Beta 3 agonists. Part 1: evolution from inception to BMS-194449.

Screening of the BMS collection identified 4-hydroxy-3-methylsulfonanilidoethanolamines as full beta 3 agonists. Substitution of the ethanolamine nitrogen with a benzyl group bearing a para hydrogen bond acceptor promoted beta(3) selectivity. SAR elucidation established that highly selective beta(3) agonists were generated upon substitution of C(alpha) with either benzyl to form (R)-1,2-diarylethylamines or with aryl to generate 1,1-diarylmethylamines. This latter subset yielded a clinical candidate, BMS-194449 (35).(1)

BMS-196085: a potent and selective full agonist of the human beta(3) adrenergic receptor.

A series of 4-hydroxy-3-methylsulfonanilido-1,2-diarylethylamines were prepared and evaluated for their human beta(3) adrenergic receptor agonist activity. SAR studies led to the identification of BMS-196085 (25), a potent beta(3) full agonist (K(i)=21 nM, 95% activation) with partial agonist (45%) activity at the beta(1) receptor. Based on its desirable in vitro and in vivo properties, BMS-196085 was chosen for clinical evaluation.

Hypoglycemic prodrugs of 4-(2,2-dimethyl-1-oxopropyl)benzoic acid.

SAH 51-641 (1) is a potent hypoglycemic agent, which acts by inhibiting hepatic gluconeogenesis. It is a prodrug of 4-(2, 2-dimethyl-1-oxopropyl)benzoic acid (2) and 4-(2, 2-dimethyl-1-hydroxypropyl)benzoic acid (3), which sequester coenzyme A (CoA) in the mitochondria, and inhibits medium-chain acyltransferase. 1-3 and 4-tert-butylbenzoic acid all cause testicular degeneration in rats at pharmacologically active doses. 14b (FOX 988) is a prodrug of 3, which is metabolized in the liver at a rate sufficient enough to have hypoglycemic potency (an ED50 of 65 micromol/kg, 28 mg/kg/day, for glucose lowering), yet by avoiding significant escape of the metabolite 3 to the systemic circulation, it avoids the testicular toxicity at doses up to 1500 micromol/kg/day. 14b was selected for clinical studies.

Effect of feed intake on antimicrobially induced increases in porcine serum insulin-like growth factor I.

This study was conducted to determine whether an antimicrobially induced (ASP-250) increase in serum IGF-I was the result of differences in feed intake. Serum IGF-I concentrations were measured in crossbred pigs that were fed a control diet or a diet supplemented with ASP-250 either for ad libitum consumption or limited to 85% of the control pigs' consumption. The pigs that consumed either diet ad libitum, control or ASP-250, consumed similar quantities of feed. The ASP-250 ad libitum-intake pigs had serum IGF-I concentrations that were greater (P<.01) than those of their ad libitum-intake control littermates. Similarly, the ASP-250 limit-fed pigs had serum IGF-I concentrations that were greater (P<.01) than those of the controls. Although the serum IGF-I concentrations of pigs fed the ASP-250-supplemented diet for ad libitum intake were greater than the serum IGF-I concentrations of the pigs limit-fed the ASP-250-supplemented diet, the differences were not significant (P<.08). The ASP-250-fed pigs had higher serum IGF binding protein (BP)-3 concentrations than did their control littermates (P<.003). A time course of antimicrobially induced alterations in serum IGF-I concentrations revealed that the effect of increased serum IGF-I levels in ASP-250-supplemented pigs (P<.02) was observed within 4 d and was maintained throughout the 4-wk study. These findings show that feed intake is not responsible for the increase in serum IGF-I observed with ASP-250 supplementation. Additionally, the antimicrobially induced increase in serum IGF-I concentrations occurs within a few days after initiation of the treatment.

Relative activities of retrovirally expressed murine prostaglandin synthase-1 and -2 depend on source of arachidonic acid.

We have developed derivatives of mouse embryonic fibroblasts (10T1/2) and Chinese hamster ovary (AS52) cells that stably express high levels of murine prostaglandin synthase-1 or -2 (PGHS-1 or -2). The cDNAs were transferred using retroviral vectors and the resulting G418-resistant clones were analyzed for prostaglandin E2 (PGE2) production. Specific expression was confirmed by Western and Northern analyses. Enzyme activities, protein, and message levels peaked 1 (10T1/2) or 2 (AS52) days after seeding but decreased as cells became density arrested. Upon subculturing, enzyme activities returned to their initial high levels. With 10 microM exogenous arachidonic acid (AA) as the substrate, PGHS-1 activities were approximately 3- to 5-fold higher than PGHS-2 activities. Conversely, when exogenous AA was left out of the medium and only endogenous AA was available as substrate, enzyme activities were lower; but PGHS-2 activities were 5-fold (10T1/2) or 1.5-fold (AS52) higher than PGHS-1 activities. Following phorbol ester treatment to stimulate endogenous AA release, PGHS-2 activities increased over time and by 6 hours, were 4-fold (10T1/2) or 2-fold (AS52) higher than PGHS-1 activities. However, when calcium ionophore A23187 was used to stimulate endogenous AA release, maximum PGHS activities occurred within 30 min of treatment; PGHS-1 activities were equal to (10T1/2) or 2-fold higher (AS52) than PGHS-2 activities. Because these cell lines allow us to measure specific PGHS activity in intact cells, we were able to demonstrate that the relative activities of the two PGHS isozymes depend on the source of AA (exogenous versus endogenous) or biochemical stimulus used to mobilize endogenous AA (A23187 versus phorbol ester). These data suggest that PGHS-1 and PGHS-2 preferentially utilize different pools of AA and may be modulated through different stimulus-initiated pathways.

Isolation of antirhinoviral sesquiterpenes from ginger (Zingiber officinale).

The dried rhizomes of Indonesian ginger, Zingiber officinale, were investigated for antirhinoviral activity in the plaque reduction test. Fractionation by solvent extraction, solvent partition, and repeated chromatography guided by bioassay, allowed the isolation of several sesquiterpenes with antirhinoviral activity. The most active of these was beta-sesquiphellandrene [2] with an IC50 of 0.44 microM vs. rhinovirus IB in vitro.

Interactions of sanguinarine and zinc on oral streptococci and Actinomyces species.

Sanguinaria extract, which contains benzophenanthridine alkaloids, has been used as a folk medicine for many years. Minimum inhibitory and minimum bactericidal concentrations (MIC and MBC values) for sanguinarine were determined for common and etiologically important plaque bacteria. Because the efficacy of sanguinarine is believed to be enhanced by zinc, isobolograms were assessed to determine their mode(s) of interaction. Hydrogen ion concentration influenced the inhibitory activity of both sanguinarine and zinc. For sanguinarine, at the optimum pH (6.5), MIC values were 4 or 8 micrograms/ml for Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguis, Actinomyces viscosus and Actinomyces naeslundii. MIC values were 0.125-0.50 mmol Zn/ml. MBC values ranged from 1 to 8 mmol Zn/ml at pH 5.5. Isobologram data revealed that sanguinarine and zinc interacted synergistically. Viadent oral rinse, which contained 300 micrograms sanguinaria extract/ml and 0.2% zinc chloride (14.9 mmol Zn/l), was inhibitory to all strains tested. MIC values were 1 or 2% (ml Viadent oral rinse/100 ml aqueous solution) for all strains except A. viscosus for which the MIC value was 12% (vol/vol).

Changes in the synthesis and phosphorylation of cellular proteins in chick fibroblasts transformed by two avian sarcoma viruses.

35S- and 32P-labeled proteins from control chick embryo fibroblasts and from fibroblasts transformed by UR2 sarcoma virus, or by a temperature-sensitive mutant (tsLA29) of Rous sarcoma virus, were separated by two-dimensional electrophoresis on giant gels to detect transformation-specific changes in protein synthesis and total phosphorylation. A nontransforming avian retrovirus, UR2-associated virus (UR2AV), was also studied. Virus-coded proteins appear in whole cell lysates of all infected cells. The structural proteins can be identified by comparison with proteins immunoprecipitated with antivirus serum. The transforming proteins pp60src and p68ros, present in cells transformed with Rous sarcoma virus and UR2, respectively, are phosphorylated in vivo. Eighteen increases and eight decreases in cellular phosphoproteins are associated with transformation, and revert toward normal levels when cells infected with tsLA29 are incubated at 42 degrees C. These changes are more extensive than previously reported, but none represent new phosphorylations, since all phosphoproteins seen in transformed cells also appear to be phosphorylated to a certain extent in control cells. Fifteen cellular proteins show increased relative rates of synthesis apparently related either to transformation or to growth at 42 degrees C. Four other proteins are increased exclusively in cells incubated at 42 degrees C, but not at 37 degrees C, whether transformed or not. Eleven additional increases in the synthesis of cellular proteins, many quite large, and one seemingly a de novo induction, appear to be specific for transformation. These changes occur in cells transformed by either UR2 or Rous sarcoma virus at 37 degrees C, do not occur with UR2AV infection, and tend to revert in cells infected with tsLA29 incubated at 42 degrees C. These 11 changes may represent increases in cellular gene expression that are related specifically to the maintenance of the transformed state.