WO1997045108A1

WO1997045108A1 - Guanidino derivatives as inhibitors of the cytotoxic effect of peroxynitrite

Info

Publication number: WO1997045108A1
Application number: PCT/US1997/008280
Authority: WO
Inventors: Garry J. Southan; Andrew L. Salzman; Csaba Szabo
Original assignee: Children's Hospital Medical Center
Priority date: 1996-05-30
Filing date: 1997-05-16
Publication date: 1997-12-04
Also published as: AU3128197A

Abstract

This invention is directed to a pharmacologically acceptable composition for inhibiting the cytotoxic effect of peroxynitrite in a mammal, which includes a guanidino derivative and a pharmaceutically acceptable carrier. The invention also concerns a method of inhibiting the cytotoxic effect of peroxynitrite, and treating various conditions where there is an advantage in inhibiting the cytotoxic effect of peroxynitrite. The method includes the step of administering to a mammal a guanidino derivative in pure form or in a pharmaceutically acceptable carrier.

Description

GUANIDINO DERIVATIVES AS INHIBITORS OF THE CYTOTOXIC EFFECT OF PEROXYNITRITE

Background of the Invention

The present invention relates to the use of guanidino

derivatives as inhibitors of the cytotoxic effect of peroxynitrite.

Peroxynitrite is a reactive oxidant produced from nitric

oxide (NO) and superoxide, which reacts with proteins, lipids and

DNA under conditions of inflammation and shock.

Immunohistochemical and biochemical evidence demonstrate

production of peroxynitrite in endotoxic and hemorrhagic shock,

chronic bowel inflammation, and in various forms of ischemia-

reperfusion injury. The reactivity and decomposition of peroxynitrite

is determined by the chemical environment, and the ratio of

superoxide vs. NO. Peroxynitrite can initiate toxic oxidative

reactions in vitro and in vivo, initiation of iipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of

glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane

Na⁺/K⁺ ATP-ase activity, inactivation of membrane sodium channels,

and other oxidative protein modifications contribute to the cytotoxic

effect of peroxynitrite. In addition, peroxynitrite is a potent trigger of

DNA strand breakage, with subsequent activation of the nuclear

enzyme poly-ADP ribosyl synthetase (PARS), with eventual severe

energy depletion of the cells.

There is now good experimental evidence that inhibition

of the formation of peroxynitrite and/or scavenging peroxynitrite is

beneficial in a number of pathophysiological conditions, including

circulatory shock and ischemia-reperfusion injury. Theoretically,

peroxynitrite-mediated cytotoxicity can be prevented in a number of

ways. First, inhibition of nitric oxide biosynthesis would reduce the

formation of nitric oxide, and, consequently, of peroxynitrite.

Second, scavenging superoxide or enhancing its decomposition (with

agents such as superoxide dismutase) also would reduce the

production of peroxynitrite. A third approach would be the use of

agents which would directly inhibit the oxidative reactions and

cytotoxic effect triggered by peroxynitrite. While various inhibitors

of the first two approaches have been patented and proposed for

therapeutic use, the available tools to directly inhibit peroxynitrite-

induced oxidative processes is limited. Therefore, it would be extremely beneficial to have a composition and/or method for directly inhibiting peroxynitrite's oxidative reactions and cytotoxic effect. Summary of the Invention

This invention is directed to a pharmacologically acceptable composition for inhibiting the cytotoxic effect of peroxynitrite in a mammal. The composition includes a guanidino derivative and a pharmaceutically acceptable carrier, with the guanidino derivative present in the composition in an effective amount to inhibit the cytotoxic effect of peroxynitrite in the mammal.

The invention also is directed to a method of inhibiting the cytotoxic effect of peroxynitrite in a mammal, which includes the

step of administering to the mammal a guanidino derivative in a pure form or in a pharmaceutically acceptable carrier.

The guanidino derivative of the composition and method is defined by a formula selected from the group consisting of:

and

or a salt thereof, wherein:

R₂ and R'₂ are independently H, lower alkyl, alkenyl, alkylene, alkenylene, amino, aminoalkyl, hydroxy, alkoxy, thioalkyiene, thioesteralkylene, phenyl or phenylalkylene, or a substituted derivative thereof;

R₃ and R'₃ are independently H, lower alkyl, alkylene,

alkenylene, amino, hydroxy, thioalkyiene, or a substituted derivative thereof; R_{1 f} R₆, R'_B# R₄ and R'₄ are independently H, alkyl, alkenyl, phenyl, alkylene, alkenylene, phenylalkylene or amino, or a substituted derivative thereof;

Alternatively, R, is R_β-Y-Z- where R_β is H, alkyl, alkenyl,

phenyl, alkylene, alkenylene, phenylalkylene, acyl, -S0₃ ^", or -P0₃ ^", or a substituted derivative thereof, and Z and Y are as defined below;

Z and Z' are independently alkylene, alkenylene, cycloalkylene or cycloalkenylene, or a substituted derivative thereof; Y and Y' are independently S or Se; When R₂ or R'₂ is alkylene, alkenylene, thioalkyiene,

amino, hydroxy or a substituted derivative thereof, said R₂ or R'₂

may be joined to any of:

(i) R₃ or R'₃, if R₃ or R'₃ is alkylene, alkenylene or

thioalkyiene;

(ii) R₄ or R'₄, if R₄ or R'₄ is alkylene or alkenylene; or

(iii) R₆ or R'₅, if R₅ or R'_ε is alkylene or alkenylene;

to form 5-, 6-, or 7-membered heterocycle;

When R₂, R₃, R'₂ or R'₃ is alkylene or alkenylene, said

R₂, R₃, R'₂ or R'₃ optionally may be joined to the adjacent Z or Z' to

form a 5- or 6-membered heterocyclic ring, with the proviso that said

heterocyclic ring optionally is substituted with a lower alkyl, alkoxy,

halo, hydroxy or amino;

When R, and R₄ are alkylene or alkenylene, said R, and

R₄ optionally may be joined together to form a 5-, 6-, or 7-membered

heterocycle;

When R, is R₆-Y-Z-, and R₆ is alkylene or alkenylene, R₆

optionally may be joined to any of:

(i) R₂, when R₂ is alkylene, alkenylene or thioalkyiene;

(ii) Z; or

(iii) R₄, when R₄ is alkylene or alkenylene;

to form a 5-, 6- or 7-membered heterocyclic ring; and a pharmaceutically acceptable carrier, said compound

present in said composition in an effective amount to inhibit the

cytotoxic effect of peroxynitrite in said mammal.

As used herein, the term "salt" refers to any addition

salt derived from any pharmaceutically acceptable organic or

inorganic acid. Examples of suitable acids include hydrochloric,

hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,

glycolic, lactic, salicylic, succinic, toluene p sulfonic, tartaric, acetic,

citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2-

sulfonic and benzenesulphonic acids. Additionally, as used herein,

any alkyl or alkylene may be straight chain, branched or cyclic, and

"halo" includes bromine, chlorine, fluorine and iodine.

In the descriptions mentioned above, a substituted

derivative of R₂, R₃, R'₂ and R'₃ refers to a derivative that may be

substituted with one or more alkoxy, halo, hydroxy and amino

groups. A substituted derivative of R_{1 (} R₅, R'₅, R₄ and R'₄ refers to a

derivative that may be substituted with one or more alkyl, alkoxy,

halo, hydroxy, amino, amino alkyl (secondary or tertiary), thio and

nitro groups.

If the R₂, R'₂, R₃ or R'₃ substituent is thioalkyiene, the

thioalkyiene preferably has a formula [-(CH₂)_n-SH] where n is

independently 1 to 4. If the R₂, or R'₂ substituent is

thioesteralkylene, the thioesteralkylene preferably has the formula [-(CH₂)_n-S-R₇] where R₇ is independently a lower alkyl and n is

independently 1 to 4.

The Z and Z' substituents of the guanidino derivative

are independently alkylene, alkenylene, cycloalkyene or

cycloalkenlyene, or a substituted derivative thereof. When such a

substituted derivative is employed, the substituent may include one

or more of lower alkyl, alkoxy, halo, amino, nitro or carboxyl.

Brief Description of the Drawings

Fig. 1 is a graph of the inhibitory effect of

mercaptoethylguanidine (MEG), S-methyl-mercaptoethylguanidine

(SMEG), guanidinoethyldisuifide (GED) and aminoguanidine (AG) on

the oxidation of dihydrorhodamine 1 23 (DHR) by peroxynitrite;

Fig. 2A is a graph of the protective effect of

aminoguanidine (AG) on mitochondrial respiration in J774

macrophages exposed to peroxynitrite;

Fig. 2B is a graph of the protective effect of

mercaptoethylguanidine (MEG), on mitochondrial respiration in J774

macrophages exposed to peroxynitrite;

Fig. 2C is a graph of the protective effect of

guanidinoethyldisuifide (GED) on mitochondrial respiration in J774

macrophages exposed to peroxynitrite; and

Fig. 3 is a graph showing the protective effect of

aminoguanidine (AG), mercaptoethylguanidine (MEG) or guanidinoethyldisulfide (GED) against the DNA single strand breakage

induced by peroxynitrite.

Fig. 4 is a graph showing the protective effect of

mercaptoethylguanidine (MEG) on the peroxynitrite-induced

suppression of vascular contractility.

Detailed Description of the Invention

This invention is directed to a pharmacologically

acceptable composition for inhibiting the cytotoxic effect of

peroxynitrite in a mammal. The composition includes a guanidino

derivative and a pharmaceutically acceptable carrier, with the

guanidino derivative present in the composition in an effective

amount to inhibit the cytotoxic effect of peroxynitrite in the mammal.

The invention also is directed to a method of inhibiting the cytotoxic

effect of peroxynitrite in a mammal, which includes the step of

administering to the mammal a guanidino derivative in pure form or

in a pharmaceutically acceptable carrier.

Suitable guanidino derivatives for use in the composition

or method are made according to the methods of synthesis taught in

the following articles which are incorporated herein in their entirety

by reference: ( 1 ) Joseph X. Khym et al., "Ion Exchange Studies of

Transguanylation Reactions. I. Rearrangement of S,2-

Aminoethylisothiourea to 2-Mercaptoethylguanidine and 2-

Aminothiazoline", Journal of the American Chemical Society, Vol. 79, pp. 5663-5666, November 5, 1 957; (2) David G. Doherty,

et al., "Synthesis of Aminoalkylisothiuronium Salts and their

Conversion to Mercaptoalkylguanidines and Thiazolines", Journal of

the American Chemical Society, Vol. 79, pp. 5667-5671 , November

5, 1 957; (3) Joseph X. Khym, et al., "Ion Exchange Studies of

Transguanylation Reactions. II. Rearrangement of 3-

Aminopropylisothiourea and N-Substituted Aminoethyl- and

Aminopropylisothioureas to Mercaptoalkylguanidines and 2-

Aminothiazolines or Penthiazolines", Journal of the American

Chemical Society, Vol. 80, pp. 3342-3349, July 5, 1 958;

(4) David G. Doherty et al. "Synthesis of D- and L-2-

Aminobutylisothiourea Dihydrobromide Isomers and their Conversion

to Guanidothiols, Disulfides, and Thiazolines", Journal of Organic

Chemistry, Vol. 28, pp. 1 339- 1 342, 1 963; (5) Shih-Hsi Chu et al.,

"Potential Antiradiation Agents. II. Selenium Analogs of 2-

Aminoethylisothiouronium Hydrobromide and Related Compounds",

Journal of the American Chemical Society, Vol. 27, pp. 2899-2901 ,

August, 1 962; (6) Tohru Hino et al., "Radiation-protective Agents.

I. Studies on N-Alkylated-2-(2-aminoethyl)thiopseudoureas and 1 , 1 -

(Dithioethylene)diguanidines", Chemical & Pharmaceutical Bulletin,

Vol. 14, No. 1 1 , pp. 1 1 93-1 201 , November, 1 966. Suitable guanidino derivatives also are made according

to the examples provided at the end of this detailed description of

the invention.

In addition, suitable guanidino derivatives are made as

indicated below:

General methods for the preparation of guanidines

involve the reaction of amines with isoureas, isothioureas, sulphinic

or sulphonic acid derivatives of thioureas, or with other guanylating

agents such as cyanamides and pyrazole-N-carboxamidines.

Relevant examples are found in Bannard et al., Can. J. Chem., 36,

1 541 -1 549 (1 958); Walton, United Kingdom Patent No. 1 084 461

(1 964); Bernatowicz et al., J. Org. Chem. , 57, 2497-2502 ( 1 992);

Maryanoff et al., J. Org. Chem. , 51 , 1 882-1 884 (1 986). Further

relevant examples are referenced in Yamamoto & Kojima in "The

Chemistry of Amidines and Imidates", J. Wiley & Sons ( 1 991 ),

pp. 485-526.

Hydroxyguanidine is prepared by the method of Walker

& Walker, J. Biol. Chem. , 234, 1481 -1484 (1 959).

1 -substituted hydroxyguanidines are prepared by the

reaction of hydroxylamine with cyanamides or 1 -substituted

isothioureas. Relevant examples are described in: Fukuto et al.,

Biochem Pharmacol. , 43, 607-61 3 (1 992); Sennequier et al., 7ef.

Lett. , 36, 6059-6062 (1 995); Yoo & Fukuto, Biochem. Pharm. , 50, 1995-2000 (1995); Aurich & Scharpenberg, Chem. Ber., 106, 1881-

1896, (1973); Butler et al., The Biology of Nitric Oxide. Part 5,

Portland Press, London, (1996), p. 179.

1,1-disubstituted hydroxyguanidines are prepared by the

reaction of hydroxylamine with cyanamides or 1,1-disubstituted

isothioureas. Relevent examples are found in: Fukuto et al.,

Biochem. Pharmacol., 43, 607-613 (1992); Belzeki et al., Bulletin De

L ' a cad a mi e Polonaise Des Sciences, Serie de sciences chimiques xviii

(7), 375-378, (1970).

1 ,3-substituted hydroxyguanidines are prepared by the

reaction of hydroxylamine with carbodiimides or N-substituted

isothioureas. Relevent examples are found in: Miller et al., Synth.

Comm., 20, 217-226 (1990); Belzecki et al., Bulletin De L'acadamie

Polonaise Des Sciences, Serie de sciences chimiques xx, 6, 499-

503, (1972).

Higher substituted hydroxyguanidines are prepared by

the reaction of hydroxylamine or N-substituted derivatives thereof

with appropriately substituted isothioureas, carbodiimides or

C-chloroformamidinium chlorides. Relevent examples are contained

in: Vob et al., Z. Chem., 13, 58 (1973); Ziman, J. Org. Chem., 41,

3258 (1976); Gross et al., J. F. Prakt. Chemie., 316, 434-442

(1974); Gross et al., Ger. Often. #2040628 (1972); also U. S.

Patent No.4,000,196. Aminoguanidines are prepared, for example, by the

reaction of amines with S-methyl or S-ethyl-isothiosemicarbazide or

N-substituted derivative thereof. Relevent examples are found in:

Lieber & Smith, Chem. Rev. , 25, 21 3-277 (1 939) , (and references

therein); Ruetten et al., Br. J. Pharmacol. , 1 1 7, (1 996), (in press);

European Patent Application No. 89 107 1 68.0 (filed April 20,

1 989); U. S. Patent No. 3,972,932; U. S. Patent No. 210,291 .

In addition, aminoguanidines are prepared by the reaction of

hydrazines, or substituted derivatives thereof, with

S-alkylisothioureas or N-substituted derivatives thereof . Relevent

examples are found in : Lieber & Smith, Chem. Rev. , 25, 21 3-277

(1 939), (and references therein); Kirsten & Smith, J. Am. Chem.

Soc , 58, 800-802 (1 936).

Aminohydroxyguanidines are prepared by the reaction of

hydroxylamine or an N-substituted derivative thereof with S-methyl

or S-ethyl- isothiosemicarbazide or N-substituted derivatives thereof.

Relevent examples are found in: Houlihan et al., U. S. Patent

No. 3,927,096; Tai et al., J. Med. Chem. , 27, 236-238 ( 1 984) .

Mercaptoalkylguanidines and their S-substituted

derivatives are prepared by reacting appropriate mercaptoalkylamine

or S-substituted mercaptoalkylamine with suitable guanylating

reagent. Relevent examples are found in: Southan et al., Br. J.

Pharmacol., 1 1 7, 619-633 (1 996). Mercaptoethyl- and mercaptopropyl-guanidine, and their

N-substituted derivatives, are prepared by neutralizing a solution of

the appropriate N^G-substituted aminoethyl- or aminopropyl-

isothiourea to induce rearrangement of the latter into the former.

Said N^G-substituted aminoethyl- or aminopropyl-isothioureas are

prepared by alkylation of thiourea with aminoalkylhalides, preferably

the bromide. Relevent examples are found in: Khym et al., J. Am.

Chem. Soc, 79, 5663-5666 (1957); Doherty & Shapira, J. Org.

Chem., 28, 1339-1342 (1963); Khym et al., J. Am. Chem. Soc,

80, 342-3349 (1958); Doherty et al., J. Am. Chem. Soc, 79, 5667-

5671 (1957); Hino et al., Chem. & Pharm. Bull., 14, 1193-1201

(1966); Shapira et al., Rad. Res., 7, 22-34 (1957); DiStephano et

al., Rad. Res., 18, 177-185 (1963); Southan et al., Br. J.

Pharmacol., 117, 619-633 (1996).

Selenoethyl- or selenopropylguanidine or their N-

substituted derivatives are prepared by neutralizing a solution of the

appropriate N^G-substituted aminoethyl- or aminopropyl-isoselenourea

to induce rearrangement of latter into the former. Said N^G-

substituted aminoethyl- or aminopropyl-isoselenoureas are prepared

by alkylation of selenourea with aminoalkylhalides, preferably the

bromide. Relevent examples are found in: Chu & Mautner, J. Org.

Chem., 27, 2899-2901 (1962); Southan et al., Life Sci., 58, 1139-

1148 (1996). Heterocyclic guanidine derivatives are prepared as

shown in: Schantl & Turk, Sci. Pharm. (scientia pharmaceutica), 57,

375-380 (1 989); Fukuto et al., Biochem. Pharmacol. , 43, 607-61 3

( 1 992); Belzecki et al., Bulletin De L 'acadamie Polonaise, Serie de

sciences chimiques xviii (7), 375-378 (1 970); Belzecki & Trojnar,

Bulletin De L 'acadamie Polonaise Des Sciences, Serie de sciences

chimiques xviii (8) , 437-440 (1 970); Belzecki & Trojnar, Tet. Lett. ,

22, 1 879-1 880 ( 1 970).

The guanidino derivative of the composition and method

is defined by a formula selected from the group consisting of:

and

or a salt thereof, wherein:

R₂ and R'₂ are independently H, lower alkyl, alkenyl,

alkylene, alkenylene, amino, aminoalkyl, hydroxy, alkoxy, thioalkylene, thioesteralkylene, phenyl or phenylalkylene, or a

substituted derivative thereof;

R₃ and R'₃ are independently H, lower alkyl, alkylene,

alkenylene, amino, hydroxy, thioalkyiene, or a substituted derivative

thereof;

R_v R₅, R'_5/ R₄ and R'₄ are independently H, alkyl,

alkenyl, phenyl, alkylene, alkenylene, phenylalkylene or amino, or a

substituted derivative thereof;

Alternatively, R, is R₆-Y-Z- where R₆ is H, alkyl,

alkenyl, phenyl, alkylene, alkenylene, phenylalkylene, acyl, -SO₃ ^", or

-PO₃ ^", or a substituted derivative thereof, and Z and Y are as defined

below;

Z and Z' are independently alkylene, alkenylene,

cycloalkylene or cycloalkenylene, or a substituted derivative thereof;

Y and Y' are independently S or Se;

When R₂ or R'₂ is alkylene, alkenylene, thioalkyiene,

amino, hydroxy or a substituted derivative thereof, said R₂ or R'₂

may be joined to any of:

(i) R₃ or R'₃, if R₃ or R'₃ is alkylene, alkenylene or

thioalkyiene;

(ii) R₄ or R'₄, if R₄ or R'₄ is alkylene or alkenylene; or

(iii) R₅ or R'₆, if R₅ or R'₅ is alkylene or alkenylene;

to form 5-, 6-, or 7-membered heterocycle; When R₂, R₃, R'₂ or R'₃ is alkylene or alkenylene, said

R₂, R₃, R'₂ or R'₃ optionally may be joined to the adjacent Z or Z' to

form a 5- or 6-membered heterocyclic ring, with the proviso that said

heterocyclic ring optionally being substituted with a lower alkyl,

alkoxy, halo, hydroxy or amino;

When R, and R₄ are alkylene or alkenylene, said R, and

R₄ optionally may be joined together to form a 5-, 6-, or 7-membered

heterocycle;

When R_T is R₆-Y-Z-, and R_e is alkylene or alkenylene, R₆

optionally may be joined to any of:

(i) R₂, when R₂ is alkylene, alkenylene or thioalkyiene;

(ii) Z; or

(iii) R₄, when R₄ is alkylene or alkenylene;

to form a 5-, 6- or 7-membered heterocyclic ring; and

a pharmaceutically acceptable carrier, said compound

present in said composition in an effective amount to inhibit the

cytotoxic effect of peroxynitrite in said mammal.

As used herein, the term "salt" refers to any addition

salt derived from any pharmaceutically acceptable organic or

inorganic acid. Examples of suitable acids include hydrochloric,

hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,

glycolic, lactic, salicylic, succinic, toluene p sulfonic, tartaric, acetic,

citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2- sulfonic and benzenesulphonic acids. Additionally, as used herein,

any alkyl or alkylene may be straight chain, branched or cyclic, and

"halo" includes bromine, chlorine, fluorine and iodine.

In the descriptions mentioned above, a substituted

derivative of R₂, R₃, R'₂ and R'₃ refers to a derivative that may be

substituted with one or more alkoxy, halo, hydroxy and amino

groups. A substituted derivative of R_{1 r} R₅, R'₆, R₄ and R'₄ refers to a

derivative that may be substituted with one or more alkyl, alkoxy,

halo, hydroxy, amino, amino alkyl (secondary or tertiary), thio and

nitro groups.

If the R₂, R'₂, R₃ or R'₃ substituent is thioalkyiene, the

thioalkyiene preferably has a formula [-(CH₂)_n-SH] where n is

independently 1 to 4. If the R₂, or R'₂ substituent is

thioesteralkylene, the thioesteralkylene preferably has the formula

[-(CH₂)_n-S-R₇] where R₇ is independently a lower alkyl and n is

independently 1 to 4.

The Z and Z' substituents of the guanidino derivative

are independently alkylene, alkenylene, cycloalkyene or

cycloalkenlyene, or a substituted derivative thereof. When such a

substituted derivative is employed, the substituent may include one

or more of lower alkyl, alkoxy, halo, amino, nitro or carboxyl.

A preferred subgroup of derivatives includes compounds

where: R₂ and R'₂ are independently H, lower alkyl, amino, aminoalkyl, hydroxy, phenyl, phenylalkylene, or a substituted

derivative thereof; R₃ and R'₃ are independently H, lower alkyl, amino

or hydroxy; R₅ and R'₅ , R₄ and R'₄ are independently H or lower

alkyl; R-i is H, lower alkyl or R₆-Y-Z- where R₆ is H, lower alkyl, acyl,

-SO₃ ^" , -PO₃ ^" , or a substituted derivative thereof; Z and Z' are

independently alkylene, optionally substituted with one or more

substituents chosen from lower alkyl or carboxylic acid; and Y and Y'

are S.

Another preferred subgroup includes guanidino

derivatives where: R, is H or alkyl; R₂ is H, amino, hydroxy,

methoxy or ethoxy; R₃ is H, lower alkyl or amino; R₄ is H; and R₅ is H

or lower alkyl. A few nonlimiting examples include aminoguanidine,

hydroxyguanidine, 1 -amino-2-hydroxyguanidine, 1 -amino-2-methyl-2-

hydroxyguanidine and diaminoguanidine.

Yet another preferred subgroup includes mercapto

derivatives where: R, is R₆-Y-Z- where R₆ is H, acyl, -S0₃ , -PO₃ ^" or

lower alkyl, R₂ and R'₂ are independently H or amino; R₃ and R'₃ are

independently H, amino or hydroxy; R₄ and R'₄ are independently H,

methyl or ethyl; Z and Z' are independently alkylene optionally

substituted with one or more methyl; and Y is S. A few nonlimiting

examples include mercaptoethylguanidine, mercaptopropylguanidine,

S-methylpropylguanidine, mercaptoethylguanidine-S-phosphoric acid

(S-(guanidinoethyl) phosphorothioic acid) and guanidinoethyldisuifide. The guanidino derivative, in pure form or in a

pharmaceutically acceptable carrier, will find benefit in treating

conditions and disorders where there is an advantage in inhibiting the

cytotoxic effect of peroxynitrite. For example, the guanidino

derivative may be used to treat a circulatory shock including its

various aspects such as vascular and myocardial dysfunction,

metabolic failure including the inhibition of mitochondrial enzymes

and cytochrome P450-mediated drug metabolism, and multiple organ

dysfunction syndrome including adult respiratory distress syndrome.

Circulatory shock may be a result of gram-negative and gram positive

sepsis, trauma, hemorrhage, burn injury, anaphylaxis, cytokine

immunotherapy, liver failure, kidney failure or systemic inflammatory

response syndrome. Guanidino derivatives also may be beneficial for

patients receiving therapy with cytokines such as TNF, IL-1 and IL-2

or therapy with cytokine-inducing agents, or as an adjuvant to short

term immunosuppression in transplant therapy.

Peroxynitrite is formed and is cytotoxic in the

reperfusion phase of various forms of ischemia-reperfusion injury.

There is, therefore, a distinct advantage in inhibiting the cytotoxic

effect of peroxynitrite in these conditions. The pathophysiological

conditions associated with ischemia-reperfusion injury include

myocardial ischemia and infarction, cardiopulmonary bypass,

noninflammatory diseases of the central nervous system (CNS) including stroke (a CNS ischemic disorder) and cerebral ischemia, and

hemorrhagic shock.

There is also evidence that peroxynitrite may be

involved in the pathophysiology of autoimmune and/or inflammatory

conditions such as arthritis, rheumatoid arthritis, inflammatory

bowel disease and myocarditis, systemic lupus erythematosus (SLE)

and insulin-dependent diabetes mellitus, and therefore, guanidino

derivatives may prove helpful in treating these conditions.

Furthermore, it is now clear that there are a number of

additional inflammatory and noninflammatory diseases and conditions

that may be associated with peroxynitrite production. Examples of

such physiological disorders include: inflammatory bowel diseases

such as ileitis and Crohn's disease; inflammatory lung disorders such

as asthma and chronic obstructive airway disease; inflammatory

disorders of the eye including corneal dystrophy, trachoma,

onchocerciasis, uveitis, sympathetic ophthalmitis and

endophthalmitis; chronic inflammatory disorders of the gum including

periodontitis; chronic inflammatory disorders of the joints including

arthritis and osteoarthritis, tuberculosis, leprosy, glomerulonephritis

sarcoid, and nephrosis; disorders of the skin including

sclerodermatitis, psoriasis and eczema; inflammatory diseases of the

central nervous system, including chronic demyelinating diseases

such as multiple sclerosis, dementia including AIDS-related neurodegeneration and Alzheimer's disease, encephaiomyelitis and

viral or autoimmune encephalitis; autoimmune diseases including

immune-complex vasculitis, systemic lupus and erythematodes; and

disease of the heart including ischemic heart disease and

cardiomyopathy. Additional diseases and conditions which may

benefit from the use of the guanidino derivative include adrenal

insufficiency; hypercholesterolemia; atherosclerosis; bone disease

associated with increased bone resorption, e.g., osteoporosis, pre¬

eclampsia, eclampsia, uremic complications, chronic liver failure,

various forms of cancer, Parkinson's disease, spinal cord trauma and

head trauma.

In addition, the guanidino derivative may be used as a

preservative solution, or as an additive to a preservative solution, for

use in preserving a harvested organ prior to transplantation.

Pharmaceutical formulations of the guanidino derivative

may include those suitable for oral, rectal, nasal, topical (including

buccal and sub-lingual), vaginal or parenteral (including

intramuscular, sub-cutaneous and intravenous) administration, or for

administration by inhalation or insufflation. The formulations may,

where appropriate, be conveniently presented in discrete dosage

units and may be prepared by any of the methods well known in the

art of pharmacy. All such pharmacy methods include the steps of

bringing into association the active compound with liquid carriers or finely divided solid carriers or both as needed and then, if necessary,

shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral

administration may conveniently be presented: as discrete units,

such as capsules, cachets or tablets, each containing a

predetermined amount of the active ingredient; as a powder or

granules; or as a solution, a suspension or as an emulsion. The

active ingredient may also be presented as a bolus electuary or

paste, and be in a pure form, i.e., without a carrier. Tablets and

capsules for oral administration may contain conventional excipients

such as binding agents, fillers, lubricants, disintegrant or wetting

agents. A tablet may be made by compression or molding, optionally

with one or more accessory ingredients. Compressed tablets may be

prepared by compressing in a suitable machine the active ingredients

in a free-flowing form such as a powder or granules, optionally mixed

with a binder, lubricant, inert diluent, lubricating, surface active or

dispersing agent. Molded tablets may be made by molding in a

suitable machine a mixture of the powdered compound moistened

with an inert liquid diluent. The tablets may be coated according to

methods well known in the art. Oral fluid preparations may be in the

form of, for example, aqueous or oily suspensions, solutions,

emulsions, syrups or elixirs, or may be presented as a dry product for

constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as

suspending agents, emulsifying agents, non-aqueous vehicles (which

may include edible oils), or preservatives. The tablets may optionally

be formulated so as to provide slow or controlled release of the

active ingredient therein.

Formulations for parenteral administration include:

aqueous and non-aqueous sterile injection solutions which may

contain anti-oxidants, buffers, bacteriostats and solutes which render

the formulation isotonic with the blood of the intended recipient; and

aqueous and non-aqueous sterile suspensions which may include

suspending agents and thickening agents. The formulations may be

presented in unit dose or multi-dose containers, for example sealed

ampoules and vials, and may be stored in a freeze-dried (lyophilized)

condition requiring only the addition of the sterile liquid carrier, for

example, saline, water-for-injection, immediately prior to use.

Alternatively, the formulations may be presented for continuous

infusion. Extemporaneous injection solutions and suspensions may

be prepared from sterile powders, granules and tablets of the kind

previously described.

Formulations for rectal administration may be presented

as a suppository with the usual carriers such as cocoa butter or

polyethylene glycol. Formulations for topical administration in the

mouth, for example buccally or sublingually, include lozenges, comprising the active ingredient in a flavored base such as sucrose

and acacia or tragacanth, and pastilles comprising the active

ingredient in a base such as gelatin and glycerin or sucrose and

acacia. For intra-nasal administration the compounds of the

invention may be used as a liquid spray or dispersible powder or in

the form of drops. Drops may be formulated with an aqueous or

non-aqueous base also comprising one or more dispersing agents,

solubilizing agents or suspending agents. Liquid sprays are

conveniently delivered from pressurized packs.

For administration by inhalation the compounds

according to the invention are conveniently delivered from an

insufflator, nebulizer pressurized packs or other convenient means of

delivering an aerosol spray. Pressurized packs may comprise a

suitable propellant such as dichlorodifluoromethane,

trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or

other suitable gas. In the case of a pressurized aerosol, the dosage

unit may be determined by providing a valve to deliver a metered

amount.

Alternatively, for administration by inhalation or

insufflation, the compounds according to the invention may take the

form of a dry powder composition, for example a powder mix of the

compound and a suitable powder base such as lactose or starch. The

powder composition may be presented in unit dosage form, in for example, capsules, cartridges, gelatin or blister packs from which the

powder may be administered with the aid of an inhalator or

insufflator.

When desired the above described formulations,

adapted to give sustained release of the active ingredient, may be

employed. The pharmaceutical compositions according to the

invention may also contain other active ingredients such as

antimicrobial agents, immunosuppressants or preservatives.

The compounds of the invention may also be used in

combination with other therapeutic agents, for example, anti-

inflammatory agents, particularly nitric oxide synthase inhibitors,

cyclooxygenase blockers, superoxide scavengers, vasodilator

prostaglandins including prostacyclin and prostaglandin E,, cancer

chemotherapeutic agents including cisplatin, NO donors or NO

inhalation therapy, or PAF-receptor antagonists.

It should be understood that in addition to the

ingredients particularly mentioned above, the formulations of this

invention may include other agents conventional in the art having

regard to the type of formulation in question, for example, those

suitable for oral administration may include flavoring agents.

Preferred unit dosage formulations are those containing

an effective dose, as recited below, or an appropriate fraction

thereof, of the active ingredient. For each of the aforementioned conditions, the

guanidino derivative may be administered orally or via injection at a

dose of from 0.1 to 250 mg/kg per day. The dose range for adult

humans is generally from 5 mg to 1 7.5 g/day, preferably 5 mg to 1 0

g/day and most preferably 1 00 mg to 3 g/day. Tablets or other

forms of presentation provided in discrete units may conveniently

contain an amount which is effective at such dosage or as a multiple

of the same, for instance, units containing 5 mg to 500 mg, usually

around 1 00 mg to 500 mg.

The pharmaceutical composition preferably is

administered orally or by injection (intravenous or subcutaneous), and

the precise amount administered to a patient will be the responsibility

of the attendant physician. However, the dose employed will depend

upon a number of factors, including the age and sex of the patient,

the precise disorder being treated, and its severity. Also the route of

administration may vary depending upon the condition and its

severity.

While not intending to be bound by any particular

theory, or to limit the scope of the invention to any such theory, we

postulate that the salutory effect of the guanidino derivatives of the

present invention is due, at least in large part, to a reaction between

the guanidino derivatives and peroxynitrite, thereby converting

peroxynitrite to less toxic, less biologically active species, including nitrite (N0₂ ) . In this manner, the guanidino compounds are acting as

scavengers of peroxynitrite, facilitating the rapid decomposition of

peroxynitrite to less biologically active species. We further believe

that this scavenging is happening in a manner, and at a rate, that is

competitive with the reaction of peroxynitrite with endogenous

biomolecules, so as to reduce or prevent: (i) the occurrence of

species otherwise produced by the reaction of peroxynitrite with

such endogenous biomolecules, or (ii) the inappropriately altered

function of such biomolecules.

It is envisaged that decomposition or reaction of

peroxynitrite can proceed either via transfer of one electron to

peroxynitrite or via direct nucleophilic attack of peroxynitrite by the

scavenger via an S_N2 type mechanism.

For example, the reaction of S-methyl

mercaptoethylguanidine with peroxynitrite may involve one electron transfer:

(a)

where G represents the guanidino group (H₂NC( = NH)NH-).

Within the solvent cage, further reaction can occur to

9've nitrite and the sulphoxide derivative of S-methyl

mercaptoethylguanidine:

(b)

The same products can be derived from an S_N2 type mechanism:

(c)

Similar reaction schemes can be drawn for the reaction of mercaptoethylguanidine with peroxynitrite:

(d)

However, an alternative pathway can result in the same

sulphenic acid and nitrite products:

(e)

(f)

in addition, the disulphide can result:

(g)

Again, an S_N2 type mechanism can lead to the same

products:

(h)

(OR)

(i)

In line with current theory regarding peroxynitrite, the

peroxynitrite species illustrated above as HOONO may be regarded

as an activated form (see Pryor and Squadrito: W. A. Pryor & G. L.

Squadrito, "The chemistry of peroxynitrite", Am. J. Physiology, 268,

699-722 (1 995)).

Although reaction schemes are drawn for neutral

reactants, it is also possible, and even probable, that the

peroxynitrite anion (ONOO ) or, for the examples illustrated, the

thiolate anion (RS ) may be the participating species. It is further

possible that intermediates other than those illustrated above are

formed. To illustrate these points, the following may be

hypothesized:

Φ

Direct reaction products may be further

metabolized/oxidized to give other products. For example, sulphenic

acid derivatives may be oxidized to the corresponding sulphinic or

sulphonic acid derivatives, or give rise to the disulphides.

The above pathways illustrate possible mechanisms for

the decomposition of peroxynitrite to nitrite by sulphur-containing

scavengers. Other scavengers, or these scavengers, may act by

similar, unrelated or multiple mechanisms, depending on the nature

of the molecule as a whole. The above reaction pathways are

intended to demonstrate that the guanidino compounds of the

invention may operate by causing the decomposition of peroxynitrite

to less reactive toxic species such as nitrite. However, the invention

is not limited to these postulated mechanisms.

The following Examples are provided by way of

illustration, and are not intended to limit the scope of the invention.

The guanidino derivatives used in Examples 1 -4 were

prepared as follows: mercaptoethylguanidine (MEG) was prepared as

shown in Example 5, guanidino ethyldisulphide (GED) was made

according to Example 6, S-methyl mercaptoethylguanidine (SMEG)

was prepared according to the method of Example 7, and

aminoguanidine (AG) was purchased from Aldrich Chemical Co.,

Milwaukee, WI, in the form of amoniguanidinehydrochloride. EXAMPIE 1

With reference to the results shown in Fig. 1 , this

example illustrates the effect of mercaptoethylguanidine (MEG), S-

methyl-mercaptoethylguanidine (SMEG), guanidinoethyldisuifide

(GED) and aminoguanidine (AG) on the in vitro oxidation of

dihydrorhodamine 1 23 (DHR) by peroxynitrite (n = 3-6). These

studies were performed in phosphate-buffered saline containing 50

μM dihydrorhodamine 1 23 at pH 7.4. The oxidation of

dihydrorhodamine 1 23 was induced by rapid mixing with

peroxynitrite (3.3 μM final concentration) in the presence of various

concentrations of the guanidino compounds. After 10 min, the

amount of rhodamine 1 23 formed (the oxidation product of

dihydrorhodamine 1 23) was measured using a fluorescent

spectrophotometer (excitation: 500 nm, emission: 536 nm).

Legend to Fig. 1 . Effect of mercaptoethylguanidine (MEG), S-methyl-

mercaptoethylguanidine (SMEG), guanidinoethyldisuifide (GED) and

aminoguanidine (AG) on the oxidation of dihydrorhodamine 1 23

(DHR) in response to peroxynitrite (PN).

EXAMPLE 2

With reference to the results shown in Figs. 2A-2C, this

example illustrates the effect of aminoguanidine (AG),

mercaptoethylguanidine (MEG), and guanidinoethyldisuifide (GED) on the decrease in mitochondrial respiration in J774 macrophages

exposed to peroxynitrite (N = 3-6). J774 macrophage cell lines

were obtained from the American Type Culture Collection (ATCC)

and were grown using standard methods in Dulbecco's Modified

Eagle Med ium (DMEM) supplemented with 10% fetal bovine serum,

glutamine, penicillin (10,000 U/l) and streptomycin (10,000 U/l).

Cells were grown in 96-well plates for measurement of mitochondrial

respiration and in 12-well plates for measurement of DNA strand

breakage (DNA strand breakage is discussed in Example 3, below).

All the experiments were carried out with 10% fetal calf serum.

Cells were pretreated with the indicated doses of the

guanidino compounds for 10 minutes. Then, cells were exposed to

peroxynitrite (1 mM final concentration). After 1 h, mitochondrial

respiration was measured. C represents a control culture, which

received no pretreatment with a guanidino compound and which was

not exposed to peroxynitrite.

Mitochondrial respiration, an indicator of cell viability,

was assessed by the mitochondrial-dependent reduction of MTT 13-

(4,5 - dimethylthiazol-2-yl) - 2,5 - diphenyltetrazolium bromide] to

formazan. Cells in 96-well plates were incubated (37°C) with MTT

(0.2 mg/ml for 60 minutes). Culture medium was removed by

aspiration and the cells solubilized in dimethylsulf oxide (DMSO) (100

μl). The extent of reduction of MTT to formazan within cells was quantitated by measurement of OD₅₅₀ using a microplate reader. The

calibration curve for the reduction of MTT to formazan was prepared

in DMSO. Formazan production by cells was expressed as a

percentage of the values obtained from untreated cells.

Cells exposed to peroxynitrite showed a markedly

suppressed mitochondrial respiration at 1 h. This suppressed

respiration was dose-dependently inhibited by the guanidino

derivatives, with mercaptoethylguanidine (MEG) being the most

potent inhibitor (see Figs. 2A-2C).

Legend to Fig. 2. Effect of aminoguanidine (AG),

mercaptoethylguanidine (MEG) and guanidinoethyldisuifide (GED) on

the suppression of mitochondrial respiration in response to

peroxynitrite (PN).

EXAMPLE 3

With reference to the results shown in Fig. 3, this

example illustrates the effect of aminoguanidine (AG),

mercaptoethylguanidine (MEG), and guanidinoethyldisuifide (GED)

(100 μM each) on the increase in DNA single strand breaks in J774

macrophages exposed to peroxynitrite (PN) ( 1 mM) (N = 3-6) . J774

macrophage cell lines were obtained and cultured as described in

Example 2. Macrophages were then exposed to peroxynitrite for 30

min. Then, the percentage of DNA single strand breaks was 80

-34- determined by the alkaline unwinding method. C represents a

control culture, which received no pretreatment with a guanidino

compound and which was not exposed to peroxynitrite. At the end

of the incubation period, cells were scraped into 0.2 ml of solution A

buffer (myoinositol 250mM, NaH₂PO₃ 1 0 mM, MgCI₂ 1 mM, pH 7.2).

The cell lysate was then transferred into plastic tubes designated T

(maximum fluorescence), P (fluorescence in sample used to estimate

extent of DNA unwinding), or B (background fluorescence). To each

tube, 0.2 ml of solution B (alkaline lysis solution: NaOH 10 mM, urea

9 M, ethylenediaminetetraacetic acid 2.5 mM, sodium dodecyl

sulfate 0.1 %) was added and incubated at 4 °C for 10 minutes to

allow cell lysis and chromatin disruption. 0.1 ml each of solutions C

(0.45 volume solution B in 0.2 N NaOH) and D (0.4 volume solution

B in 0.2 N NaOH) was then added to the P and B tubes. 0.1 ml of

solution E (neutralizing solution: glucose 1 M, mercaptoethanol 14

mM) was added to the T tubes before solutions C and D were added.

From this point incubations were carried out in the dark. A 30-

minute incubation period at 0 °C was then allowed during which the

alkali diffused into the viscous lysate. Since the neutralizing

solution, solution E, was added to the T tubes before addition of the

alkaline solutions C and D, the DNA in the T tubes was never

exposed to a denaturing pH. At the end of the 30 minute incubation,

the contents of the B tubes were sonicated for 30 seconds to ensure rapid denaturation of DNA in the alkaline solution. All tubes were

then incubated at 1 5 °C for 1 0 minutes. Denaturation was stopped

by chilling to 0 °C and adding 0.4 ml of solution E to the P and B

tubes. 1 .5 ml of solution F (ethidium bromide 6.7 μg/ml in 1 3.3 mM

NaOH) was added to all the tubes and fluorescence (excitation: 520

nm, emission: 590 nm) was measured spectrophometrically. Under

the conditions used, in which ethidium bromide binds preferentially

to double stranded DNA, the percentage of double stranded DNA (D)

may be determined using the equation: % D = 100 X [F(P) -

F(B)]/[F(T) - F (B)]; where F(P) is the fluorescence of the sample, F(B)

the background fluorescence, i.e. fluorescence due to all cell

components other than double stranded DNA, and F(T) the maximum

fluorescence.

Fig. 3 shows that pretreatment of the cells with

aminoguanidine (AG), mercaptoethylguanidine (MEG) and

guanidinoethyldisuifide (GED) (100 μM each) protected against the

peroxynitrite (PN) (1 mM)-induced single strand breakage.

Legend to Fio. 3. Percentage of DNA single strand breaks in control

(C, untreated macrophages), in cells exposed to peroxynitrite (PN),

and in cells exposed to peroxynitrite in the presence of

aminoguanidine (AG), mercaptoethylguanidine (MEG) or

guanidinoethyldisuifide (GED) (n = 6). US97/08280

-36- EXAMPLE 4

With reference to the results shown in Fig. 4, this

example illustrates the effect of mercaptoethylguanidine (MEG) on

the peroxynitrite-induced suppression of contractility in vascular

rings. Thoracic aortae from rats were cleared of adhering

periadventitial fat and cut into rings of 1 -4 mm width. Rings in

Krebs' solution were exposed to peroxynitrite (ONOO) (300 μM) or

vehicle control, in the presence or absence of 100 μM MEG >

following a 30 minute incubation, rings were set up for the

measurements of isometric contractility. The rings were mounted in

organ baths (5 ml) filled with warmed (37 °C) oxygenated

(95% O₂/5%CO₂ Krebs' solution (pH 7.4) consisting of (mmol/L):

NaCI 1 18, KCl 4.7, KH₂PO₄ 1 .2, MgS0₄ 1 .2, CaCI₂ 2.5, NaHCO₃ 25

and glucose 1 1 .7, in the presence of indomethacin (10 μmol/L).

Isometric force was measured with isometric transducers (Kent

Scientific Corp. Litchfield, CT, USA), digitalized using a Maclab A/D

converter (AD Instruments, Milford, MA, USA), and stored and

displayed on a Macintosh personal computer. A tension of 1 g was

applied and the rings were equilibrated for 60 min. Fresh Krebs'

solution was provided at 1 5 minute intervals. After the incubation

period, concentration-response curves to noradrenaline (10 ⁹-10^'5

mol/L) were obtained. Peroxynitrite induced a marked suppression of vascular

contractility. However, MEG caused a partial protection against the

peroxynitrite-induced suppression of vascular contractility.

Legend to Figure 4. Concentration-response curves to noradrenaline

for control vascular rings, for rings exposed to peroxynitrite (PN), and

for rings exposed to peroxynitrite (PN) in the presence of

mercaptoethylguanidine (MEG).

EXAMPLE 5 This example illustrates a method for synthesizing

mercaptoethylguanidine sulfate. Mercaptoethylamine hydrochloride

(2g) was dissolved in methanol (5 ml) and cooled in a salt/ice bath.

A cold solution of potassium hydroxide (0.99 g) in methanol (1 0 ml)

was added and the mixture stirred. After 1 hour, the solution was

filtered and S-methylisothiourea (2g) was added to 1 2 ml of the

filtrate. The solution was stirred at room temperature (1 8°C) for 1 6

hours under nitrogen. The solution was then filtered and ether was

added to precipitate the crude product which was then recrystallized

from an ether/ethanol mixture.

EXAMPLE 6

A further example is for the preparation of

guanidinoethyldisulphide (GED) dihydrochloride as follows: to a

solution of cystamine dihydrochloride (.5 g, 2.2 mmol) in water (25 ml) was added 10 ml of Amberlite IRA 402 (OH) resin, followed by

1 H-pyrozole-1 -carboxamide HCI (0.98 g, 6.6 mmol) . The mixture

was stirred for 1 6 h at room temperature, the resin removed and the

filtrate extracted with ethylacetate. The aqueous layer was acidified

with HCI to pH2 and lyophilised to afford 0.56 g GED

dihydrochloride.

EXAMPLE 7

2-(Methylthio)ethylguanidine sulphate was prepared as

follows: to a solution of 0.695 g S-methylisothiourea in 1 5 ml of

90% methanol was added 0.456 g 2-(methylthio)ethylamine. The

solution was stirred for 20 h at room temperature, filtered and the

solvent removed in vacuo. The residue was crystallized from a

mixture of methanol and ether.

EXAMPLE 8 2-(ethylthio)ethylguanidtne sulphate was prepared using

the procedure of example 5; however, 0.5 g of 2-

(ethylthio)ethylamine was used instead of 2-(methylthio)ethylamine.

EXAMPLE 9

N-amidinylthiomorpholine sulphate was prepared as

follows: thiomorpholine (3 ml) was added to a solution of 4.1 7 g S-

methylisothiourea in 30 ml of 25% aqueous methanol and the

solution was stirred overnight. The solvent was removed under

reduced pressure and the residue taken up in warm methanol and filtered. The volume was reduced and the solution was left for 2

days after which the solid was collected.

EXAMPLE 10

N-amidinylthiazolidine sulphate was prepared as follows:

thiazolidine ( 1 g) was added to a solution of 1 .56 g S-

methylisothiourea in 1 5 ml of 25% aqueous methanol and the

solution was stirred overnight. The solvent was removed under

reduced pressure and the residue recrystallized from methanol/water

to give a white solid in low yield.

The detailed description of the invention presented

above is provided by way of illustration, and it is not intended to

limit the scope of the invention which is to be determined by the

following claims.

WHAT IS CLAIMED IS:

Claims

1 ■ A pharmacologically acceptable composition for inhibiting the cytotoxic effect of peroxynitrite in a mammal, comprising: a compound having a formula selected from the group consisting of:

and

or a salt thereof, wherein:

R₃ and R'₃ are independently H, lower alkyl, alkylene, alkenylene, amino, hydroxy, thioalkyiene, or a substituted derivative

thereof; R_v R₅, R'₅, R₄ and R'₄ are independently H, alkyl,

alkenyl, phenyl, alkylene, alkenylene, phenylalkylene or amino, or a

substituted derivative thereof;

Alternatively, R, is R₆-Y-Z- where R₆ is H, alkyl, alkenyl,

phenyl, alkylene, alkenylene, phenylalkylene, acyl, -SO₃ ^", or -PO₃ ^", or

a substituted derivative thereof, and Z and Y are as defined below;

Z and Z' are independently alkylene, alkenylene,

cycloalkylene or cycloalkenylene, or a substituted derivative thereof;

Y and Y' are independently S or Se;

When R₂ or R'₂ is alkylene, alkenylene, thioalkyiene,

amino, hydroxy or a substituted derivative thereof, said R₂ or R'₂

may be joined to any of:

(i) R₃ or R'₃, if R₃ or R'₃ is alkylene, alkenylene or

thioalkyiene;

(ii) R₄ or R'₄, if R₄ or R'₄ is alkylene or alkenylene; or

(iii) R₅ or R'₅, if R₅ or R'₅ is alkylene or alkenylene;

to form 5-, 6-, or 7-membered heterocycle;

When R₂, R₃, R'₂ or R'₃ is alkylene or alkenylene, said

R₂, R₃, R'₂ or R'₃ optionally may be joined to the adjacent Z or Z' to

form a 5- or 6-membered heterocyclic ring, with the proviso that said

heterocyclic ring optionally is substituted with a lower alkyl, alkoxy,

halo, hydroxy or amino; When R₁ and R₄ are alkylene or alkenylene, said R, and

R₄ optionally may be joined together to form a 5-, 6-, or 7-membered

heterocycle;

When R, is R₆-Y-Z-, and R₆ is alkylene or alkenylene, R₆

optionally may be joined to any of:

(i) R₂, when R₂ is alkylene, alkenylene or thioalkyiene;

(ii) Z; or

(iii) R₄, when R₄ is alkylene or alkenylene;

to form a 5-, 6- or 7-membered heterocyclic ring; and

a pharmaceutically acceptable carrier, said compound

present in said composition in an effective amount to inhibit the

cytotoxic effect of peroxynitrite in said mammal.

2. The composition of claim 1 wherein said substituted

derivative of R₂, R₃, R ₂ or R' ₃ is independently selected from the

group consisting of one or more of alkoxy, halo, hydroxy and amino.

3. The composition of claim 1 wherein said substituted

derivative of R, , R₅, R'₅, R₄ or R'₄ is independently selected from the

group consisting of one or more of alkyl, alkoxy, halo, hydroxy,

amino, amino alkyl (secondary or tertiary), thio and nitro.

4. The composition of claim 1 wherein said R₂, R₃, R'₂ or

R'₃ thioalkyiene has a formula [-(CH₂)_n-SH] where n is independently

1 to 4.

5. The composition of claim 1 wherein said R₂ or R'₂

thioesteralkylene has a formula I-(CH₂)_n-S-R₇] where R₇ is

independently a lower alkyl and n is independently 1 to 4.

6. The composition of claim 1 wherein said substituted

derivative of Z or Z' is independently selected from the group

consisting of one or more of lower alkyl, alkoxy, halo, amino, nitro

and carboxyl.

7. The composition of claim 1 wherein:

R₂ and R'₂ are independently selected from the group

consisting of H, lower alkyl, amino, aminoalkyl, hydroxy, phenyl,

phenylalkylene and a substituted derivative thereof;

R₃ and R'₃ are independently selected from the group

consisting of H, lower alkyl, amino and hydroxy;

R₅/ RV R₄ ^and R'₄ ^are independently selected from the

group consisting of H and lower alkyl;

R, is selected from the group consisting of H, lower

alkyl and R₆-Y-Z-, where R₆ is selected from the group consisting of

H, lower alkyl, acyl, -SO₃ ^{" '}, -PO₃ ^" and a substituted derivative

thereof;

Z and Z' are independently alkylene, optionally

substituted with one or more substituents selected from the group

consisting of lower alkyl and carboxyl; and

Y and Y' are S.

8. The composition of claim 1 wherein:

R, is selected from the group consisting of H and alkyl;

R₂ is selected from the group consisting of H, amino,

hydroxy, methoxy and ethoxy;

R₃ is selected from the group consisting of H, lower

alkyl and amino;

R₄ is H; and

R₅ is selected from the group consisting of H and lower

alkyl.

9. The composition of claim 1 wherein said compound is

selected from the group consisting of aminoguanidine,

hydroxyguanidine, 1 -amino-2-hydroxyguanidine, 1 -amino-2-methyl-2-

hydroxyguanidine and diaminoguanidine.

1 0. The composition of claim 1 wherein:

R₁ is R₆-Y-Z-;

R₆ is selected from the group consisting of H, acyl,

-SO₃ ^", -PO₃ ^" and lower alkyl;

R₂ and R'₂ are independently selected from the group

consisting of H and amino;

R₃ and R'₃ are independently H, amino and hydroxy;

R₄ and R'₄ are independently H, methyl and ethyl;

Z and Z' are independently alkylene optionally

substituted with one or more methyl; and

Y is S.

1 1 . The composition of claim 1 wherein said compound is

selected from the group consisting of mercaptoethylguanidine,

mercaptopropylguanidine, S-methyl-mercaptoethyiguanidine, S-

methyl-mercaptopropylguanidine, 2-mercapto-2-

methylpropylguanidine, mercaptoethylguanidine-S-phosphoric acid

and guanidinoethyldisuifide.

1 2. The composition of claim 1 wherein said compound

reacts with the peroxynitrite, thereby inhibiting the cytotoxic effect

of the peroxynitrite.

13. The composition of claim 1 for inhibiting peroxynitrite-

induced oxidative reactions, thereby inhibiting the cytotoxic effect of

the peroxynitrite.

1 4. The composition of claim 1 for inhibiting peroxynitrite-

induced suppression of cellular respiration.

1 5. The composition of claim 1 for inhibiting peroxynitrite-

induced DNA strand breakage.

1 6. The composition of claim 1 for inhibiting peroxynitrite-

induced suppression of vascular smooth muscle contractility.

1 7. The composition of claim 1 wherein said compound is

present in an amount sufficient to treat a condition where there is an

advantage in inhibiting the cytotoxic effect of peroxynitrite.

1 8. The composition of claim 1 7 wherein said condition is

selected from the group consisting of circulatory shock, systemic

inflammatory response syndrome, therapy with cytokines, ischemia-

reperfusion injury of the heart, ischemia-reperfusion injury of the

brain, therapy with cytokine-inducing agents, transplantation,

transplant rejection, local inflammatory responses, systemic

inflammation, autoimmune diseases, adult respiratory distress

syndrome, arthritis, rheumatoid arthritis, diabetes mellitus, ileitis,

ulcerative colitis, Crohn's disease, asthma, periodontitis, nephrosis,

chronic demyelinating diseases of the nervous system, multiple

sclerosis, AIDS-related complications, Alzheimer's disease,

cardiomyopathy, adrenal insufficiency, hypercholesterolemia,

atherosclerosis, bone diseases associated with increased bone

resorption, pre-eclampsia, eclampsia, uremic complications, chronic

liver failure, stroke, and cancer.

1 9. The composition of claim 1 7 wherein said condition is

selected from the group consisting of circulatory shock, myocardial

ischemia and a central nervous system ischemic disorder.

20. The composition of claim 1 formulated for oral, rectal,

nasal, topical, buccal, sub-lingual, vaginal, parenteral, intramuscular,

sub-cutaneous, intravenous, inhalation or insufflation administration.

21 . The composition of claim 1 formulated for oral

administration, said carrier including an ingredient selected from the

group consisting of a binding agent, filler, lubricant, disintegrant,

wetting agent, inert diluent, surface active agent, dispersing agent,

suspending agent, emulsifying agent, edible oil, flavoring agent and

mixtures thereof.

22. The composition of claim 1 formulated for topical

administration in the mouth, said carrier including an ingredient

selected from the group consisting of a flavor, sucrose, acacia,

tragacanth, gelatin, glycerin and mixtures thereof.

23. The composition of claim 1 formulated for nasal

administration, said carrier including an ingredient selected from the

group consisting of a dispersing agent, solubilizing agent, suspending

agent and mixtures thereof.

24. The composition of claim 1 formulated for

administration by inhalation, said carrier including a propellant.

25. The composition of claim 24 wherein said propellant is

selected from the group consisting of dichlorodifluoromethane,

trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide and

mixtures thereof.

26. The composition of claim 1 formulated for

administration by inhalation or insufflation, said carrier including an

ingredient selected from the group consisting of lactose, starch and

mixtures thereof.

27. The composition of claim 1 formulated for parenteral

administration, said carrier including an ingredient selected from the

group consisting of an anti-oxidant, buffer, bacteriostat, suspending

agent, thickening agent, saline, water and mixtures thereof.

28. The composition of claim 1 formulated for rectal

administration, said carrier including an ingredient selected from the

group consisting of cocoa butter, polyethylene glycol and mixtures

thereof.

29. The composition of claim 1 formulated to include an

ingredient selected from the group consisting of an antimicrobial

agent, an immunosuppressant, a preservative and mixtures thereof.

30. The composition of claim 1 formulated for

administration at a dose of from about 5 mg to about 1 7.5 g/day of

said compound.

31 . The composition of claim 30 formulated for

administration at a dose of from about 5 mg to about 10 g/day of

said compound.

32. The composition of claim 31 formulated for

administration at a dose of from about 100 mg to about 3 g/day of

said compound.

33. A method for inhibiting the cytotoxic effect of peroxynitrite in a mammal comprising: administering to the mammal an effective amount of a compound to inhibit the cytotoxic effect of peroxynitrite in the mammal, said compound having a formula selected from the group consisting of:

and

or a salt thereof, wherein:

R₃ and R'₃ are independently H, lower alkyl, alkylene, alkenylene, amino, hydroxy, thioalkyiene, or a substituted derivative thereof; R₃ and R'₃ are independently H, lower alkyl, alkylene,

alkenylene, amino, hydroxy, thioalkyiene, or a substituted derivative

thereof;

R,, R₅, R'₅, R₄ and R'₄ are independently H, alkyl,

alkenyl, phenyl, alkylene, alkenylene, phenylalkylene or amino, or a

substituted derivative thereof;

Alternatively, R, is R₆-Y-Z- where R₆ is H, alkyl, alkenyl,

phenyl, alkylene, alkenylene, phenylalkylene, acyl, -SO₃ ^" , or -PO₃ ^", or

a substituted derivative thereof, and Z and Y are as defined below;

Z and Z' are independently alkylene, alkenylene,

cycloalkylene or cycloalkenylene, or a substituted derivative thereof;

Y and Y' are independently S or Se;

When R₂ or R'₂ is alkylene, alkenylene, thioalkyiene,

amino, hydroxy or a substituted derivative thereof, said R₂ or R'₂

may be joined to any of:

(i) R₃ or R'₃, if R₃ or R'₃ is alkylene, alkenylene or

thioalkyiene;

(ii) R₄ or R'₄, if R₄ or R'₄ is alkylene or alkenylene; or

(iii) R₅ or R'₅, if R₅ or R'₅ is alkylene or alkenylene;

to form 5-, 6-, or 7-membered heterocycle;

When R₂, R₃, R'₂ or R'₃ is alkylene or alkenylene, said

R₂, R₃, R'₂ or R'₃ optionally may be joined to the adjacent Z or Z' to

form a 5- or 6-membered heterocyclic ring, with the proviso that said heterocyclic ring optionally being substituted with a lower alkyl,

alkoxy, halo, hydroxy or amino;

When R, and R₄ are alkylene or alkenylene, said R, and

R₄ optionally may be joined together to form a 5-, 6-, or 7-membered

heterocycle; and

When R, is R₆-Y-Z-, and R₆ is alkylene or alkenylene, R₆

optionally may be joined to any of:

(i) R₂, when R₂ is alkylene, alkenylene or thioalkyiene;

(ii) Z; or

(iii) R₄, when R₄ is alkylene or alkenylene;

to form a 5-, 6- or 7-membered heterocyclic ring.

34. The method of claim 33 wherein said substituted

derivative of R₂, R₃, R'₂ or R'₃ is independently selected from the

group consisting of one or more of alkoxy, halo, hydroxy and amino.

35. The method of claim 33 wherein said substituted

derivative of R_{1 #} R₅, R'₅, R₄ or R'₄ is independently selected from the

group consisting of one or more of alkyl, alkoxy, halo, hydroxy,

amino, amino alkyl (secondary or tertiary), thio and nitro.

36. The method of claim 33 wherein said R₂, R₃, R'₂ or R'₃

thioalkyiene has a formula [-(CH₂)_n-SH] where n is independently

1 to 4.

37. The method of claim 33 wherein said R₂ or R'₂

thioesteralkylene has a formula [-(CH₂)_π-S-R₇] where R₇ is

independently a lower alkyl and n is independently 1 to 4.

38. The method of claim 33 wherein said substituted

derivative of Z or Z' is independently selected from the group

consisting of one or more of lower alkyl, alkoxy, halo, amino, nitro

and carboxyl.

39. The method of claim 33 wherein:

R₂ and R'₂ are independently selected from the group

consisting of H, lower alkyl, amino, aminoalkyl, hydroxy, phenyl,

phenylalkylene and a substituted derivative thereof;

R₃ and R'₃ are independently selected from the group

consisting of H, lower alkyl, amino and hydroxy;

R₅, R'₅, R₄ and R'₄ are independently selected from the

group consisting of H and lower alkyl;

R, is selected from the group consisting of H, lower

alkyl and R₆-Y-Z-, where R₆ is selected from the group consisting of

H, lower alkyl, acyl, -SO₃ ^" , -PO₃ ^" and a substituted derivative

thereof;

Z and Z' are independently alkylene, optionally

substituted with one or more substituents selected from the group

consisting of lower alkyl and carboxylic acid; and

Y and Y' are S.

40. The method of claim 33 wherein:

R, is selected from the group consisting of H and alkyl;

R₂ is selected from the group consisting of H, amino,

hydroxy, methoxy and ethoxy;

R₃ is selected from the group consisting of H, lower

alkyl and amino;

R₄ is H; and

R₅ is selected from the group consisting of H and lower

alkyl.

41 . The method of claim 33 wherein said compound is

selected from the group consisting of aminoguanidine,

hydroxyguanidine, 1 -amino-2-hydroxyguanidine, 1 -amino-2-methyl-2-

hydroxyguanidine and diaminoguanidine.

42. The method of claim 33 wherein:

R, is R₆-Y-Z-;

R₆ is selected from the group consisting of H, acyl,

-SO₃ ^" , -PO₃- and lower alkyl;

R₂ and R'₂ are independently selected from the group

consisting of H and amino;

R₃ and R'₃ are independently H, amino and hydroxy;

R₄ and R'₄ are independently H, methyl and ethyl;

Z and Z' are independently alkylene optionally

substituted with one or more methyl; and

Y is S.

43. The method of claim 33 wherein said compound is

selected from the group consisting of mercaptoethylguanidine,

mercaptopropylguanidine, S-methyl-mercaptoethylguanidine, S-

methyl-mercaptopropylguanidine, 2-mercapto-2-

methylpropylguanidine, mercaptoethylguanidine-S-phosphoric acid

and guanidinoethyldisuifide.

44. The method of claim 33 wherein said compound reacts

with the peroxynitrite, thereby inhibiting the cytotoxic effect of the

peroxynitrite.

45. The method of claim 33 for inhibiting peroxynitrite-

induced oxidative reactions, thereby inhibiting the cytotoxic effect of

the peroxynitrite.

46. The method of claim 33 for inhibiting peroxynitrite-

induced suppression of cellular respiration.

47. The method of claim 33 for inhibiting peroxynitrite-

induced DNA strand breakage.

48. The method of claim 33 for inhibiting peroxynitrite-

induced suppression of vascular smooth muscle contractility.

49. The method of claim 33 conducted for treating a

condition where there is an advantage in inhibiting the cytotoxic

effect of peroxynitrite.

50. The method of claim 49 wherein said condition is

selected from the group consisting of circulatory shock, systemic

inflammatory response syndrome, therapy with cytokines, ischemia-

reperfusion injury of the heart, ischemia-reperfusion injury of the

brain, therapy with cytokine-inducing agents, transplantation,

transplant rejection, local inflammatory responses, systemic

inflammation, autoimmune diseases, adult respiratory distress

syndrome, arthritis, rheumatoid arthritis, diabetes mellitus, ileitis,

ulcerative colitis, Crohn's disease, asthma, periodontitis, nephrosis,

chronic demyelinating diseases of the nervous system, multiple

sclerosis, AIDS-related complications, Alzheimer's disease,

cardiomyopathy, adrenal insufficiency, hypercholesterolemia,

atherosclerosis, bone diseases associated with increased bone

resorption, pre-eclampsia, eclampsia, uremic complications, chronic

liver failure, stroke, and cancer.

51 . The method of claim 49 wherein said condition is

selected from the group consisting of circulatory shock, myocardial

ischemia and a central nervous system ischemic disorder.

52. The method of claim 33 by administering said

compound by a method selected from the group consisting of oral,

rectal, nasal, topical, buccal, sub-lingual, vaginal, parenteral,

intramuscular, sub-cutaneous, intravenous, inhalation and insufflation

administration.

53. The method of claim 33 by orally administering said

compound in a pharmacologically acceptable carrier, said carrier

including an ingredient selected from the group consisting of a

binding agent, filler, lubricant, disintegrant, wetting agent, inert

diluent, surface active agent, dispersing agent, suspending agent,

emulsifying agent, edible oil, flavoring agent and mixtures thereof.

54. The method of claim 33 by topically administering in the

mouth, said carrier including an ingredient selected from the group

consisting of a flavor, sucrose, acacia, tragacanth, gelatin, glycerin

and mixtures thereof.

55. The method of claim 33 by nasally administering said

compound in a pharmacologically acceptable carrier, said carrier

including an ingredient selected from the group consisting of a

dispersing agent, solubilizing agent, suspending agent and mixtures

thereof.

56. The method of claim 33 by administering said

compound in a pharmacologically acceptable carrier by inhalation,

said carrier including a propellant.

57. The method of claim 56 wherein said propellant is

selected from the group consisting of dichlorodifluoromethane,

trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide and

mixtures thereof.

58. The method of claim 33 by administering said

compound in a pharmacologically acceptable carrier by inhalation or

insufflation, said carrier including an ingredient selected from the

group consisting of lactose, starch and mixtures thereof.

59. The method of claim 33 by administering said

compound in a pharmacologically acceptable carrier parenterally, said

carrier including an ingredient selected from the group consisting of

an anti-oxidant, buffer, bacteriostat, suspending agent, thickening

agent, saline, water and mixtures thereof.

60. The method of claim 33 by administering said

compound in a pharmacologically acceptable carrier rectally, said

carrier including an ingredient selected from the group consisting of

cocoa butter, polyethylene glycol and mixtures thereof.

61 . The method of claim 33 wherein said compound is in

combination with an ingredient selected from the group consisting of

an antimicrobial agent, an immunosuppressant, a preservative and

mixtures thereof.

62. The method of claim 33 wherein said compound is

administered at a dose of from about 5 mg to about 1 7.5 g/day.

63. The method of claim 62 wherein said compound is

administered at a dose of from about 5 mg to about 10 g/day.

64. The method of claim 63 wherein said compound is

administered at a dose of from about 1 00 mg to about 3 g/day.