Transformation of heterocyclic reversible monoamine oxidase-B inactivators into irreversible inactivators by N-methylation.

3-[4-[(3-Chlorophenyl)methoxy]phenyl]-5-[(methylamino)methyl]- 2-oxazolidinone (1) is a secondary amine known to be a potent time-dependent irreversible inactivator of monoamine oxidase B (MAO-B). The primary amine analogues of derivatives of 1, as well as of the corresponding dihydrofuranone and pyrrolidinone, had been shown to be time-dependent, but reversible, inhibitors of MAO-B. Here it is shown that the primary amine analogue of 1 is a time-dependent reversible inhibitor of MAO-B and that the secondary and tertiary amine analogues of the corresponding oxazolidinone, dihydrofuranone, and pyrrolidinone are time-dependent irreversible inhibitors of MAO-B. The reaction leading to the irreversible enzyme adduct formation with 1 can be reversed by increasing the temperature. These results are consistent with a stabilizing stereoelectronic effect on the enzyme adduct caused by N-methylation which hinders free rotation and prevents the sp3-orbital containing the nitrogen nonbonded electrons from being trans to the active site amino acid leaving group.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine analogues. Inactivation of monoamine oxidase by conformationally rigid analogues of N,N-dimethylcinnamylamine.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a potent neurotoxin and also an inactivator of monoamine oxidase (MAO). Since MPTP is a conformationally rigid analogue of N,N-dimethylcinnamylamine, other conformationally rigid analogues of N,N-dimethylcinnamylamine were synthesized and tested as inhibitors and inactivators of MAO. (E)-2-(Phenylmethylene)cyclohexanamine (5a), (E)-N,N-dimethyl-2-(phenylmethylene)cyclohexanamine (5b), 3-phenyl-2-cyclohexen-1-amine (6a), N,N-dimethyl-3-phenyl-2-cyclohexen-1-amine (6b), and (E)- and (Z)-N-methyl-3-(phenylmethylene)piperidine (7 and 8) are all inhibitors and time-dependent inactivators of MAO B, but none is as potent as MPTP. alpha-Methylation and methylation of the amino group in all cases increases the Ki value relative to that for the parent compound. Compounds 5a, 5b, 6a and 6b are highly cytotoxic, but cytotoxicity is not prevented by pretreatment of the cells with pargyline. There does not appear to be a correlation between the configuration of the N,N-dimethylcinnamylamine analogue and its potency as a MAO inactivator.

New analogues of N-(2-aminoethyl)-4-chlorobenzamide (Ro 16-6491). Some of the most potent monoamine oxidase-B inactivators.

A series of halo- and nitro-substituted analogues of N-(2-aminoethyl)benzamide has been synthesized. All of the compounds are competitive, time-dependent inhibitors of monoamine oxidase-B (MAO-B), but upon dialysis complete return of enzyme activity is observed for all compounds. Therefore, these are mechanism-based reversible inhibitors of MAO-B. The relative potencies of the compounds are rationalized in terms of steric and hydrophobic effects.

5-(Aminomethyl)-3-aryldihydrofuran-2(3H)-ones, a new class of monoamine oxidase-B inactivators.

Both cis- and trans-5-(aminomethyl)-3-aryldihydrofuran-2(3H)-one hydrochloride salts (9 and 10) were synthesized efficiently in a 5-step sequence from arylacetic acids. Both compounds were found to be irreversible inactivators of monoamine oxidase B. These compounds constitute the first members of a new class of monoamine oxidase inactivators.

Design of potential anticonvulsant agents: mechanistic classification of GABA aminotransferase inactivators.

Because of the importance of the inactivation of GABA aminotransferase to the design of anticonvulsant agents, a seemingly wide variety of inactivators has been investigated; all of the compounds, however, are analogues of GABA, beta-alanine, or delta-aminovaleric acid, which are substrates for the enzyme. Relatively minor modifications in the inactivator structures result in major differences in inactivation mechanisms and enzyme adduct structures. Compounds that inactivate GABA aminotransferase by a Michael addition mechanism, leading to modification of an active-site residue are Class I inactivators. Those that proceed by an enamine mechanism and give ternary adducts are Class II inactivators. Class III inactivators modify only the PLP cofactor; if the inactivation involves aromatization of the inactivator, it is a Class IIIA inactivation, and if no aromatization is involved, then it is a Class IIIB inactivation. The last class of inactivators (Class IV) are not classified on the basis of the mechanism, but, rather, that they require the enzyme to be in the PMP form. There appears to be no trend in partition ratio values when comparing Class I with Class II inactivators. Class III inactivations alter only the cofactor, so it may be possible for these adducts to diffuse slowly out of the active site; reactivation of the apoenzyme would require additional PLP. These inactivators also inactivate a variety of other PLP-dependent enzymes. At this point there does not seem to be a therapeutic advantage of one class of inactivators over another, although the only current example of these inactivators to be useful clinically is gamma-vinyl GABA (vigabatrin), a Class I inactivator recently approved for the drug market in France and the U.K. There is a mechanistic significance, however, for one class over another. If labeling of an active-site amino acid residue is desired, then Class I inactivators should be selected; desire for attachment of the inactivator to both the protein and the cofactor or just to the cofactor would determine whether Class II or Class III inactivators would be chosen. The classification presented here should allow us to think about inactivator structures in terms of their mechanistic potential and, as a result of this, should afford us the opportunity to be able to make predictions regarding inactivation mechanisms for hypothetical new structural classes of inactivators. Since the different mechanistic pathways lead to different types of enzyme adducts, inactivator design may be driven by the class of adduct that is desired.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Slow-binding inhibition of gamma-aminobutyric acid aminotransferase by hydrazine analogues.

(3-Hydroxybenzyl)hydrazine and methylhydrazine have been found to be potent slow-binding inhibitors of the pyridoxal 5-phosphate (PLP)-dependent enzyme gamma-aminobutyric acid aminotransferase (GABA-AT). Both compounds follow mechanism A (Morrison, J.F.; Walsh, C. T. Adv. Enzymol. 1988, 61, 201-301) which does not involve formation of a rapidly reversible enzyme-inhibitor complex before the formation of the final tight complex. The rate constant for formation of the enzyme-inhibitor complex determined from the slow-binding kinetics was 2.08 x 10(3) and 1.98 x 10(4) M-1 min-1 for methylhydrazine and (3-hydroxybenzyl)hydrazine, respectively. The rate constant for dissociation of the enzyme--inhibitor complex determined for the slow-binding kinetics was 4.6 x 10(-3) and 5 x 10(-3) min-1, respectively. The inhibition constants calculated from the slow-binding inhibition kinetics are 2.2 microM for methylhydrazine and 0.3 microM for (3-hydroxybenzyl)hydrazine. Reactivation of the inhibited enzyme was not first order, perhaps due to a side reaction of the hydrazine, but was consistent with the results obtained from the slow-binding kinetics. Inhibition constants were calculated from the level of enzyme activity at equilibrium inhibition. These constants are 2.8 and 0.46 microM for methylhydrazine and (3-hydroxybenzyl)hydrazine, respectively, in good agreement with those calculated from the slow-binding inhibition kinetics. 3-Hydrazinopropionate also behaved as a slow-binding inhibitor. However, the dependence of its kinetics on the concentration of inhibitor could not be described by the slow-binding or slow, tight-binding inhibition models. These kinetics could not be described by the tight-binding character of the inhibition because the addition of the competitive inhibitor propionic acid at 100 times its Ki did not affect the shape of the curve for inhibitor concentration dependence. The slow-binding inhibition appeared to require 2-4 molecules of 3-hydrazinopropionate/enzyme. The reactivation of enzyme inhibited by 3-hydrazinopropionate was first order with a rate constant of 6.9 x 10(-3) min-1. Its equilibrium inhibition constant was calculated to be < 20 nM. However, the inhibition constant calculated was dependent on the concentration of inhibitor because of the unusual character discussed above and may be much lower. Only 1 PLP/enzyme dimer reacted with methylhydrazine or (3-hydroxybenzyl)hydrazine, as indicated by Scatchard plots, or with 3-hydrazinopropionate, as shown by a spectrophotometric titration. Slow-binding inhibition does not appear to be the result of a significant enzyme conformational change because there is no change in the tryptophan fluorescence of GABA-AT upon binding either methylhydrazine or 3-hydrazinopropionate. Implications for the design of hydrazine inhibitors of GABA-AT are discussed.

Selective inhibition of gamma-aminobutyric acid aminotransferase by (3R,4R),(3S,4S)- and (3R,4S),(3S,4R)-4-amino-5-fluoro-3-phenylpentanoic acids.

(3R,4R),(3S,4S)- and (3R,4S),(3S,4R)-4-amino-5-fluoro-3-phenylpentanoic acid (1a and 1b) were synthesized and studied as selective inactivators of gamma-aminobutyric acid (GABA) aminotransferase. Neither compound caused time-dependent inactivation of the enzyme. Neither compound underwent enzyme-catalyzed transamination nor was fluoride ion eliminated from either compound by the enzyme. No 3-phenyllevulinic acid, the product of elimination of HF followed by enamine hydrolysis, was detected. However, both 1a and 1b were competitive reversible inhibitors of GABA aminotransferase; the Ki for 1a was smaller than the Km for GABA. These results suggest that 1a and 1b bind to the active site of GABA aminotransferase, but gamma-proton removal does not occur. Whereas (S)-4-amino-5-fluoropentanoic acid (AFPA) is a potent inhibitor of L-glutamic acid decarboxylase (GAD), neither 1a nor 1b at concentrations 40 times the Ki of AFPA caused any detectable competitive inhibition of GAD. Therefore, the incorporation of a phenyl substituent at the 3-position of AFPA confirms selective inhibition of GABA aminotransferase over GAD.

2-(Fluoromethyl)-3-phytyl-1,4-naphthoquinone and its 2,3-epoxide. Inhibition of vitamin K epoxide reductase.

2-(Fluoromethyl)-3-phytyl-1,4-naphthoquinone (7) was synthesized from the known compound 2-bromo-3-methyl-1,4-dimethoxynaphthalene by N-bromosuccinimide bromination of the 3-methyl group, conversion to the corresponding 3-fluoromethyl compound with silver fluoride, attachment of the 3-phytyl substitutent via the lithium diaryl cuprate and phytyl bromide, and then silver oxide oxidation to 7. Epoxidation with basic hydrogen peroxide gave the corresponding 2,3-oxide (1) in a very low yield. Compound 1 was not a time-dependent inhibitor of beef liver microsomal vitamin K epoxide reductase, but it was a competitive, reversible inhibitor. It was not possible to determine if 1 was a substrate for the enzyme because the expected product of reduction, namely 7, rapidly decomposed under the assay conditions.

1-Benzylcyclopropylamine and 1-(phenylcyclopropyl)methylamine: an inactivator and a substrate of monoamine oxidase.

1-Benzylcyclopropylamine (1) and 1-(phenylcyclopropyl)methylamine (2), cyclopropane analogues of phenethylamine, were tested as inactivators for monoamine oxidase (MAO). Compound 1 is a potent competitive reversible inhibitor of the oxidation of benzylamine and also is a mechanism-based inactivator. It requires 2.3 equiv of 1 to inactivate 1 equiv of MAO. The excess equivalents of 1 are converted into benzyl vinyl ketone. A one-electron mechanism of inactivation is proposed. Compound 2 is a substrate for MAO and is converted into 1-phenylcyclopropanecarboxaldehyde without inactivation of the enzyme. Mechanistic consequences are discussed as a result of this observation.

Inactivation of gamma-aminobutyric acid aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid and effect on the enzyme of (E)-3-(1-aminocyclopropyl)-2-propenoic acid.

(S,E)-4-Amino-5-fluoropent-2-enoic acid (6) is synthesized in six steps starting from the known gamma-aminobutyric acid aminotransferase (gamma-Abu-T) inactivator, (S)-4-amino-5-fluoropentanoic acid (1). Compound 6 is a mechanism-based inactivator of gamma-Abu-T: time-dependent inactivation is saturatable and protected by substrate; thiols do not protect the enzyme from inactivation; no enzyme activity returns upon dialysis. This compound (6) binds 50 times more tightly to gamma-Abu-T than does the saturated analogue (1). No transamination of 6 occurs prior to inactivation. However, five molecules of 6 are required to inactivate the enzyme with concomitant release of five fluoride ions. Therefore, four molecules are being converted to product for each inactivation event. (E)-3-(1-Aminocyclopropyl)-2-propenoic acid is synthesized in seven steps from 1-aminocyclopropanecarboxylic acid. It is prepared as a cyclopropyl derivative of the proposed intermediate in the inactivation of gamma-Abu-T by 6. The cyclopropyl derivative, however, is a noncompetitive inhibitor and does not inactivate the enzyme. This study shows the usefulness and hazards of incorporation of a trans double bond into potential gamma-Abu-T inactivators.

Inactivation of monoamine oxidase B by analogues of the anticonvulsant agent milacemide (2-(n-pentylamino)acetamide).

Analogues of the anticonvulsant agent milacemide (1,2-(n-pentylamino)acetamide), in which the carboxamide group is changed to a nitrile (2), a carbethoxy group (3), a carboxylic acid (4), a cyanomethyl group (5), and a trifluoromethyl group (6), were synthesized and tested as substrates and inactivators of monoamine oxidase B (MAO B). The carboxylic acid was neither a substrate nor an inactivator. The trifluoromethyl compound was not soluble in buffer even when organic cosolvents were added, so it could not be tested. All of the other compounds were both substrates and time-dependent irreversible inactivators of MAO B. A plot of the logarithm of kcat/k(inact) (a measure of the efficiency of the inactivators) versus sigma I (Figure 1) shows a linear free energy relationship between the inactivator efficiency and the electron-withdrawing ability of the substituent. As the electron-withdrawing ability increases, the partition ratio decreases indicating that inactivation is becoming more efficient relative to substrate turnover to product. Milacemide was the least efficient of the compounds tested; the nitrile 2 was the most efficient.

Inhibition and substrate activity of conformationally rigid vigabatrin analogues with gamma-aminobutyric acid aminotransferase.

Several cyclopentene GABA analogues were synthesized as conformationally rigid analogues of the epilepsy drug vigabatrin and tested as inhibitors and substrates of gamma-aminobutyric acid aminotransferase (GABA-AT). None of these compounds produced time-dependent inhibition. (1R, 4S)-(+)-4-Amino-2-cyclopentene-1-carboxylic acid ((+)-3), (4R)-(-)-4-amino-1-cyclopentene-1-carboxylic acid ((-)-4), and d, l-3-amino-1-cyclopentene-1-carboxylic acid (6) are good substrates. The K(m) and k(cat) values for the latter two compounds are very similar to those of GABA, suggesting that they bind in an orientation similar to that of GABA. The K(m) value for (+)-3 is 24 times lower than that for GABA, although its k(cat) value is only one-fourth that for GABA; nonetheless, it is a better substrate for GABA-AT than is GABA. All of these compounds, as well as the enantiomers of 3 and 4 and d, l-trans-4-amino-2-cyclopentene-1-carboxylic acid (5), are competitive inhibitors of GABA-AT. These results demonstrate the effects of the carboxylate group orientation and the stereochemistry of the amino and carboxylate groups on the substrate activity and inhibitor activity, and this should be important to the future design of inhibitors of GABA-AT.

Synthesis and evaluation of peptidomimetics as selective inhibitors and active site probes of nitric oxide synthases.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO). Selective inhibition of the isoforms of NOS could have great therapeutic potential in the treatment of certain disease states arising from pathologically elevated synthesis of NO. Recently, we reported dipeptide amides containing a basic amine side chain as potent and selective inhibitors of neuronal NOS (Huang, H.; Martasek, P.; Roman, L. J.; Masters, B. S. S.; Silverman, R. B. J. Med. Chem. 1999, 42, 3147). The most potent nNOS inhibitor among these compounds is L-ArgNO2-L-Dbu-NH2 (1) (Ki = 130 nM), which also exhibits the highest selectivity over eNOS (>1,500-fold) with excellent selectivity over iNOS (190-fold). Here we describe the design and synthesis of a series of peptidomimetic analogues of this dipeptide as potential selective inhibitors of nNOS. The biochemical evaluation of these compounds also revealed the binding requirements of the dipeptide inhibitors with NOS. Incorporation of protecting groups at the N-terminus of the dipeptide amide 1 (compounds 4 and 5) resulted in dramatic decreases in the inhibitory potency of nNOS. Masking the NH group of the peptide bond (peptoids 6-8 and N-methylated compounds 9-11) also gave much poorer nNOS inhibitors than 1. Both of the results demonstrate the importance of the alpha-amine of the dipeptide and the NH moiety of the peptide bond for binding at the active site. Modifications at the C-terminus of the peptide included converting the amide to the methyl ester (12), tert-butyl ester (13), and carboxylic acid (14) and also descarboxamide analogues (15-17), which revealed less restricted binding requirements for the C-terminus of the dipeptide. Further optimization should be possible when we learn more about the binding requirements at the active sites of NOSs.

Transformation of monoamine oxidase-B primary amine substrates into time-dependent inhibitors. Tertiary amine homologues of primary amine substrates.

A family of N-methylated and N,N-dimethylated alkyl and arylalkylamines was prepared and more than half of the analogues were shown to be time-dependent pseudo-first-order inhibitors of monoamine oxidase-B. Some of the time-dependent inactivators were reversible and others were irreversible with respect to prolonged dialysis following inactivation. Partition ratios ranged from zero to 11,000. These results are rationalized in terms of a combination of an inductive effect and a stereoelectronic effect as a result of hindered rotation of an active site covalent adduct. A molecular mechanics calculation indicates that there is at least 10 kcal/mol of torsional energy to be overcome in order for the enzyme adduct to be released. These findings show that tertiary amine homologues of primary amine substrates of monoamine oxidase are time-dependent inhibitors, and this should be useful in the design of new inactivators of this enzyme.

4-Amino-2-(substituted methyl)-2-butenoic acids: substrates and potent inhibitors of gamma-aminobutyric acid aminotransferase.

4-Amino-2-(substituted methyl)-2-butenoic acids, where X (the substituted group) = F, Cl, OH, are synthesized from Cbz-protected tert-butyl 4-aminobutanoate. Successive substitutions at the alpha-carbon by phenylseleno and hydroxymethyl groups, followed by elimination of the selenoxide and halide substitution at the hydroxymethyl group, afford the compounds in good yields. An unexpected degree of stereoselectivity is observed in the selenoxide elimination step, which yields the desired E isomer as the sole product. These compounds complement two previously reported series of compounds (Silverman, R. B.; Levy, M. A. Biochem. Biophys. Res. Commun. 1980, 95, 250-255; J. Biol. Chem. 1981, 256, 11 565-11 568) and are used in an approach to map a section of the active site of gamma-aminobutyric acid aminotransferase (GABA-T). None of these compounds is a time-dependent inactivator of GABA-T, but all are potent competitive reversible inhibitors; the hydroxy compound has a Ki value of 5 microM. That these compounds are not inactivators suggests that either elimination of X does not occur or that there is no active site nucleophile in the appropriate position for reaction following elimination. With use of the fluoro analogue, enzyme-catalyzed fluoride ion release is demonstrated, indicating that elimination does occur. Unlike the previous two series of compounds (op. cit.) in which exclusive elimination occurs when the substituent is a halogen but exclusive transamination prevails for the hydroxyl-substituted analogues, in the series described here, the fluoro analogue gives a 4:1 ratio of elimination to transamination. This suggests that the 2,3-double bond stabilizes the product of azallylic isomerization of the Schiff base between the fluoro compound and pyridoxal phosphate. The results described here indicate that the design of a mechanism-based inactivator for GABA-T should not be based on electrophile generation near the 2-position of enzyme-bound GABA. Furthermore, substitution of an inhibitor with a 2-hydroxymethyl group (or other hydrogen-bonding substituent) and a 2,3-double bond may lend auspicious binding properties to the molecule for GABA-T.

Selective inhibition of neuronal nitric oxide synthase by N omega-nitroarginine-and phenylalanine-containing dipeptides and dipeptide esters.

A series of N omega-nitroarginine (ArgNO2)- and phenylalanine-containing dipeptides and dipeptide esters were synthesized as potential selective inhibitors of neuronal nitric oxide synthase (nNOS). All of the dipeptides and dipeptide esters are competitive inhibitors of nNOS, macrophage nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS), except for the ones that contain D-ArgNO2 (8-10, 12, 13), which are uncompetitive inhibitors of iNOS but competitive inhibitors of nNOS and eNOS. None of the dipeptides or dipeptide esters tested (1, 2, 12, 13) exhibited time-dependent inhibition of any of the NOS isoforms, unlike N omega-nitro-L-arginine itself, which does, although it is reversible. The order of the amino acids in the dipeptide or dipeptide ester is important to selectivity, and the selectivity depends on the chirality of the amino acids. In the case of the corresponding benzyl esters (5 vs 6), both dipeptides favor iNOS over nNOS and eNOS inhibition. All of the dipeptide methyl esters containing a D-amino acid, however, exhibit an inhibitory preference for nNOS over iNOS and eNOS. The most impressive selectivities observed are 1800- and 800-fold for 12 and 13, respectively, in favor of nNOS over iNOS; unfortunately, the selectivities of these compounds for nNOS over eNOS are only 2.5 and 5.3, respectively.

Substituted vitamin K epoxide analogues. New competitive inhibitors and substrates of vitamin K1 epoxide reductase.

2- and 3-substituted vitamin K 2,3-epoxide analogues were synthesized and tested as inactivators, inhibitors, and substrates for beef liver microsomal vitamin K1 epoxide reductase. 2-(X)-3-phytyl-1,4-naphthoquinone 2,3-epoxides, where X is hydroxymethyl, chloromethyl, fluoromethyl, difluoromethyl, and formyl were all competitive inhibitors, but none was an inactivator. Only the 2-hydroxymethyl analogue was reduced to a quinone that was stable enough under the conditions of the experiment to be detected. Vitamin K1 epoxide analogues with modified phytyl chains (1'-hydroxy, 3'-fluoro with isomerized double bond, 1'-hydroxy and 1'-fluoro with saturated double bond, and the corresponding unsubstituted chains) were synthesized. All of the analogues were competitive inhibitors of vitamin K1 epoxide reductase. The nonfluorinated analogues also were shown to be substrates, being reduced to the corresponding quinone without enzyme inactivation. At least one other enzyme besides vitamin K1 epoxide reductase in beef liver microsomes also metabolizes all of these analogues.

Reduced amide bond peptidomimetics. (4S)-N-(4-amino-5-[aminoakyl]aminopentyl)-N'-nitroguanidines, potent and highly selective inhibitors of neuronal nitric oxide synthase.

Selective inhibition of the isoforms of nitric oxide synthase (NOS) could be therapeutically useful in the treatment of certain disease states arising from the overproduction of nitric oxide. Recently, we reported nitroarginine-containing dipeptide amides (Huang, H; Martasek, P.; Roman, L. J.; Masters, B. S. S.; Silverman, R. B. J. Med. Chem. 1999, 42, 3147.) and some peptidomimetic analogues (Huang, H; Martasek, P.; Roman, L. J.; Silverman, R.B. J. Med Chem. 2000, 43, 2938.) as potent and selective inhibitors of neuronal NOS (nNOS). Here, reduced amide bond pseudodipeptide analogues are synthesized and evaluated for their activity. The deletion of the carbonyl group from the amide bond either preserves or improves the potency for nNOS. Significantly, the selectivities for nNOS over eNOS (endothelial NOS), and iNOS (inducible NOS) are greatly increased in these series. The most potent nNOS inhibitor among these compounds is (4S)-N-(4-amino-5-[aminoethyl]aminopentyl)-N'-nitroguanidine (7) (K(i) = 120 nM), which also shows the highest selectivity over eNOS (greater than 2500-fold) and 320-fold selectivity over iNOS. The reduced amide bond is an excellent surrogate of the amide bond, and it will facilitate the design of new potent and selective inhibitors of nNOS.

N(omega)-Nitroarginine-containing dipeptide amides. Potent and highly selective inhibitors of neuronal nitric oxide synthase.

Selective inhibition of the isoforms of nitric oxide synthase (NOS) could be therapeutically useful in the treatment of certain disease states arising from the overproduction of nitric oxide (NO). Recently, we reported the dipeptide methyl ester, D-Phe-D-Arg(NO)()2-OMe (19), as a modest inhibitor of nNOS (K(i) = 2 microM), but with selectivity over iNOS as high as 1800-fold (Silverman, R. B.; Huang, H.; Marletta, M. A.; Martasek, P. J. Med. Chem. 1997, 40, 2813-2817). Here a library of 152 dipeptide amides containing nitroarginine and amino acids other than Phe are synthesized and screened for activity. Excellent inhibitory potency and selectivity for nNOS over eNOS and iNOS is achieved with the dipeptide amides containing a basic amine side chain (20-24), which indicates a possible electrostatic (or hydrogen bonding) interaction at the enzyme active site. The most potent nNOS inhibitor among these compounds is L-Arg(NO)()2-L-Dbu-NH(2) (23) (K(i) = 130 nM), which also exhibits the highest selectivity over eNOS (>1500-fold) with a 192-fold selectivity over iNOS. These compounds do not exhibit time-dependent inhibition. The order and the chirality of the amino acids in the dipeptide amides have profound influences on the inhibitory potency as well as on the isoform selectivity. These dipeptide amide inhibitors open the door to the design of potent and highly selective inhibitors of nNOS.