2-(4-Amino-4-carboxybutyl)aziridine-2-carboxylic acid. A potent irreversible inhibitor of diaminopimelic acid epimerase. Spontaneous formation from alpha-(halomethyl)diaminopimelic acids.

2-(4-Amino-4-carboxybutyl)aziridine-2-carboxylic acid (3) (aziridino-DAP) was identified as the product of spontaneous hydrolysis of alpha-(halomethyl)diaminopimelic acids (alpha-halomethyl-DAPs) 2a-c. Under physiological conditions, 3 is an extremely potent irreversible inhibitor of the bacterial enzyme diaminopimelic acid epimerase (DAP-epimerase; EC 5.1.1.7). This unusual mode of action of an alpha-halomethyl amino acid with a non-pyridoxal enzyme is investigated. Synthesis and characterization of 2a-c and 3, kinetics of spontaneous formation of 3 from alpha-halomethyl-DAPs, and kinetics of enzyme inhibition by both 3 and by alpha-halomethyl-DAPs are reported.

Ornithine aminotransferase activity, liver ornithine concentration and acute ammonia intoxication.

5-Fluoromethylornithine (5FMOrn) is a specific inactivator of L-ornithine:2-oxoacid aminotransferase (OAT). Inactivation of OAT causes the enhancement of L-ornithine (Orn) concentrations in all tissues. Intraperitoneal or oral administration of 10-50 mg/kg of 5FMOrn per day to albino mice rendered partial protection against lethal intoxication with 26 mmol/kg of ammonium acetate. The protective effect was maximal around 16 h after 5FMOrn administration, at the time when endogenous Orn concentrations were maximal. At this time protection by 5FMOrn against acute ammonia intoxication was comparable to that observed 1 h after the intraperitoneal administration of 10 mmol/kg of L-arginine. Pretreatment with 5FMOrn prevented the enhancement of excessive urinary excretion of orotic acid by ammonia intoxicated mice, and it enhanced urea formation in the liver. These biochemical effects demonstrate that 5FMOrn shifts Orn into the urea cycle, Orn which normally would be transaminated. Since even long-term treatment of mice with 5FMOrn did not reveal toxic effects, this compound may be considered for the treatment of certain conditional deficiencies of Orn or arginine.

Activation of rat liver S-adenosylmethionine decarboxylase by putrescine and 2-substituted 1,4-butanediamines.

1. The activation of S-adenosyl-L-methionine decarboxylase (SAM-DC) by putrescine and a series of 1,4-butanediamines with a substituent in position 2 was studied. 2. Kinetic data show the activation of SAM-DC by putrescine is essentially uncompetitive. 3. All 2-substituted 1,4-butanediamines were activators, although not as potent as putrescine itself. 4. At high concentrations of SAM activation of SAM-DC by putrescine and putrescine analogs deviated considerably from uncompetitive activation kinetics. 5. In order to explain the experimental data, especially the non-linearity of the "fractional velocity plots", it was necessary to postulate two independent, but equivalent activator binding sites, for which substrate (SAM) and activator compete. 6. Based on this kinetic model an equation was derived which describes the rate of SAM decarboxylation as a function of substrate and activator concentrations. 7. From the simulated curves, approximate values for equilibrium constants for the binding of activator and substrate to the activator binding sites, and relative rate constants for the product forming steps were calculated. 8. Even a minor change of the structure, such as the substitution of one hydrogen atom by fluorine in the 2-position of putrescine had a very considerable effect on the potency of activation. 9. It is apparent that the structural requirements of an activator of SAM-DC are highly specific.

Fluorinated analogues of spermidine as substrates of spermine synthase.

A series of mono- and geminal difluorinated analogues of spermidine (4-azaoctane-1,8-diamine) have been tested as potential substrates of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase (EC 2.5.1.22). Substitution of the hydrogen atoms of the methylene group at position 7 by one or two fluorine atoms decreases 8-fold and 160-fold the apparent Km values for the HTC cell enzyme respectively. Similarly, the Km values of 7-monofluoro and 7,7-difluorospermidine for the pure bovine enzyme are reduced 8-fold and 100-fold respectively, in comparison with spermidine. Di-, but not monofluoro substitution, in the 6-position causes a 5-fold reduction in the affinity for the HTC cell enzyme. Gem-fluorine substitution in the 2-position abolishes substrate capability. In addition to their high affinity for spermine synthase, 7-monofluorospermidine and 7,7-difluorospermidine cause substrate inhibition. This phenomenon, which is more pronounced in the case of the difluorinated analogues is pH-dependent. These enzymatic findings are discussed with regard to the protonation sites of the spermidine analogues, determined by potentiometric titration, which vary as a function of the number and position of the fluorine substituents relative to the basic amino groups.

5-Fluoromethylornithine, an irreversible and specific inhibitor of L-ornithine:2-oxo-acid aminotransferase.

5-Fluoromethylornithine (5-FMOrn) is the first specific irreversible inhibitor of L-ornithine:2-oxoacid aminotransferase (OAT) found. Single doses (greater than 10 mg/kg) of this compound inactivate OAT to a residual OAT-like activity. This activity (10-20% of total activity) is resistant to further inactivation by higher or repeated doses of 5-FMOrn, or incubation with the inactivator in vitro. Ornithine concentrations are greatly enhanced in various tissues, and urinary ornithine is dramatically increased, but no other amino acid is affected after acute treatment with 5-FMOrn. Repeated administration decreases carnosine and homocarnosine concentrations in brain. Toxic effects were not observed. The new inactivator is considered as a tool in the establishment of functions of OAT under physiological and pathological conditions.

Synthesis and biological properties of alpha-mono- and alpha-difluoromethyl derivatives of tryptophan and 5-hydroxytryptophan.

The syntheses of alpha-mono- and alpha-difluoromethyl derivatives of tryptophan and 5-hydroxytryptophan are described. In an attempt to selectively regulate serotonin synthesis, alpha-(mono- and difluoromethyl)tryptophan were tested in vivo as precursors (or prodrugs) of their 5-hydroxy analogues. Although alpha-(mono- and difluoromethyl)-5-hydroxytryptophans are potent irreversible inhibitors of aromatic amino acid decarboxylase (equipotent to alpha-difluoromethyl-Dopa), only alpha-(monofluoromethyl)tryptophan affects the level of serotonin in vivo (small decrease), alpha-(difluoromethyl)tryptophan being a very poor substrate of the activating (or helper) enzyme, tryptophan hydroxylase.

Fluorine-containing polyamines: biochemistry and potential applications.

Investigations with the fluorinated spermidine analogues show clearly that these compounds have significant potential for studying the metabolism and functions of the polyamines. However, the biochemical and biological properties of these analogues are dissimilar. This is due to the influence of the fluorine substituent(s) on the basicity of the amine function proximal to the fluoromethylene group, this effect being amplified by geminal disubstitution. The monofluorinated spermidine analogues compare well with the natural amine in their ability to regulate the expression of the decarboxylase enzymes, to be substrates of spermine synthase and to support growth of polyamine-deficient cells. It is also likely that 6-monofluorospermine, formed biochemically in situ, shares with spermine similar functions. These findings raise the possibility of using these spermidine analogues to study the metabolism and pharmacology of polyamines in vivo but also to provide more insight into the regulatory role of spermidine in ODC and SAM-DC expression. Another potential application may be the use of these analogues as probes in tumor imaging and therapy control. This indication has been inferred by studies in tumor-bearing animals, using 19F-NMR spectroscopy determination of tissue fluorospermidine and fluorospermine, formed biochemically from the precursors 2-fluoro or 2,2-difluoroputrescine, and which demonstrate preferential accumulation in tumor versus normal tissue. Finally, these monofluorinated spermidine analogues may exert beneficial effects in pathological states associated with polyamine deficiency. These diseases remain however to be identified. Among the difluorinated spermidine analogues, 7,7-difluorospermidine possesses the most interesting properties. This spermidine analogue still possesses ODC and SAM-DC repressing activities although at much higher concentration than spermidine. More importantly it is a potent inhibitor of spermine synthesis both in cultured cells and in vivo due to its efficient competition with spermidine in the spermine synthase reaction. This compound not only depletes tumor cell of its spermine content but, in addition, appears to exert by itself and/or via 6,6-difluorospermine, the product of its metabolism, polyamine antagonist effects. Combined with MAP but also with DFMO, two potent irreversible inhibitors of ODC which block the synthesis of the natural endogenous polyamines, 7,7-difluorospermidine causes an immediate decrease of viability in cultured HTC cells and promotes tumor regression and stabilization in hepatoma-bearing rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Chain-fluorinated polyamines as tumor markers. II. Metabolic aspects in normal tissues.

The objective of this work was to study certain metabolic aspects of fluorine-substituted analogues of natural polyamines in healthy experimental animals, with the aim of exploring their potential application as tumor markers. Tissue polyamine concentrations were more effectively depleted by combined treatment with D,L-alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, and N1,N4-bis-allenylputrescine, an inactivator of polyamine oxidase, than with either inhibitor alone. This suggests the general importance of polyamine interconversion as a metabolic source of putrescine. Administration of 2,2-difluoroputrescine after 2 weeks pretreatment with the two inhibitors caused the formation of 6,6-difluorospermidine and 6,6-difluorospermidine in nearly all tissues. Highest concentrations of the chain-fluorinated polyamines were observed in the small intestine. At 24 h after 2,2-difluoroputrescine administration the amount was about 8% of the normal endogenous polyamine pool in the small intestine, but lower in all other tissues. Replenishment of endogenous polyamine pools is a relatively slow process. Approximately 9 days after cessation of treatment with the two inhibitors normal values had been reestablished. The rate of formation of endogenous polyamines was not affected by the presence of their difluoro analogues. Elimination of the chain-fluorinated polyamines from tissues seems not to follow normal polyamine metabolic patterns. Their most rapid elimination coincides with the enhancement of endogenous polyamines, indicating that the fluoro analogues are displaced by the natural polyamines. Most of the 2,2-difluoroputrescine was rapidly excreted in the urine, and formation of a conjugate was detected. 6,6-Difluorospermidine was also a urinary excretion product. However, the metabolic fate of 6,6-difluorospermine could not be clarified. It was not found in urine, either free or as conjugate. The relatively low accumulation of chain-fluorinated polyamines, together with their rapid elimination from normal tissues are characteristics which together with their previously established selective uptake into rapidly proliferating tissues recommend them as potential tumor markers that can be determined by 19F-NMR spectroscopy.

Chain-fluorinated polyamines as tumor markers--I. In vivo transformation of 2,2-difluoroputrescine into 6,6-difluorospermidine and 6,6-difluorospermine.

1. Injection of 2,2-difluoroputrescine (DFPut) into the yolk sac of chick embryos causes the formation of 6,6-difluorospermidine (6,6DFSpd) and 6,6-difluorospermine (6,6DFSpm), demonstrating that the difluoroanalogs of putrescine and spermidine are in vivo substrates of spermidine and spermine synthase, respectively. 2. Depletion of tissue putrescine and spermidine concentrations by administration of D,L-alpha-difluoromethylornithine (DFMO, Ornidyl) causes a very marked enhancement of difluoropolyamine formation from DFPut. 3. The major accumulation of 6,6DFSpd and 6,6DFSpm in DFMO-pretreated rodents occurs in small intestines and tumors, i.e. in tissues with high cell proliferation rates, which are also the most susceptible to polyamine depletion by inhibition of ornithine decarboxylase. 4. Their preferential accumulation in tumors and the fact that DFPut and its metabolites seem not to exert toxic effects, suggest DFPut as a serious candidate for the use as probe in 19F-NMR imaging of tissues with a high proliferation rate and a high rate of polyamine biosynthesis.

Left or right transaxillary approach in lower extremity arteriography for severe occlusive vascular disease. An anatomic study of a roentgenologic problem.

In an anatomic study of 46 specimens, the angle of the brachiocephalic trunk and the left subclavian artery to the tangent of the aortic arch was measured in an effort to decide which route offers the quickest access to the descending aorta in lower extremity angiography for severe occlusive vascular diseases. The study shows that the average angles (77.1 +/- 16.0 degrees for the brachiocephalic trunk/77.7 +/- 16.3 degrees for the left subclavian artery) are not significantly different. Since the right axillary approach incurs other risks that could only be compensated by significantly quicker access, i.e. shorter maneuvering time in the aortic arch we conclude that in patients of all ages the left side should be the access route of choice.

Marked and prolonged inhibition of mammalian ornithine decarboxylase in vivo by esters of (E)-2-(fluoromethyl)dehydroornithine.

(E)-2-(fluoromethyl)dehydroornithine, a new enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC) is no more effective than alpha-difluoromethylornithine (DFMO) at inhibiting polyamine biosynthesis in rat hepatoma tissue culture (HTC) cells and in rat organs even though its potency is over 15 times higher than that of DFMO in vitro. The methyl, ethyl, octyl and benzyl esters of (E)-2-(fluoromethyl)dehydroornithine were synthesized as potential prodrugs of the amino acid. When tested at concentration equivalent to the Ki value of the amino acid, they are devoid of ODC-inhibitory property. When measured 6 hr after its addition to the HTC cell culture medium, the absorption of methyl ester was 20 times higher than that of the parent amino acid or that of DFMO, and was accompanied by a more marked intracellular accumulation of (E)-2-(fluoromethyl)dehydroornithine than that achieved by the addition of the parent amino acid. The methyl ester used at 10 times lower concentrations is as effective as its parent amino acid or as DFMO at inhibiting polyamine biosynthesis in HTC cells. Similarly, the methyl and the ethyl esters of (E)-2-(fluoromethyl)dehydroornithine used at 10 times lower doses are as effective as the parent amino acid and as DFMO at inhibiting ODC in the ventral prostate of rat, 6 hr after oral administration. All the esters of (E)-2-(fluoromethyl)dehydroornithine produce a particularly long duration of ODC inhibition in the ventral prostate and in the testes. Repeated administration (25 mg/kg given once a day by gavage) of the methyl ester of (E)-2-(fluoromethyl)dehydroornithine for 8 days to rats results in a constant 80% inhibition of ODC over a 24-hr period, accompanied by a 90% decrease of putrescine and spermidine concentrations in the ventral prostate.

alpha-(Fluoromethyl)dehydroornithine and alpha-(fluoromethyl)dehydroputrescine analogues as irreversible inhibitors of ornithine decarboxylase.

(E)-Dehydro analogues of alpha-(fluoromethyl)putrescine and -ornithine derivatives were synthesized and evaluated in vitro as irreversible inhibitors of a preparation of ornithine decarboxylase (ODC, EC 4.1.1.17) obtained from rat liver. The key step in the synthesis of (E)-alpha-(fluoromethyl)dehydroornithine (17) and -putrescine (14) was the addition of propenylmagnesium bromide to fluoroacetonitrile. The resulting unstable conjugated imine salt was reduced regioselectively in situ with NaBH4 or was quenched with a solution of NaCN to give the corresponding unsaturated alpha-(fluoromethyl) amine and alpha-amino nitrile, respectively. These were transformed into 17 and 14 via a four-step sequence involving (a) phthaloyation of the amine function; (b) allylic bromination of the methyl group; (c) Gabriel reaction; and (d) hydrolytic cleavage of the protective groups. (E)-alpha-(Difluoromethyl)dehydroornithine (10) and -putrescine (7) were prepared from ethyl tert-butyl 2-(difluoromethyl)-2-(2-propenyl)malonate and di-tert-butyl 2-(difluoromethyl)-2-(2-propenyl)malonate, respectively, via a sequence similar to that reported previously for the synthesis of the saturated analogues. Compounds 17, 14, 10, and 7 proved to be much more potent enzyme-activated irreversible inhibitors of ODC than the corresponding saturated analogues. The increase in potency is particularly marked in the alpha-fluoromethyl series. The apparent dissociation constants (KI) and the times of half-inactivation of enzyme (tau 50) at infinite concentration of inhibitors are 2.7 microM and 2.6 min for 17 and 42 microM and 0.2 min for 14. The KI and tau 50 of the corresponding saturated analogues are 75 microM and 1.6 min for the ornithine derivative and 56 microM and 4.4 min for the putrescine derivative.

Effect of prolonged inhibition of histidine decarboxylase on tissue histamine concentrations.

In rats, chronic infusion of alpha-fluoromethyl histidine, a selective irreversible inhibitor of mammalian histidine decarboxylase, caused a marked depletion of histamine in all tissues examined. There were no gross pharmacological effects associated with this depletion.

alpha-Fluoromethyl histidine. Inhibition of histidine decarboxylase in pylorus ligated rat.

alpha-Fluoromethyl histidine is an irreversible inhibitor of histidine decarboxylase. The injection of a single dose to pyloric-ligated rats inhibits gastric mucosal histidine decarboxylase in a dose-dependent manner but does not modify histamine content and gastric acid secretion even at the highest dose used. Administration of cimetidine increases histidine decarboxylase activity, decreases histamine level in gastric mucosa and inhibits gastric acid secretion. The co-administration of alpha-fluoromethyl histidine blocks the augmentation in enzyme activity, maintains lowered histamine level and prolongs the antisecretory action of cimetidine.

Histamine receptor blockade (H2) versus inhibition of histamine synthesis in stress ulceration in rats.

Cold and restraint stress in rats induced gastric mucosa lesions, increased gastric mucosa histidine decarboxylase activity and elevated hypothalamic content. Cimetidine did not modify the biochemical effects induced by stress but partially protected against gastric ulceration. alpha-Fluoromethyl histidine inhibited the increase of histidine decarboxylase activity in the gastric mucosa, inhibited the rise of hypothalamic histamine content and decreased the incidence of gastric lesions to the same extent as did cimetidine.

Inhibition of histamine biosynthesis and gastric function in the rat: effect on pentagastrin-induced gastric acid secretion.

A very simple and rapid method to measure gastric acid secretion in the conscious unoperated fasted rat is reported. Antisecretory activity of cimetidine and hypersecretory activity of pentagastrin are detected by this procedure. Pentagastrin induces gastric acid secretion and causes an increase by 2-fold in histidine decarboxylase activity. There is a delay between the increase of gastric acid output and the appearance of histidine decarboxylase activity. Co-administration of monofluoromethyl histidine, a specific irreversible inhibitor of histidine decarboxylase, does not modify the peak response to pentagastrin but shortens the duration of stimulation. We suggest from this data that histamine biosynthesis is needed for maintenance of elevated gastric acid secretion.

omega-Fluoromethyl analogues of omega-amino acids as irreversible inhibitors of 4-aminobutyrate:2-oxoglutarate aminotransferase.

omega-Monofluoromethyl and omega-difluoromethyl analogues of the known substrates of GABA-T, beta-alanine, gamma-aminobutyric acid, and 5-aminopentanoic acid, are time-dependent inhibitors of purified 4-aminobutyrate: 2-oxoglutarate aminotransferase (GABA-T). The inhibitory activity decreases with increasing chain length. In vitro, inhibitory activity decreases with increasing fluorine substitution of the methyl group. In vivo, beta-difluoromethyl-beta-alanine and 2,4-difluoro-3-aminobutyric acid are the most potent GABA-T inhibitors ever reported. Trifluoromethyl derivatives are devoid of GABA-T inhibitory activity in vitro or in vivo.