H by methyl and ethyl groups drastically decreased inhibitory activity of 6-(benzylamino)- and 6-p-toluidinouracils, but 5-bromo and 5-iodo analogues were equipotent with the parent compounds. These results indicate that the phenyl rings of these compounds must exist in conformations in which they are perpendicular to the pyrimidine ring plane and that charge-transfer stabilization of such "active conformations" may compensate for steric barriers from 5-halo groups in the inhibitor-enzyme complex.

2-(p-n-Butylanilino)adenine (BuAA), an homolog of the DNA polymerase alpha (pol alpha)-specific inhibitor, N2-(p-n-butylphenyl)guanine (BuPG), was transformed to its 2'-deoxyribonucleoside, BuAdA, and the corresponding 2'-deoxyribonucleoside 5'-phosphates, BuAdAMP, BuAdADP, and BuAdATP. All five forms of BuAA are highly selective inhibitors of mammalian pol alpha, and the action of each is subject to specific competitive antagonism by dATP. BuAdADP, and BuAdATP, like the corresponding forms of BuPG, are very potent pol alpha inhibitors, displaying apparent Ki's of less than 3 nanomolar on natural activated templates. BuAdATP, like BuPdGTP, also inhibits pol alpha-catalysed reactions directed by non-complementary, thymine-deficient templates, and it does so via a mechanism subject to specific antagonism by its natural homolog, dATP. The results of the BuAdATP-homopolymer experiments complement those of analogous experiments with BuPdGTP and the dCTP-specific pol alpha inhibitor, aphidicolin, and strengthen the suggestion that mammalian pol alpha contains dNDP and dNTP binding sites which can recognize specific bases without direction by templates.

The active forms of 6-( p -hydroxyphenylazo)-uracil and 6-( p -hydroxyphenylazo)-isocytosine were isolated and identified as their respective hydrazino derivatives. These arylhydrazino pyrimidines selectively inhibited a chromatographically distinct DNA polymerase from Bacillus subtilis . The actions of the reduced drugs on this polymerase were identical to those observed on ATP-dependent DNA synthesis in toluene-treated cells; dGTP competitively antagonized the inhibitory activity of the uracil derivative, and dATP competitively antagonized that of the isocytosine derivative. Analysis of the interactions of the arylhydrazinopyrimidines and nucleic acid bases by nuclear magnetic resonance suggested that hydroxyphenylhydrazino-uracil and hydroxyphenylhydrazino-isocytosine pair in a novel manner with, respectively, cytosine and thymine. A mechanism of inhibitor action, involving binding of the reduced drugs to enzyme and the pyrimidines of DNA template, is proposed.

Background Diffuse large B-cell lymphoma is a clinically and molecularly heterogeneous disease. Gene expression profiling studies have shown that the tumor microenvironment affects survival and that the angiogenesis-related signature is prognostically unfavorable. The contribution of histopathological microvessel density to survival in diffuse large B-cell lymphomas treated with immunochemotherapy remains unknown. The purpose of this study is to assess the prognostic impact of histopathological microvessel density in two independent series of patients with diffuse large B-cell lymphoma treated with immunochemotherapy. Design and Methods One hundred and forty-seven patients from the Leukemia Lymphoma Molecular Profiling Project (training series) and 118 patients from the Catalan Lymphoma-Study group-GELCAB (validation cohort) were included in the study. Microvessels were immunostained with CD31 and quantified with a computerized image analysis system. The stromal scores previously defined in 110 Leukemia Lymphoma Molecular Profiling Project cases were used to analyze correlations with microvessel density data. Results Microvessel density significantly correlated with the stromal score (r = 0.3209; P < 0.001). Patients with high microvessel density showed significantly poorer overall survival than those with low microvessel density both in the training series (4-year OS 54% vs. 78%; P = 0.004) and in the validation cohort (57% vs. 81%; P = 0.006). In multivariate analysis, in both groups high microvessel density was a statistically significant unfavorable prognostic factor independent of international prognostic index [training series: international prognostic index (relative risk 2.7; P = 0.003); microvessel density (relative risk 1.96; P = 0.002); validation cohort: international prognostic index (relative risk 4.74; P < 0.001); microvessel density (relative risk 2.4; P = 0.016)]. Conclusions These findings highlight the impact of angiogenesis in the outcome of patients with diffuse large B-cell lymphoma and the interest of evaluating antiangiogenic drugs in clinical trials.

Both bacteriophage T4 and human type I DNA ligases in the presence of a mixture of ATP, AMP and PPi altered the topological properties of a supercoiled substrate by a step-wise reaction eventually leading to a population of fully relaxed, covalently closed products. In the presence of only AMP and PPi DNA products containing nicks with 3'OH/5'P termini accumulated in the presence of bacteriophage T4 DNA ligase, suggesting reversal of the entire joining reaction, but not in the presence of human DNA ligase I. Both DNA ligases became deoxyadenylylated in the presence of dATP, but the joining reaction did not proceed to completion. However, with both enzymes the full relaxing reaction took place in the presence of dAMP alone and in the presence of a mixture of dATP, dAMP and PPi. In no case could the joining reaction be reversed by dAMP and PPi. Related experiments with modified derivatives of deoxyribonucleoside 5'-triphosphates and PPi gave results in accord with these observations. The AMP dependent DNA relaxation catalysed by DNA ligases was insensitive to the presence of exonuclease III. These results indicate that controlled relaxation of the substrate by both DNA ligases occurs as a separate reaction rather than by simple reversal of the joining reaction. These findings support the hypothesis that in vivo the DNA topoisomerising ligases relax their substrate at the replication fork both during and separately from ligation of a pre-existing nick.

Mediastinal involvement is considered essential for the diagnosis of primary mediastinal large B-cell lymphoma (PMBL). However, we have observed cases of diffuse large B-cell lymphoma (DLBCL) with features of PMBL but without detectable mediastinal involvement. The goal was to assess our previously established gene expression profiling (GEP) signature for PMBL in classifying these cases. In a large series of DLBCL cases, we identified 24 cases with a GEP signature of PMBL, including 9 cases with a submission diagnosis of DLBCL consistent with PMBL (G-PMBL-P) and 15 cases with a submission diagnosis of DLBCL. The pathology reviewers agreed with the diagnosis in the 9 G-PMBL-P cases. Among the other 15 DLBCL cases, 11 were considered to be PMBL or DLBCL consistent with PMBL, 3 were considered to be DLBCL, and 1 case was a gray-zone lymphoma with features intermediate between DLBCL and classical Hodgkin lymphoma. All 9 G-PMBL-P and 9 of the 15 DLBCL cases (G-PMBL-M) had demonstrated mediastinal involvement at presentation. Interestingly, 6 of the 15 DLBCL cases (G-PMBL-NM) had no clinical or radiologic evidence of mediastinal involvement. The 3 subgroups of PMBL had otherwise similar clinical characteristics, and there were no significant differences in overall survival. Genetic alterations of CIITA and PDL1/2 were detected in 26% and 40% of cases, respectively, including 1 G-PMBL-NM case with gain of PDL1/2. In conclusion, PMBL can present as a nonmediastinal tumor without evidence of mediastinal involvement, and GEP offers a more precise diagnosis of PMBL.

In a recent paper the first selective inhibitors of HSV1 uracil-DNA glycosylase (UDG) acting in the micromolar range have been reported 1. A 28.5 kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved2. Starting with the optimized model of binding between 6-(4′-n-octylanilino)uracil (octAU) and UDG some new derivatives have been predicted to be active. In vitro studies with the novel synthetized compounds confirm the plausibility of the model and define the structure features for UDG inhibitors.

Abstract 1948 Poster Board I-971 Survival after treatment of diffuse large B-cell lymphoma (DLBCL) is influenced by differences in the tumor microenvironment. Gene expression profiling (GEP) studies have shown that the angiogenesis-related signature (stromal-2 signature) is prognostically unfavorable. However, the clinical and biological significance of angiogenesis quantified in tumor tissue sections of DLBCL from patients treated with rituximab plus chemotherapy (R-CT) is not yet fully explored. CD31, the platelet adhesion molecule PECAM1, is one of the genes included in the “stromal-2 signature” reported in the GEP studies. The objective of this study was to determine whether the microvessel density (MVD) and microvessel number (MVN) in DLBCL patients treated with R-CT were associated with the clinicopathological features of the tumors and related to the outcome of the patients. The MVD and MVN were assessed in a series of 160 patients with DLBCL from the Leukemia Lymphoma Molecular Profiling Project consortium (LLMPP) 86M /74F; median age 64 yrs. The GEP was investigated in 116 of these including 50 germinal center B (GCB), 55 activated B-cell (ABC) and 11 unclassifiable cases. An independent series of 129 patients from the Catalan Lymphoma-Study Group (GELCAB) (67M/62F; median age 64 yrs) was used to validate the results. Front-line treatment was R-CT in all cases of both series. Tissue Microarrays (TMA) were constructed from pretreatment biopsy specimens of de novo DLBCL. High grade B cell lymphoma otherwise unclassifiable, primary mediastinal B cell lymphoma, T-cell-rich B cell lymphoma, and tumors associated with immunodeficiency were excluded. All cases were stained in an automated immunostainer with an antibody against CD31 (DAKO). The MVD and MVN were quantified using digitalized images of the tumor using Olympus Cell B Basic Imaging Software. Microvessel areas were defined as vascular areas delineated by CD31+ staining. The MVD was calculated as the sum of all microvessel areas divided by the total area analyzed. The MVN was the sum of all identified individual vessels, divided by the total area analyzed. TMAs were independently scored by two observers and discrepancies were resolved over a double-headed microscope. To determine whether the angiogenic values scored using the TMA were representative of the tumor sample, whole tissue sections and TMA cores from the same tumor were evaluated in 40 cases and compared by a linear regression analysis. MVD and MVN were grouped in quartiles when necessary and considered high or low when above or below the 50th percentile, respectively. Linear correlation analysis between the CD31 (+) MVD results on TMA cores and on the corresponding whole tissue sections in 40 cases showed a good correlation (R2=0.81). In the LLMPP cohort, DLBCL with an ABC profile showed higher MVD than those with GCB profile (p=0.05). In addition, higher MVD was observed in patients with advanced stage (p Disclosures: Gascoyne: Roche Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Connors:Roche Canada: Research Funding. Rimsza:High Throughput Genomics: .

Introduction: Germinal center B-cell (GCB)-like and activated B-cell (ABC)-like diffuse large B-cell lymphomas (DLBCL) are characterized by different recurrent chromosomal imbalances. In a series of 177 untreated de novo DLBCL analyzed by comparative genomic hybridization (CGH), GCB-like DLBCL had shown more frequent gains of chromosome 12p/12cen-q14 (22% vs 5%, p=.0025), whereas ABC-like DLBCL had shown more frequent gains of 3/3q27-qter (34% vs 6%; p Results: Gains/amplifications of chromosomes 2p, 3q, 12q and 18q resulted in different gene expression patterns depending on the array-defined DLBCL subtype. For example, 18q gains/amplifications correlated with overexpression of P15RS, MADH2, MADH4, LOC51320 and PMAIP1 in GCB-like DLBCL, whereas P15RS, MIZ1, MADH2, ME2, MADH4, LOC51320, MALT1, PMAIP1, BCL2, FVT1 and NFATC1 were overexpressed in ABC-like DLBCL with 18q gains/amplifications (p Conclusion: In GCB-like and ABC-like DLBCL, chromosomal imbalances lead to subgroup-specific overexpression of genes located within the gained/amplified regions. Chromosomal imbalances furthermore are associated with profound changes in the gene expression signatures in both GCB- and ABC-like DLBCL. Importantly, consideration of chromosomal imbalances significantly improves on the previously-defined gene expression based outcome predictors for DLBCL patients.

The action of acid on 6-(p-tolylhydrazino)-2-thiouracil gives appreciable amounts of the stable Fischer intermediate, 5-(2 -amino-5 -tolyl)-6-amino-2-thiouracil. This compound undergoes intramolecular cyclization at high temperatures, and represents the first reported isolation of a diamine intermediate derived from a Fischer-type indolization reaction.

The final product of the irradiation of reserpine in a chloroform solution was isolated and shown to be 3,4,5,6‐tetradehydroreserpine. The structure was determined by NMR spectroscopy as well as by chemical oxidation of reserpine. 3,4‐Dehydroreserpine was found to be an intermediate in both the chemical and photooxidation reactions. TLC examination of several reserpine tablets showed the presence of small amounts of dehydroreserpine but no tetradehydroreserpine.

Ample evidence indicates that the cytotoxicity of MTX depends on an increase in [dUTP]/[dTTP], uracil misincorporation into DNA, and uracil excision without adequate repair. The result is extensive DNA fragmentation and cell death. In a survey of many cell lines we found notable differences in their levels of dUTPase activity. When 7 cell lines selected for their wide differences in dUTPase content were assayed for (1) misincorporation of uracil due to MTX, (2) sensitivity to growth inhibition by MTX, and (3) effect of MTX on [dUTP]/[dTTP], the data suggested an apparent inverse correlation between dUTPase levels and sensitivity to MTX. The evidence that low dUTPase level may be associated with higher MTX sensitivity led to a search for dUTPase inhibitors that would enhance the cytotoxicity of MTX. Studies were performed on 44 uracil analogues (mainly 5- and 6-substituted derivatives). Many inhibited dUTPase in cells and sonicates at concentrations of 0.25-3.0 mM. A few were effective inhibitors at 0.1 mM. The most interesting compounds did enhance MTX cytotoxicity as measured by cell growth, uracil misincorporation, and alkaline elution of DNA fragments. No uracil analogue tested inhibited purified dUTPase but added ATP increased inhibition of dUTPase by analogues in sonicates. Thus active inhibitors are probably nucleotide derivatives of uracil analogues.

The structure of a rearrangement product of 6-(phenylhydrazino)uracil + formic acid has been proved to be 1-phenyl-3-carboxamidomethyl-1,2,4-triazole: hydrolysis of this compound and thermal decarboxylation of the intermediate acid gave the known 1-phenyl-3-methyl-1,2,4-triazole. Yields of triazoles from substituted 6-(phenylhydrazino)uracils follow inversely yields of pyrimidoindoles derived from Fischer-type cyclization [J. Heterocyclic Chem., 13, 539 (1976)]. The lack of formation of a triazole from 3-methyl-6-(phenylhydrazino)uracil suggests that an intermediate in this rearrangement, ie., that resulting from formylation of the anilino nitrogen atom followed by intramolecular cyclization involving the uracil 1-nitrogen atom, must undergo conjugate elimination of water to generate the triazole ring.