Dimetalation of pyrazines. a one-pot synthesis of multisubstituted pyrazine C-nucleosides.

As a part of our efforts to pursue direct, convergent, and concise methodologies for the synthesis of pyrazine C-nucleosides, we have successfully established a sequential dilithiation-addition method, which allows one to introduce two different functional groups to a pyrazine ring in a one-pot fashion. 2,6-Dichloropyrazine was dilithiated at -100 degrees C and then allowed to react with an electrophile, such as bromine, iodine, or disulfides, followed by a reaction with a protected ribonolactone to afford C-nucleosides. After reduction and deprotection, tetrasubstituted pyrazine C-nucleosides, including 2,6-dichloro-3-iodo-5-(beta-D-ribofuranosyl)pyrazine and 2-bromo-3,5-dichloro-6-(beta-D-ribofuranosyl)pyrazine, were obtained. A tandem reaction sequence occurred when disulfides were used, resulting in the formation of 5,6-bis-methylthio-2-chloro-3-(beta-D-ribofuranosyl)pyrazine and 6-(beta-D-ribofuranosyl)-2,3,5-tris-phenylthiopyrazine.

The oxidative DNA lesion 8,5'-(S)-cyclo-2'-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells.

Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cell's capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.

Synthesis and antiviral evaluation of certain novel pyrazinoic acid C-nucleosides.

Pyrazine (1,4-diazine) C-nucleosides constitute a rare class of nucleic acid analogues that has only recently been reported in the literature. As part of our ongoing investigation into the synthesis and reactivity of these compounds, we have developed an electrophilic esterification of a lithiated pyrazine C-nucleoside (1) to give, following deprotection, the versatile intermediate ethyl 3,5-dichloro-6-(beta-d-ribofuranosyl)pyrazine-2-carboxylate (4). This intermediate was subjected to a variety of reaction conditions to generate a series of pyrazinoic acid C-nucleosides. These compounds, along with 3, 5-dichloro-2-(beta-d-ribofuranosyl)pyrazine (2) and 4, were evaluated for antiviral activity and cytotoxicity. No significant activity was observed for compounds 2 and 5-9, but 4 was active against two herpes viruses and cytotoxic in the micromolar range.

Synthesis and evaluation of 6-(dibromomethyl)-5-nitropyrimidines as potential antitumor agents.

A series of 2,4,6-trisubstituted-5-nitropyrimidines have been prepared and evaluated for inhibition of proliferation of L1210 and H.Ep.2 cells in vitro. The most potent compound was 6-(dibromomethyl)-2-methoxy-4-morpholino-5-nitropyrimidine (11) (L1210, IC50 = 0.32 microM; H.Ep.2, IC50 = 1.6 microM). Of the 6-substituents incorporated, only CHBr2, CH2Br, and CHO were compatible with antiproliferative activity, while a wider variety of 4-substituents were tolerated. At concentrations near the IC50 for antiproliferative activity, a delayed resumption of cell proliferation in L1210 cultures indicated that the activity of the compounds was short-lived and suggested they might act by an alkylation mechanism.

Synthesis of 2,4-disubstituted thiazoles and selenazoles as potential antitumor and antifilarial agents: 1. Methyl 4-(isothiocyanatomethyl)thiazole-2-carbamates, -selenazole-2- carbamates, and related derivatives.

Methyl 4-(isothiocyanatomethyl)thiazole-2-carbamate and methyl 4-(isothiocyanatomethyl)selenazole-2-carbamate have been prepared via chemical transformations involving 2-amino-4-(chloromethyl)thiazole (1) and 2-amino-4-(chloromethyl)selenazole (2), respectively, as starting materials. The homoanalog, methyl 4-(2-isothiocyanatoethyl)thiazole-2-carbamate, was prepared from (2-aminothiazol-4-yl)acetic acid. All compounds prepared were evaluated for their ability to inhibit leukemia L1210 cell proliferation. Methyl 4-(isothiocyanatomethyl)thiazole-2-carbamate (7) was the most active compound in this screen, inhibiting the growth of L1210 leukemic cells with an IC50 = 3.2 microM. Mitotic blocking appears to be its primary mechanism of cytotoxic activity. Compound 7 also was the only compound which demonstrated significant in vivo antifilarial activity against the adult worms of Acanthocheilonema viteae in experimentally infected jirds. This compound was inactive against Brugia pahangi at a dosage of 100 mg/kg x 5 days.

Synthesis of 2,4-disubstituted thiazoles and selenazoles as potential antifilarial and antitumor agents. 2. 2-Arylamido and 2-alkylamido derivatives of 2-amino-4-(isothiocyanatomethyl)thiazole and 2-amino-4-(isothiocyanatomethyl)selenazole.

The synthesis of a series of 2-arylamido and 2-alkylamido derivatives of 2-amino-4-(isothiocyanatomethyl)thiazole and 2-amino-4-(isothiocyanatomethyl)selenazole is described. In vitro antiproliferative evaluations were carried out using L1210 cells. The 2-(alkylamido)thiazole derivatives were moderately antiproliferative, with IC50's of 4-8 microM. A significant increase in activity was obtained for the arylamido derivatives, with IC50's of 0.2-1 microM. The results obtained for the selenazoles were similar to those for the thiazoles. 2-Benzamido-4-(isothiocyanatomethyl)-thiazole (19) was found to be a potent inhibitor of GMP synthetase. None of the compounds prepared in this study demonstrated antifilarial activity.

Synthesis and biological activity of certain alkyl 5-(alkoxycarbonyl)-1H-benzimidazole-2-carbamates and related derivatives: a new class of potential antineoplastic and antifilarial agents.

A series of methyl and ethyl 5-(alkoxycarbonyl)-1H-benzimidazole-2-carbamates (7-19) and methyl 5-carbamoyl-1H-benzimidazole-2-carbamates (24-34) have been synthesized via the reaction of an appropriate alcohol or amine with the acid chloride derivatives 6a or 6b at room temperature. Reaction of an alcohol with acid chloride 6a at reflux temperature afforded transesterified products 20-23 in good yield. Treatment of methyl 5-amino-1H-benzimidazole-2-carbamate with substituted benzoyl chlorides furnished the methyl 5-benzamido-1H-benzimidazole-2-carbamates (36-38). Compounds 9, 16, 20, and 22 demonstrated significant growth inhibition in L1210 cells with IC50's less than 1 microM. Growth inhibition by this series of compounds appears to be associated with mitotic spindle poisoning. All the compounds tested, 9, 10, 19, 20, 22, and 23, caused significant accumulation of L1210 cells in mitosis. Compounds 7, 9, 19, 25, 26, 27, and 36 showed significant in vivo antifilarial activity against adult worms of Brugia pahangi, Litomosoides carinii, and Acanthocheilonema viteae in experimentally infected jirds.

The synthesis and chemistry of certain anthelmintic benzimidazoles.

A basis for interest in the benzimidazole ring system as a nucleus from which to develop potential chemotherapeutic agents was established in the 1950s when it was found that 5,6-dimethyl-l-(alpha-D-ribofuranosyl)benzimidazole (I) was an integral part of the [structure: see text] structure of vitamin B(12). As a result of these interests and extensive studies, one health related arena that has benefited greatly has been the treatment of parasitic diseases. The discovery of thiabendazole in 1961 further spurred chemists around the world to design and synthesize several thousand benzimidazoles for screening for anthelmintic activity but less than twenty of them have reached commercial use. Much of this work has been done by pharmaceutical companies and is only reported in the patent literature. In this paper, Leroy Townsend and Dean Wise review the development of some of the synthetic methods that have been critical to the preparation of the benzimidazoles of anthelmintic importance. Only a few molecules that demonstrate the processes are discussed here, but numerous reviews of the synthesis and chemistry of other benzimidazoles are available.

A rapid and sensitive screening method for the detection of anti-2-acetylaminofluorene immunoglobulins.

A method is described in which anti-2-acetylaminofluorene immunoglobulins may be detected using a simple and sensitive screening procedure. The method is based on immunoglobulin binding of an 125I derivatized 2-aminofluorene radiotracer. Tracer binding is not isotype specific, and thus the method is useful for the detection of either IgG or IgA. Competitive binding experiments with the radiotracer were used to determine the specificity of immunoglobulin response by measurement of cross-reactivity with related ligands. This method allows quantitation of the immune response to the carcinogen in serum and other biological fluids (i.e., intestinal secretions).

Synthesis and biological activity of a novel adenosine analogue, 3-beta-D-ribofuranosylthieno[2,3-d]pyrimidin-4-one.

The title nucleoside 5 was prepared by a condensation of the silylated heterocycle thieno[2,3-d]pyrimidin-4-one (1) with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose (2a) in the presence of a Lewis acid or with 2,3,5-tri-O-acetyl-D-ribofuranosyl bromide (2b) in the presence of mercuric oxide and mercuric bromide. The site of ribosylation and anomeric configuration of this nucleoside were established by 1H NMR. The synthesis of 3-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidin-4-one (8), 1-phenyl-5-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4-one (9), 5-methyl-3-beta-D-ribofuranosylthieno[2,3-d]pyrimidin-4-one (10), and 2-methyl-6-beta-D-ribofuranosyltriazolo[5,4-d]pyrimidin-7-one (11) is also described. The title compound inhibited the growth of murine L-1210 leukemic cells in vitro with an ID50 of 3 X 10(-5)M. The growth inhibition could not be prevented by uridine, cytidine, thymidine, deoxycytidine, cytosine, hypoxanthine, or uridine and hypoxanthine together. On the other hand, inhibition of adenosine kinase by 10(-7) M 5-iodotubercidin prevented the cytotoxic effect. Also a subline of L-1210 cells resistant to several cytotoxic adenosine analogues was also resistant to this nucleoside. Thus it appears that this compound 5 may act as an adenosine analogue.

Synthesis of potential antifilarial agents. 1-(5-Benzoylbenzimidazol-2-yl)-3-alkyl- and -arylureas.

A series of 1-(5-benzoylbenzimidazol-2-yl)-3-substituted ureas have been synthesized by reacting an appropriate isocyanate with 2-amino-5-benzoylbenzimidazole or by reacting methyl (5-benzoylbenzimidazol-2-yl)carbamate with various amines. Several of the compounds have demonstrated antifilarial activity against Brugia pahangi and Litomosoides carinii.

Synthesis of pyridazine analogues of the naturally occurring nucleosides cytidine, uridine, deoxycytidine, and deoxyuridine.

The condensation of 4,5-dichloro-3-[(trimethylsilyl)oxy]pyridazine (5) with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose (6) was accomplished by the stannic chloride procedure to yield 4,5-dichloro-1-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)pyridazin-6-one (7). Procedures used to unequivocally determine the site of ribosylation and anomeric configuration of 7 are discussed. Treatment of 7 with liquid ammonia effected a concomitant removal of the blocking groups and selective nucleophilic displacement of the 4-chloro group. Subsequent dehalogenation yielded 4-amino-1-(beta-D-ribofuranosyl)pyridazin-6-one (11, 6-aza-3-deazacytidine). Treatment of 7 with methanolic sodium methoxide, followed by dehalogenation and hydrolysis with aqueous alkali, yielded 4-hydroxy-1-beta-D-ribofuranosylpyridazin-6-one (6-aza-3-deazauridine, 15). The syntheses of various nucleosides derived from 7, 11, and 15 are described. Condensation of 5 with 3,5-di-O-p-toluoyl-2-deoxy-D-erythro-pentofuranosyl chloride (27) gave a mixture of the blocked anomeric 2'-deoxynucleosides 28 and 29. Nucleoside 28, the beta anomer, was treated in the same manner as 7 to yield 4-amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)pyridazin-6-one (32, 6-aza-3-deaza-2'-deoxycytidine) and 4-hydroxy-1-(2-deoxy-beta-D-erythro-pentofuranosyl)pyridazin-6-one (32, 6-aza-3-deaza-2'-deoxycytidine) and 4-hydroxy-1-(2-deoxy-beta-D-erythro-pentofuranosyl)pyridazin-6-one (34, 6-aza-3-deaza-2'-deoxy-uridine). 6-Aza-3-deazauridine (15) was found to inhibit the growth of L1210 cells with an ID50 of about 7 x 10(-5) M.

Relative locations of the beta and delta chains of the acetylcholine receptor determined by electron microscopy of isolated receptor trimer.

The monomeric unit of the acetylcholine receptor of electric tissue of Torpedo californica has previously been shown to have a subunit composition of alpha 2 beta gamma delta. Receptor in membrane isolated from Torpedo electric tissue occurs as both monomer and dimer. In the dimer which is the predominant form, the monomeric units are cross-linked via a disulfide bond between delta chains. The addition of diamide to receptor-rich membrane causes the formation of trimer and higher oligomers in which the monomeric units are linked by disulfide bonds alternately between pairs of delta chains and between pairs of beta chains. We have isolated receptor trimer and determined the relative locations of the monomeric units by scanning transmission electron microscopy of negatively stained preparations. In face view, the trimer appears as three approximately 90 A disks, each with a central, densely staining pit. From the angles of the triangle formed by the lines connecting the centers of the monomers in the trimer, we infer that the beta-beta disulfide bond is separated from the delta-delta disulfide bond by an angle in the range of 50-80 degrees.

Structure of acetylcholine receptor dimer determined by neutron scattering and electron microscopy.

Previous work has shown that the predominant native form of the acetylcholine receptor from the electric tissue of Torpedo californica is a dimer of Mr = 500,000, cross-linked by a disulfide bond between the largest (delta) of the five chains (alpha 2 beta gamma delta) that comprise the monomer. Small-angle neutron scattering of purified receptor dimer in Triton X-100 solution containing 18% D2O, in which the Triton X-100 is contrast-matched, yields a radius of gyration of the dimer of 66 A. Based on the assumptions that the dimer is symmetrical and that the radius of gyration of the monomer does not change in forming dimer, this value, together with the radius of gyration of the receptor monomer (46 A), determined previously, allows the calculation of the distance separating the centers of neutron scattering density of monomers in a dimer; the result is 96 A. Electron microscopy of negatively stained dimers permits an independent measurement of the distance between the apparent centers of mass of the monomers; the average is 96 A, in agreement with the result of the neutron scattering analysis. The electron micrographs of dimer also permit the location of the delta chains at the region of contact of the monomers. A model for the receptor dimer consistent with all available structural information is presented.

An analysis by low-angle neutron scattering of the structure of the acetylcholine receptor from Torpedo californica in detergent solution.

The acetylcholine receptor from the electric tissue of Torpedo californica is a large, integral membrane protein containing four different types of polypeptide chains. The structure of the purified receptor in detergent solution has previously been investigated by sedimentation analysis and gel filtration. Sedimentation analysis yielded a molecular weight of 250,000 for the protein moiety of the receptor monomer-detergent complex; hydrodynamic characteristics such as the Stokes radius, however, refer to the receptor-detergent complex. In this paper we report the results of our use of low-angle neutron scattering to investigate the shape of the receptor-detergent (Triton X-100 from Rohm & Haas Co., Philadelphia, Pa.) complex and separately of its protein and detergent moieties. By adjustment of the neutron-scattering density of the solvent with D2O to match that of one or the other of the moieties, its contribution to the scattering can be nearly, if not completely, eliminated. Neutron scattering from Triton X-100 micelles established that this detergent is contrast matched in approximately 18% D2O. Scattering measurements on the receptor-detergent complex in this solvent yielded a radius of gyration of the acetylcholine receptor monomer of 46 +/- 1A. The radius of gyration and molecular volume (305,000 A3) of the receptor are inconsistent with a compact spherical shape. These parameters are consistent with, for example, a prolate cylinder of dimensions (length x diameter) approximately 150 x approximately 50 A or an oblate cylinder, approximately 25 x approximately 130 A. More complex shapes are possible and in fact seem to be required to reconcile the present results with previous electron microscopic and x-ray analyses of receptor in membrane and with considerations of the function of the receptor in controlling ion permeability. The neutron-scattering data yield, in addition, an independent determination of the molecular weight of the receptor protein (240,000 +/- 40,000), the extent of Triton X-100 binding in the complex (approximately 0.4 g/g protein), and from the extended scattering curve, an approximation to the shape of the receptor-Triton X-100 complex, namely an oblate ellipsoid of axial ratio 1:4.

Conformation of pyridazine nucleosides: the molecular structure of 4-hydroxy-1-(beta-D-ribofuranosyl)pyridazin-6-one (3-deaza-6-azauridine).

The glycosyl torsiona angle in 4-hydroxy-1-(beta-D-ribofuranosyl)-pyridazin-6-one (or 3-deaza-6-azauridine) is in the "high-anti" region. This is similar to the torsional angles observed for 6-azapyrimidine and 8-azapurine nucleosides, but in marked contrast to those found in uridine, 3-deazauridine and other pyrimidine nucleosides.