Polymers containing stable free radicals, 4. Preparation of 2,4-diphenyl-6-(3-maleoyliminophenyl)-3,4-dihydro-s-tetrazin-1(2H)-yl and its anionic polymerization

2,4-Diphenyl-6-(3-maleoyliminophenyl)-3,4-dihydro-s-tetrazin-1(2H)-yl ( 6 ) was synthesized and anionically polymerized with a catalytic amount of sodium 1,4-dihydro-1,4-naphthalenediide, with a conversion up to 96%. The polymer was a dark green powder with a content of radical groups of about 58% (based on unit mol), and its relative molecular mass was about 4 800, corresponding to 12 monomeric units.     

Polymers from carbohydrate-based monomers, 1. Poly(acylhydrazone)s from D-glucarodihydrazide and related compounds

The synthesis of poly(acylhydrazone)s from aldarodihydrazides, principally D -glucarodihydrazide, was studied. Polymers were produced on reaction of the dihydrazides with dialdehydes and with aliphatic ketoaldehydes and diketones, but not with aromatic ketoaldehydes or aromatic diketones. The polymers, which have only limited solubilities that are not appreciably improved by copolymerization, undergo a tautomeric change on treatment with acid or alkali to give yellow-coloured polymers. Of the poly(acylhydrazone)s prepared, that from D -glucarodihydrazide and 2,3-butanedione had the best overall properties, giving strong, flexible, colourless films on casting from DMSO solution.     

Synthesis and in vitro evaluation of the anti-viral activity of N-[4-(1H(2H)-benzotriazol-1(2)-yl)phenyl]alkylcarboxamides

A series N-[4-(1H(2H)-benzotriazol-1(2)-yl)phenyl]alkylcarboxamides (8e-k, 9e-i, k, l) and their parent amines (5a-c and 6a-d) were prepared according to Schemes (1 and 2). Compounds were evaluated in vitro for cytotoxicity and antiviral activity against a wide spectrum of RNA (positive- and negative-sense) viruses, like [Bovine Viral Diarrhea Virus (BVDV), Yellow Fever Virus (YFV), Coxsackie Virus B (CVB-2), Polio Virus (Sb-1), Human Immunodeficiency Virus (HIV-1), Respiratory Syncytial Virus (RSV)] or double-stranded (dsRNA) virus, like Reoviridae (Reo-1). The Entero (CVB-2 and Sb-1) were the only viruses inhibited by title compounds. In particular, two of them emerged for their selective, although not very potent, antiviral activity: 8i, which was the most active against CVB-2 (CC50 >100 microM; EC50 = 10 microM) and 9l, which was the most active against Sb-1 (CC50 90 microM; EC50 = 30 microm). Title compounds were evaluated in silico against the Sb-1 helicase, as the crystal structure of this enzyme was not available, the corresponding 3D model was obtained by homology techniques (see Fig. 2).     

A common antigenic epitope in influenza A virus (H1, H2, H5, H6) hemagglutinin]

Avian influenza A viruses belonging to hemagglutinin (HA) subtypes H5 and H6 were studied in the infectivity neutralization test and radioimmunoprecipitation assay (RIPA) with monoclonal antibody MAb C179. This MAb recognizes a conformational antigenic epitope in the stem region of HA formed by two regions (amino acid positions 318-322 in HA1 subunit and 47-58 in HA2), conserved in all H1 and H2 influenza viruses. MAb C179 reacts with HA of H5 viruses in RIPA and neutralizes these strains as efficiently as H2 viruses. C179 precipitates H6 subtype HA but does not neutralize the infectivity of these viruses. Comparison of amino acid sequences of H2, H5, and H6 strains showed identical epitope recognized by MAb C179 in H5 and H6 HAs, which differs from epitopes of H1 and H2 by two amino acids in the HA2 subunit. Causes of disagreement between immunoprecipitation of H6 HA by MAb C179 and neutralization of this serosubtype by this MAb are discussed.     

Polymers of carbonic acid, 6. Polymerization of trimethylene carbonate (1,3-dioxan-2-one) with complexation catalysts

Numerous bulk polymerizations of trimethylene carbonate (1,3-dioxan-2-one, 2 ) were conducted at 90, 120 and 150°C. Tributyltin methoxide, tributyltin acetate, dibutyltin dibromide, tin(II) 2-ethylhexanotate, bismuth(III) 2-ethylhexanoate and zinc stearate were used as initiators. On the basis of GPC measurements with universal calibration, weight-average molecular weights up to approx. 22000 were found. The chemical structure of the resulting polycarbonates was examined by elemental analyses and ¹H NMR spectroscopy. In contrast to similar polymerizations of ethylene carbonate, either groups were never found. However, all polycarbonates contain a CH₂OH end-group. Furthermore, methylcarbonate, acetate, 2-ethylhexanoate or stearate end-groups were found. Cyclic oligomers were not detectable in the gel-permeation chromatograms of reaction mixtures. IR spectra indicate complexation of the carbonyl group by the initiators. The reaction mechanism is discussed.     

Synthesis and anti-picornaviridae in vitro activity of a new class of helicase inhibitors the N,N'-bis[4-(1H(2H)-benzotriazol-1(2)-yl)phenyl] alkyldicarboxamides

A series N,N'-bis[4-(1H(2H)-benzotriazol-1(2)-yl)phenyl]alkyldicarboxamides (3a-f and 5a-j) were prepared starting from their already known (1a-d) and (4a-c) or new (4d) amine parents. Because of the antiviral activity of several N-[4-(1H(2H)-benzotriazol-1(2)-yl)phenyl]alkylcarboxamides previously reported, title compounds were evaluated in vitro for cytotoxicity and antiviral activity against viruses representative of Picornaviridae, [i.e. Enterovirus Coxsackie B2 (CVB-2) and Polio (Sb-1)] and of two of the three genera of the Flaviviridae [Bovine Viral Diarrhea Virus (BVDV) and Yellow Fever Virus (YFV)]. Furthermore, because of the in silico activity against the RNA-dependent RNA-helicase of Polio 1 previously reported, title compounds were evaluated against the 3D model of the Sb-1 helicase and against the 2D model of the CVB-2 helicase. As a reference we used the antiviral and in silico activities of an imidazo counterpart of the title compounds, N,N'-bis[4-(2-benzimidazolyl)phenyl]alkyldicarboxamides (III) that other authors reported to be able to inhibit the corresponding enzyme of Hepatitis C Virus (HCV). In cell-based antiviral assays, N,N'-bis[4-(1H-benzotriazol-1-yl)phenyl]alkyldicarboxamides (3a-f) resulted completely inactive whereas the bis-5,6-dimethyl-benzotriazol-2-yl derivatives (5d-f) exhibited good activity against the Enteroviruses, (EC(50)s ranged between 7 and 11 microM against CVB-2 and 19-52 against Sb-1). Interestingly, bis-5,6-dichloro-benzotriazol-2-yl derivatives (5h-j) showed very selective activity against CVB-2 (EC(50)s = 4-11 microM) whereas they resulted completely inactive against all the other viruses screened. In general, all title compounds showed a good cytotoxicity profile in MT-4 cells. Molecular modeling investigations showed that active compounds may interact with the binding site of the Sb-1 helicase and that their free binding energy values are in agreement with their EC(50)s values.     

Structural study of compounds modelling elementary polymer units, 1. Molecular and crystal structures of 1,3,5-tris(4-biphenylyl)-benzene and 1,3,5-tris[4-(C-o-carboranylmethyl)phenyl]benzene

For elucidating the structural features of elementary units of polyphenylene type polymers, prepared by polycondensation of di- and mono-acetylaromatic compounds, and X-ray study of two model compounds was performed, viz. 1,3,5-tris(4-biphenylyl)benzene (1) and 1,3,5-tris[4-(C-o- carboranylmethyl)phenyl]benzene (2) (diffractometer: R = 0,063 (1) and 0,061 (2) )· 1 0,5 C₆H₆ crystals were found to be rhombohedral, a = 20,168 (3), c = 13,678 (3) ǎ, space group R3c, Z = 6;2· CHCl₃ crystals are triclinic, a = 15,7528 (2), b = 12,7004 (6), c = 15,460 (2) Å, α = 109,97(1) β = 111,92(1) γ = 100,69 (2) °, space group P1, Z = 2. In the crystal the molecule 1 has a 32 symmetry with a propeller-like orientation of the biphenyl fragments with respect to the central ring (the rotation of the disubstituted benzene rings relative to the central one is 47,2° and relative to the therminal ones, it is 35,9°). In 2 the disubstituted benzene rings are rotated with respect to the central one by different values (dihedral angeles are 31,7 - 30,7, and 52,5°). In all benzene rings of both molecules a decrease of the endocyclic bond angles at the ipso-atoms is observed which in 2 depends on the dihedral angeles between the planes of bonded benzene rings. All there carboranylmethylphenyl fragements in 2 have the same conformation: the CH-group of the carborane skeleton “hangs” over the benzene ring plane. The crystal packing of 1 and 2 is characterized by sufficiently large cavities which are occupied by solvent molecules. In crystals of 2 the carborane parts of the molecules are associated into separate structural motives.     

Comparison of colorimetric,fluorometric, and visual methods for determining anti-influenza (H1N1 and H3N2) virus activities and toxicities of compounds

Methods have been developed previously for rapid evaluation of compounds for antiviral activity in 96-well microplates, which include visual quantitation of antiviral activity based upon inhibition of virus-induced cytopathic effect (CPE) or by less subjective colorimetric or fluorometric means. In the present studies we compared a number of colorimetric (crystal violet, MTT [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide], and neutral red) and fluorometric (Alamar Blue, bisbenzimide [Hoechst 33258], fluorescein diacetate, and rhodamine 6G) methods to visual scoring of antiviral activity in influenza A virus infections in Madin Darby canine kidney (MDCK) cells. Toxicity determinations using these same methods were also made for anti-influenza inhibitors and other compounds known to inhibit cell proliferation. Against influenza A/Texas/36/91 (H1N1) and A/Sydney/05/97 (H3N2) viruses, visual scoring and dye or stain methods produced results that were not significantly different from each other in deriving 50% virus-inhibitory concentrations (EC(50) values) for six anti-influenza compounds (amantadine, rimantadine, ribavirin, RWJ-270201 [BCX-1812], oseltamivir carboxylate, and zanamivir), with the exception of Alamar Blue which quantified lower EC(50) values than expected. In uninfected replicating cells, the visual and Alamar Blue methods underestimated the 50% cytotoxic concentrations (IC(50) values) of ribavirin, 1-beta-D-arabinofuranosylcytosine, and 6-azauridine, but more accurately assessed the toxicities of amantadine, rimantadine, and cycloheximide. Visual scoring, coupled with the use of one of these dyes or stains except Alamar Blue, can be used to accurately and rapidly quantify the anti-influenza virus activities and toxicities of potential new influenza virus inhibitors. These methods should also be applicable to evaluating antiviral effects against other lytic virus infections.     

Polymers containing enzymatically degradable bonds, 1. Chymotrypsin catalyzed hydrolysis of p-nitroanilides of phenylalanine and tyrosine attached to side-chains of copolymers of N-(2-hydroxypropyl)methacrylamide

A series of copolymers of N-(2-hydroxypropyl)methacrylamide were prepared, which contained side chains of the general formula -Gly-X-Y-NAp, where Gly ¨ glycine; X ¨ glycine, alanine, β-alanine, valine, leucine, isoleucine, phenylalanine; Y ¨ phenylalanine or tyrosine; NAp ¨ p-nitroanilide, the latter modelling biologically active compounds. The rates of chymotrypsin-catalyzed hydrolysis of p-nitroanilide groups at pH = 8,0 and 25°C were determined over a range of substrate concentrations to derive values for k_cat and K_M. The results allowed us to determine the influence of the structure of side chains on the rate of cleavage of Y-NAp. The increase in the susceptibility to chymotrypsin attack with an increasing spacing of the Y-NAp residue from the backbone of the polymer chains is demonstrated by comparing the kinetic data of copolymers containing -Gly-Gly-Phe-Phe-NAp, -Gly-Gly-Phe-NAp and -Gly-Phe-NAp side chains. Results obtained with α-chymotrypsin were compared with the cleavage of the above polymer substrates with chymotrypsin covalently bound to a copolymer of N-(2-hydroxypropyl)methacrylamide.     

Polymers containing enzymatically degradable bonds, 2. Poly[N-(2-hydroxypropyl)methacrylamide] chains connected by oligopeptide sequences cleavable by chymotrypsin

Copolymers of N-(2-hydroxypropyl)methacrylamide ( 1 ) with p-nitrophenyl esters of N-methacryloylated oligopeptides ( 2–16 ) were prepared. These copolymers were crosslinked below the gel point by diamines ( 17–27 ). The crosslinks connecting poly[N-(2-hydroxypropyl)methacrylamide] chains contained an oligopeptidic sequence of 2–4 amino acids, cleavable by α-chymotrypsin: -Gly-X-Y- (X… Gly, Ala, β-Ala, Val, Leu, Ile, Phe, D -Phe; Y… Phe, Tyr), -Gly-Gly-Phe-Y-; -U-Gly-Val-Phe- (U… Ala, Gly); -Gly-Phe-W- (W… Ala, Gly, D -Phe); -Gly-Phe-Ala-U-. The changes in the distribution of molecular weights of the studied copolymers, after incubation with α-chymotrypsin, allowed us to estimate the amount of degradable crosslinks and to determine the relationship between the structure and cleavability. The results are interpreted from the viewpoint of the contribution of subsite (S-P) interactions to the degradability of the studied polymers.     

Structural study of compounds modelling elementary polymer units, 3. Molecular and crystal structure of 1,3,5-tris[4′-(C-o-carboranyl)biphenyl-4-yl]benzene

Following the studies on elucidating the main structural features of elementary units of polyphenylene-type polymers prepared by condensation of di- and monoacetylaromatic compounds, an X-ray structural analysis of 1,3,5-tris[4′-(C-o-carboranyl)biphenyl-4-yl]benzene ( 2 ) (systematic name: 1,3,5-tris[)1,2-dicarba-closo-dodecarborane( 12 )-1-yl(biphenyl-4-yl]benzene), which is the low-molecular-weight analog of one of the polymers in the given series, was performed. Compound 2 crystallizes as unstable solvate 1:1:1 with chloroform and benzene. The crystals are monoclinic, a = 17,060 Å, b = 20,220 Å, c = 18,448 Å, β = 102,79°, space group P2₁/n, Z = 4. Molecule 2 exhibits asymmetric conformation with substantial distortions of the linear geometry of polyphenylene units. The crystal is built of layers of molecules 2 , the solvate molecules being situated between these molecules in the channels and cavities with a population of 2/3. The benzene molecules participate in stacking-interaction with one of the carboranyl-substituted (i.e., the strongest electron-acceptor) benzene rings of the host molecule 2 ; chloroform molecules pack the channels without a specific interaction with the host molecules.     

Vaccination of an adolescent group with A (H3N2) + A(HSW1N1) bivaccine before an A (H1N1) influenza epidemic.

A group of 35 adolescents aged 18--22 years were vaccinated intradermally with 0.2 ml of a mixed A (H3N2) + A (Hsw1N1) formolized vaccine and revaccinated with A (Hsw1N1) monovaccine 10 days before an A (H1N1) influenza epidemic. The vaccination had no effect on morbidity or the clinical course of disease. Serology revealed a primary immune response to A (Hsw1N1) and a booster response to A (H3N2). Apparently, prevention of a new influenza subtype by formolized vaccine possessing only the corresponding neuraminidase type is ineffective.     

Identification of polymers by mass spectrometry, 2. Polymers containing 1-trifluoromethylvinylene, 1,4-phenylene,and/or 2,5-thiophenediyl units

Polymers containing 1-trifluoromethylvinylene, 1,4-phenylene, and/or 2,5-thiophenediyl units ( 6a , b and 8a - d ), obtained by base-free Wittig condensation reactions with phase transfer catalysts, were investigated combining direct evaporation in the ion source of a double focusing mass spectrometer with the “Linked-Scan” metastable technique. This method could be shown to allow the unequivocal identification and differentiation of components in polymers up to high mass regions (m/z ≤ 3000). The degree of polycondensation up to 18 aromatic/heteroaromatic units in the chain was determined by mass chromatography, which correlated with the HPLC results. Terminal groups and sequence ions were characterized by comparison of metastable transitions of selected ions with model compounds. Detailed information on the polycondensation reaction was obtained. In addition to linear products, cyclic species with 3 to 18 arylene-1-trifluoromethylvinylene units were formed. The main fragmentations may be explained by sterical effects of both the α-trifluoromethylstilbene moiety and the heteroaromatic analogues in the polymeric chains.     

Polymerization of lactams, 84. Anionic polymerization of lactams accelerated with N-iminolactams, 3. The role of 1-(1-pyrrolin-2-yl)-2-pyrrolidone in the anionic polymerization of 2-pyrrolidone

The anionic polymerization of 2-pyrrolidone was accelerated with 1-(1-pyrrolin-2-yl)-2-pyrrolidone (1) , labelled with nuclide ¹⁵N. The incorporation of 1 into the polymer was followed by the change of isotope concentration, and the apparent rate constants were derived for some elementary reactions in the investigated polyreaction. The interchange reaction between 1 and monomer was also measured. 1 can be considered a slow activator, each molecule incorporating into the polymer being the precursor of a growth centre.     

Polymers of 1-azabicyclo[4.2.0]octane, 2. Polystyrene-block-poly(1-azabicyclo[4.2.0]octane) by mixed-mechanism technique

By endcapping of living polystyrene with ethylene oxide and subsequent reaction with bromoacetyl bromide a polymer with a bromoacetoxy endgroup was obtained. This was used as a macroinitiator for the living cationic polymerization of 1-azabicyclo[4.2.0]octane. The structure and composition of the so formed two-block copolymer was elucidated by different methods. The ratio of the two block and their lengths were varied within broad limits.     

4,4′-二(1″,1″,1″–三氟-2″,4″-丁二酮-6″-酰基)-氯磺化邻-三联苯的合成与表征

以邻-三联苯为原料,经傅氏酰基化反应、酯酮缩合反应和氯磺化反应,合成了双功能螯合剂4,4′-二(1″,1″,1″–三氟-2″,4″-丁二酮-6″-酰基)-氯磺化邻-三联苯(BTBCT),经红外光谱、熔点、元素成份等表征分析,证实其结构;通过标记技术和荧光光谱分析确认BTBCT具有双向功能.BTBCT与BSA偶联,经UA仪监定后,再与Eu3+制成BSA-BTBCT-Eu3+.BTBCT可以作为新型固相TRFIA体系的双功能螯合剂.     

Polymers containing enzymatically degradable bonds, 8. Degradation of oligopeptide sequences in N-(2-hydroxypropyl)methacrylamide copolymers by bovine spleen cathepsin B

Three types of copolymers of N-(2-hydroxypropyl)methacrylamide(HPMA) were prepared which contain oligopeptide sequences: (a) HPMA copolymers containing oligopeptide side-chains terminated with p-nitroaniline; (b) soluble HPMA copolymers containing oligopeptide sequences in crosslinks connecting two poly(HPMA) chains; (c) a hydrophilic gel, i.e. a three dimensional copolymer of HPMA containing an oligopeptide sequence in the crosslinks. These polymeric substrates (suitable as drug carriers) containing potentially degradable oligopeptide sequences were incubated with an intracellular proteolytic enzyme, bovine spleen cathespin B. The degradation process of the substrates made it possible to reveal the relationship between the structure of oligopeptide sequences and their degradability. The results suggest an important role played by cathepsin B in the degradation of polymeric substrates investigated in this study under physiological conditions.