[Course of the anaesthesiology and intensive care unit residents in Lorraine, France]. / Parcours des anesthésistes-réanimateurs formés en Lorraine depuis 1996.

OBJECTIVE: The anaesthesiology and intensive care physician demography is becoming critical in most French regions; more than 56% of the residents trained in Lorraine are leaving the area after they graduate. METHODOLOGY: A descriptive and retrospective survey was carried out. The aims of the research were threefold: to ascertain firstly, why residents chose Lorraine in the first instance, secondly their experiences of the training and thirdly their expectations. The target group consisted of 76 former anaesthesia residents dating back to 1996. RESULTS: The response rate was 72.4%. Choosing Lorraine was based on its well-established teaching program (64%) and also on results achieved at National Medical Examination (85%). The academic medical training and working conditions were well-rated. A position was offered to 93% of the residents. Fifty-three percent stated having had previous work experience in the hospital as a decisive factor in taking up a position. By the end of the residency program, only 43.5% of anesthesia physicians remained in Lorraine; i.e., 79% were native to Lorraine and 25% born outside. Reasons given for leaving Nancy were: family (81%), more favourable climate (51%) and to go back to their region of origin (45%). CONCLUSION: Desired improvements would be: better performance of the Lorraine born students at the National Medical Examination, earlier and well-defined job offers, better communication within the team and consideration of the family situation as a whole.

Structure of the product from a novel cyclization reaction involving a C(6)-substituted uridine analog.

5,6,7,7a,9,10,14b,14c-Octahydro-4-[2,3-O-(1-methylethylidene )-beta-D- ribofuranosyl]cyclopenta-[4,5]pyrimido[5',4':3,4]pyrrolo[2,1- a]isoquinoline-1,3(2H,4H)-dione, C25H31N3O6, M(r) = 469.54, tetragonal, P4(3)2(1)2, a = 12.577 (2), b = 12.577 (2), c = 29.893 (4) A, V = 4729 (1) A3, Z = 8, Dx = 1.319 g cm-3, lambda (Cu K alpha) = 1.5418 A, mu = 7.9 cm-1, F(000) = 2000, T = 298 K, final R = 0.038, wR = 0.064 for all 1884 independent reflections and 432 variables. The crystal structure shows a syn conformation around the N(1)--C(1') single bond, consistent with other C(6)-substituted uridine analogs, and an unusual O(1') endo conformation of the ribose ring. The stereochemistries of the three newly created chiral centers are 14cR, 4aR, 7aS.

Linear free energy relationship studies of enzyme active site binding: thymidylate synthase.

The requirements for active-site binding of thymidylate synthase from three sources, Lactobacillus casei, murine leukemia L1210, and human lymphoblast (Molt/4F), were investigated by analyzing the binding of a series of 5-(p-substituted phenyl)-2'-deoxyuridylates (N1-substituted 5-aryl-2, 4-dioxopyrimidines) to the enzyme. Multiple regression analysis revealed that an increase in electron density of the heterocyclic ring and hydrophobic substituents enhance affinity. Correlations of biological results with spectral data indicated that higher electron densities at the oxygen atoms are responsible for increase in binding. These results support the presence of both a cationic binding site and a hydrophobic region. In addition, the results revealed an unusual reversal of electronic requirements for binding and catalysis. The formation of the binary complex is enhanced by electron-donating substituents, while the initial catalytic reaction, formation of the covalent ternary complex, is promoted and stabilized by electron-withdrawing substituents.

5-Quinone derivatives of 2'-deoxyuridine 5'-phosphate: inhibition and inactivation of thymidylate synthase, antitumor cell, and antiviral studies.

Both photochemical aromatic substitution and palladium (0)-catalyzed biaryl coupling reactions have been employed in the synthesis of 5-substituted 2'-deoxyuridines. The former procedure was useful in the preparation of the 3,4-dimethyl-2,5-dimethoxyphenyl derivative 12a and the 3,4,6-trimethyl-2,5-dimethoxyphenyl derivative 12b. The latter reaction was efficient in the preparation of the 2-(3-methyl-1,4-dimethoxynaphthyl) derivative 14. These compounds and their nucleotides (20a-c) were converted to the corresponding quinone nucleosides 19a-c and nucleotides 6-8 by an oxidative demethylation reaction using ceric ammonium nitrate and silver(II) oxide, respectively. The kinetics and products of the reaction of the quinone nucleosides 19a,b with methyl thioglycolate showed rapid addition to the quinone ring in the trisubstituted derivative 19a and somewhat slower redox reactions with the tetrasubstituted quinones 19b and 19c. All six nucleotides had high affinity for the title enzyme from Lactobacillus casei with Ki values ranging from 0.59 to 3.6 microM; the most effective compounds were the dimethyl quinone 6 and the naphthoquinone 8. Somewhat higher inhibitory constants were observed with the quinones against the L1210 enzyme. The dimethyl quinone nucleotide 6 showed time-dependent inactivation (kinact = 0.015 s-1) against the L. casei enzyme, a rate saturation effect, and substrate protection in accord with the kinetic expression for an active-site-directed alkylating agent. The apparent second-order rate of this reaction (2.5 X 10(4) M-1 s-1) is one-twentieth the rate (kcat.) of the normal enzymatic reaction leading to product. None of the compound exhibited sufficient activity in the antitumor cell or antiviral assays to warrant further study.

Thiol addition to quinones: model reactions for the inactivation of thymidylate synthase by 5-p-benzoquinonyl-2'-deoxyuridine 5'-phosphate.

The reaction of methyl mercaptoacetate (5) with phenyl-p-benzoquinone (6) or 5-p-benzoquinonyl-3',5'-di-O-acetyl-2'-deoxyuridine (10) resulted in the formation of the three possible adducts to the quinone rings of 6 and 10; an additional product in the reaction with 10 was the unsubstituted hydroquinone (14). Both reactions were found to be solvent dependent; in buffered aqueous acetonitrile the meta and para adducts of 10 were formed in the ratio of 2:1. In ethyl acetate the ortho adduct and the reduction product of 10 were isolated in a ratio of 2:3. The second-order rate constant for the reaction of 5 with 10 in acetonitrile was 0.53 M-1 s-1; the reaction was accelerated by the addition of water. Although the initially proposed mechanism-based enzyme inactivation cannot be excluded, the results of the model reactions support the alternative mechanism, active-site thiol addition to the quinone ring. If this is true the title compound would be classed as an affinity label, not a mechanism-based inhibitor.

Palladium (II)-catalyzed olefin-coupling reactions of kainic acid: effects of substitution on the isopropenyl group on receptor binding.

Two palladium-catalyzed carbon-carbon bond forming reactions were found to be useful for the modification of a protected amino acid derivative containing a sterically hindered isopropenyl group. Arylation of the terminal methylene group of the dimethyl ester of N-(ethoxycarbonyl)kainic acid (3) was accomplished by treatment with an aromatic amine, palladium(II) acetate, and tert-butyl nitrite. Substitution of the allylic methyl group of 3 was accomplished by conversion to the pi-(allyl)palladium complex (5) which, on subsequent treatment with the carbanions of tert-butyl acetoacetate or phenylthioacetone, gave the alkylated products. Both the (Z)- and (E)-3-nitrophenyl derivatives (8a,b) of kainic acid were active in the standard binding assay. Unexpectedly, the cis compound in the nitrophenyl series (8a), which more closely resembles the extended conjugation found in domoic acid, was found to be 20 times less potent than the trans derivative 8b. The latter had one-fifth the receptor-binding affinity of kainic acid.

5-p-benzoquinonyl-2'-deoxyuridine 5'-phosphate: a possible mechanism-based inhibitor of thymidylate synthetase.

The title compound (1), designed as a suicide inhibitor of thymidylate synthetase, can be prepared by silver(II) oxide oxidative demethylation of the corresponding dimethoxyphenyl derivative. Compound 1 shows time-dependent inactivation of thymidylate synthetase (methotrexate-resistant Lactobacillus casei) and saturation kinetics, and the inactivation is responsive to substrate protection. The inactivation is not reversible on prolonged dialysis in attempts to remove the inhibitor. The rate constant for inactivation is 0.065 s-1; the dissociation constant (Ki) was estimated to be 2 microM. The kinetics of this inactivation are compared to inactivation caused by model thiol reagents that do not have affinity for the active site of thymidylate synthetase.

5-Substituted 2'-deoxyuridines: correlation between inhibition of tumor cell growth and inhibition of thymidine kinase and thymidylate synthetase.

A large variety of 5-substituted 2'deoxyuridines (dUrds) and 2'-deoxyuridylates (dUMPs) have been evaluated for their inhibitory effects on the thymidine (dThd) kinase or thymidylate (dTMP) synthetase isolated from mouse leukemia L1210 cells. The most potent inhibitors of dThd kinase were 5-chloro-, 5-bromo- and 5-iodo-dUrd. Their Ki/Km values ranged from 0.57 to 0.82. All dUrd analogs tested showed competitive kinetics with respect to dThd. However, there was little, if any, correlation between the inhibitory effects of the compounds on L1210 cell growth and their inhibitory activities against dThd kinase (r = 0.16). The most potent inhibitors of dTMP synthetase were (in order of decreasing activity): 5-nitro-dUMP greater than 5-formyl-dUMP greater than 5-fluoro-dUMP greater than 5-oxime of 5-formyl-dUMP greater than 5-azidomethyl-dUMP greater than (E)-5-(2-bromovinyl)-dUMP. The ki/Km values for these compounds ranged from 0.001 to 0.665. All dUMP analogs tested showed competitive kinetics with respect to dUMP (if not preincubated with the enzyme at 37 degrees). There was a strong correlation (r = 0.833) between the inhibitory effects of these compounds on L1210 cell growth and their inhibitory activities against dTMP synthetase. Thus, the suppressive action of 5-substituted dUrd derivatives on tumor cell growth would involve prior conversion of the nucleoside analogs to the corresponding 5'-monophosphates followed by an inhibition of dTMP synthetase.

Role of thymidine kinase in the inhibitory activity of 5-substituted-2'-deoxyuridines on the growth of human and murine tumor cell lines.

Twenty-four 5-substituted 2'-deoxyuridines have been evaluated for their inhibitory effects on the growth of three human lymphoblast cell lines (Namalva, RAji and TK- (thymidine kinase deficient) Raji) and these inhibitory effects were compared to those for two murine leukemia cell lines (L1210/0 and L1210/BdUrd). The latter was selected from the parental L1210/0 cell line by its ability to grow at high concentrations of 5-bromo-dUrd and could also be considered as TK-. There was a close correlation between the inhibitory effects of the deoxyuridine analogs on Namalva, Raji and L1210 cells: the correlation coefficient (r) for log ID50 (median inhibitory dose) for L1210 cell growth, on the one hand, and log ID50 for Namalva or Raji cell growth, on the other hand, was 0.902 and 0.929, respectively. There was also a strong correlation (r = 0.936) between the log ID50 values for the two human lymphoblast cell lines. However, there was no significant correlation (r less than 0.40) either between the log ID50 for the TK- Raji cells and the parental TK+ Raji cells, or between the log ID50 for the TK- L1210/BdUrd cells and the parental TK+ L1210/0 cells. We may conclude therefore, that (i) the murine leukemia L1210 cell system is predictive for the growth-inhibitory effects of 5-substituted 2'-deoxyuridines on human lymphoblast cell lines, and (ii) the antitumor cell activity of the 5-substituted 2'-deoxyuridines is, to a large extent, dependent on the thymidine kinase activity of the tumor cells.

Oxime and dithiolane derivatives of 5-formyl-2'-deoxyuridine and their 5'-phosphates: antiviral effects and thymidylate synthetase inhibition.

5-Formyl-2'-deoxyuridine (2a), an effective inhibitor of herpes simplex virus type 1 or 2 (HSV-1, HSV-2) and vaccinia virus, was converted to the oxime (3a) and dithiolane (4a) derivatives. The oxime (3a) was equally as potent as the formyl compound against HSV-1, but one-fifth as active against HSV-2, 100 times less effective against vaccinia, and 25 times less toxic for the host cells. In addition, compound 3a was about 10 times less active than 2a in inhibiting thymidylate synthetase in vivo (as reflected by a differential inhibition of dThd and dUrd incorporation into host cell DNA). The dithiolane (4a) did not exert an appreciable effect on either virus multiplication or dThd or dUrd incorporation, nor was it cytotoxic. All these compounds as their 5'-phosphate derivatives were potent in vitro inhibitors of thymidylate synthetase (Lactobacillus casei). The inhibition was competitive with substrate with Ki/Km ratios of 0.05 for the formyl 2b, 0.5 for the oxime 3b, and 0.2 for the dithiolane 4b. Thus, 3b was 10 times less active than 2b as an in vitro inhibitor of thymidylate synthetase, which appears to corroborate the in vivo findings.

Synthesis of 5-[(methylthio)methyl]-2'-deoxyuridine, the corresponding sulfoxide and sulfone, and their 5'-phosphates: antiviral effects and thymidylate synthetase inhibition.

Substitution on the alpha position of thymidine with methylthio (3) and methylsulfonyl (5) groups gave antiviral agents that were specific and relatively nontoxic inhibitors of herpes simplex virus replication in cell culture. The thioether (3) was effective against both types 1 and 2 of herpes simplex virus, whereas the activity of the sulfone derivative (5) was restricted to herpes simplex virus type 1. The sulfoxide derivative 1-(2-deoxy-beta-D-ribofuranosyl)-alpha-(methylsulfinyl)thymine (4) was inactive as an antiviral agent. The 5'-phosphates of these three thymidine derivatives were relatively potent inhibitors of thymidylate synthetase (Ki values range from 7.8 to 1.9 microM). It is improbable that the inhibition of this enzyme accounts for the anti-herpes activity of compounds 3 and 5.

5-(alpha-Bromoacetyl)-2'-deoxyuridine 5'-phosphate: an affinity label for thymidylate synthetase.

5-(alpha-Bromoacetyl)-2'-deoxyuridine 5'-phosphate (1) is an active-site-directed irreversible inhibitor of thymidylate synthetase from Lactobacillus casei. Analysis of the rate of inactivation of the enzyme in the presence of substrate confirmed the intermediate formation of a reversible enzyme-inhibitor complex.

5-[(4-Methyl-1,2,3,4-tetrahydroquinoxalyl)methyl]-2'-deoxyuridine 5'-phosphate: an analogue of a proposed intermediate in thymidylate synthetase catalysis.

In a study of the sequence steps involved in the mechanism of thymidylate synthetase catalysis, 5-[(N-methyl-piperazinyl)methyl]- (5) and 5-[(4-methyl-1,2,3,4-tetrahydroquinoxalyl)methyl]-2'-deoxyuridine 5'-phosphate (6) were synthesized. Compound 6 has high affinity for the Lactobacillus casei enzyme (Ki = 0.75 microM, KI/Km - 0.23), which is 50 times stronger than that of the piperazinyl derivative 5. Compound 6, a possible multisubstrate inhibitor, is an analogue of a proposed intermediate in the reaction mechanism wherein the enzyme is eliminated from the covalent complex (enzyme--substrate--cofactor) prior to the redox reaction leading to the products 2'-deoxythymidine 5'-phosphate and 7,8-dihydrofolic acid.

5-Cyano-2'-deoxyuridine 5'-phosphate: a potent competitive inhibitor of thymidylate synthetase.

The 5'-phosphate (1) of the antiviral nucleoside 5-cyano-2'-deoxyuridine was synthesized and evaluated for inhibition of thymidylate synthetase purified from methotrexate-resistant Lactobacillus casei. Compound 1 was a potent competitive inhibitor with a K1 of 0.55 microns. Irreversible enzyme inhibition by this compound could not be detected.