Synthesis and angiotension converting enzyme inhibitory activity of L-lysyl-N-substituted glycine derivatives

The synthesis and biological activity of novel L-lysyl-N-substituted glycine derivatives which exhibit in vitro and in vivo angiotensin converting enzyme (ACE) inhibition, are described. Particularly, N-[N alpha-(1-carboxy-3-phenylpropyl)-L-lysyl]-N-(4-phenylcyclohexyl)glycine (11e), N-[N alpha-(1-carboxy-3-cyclopentylpropyl)-N epsilon-carbobenzoxy-L- lysyl]-N-cyclopentyl glycine (9h) and N-[N alpha-[1(S)-carboxy-3-cyclohexylpropyl]-L-lysyl]-N-cyclopentyl glycine (19a) showed a strong inhibitory activity at IC50 as low as 0.65, 0.64 and 0.11 nmol/l, respectively. The most potent activity in vivo was observed with the compound 19a after i.v. administration in the rat.     

Pharmacological properties of the new non-sulfhydryl angiotensin converting enzyme inhibitor N-[8-amino-1(S)-carboxyoctyl]-L-alanyl-L-proline

AB-47 (N-[8-amino-1(S)-carboxyoctyl]-L-alanyl-L-proline) is a non-sulfhydryl angiotensin converting enzyme (ACE) inhibitor with omega-aminoalkyl group. AB-47 was slightly more potent than enalaprilat in inhibiting rabbit lung ACE. The ACE inhibition and bradykinin (BK) potentiation by AB-47 in guinea-pig ileal longitudinal muscle were as potent as with enalaprilat. In conscious rat, AB-47 (i.v.) inhibited angiotensin I (A-I)-induced pressor response and augmented BK-induced depressor response more potently and in a long-lasting manner than enalaprilat. Furthermore, AB-47 exhibited higher selectivity for ACE inhibition than for BK inactivation and higher selectivity than enalaprilat and captopril. The inhibition of A-I-induced pressor response by AB-47 (p.o.) was as potent as that of enalapril. These results suggest that AB-47 is a highly potent, long-lasting and relatively A-I-selective ACE inhibitor.     

Studies on angiotensin converting enzyme inhibitors. 4. Synthesis and angiotensin converting enzyme inhibitory activities of 3-acyl-1-alkyl-2-oxoimidazolidine-4-carboxylic acid derivatives

(4S)-1-Alkyl-3-[[N-(carboxyalkyl)amino]acyl]-2-oxoimidazolidine-4- carboxylic acid derivatives (3) were prepared by two methods. Their angiotensin converting enzyme (ACE) inhibitory activities and antihypertensive effects were evaluated, and the structure-activity relationships were discussed. The dicarboxylic acids 3a-n possessing S,S,S configuration showed potent in vitro ACE inhibitory activities with IC50 values of 1.1 X 10(-8)-1.5 X 10(-9) M. The most potent compound in this series, monoester 3p, had an ID50 value of 0.24 mg/kg, po for inhibition of angiotensin I induced pressor response in normotensive rats and produced a dose-dependent decrease in systolic blood pressure of spontaneously hypertensive rats (SHRs) at doses of 1-10 mg/kg, po.     

Inhibitory effect of reactive oxygen species on angiotensin I-converting enzyme (kininase II)

1. Somatic angiotensin I-converting enzyme (ACE) is a protein that contains two similar domains (N- and C-terminal), each possessing an active site. We have examined the effects of a generator of hydroxyl radicals (g*OH: 2,2'-azo-bis(2-amidinopropane)) and hydrogen peroxide (H2O2) on ACE using an in vitro approach. 2. The generator of hydroxyl radicals inactivated ACE in a time (2-6 h)- and concentration (0.3-3 mmol/L)-dependent manner at 37 degrees C. When ACE was coincubated for 4 h with g*OH (3 mmol/L), its activity decreased by 70%. Addition of dimethylthiourea or mannitol + methionine, two *OH scavengers, resulted in a significant protection of ACE activity. Mercaptoethanol and dithiotreitol, two thiol-reducing agents, also efficiently protected ACE activity. 3. The hydrolysis of two natural and domain-specific substrates was explored. The hydrolysis of angiotensin I, preferentially cleaved by the C-domain, was significantly inhibited (57-58%) after 4 h exposure to g*OH (0.3-1 mmol/L). Under the same conditions of exposure, the hydrolysis of N-acetyl-Ser-Asp-Lys-Pro, a specific substrate for the N-domain, was only slightly inhibited by 1 mmol/L g*OH. 4. Hydrogen peroxide, another source of *OH, was used. After exposure to H2O2 (3 mmol/L; 4 h), an 89% decrease in ACE activity was observed. Pretreatment with the iron chelator deferoxamine (1 mmol/L) attenuated H2O2-mediated ACE inactivation, demonstrating that the effect of H2O2 was partly due to its conversion into *OH (Fenton reaction). 5. In summary, our findings demonstrate that g*OH and H2O2 inhibit ACE activity and suggest a preferential action of g*OH on the C-domain of the enzyme.     

Mechanism-based inactivation of cytochrome P450 2B6 by a novel terminal acetylene inhibitor.

N-(3,5-Dichloro-4-pyridyl)-3-(cyclopentyloxy)-4-methoxybenzamide (DCMB) is a known marker substrate for cytochrome p450 2B6. Based on the chemical template of DCMB, a novel terminal acetylene compound, N-(3,5-dichloro-4-pyridyl)-4-methoxy-3-(prop-2-ynyloxy)benzamide (TA) was synthesized and evaluated as a mechanism-based inactivator of p450 2B6. The pseudo first-order inactivation of expressed p450 2B6 by TA was both substrate and time-dependent. The kinetics of inhibition resulted in a maximal rate constant (k(inactivation)) of 0.09 min(-1) and an apparent K(I) of 5.1 microM. Incubation of expressed p450 2B6 with TA and NADPH resulted in a 68% loss in enzyme activity and a concurrent 62% loss in the formation of a reduced carbon monoxide complex, suggesting that heme destruction is the primary mode of enzyme inactivation. Enzyme inactivation of p450 2B6 was not reduced by the presence of 10 mM glutathione and was protected by incubation of excess DCMB with TA. The production of the carboxylic acid metabolite, N-(3,5-Dichloro-4-pyridyl)-3-(2-carboxyethoxy)-4-methoxybenzamide (TA-COOH), during the incubation of TA with 2B6 suggests that inactivation proceeds through a ketene intermediate. For 2B6 inactivation, the partition ratio was approximately 1.5 nmol TA-COOH formed/nmol P450 inactivated. Finally, TA was evaluated for mechanism-based inactivation of p450 3A4, 2C9, 2C19, 2D6, and 2E1 using human liver microsomes. In addition to 2B6, p450 2C forms were also found to be sensitive to TA-mediated inactivation, suggesting that subtle changes in the O-alkyl chain of the parent may be critical for the selectivity of enzyme inactivation.     

N-(顺式-4-异丙基环己基-1-甲酰基)-D-苯丙氨酸和N-(反式-4-异丙基环己基-1-甲酰基)-L-苯丙氨酸的合成

目的合成N-(顺式-4-异丙基环己基-1-甲酰基)-D-苯丙氨酸和N-(反式-4-异丙基环己基-1-甲酰基)-L-苯丙氨酸.方法以(4-异丙基)环己基甲酸为原料,在二环己基碳二亚胺(DCC)作用下,与N-羟基琥珀酰亚胺反应得到(4-异丙基)环己基甲酸琥珀酰亚胺酯(3),柱色谱分离化合物3得到顺式和反式异构体.顺式体与D-苯丙氨酸甲酯发生酰化反应,碱水解后即得到N-(顺式-4-异丙基环己基-1-甲酰)-D-苯丙氨酸;而反式体与L-苯丙氨酸甲酯发生酰化反应,水解后得到N-(反式-4-异丙基环己基-1-甲酰)-L-苯丙氨酸.结果与讨论成功合成了目标化合物,反应总收率分别为39%和31%.     

Acute and chronic effects of a novel dihydrobenzofuran analogue and enalapril on blood pressure and plasma and tissue angiotensin converting enzyme activity in the sodium deficient normotensive rat

1. Changes occurring in plasma and tissue angiotensin converting enzyme (ACE) activity have been examined in relation to blood pressure response following acute and chronic administration of N-[N-[[4-(2,3-dihydro-2-benzofuranyl)-1- (ethoxycarbonyl)]-butyl]-(s)-alanyl]-(s)-proline (BRL 36378) and enalapril in the sodium deficient normotensive rat. 2. Both BRL 36378 and enalapril produced a reduction in blood pressure which was evident at 2 and 24 h after acute administration, or at 24 h after chronic (21 days) administration. This was accompanied by inhibition of ACE activity in both plasma and tissues. 3. The magnitude of ACE inhibition was greater following enalapril administration than achieved after BRL 36378 treatment; this was reflected by the greater fall in blood pressure evoked by enalapril. 4. Removal of the respective ACE inhibitors revealed an apparent increase in total enzyme in the plasma of animals dosed chronically with BRL 36378 and enalapril. The onset of this increase in total enzyme was rapid, as it was apparent in plasma at 24 h after a single oral dose of BRL 36378 and enalapril. 5. The increase in total enzyme in plasma may be related to the degree of ACE inhibition, since the increase in total enzyme was of greater magnitude after 21 days treatment with enalapril than following corresponding dosing with BRL 36378. 6. No consistent effects on total enzyme were observed in tissues following acute and chronic administration with the ACE inhibitors. 7. Stimulation of drinking behaviour was observed throughout the periods of chronic (7 and 21 days) administration with both BRL 36378 and enalapril.(ABSTRACT TRUNCATED AT 250 WORDS)     

CGS 35601, a triple inhibitor of angiotensin converting enzyme, neutral endopeptidase and endothelin converting enzyme

CGS 35601 (L-tryptophan, N-[[1-[[(2S)-2-mercapto-4-methyl-1-oxopentyl]amino]-cyclopentyl]carbonyl]) is one of a few single molecules capable of inhibiting the activities of angiotensin-converting enzyme (ACE), neutral endopeptidase (NEP) and endothelin converting enzyme (ECE) simultaneously, with IC(50) values of 22, 2, and 55 nM, respectively. Through the inhibition of ACE and ECE, it blocks the conversion of angiotensin I (AI) and big endothelin-1 (big ET-1) into the two most potent peptidic vasoconstrictors, angiotensin II (AII) and ET-1, respectively. By inhibiting NEP, CGS 35601 also prevents the degradation of peptidic vasodilators such as bradykinin (BK), natriuretic peptides (NPs) and adrenomedullin (ADM) and, hence, modulates the secondary release of other vasoactive mediators such as nitric oxide (NO) and prostaglandins. In chronic (30 days) experiments, CGS 35601 is well tolerated with a very good safety profile in healthy normotensive, hypertensive and type 2 diabetic rats. The antihypertensive efficacy of CGS 35601 was demonstrated in chronically instrumented, unrestrained and conscious rat models of hypertension (SHR and DSS) and type 2 diabetes (ZDF-fatty). It lowered blood pressure effectively as well as modulated plasma concentrations of a number of circulating vasoactive peptidic mediators that are keys to the regulation of the vascular tone. These data suggest that CGS 35601, a triple vasopeptidase inhibitor (VPI), may represent a novel class of antihypertensive drugs and may have the potential to reduce morbidity and mortality from cardiovascular disorders, diabetes and subsequent renal complications. Similar in vivo ACE, NEP, and ECE inhibitory activities were also observed with the orally active prodrug, CGS 37808 (L-tryptophan, N-[[1-[[(2S)-2-(acetylthio)-4-methyl-1-oxopentyl]amino]cyclopentyl]-carbonyl]-, methyl ester.     

O-acetylsalicylhydroxamic acid, a novel acetylating inhibitor of prostaglandin H2 synthase: structural and functional characterization of enzyme-inhibitor interactions.

Aspirin is unique among clinically used nonsteroidal antiinflammatory drugs in that it irreversibly inactivates prostaglandin (PG) H2 synthase (PGHS) via acetylation of an active-site serine residue. We report the synthesis and characterization of a novel acetylating agent, O-acetylsalicylhydroxamic acid (AcSHA), which inhibits PGE2 synthesis in vivo and blocks the cyclooxygenase activity of PGHS in vitro. AcSHA requires the presence of the active-site residue Ser-529 to be active against human PGHS-1; the S529A mutant is resistant to inactivation by the inhibitor. Analysis of PGHS inactivation by AcSHA, coupled with the X-ray crystal structure of the complex of ovine PGHS-1 with AcSHA, confirms that the inhibitor elicits its effects via acetylation of Ser-529 in the cyclooxygenase active site. The crystal structure reveals an intact inhibitor molecule bound in the enzyme's cyclooxygenase active-site channel, hydrogen bonding with Arg-119 of the enzyme. The structure-activity profile of AcSHA can be rationalized in terms of the crystal structure of the enzyme-ligand complex. AcSHA may prove useful as a lead compound to facilitate the development of new acetylating inhibitors.     

Synthesis and angiotensin converting enzyme inhibitory activity of N-carboxymethyldipeptides with an omega-aminoalkyl group

A series of novel L-alanyl- and L-lysyl-L-proline derivatives having an omega-amino-1-carboxyalkyl group was prepared, and assayed for their inhibitory activity against angiotensin converting enzyme (ACE). The dicarboxylic acids possesing S,S,S configuration showed potent in vitro ACE inhibitory activity with IC50 values of 0.68-1.4 nmol/l. The length of the carbon chain in the omega-aminoalkyl moiety was varied from 6 to 9 to investigate the optimal structure for long-acting ACE inhibitors. The most prolonged activity in vivo was observed with N-[8-amino-1(s)-carboxyoctyl]-L-alanyl-L-proline upon i.v. and p.o.     

Synthesis and angiotensin converting enzyme inhibitory activity of N-carboxymethyldipeptides with omega-(4-piperidyl)alkyl group

The synthesis of a series of novel, potent angiotensin converting enzyme (ACE) inhibitors containing 1(S)-carboxy-omega-(4-piperidyl)alkyl group at the N-terminal of the dipeptide is described. These 1-carboxy-omega-(4-piperidyl)alkyl derivatives possess greater or equivalent in vitro potency and in vivo efficacy than captopril and enalapril. The length (n) of the carbon chain in the omega-(4-piperidyl)alkyl moiety was varied from two to six to investigate the optimal structure for long-acting ACE inhibitors. 1-[N-[1(S)-Carboxy-6-(4- piperidyl)hexyl]-L-alanyl]-(2a,3a beta, 7a beta)-octahydro- 1H-indole-2-carboxylic acid (9b), the most potent member of the series, had an in vivo area under the curve (AUC) of 685, which was calculated by the inhibition of angiotensin I-induced pressor response vs. time curves (0 to 8 h) after p.o. administration.     

N-4-Substituted 1-(2-arylethyl)-4-piperidinyl-N-phenylpropanamides, a novel series of extremely potent analgesics with unusually high safety margin.

The intravenous analgesic activity and toxicity of a novel series of N-[4-substituted 1-(2-arylethyl)-4-piperidinyl]-N-phenylpropanamides was studied in rats. Onset, potency and duration of analgesic action were assessed in the tail withdrawal test and compared with the activity of fentanyl, (+)-cis-3-methylfentanyl (R 26 800), morphine, and pethidine. All compounds studied were found to be extremely potent analgesics characterized by an unusually high safety margin. Methyl 4-[N-(1-oxopropyl)-N-phenyl-amino]-1-(2-phenylethyl)-4-piperidinecarboxylate (R 31 833; lowest ED50 = 0.00032 mg/kg) is the most potent compound (10 031 times morphine). cis-Methyl 3-methyl-4-[N-(1-oxopropyl)-N-phenylamino]-1-(2-phenylethyl)-4-piperidine carboxylate (R 32 792) is the longest acting compound (more than 8 h at 4 times the lowest ED50) and N-[4-(1-oxopropyl)-1-[2-(2-thienyl)ethyl]-4-piperidinyl]-N-phenylpropanamide (R 33 352) is the shortest acting compound (0.74 h at 4 times the lowest ED50) of the 4-substituted fentanyl derivatives. N-[4-(Methoxymethyl)-1-[2-(2-thienyl)ethyl]-4-piperidinyl]-N-phenylpropanamide (R 30 730) was selected for further investigation. R 30 730 has a rapid onset of action and is 4521 times more potent than morphine at the time of peak effect; it has a relatively short duration of action comparable to that of fentanyl and its safety margin (LD50/lowest ED50 = 25 211) is unusually high.     

Angiotensin converting enzyme inhibitors. (Mercaptoaroyl)amino acids

A series of (mercaptoaroyl)amino acids and related compounds was synthesized and tested for ability to inhibit angiotensin converting enzyme (ACE). The most active compound was N-(3-chloro-2-mercaptobenzoyl)-N-cyclopentylglycine, having an in vitro I50 = 0.28 microM. Substitution of the aromatic 3-position by small polar groups enhanced ACE inhibitory activity, whereas bulky groups diminished it. Alteration of the beta relationship between the mercaptan and amide carbonyl or masking of the thiol by acylation reduced activity. Replacement of the thiol by nitro, hydroxy, or carboxy gave compounds lacking ACE inhibitory activity.     

Antihypertensive activity during inhibition of neutral endopeptidase and angiotensin converting enzyme.

The specific neutral endopeptidase (NEP) inhibitor, SQ 29,072 (7-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid), was studied in conscious spontaneously hypertensive rats (SHRs) and in DOCA/salt hypertensive rats during inhibition of angiotensin-converting enzyme (ACE) activity with captopril or SQ 27,519 (the free acid of fosinopril). In the SHR, the maximal depressor responses to the combination of SQ 29,072 and SQ 27,519 (-44 +/- 4 mm Hg) were greater than the responses to any of the inhibitors given alone (-26 +/- 5, -40 +/- 10, and -28 +/- 6 mm Hg for SQ 29,072, captopril, and SQ 27,519, respectively). In contrast, the maximal antihypertensive activities of SQ 29,072 were the same in conscious DOCA/salt hypertensive rats infused with saline, captopril, or SQ 27,519 (-54 +/- 10, -51 +/- 8, and -58 +/- 11 mm Hg, respectively), indicating a lack of synergism in this model. In agreement, SQ 28,133 [N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-L-leucine], a compound that inhibits both NEP and ACE, elicited significant depressor activities in both SHR and DOCA/salt hypertensive rats. In conclusion, a selective NEP inhibitor enhanced the depressor activity of ACE inhibitors in the conscious SHR, indicating that these agents may be effectively combined for treatment of some types of hypertension.     

Humoral and blood pressure effects of the angiotensin converting enzyme inhibitor ramipril in essential hypertension

The humoral and antihypertensive activities of the angiotensin converting enzyme (ACE) inhibitor 2-[N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl]-(1S, 3S, 5S)-2-azabicyclo[3.3.0] octane-3-carboxylic acid (ramipril, Hoe 498) were investigated in 10 patients with essential hypertension (WHO stage I or II). After a 7-day placebo period, the patients were treated with 5 mg ramipril orally once daily for 14 days. Peak serum concentrations of the active metabolite M1 (dicarboxylic acid) of 5.4-62.0 ng/ml were observed 2-6 h after the first oral dose. The maximum ACE inhibition of 95% was reached 2-4 h after the first oral dose, inhibition exceeded 70% 24 h after dosing. The maximum drop in the systolic and diastolic blood pressure (random zero sphygmomanometer) was measured 4 h after ramipril (p less than 0.02, p less than 0.01), but blood pressure on days 7 and 14 of the treatment period was not different from pretreatment values. Automatically recorded blood pressure results showed a marked reduction of both systolic and diastolic blood pressure during treatment compared to placebo. No side effects occurred. From the present data it is concluded that ramipril is a potent ACE inhibitor in hypertensive patients and that further controlled studies are required for the evaluation of the antihypertensive effect of 5 mg ramipril in essential hypertension.     

Studies of metabolism and disposition of potent human immunodeficiency virus (HIV) integrase inhibitors using 19F-NMR spectroscopy

(19)F-nuclear magnetic resonance (NMR) has been extensively used in a drug-discovery programme to support the selection of candidates for further development. Data on an early lead compound, N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(4-methylmorpholin-3-yl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide (compound A (+)), and MK-0518 (N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(1-methyl-1-{[(5-methyl-1,3,4-oxadiazol-2-yl)carbonyl]amino}ethyl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide), a potent inhibitor of this series currently in phase III clinical trials, are described. The metabolic fate and excretion balance of compound A (+) and MK-0518 were investigated in rats and dogs following intravenous and oral dosing using a combination of (19)F-NMR-monitored enzyme hydrolysis and solid-phase extraction chromatography and NMR spectroscopy (SPEC-NMR). Dosing with the (3)H-labelled compound A (+) enabled the comparison of standard radiochemical analysis with (19)F-NMR spectroscopy to obtain quantitative metabolism and excretion data. Both compounds were eliminated mainly by metabolism. The major metabolite identified in rat urine and bile and in dog urine was the 5-O-glucuronide.     

Chronic oral toxicity of N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-L-phenylalanine 1-methyl ester, monohydrate (advantame) in the dog

Advantame (N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-L-phenylalanine 1-methyl ester, monohydrate), an N-substituted analog of aspartame, has been developed as a high-intensity sweetener. Groups of 4 dogs of each sex were treated at 0, 2000, 10,000, or 50,000 ppm of advantame in the diet for 52 weeks. Additional groups of 2 dogs/sex at the control, and mid- and high-dose groups were treated for 52 weeks followed by a 6-week recovery period. There was no effect of treatment on mortality, body weight, organ weights, food consumption, or the results of ophthalmological, electrocardiographic, haematological, clinical chemistry or urinalysis examinations. No histopathological changes were associated with advantame treatment. The NOAEL was considered to be 50,000 ppm, the highest concentration tested, which was equivalent to 2057 and 2139 mg/kg body weight/day in males, and females, respectively. The results of the study support the safety of advantame for use as a high-intensity sweetener.     

Angiotensin converting enzyme inhibitors: N-substituted monocyclic and bicyclic amino acid derivatives

The synthesis of N-(3-mercaptopropionyl)-N-arylglycines (14a-x),- N-arylalanines (15a,b),-N-cycloalkylglycines (16a-k), and -1,2,3,4-tetrahydroisoquinoline-3-carboxylic acids (17a-d), -1,2,3,4-tetrahydroquinoline-2-carboxylic acids (18a-f), and -indoline-2-carboxylic acids (19a-k) is described. In vitro inhibition of angiotensin converting enzyme (ACE) is reported for each compound, and the structure--activity relationship for each series is discussed. The in vivo inhibition of ACE and antihypertensive effects of representative compounds from each series are discussed. The most potent compound, 19d, had an in vitro ACE IC50 of 2.6 X 10(-9) M and lowered blood pressure in spontaneous hypertensive rats 85 mm at a dose of 10 mg/kg po.     

Different in vivo functions of the two catalytic domains of angiotensin-converting enzyme (ACE)

Angiotensin-converting enzyme (ACE) can cleave angiotensin I, bradykinin, neurotensin and many other peptide substrates in vitro. In part, this is due to the structure of ACE, a protein composed of two independent catalytic domains. Until very recently, little was known regarding the specific in vivo role of each ACE domain, and they were commonly regarded as equivalent. This is not true, as shown by mouse models with a genetic inactivation of either the ACE N- or C-domain. In vivo, most angiotensin II is produced by the ACE C-domain. Some peptides, such as the anti-fibrotic peptide AcSDKP, are substrates only of the ACE N-domain. Knowing the in vivo role of each ACE domain has great significance for developing ACE domain-specific inhibitors and for understanding the full effects of the anti-ACE pharmaceuticals in widespread clinical use.     

A new class of conformationally rigid analogues of 4-amino-5-halopentanoic acids, potent inactivators of gamma-aminobutyric acid aminotransferase

Recently, we found (Qiu, J.; Pingsterhaus, J. M.; Silverman, R. B. J. Med. Chem. 1999, 42, 4725-4728) that conformationally rigid analogues of the GABA aminotransferase (GABA-AT) inactivator vigabatrin were not inactivators of GABA-AT. To determine if this is a general phenomenon of GABA-AT inactivators, several mono- and di-halogen-substituted conformationally rigid analogues (7-15) of other GABA-AT inactivators, 4-amino-5-halopentanoic acids, were synthesized as potential inactivators of GABA-AT. Four of them, (+)-7, (-)-9, (+)-10, and (+)-15, were inactivators, although not as potent as the corresponding open-chain analogues. The maximal inactivation rate constants, k(inact), for the fluoro- and bromo-substituted analogues were comparable, indicating that cleavage of the C-X bond is not rate determining. Consistent with that observation is the finding that [3-(2)H]-10 exhibits a deuterium isotope effect on inactivation of 3.3, suggesting that C-H bond cleavage is the rate-determining step. The rate of inactivation of GABA-AT by the fluorinated analogue 7 is 1/15 that of inactivation by the corresponding open-chain analogue, 4-amino-5-fluoropentanoic acid (3a). Whereas inactivation by 3a releases only one fluoride ion, inactivation by 7 releases 148 fluoride ions, accounting for the less efficient inactivation rate. Inactivation leads to covalent attachment of 2 equiv of inactivator after gel filtration; upon urea denaturation, 1 equiv of radioactivity remains bound to the enzyme. This suggests that, unlike the open-chain anlogue, the conformationally rigid analogue becomes, at least partially, attached to an active-site residue. It appears that the conformational constraint has a larger effect on inactivators that inactivate by a Michael addition mechanism than by an enamine mechanism.