Spontaneous rearrangement of aminoalkylisothioureas into mercaptoalkylguanidines, a novel class of nitric oxide synthase inhibitors with selectivity towards the inducible isoform

Garry J. Southan, Basilia Zingarelli, Michael O'Connor, Andrew L. Salzman, Csaba Szabo

Research output: Contribution to journalArticle

99 Citations (Scopus)

Abstract

1 The generation of nitric oxide (NO) from L-arginine by NO synthases (NOS) can be inhibited by guanidines, amidines and S-alkylisothioureas. Unlike most L-arginine based inhibitors, however, some guanidines and S-alkylisothioureas, in particular aminoethylisothiourea (AETU), show selectivity towards the inducible isoform (iNOS) over the constitutive isoforms (endothelial, ecNOS and brain isoform, bNOS) and so may be of therapeutic benefit. In the present study we have investigated the effects of AETU and other aminoalkylisothioureas on the activities of iNOS, ecNOS and bNOS. 2 AETU, aminopropylisothiourea (APTU) and their derivatives containing alkyl substituents on one of the amidino nitrogens, potently inhibit nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values ranging from 6-30 μM (EC50 values for N(G)-methyl-L-arginine (L-NMA) and N(G)-nitro-L-arginine were 159 and > 1000 μM, respectively). The inhibitory effects of these aminoalkylisothioureas (AATUs) were attentuated by L-arginine in the incubation medium, indicating that these agents may compete with L-arginine for its binding site on NOS. 3 The above AATUs undergo chemical conversion in neutral or basic solution (pH 7 or above) as indicated by (1) the disappearance of AATUs from solution as measured by h.p.l.c., (2) the generation of free thiols not previously present and (3) the isolation of species (as picrate and flavianate salts) from neutral or basic solutions of AATUs that are different from those obtained from acid solutions. 4 Mercaptoalkylguanidines (MAGs) were prepared and shown to be potent inhibitors of iNOS activity with EC50s comparable to those of their isomeric AATUs. 5 These findings suggest that certain AATUs exert their potent inhibitory effects through intramolecular rearrangement to mercaptoalkylguanidines (MAGs) at physiological pH. Those AATUs not capable of such rearrangement do not exhibit the same degree of inhibition of iNOS. 6 In contrast to their potent effects on iNOS, some AATUs and MAGs were 20-100 times weaker than N(G)-methyl-L-arginine and N(G)-nitro-L-arginine as inhibitors of ecNOS as assessed by their effects on the conversion of L-arginine to L-citrulline in homogenates of bovine endothelial cells and by their presser effects in anaesthetized rats. Thus mercaptoalkylguanidines represent a new class of NOS inhibitors with preference towards iNOS. 7 AETU and mercaptoethylguanidine (MEG), when given as infusions, gave slight decreases in MAP in control rats. However, infusions of AETU or MEG to endotoxin-treated rats caused an increase in MAP and restored 80% of the endotoxin-induced fall in MAP. 8 High doses of MEG (30-60 mg kg-1) caused a decrease in MAP of normal rats. This depressor effect may be a consequence of the in vivo oxidation of MEG to the disulphide, guanidinoethyldisulphide (GED), which caused pronounced, transient hypotensive responses in anaesthetized rats and caused endothelium-independent vasodilator responses in precontracted rat aortic rings in vitro. 9 In some cases, slight differences were observed in the activities of AATUs and the corresponding MAGs. These may be explained by the formation of other species from AATUs in physiological media. For example, AETU can give rise to small amounts of the potent ecNOS inhibitor, 2-aminothiazoline, in addition to MEG. This may account for the differences in the in vitro and in vivo effects of AETU and MEG. 10 In conclusion, the in vitro and in vivo effects of AETU and related aminoalkylisothioureas can be explained in terms of their intramolecular rearrangement to generate mercaptoalkylguanidines, a novel class of selective inhibitors of iNOS.

Original languageEnglish (US)
Pages (from-to)619-632
Number of pages14
JournalBritish Journal of Pharmacology
Volume117
Issue number4
StatePublished - 1996
Externally publishedYes

Fingerprint

Nitric Oxide Synthase
Arginine
Protein Isoforms
Guanidines
Endotoxins
Amidines
Citrulline
Nitrites
Vasodilator Agents
Sulfhydryl Compounds
Disulfides
Endothelium
2-mercaptoethylguanidine
Nitric Oxide
Nitrogen
Endothelial Cells
Salts
Macrophages
Binding Sites
Acids

Keywords

  • Blood pressure
  • EDRF
  • Endotoxin
  • Isoform-selective inhibition
  • Isothioureas
  • Mercaptoalkylguanides
  • Nitric oxide
  • Shock
  • Vasoconstriction
  • Vasodilatation

ASJC Scopus subject areas

  • Pharmacology

Cite this

Spontaneous rearrangement of aminoalkylisothioureas into mercaptoalkylguanidines, a novel class of nitric oxide synthase inhibitors with selectivity towards the inducible isoform. / Southan, Garry J.; Zingarelli, Basilia; O'Connor, Michael; Salzman, Andrew L.; Szabo, Csaba.

In: British Journal of Pharmacology, Vol. 117, No. 4, 1996, p. 619-632.

Research output: Contribution to journalArticle

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abstract = "1 The generation of nitric oxide (NO) from L-arginine by NO synthases (NOS) can be inhibited by guanidines, amidines and S-alkylisothioureas. Unlike most L-arginine based inhibitors, however, some guanidines and S-alkylisothioureas, in particular aminoethylisothiourea (AETU), show selectivity towards the inducible isoform (iNOS) over the constitutive isoforms (endothelial, ecNOS and brain isoform, bNOS) and so may be of therapeutic benefit. In the present study we have investigated the effects of AETU and other aminoalkylisothioureas on the activities of iNOS, ecNOS and bNOS. 2 AETU, aminopropylisothiourea (APTU) and their derivatives containing alkyl substituents on one of the amidino nitrogens, potently inhibit nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values ranging from 6-30 μM (EC50 values for N(G)-methyl-L-arginine (L-NMA) and N(G)-nitro-L-arginine were 159 and > 1000 μM, respectively). The inhibitory effects of these aminoalkylisothioureas (AATUs) were attentuated by L-arginine in the incubation medium, indicating that these agents may compete with L-arginine for its binding site on NOS. 3 The above AATUs undergo chemical conversion in neutral or basic solution (pH 7 or above) as indicated by (1) the disappearance of AATUs from solution as measured by h.p.l.c., (2) the generation of free thiols not previously present and (3) the isolation of species (as picrate and flavianate salts) from neutral or basic solutions of AATUs that are different from those obtained from acid solutions. 4 Mercaptoalkylguanidines (MAGs) were prepared and shown to be potent inhibitors of iNOS activity with EC50s comparable to those of their isomeric AATUs. 5 These findings suggest that certain AATUs exert their potent inhibitory effects through intramolecular rearrangement to mercaptoalkylguanidines (MAGs) at physiological pH. Those AATUs not capable of such rearrangement do not exhibit the same degree of inhibition of iNOS. 6 In contrast to their potent effects on iNOS, some AATUs and MAGs were 20-100 times weaker than N(G)-methyl-L-arginine and N(G)-nitro-L-arginine as inhibitors of ecNOS as assessed by their effects on the conversion of L-arginine to L-citrulline in homogenates of bovine endothelial cells and by their presser effects in anaesthetized rats. Thus mercaptoalkylguanidines represent a new class of NOS inhibitors with preference towards iNOS. 7 AETU and mercaptoethylguanidine (MEG), when given as infusions, gave slight decreases in MAP in control rats. However, infusions of AETU or MEG to endotoxin-treated rats caused an increase in MAP and restored 80{\%} of the endotoxin-induced fall in MAP. 8 High doses of MEG (30-60 mg kg-1) caused a decrease in MAP of normal rats. This depressor effect may be a consequence of the in vivo oxidation of MEG to the disulphide, guanidinoethyldisulphide (GED), which caused pronounced, transient hypotensive responses in anaesthetized rats and caused endothelium-independent vasodilator responses in precontracted rat aortic rings in vitro. 9 In some cases, slight differences were observed in the activities of AATUs and the corresponding MAGs. These may be explained by the formation of other species from AATUs in physiological media. For example, AETU can give rise to small amounts of the potent ecNOS inhibitor, 2-aminothiazoline, in addition to MEG. This may account for the differences in the in vitro and in vivo effects of AETU and MEG. 10 In conclusion, the in vitro and in vivo effects of AETU and related aminoalkylisothioureas can be explained in terms of their intramolecular rearrangement to generate mercaptoalkylguanidines, a novel class of selective inhibitors of iNOS.",
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T1 - Spontaneous rearrangement of aminoalkylisothioureas into mercaptoalkylguanidines, a novel class of nitric oxide synthase inhibitors with selectivity towards the inducible isoform

AU - Southan, Garry J.

AU - Zingarelli, Basilia

AU - O'Connor, Michael

AU - Salzman, Andrew L.

AU - Szabo, Csaba

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N2 - 1 The generation of nitric oxide (NO) from L-arginine by NO synthases (NOS) can be inhibited by guanidines, amidines and S-alkylisothioureas. Unlike most L-arginine based inhibitors, however, some guanidines and S-alkylisothioureas, in particular aminoethylisothiourea (AETU), show selectivity towards the inducible isoform (iNOS) over the constitutive isoforms (endothelial, ecNOS and brain isoform, bNOS) and so may be of therapeutic benefit. In the present study we have investigated the effects of AETU and other aminoalkylisothioureas on the activities of iNOS, ecNOS and bNOS. 2 AETU, aminopropylisothiourea (APTU) and their derivatives containing alkyl substituents on one of the amidino nitrogens, potently inhibit nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values ranging from 6-30 μM (EC50 values for N(G)-methyl-L-arginine (L-NMA) and N(G)-nitro-L-arginine were 159 and > 1000 μM, respectively). The inhibitory effects of these aminoalkylisothioureas (AATUs) were attentuated by L-arginine in the incubation medium, indicating that these agents may compete with L-arginine for its binding site on NOS. 3 The above AATUs undergo chemical conversion in neutral or basic solution (pH 7 or above) as indicated by (1) the disappearance of AATUs from solution as measured by h.p.l.c., (2) the generation of free thiols not previously present and (3) the isolation of species (as picrate and flavianate salts) from neutral or basic solutions of AATUs that are different from those obtained from acid solutions. 4 Mercaptoalkylguanidines (MAGs) were prepared and shown to be potent inhibitors of iNOS activity with EC50s comparable to those of their isomeric AATUs. 5 These findings suggest that certain AATUs exert their potent inhibitory effects through intramolecular rearrangement to mercaptoalkylguanidines (MAGs) at physiological pH. Those AATUs not capable of such rearrangement do not exhibit the same degree of inhibition of iNOS. 6 In contrast to their potent effects on iNOS, some AATUs and MAGs were 20-100 times weaker than N(G)-methyl-L-arginine and N(G)-nitro-L-arginine as inhibitors of ecNOS as assessed by their effects on the conversion of L-arginine to L-citrulline in homogenates of bovine endothelial cells and by their presser effects in anaesthetized rats. Thus mercaptoalkylguanidines represent a new class of NOS inhibitors with preference towards iNOS. 7 AETU and mercaptoethylguanidine (MEG), when given as infusions, gave slight decreases in MAP in control rats. However, infusions of AETU or MEG to endotoxin-treated rats caused an increase in MAP and restored 80% of the endotoxin-induced fall in MAP. 8 High doses of MEG (30-60 mg kg-1) caused a decrease in MAP of normal rats. This depressor effect may be a consequence of the in vivo oxidation of MEG to the disulphide, guanidinoethyldisulphide (GED), which caused pronounced, transient hypotensive responses in anaesthetized rats and caused endothelium-independent vasodilator responses in precontracted rat aortic rings in vitro. 9 In some cases, slight differences were observed in the activities of AATUs and the corresponding MAGs. These may be explained by the formation of other species from AATUs in physiological media. For example, AETU can give rise to small amounts of the potent ecNOS inhibitor, 2-aminothiazoline, in addition to MEG. This may account for the differences in the in vitro and in vivo effects of AETU and MEG. 10 In conclusion, the in vitro and in vivo effects of AETU and related aminoalkylisothioureas can be explained in terms of their intramolecular rearrangement to generate mercaptoalkylguanidines, a novel class of selective inhibitors of iNOS.

AB - 1 The generation of nitric oxide (NO) from L-arginine by NO synthases (NOS) can be inhibited by guanidines, amidines and S-alkylisothioureas. Unlike most L-arginine based inhibitors, however, some guanidines and S-alkylisothioureas, in particular aminoethylisothiourea (AETU), show selectivity towards the inducible isoform (iNOS) over the constitutive isoforms (endothelial, ecNOS and brain isoform, bNOS) and so may be of therapeutic benefit. In the present study we have investigated the effects of AETU and other aminoalkylisothioureas on the activities of iNOS, ecNOS and bNOS. 2 AETU, aminopropylisothiourea (APTU) and their derivatives containing alkyl substituents on one of the amidino nitrogens, potently inhibit nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values ranging from 6-30 μM (EC50 values for N(G)-methyl-L-arginine (L-NMA) and N(G)-nitro-L-arginine were 159 and > 1000 μM, respectively). The inhibitory effects of these aminoalkylisothioureas (AATUs) were attentuated by L-arginine in the incubation medium, indicating that these agents may compete with L-arginine for its binding site on NOS. 3 The above AATUs undergo chemical conversion in neutral or basic solution (pH 7 or above) as indicated by (1) the disappearance of AATUs from solution as measured by h.p.l.c., (2) the generation of free thiols not previously present and (3) the isolation of species (as picrate and flavianate salts) from neutral or basic solutions of AATUs that are different from those obtained from acid solutions. 4 Mercaptoalkylguanidines (MAGs) were prepared and shown to be potent inhibitors of iNOS activity with EC50s comparable to those of their isomeric AATUs. 5 These findings suggest that certain AATUs exert their potent inhibitory effects through intramolecular rearrangement to mercaptoalkylguanidines (MAGs) at physiological pH. Those AATUs not capable of such rearrangement do not exhibit the same degree of inhibition of iNOS. 6 In contrast to their potent effects on iNOS, some AATUs and MAGs were 20-100 times weaker than N(G)-methyl-L-arginine and N(G)-nitro-L-arginine as inhibitors of ecNOS as assessed by their effects on the conversion of L-arginine to L-citrulline in homogenates of bovine endothelial cells and by their presser effects in anaesthetized rats. Thus mercaptoalkylguanidines represent a new class of NOS inhibitors with preference towards iNOS. 7 AETU and mercaptoethylguanidine (MEG), when given as infusions, gave slight decreases in MAP in control rats. However, infusions of AETU or MEG to endotoxin-treated rats caused an increase in MAP and restored 80% of the endotoxin-induced fall in MAP. 8 High doses of MEG (30-60 mg kg-1) caused a decrease in MAP of normal rats. This depressor effect may be a consequence of the in vivo oxidation of MEG to the disulphide, guanidinoethyldisulphide (GED), which caused pronounced, transient hypotensive responses in anaesthetized rats and caused endothelium-independent vasodilator responses in precontracted rat aortic rings in vitro. 9 In some cases, slight differences were observed in the activities of AATUs and the corresponding MAGs. These may be explained by the formation of other species from AATUs in physiological media. For example, AETU can give rise to small amounts of the potent ecNOS inhibitor, 2-aminothiazoline, in addition to MEG. This may account for the differences in the in vitro and in vivo effects of AETU and MEG. 10 In conclusion, the in vitro and in vivo effects of AETU and related aminoalkylisothioureas can be explained in terms of their intramolecular rearrangement to generate mercaptoalkylguanidines, a novel class of selective inhibitors of iNOS.

KW - Blood pressure

KW - EDRF

KW - Endotoxin

KW - Isoform-selective inhibition

KW - Isothioureas

KW - Mercaptoalkylguanides

KW - Nitric oxide

KW - Shock

KW - Vasoconstriction

KW - Vasodilatation

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