Large, activated B cells are the primary B-cell target of 8-bromoguanosine and 8-mercaptoguanosine

Previous studies have documented the ability of 8-bromoguanosine (8-BrGuo) and 8-mercaptoguanosine (8-MGuo) to induce polyclonal proliferation and differentiation of B cells from a variety of mouse strains. In the present study, we have defined the cellular target of this mitogenic activity. Using B cells fractionated according to size, we have found that large B cells are responsive to 8-BrGuo- and 8-MGuo-induced proliferation and differentiation whereas small, resting B cells are relatively unresponsive to these compounds. Addition of splenic adherent cells to the small B-cell fraction partially restored the proliferative but not the differentiative responses to 8-BrGuo and 8-MGuo. Although small B cells alone did not proliferate or differentiate in response to 8-BrGuo and 8-MGuo, cell surface expression of Ia antigens increased following incubation with these compounds. Thus, the biological activity of 8-BrGuo and 8-MGuo appears to be dictated by the cell type upon which it is acting. Small B cells are activated as evidenced by increased levels of surface Ia whereas large B cells are not only activated but are also induced to proliferate and differentiate.     

Effects of the interferon-inducer ABPP on colon cancer in rats; importance of tumor load and tumor site

Summary ABPP (2-amino-5-bromo-6-phenyl-4-pyrimidinone) is a pyrimidinone with known interferon-inducing, natural killer (NK) cell activity enhancing, antiviral and antitumor properties in several animal species. Its effect on CC531, a dimethylhydrazine-induced, transplantable, weakly immunogenic adenocarcinoma of the colon in WAG rats, was studied. ABPP was found to have no direct cytotoxic effect on CC531 cells in vitro. When small cubes of tumor of equal weight were implanted under the renal capsule, administration of 250 mg/kg of ABPP i. p. on day 0 and +1 led repeatedly to significant (p<0.02 up to p<0.001) inhibition of tumor growth, when measured on day +7. Lower doses or a single dose of ABPP did not achieve this effect. Late administration (on day +6 and +7) of 250 mg/kg of ABPP in this model was found to have no effect on tumor growth when measured on day +13. When 5×10⁵ tumor cells were injected in the portal vein, administration of 250 mg/kg of ABPP i.p. on day 0 and +1 reduced significantly (p=0.002) the number of liver metastases, when counted on day +30. Survival in this group was significantly prolonged (p<0.01). However when ABPP was given on day +6 and +7, significantly more (p<0.02) metastases in the liver were counted on day +30. The results show a significant antitumor effect of ABPP against tumor CC531 in the subrenal capsule assay (SRCA) model as well as in the liver metastasis model when administered at the time of tumor inoculation. Late administration of ABPP did not inhibit tumor growth in the SRCA and significantly enhanced the development of liver metastases. The role of timing, tumor site, and the mechanisms by which this dual outcome of immunotherapy with ABPP is mediated are discussed. The results of these experiments may have important implications for the design of clinical studies with ABPP.This study was supported in part by a grant from The Upjohn Company, Kalamazoo, Michigan, USA     

The synthesis, physicochemical properties and immunological activity of 5-amino-3-methylisoxazolo[5,4-d]4-pyrimidinone derivatives

A series of 5-amino-3-methylisoxazole[5,4-d]4-pyrimidinone derivatives were obtained by reacting substituted 5-amino-3-methylisoxazol-4-carboxylic acid hydrazide with ethyl ortho-formate. The compounds were tested using the models of in vivo cellular and humoral immune response in mice and pokeweed mitogen-induced (PWM-induced) polyclonal antibody production in a culture of human peripheral blood mononuclear cells (PBMC). The compounds exhibited differential inhibitory activities in the described models, depending on the character and location of the substituted groups. We suggest that the compounds affect the early stages of the immune response.     

Synthesis and biological relationships of 3',6-substituted 2-phenyl-4-quinolone-3-carboxylic acid derivatives as antimitotic agents

As part of a continuing search for potential anticancer drug candidates in the 2-phenyl-4-quinolone series, 3',6-substituted 2-phenyl-4-quinolone-3-carboxylic acid derivatives and their salts were synthesized and evaluated. Preliminary screening showed that carboxylic acid analogs containing a m-fluoro substituted 2-phenyl group displayed the highest in vitro anticancer activity. Activity decreased significantly if a chlorine or methoxy group replaced the fluorine atom. 3'-Fluoro-6-methoxy-2-phenyl-4-quinolone-3-carboxylic acid (68) had the highest in vitro cytotoxic activity among all tested carboxylic acid derivatives and their salts. The mechanism of action may be similar, but not identical, to that of tubulin binding drugs, such as navelbine and taxol. Compound 68 merits further investigation as a novel hydrophilic antimitotic agent.     

Structural studies on bioactive compounds. Part 29: palladium catalysed arylations and alkynylations of sterically hindered immunomodulatory 2-amino-5-halo-4,6-(disubstituted)pyrimidines

The immunological agent bropirimine 5 is a tetra-substituted pyrimidine with anticancer and interferon-inducing properties. Synthetic routes to novel 5-aryl analogues of bropirimine have been developed and their potential molecular recognition properties analysed by molecular modelling methods. Sterically challenged 2-amino-5-halo-6-phenylpyrimidin-4-ones (halo = Br or I) are poor substrates for palladium catalysed Suzuki cross-coupling reactions with benzeneboronic acid because the basic conditions of the reaction converts the amphoteric pyrimidinones to their unreactive enolic forms. Palladium-mediated reductive dehalogenation of the pyrimidinone substrates effectively competes with cross-coupling. 2-Amino-5-halo-4-methoxy-6-phenylpyrimidines can be converted to a range of 5-aryl derivatives with the 5-iodopyrimidines being the most efficient substrates. Hydrolysis of the 2-amino-5-aryl-4-methoxy-6-phenylpyrimidines affords the required pyrimidin-4-ones in high yields. Semi-empirical quantum mechanical calculations show how the nature of the 5-substituent influences the equilibrium between the 1H- and 3H-tautomeric forms, and the rotational freedom about the bond connecting the 6-phenyl group and the pyrimidine ring. Both of these factors may influence the biological properties of these compounds.     

Synthesis of novel 2-amino-4-(5′-substituted 2′-phenyl-1H-indol-3′-yl)-6-aryl-4H-pyran-3-carbonitrile derivatives as antimicrobial and antioxidant agents

As a part of systematic investigation of synthesis and biological activities of indole analogues linked to various heterocyclic systems, we have synthesized new compounds viz., 2-amino-4-(5′-substituted 2′-phenyl-1H-indol-3′-yl)-6-aryl-4H-pyran-3-carbonitriles (2a–i), 4,5-diamino-6-(5′-substituted 2′-phenyl-1H-indol-3′-yl)-8-aryl-2-oxo-2,6-dihydrodipyrano [2,3-b:3,2-e]pyridine-3-carbonitriles (3a–i), 4-amino-5-(5′-substituted 2′-phenyl-1H-indol-3-yl)-7-aryl-1H-pyrano[2,3-d]pyrimidin-2(5H)-ones (4a–i), 4-amino-5-(5′-substituted 2′-phenyl-1H-indol-3′-yl)-7-aryl-1H-pyrano[2,3-d]pyrimidin-2(5H)-thiones (5a–i), 4-(5′-subtituted 2′-phenyl-1H-indol-3′-yl)-6-aryl-1,4-dihydropyrano[2,3-c]pyrazol-3-amines (6a–i) and 5-(5′-substituted 2′-phenyl-1H-indol-3′-yl)-7-aryl-3H-pyrano[2,3-d]pyrimidin-4(5H)-ones (7a–i). Antibacterial activity results revealed that, compound 6a showed promising activity versus Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. Compound 6d exhibited good activity against S. aureus, K. pneumoniae and Pseudomonas aeruginosa. Antifungal activity results indicated that, compound 4d exhibited maximum zone of inhibition against Aspergillus oryzae and Aspergillus flavus. In case of antioxidant activity, compound 4a showed promising radical scavenging activity, ferric ions (Fe³⁺) reducing antioxidant power (FRAP) and metal chelating activity.     

Synthesis and pharmacological assessment of derivatives of isoxazolo[4,5-d]pyrimidine

A series of new 5-alkyl and 5-arylisoxazolo[4,5-d]pyrimidinones (5a-g, 6-8) were prepared from 4-amino-3-oxo-isoxazolidine-5-carboxylic acid amide. Some of the aryl derivatives of isoxazolo[4,5-d]pyrimidine were tested pharmacologically in comparison with Diazepam. Compounds 5b-d and 7 demonstrated interesting anxiolytic activity.     

Synthesis and in vitro cytotoxicity of 5-substituted 2-cyanoimino-4-imidazodinone and 2-cyanoimino-4-pyrimidinone derivatives

A series of 5-substituted 2-cyanoimino-4-imidazodinone and 2-cyanoimino-4-pyrimidinone derivatives were synthesized and their anticancer cytotoxicity were evaluated in in vitro assay. It was found that the bulky aryl functionality in the 5-position of the 2-cyanoimino-4-imidazolidinone compounds was essential for the cytotoxicity of these heterocyclic compounds. Some of the derivatives exhibited modest cytotoxicity against a variety of cancer cell lines. One of the derivatives, [1-[6-(4-chlorophenoxy)hexyl]-5-oxo-4-phenyl-3-(4-pyridyl)tetrahydro-1H-2-imidazolyliden]aminomethanenitrile (Compound 11), exhibited the most potent cytotoxic activity with IC(50) in the nanomolar range. The cytotoxicity of these derivatives was selection with no apparent toxic effect toward normal fibroblasts.     

Ring opening is not rate-limiting in the GTP cyclohydrolase I reaction

GTP cyclohydrolase I catalyzes a mechanistically complex ring expansion affording dihydroneopterin triphosphate and formate from GTP. Single turnover quenched flow experiments were performed with the recombinant enzyme from Escherichia coli. The consumption of GTP and the formation of 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate, formate, and dihydroneopterin triphosphate were determined by high pressure liquid chromatography analysis. A kinetic model comprising three consecutive unimolecular steps was used for interpretations where the first intermediate, 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone 5'-triphosphate, was formed in a reversible reaction. The rate constant k(1) for the reversible opening of the imidazole ring of GTP was 0.9 s(-1), the rate constant k(3) for the release of formate from 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate was 2.0 s(-1), and the rate constant k(4) for the formation of dihydroneopterin triphosphate was 0.03 s(-1). Thus, the hydrolytic opening of the imidazole ring of GTP is rapid by comparison with the overall reaction.     

Conversion of substituted naphthalenesulfonates by Pseudomonas sp. BN6

The range of substituted naphthalenesulfonates which are metabolized by Pseudomonas sp. BN6 were investigated. Resting cells from strain BN6 oxidized 1- and 2-naphthalenesulfonate, 1-hydroxynaphthalene-2-sulfonate, 2,6-naphthalenedisulfonate and all monosulfonated naphthalene-2-sulfonates which carry one or two substitutents in the positions 4-, 5-, 6-, 7- or 8- of the naphthalene ring-system. With the exception of (substituted) 4- or 5-amino- and 4-hydroxynaphthalene-2-sulfonates these compounds were converted to the corresponding salicylates. Strain BN6 did not oxidize substituted naphthalene-1-sulfonates, 3-substituted naphthalenesulfonates and substituted naphthalenedisulfonates. Turnover of 4-amino- or 4-hydroxynaphthalene-2-sulfonates resulted in the accumulation of the corresponding naphthoquinones in the culture medium. Thus, degradation of 4-amino- and 4-hydroxynaphthalenesulfonates was restricted by the rapid autoxidation of the substituted 1,2-dihydroxynaphthalenes formed as metabolites. Catabolic activities of strain BN6 for naphthalenesulfonates were induced by salicylate, 3- or 6-hydroxysalicylate, and 3-, 4- or 5-aminosalicylate but not by 4- and 5-hydroxysalicylate. All naphthalenesulfonates that were not converted into the corresponding salicylates, were found to be inefficient as effectors. It was therefore concluded that (substituted) salicylates are the inducers of the relevant enzymes. The degradation of 2-naphthalene-sulfonate by a pure culture of strain BN6 was prevented by the toxicity of the dead-end product salicylate. Substituted salicylates were less toxic and allowed growth of strain BN6 in axenic culture with various substituted naphthalenesulfonates.Abbreviations AB aminobenzoate - ANS aminonaphthalenesulfonate - DHN dihydroxynaphthalene - DHNC dihydroxynaphthalene-carboxylate - DHNDO 1,2-dihydroxynaphthalene dioxygenase - HBPA 2-hydroxybenzalpyruvate aldolase - HNS hydroxynaphthalenesulfonate - HS hydroxysalicylate - Ind-C indolecarboxylate - Ind-S indolesulfonate - MANS N-methylaminonaphthalenesulfonate - NC naphthalenecarboxylate - NDS naphthalenedisulfonate - NQ naphthoquinone - NS naphthalenesulfonate - NSDO naphthalenesulfonate dioxygenase - R_t retention time - SADH salicylaldehyde dehydrogenase - THN trihydroxynaphthalene (hydroxy-1,2-dihydroxynaphthalene)     

Synthesis and enzymatic evaluation of pyridinium-substituted uracil derivatives as novel inhibitors of thymidine phosphorylase

A series of water soluble N(1)- and C(6)-substituted uracil pyridinium compounds were prepared as potential inhibitors of thymidine phosphorylase (TP). The C(6)-uracil substituted derivatives were the most active. 1-[(5-Chloro-2,4-dihydroxypyrimidin-6-yl)methyl]pyridinium chloride, was identified as the best inhibitor being 5-fold more potent than the known inhibitor, 6-amino-5-bromouracil.     

Dissociation of inductive from differentiative signals transmitted by C8-substituted guanine ribonucleosides to B cells from SJL mice

A hitherto unknown defect in the immune responsiveness of B lymphocytes from SJL mice has enabled us to distinguish two qualitatively distinct classes of signal delivered to B cells by C8-substituted guanine ribonucleosides. This defect renders B cells from SJL mice unresponsive to the inductive (early acting) signal of 8-mercaptoguanosine (8MGuo) that culminates in mitogenesis and nonspecific secretion of immunoglobulin. Unresponsiveness is not attributable to a shift in either the dose-response or kinetic profiles, nor can the presence of suppressor cells be demonstrated. In striking contrast, however, SJL B cells exhibit normal responsiveness to the differentiative (T cell-like, or late acting) signal provided by the substituted nucleoside. This signal enables SJL B cells, depleted of T cells, to respond to T cell-dependent antigens, and synergizes with T cell-derived lymphokines. These data suggest 1) that nonspecific secretion of immunoglobulin is dependent on both inductive and differentiative signals, 2) that antigen alone can supply an effective inductive signal for antigen-specific responses, and 3) that the SJL mouse will provide a useful model for selective study of inductive vs differentiative events.     

Stimulation of natural killer cells in two random-bred strains of athymic rats by interferon-inducing pyrimidinone

We tested the effect of 2-amino-5-bromo-6 phenyl-4 pyrimidinol (ABPP), an interferon (IFN) inducer, on NK cell cytotoxicity in various tissues of athymic and euthymic rats. Significant augmentation of NK cell cytotoxicity was observed in SP, PB, PE, lung, and liver of adult rats. Cytotoxic potential was also induced by ABPP in young rats, not exhibiting cytotoxicity before the treatment. Cytotoxic cells were detected in nylon wool-filtered fraction, and were not removed by carbonyl iron treatment. Separation on Percoll gradient indicated that ABPP-stimulated cytotoxic SPC resided in LGL-enriched fractions and some of them exhibited less dense characteristics than SPC from untreated rats. The ABPP-mediated cytotoxicity was thymus-independent; both athymic and euthymic rats were equally augmented. The observation that only tissues displaying NK cell augmentation produced significant levels of IFN suggest possible involvement of IFN in ABPP-mediated augmentation.     

The pyrimidine nucleotide reductase step in riboflavin and F(420) biosynthesis in archaea proceeds by the eukaryotic route to riboflavin

The Methanococcus jannaschii gene MJ0671 was cloned and overexpressed in Escherichia coli, and its gene product was tested for its ability to catalyze the pyridine nucleotide-dependent reduction of either 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (compound 3) to 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate (compound 4) or 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (compound 7) to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (compound 5). Only compound 3 was found to serve as a substrate for the enzyme. NADPH and NADH functioned equally well as the reductants. This specificity for the reduction of compound 3 was also confirmed by using cell extracts of M. jannaschii and Methanosarcina thermophila. Thus, this step in riboflavin biosynthesis in these archaea is the same as that found in yeasts. The absence of the other genes in the biosynthesis of riboflavin in Archaea is discussed.     

N6-substituted 9-methyladenines: a new class of adenosine receptor antagonists

A series of 15 N6-substituted 9-methyladenines have been assessed as antagonists of A2-adenosine receptor-mediated stimulation of adenylate cyclase in membranes of human platelets and rat PC12 cells and of A1-adenosine receptor-mediated inhibition of adenylate cyclases in membranes of rat fat cells and as inhibitors of binding of N6-R-[3H]phenylisopropyladenosine to A1-adenosine receptors in rat brain membranes. N6 substitution can markedly increase the potency of 9-methyladenine at A1 receptors, while having lesser effects or even decreasing potency at A2 receptors. Effects of N6 substituents on adenosine receptor activity of the 9-methyladenines are reminiscent of effects of N6 substituents on activity of adenosine, suggesting that N6 substituted 9-methyladenines bind to adenosine receptors in the same orientation as do N6-substituted adenosines. N6-Cyclopentyl-9-methyladenine with Ki values at the A1 receptors of 1.3 microM (fat cells) and 0.5 microM (brain) is at least 100-fold more potent than 9-methyladenine (Ki 100 microM, both receptors), while at the A2 receptors KB values of 5 microM (platelets) and 25 microM (PC12 cells) make it 5-fold more potent and equipotent, respectively, compared to 9-methyladenine (KB 24 microM, both receptors). N6-Cyclopentyl and several other N6-alkyl and N6-cycloalkyl analogs are selective for A1 receptors while 9-methyladenine is the most A2 receptor selective antagonist. The N6-R- and N6-S-(1-phenyl-2-propyl)-9-methyladenines, analogous to N6-R- and N6-S-phenylisopropyladenosines, exhibit stereoselectivity at both A1 and A2 receptors. Marked differences in potency of certain N6-substituted 9-methyladenines at the A2 receptors of human platelets and rat PC12 cells provide evidence that these are not identical receptors.     

The interplay of hydrogen bonds and halogen bonds in the structure of NH-pyrazoles bearing C-aryl and C-halogen substituents

The behavior in solution and in the solid state of 3(5)-phenyl-1H-pyrazole (7), 3(5)-phenyl-4-chloro-1H-pyrazole (6), 3(5)-phenyl-4-bromo-1H-pyrazole (1), and 3(5)-p-chlorophenyl-4-bromo-1H-pyrazole (8) is discussed in relation to their 3-phenyl (a)/5-phenyl (b) annular tautomerism. Two new X-ray structures are reported: a new polymorph of 1 and the structure of 6. The new polymorph is a 3-phenyl-1H-pyrazole 1a′ trimer while the new structure is a 5-phenyl-1H-pyrazole 6b trimer. The combined use of NMR at low temperature and DFT calculations allows to discuss the tautomerism of the first three pyrazoles and to predict that the fourth one should be a tetramer formed by both tautomers, 8a and 8b.     

Synthesis of base substituted 2-hydroxy-3-(purin-9-yl)-propanoic acids and 4-(purin-9-yl)-3-butenoic acids

Alkylation of 6-chloropurine and 2-amino-6-chloropurine with bromoacetaldehyde diethyl acetal afforded 6-chloro-9-(2,2-diethoxyethyl)purine (3a) and its 2-amino congener (3b). Treatment of compounds 3 with primary and secondary amines gave the N6-substituted adenines (5a-5c) and 2,6-diaminopurines (5d-5f). Hydrolysis of 3 resulted in hypoxanthine (6a) and guanine (6b) derivatives, while their reaction with thiourea led to 6-sulfanylpurine (7a) and 2-amino-6-sulfanylpurine (7b) compounds. Treatment with diluted acid followed by potassium cyanide treatment and acid hydrolysis afforded 6-substituted 3-(purin-9-yl)- and 3-(2-aminopurin-9-yl)-2-hydroxypropanoic acids (8-10). Reaction of compounds 3 with malonic acid in aqueous solution gave exclusively the product of isomerisation, 6-substituted 4-(purin-9-yl)-3-butenoic acids (15).     

Synthesis and Herbicidal Activity of 6-Phenyl-5-Propylamino-2-Pyrazinecarbonitriles and Related Compounds

6-Phenyl- and 5-phenyl-2-pyrazinecarbonitriles with or without a propylamino group at the 3-, 5- or 6-position of the pyrazine ring were prepared together with some related compounds from the corresponding 2,3-pyrazinedicarbonitriles. Their herbicidal activities against barnyardgrass and broadleaf weeds were examined in pot tests. The 6-phenyl-2-pyrazinecarbonitriles were relatively potent compared with the 5-phenyl derivatives. Moreover, the presence of a propylamino group at the 5-position of the 6-phenyl-2-pyrazinecarbonitriles was closely related to an increase in activity.     

Biosynthesis of vitamin B2.

GTP cyclohydrolase II catalyzes the hydrolytic release of formate and pyrophosphate from GTP producing 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, the first committed intermediate in the biosynthesis of riboflavin. The enzyme was shown to contain one zinc ion per subunit. Replacement of cysteine residue 54, 65 or 67 with serine resulted in proteins devoid of bound zinc and unable to release formate from the imidazole ring of GTP or from the intermediate analog, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate. However, the mutant proteins retained the capacity to release pyrophosphate from GTP and from the formamide-type intermediate analog. The data suggest that the enzyme catalyzes an ordered reaction in which the hydrolytic release of pyrophosphate precedes the hydrolytic attack of the imidazole ring. Ring opening and formate release are both dependent on a zinc ion acting as a Lewis acid, which activates the two water molecules involved in the sequential hydrolysis of two carbon-nitrogen bonds.     

Synthetic studies towards a new scaffold, spirobicycloimidazoline

A new scaffold consisting of a carbocycle and a substituted imidazoline in an orthogonal arrangement was synthesized as a potential specific inhibitor of glycosidases. The spirobicycloimidazoline, (5R,6R,7R,8R)-8-(hydroxymethyl)-2-phenyl-1,3-diazaspiro[4.4]non-1-ene-6,7-diol, was synthesized from methyl 2-O-p-methoxybenzyl-3,4-di-O-benzyl-alpha/beta-D-gluco-6-enopyranoside via (1R,2S,3S,4R,5S)-3,4-bis(benzyloxy)-2-(4-methoxybenzyloxy)-5-vinyl-cyclopentanol. The ring contraction of the 6-enopyranoside in the presence of zirconocene equivalent ('Cp(2)Zr') reagent gave exclusively the corresponding cyclopentanol without cleavage of the PMB protecting group. In the course of the study, a new alpha-mannosidase inhibitor, (1R,2R,3R,5R)-5-amino-3-hydroxymethyl-cyclopentane-1,2-diol, was also discovered.