Arylacetamides as peripherally restricted kappa opioid receptor agonists.

Analogues of the kappa (kappa) opioid receptor agonist, ICI 199441, were prepared. Ki values for these analogues at the cloned human kappa opioid receptor ranged from 0.058 to 25 nM. Trifluoromethylaryl derivatives were potent analgesics when administered subcutaneously in the rat and were more peripherally restricted than the parent compound, ICI 199441.

Mechanism of inhibition of human leucocyte elastase by beta-lactams. 2. Stability, reactivation kinetics, and products of beta-lactam-derived E-I complexes.

The monocyclic beta-lactams reported by Knight et al. [Knight, W. B., et al. (1992) Biochemistry 31, 8160; Chabin, R., et al. (1993) Biochemistry 32, 8970] as inhibitors of human leucocyte elastase (HLE) produce stable HLE-inhibitor complexes that slowly reactivate with half-lives ranging from less than 1 to 15 h at 37 degrees C. The complexes produced between PPE and two C-3 dimethyl-substituted beta-lactams are less stable than those produced between HLE and analogous C-3 diethyl-substituted lactams. The stability of the HLE-I complexes is governed primarily by the structure of the substituted urea portion of the inhibitors and not by the identity or presence of a leaving group at C-4 of the lactam ring. In some cases substitutions on the urea portion of the inhibitors yielded complexes that displayed biphasic reactivation kinetics. This suggests the presence of at least two different complexes. The stereochemistry of the leaving group at C-4 has a small effect on the stability of the final complex (1.3-2-fold); therefore, the identity of the final complex is dependent upon the initial stereochemistry at that position. The stability of the complexes was relatively insensitive to hydroxylamine, which suggests that the acyl-enzymes are protected from nucleophilic "rescue". The rate of reactivation of the complex derived from L-680,833,[S-R*,S*)]-4-[(1-(((1-(4- methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-2-oxo-4-azetidinyl)ben zeneacetic acid, was pH independent, while the L-684,481, (R)-(1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-2-azeti din one generated complex displayed a pH-dependent reactivation rate. In the latter case, the increase in reactivation rate with pH displayed a pKa of 7.2. This is consistent with the requirement for base catalysis by the active site histidine to regenerate enzymatic activity. Reactivation of the L-680,833-derived complex produced different products as a function of pH, suggesting two different pH-dependent routes of reactivation. At low pH a route that produced primarily the substituted urea is favored, while at higher pH production of two six-membered ring diastereomers competes with urea generation. Thus, the apparent pH independence of the return of activity is the result of two offsetting pathways. Other compounds such as L-670,258, (S)-4-[((((2-naphthylmethyl)amino)carbonyl)-3,3-diethyl-4-oxo-2- azetidinyl)oxy]benzoic acid, reactivate by these two routes as well as by aminolysis by the other urea nitrogen to produce an additional regioisomer. The temperature dependence of the reactivation of the complexes derived from L-684,481 and L-680,833 suggests different mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Orally active beta-lactam inhibitors of human leukocyte elastase. 3. Stereospecific synthesis and structure-activity relationships for 3,3-dialkylazetidin-2-ones.

The stereospecific synthesis of several 4-[(4-carboxyphenyl)oxy]- 3,3-dialkyl-1-[[(1-phenylalkyl)-amino]carbonyl]azetidin-2-on es 3 is described in which the C-3 alkyl groups were varied from methyl to butyl as well as allyl, benzyl and methoxymethyl. The structure-activity relations for these compounds are discussed in terms of the hydrolytic stability of the beta-lactam ring, their in vitro inhibitory potency for human leukocyte elastase (HLE), and their in vivo oral efficacy in an HLE-mediated hamster lung hemorrhage assay. Further alkyl substitution on the benzylic urea moiety, especially in the R configuration, afforded enhanced HLE inhibition and in vivo efficacy. The stereochemical assignments for (3R,4S)-4-[(4-carboxyphenyl)oxy]-3-ethyl-3-methyl-1-[[((R)-1- phenylpropyl)amino]carbonyl]azetidin-2-one (42a) (kobs/[I] = 91,000 M-1 s-1) were confirmed with an X-ray structure determination, which was also utilized to develop an HLE inhibition model.

Mechanism of inhibition of human leucocyte elastase by monocyclic beta-lactams.

The kinetic and catalytic mechanisms of time-dependent inhibition of human polymorphonuclear leukocyte elastase (HLE) by the monocyclic beta-lactams described by Knight et al. [Knight, W.B., et al. (1992) Biochemistry 31, 8160] are investigated in this work. The dependence of the pseudo-first-order rate constant (k(obs)) on inhibitor concentration was saturable. The individual kinetic constants for the inhibition by L-680,833, [S-(R*,S*)]-4-[(1-(((1-(4- methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-4-oxo-2- azetidinyl)oxy]benzeneacetic acid, and L-683,845, [S-(R*,S*)]-4-[(1-(((1-(5-benzofuranyl)butyl)amino)carbonyl)- 3,3-diethyl-4-oxo-2-azetidinyl)oxy]benzeneacetic acid, at pH 7.5 were k(inact) = 0.08 and 0.06 s-1 and Ki = 0.14 and 0.06 microM, respectively. The relative potency of this class of compounds as measured by k(inact)/Ki is primarily controlled by the Ki, term which ranged from 6 nM to 8 mM, while K(inact) was relatively insensitive to structural changes and varied by only an order of magnitude. Inactivation by the beta-lactams was efficient, requiring only 1.3 and 1.7 equiv of L-680,833 and L-683,845 to inactivate HLE. These values are indicative of some partitioning between turnover of inhibitor and inactivation. The partition ratio ranged as high as 3.5:1 depending upon the structure of the inhibitors, but this ratio was essentially independent of the availability and identity of a leaving group at C-4 of the lactam ring. Inactivation and partitioning liberate the leaving group when present at C-4. p-Hydroxy-m-nitrophenylacetic acid is liberated from this position at a rate similar to that for enzyme inactivation, suggesting kinetic competence of this process. Other products observed during the interaction of L-680,833 with HLE include a substituted urea, a species previously observed during the base-catalyzed decomposition of this class of compounds, and small amounts of products observed during reactivation of beta-lactam-derived HLE-I complexes. Both the pH dependence of k(inact)/Ki for the inactivation of HLE by [S-(R*,S*)]-4-[(1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-3,3-diethyl - 4-oxo-2-azetidinyl)oxyl]benzoic acid and V/K for HLE-catalyzed substrate hydrolysis indicate that a single ionizable group with a pK of approximately 7 must be deprotonated for both processes. This group is likely the active site histidine. The data are consistent with initial formation of a Michaelis complex, acylation of the catalytic serine, and loss of the leaving group at C-4 of the original beta-lactam ring followed by partitioning between regeneration of active enzyme and production of a stable enzyme-inhibitor complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Orally active beta-lactam inhibitors of human leukocyte elastase. 2. Effect of C-4 substitution.

The effect of changing the C-4 substituent of 3,3-diethyl-1-[(benzylamino)carbonyl]-2-azetidinone on inhibition of HLE and in a model of HLE-induced lung damage in hamsters was explored. Substituents at this position do not appear to interact strongly with HLE with the most potent compounds having k(obs)/[I] = 6900 M-1 s-1. However, substituents at this position had a marked effect on in vivo activity. The greatest oral activity in the lung hemorrhage assay was achieved with C-4 aryl carboxylic acid ethers (60-85% inhibition at 30 mg/kg po). Based upon the established mechanism of inhibition by these compounds, the C-4 substituent would be released, and therefore, the pharmacological potential of these C-4 substituents was of considerable concern. Fortunately, compounds containing 4-hydroxybenzoic acid and 4-hydroxyphenylacetic acid ethers at C-4 were among the most active analogs. These phenolic acids are also found as urinary metabolites in healthy humans. Other heteroaryls at C-4 were also orally active in this model despite relatively modest enzyme activity.

Specificity, stability, and potency of monocyclic beta-lactam inhibitors of human leucocyte elastase.

Stable, potent, highly specific, time-dependent monocyclic beta-lactam inhibitors of human leucocyte elastase (HLE) are described. The heavily substituted beta-lactams are stable under physiological conditions including in the presence of enzymes of the digestive tract. The beta-lactams were unstable in base. At pH 11.3 and 37 degrees C they were hydrolyzed with half-lives of 1.5-2 h. Hydrolysis produced characteristic products including the substituent originally at C-4 of the lactam ring, a substituted urea, and products resulting from decarboxylation of the acid after ring opening. The most potent beta-lactam displayed only 2-fold less activity versus HLE than alpha 1PI, the natural proteinaceous inhibitor. The compounds were more potent against the human and primate PMN elastases than versus either the dog or rat enzymes. Differences in the structure-activity relationships of the human versus the rat enzymes suggest significant differences between these two functionally similar enzymes. The specificity of these compounds toward HLE versus porcine pancreatic elastase (PPE) is consistent with the differences in substrate specificity reported for these enzymes [Zimmerman & Ashe (1977) Biochim. Biophys. Acta 480, 241-245]. These differences suggest that the alkyl substitutions at C-3 of the lactam ring bind in the S1 specificity pocket of these enzymes. The dependence of the stereochemistry at C-4 suggests additional differences between HLE and PPE. Most of the compounds do not inhibit other esterases or human proteases. Weak, time-dependent inhibition of human cathepsin G and alpha-chymotrypsin by one compound suggested a binding mode to these enzymes that places the N-1 substitution in the S1 pocket.

Mechanism of inhibition of human leukocyte elastase by two cephalosporin derivatives.

The cephalosporin derivatives L 658758 [1-[[3-(acetoxymethyl)-7 alpha-methoxy-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-en-2-yl]carbonyl]proline S,S-dioxide] and L 659286 [1-[[7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo- 1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-aza-(6R)-bicyclo[4.2.0]-o ct-2-en-2-yl]carbonyl]pyrrolidine S,S-dioxide] are mechanism based inhibitors of human leukocyte elastase (HLE). The mechanism involves initial formation of a Michaelis complex followed by acylation of the active site serine. The group on the 3'-methylene is liberated during the course of these reactions, followed by partitioning of an intermediate between hydrolysis to regenerate active enzyme and further modification to produce a stable HLE-inhibitor complex. The partition ratio of 2.0 obtained for the reaction with L 658758 approaches that of an optimal inhibitor. These compounds are functionally irreversible inhibitors as the recovery of activity after inactivation is slow. The half-lives at 37 degrees C of the L 658758 and L 659286 derived HLE-I complexes were 9 and 6.5 h, respectively. The complexes produced by both inhibitors are similar chemically since the thermodynamic parameters for activation to regenerate active enzyme are essentially identical. The free energy of activation for this process is dominated primarily by the enthalpy term. The stability of the final complexes likely arises from Michael addition on the active site histidine to the 3'-methylene.

Inhibition of human leukocyte elastase. 2. Inhibition by substituted cephalosporin esters and amides.

A variety of 7 alpha-methoxycephalosporin ester and amide sulfones were prepared and tested to determine the structure-activity relations for inhibition of human leukocyte elastase (HLE), a serine protease which has been implicated in several degenerative lung and tissue diseases. The most potent IC50 values were obtained with neutral, lipophilic derivatives, with the esters being more active than the amides. However, the best time-dependent inhibition in this series was observed with the p- and m-carboxybenzyl esters 7b and 7c. These results are discussed in terms of the proposed mechanism of inhibition as well as a molecular modeling study using the recently solved X-ray crystal structure of HLE.

Inhibition of human leukocyte elastase. 3. Synthesis and activity of 3'-substituted cephalosporins.

Several 3'-substituted cephalosporin sulfones were synthesized from 3-(hydroxymethyl)cephalosporin, which was prepared by Ti(OiPr)4 hydrolysis of the corresponding acetate. A method was also developed to prepare a 3-vinylcephalosporin. Some of these compound were found to be potent time-dependent inhibitors of human leukocyte elastase (HLE). The HLE inhibitory activity was correlated with sigma 1 and it was concluded that the potency was determined by the electron-withdrawing ability as well as the size of the substituent. A mechanism for inhibition of HLE by cephalosporin sulfones is proposed.

Synthesis and structure-activity relationships of a novel class of 5-lipoxygenase inhibitors. 2-(Phenylmethyl)-4-hydroxy-3,5-dialkylbenzofurans: the development of L-656,224.

The synthesis of a series of 2-(phenylmethyl)-4-hydroxy-3,5-dialkylbenzofurans and their inhibitory effects against leukotriene biosynthesis and 5-lipoxygenase activity in vitro are described. Many compounds in this series were found to be potent inhibitors of LTB4 production by human polymorphonuclear leukocytes with IC50 values ranging from 7 to 100 nM. Structure-activity relationships of the series are presented. Within this series, 2-[(4'-methoxyphenyl)methyl]-4-hydroxy-3-methyl-5-propyl-7-chlorobenz ofuran (L-656,224) showed extremely potent activity, inhibiting leukotriene biosynthesis in intact human leukocytes (IC50 = 11 nM), as well as the 5-lipoxygenase reaction catalyzed by cell-free preparations from rat leukocytes (IC50 = 36 nM), human leukocytes (IC50 = 0.4 microM), and the purified enzyme from porcine leukocytes (IC50 = 0.4 microM). The compound also shows oral activity in a number of animal models in vivo.

Development of enzyme-linked immunosorbent assays for measurement of leukotrienes and prostaglandins.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the leukotrienes LTC4 and LTB4 and the prostaglandins 6-keto PGF1 alpha and thromboxane (TxB2). In an indirect assay procedure for all 4 eicosanoids a BSA conjugate of the leukotrienes or an ovalbumin conjugate of the prostaglandins was absorbed to polystyrene microtiter plates. Samples containing the respective eicosanoids were incubated in the coated wells with specific rabbit antisera. The wells were then incubated successively with a goat anti-rabbit antibody linked to fluorescein and a rabbit anti-fluorescein antibody linked to alkaline phosphatase. The resultant assays for LTC4, LTB4, 6-keto PGF1 alpha, and TxB2, gave steep, sensitive inhibition curves; IC50s were 0.2, 10, 1, and 0.4 pmol respectively with minimal cross-reactivity to other eicosanoids. The sensitivities and specificities were comparable to those found in the RIA, and the levels determined in this assay correlate well with those determined in non-immunological assays.

Inhibition of histamine synthesis in vitro and in vivo by S-alpha-fluoromethylhistidine.

(S)-alpha-Fluoromethylhistidine (alpha-FMH) is a Kcat or "suicide-substrate" inhibitor of partially purified mammalian histidine decarboxylase; i.e. the agent is converted enzymatically to a more active form which effects a time-dependent, irreversible inhibition. Incubation of a alpha-FMH[4-3H] with enzyme and pyridoxal phosphate resulted in an apparently irreversible labeling of protein, with no demonstratable formation of free-amine product, suggesting a very low to non-existent turnover ratio. alpha-FMH was accumulated in isolated mastocytoma cells and effected a time-dependent inhibition of the conversion histidine[3H]----histamine[3H], the latter product having a markedly different distribution between cells and medium than the pre-existing histamine pool. Inhibition of whole-body histidine decarboxylase activity, as specifically measured by alpha-methylhistidine-14COOH----14CO2, was also time dependent. Concomitant reduction in histamine levels was seen only in the rapidly turning-over pools of stomach and brain. However, over the course of 13 weeks of chronic treatment, depletion of the relatively inert mast-cell histamine pool(s) was seen as well.

The development of sensitive and specific radioimmunoassays for leukotrienes.

Radioimmunoassays for leukotriene C4 (LTC4) and for leukotriene B4 (LTB4) have been developed. LTC4 was conjugated with thiolated hemocyanin (Keyhole Limpet) (KLH) using 6-(N-maleimido)hexanoic acid chloride as coupling agent. LTB4 was converted to its hydrazide derivative, via the delta-lactone and the hydrazide was similarly coupled with thiolated KLH using 6-(N-maleimido)hexanoic acid chloride as coupling agent. These conjugates were used to consistently raise high titres of anti-leukotriene antibodies in rabbits. 14,15-[3H]-LTC4 was prepared by total synthesis via two routes. 14,15-[3H]-LTB4 was prepared by total synthesis. The assay for LTC4 recognizes LTC4, LTD4 and LTF4, and to a lesser extent, LTE4 with a detection limit of ca. 0.1 pmoles LTC4 per mL of sample. The assay for LTB4 is highly specific and has a similar detection limit.

Measuring leukotrienes of slow reacting substance of anaphylaxis: development of a specific radioimmunoassay.

Rabbits were immunized with leukotriene C4 (LTC4) coupled to thiolated keyhole limpet hemocyanin (KLH) by using 6-N-maleimidohexanoic acid as a spacer molecule. Immune serum was obtained with 7.9 nmol of LTC4-specific immunoglobulin per milliliter and a mean association constant of 2.1 X 10(9) M-1. A radioimmunoassay was developed that detected 0.1 pmol of LTC4 per 1-ml sample. LTD4 and LTE4, three isomers of LTC4, the sulfones of LTC4, LTD4, and LTE4, and one isomer of LTD4 reacted to varying degrees in the assay. A number of other structurally related compounds, such as LTB4 and 5-HETE, did not react. Conditions were established to determine LTC4 levels in human plasma without loss of LTC4 during sample preparation and without the need for extraction procedures before the measurement of LTC4.

Leukotriene A4: preparation and enzymatic conversion in a cell-free system to leukotriene B4.

Treatment of leukotriene A4 (LTA4) methyl ester with sodium hydroxide in aqueous methanol at 4 degrees C afforded LTA4, the presence of which was inferred from the UV spectrum of the compound, its rate of reaction with water, and the identity of the hydration products obtained. The half-life of LTA4 in water (pH 7.4, room temperature) was increased from 14 to 500 s by 1 mg/ml of bovine serum albumin. This stabilized (chiral) LTA4 was converted to LTB4 by an epoxide hydrolase activity in the 100,000 x g supernatant fraction from sonified rat basophilic leukemia cells. Neither the ester of LTA4 nor the biologically incorrect enantiomer of LTA4 was metabolized to LTB4 under these conditions.

A new class of angiotensin-converting enzyme inhibitors.

Much current attention focuses on the renin-angiotensin system in relation to mechanisms controlling blood pressure and renal function. Recent demonstrations (ref. 1, ref. 2 and refs therein) that angiotensin-converting enzyme inhibitors show promising clinical antihypertensive properties have been of particular interest. We now report on the design of a novel series of substituted N-carboxymethyl-dipeptides which are active in inhibiting angiotensin-converting enzyme at nanomolar levels. We suggest that these compounds are transition-state inhibitors and that extensions of this design to other metalloendopeptidases merit further study.

Inactivation of 3-(3,4-dihydroxyphenyl)alanine decarboxylase by 2-(fluoromethyl)-3-(3,4-dihydroxyphenyl)alanine.

2-(Fluoromethyl)-3-(3,4-dihydroxyphenyl)alanine [alpha-FM-Dopa (I)] causes rapid, time-dependent, stereospecific, and irreversible inhibition of hog kidney aromatic amino acid (Dopa) decarboxylase. The inactivation occurs with loss of both the carboxyl carbon and fluoride from I and results in the stoichimetric formation of a covalent enzyme-inhibitor adduct. The data are consistent with I being a suicide inactivator of the enzyme, and a plausible mechanism for the inactivation process is presented. The inactivation is highly efficient in that there is essentially no enzymatic turnover of I to produce the corresponding amine, 1-(fluoromethyl)-2-(3,4-dihydroxyphenyl)ethylamine [alpha-FM-dopamine (II)]. Amine II is also a potent inactivator of the enzyme. In vivo compound I is found to inactivate both brain and peripheral (liver) Dopa decarboxylase activity. The possible significance of these data with respect to the known antihypertensive effect of I is discussed.