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The inhibition of key receptor tyrosine kinases (RTKs) that are implicated in tumor vasculature formation and maintenance, as well as tumor progression and metastasis, has been a major focus in oncology research over the last several years. Many potent small molecule inhibitors of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) kinases have been evaluated. More recently, compounds that act through the complex inhibition of multiple kinase targets have been reported and may exhibit improved clinical efficacy. We report herein a series of potent, orally efficacious 4-amino-3-benzimidazol-2-ylhydroquinolin-2-one analogues as inhibitors of VEGF, PDGF, and fibroblast growth factor (FGF) receptor tyrosine kinases. Compounds in this class, such as 5 (TKI258), are reversible ATP-competitive inhibitors of VEGFR-2, FGFR-1, and PDGFRbeta with IC(50) values

The desorption of CO from a Pd(111) surface following absorption of 120 fs pulses of 780 nm light occurs on two distinct and well-separated time scales. Two-pulse correlation measurements show a fast subpicosecond decay followed by a slower, approximately 40 ps decay. Simulations based on the two-temperature model of electron and phonon heat baths within the substrate, and an empirical friction model to treat coupling to the adsorbate, support the assignment of the desorption mechanism as an electron-mediated process. The photodesorption yield and overall width of the temporal response exhibit a marked dependence on the initial surface temperature in the 100-375 K range despite the much higher transient electronic temperatures (approximately 7000 K) achieved. The observed temperature dependences can be attributed directly to variations in the initial temperature within the frictional coupling picture. Simulations of this extended data set imply that the activation barrier to photoinduced desorption is equal in magnitude to that derived from thermal desorption experiments for this system within the limits of a one-dimensional Arrhenius desorption model. The simulations also imply that the slower decay is not the result of phonon-driven desorption. Though we cannot unambiguously determine the strength of the adsorbate-phonon coupling, our results suggest that its role is to moderate the degree of the adsorbate excitation.

Purpose: To evaluate the therapeutic and biological effects of CHIR-258, an orally bioavailable, potent inhibitor of class III-V receptor tyrosine kinases, in colon cancer models. Experimental Design: The pharmacologic activity of CHIR-258 was characterized by monitoring target modulation as well as by evaluating the antitumor and antiangiogenic effects in human colon xenograft models. Results: CHIR-258 inhibits vascular endothelial growth factor receptor 1/2, fibroblast growth factor receptor 1/3, and platelet-derived growth factor receptor β (PDGFRβ) and shows both antitumor and antiangiogenic activities in vivo. Treatment of KM12L4a human colon cancer cells with CHIR-258 resulted in a dose-dependent inhibition of vascular endothelial growth factor receptor 1 and PDGFRβ phosphorylation and reduction of phosphorylated extracellular signal-regulated kinase (ERK) levels, indicating modulation of target receptors and downstream signaling. In vivo administration of CHIR-258 resulted in significant tumor growth inhibition and tumor regressions, including large, established tumors (500-1,000 mm3). Immunohistochemical analysis showed a reduction of phosphorylated PDGFRβ and phosphorylated ERK in tumor cells after oral dosing with CHIR-258 compared with control tumors. These changes were accompanied by decreased tumor cell proliferation rate and reduced intratumoral microvessel density. CHIR-258 inhibited the phosphorylation of PDGFRβ and ERK phosphorylation in tumors within 2 hours following dosing and the inhibitory activity was sustained for >24 hours. Significant antitumor activity was observed with intermittent dosing schedules, indicating a sustained biological activity. Conclusion: These studies provide evidence that biological activity of CHIR-258 in tumors correlates with efficacy and aids in the identification of potential biomarkers of this multitargeted receptor tyrosine kinase inhibitor. CHIR-258 exhibits properties that make it a promising candidate for clinical development in a variety of solid and hematologic malignancies.

Deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine by LpxC is the first committed step in the Pseudomonas aeruginosa biosynthetic pathway to lipid A; homologous enzymes are found widely among Gram-negative bacteria. As an essential enzyme for which no inhibitors have yet been reported, the P. aeruginosa LpxC represents a highly attractive target for a novel antibacterial drug. We synthesized several focused small-molecule libraries, each composed of a variable aromatic ring, one of four heterocyclic/spacer moieties, and a hydroxamic acid and evaluated the LpxC inhibition of these compounds against purified P. aeruginosa enzyme. To ensure that the in vitro assay would be as physiologically relevant as possible, we synthesized a tritiated form of the specific P. aeruginosa glycolipid substrate and measured directly the enzymatically released acetate. Several of our novel compounds, predominantly those having fluorinated substituents on the aromatic ring and an oxazoline as the heterocyclic moiety, demonstrated in vitro IC(50) values less than 1 microM. We now report the synthesis and in vitro evaluation of these P. aeruginosa LpxC inhibitors.

Z-Leu-Arg-(7-methoxynaphthyl)amide (1) is a substrate for calpain I. The specificity constant for 1 ( k cat K m = 1405 ± 40 M−1s−1) is 10x greater than for any previously reported fluorogenic substrate. Using this substrate, a sensitive, continuous fluorogenic assay was developed permitting the identification of Z-(D)Ala-Leu-Phe-(OCO-2,6-Fl2-Ph) (69) as the first selective (>100-fold versus cathepsins B and L) time-dependent inhibitor of the enzyme.

1,4-Dihydro-4-oxo-3-quinolinecarboxamides are a class of non-peptide reversible inhibitor of human erythrocyte Calpain I. The preparation and in vitro evaluation of these compounds are discussed.

1,4-Dihydro-4-oxo-3-quinolinecarboxamides are a class of non-peptide reversible inhibitor of human erythrocyte Calpain I. The preparation and in vitro evaluation of these compounds are discussed.

Azapeptide analogues ii of the α-halomethyl ketones i were synthesized and evaluated as potential inhibitors of serine and cysteine proteases. Inhibitors ii discriminate between the two classes of protease, demonstrating selectivity for cysteine proteases. Azaglycines 1–3 and 5 displayed time-dependent inactivation (kobs/[I] = 200–1500 M−1s−1) of cathepsin B and calpain.

Ipazilide fumarate is a novel antiarrhythmic currently undergoing clinical evaluation. Since most antiarrhythmics have adverse cardiovascular side effects, we examined the potential effects of ipazilide on cardiac muscle and vascular hemodynamic function utilizing in vivo and in vitro models. The cardiovascular effects of ipazilide and selected reference antiarhythmics, disopyramide and sotalol, at relevant antiarrhythmic concentrations were compared. All agents decreased cardiac contractility, as evident by reductions in dP/dt and cardiac output in anesthetized dogs, and by decreases in contractile force in isolated guinea pig papillary muscles. In vivo, ipazilide was approximately 3–10 × less potent than disopyramide or sotalol while in vitro, ipazilide produced a greater decrease in contractility at a relatively high concentration (30 μM). All three agents increased cardiac preload as reflected by increases in left ventricular end diastolic pressure and right atrial pressure, with ipazilide 2–3 × less potent than disopyramide or sotalol. Differential effects on vascular function were also evident with the three agents. Disopyramide and sotalol significantly increased systemic vascular resistance, and either pulmonary vascular resistance (disopyramide) or left coronary arterial vascular resistance (sotalol); ipazilide did not significantly affect any of these parameters. In isolated rat arterial or canine coronary arterial smooth muscle, ipazilide produced concentration-related vasorelaxation, with EC50 values ranging from 11–300 μM. Efficacy of ipazilide was higher in depolarized vascular tissue. In conclusion, since previous preclinical studies have indicated equal or greater antiarrhythmic activity of ipazilide relative to disopyramide or sotalol, the current data indicate the possibility for lessened in vivo hemodynamic effects of ipazilide at efficacious antiarrhythmic dosages. © 1992 Wiley-Liss, Inc.

Considerable evidence suggests that protein kinase C activation participates in the regulation of vascular smooth muscle tone. The objective of the current study was to examine the relations between inhibition of protein kinase C (PKC) and myosin light-chain kinase (MLCK) and vasorelaxation and blood pressure regulation in spontaneously hypertensive rats (SHR). Putative PKC inhibitors from two chemical classes, staurosporinelike (staurosporine and K252A) and isoquinolinesulfonamides (H7 and HA1004), were tested for their ability to 1) inhibit PKC and MLCK from SHR aorta, 2) relax isolated SHR aorta, and 3) lower blood pressure in conscious SHR. A rank order of potency for the inhibition of PKC and MLCK was established, with the staurosporinelike compounds (staurosporine PKC IC50 = 54 nM) clearly more potent than the isoquinolinesulfonamides (H7 PKC IC50 = 128 microM). The rank order of potency for inhibition of PKC was retained for inhibition of MLCK for all compounds. Staurosporine (EC50 = 75 nM) and H7 (EC50 = 2 microM) caused concentration-dependent relaxation of SHR aorta, but only staurosporine produced vasorelaxation at concentrations consistent with the inhibition of PKC or MLCK. Dose-dependent reductions in arterial pressure of SHR were demonstrated after intravenous injection of staurosporine and HA1004. A single intravenous injection of staurosporine (0.3 mg/kg) lowered blood pressure for more than 10 hours. Staurosporine also lowered blood pressure after oral administration. The depressor response to staurosporine was unaffected by sympathetic beta-adrenergic blockade. In conclusion, the vasorelaxant and antihypertensive actions of staurosporine in SHR are consistent with the inhibition of PKC but could also be equally related to inhibition of MLCK. Not all PKC inhibitors produce vasorelaxation and lower blood pressure. Moreover, the lack of correlation between in vitro vasodilation and PKC or MLCK inhibition for the isoquinolinesulfonamide protein kinase inhibitors H7 and HA1004 suggests that these agents do not cause vasorelaxation in SHR by inhibition of these enzymes.

This study compared oral renal excretory effects of several agents in three rat strains with that reported in man to assess predictive value of this species in cardiorenal drug development. Sprague-Dawley (SD), Wistar-Kyoto (WK), and spontaneously hypertensive rats (SHR) were hydrated and 3 hr urine volume, Na+, and K+ were measured, Normotensive SD and WK rats responded similarly, i.e., hydrochlorothiazide (HCTZ) and aminophylline were diuretic, while hydralazine and milrinone caused Na+ retention. Accordingly the more economical SD rat was then compared with SHR. Aminophylline (3–30 mg/kg) and the more effective HCTZ (1–30 mg/kg) achieved dose-related diuresis and kaliuresis in both strains, increasing Na+ excretion by a maximum of 5–14-fold, whereas the vasodilators hydralazine (3–30 mg/kg) and milrinone (1–10 mg/kg) reduced Na+, K+ and volume dose-relatedly with Na+ output being 5–50% of control values. Verapamil promoted Na+ excretion in both strains by 1.5- to 4-fold at 30 mg/kg, but was antinatriuretic at 80 mg/kg. Conversely, nifedipine had qualitatively differents effects in SHR and SD rats. That is, it elicited diuresis in the SHR at 10mg/kg (Δ Na+ = +155%) and no change in urine volume or Na+ and K+ cutput at 30 mg/kg, where as the Ca++-antagonist provoked dose-related volume, Na+, and K+ retention in the normotensive rat with 3 hr output being ca. 15–22% of that of vehicle-treated controls. Accordingly the SD rat predicted the renal effects typically seen clinically for all agents except milrinone (Cody et al.: Clinical Pharmacology and Therapeutics 39: 128–135, 1986), supporting use of the economical SD rat as a primary assay for effects on renal excretion with the SHR strain reserved for assessing Ca++-channel blockers in the hypertensive state.

Cicletanine is an antihypertensive/vasorelaxant/natriuretic agent of unknown mechanism. We wished (a) to determine if cicletanine interacts with guanylate cyclase activators that modulate vasomotor tone and sodium balance [i.e., atriopeptin II (AP II), endothelium-derived relaxing factor (EDRF), and sodium nitroprusside (SNP)], and (b) to define the subcellular basis for this interaction by quantitating the effects of cicletanine on low Km cyclic GMP phosphodiesterase (PDE) activity. In phenylephrine-contracted rat aortic smooth muscle, the vasorelaxant potency of cicletanine was increased twofold in the presence of a threshold-relaxant concentration of AP II, and functional cyclic GMP PDE inhibition was also evident from the three- to sixfold potentiation by cicletanine of AP II- or SNP-induced vasorelaxation. Vasorelaxation produced by cicletanine was not endothelium dependent, however. In further studies, intravenous (i.v.) administration of cicletanine or the low Km cyclic GMP PDE inhibitor, zaprinast, decreased blood pressure (BP) greater than or equal to 20% in conscious spontaneously hypertensive rats (SHR). These results are consistent with the additional finding that cicletanine inhibited Ca2(+)-calmodulin (CaM) cyclic GMP PDE and zaprinast-sensitive cyclic GMP specific PDE over a concentration range (10-600 microM) similar to that for vasorelaxation. Thus, inhibition of low Km cyclic GMP PDEs by cicletanine may be partly responsible for the vasorelaxant effect of cicletanine as well as the potentiation by cicletanine of the vasorelaxant actions of guanylate cyclase activators. The extent to which this mechanism contributes to the antihypertensive efficacy of cicletanine has not yet been fully determined.

Medorinone is a structurally novel positive inotropic/vasodilator agent that selectively inhibits cardiovascular low Km cAMP phosphodiesterase (PDE). As inhibitors of this PDE isozyme would be expected to activate the cAMP system, the objectives of the present study were to quantitate changes in cAMP content, activation of cAMP protein kinase (cAPK), and positive inotropy in isolated guinea pig papillary muscles or vasorelaxation in aortic smooth muscle by medorinone. In papillary muscles, positive and significant correlations were evident between isometric force development as a function of cAMP content (r = 0.92; P

The vasorelaxant effects of milrinone and amrinone in canine coronary, cerebral, and renal arterial rings or strips contracted by either K+-depolarization, U46619 (a thromboxane mimetic), or prostaglandin F2 alpha (PGF 2 alpha) were quantitated. Milrinone was more potent as a vasorelaxant in coronary arteries relative to cerebral or renal arteries regardless of the mode of contraction; amrinone was coronary selective with K+ contraction only. When comparing potency in arteries contracted by different agonists, milrinone was significantly more potent as a vasorelaxant in all three arteries contracted by either U46619 or PGF2 alpha than in arteries contracted by K+ depolarization, whereas amrinone was only selective for U46619-induced contractions in cerebral arteries. This profile of activity for milrinone was similar to that of sodium nitrite and isoproterenol and dissimilar from the calcium entry blocking agents nimodipine and nifedipine. In conclusion, this study shows that coronary vascular selectivity exists for milrinone and amrinone. Moreover, the relaxant profiles of milrinone and amrinone, with different sources of vascular smooth muscle, are unlike those of calcium entry blocking agents and more similar to the profiles of agents that modulate cyclic nucleotide levels.

The purpose of this study was to investigate the effect of ouabain pretreatment (1 microM) on relaxation induced by milrinone and other reference vasorelaxants in guinea pig aorta. Pretreatment with ouabain for 1 h significantly reduced the threshold concentration and increased the vasoconstrictor potency of phenylephrine. Relaxation by milrinone of aortic rings contracted by phenylephrine or by an equieffective concentration of phenylephrine in the presence of ouabain was significantly attenuated in the presence of ouabain. The effect of all other vasorelaxants tested, which included isosorbide dinitrate, hydralazine, sodium nitroprusside, forskolin, HA 1004, isoproterenol and verapamil, were also significantly reduced in the presence of ouabain. The vasorelaxant effects of milrinone and verapamil were also evaluated in K+-contracted guinea pig aorta. In contrast to results obtained with phenylephrine-contracted vessels, milrinone and verapamil were equipotent as vasorelaxants in K+-contracted vessels in the presence or absence of ouabain. The results show that ouabain not only potentiates the effect of the vasoconstrictor phenylephrine, but also reduces the potency of drugs that cause vasorelaxation in phenylephrine-contracted tissues, regardless of the mechanism of action of the vasorelaxant. These data may have clinical relevance to the concomitant use of vasodilators and digitalis in the treatment of congestive heart failure.

Milrinone and enalapril, which inhibit PDE-III and ACE, respectively, are able to prolong survival of myocardially infarcted (MI) rats. This study sought to identify oral hemodynamic effects of these agents which could underlie such efficacy in this heart failure model. Four weeks after ligation of the left main coronary artery, basal left ventricular (LV) systolic pressure and dP/dtmax, heart rate and mean blood pressure of the MI rats were significantly less than that of sham-operated controls, and LV end-diastolic pressure (LVEDP) was markedly elevated. Milrinone, at 2.0 mg/kg, reduced LVEDP and renal blood flow of these 4-week MI rats by an average of 39 and 18%, respectively (P less than 0.05) within 1 h. At 4.0 mg/kg, it reduced LVEDP by 46% and raised heart rate by 16% (P less than 0.05). Enalapril (1.0 mg/kg) increased small intestine blood flow of these compromised rats by 16% (P less than 0.05), and tended to reduce LVEDP (-28%) within 1.5 h. Treatment with milrinone (2.0 mg/kg) plus enalapril (1.0 mg/kg) promoted LV dP/dtmax, coronary blood flow, and heart rate by 48, 40 and 13%, and reduced LVEDP by 40% (P less than 0.05 for all effects). Thus these agents can reduce LVEDP and redistribute cardiac output of MI rats. Furthermore, the combination of enalapril and milrinone can restore LVEDP and LV dP/dtmax of MI rats to near normal and promote coronary blood flow without compromising cardiac output or renal blood flow. Such effects, it timely or sustained, may prolong survival.

Protein kinase C (PKC) activity in aortic and renal arterial smooth muscle form SHR (20–23 wk male; mean arterial pressure = 178 mm Hg) and WKY (age/sex matched; mean arterial pressure = 126 mm Hg) was quantitated. Activity was greatest in the particulate fractions relative to the soluble fractions in all sources. The only difference between SHR and WKY was in the soluble fraction from SHR renal arteries, which had 2 fold more activity (255 pmol/mg/min) when compared with WKY (136 pmol/mg/min). This difference was not apparently related to force modulation, since the magnitude of isometric force development in renal arteries in response to phorbol 12, 13-dibutyrate was not different between SHR and WKY. The magnitude of force developed in response to phorbol 12, 13-dibutyrate and PKC activity in the particulate fraction was greatest in aorta vs. renal arteries in both WKY and SHR. These results suggest that regional vascular differences in the amount of PKC activity may exist which are not apparently related to a disease state (i.e., hypertension). These differences may be related to differential sensitivity to phorbol ester-mediated contractions in isolated smooth muscle.