Design, Synthesis, and Structure-Activity Relationship Studies of 3-(Phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine Derivatives as a New Class of Src Inhibitors with Potent Activities in Models of Triple Negative Breast Cancer.

A series of 3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine derivatives were designed and synthesized. Structure-activity relationship (SAR) analysis of these compounds led to the discovery of compound 1j, which showed the highest inhibitory potency against the Src kinase and the most potent antiviability activity against the typical TNBC cell line MDA-MB-231 among all the synthesized compounds. Further kinase inhibition assays showed that compound 1j was a multikinase inhibitor and potently inhibited Src (IC50 = 0.0009 µM) and MAPK signaling protein kinases B-RAF and C-RAF. In an MDA-MB-231 xenograft mouse model, a once-daily dose of compound 1j at 30 mg/kg for 18 days completely suppressed the tumor growth with a tumor inhibition rate larger than 100% without obvious toxicity. It also displayed good pharmacokinetic properties in a preliminary pharmacokinetic assay. Western blot and immunohistochemical assays revealed that compound 1j significantly inhibited Src and MAPK signaling and markedly induced apoptosis in tumor tissues.

Comparison of thoracic bioimpedance with acetylene uptake for measuring cardiac output.

UNLABELLED: Cardiac output is shown to be a key determinant for oxygen transport, performance and health. Reliable and accurate non-invasive measurements of cardiac output, especially during exercise, are therefore of importance. The present study compared a new thoracic bioimpedance method with the established single-breath acetylene uptake method. We assessed cardiac output in 20 (24±4 years.) moderately trained males, at rest and during cycling. Both methods showed good test-retest reliabilities with ±2 SD limits of agreement of 3.67 and -4.50 L ∙ min(-1) (thoracic bioimpedance) and 4.46 and -5.69 L ∙ min(-1) (single breath), respectively. When thoracic bioimpedance was compared with single breath, the ±2 SD limits of agreement were poor (-6.05 and 9.57 L ∙ min(-1)). Thoracic bioimpedance displayed significantly lower (p<0.05) absolute cardiac output values than single breath, and the cardiac output-oxygen consumption slopes (y=5.7x+5.5 (single breath) and y=5.0x+5.0 (thoracic bioimpedance) tended (p=0.08) to show less increase for thoracic bioimpedance. CONCLUSIONS: Results from the single-breath method are in line with previous findings, showing a good reliability. Although thoracic bioimpedance showed a similar reliability as the single-breath method, and is easier to use, the agreement with single breath was poor, and thoracic bioimpedance seems not to be able to replace it.

Novel TNF-α converting enzyme (TACE) inhibitors as potential treatment for inflammatory diseases.

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors has led to an acetylene containing series that demonstrates sub-nanomolar potency (K(i)) as well as excellent activity in human whole blood. These studies led to the discovery of highly potent TACE inhibitors with good DMPK profiles.

Acetylene in breath: background levels and real-time elimination kinetics after smoking.

We have measured the acetylene concentration in the exhaled breath of 40 volunteers (31 non-smokers, nine smokers) using near-infrared cavity ring-down spectroscopy. The acetylene levels were found to be the same as in ambient air for non-smokers, whereas elevated levels were observed for smokers. Real-time measurements with sub-second time resolution have been applied to measure the elimination kinetics of acetylene in breath after exposure to tobacco smoke. Three exponential time constants can be distinguished from the data and these can be used to define the residence times for different compartments, according to the multi-compartment model of the human body.

The relationship between resting lung-to-lung circulation time and peak exercise capacity in chronic heart failure patients.

BACKGROUND: Peak exercise capacity (VO2peak) is a measure of the severity of chronic heart failure (CHF); however, few indices of resting cardiopulmonary function have been shown to predict VO2peak. A prolonged circulation time has been suggested as an index of increased severity of CHF. The aim of this study was to investigate the relationship between resting lung-to-lung circulation time (LLCT) and VO2peak in CHF. METHODS AND RESULTS: Thirty CHF patients (59 +/- 13 years, New York Heart Association: 1.9 +/- 1.0) undertook the study. Each subject completed resting pulmonary and echocardiography measures and an incremental exercise test. LLCT was measured using the reappearance of end-tidal acetylene (P(ET),C2H2) after a single inhalation. Univariate and multivariate stepwise linear regression was used to determine the predictors of VO2peak. Univariate correlates of VO2peak (group mean 1.53 +/- 0.44 L/min(-1)) included LLCT (r = -0.75), inspiratory capacity (r = 0.41), ejection fraction (r = 0.33), peak early flow velocity (r = -0.39), and the ratio of early to late flow velocity (r = -0.31). LLCT was the only independent predictor where VO(2peak) = 3.923-0.045 (LLCT); r2 = 54%. CONCLUSIONS: These results suggest that resting LLCT determined using the soluble inert gas technique represents a simple, noninvasive method that provides additional information regarding exercise capacity in CHF.

New diphenylacetylenes as probes for positron emission tomographic imaging of amyloid plaques.

A series of 18F fluoropegylated diphenylacetylenes as probes for binding to Abeta plaques were successfully prepared. These relatively rigid acetylenes, 12a, 12b, 14a, and 14b, displayed high binding affinities in postmortem AD brain homogenates (Ki ranging from 1.2 to 2.9 nM). In vivo biodistribution in normal mice exhibited excellent initial brain penetrations (4.42, 4.55, 5.41, and 6.78% dose/g at 2 min for [18F]12a, 12b, 14a, and 14b, respectively). [18F]12b and [18F]14b, with a longer fluoropegylated unit, that is, n=3, showed faster brain washout at 30 min postinjection (0.42 and 1.57% dose/g) as compared to the shorter fluoropegylated chain ligands, that is, [18F]12a and [18F]14a (1.03 and 3.69% dose/g). Autoradiography and homogenate binding confirmed the high binding signal due to Abeta plaques. These preliminary results suggest that the novel diphenylacetylenes may be potentially useful for imaging of Abeta plaques in the brain of patients with Alzheimer's disease.

Mechanism of delta-aminolevulinate dehydratase inhibition by phenyl selenoacetylene involves its conversion to diphenyl diselenide.

The mechanism of delta-aminolevulinate dehydratase (delta-ALA-D) inhibition by phenyl selenoacetylene in vitro was investigated in this study. Phenyl selenoacetylene (40-400 microM) inhibition of delta-aminolevulinate dehydratase from rat liver (low speed supernatant fraction, S1 fraction) was partially prevented by incubation under argon atmosphere and completely prevented by dithiothreitol. After incubation with S1 fraction from rat liver or cysteine (40 mM), phenyl selenoacetylene was partially converted into diphenyl diselenide, which is a stronger inhibitor of delta-aminolevulinate dehydratase than phenyl selenoacetylene. Diphenyl diselenide increased the rate of oxidation of -SH groups, while phenyl selenoacetylene did not affect such oxidation. delta-Aminolevulinate dehydratase purified from bovine liver (Sigma) was less sensitive to phenyl selenoacetylene and diphenyl diselenide than the enzyme from S1 fraction. We propose that the lower sensitivity of purified enzyme to selenides could be related to the formation of selenols due to the presence of dithiothreitol (a reducing agent) in the incubation medium. In agreement, incubation of purified enzyme (Sigma) with diphenyl diselenide (2 microM) and sodium borohydride (a reducing agent) under argon atmosphere significantly increased enzyme activity. Results obtained suggest that delta-aminolevulinate dehydratase inhibition by phenyl selenoacetylene is dependent on its conversion into diphenyl diselenide that induces oxidation of essential -SH groups of delta-aminolevulinate dehydratase. We propose that oxygen could be important in the regeneration of diphenyl diselenide leading to a catalytic oxidation of the enzyme.

Phloretin and 6-ketocholestanol: membrane interactions studied by a phospholipid/polydiacetylene colorimetric assay and differential scanning calorimetry.

The aim of this study was to investigate membrane interactions of phloretin and 6-ketocholestanol using different methods. A previously reported colorimetric assay with phospholipid/polydiacetylene (PDA) vesicles was used to examine a possible interaction of phloretin and 6-ketocholestanol with this target. During this interaction the used aggregates of lipids and conjugated PDA undergo a visible and quantifiable blue to red color transition. A positive result is indicative for a reaction response with membrane lipids of a simplified bilayer structure instead of the complex bilayer system of the stratum corneum. Results of this test confirm previous proposed membrane interactions by skin diffusion studies. Additional differential scanning calorimetry studies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes confirm a membrane interaction and indicates that phloretin and 6-ketocholestanol interact with the lipid layer and change structural parameters. They strongly decrease the lipid phase transition temperature of DMPC and DPPC liposomes by at least about 6.6 degrees C and maximally about 13.9 degrees C which refers to a higher fluidity of the membrane.

Use of acetylene breathing to determine cardiac output in young and older adults.

PURPOSE: The aims of this investigation were: 1). to establish the day-to-day reproducibility of open-circuit acetylene breathing for measuring exercise cardiac output (Q(c)) in young and older adults; and 2). to compare estimates of Q(c) from open-circuit acetylene breathing with estimates of Q(c) from previously established closed-circuit acetylene rebreathing. METHODS: Twenty men (10 young: 28 +/- 1 yr; 10 older: 61 +/- 1 yr (mean +/- SE)) performed cycle ergometry exercise on 3 separate days. Q(c) was estimated using open-circuit acetylene breathing on 2 d, and closed-circuit acetylene rebreathing on 1 d. RESULTS: Open-circuit acetylene breathing was highly reproducible (young: standard error of measurement (SEM) = 1.52 L.min (-1) limits of agreement (LOA) = 0.2 +/- 4.2 L.min (-1), coefficient of variation 6% < CV < 8%, day 2 = (0.9 x day 1) + 2.4, r = 0.90, P< 0.001, r (2)= 0.82; older: SEM = 0.94 L.min (-1), LOA = 0.1 +/- 2.8 L.min (-1), 4% < CV < 10%, day 2 = (1.0 x day 1) + 0, r = 0.91, < 0.001, r(2) = 0.82). Estimates of Q(c) from open-circuit acetylene breathing demonstrated good agreement with closed-circuit acetylene rebreathing (young: SEM = 1.52 L.min (-1), LOA = 0.9 +/- 4.4 L.min (-1), 5% < CV < 10%, open-circuit = (1.0 x closed-circuit) + 1.5, r = 0.89, < 0.001, r (2) = 0.79; older: SEM = 1.13 L.min (-1), LOA = 0.1 +/- 3.2 L.min (-1), 5% < CV < 9%, open-circuit = (0.9 x closed-circuit) + 1.6, r = 0.88, < 0.001, r(2) = 0.78). CONCLUSION: These results demonstrate that open-circuit acetylene breathing provides reproducible measurements of Q(c) during exercise that demonstrate good agreement with values obtained from the acetylene rebreathing procedure in young and older healthy men.

Cardiac output during exercise by the open circuit acetylene washin method: comparison with direct Fick.

An open-circuit (OpCirc) acetylene uptake cardiac output (QT) method was modified for use during exercise. Two computational techniques were used. OpCirc1 was based on the integrated uptake vs. end-tidal change in acetylene, and OpCirc2 was based on an iterative finite difference modeling method. Six subjects [28-44 yr, peak oxygen consumption (VO(2)) = 120% predicted] performed cycle ergometry exercise to compare QT using OpCirc and direct Fick methods. An incremental protocol was repeated twice, separated by a 10-min rest, and subsequently subjects exercised at 85-90% of their peak work rate. Coefficient of variation of the OpCirc methods and Fick were highest at rest (OpCirc1, 7%, OpCirc2, 12%, Fick, 10%) but were lower at moderate to high exercise intensities (OpCirc1, 3%, OpCirc2, 3%, Fick, 5%). OpCirc1 and OpCirc2 QT correlated highly with Fick QT (R(2) = 0.90 and 0.89, respectively). There were minimal differences between OpCirc1 and OpCirc2 compared with Fick up to moderate-intensity exercise (<70% peak VO(2)); however, both techniques tended to underestimate Fick at >70% peak VO(2). These differences became significant for OpCirc1 only. Part of the differences between Fick and OpCirc methods at the higher exercise intensities are likely related to inhomogeneities in ventilation and perfusion matching (R(2) = 0.36 for Fick - OpCirc1 vs. alveolar-to-arterial oxygen tension difference). In conclusion, both OpCirc methods provided reproducible, reliable measurements of QT during mild to moderate exercise. However, only OpCirc2 appeared to approximate Fick QT at the higher work intensities.

Measurement of cardiac output during exercise by open-circuit acetylene uptake.

Noninvasive measurement of cardiac output (QT) is problematic during heavy exercise. We report a new approach that avoids unpleasant rebreathing and resultant changes in alveolar PO(2) or PCO(2) by measuring short-term acetylene (C(2)H(2)) uptake by an open-circuit technique, with application of mass balance for the calculation of QT. The method assumes that alveolar and arterial C(2)H(2) pressures are the same, and we account for C(2)H(2) recirculation by extrapolating end-tidal C(2)H(2) back to breath 1 of the maneuver. We correct for incomplete gas mixing by using He in the inspired mixture. The maneuver involves switching the subject to air containing trace amounts of C(2)H(2) and He; ventilation and pressures of He, C(2)H(2), and CO(2) are measured continuously (the latter by mass spectrometer) for 20-25 breaths. Data from three subjects for whom multiple Fick O(2) measurements of QT were available showed that measurement of QT by the Fick method and by the C(2)H(2) technique was statistically similar from rest to 90% of maximal O(2) consumption (VO(2 max)). Data from 12 active women and 12 elite male athletes at rest and 90% of VO(2 max) fell on a single linear relationship, with O(2) consumption (VO(2)) predicting QT values of 9.13, 15.9, 22.6, and 29.4 l/min at VO(2) of 1, 2, 3, and 4 l/min. Mixed venous PO(2) predicted from C(2)H(2)-determined QT, measured VO(2), and arterial O(2) concentration was approximately 20-25 Torr at 90% of VO(2 max) during air breathing and 10-15 Torr during 13% O(2) breathing. This modification of previous gas uptake methods, to avoid rebreathing, produces reasonable data from rest to heavy exercise in normal subjects.

Noninvasive measurement of cardiac output by an acetylene uptake technique and simultaneous comparison with thermodilution in ICU patients.

A simple, accurate, and noninvasive method of cardiac output measurement can be an extremely useful tool for the clinician and researcher. This study used the acetylene gas uptake technique to measure the absorption of acetylene into the pulmonary circulation during a constant exhalation, which is proportional to the pulmonary capillary blood flow and to the cardiac output, assuming no anatomic shunts. We compared cardiac output measured simultaneously by this and by the standard thermodilution (TD) technique in 21 patients in the ICU with a variety of medical and surgical conditions and a wide range of cardiac outputs. We also compared the two techniques in 19 ambulatory patients with a 2-h interval between the invasive and noninvasive test to assess variability over time. The two tests had an excellent correlation when done simultaneously with a correlation coefficient of 0.89 (p < 0.001). With a 2-h interval between the two tests, the correlation coefficient was 0.66 (p = 0.0018). Nine patients in the simultaneous group had cardiomyopathy. When they were excluded, the correlation coefficient increased to 0.96. Most of these patients had documented tricuspid regurgitation (TR), which may underlie the greater difference between acetylene uptake and TD values, with consistently higher TD values in these patients. This study confirms the correlation between the acetylene uptake and the standard invasive TD techniques in sick patients with various medical and surgical conditions and a wide range of cardiac outputs. Furthermore, we believe this would be a more accurate method for measuring cardiac output in patients with cardiomyopathy and TR because it is based only on pulmonary capillary blood flow.

Cardiac output during exercise measured by acetylene rebreathing, thermodilution, and Fick techniques.

In dogs during exercise, respiratory rate can reach 200 breaths/min, blood temperature can exceed 42 degrees C, and hematocrit can approach 60%. To determine whether these changes significantly affect the measurement of cardiac output by the acetylene rebreathing method (QcRB), we compared estimates of QcRB with those measured by thermodilution and Fick (QcFI) techniques in nine dogs at rest and during steady-state exercise on a treadmill up to near-maximal workloads. Solubility of acetylene in blood was corrected to the simultaneously measured blood temperature and hematocrit. Results were also adjusted for mixing efficiency. Up to a QcFI of 20 l/min, QcRB was not significantly different from QcFI (P > 0.05). However, cardiac output measured by thermodilution was consistently higher than those measured by the other techniques (P < 0.0001). We conclude that the overall agreement between QcRB and QcFI estimates supports the validity of the rebreathing technique under exercise conditions where body temperature and hematocrit are changing rapidly and the breathing pattern is unrestrained. Systematic error by the thermodilution technique may be related to a variety of methodological issues as well as possible dissipation of cooling into the myocardial tissue and subsequent incomplete washout.

Measurement of cardiac output by automated single-breath technique, and comparison with thermodilution and Fick methods in patients with cardiac disease.

Accurate noninvasive methods are needed for determination of cardiac output. Current methods are generally complex or may be unreliable. A previously described method, based on absorption of acetylene gas during a constant exhalation that enables calculation of cardiac output by estimating pulmonary capillary circulation, is incorporated in a new, automated commercial product (SensorMedics 2200). In this study, cardiac output by single-breath acetylene blood flow measured with this device was compared with the standard thermodilution and direct Fick methods in 20 patients undergoing cardiac or pulmonary artery catheterization. Patients inhaled test gas mixture to total lung capacity and exhaled at a constant rate through an adjustable resistor. Lung volumes and noninvasive acetylene blood flow value were calculated automatically. Correlation between the automated single-breath technique and both thermodilution and Fick cardiac output determinations was very high (correlation coefficients were 0.90 and 0.92, respectively), regression slopes were close to identity (0.98 and 0.90), and bias (-0.39 and -0.79 liter/min) and precision (0.94 and 1.02) were good; when shunt correction was applied, bias was reduced to 0.06 and 0.35 liter/min, respectively. Rapid, accurate, noninvasive measurement of cardiac output was easily obtained using the automated device. This technique may have a wide applicability for noninvasive evaluation of patients with cardiac disease and for monitoring effects of therapeutic interventions.

Brain uptake of S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine, the glutathione and cysteine S-conjugates of the neurotoxin dichloroacetylene.

Dichloroacetylene causes trigeminal neuropathy in humans and animals. Glutathione conjugation of dichloroacetylene affords S-(1,2-dichlorovinyl)glutathione (DCVG), which is hydrolyzed to S-(1,2-dichlorovinyl)-L-cysteine (DCVC). This study was undertaken to test the hypothesis that the neurotoxicity of dichloroacetylene may be associated with glutathione S-conjugate formation and brain uptake and bioactivation of the dichloroacetylene-derived S-conjugates. With the Oldendorf technique, the Brain Uptake Index for [35S]DCVC and [35S]DCVG was determined and compared with the uptake of [35S]methionine and [14C]sucrose. Brain uptake of DCVC exceeded uptake of methionine and DCVG uptake was comparable to methionine uptake. Both [35S]DCVC and [35S]DCVG were recovered intact in brain tissue. The uptake of the 35S-labeled S-conjugates was inhibited by unlabeled DCVC and DCVG in a concentration-dependent manner. The data indicated that DCVC, but not DCVG, was transported by the sodium-independent system-L transporter for neutral amino acids. In vitro studies revealed that DCVG can be hydrolyzed to DCVC by brain tissue in a concentration-dependent manner.

Metabolism of 14C-dichloroethyne in rats.

1. The metabolism of 14C-dichloroethyne was studied in rats by inhalation in a dynamic nose-only exposure system. 14C-Dichloroethyne was generated in 95-99% yield from 14C-trichloroethene by alkaline dehydrochlorination. 2. After inhalation of 20 ppm and 40 ppm dichloroethyne for 1 h, the retention rates were 17.6% and 15.6% of the radioactivity introduced into the exposure system, respectively. During the period of observation (96 h), almost quantitative elimination of the dose was observed. Elimination with urine accounted for 60.0% (40 ppm) and 67.8% (20 ppm) of absorbed radioactivity and elimination with faeces for 27% (40 ppm) and 27.7% (20 ppm), 3.4-3.5% remained in the carcasses. 3. Metabolites of dichloroethyne identified are: N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, dichloroethanol, dichloroacetic acid, oxalic acid and chloroacetic acid in urine; N-acetyl-S-(1,2-dichlorovinyl-L-cysteine in faeces. 4. In bile of rats exposed to 40 ppm of dichloroethyne, S-(1,2-dichlorovinyl)glutathione was the only metabolite identified. Biliary cannulation did not influence the renal excretion of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, indicating that glutathione conjugate formation occurs in the kidney. 5. The results suggest that two metabolic pathways are operative in dichloroethyne metabolism in vivo. Cytochrome P450-dependent oxidation represents a minor pathway accounting for the formation of 1,1-dichloro compounds after chlorine migration. The major pathway is the biosynthesis of toxic glutathione conjugates. Organ-specific toxicity and carcinogenicity of dichloroethyne is due most likely to the topographical distribution of gamma-glutamyl transpeptidase which is concentrated mainly in the kidney in rats.

Effects of environmental O2 on blood flow and diffusing capacity in amphibian skin.

The effects of local environmental PO2 on cutaneous blood flow (Q) and the membrane diffusing capacity of the skin (D) were investigated in the leopard frog, Rana pipiens, and the lungless salamander, Desmognathus quadramaculatus. Halothane anesthetized animals were equilibrated with Freon-22 (Fr) and acetylene (Ac) in a box. A gas mixture containing either 0, 20 or 40% O2, respectively, in N2 and initially free of Fr and Ac was drawn through a small sample chamber on the abdomen. The excretion of Fr and Ac into the chamber was analyzed with a mass spectrometer. These conditions allowed the determination of the cutaneous conductance to Fr (GFr) and Ac (GAc) at the 3 levels of local environmental O2. GFr and GAc of the isolated skin and the blood solubilities of the 2 gases were also determined. To estimate Q, DAc and DFr, the data were analyzed with a homogeneous single capillary model. In the frog, Q varied directly with sample chamber [O2], but chamber [O2] had no effect on D. In the salamander, O2 had no effect on either Q or D. The results indicate that regulation of cutaneous gas exchange in the frog by local environmental O2 only involves alterations in Q. Similar control of cutaneous gas exchange is absent in the lungless salamander.

Elimination kinetics of acetylene and Freon 22 in resting and active lungless salamanders.

To quantify diffusion limitation in cutaneous gas exchange, the elimination of two inert gases of different diffusivity, Freon 22 (CHC1F2) and acetylene (C2H2), was measured simultaneously in exclusively skin-breathing lungless salamanders, Desmognathus quadramaculatus. In resting salamanders, elimination of both gases could be described as the sum of three exponential terms. For both the medium and the slow exponential component, the ratio of the respective rate constants (k) for acetylene and Freon averaged 1.77. This value is between the values expected for perfusion limitation (1.00) and diffusion limitation (1.94), indicating combined diffusion and perfusion limitation. In salamanders stimulated to run on a treadmill, the elimination rates and the rate constants increased more for Freon than for acetylene. During spontaneous activity, the increase in elimination of Freon was larger than that of acetylene. These findings suggest an increase in the diffusing capacity of the skin during exercise. Thus the diffusing capacity of salamander skin for gases appears to be variable and to be adjusted to meet the increased O2 requirement during exercise.