5-Fluorouracil derivatives. XXII. Synthesis and antitumor activities of 1-carbamoyl-5-fluorouracils.

Fifty-four 1-carbamoyl-5-fluorouracils were synthesized from 5-fluorouracil and isocyanate or amine. Antitumor activity was tested in the L-1210 tumor system, and 11 compounds gave better values of therapeutic ratio than HCFU (1-hexylcarbamoyl-5-fluorouracil). 1-(4-Methoxycyclohexylcarbamoyl)-5-fluorouracil gave the best result.

Short-circuit current overshoot in epithelial sodium channels following apical sodium jump.

Following a jump in the sodium concentration of the solution bathing the apical surface of frog skin, the inward sodium current rises rapidly to a peak and then falls to a steady-state plateau. Lindemann suggested that this fall is due to rapid closing (in 2 to 3 s) of Na channels. However, the lack of a corresponding corner frequency in the sodium-noise spectrum indicates a much slower closing. We propose a compartmental mechanism for the overshoot: the inward Na current causes Na to accumulate in the intracellular region adjacent to the sodium channel--a virtual compartment--thereby decreasing the outside/inside [Na] ratio. As that ratio falls with rising [Na] in the virtual compartment, the force driving the current falls. The predictions of such a model have been curve-fitted to the time-course of the current overshoot. The differential equation describing the rate of change of [Na] in the virtual compartment has several time constants: a filling time for the compartment, a leakage time for escape of Na into the larger intracellular space, a mixing time in the apical bathing solution, and, of course, the channel-closing time. This curve fitting shows that channel closing becomes important only in the tail of the overshoot (> 15 s) with mean open times in a range from 7 s to 3 min. Similarly, the time-course of the current after washout of apical [Na] was fitted using the same differential equation, with the channel-closing time replaced with a channel-opening time. Other phenomena explainable by this compartmental model but not by fast channel closing include the open-circuit-potential overshoot, current overshoot through nystatin channels, and the less-than-59-mV-per-decade slopes of semilog plots of open-circuit potential vs. [Na].

Responsible conduct of scientific research: a one-semester course for graduate students.

This course was developed to satisfy in part the requirement that "a program in the principles of scientific integrity" be a part of any training program funded by the National Institutes of Health (NIH) or the Alcohol, Drug Abuse, and Mental Health Administration (NIH "Guide for Grants and Contracts," vol. 18, no. 45, 1989). The booklet On Being A Scientist (National Academy of Sciences, 1989) was chosen as required reading. The issues were presented in 15 1-h sessions. Public policy issues were not addressed. A danger was to be overly critical of scientific misconduct and stimulate a cynical skepticism of the integrity of the working scientist. One problem is that there are only general procedures a student can follow when encountering misconduct. Numerous guest speakers provided depth, objectivity, and authenticity to the discussion.

The synthesis of trans-3,4-bis-(3,4-dihydroxyphenyl)pyrrolidine, a novel DA1 agonist.

The synthesis of trans-3,4-bis-(3,4-dihydroxyphenyl)pyrrolidine by the Michael reaction of esterenolate with nitro olefin is described. This compound was expected to be the agonist toward dopamine (DA1) receptor with equipotent affinity and to be more selective than dopamine itself.

Pharmacokinetics of a novel dopamine DA1 receptor agonist, E4101, and an improvement of its bioavailability in dogs.

Pharmacokinetic parameters of E4101,(-)-(3R,4R)-3-(2-chloro-3-hydroxyphenyl)- 4-(3,4-dihydroxyphenyl)-pyrrolidine hydrochloride, in plasma were determined in conscious beagles. E4101 was extracted from plasma using a Bond Elute C18 cartridge and determined by HPLC connected to a reverse phase ODS column and an electrochemical detector. After an i.v. administration of 1 mg/kg, the plasma level of E4101 decreased according to the three compartment model. The half lives of the alpha, beta and gamma phases were 1.3, 8.1 and 27 min, respectively. The mean residence time (MRT) and total clearance were 14.5 min and 78 ml/min/kg, respectively. When 3 mg/kg of E4101 were administered orally under fasting, E4101 in plasma reached the peak (Cmax: 22.4 ng/ml) 45 min after administration. The bioavailability was 3.6%. When 3 mg/kg of E4101 in combination with 15 mg/kg of ascorbic acid was orally administered in capsule, the area under concentration curve (AUC) was increased 7-fold compared with that in a matched control study. A combination with 15 mg/kg of tartaric acid, citric acid, L-aspartic acid or L-cysteine also increased the AUC, byt they were less effective than ascorbic acid. When 10 mg/kg of E4101 in combination with 25 mg/kg of ascorbic acid as a sustained-release dosage form using oily somisolid matrix orally administered to non-fasted beagles, the duration of effective plasma concentration (2 ng/ml plasma in i.d. administration) was 8.4 +/- 1.0 hr (mean +/- SE, n-4). The Cmax was 16.2 +/- 2.5 ng/ml.

Effects of basolateral ouabain, amphotericin B, cyanide and potassium on amiloride noise during voltage clamp of Rana pipiens skin support sodium-amiloride competition.

In a previous study, the amiloride-induced corner frequency (fc) was found to decrease as apical sodium was increased. This effect was small or absent when the basolateral surface was exposed to high potassium. It has been suggested that the apical sodium effect may be indirect, due either to increased intracellular [Na+] which repelled amiloride or to an increased potential at the apical surface which reduced amiloride affinity. High basolateral K+ might then suppress the sodium effect either by preventing intracellular [Na+] from increasing or by allowing a better clamp of the apical membrane potential by reducing basolateral membrane resistance and potential. We checked the effects of basolateral [K+], of cyanide and of ouabain at concentrations known to increase intracellular [Na+]. We found only negligible effects on fc. In addition, amphotericin B added to the basolateral bathing solution either in 115 mM Na+ or in 120 mM K+ had no significant effect on fc. We found that relatively wide variation in clamp potential under all conditions, even with active transport severely inhibited, left fc virtually constant. Since the amiloride kinetics were independent of clamp potential, we were able to measure paracellular and transcellular conductances separately by examining the voltage dependence of clamp current (linear) and amiloride noise power (quadratic). This made possible estimation of channel density and single-channel current.

Effect of sodium on amiloride- and triamterene-induced current fluctuations in isolated frog skin.

The apparent association constants of two agents, amiloride and triamterene, that block the Na-selective channel of apical membrane of frog skin are shown to decrease as the Na concentration is increased in the apical bathing solution in isolated skin of the frog, Rana temporaria, Rana esculenta, and Rana pipiens. These results were obtained in "normally polarized" skins. These effects were independent of the anion used (chloride or methylsulfate) or the cation used as the Na substitute (Tris, DDA, or K ion). When NaCl was replaced with mannitol, the Na effect on the amiloride association rate constant persisted, which shows that ionic strength was not critically involved. The amiloride corner frequency was unaffected when the clamp potential was altered from +100 to -60 mV. The Na dependence was greatly attenuated or absent when the serosal surface was bathed in 120 mM K Ringer's, an effect that appears to be attributable to some pharmacological effect of high serosal K. A previously described three-state model is used to analyze the inhibitory effect of Na on the blocker association rate constant.

Synthesis of N1- and N3-glycosyl pyrimidines from protected pyrimidines and halo sugars in the presence of tert-amine.

The reaction of N1- and N3-(octylthio)carbonyl pyrimidines with acylglycosyl halides in the presence of tert-amine affords fully protected nucleosides with (N1-C1') and (N3-C1') glycosidic linkages in good yields. The (octylthio)carbonyl and acyl groups are removed stepwise or at once.

Competitive blocking of apical sodium channels in epithelia.

Apical sodium-selective channels in frog skin, when blocked by amiloride or triamterene, exhibit fluctuations in current, the spectra of which are Lorentzian. These effects have been modeled previously with two-state and three-state models by Lindemann and Van Driessche. A recent observation by Hoshiko and Van Driessche that corner frequencies are lowered by increasing the apical sodium concentration cannot be accounted for by these models. We explore the possibility that sodium (S) and amiloride (A) compete for a site at the mouth of the channel. A new three-state channel model (sodium-occupied, open/unoccupied, open/amiloride-blocked) is analyzed. Its corner frequency is of the form fc = fco [1 + (A/KA)/(1 + S/KS)], consistent with the observed sodium dependence of the corner frequency. The minimum frequency, fco, and the inhibition constants, KA and KS, are expressed in terms of the rate constants of the model. To account for sodium self-inhibition, we postulate that two sodium ions in the channel may result in clogging--a fourth state. The two corner frequencies are calculated; so are the plateau values of the noise power. The noise power shows a maximum as a function of blocker concentration, as observed previously using triamterene. The four-state model predicts the observed suppression by small amounts of blocker of the low-frequency sodium (clogging) noise.

5-Fluorouracil derivatives. III. A simple method to prepare 5-fluoro-2'-deoxyuridine derivatives.

3',5'-Diacyl-2'-bromo-5-fluoro-2'-deoxyuridine (4) was obtained by the reaction of 5, 6-dihydro-6-hydroxy-5-fluorouridine (2) and acyl bromide. Because the route from uridine (1) to 2, the route from 4 to 3',5'-diacyl-5-fluoro-2'-deoxyuridine (5), and the route from 5 to 5-fluoro-2'-deoxyuridine (FUDR, 6) are known reactions, the three step synthesis from uridine to 5 and four step synthesis from uridine to FUDR have been accomplished.

Power density spectra of frog skin potential, current and admittance functions during patch clamp.

Clamp current fluctuations in frog skin of areas down to 0.07 cm2 are dominated by low frequency components (less than 100 Hz). Patch clamp of 0.001 cm2 under high density fluorosilicone oil exhibits components up to 5000 Hz, often including a peak in the current power density spectrum. The admittance spectrum also exhibits a peak at the same frequency. In some skins no peak was observed, but the break in the curve was too sharp to be Lorentzian. In all instances the final limiting slope approached 1/f2. The resonance peak was observed in either Cl- or SO = 4 Ringer's but disappeared when Na+ was replaced with K+. Resonance frequency varied from 100 to 700 Hz.