Synthesis and evaluation of substituted 4-aryloxy- and 4-arylsulfanyl-phenyl-2-aminothiazoles as inhibitors of human breast cancer cell proliferation

Several substituted 4-aryloxy- and 4-arylsulfanyl-phenyl-2-aminothiazoles were synthesized and evaluated for cytotoxic activity against estrogen-positive, estrogen-negative, and adriamycin-resistant human breast cancer cell lines. 4-[4'-(3,4-Dichlorophenoxy)-phenyl]-thiazol-2-yl ammonium iodide demonstrated potent activity against both estrogen-positive and negative breast cancer cell lines with low micromolar (microM) GI(50) values. In addition, we have identified several 2-aminothiazoles that demonstrated selective potency for the adriamycin-resistant and estrogen-negative breast cancer cell lines. The results suggest that these 2-aminothiazoles represent lead compounds for evaluation in animal models of breast cancer.     

Synthesis and biological evaluation of 2-aminothiazoles and their amide derivatives on human adenosine receptors. Lack of effect of 2-aminothiazoles as allosteric enhancers

A number of 2-aminothiazoles (2a-e) and their amide derivatives (4-10) were prepared. The 2-aminothiazoles themselves were tested as allosteric enhancers of agonist binding to human adenosine A(1) receptors. In a variety of experimental set-ups the compounds did not show any such effect, in contrast to earlier findings by another research group. Subsequently the 2-aminothiazoles were used as intermediates in the synthesis of a number of amide derivatives of either aromatic (4-6) or aliphatic nature (7-10). Some of the compounds emerged as moderately active antagonists on human adenosine A(1) and/or A(2A) receptors with lower or negligible potency at adenosine A(3) receptors.     

Dibenzofuran,dibenzothiophene and N-methyl carbazole tethered 2-aminothiazoles and their cinnamamides as potent inhibitors of Mycobacterium tuberculosis

Herein described the design, synthesis and antitubercular evaluation of novel series of dibenzofuran, dibenzothiophene and N-methyl carbazole tethered 2-aminothiazoles and their cinnamamide analogs. One pot condensation of N-methyl carbazole, dibenzofuran and dibenzothiophene methyl ketones with thiourea in the presence of Iodine and CuO gave respective 2-aminothiazoles 4–6 in very good yields. Aminothiazoles were further coupled with substituted cinnamic acids using acid-amine coupling conditions to give desired cinnamamide analogs 8a–e, 9a–e and 10a–e. All the newly synthesized compounds were fully characterized by their NMR and mass spectral analysis. In vitro screening of new derivatives against Mycobacterium tuberculosis H37Rv (Mtb) resulted 8c, 10d and 10e (MIC: 0.78?µg/mL) and 2-aminothiazoles 5 and 6 (MIC: 1.56?µg/mL) as potent compounds with lower cytotoxicity profile.     

Synthesis of stable and selective inhibitors of human galectins-1 and -3

The syntheses of glycolytically stable galactosides and lactosides have been made toward the selective inhibition of human galectins-1 and -3. Transition metal-catalyzed cross-coupling reactions were used to create carbon-carbon bond formation (Sonogashira, Suzuki, Heck, Glaser). Additionally, Hantzsch condensation was used to create novel 2-aminothiazoles which reacted with a panel of acylating and sulfonylating reagents. Moreover, dimeric galactosides and lactosides bearing triazoles, regiospecifically prepared using copper-catalyzed Huisgen azide-alkyne [1,3]-dipolar cycloaddition, provided efficient galectins-1 and -3 inhibitors. Best monovalent inhibitor among the tested series was (E)-methyl 2-phenyl-4-(beta-D-galactopyranosyl)-but-2-enoate 15 with inhibitory potency of 313 microM against galectin-1 and best dimers were bis-lactoside 68 and 75 having both inhibitory properties of 160 microM against Galectin-3.     

Monkey rotavirus binding to alpha2beta1 integrin requires the alpha2 I domain and is facilitated by the homologous beta1 subunit

Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infection by simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the alpha2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-alpha2beta1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from alpha2beta1 integrin, and SA11 precipitated beta1 from alpha2beta1-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160- to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the alpha2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human alpha2 or alpha2beta1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human alpha2beta1 and, to a lesser extent, human alpha2 combined with hamster beta1. Binding was inhibited by anti-alpha2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to alpha2, and required the presence of the alpha2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the alpha2 I domain that are necessary for type I collagen binding to alpha2beta1 were not essential for rotavirus binding. Rotavirus-alpha2beta1 binding led to increased virus infection and RRV growth. SA11 and RRV require the alpha2 I domain for binding to alpha2beta1, and their binding to this integrin is distinguishable from that of collagen.     

Synthesis and biological activity of 2-aminothiazoles as novel inhibitors of PGE2 production in cells

This Letter presents the synthesis and biological evaluation of a collection of 2-aminothiazoles as a novel class of compounds with the capability to reduce the production of PGE(2) in HCA-7 human adenocarcinoma cells. A total of 36 analogs were synthesized and assayed for PGE(2) reduction, and those with potent cellular activity were counter screened for inhibitory activity against COX-2 in a cell free assay. In general, analogs bearing a 4-phenoxyphenyl substituent in the R(2) position were highly active in cells while maintaining negligible COX-2 inhibition. Specifically, compound 5l (R(1)=Me, R(2)=4-OPh-Ph, R(3)=CH(OH)Me) exhibited the most potent cellular PGE(2) reducing activity of the entire series (EC(50)=90 nM) with an IC(50) value for COX-2 inhibition of >5 μM in vitro. Furthermore, the anti-tumor activity of analog 1a was analyzed in xenograft mouse models exhibiting promising anti-cancer activity.     

Biochemical evidence that equine leucocyte antigens W13, W22 and W23 are present on horse major histocompatibility complex class II molecules

A number of horse alloantisera were characterized biochemically as being directed against MHC class I or class II antigens by immunoprecipitation of the corresponding antigens from lysates of biosynthetically radioactively labelled lymphocytes and determination of their molecular weights by SDS-PAGE and fluorography. Sera recognizing A2 and A3 specificities precipitated antigens of 44,000 Daltons molecular weight (class I heavy chain), whereas sera with specificities W13, W22 and W23 precipitated antigens corresponding to class II dimers (30,000 and 32,000 Daltons). Comparison with antigens precipitated from horse lymphocyte lysates using (cross-reacting) antibodies to human class I and class II MHC molecules confirmed the results obtained.     

Characterization of class I bovine lymphocyte antigens (BoLA) by one-dimensional isoelectricfocusing

BoLA class I antigens were characterized in a group of British and Dutch Friesian cattle by one-dimensional isoelectric focusing (1D-IEF) and the results compared with serology using alloantisera and microcytotoxicity. For IEF analysis, non-stimulated peripheral blood mononuclear cells (PBM) were metabolically labelled with 35S methionine, detergent lysates were prepared and MHC molecules precipitated with the monoclonal antibodies (mAbs) W6/32 or B1.1G6. Staphylococcus protein A precipitated antigens were separated on a vertical slab gel under denaturing conditions. The banding patterns seen for the W6/32 precipitated molecules obtained by 1D-IEF were compared with the serological specificities. Characteristic banding patterns were observed for most serological specificities as well as workshop undefined haplotypes. These patterns were seen both in families and the outbred population. In families IEF haplotypes segregated with serotypes. Additional MHC class I products were suggested by variable banding patterns for different w10 haplotypes and when using the different mAbs. A pulse chase experiment with a w12 animal also suggested more than one expressed product. The w2 and w5 specificities were not precipitated by either W6/32 or B1.1G6 and w6.2 and w6.4 were precipitated by W6/32 but not by B1.1G6. These results show that 1D-IEF is useful for BoLA typing. For the characterization of class I antigens, however, much depends on the mAbs used.     

The Design of a Pharmaceuttcally Acceptable Liposomal Formulation of Recombinant Interleukin-2 (Ril-2) for Locoregional Anticancer Immunotherapy

Abstract

Liposomes composed of egg-phosphatidylcholine and egg-phosphati-dylglycerol containing recombinant interleukin-2 (rIL-2) were prepared and characterised for use in locoregional anticancer immunotherapy. During the encapsulation studies it was observed that the protein precipitated. Therefore, the impact of the precipitation phenomenon on the characteristics and in vivo performance of rIL-2 liposomes was studied. Recombinant IL-2 was diluted in various aqueous media and the amount of precipitated protein determined. Also, the in vitro bioactivity, chemical stability, and in vivo antitumor efficacy of liposomes prepared with precipitated rIL-2 or non-precipitated rIL-2 were assessed.

Massive protein precipitation (60 up ± 95% of total protein) was observed upon dilution of rIL-2 in salt-containing media, but not upon dilution in 5% glucose or water. Liposomes prepared with precipitated rIL-2 were shown to release 50% of the entrapped rIL-2 over a three-day period at 37° in protein-containing media. Loss of rIL-2 bioactivity and chemical integrity was observed during storage at 4° over a 4-week period. Locoregional administration of precipitated rIL-2 in the guinea pig Line 10 tumor model resulted in significantly more tumor growth inhibition than administration of non-precipitated rIL-2. Liposomes containing non-precipitated rIL-2 were found to elicit similar antitumor effects as precipitated rIL-2. The results point to the importance of proper characterisation of new rIL-2 formulations as the physical properties of formulated rIL-2 may strongly influence its bioactivity.     

2-Aminothiazole based P2Y1 antagonists as novel antiplatelet agents

ADP receptors, P2Y₁ and P2Y₁₂ have been recognized as potential targets for antithrombotic drugs. A series of P2Y₁ antagonists that contain 2-aminothiazoles as urea surrogates were discovered. Extensive SAR of the thiazole ring is described. The most potent compound 7j showed good P2Y₁ binding (K_i = 12 nM), moderate antagonism of platelet aggregation (PA IC₅₀ = 5.2 μM) and acceptable PK in rats.     

Molecular basis of a unique tumor antigen of radiation leukemia virus-induced leukemia B6RV2: its relation to MuLV gp70 of xenotropic class

Hybridomas secreting monoclonal antibodies that reacted with the B6 radiation leukemia virus (RadLV)-induced leukemia B6RV2 were produced by fusion of BALB/c NS-1 myeloma cells with spleen cells from (BALB/c X B6)F1 mice immunized with B6RV2. By direct and absorption analyses with 28 B6 and BALB/c leukemias, the monoclonal antibodies NU7-4 and NU7-99 were shown to react only with B6RV2, indicating that they recognized an individually distinct antigen on B6RV2 that was identified previously with conventional (BALB/c X B6)F1 anti-B6RV2 serum. Another monoclonal antibody, NU1-132, showed relatively restricted reactivity with B6 RadLV leukemias. These three monoclonal antibodies all precipitated material of approximately 80,000 daltons, which is the same size as that precipitated by anti-xenotropic MuLV gp70 serum. Sequential immunoprecipitation analysis revealed that the molecules precipitated by NU7-4 were not removed by pretreatment of NU7-99 or NU1-132 and that the molecules precipitated by NU7-99 were not removed by NU7-4 or NU1-132. The molecules precipitated by NU1-132 were partially removed by pretreatment with NU7-4, but not with NU7-99. The molecules precipitated by these three monoclonal antibodies were removed by pretreatment with anti-xenotropic gp70. These results suggested heterogeneity of the xenotropic MuLV gp70-related molecules expressed on B6RV2 and a possible relation between serologically defined unique tumor antigens and gp70-related molecules.     

The determination of the activity of immobilised amidases using an ammonia-sensitive glass electrode.

Ribosomal proteins, separated from rRNA by the LiCl-urea method, are then precipitated by adding one volume of 20% trichloroacetic acid. We show here that this precipitate can be dissolved directly into unbuffered Tris-OH for electrophoresis on acrylamide gels without further washing of the pellet or dialyzing of the solution.Ribosomal proteins can also be separated from rRNA by using 66% acetic acid in presence of 0.1 m Mg²⁺. We show here that the ribosomal proteins can be precipitated directly from the acetic acid by adding ten volumes of 10% trichloroacetic acid. This precipitate is then easily dissolved in unbuffered Tris-OH to prepare the proteins for acrylamide-gel electrophoresis without further treatment.     

Morphoanalysis of Bacterially Precipitated Subaqueous Calcium Carbonate from Weebubbie Cave,Australia

In this article, we present a previously unreported morphology of bacterially precipitated calcite (determined using XRD, FTIR, and SAED) occurring subaqueously in Weebubbie Cave. Observations using FESEM and TEM revealed spindle-shaped crystals with curved [hk.0] faces lying parallel to the c-axis. Calcite precipitated under conditions designed to mimic the inorganic solution chemistry of the cave revealed a different morphology. These differences between the crystals suggest that the formation of the cave crystals is a consequence of biologically activity.     

Production and preliminary characterization of monoclonal antibodies to rat plasma fibronectin

We have produced several monoclonal antibodies which appear to be directed against different antigenic determinants of rat plasma fibronectin. Fibronectin was purified from rat plasma by affinity chromatography on gelatin-Sepharose and arginine-Sepharose columns. Mice were immunized and hybridomas were prepared by fusing spleen cells with Sp2/0-Ag14 myeloma cells using poly(ethylene glycol). Three hybridomas (RFN1, RFN2 and RFN3) were selected for characterization. All are IgG molecules, one is IgG2a, one IgG2b and one IgG1. Titers of ascites fluids produced using these hybridomas range from 102 400 to greater than 409 600. The antibodies cross-reacted to different degrees with human fibronectin. Rat fibronectin was radioactively labeled and cleaved using human polymorphonuclear leukocyte elastase. Four major peptides, Mr approx. 160 000, 140 000, 60 000 and 30 000 were produced. Each of the hybridoma antibodies immunoprecipitated different elastase peptides. RFN1 precipitated the Mr 160 000 peptide, RFN2 precipitated the Mr 160 000 and the Mr 140 000 peptide and RFN3 precipitated the Mr 60 000 peptide as well as low molecular weight material migrating at the buffer front. These antibodies will be useful in studies of structure/function relationships of rat fibronectin.     

Partial purification and cytotoxic effects of Salmonella proteinous moieties on chick embryo fibroblasts

Salmonella enterica serovars, viz., S. Weltevreden, S. Typhimurium, S. Gallinarum and S. Bareilly were treated with cephotaxime to release of intracellular proteins. The cephotaxime extract (CE) was salt precipitated with ammonium sulphate (45-70%) and dialyzed, and denoted as precipitated dialyzed proteins (PDP). Further, both CE and PDP of Salmonella Weltevreden and PDP of rest of the serovars were subjected to gel filtration using Sephacryl S-200HR. Different fractions along with CE and PDP were studied for their cytotoxicity using chicken embryo fibroblast (CEF). All the CE and PDP exerted cytotoxic effects, characterized by rounding, detachment, shrinkage and clumping of cells with syncytia formation. Also, the fractions eluted in the 2nd and 3rd peaks through Sephacryl S-200HR column invariably had cytotoxic activity. It was concluded that in place of Vero cell line, CEF cells could also be used to test cytotoxicity.     

Monoclonal antibodies to two glycoproteins of herpes simplex virus type 2.

Monoclonal antibodies to herpes simplex virus type 2 were found to precipitate different numbers of radiolabeled polypeptides from lysates of virus-infected cells. Antibodies directed against two viral glycoproteins were characterized. Antibodies from hybridoma 17 alpha A2 precipitated a 60,000-molecular-weight polypeptide which chased into a 66,000- and 79,000-molecular-weight polypeptide. All three polypeptides labeled in the presence of [3H]glucosamine and had similar tryptic digest maps. The 60,000-molecular-weight polypeptide also chased into a 31,000-molecular-weight species which did not label with [3H]glucosamine. Antibodies from hybridoma 17 beta C2 precipitated a 50,000-molecular-weight polypeptide which chased into a 56,000- and 80,000-molecular weight polypeptide. These polypeptides also shared a similar tryptic digest map and labeled with [3H]glucosamine. Both monoclonal antibodies were herpes simplex virus type 2 specific. The viral proteins precipitated by 17 alpha A2 antibodies had characteristics similar to those reported for glycoprotein E, whereas the proteins precipitated by 17 beta C2 antibodies appeared to represent a glycoprotein not previously described. This glycoprotein should be tentatively designated glycoprotein F.     

Monoclonal antibodies against a specific surface determinant on malarial (Plasmodium knowlesi) merozoites block erythrocyte invasion

Twelve hybridoma cell lines secreting monoclonal antibodies against Plasmodium knowlesi merozoites have been produced. Antibodies from 3 of the 12 lines agglutinated merozoites. The 2 monoclonal antibodies (13C11 and 16F8) that markedly agglutinated merozoites blocked merozoite invasion of erythrocytes. Of these 2 lines, the one that induced the most agglutination also blocked invasion most effectively. The third monoclonal antibody (53B3) caused minimal agglutination of merozoites and did not block invasion, nor did the other 9 nonagglutinating antibodies. The 2 blocking monoclonal antibodies bound to antigens around the entire surface of merozoites, as demonstrated by immunoelectron microscopy, and precipitated a single biosynthetically labeled protein of apparent m.w. of 250,000. None of the nonagglutinating lines precipitated this protein. Monoclonal antibodies 13C11 and 16F8 reacted with a common antigenic determinant on a Malaysian and a Philippine strain of P. knowlesi in that they blocked invasion and precipitated a 250,000 m.w. protein from both. Sera from immune monkeys also precipitated this 250,000 m.w. protein.     

Optimum conditions for the precipitation of chitosan oligomers with DP 5–7 in concentrated hydrochloric acid at low temperature

Chitosan was partially hydrolysed with 35% hydrochloric acid for 2 h at 80°C and the hydrolysate stored at −20°C after dilution with water to precipitate higher molecular weight (MW) chitosan oligomers. When the hydrolysate was not diluted with water, no precipitate was formed but 7.3% chitosan oligomers were precipitated at a dilution ratio of 1.0 (ml water/ml hydrolysate). The time for precipitation was not significantly changed after storing the hydrolysate at −20°C for 1 day. In addition, the precipitation yield was not significantly influenced by the concentration of HCl used for the hydrolysis except at less than 5.0 (ml HCl/g chitosan). However, the yield of precipitated oligomers changed with partial hydrolysis time. For 0.5 and 2 h hydrolysis, 10.1 and 7.3% of the oligomers were precipitated, respectively, but only 3.1% of the oligomers were obtained after a 4 h reaction. When methanol was added to the hydrolysate, the precipitation yield increased up to 70% but the amounts of lower MW chitosan oligomers in the precipitated oligomers also increased with the increase of higher MW. The precipitated oligomers were mainly composed of pentamers and hexamers.     

Identification, isolation and purification of neurotoxic phospholipases A2 from Vipera russelli venom using polyclonal antibodies.

All the neurotoxic phospholipases A2 present in whole Vipera russelli venom were precipitated selectively from other non-neurotoxic phospholipases A2 and non-phospholipases A2 fractions using antibodies (anti PL-V Ig) raised against one of the purified neurotoxic phospholipases A2 (VRV PL-V). These neurotoxins were identified and isolated in their homogeneous form by chromatographic and electrophoretic methods. The present report of selective isolation and purification of all the neurotoxic phospholipases A2 of V. russelli venom is first of its kind.