Synthesis and biological activity of anthelmintic thiadiazoles using an AF-2 receptor binding assay.

Following our discovery of the strong binding of thiadiazole 1 to the AF-2 neuropeptide receptor of gastrointestinal nematodes (e.g., Ascaris suum), we prepared two series of analogs. Only the series containing the thiadiazole ring had potencies comparable to that of compound 1. Analog 50 exhibited an apparent potency in the AF-2 binding assay 300 times that of compound 1.

5-HT1D receptor agonist properties of novel 2-[5-[[(trifluoromethyl)sulfonyl]oxy]indolyl]ethylamines and their use as synthetic intermediates.

2-[5-[[(trifluoromethyl)sulfonyl]oxy]-1H-indol-3-yl]ethylamine (18), its N,N-di-n-propyl (12), N,N-diethyl (13), and N,N-dimethyl (14) derivatives, and 4-[3-[2-(N,N-dimethylamino)ethyl]-1H-indol-3-yl]-N-(p-methoxybenzyl) acrylamide (GR46611, 19) were synthesized and tested for binding affinities to cloned 5-HT1A, 5-HT1D alpha, 5-HT1D beta, and D2 receptors. In addition, the intrinsic efficacy was measured as the reduction of forskolin-stimulated cAMP in cells transfected with 5-HT1D alpha and 5-HT1D beta receptors in vitro. The 5-substituted indolyethylamines investigated displayed agonist activity at the 5-HT1D receptors with varying degrees of preference for the 5-HT1D alpha vs the 5-HT1D beta receptors. The primary amine and N,N-dimethyl substitution seemed to be optimal for 5-HT1D alpha affinity. Furthermore, the N,N-diethyl (13) and N,N-dimethyl (14) derivatives showed a 10-25 times preference for the 5-HT1D alpha vs the 5-HT1D beta receptor. In addition, all of the novel compounds showed affinity for the 5-HT1A receptor in vitro (Ki values ranging from 18 to 40 nM). The most promising derivative 14 was virtually devoid of central 5-HT1A agonist activity in rats, as determined by in vivo biochemical assays. Paradoxically, 14, like 19, induced a hypothermic response and a decrease in 5-HIAA levels in the prefrontal cortex and hypothalamus in guinea pigs after systemic administration. Sumatriptan failed to produce either of these effects due to a poor brain penetration.

Characterization of U-92016A as a selective, orally active, high intrinsic activity 5-hydroxytryptamine1A agonist.

The purpose of the present study was to characterize U-92016A [(+)-R)-2-cyano-N,N-dipropyl-8-amino-6,7,8,9-tetrahydro-3H-benz[e] indole] as a 5-hydroxytryptamine (5-HT)1A receptor agonist and to compare its activity with that of standard 5-HT1A receptor agonists. U-92016A binds with high affinity to human 5-HT1A receptors expressed in Chinese hamster ovary cells (Ki = 0.2 nM). Radioligand binding studies also indicate that U-92016A is selective for the 5-HT1A receptor over other biogenic amine receptors. In Chinese hamster ovary cells expressing the human 5HT1A receptor, U-92016A decreased the forskolin-induced increase in cyclic AMP synthesis and had an intrinsic activity of 0.82 relative to 5-HT. U-92016A potently decreased rectal temperature in mice. The maximum temperature decrease was significantly greater than that observed for 8-hydroxy-di-n-propyl aminotetralin, buspirone, gepirone, ipsapirone or flesinoxan. U-92016A also elicited the 5-HT-mediated syndrome in rats and resulted in a dose-related decrease in 5-hydroxytryptophan accumulation. The compound also decreased arterial blood pressure in spontaneously hypertensive rats and inhibited sympathetic nerve activity in cats. In these assays U-92016A displayed excellent potency and a long duration of action. U-92016A also inhibited the firing of dorsal raphe 5-HT neurons and was active in two social interaction assays. The p.o. bioavailability of U-92016A was calculated to be 45%. Taken together, these data indicate that U-92016A is a metabolically stable, p.o. active 5-HT1A receptor agonist with an exceptionally high degree of intrinsic activity.

Regulation of expression of IL-1 receptor antagonist protein in human synovial and dermal fibroblasts.

The IL-1R antagonist protein (IRAP) is a competitive inhibitor of IL-1, which is predominantly synthesized by monocytes. We show that this molecule is also expressed in human synovial fibroblasts and dermal fibroblasts (CRL 1445). IRAP mRNA was regulated in a time- and dose-dependent manner by IL-1 alpha, TNF-alpha, LPS, and PMA. Maximal induction of IRAP mRNA was observed between 8 and 16 h after stimulation with IL-1 alpha (1 U/ml), TNF-alpha (10 U/ml), LPS (50 ng/ml), and PMA (10 ng/ml). Their relative efficacy was as follows: PMA > LPS > IL-1 alpha > TNF-alpha. Potentiation was observed when fibroblasts were treated with IL-1 alpha plus basic fibroblast growth factor and IL-1 alpha plus platelet-derived growth factor-BB homodimer. Although LPS and PMA were the best inducers of IRAP mRNA, quantitation of the IRAP protein revealed that its synthesis and release were differentially regulated. Immunoprecipitation and SDS-PAGE of culture supernatant from LPS-treated cells and cell lysates of fibroblasts treated with LPS or PMA showed a single IRAP band with a molecular mass of approximately 22 kDa. Very little IRAP was detected in culture supernatants of cells treated with PMA. Quantitation of IRAP revealed that LPS induced the synthesis of secreted IRAP that was released, whereas the majority of the protein induced by PMA remained cell-associated. Reverse transcriptase-polymerase chain reaction amplification demonstrated that although LPS and PMA induced both transcripts, LPS preferentially induced secreted IRAP, whereas PMA differentially induced intracellular IRAP mRNA. Fibroblasts synthesize at least two different forms of IRAP depending on the inducing signal, and may regulate the inflammatory response by dampening the proinflammatory effects of IL-1 via a negative feedback mechanism with IRAP. The relative importance of fibroblast sIRAP vs intracellular IRAP in regulating the inflammatory response by the connective tissue remains to be determined.

Maggiemycin and anhydromaggiemycin: two novel anthracyclinone antitumor antibiotics. Isolation, structures, partial synthesis and biological properties.

Two new anthracyclinone antitumor antibiotics, maggiemycin (6, NSC-D344012) and anhydromaggiemycin (8) have been isolated from a culture of an unspeciated Streptomyces (ATCC No. 39235). Bioautography against Bacillus subtilis was used for the preliminary detection of these anthracyclinones. Structures were proposed based on their UV-visible, IR, 1H NMR, 13C NMR spectra, electron impact (EI) and high-resolution EI-MS and confirmed by partial synthesis and a direct correlation with epsilon-rhodomycinone. Both the anthracyclinones are active against KB, P388 and L1210 murine tumor cell lines; however, anhydromaggiemycin was more active than maggiemycin. A number of related anthracyclinones have also been prepared and their biological activity has been determined. The structure-activity relationship of these new anthracyclinones is also discussed.

Monoclonal antibodies specific for type 3 protein kinase C recognize distinct domains of protein kinase C and inhibit in vitro functional activity.

Monoclonal antibodies (mAbs) which distinguish Type 3 protein kinase C (PKC) from Types 1 and 2 have been obtained from mice immunized with purified Type 3 PKC from rabbit brain cytosol. Most of these mAbs (seven out of eight) selectively recognize Type 3 versus Types 1 and 2 PKC in both enzyme-linked immunosorbent and immunoblot assays. Trypsin treatment of Type 3 PKC reduced the immunoreactivity with 82-kDa PKC and generated immunoreactive fragments of 45 and 35 kDa. The mAbs can be divided into two classes based on their ability to recognize the 45-kDa catalytic fragment (5/8) or the 35 kDa regulatory domain fragment (3/8). Each of the mAbs inhibits phosphorylation of histone or lipocortin by PKC, although the extent of the inhibition varied. Only those mAbs that recognize the 35-kDa regulatory domain inhibited phorbol ester binding. The inhibition of both kinase and binding activities by this group of mAbs was sensitive to the concentration of phospholipid used in the assay. This functional inhibition suggests that these mAbs may be useful for defining the phospholipid binding domain(s) of Type 3 PKC. The mAbs recognized 82-kDa PKC in a variety of cell types; the presence of smaller molecular weight fragments was not consistently found. Distinct immunofluorescence staining patterns were observed with mAbs directed toward different epitopes, suggesting that there may be heterogeneity in the subcellular localization of PKC. The type specificity of these mAbs will make them valuable tools for studying activation and regulation of Type 3 PKC in cell culture model systems.

Ia binding ligands and cAMP stimulate nuclear translocation of PKC in B lymphocytes.

Altered subcellular distribution and activity of protein kinase C (PKC) is associated with transmembrane signalling in a variety of systems in which receptor occupancy leads to increased hydrolysis of polyphosphoinositides. Here we report evidence that in B lymphocytes, cyclic-cAMP-generating signal transduction pathways can activate translocation of PKC from the cytosol to the nucleus. Elevated cAMP levels and translocation of PKC to the nucleus are induced by antibodies against Ia antigens in normal B lymphocytes. Further, cAMP analogues mediate the translocation of PKC to the nucleus of these cells. These findings suggest that in physiological situations, ligation of B-lymphocyte Ia molecules by helper T cells leads to increased cAMP production which in turn causes PKC translocation to the nucleus. In view of recent observations that antibodies against Ia antigens induce differentiation of B cells, we conclude that nuclear PKC may function in the regulation of gene expression.

Transmembrane signaling through B cell MHC class II molecules: anti-Ia antibodies induce protein kinase C translocation to the nuclear fraction.

Among receptors of immunologic importance, antigen receptors on B cells (mIg) and T cells (Ti/T3), as well as receptors for IL 2 and IL 3, appear to utilize a transmembrane signal transduction mechanism that involves protein kinase C (PKC) translocation from the cytosol to the plasma membrane. Here we report evidence that B cell surface type II major histocompatibility molecules (I-A and I-E) can act as signal transducing molecules, as evidenced by the ability of soluble ligands that bind these molecules to stimulate rapid translocation (less than 2 min) of cytosolic PKC. This phenomenon differs qualitatively from translocation induced by anti-Ig antibodies and all other ligands thus far studied, in involving an apparent translocation of protein kinase C to the nucleus. Data suggest that I-A/E molecules may play an active role in T cell-dependent B cell activation. They also provide an explanation for the previously observed suppressive effects of soluble I-A and I-E binding ligands on B cell activation in demonstrating that these ligands (anti-IA/E) deplete the cytosolic reservoir of PKC normally translocated to the plasma membrane after anti-Ig or LPS stimulation.

The role of cyclooxygenase and lipoxygenase pathways in the adhesive interaction between bovine polymorphonuclear leukocytes and bovine endothelial cells.

The effects of arachidonic acid metabolites, analogues and cyclooxygenase/lipoxygenase inhibitors were tested on an "in vitro" bovine model of endothelial cell (EC)-polymorphonuclear leukocyte (PMN) adhesion. Arachidonic acid blocked adhesion at 10(-5)M, a dose which also induced aggregation of PMN. Lower doses did not affect either EC-PMN adhesion or PMN aggregation. Various cyclooxygenase pathway metabolites were inactive in the EC-PMN adhesion assay, with the exception of prostaglandin A2 and prostaglandin B2 which significantly suppressed adhesion at 10(-5)M. Of the synthetic analogs tested, 6 alpha-carbaprostacyclin I2, (5Z)-9 beta-ethynyl-calcium salt (U-64,567E) was inhibitory at 10(-5)M. The cyclooxygenase inhibitors acetylsalicylic acid, indomethacin and ibuprofen were inactive. Products of the lipoxygenase pathways, leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (5-HETE) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) exhibited variable inhibitory activity at 10(-5)M only. Paradoxical effects were observed with the putative lipoxygenase inhibitors 4,7,10,13-eicosatetraynoic acid (4,7,10,13 ETYA), 5,8,11,14-eicosatetraynoic acid (5,8,11,14 ETYA) and nordihydroguairetic acid (NDGA), which also suppressed EC-PMN adhesion at 10(-5)M. The dual cyclooxygenase-lipoxygenase inhibitor, BW755C was inactive. Bovine PMNs did not respond chemotactically to LTB4 although they were able to synthesize the 5-lipoxygenase products LTB4 and 5-HETE.

Production of prostaglandin D2 by murine macrophage cell lines.

Several tumor-derived murine macrophage cell lines were evaluated in vitro as cloned prototypes of tissue macrophages for their ability to metabolize arachidonic acid. Unexpectedly, two cell lines, J774A.1 and WR19M.1, rapidly converted exogenous 14C-arachidonic acid (AA) to a single major prostaglandin metabolite. The compound, PGD2, was positively identified by TLC, HPLC, and GC-MS. The enzymatic formation of the PGD2 was shown by inhibition of its formation by indomethacin and reduced formation of 14C-PGD2 from 14C-PGH2 in boiled cells. When J774A.1 cells were prelabeled with 3H-AA, cultured for 24 hours, and stimulated with lipopolysaccharide (LPS), PGD2 was again the predominant product. No other tumor derived cell lines, including several other murine macrophage lines, produced significant amounts of PGD2. Elicited and activated murine peritoneal macrophages produced only small amounts of PGD2, but resident peritoneal macrophages produced modest amounts of PGD2. Exaggerated formation of PGD2 by J774A.1 and WR19M.1 cells may be a consequence of neoplastic transformation or the clonal expansion of a minor subpopulation of normal tissue macrophages.

Metabolism of arachidonic acid by human neutrophils. Characterization of the enzymatic reactions that lead to the synthesis of leukotriene B4.

Human neutrophils stimulated with calcium ionophore A23187 synthesized 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4. Time-course studies showed that the concentrations of both products reached a maximum after 2 min after which the products were rapidly removed. In longer incubations, 5-HETE was esterified into membrane lipids, and leukotriene B4 was converted to 20- hydroxyleukotriene B4 and/or 20- carboxyleukotriene B4. The reaction is apparently self-limiting. After the maximum was reached, addition of fresh ionophore, Ca2+ or oxygen had little effect. Fresh arachidonic acid increases the yields of 5-HETE and delta 6-trans-leukotriene B4 but not additional leukotriene B4. Only the addition of fresh neutrophils gave additional leukotriene B4. This finding suggests that leukotriene B4 synthesis is limited by both substrate availability and enzyme inhibition by hydroperoxide intermediate. Exogenous arachidonic acid added with ionophore had different effects on the syntheses of leukotriene B4, delta 6-trans-leukotriene B4, and 5-HETE. As the arachidonic acid concentration increases, product formation increases in the following order: 5-HETE greater than delta 6-trans-leukotriene B4 greater than leukotriene B4. At a high concentration (more than 10 microM) of arachidonic acid, the synthesis of delta 6-trans-leukotriene B4 was greater than leukotriene B4 itself. Since delta 6-trans-leukotriene B4 represents the nonenzymatic decomposition of leukotriene A4, we suggest that one of the rate-limiting steps in the synthesis of leukotriene B4 is the leukotriene A4 hydrolase. Our data suggest the synthesis of leukotriene B4 is under the control of three factors: (1) substrate availability; (2) limited capacity of the leukotriene A4 hydrolase, and (3) enzyme inactivators generated during the reaction, such as hydroperoxide intermediate. The tightly controlled system assures only a finite amount of this powerful bioactive substance will be produced under most conditions.

Inhibition of human neutrophil arachidonate 5-lipoxygenase by 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1 (U-60257).

6,9-Deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1 (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both in vitro and in vivo. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB4 synthesis in human peripheral neutrophils with an ID50 of 1.8 and 0.42 microM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 microM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester are potent and selective inhibitors of arachidonate 5-lipoxygenase.

6,9-deepoxy-6,9,-(phenylimino)-delta 6,8-prostaglandin I1, (U-60,257), a new inhibitor of leukotriene C and D synthesis: in vitro studies.

Addition of the calcium inophore, A 23187, and cysteine to isolated mononuclear cells from rat peritoneal washings causes a marked increase in the formation of thromboxane B2 (TxB2) along with the formation of leukotrienes C and D (LT's). The formation of LT's in this system was inhibited by 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1, U-60,257, or its methyl ester, U-56,467, (ID50 4.6 and 0.31 microM, respectively). There was no inhibition of TxB2 formation. By contrast, two structurally-related compounds, PGI2 and its stable analog, 6-beta-PGI1, did not affect the formation of either LT's or TxB2. The inhibition of LT formation by U-60,257 was rapidly reversed after removal of this compound from the cells. U-60,257 did not inhibit the cyclooxygenase of human polymorphonuclear leukocytes. Nor did it inhibit formation of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) in human platelets. On the other hand, U-60,257 inhibited glutathione S-transferase activity of rat basophil leukemia cells (ID50, 37 microM), suggesting that this compound may inhibit the last step in LTC biosynthesis. In addition to inhibiting LT synthesis, U-60,257 also appears to be a competitive inhibitor of the action of LT on the guinea pig ileum, although this inhibition requires a higher drug concentration than those ordinarily encountered during assay for LT's in U-60,257-treated incubations.