WO1996040746A1

WO1996040746A1 - Lysyloxidase inhibitors

Info

Publication number: WO1996040746A1
Application number: PCT/US1996/009102
Authority: WO
Inventors: Kaliappanadar Nellaiappan; Edward G. Bernstine; Herbert M. Kagan; Carl S. Apstein
Original assignee: Trustees Of Boston University; Fibromed, Inc.
Priority date: 1995-06-07
Filing date: 1996-06-06
Publication date: 1996-12-19
Also published as: AU6049796A

Abstract

Compounds having anti-fibrotic effects are provided. Also provided is a method for treating disorders, diseases or conditions associated with pathological fibrotic states. The compounds useful in the present invention are homocysteine thiolactone and selected derivatives thereof.

Description

LYSYLOXIDASE INHIBITORS

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to a method for treating diseases and conditions associated with the abnormal deposition of collagen in a patient. The invention is also directed to anti-fibrotic agents that are useful in controlling or treating various pathological fibrotic disorders or abnormalities.

2. Description of the Prior Art Fibrosis, the formation of excessive amounts of scar tissue, is a central issue in medicine. Scar tissue blocks arteries, immobilizes joints and damages internal organs, wreaking havoc on the body's ability to maintain vital functions. Every year 1.3 million people are hospitalized due to the damaging effects of fibrosis, yet doctors have few therapeutics to help them control this dangerous condition. As a result, they often see patients crippled, disfigured or killed by unwanted masses of uncontrollable scars. Fibrosis can follow surgery in the form of adhesions, keloid tumors or hypertrophic (very severe) scarring. Fibrosis causes contractures and joint dislocation following severe burns, wounds or orthopaedic injuries; it can occur in any organ and accompanies many disease states, such as hepatitis (liver cirrhosis), hypertension (heart failure), tuberculosis (pulmonary fibrosis), scleroderma (fibrotic skin and internal organs), diabetes (nephropathy) and atherosclerosis (fibrotic blood vessels).

Scar tissue, like all connective tissue, is composed largely of collagen. Collagen is the body's most abundant protein and forms the basis for its architectural framework. Bones, skin, ligaments, cartilage, organ casings and tendons are all composed of this tough, fibrous protein. Collagen is synthesized inside connective tissue cells such as fibroblasts and arterial smooth muscle cells. Each collagen molecule is formed from three polypeptide chains that twist around one another to make a stiff, rope-like structure with loose ends. These ends are called terminal extension peptides, and they help prevent the collagen molecules from assembling prematurely into larger fibrils.

After molecular synthesis is complete, collagen deposition takes place. The immature strands of collagen, known as procollagen, are secreted into the extra-cellular matrix where the extension peptides are cut off by specific enzymes. The now mature collagen molecules, called tropocollagen, spontaneously align end to end in staggered parallel rows ready to cross-link. The enzyme lysyl oxidase then acts on specific lysine residues of the collagen molecules and changes their chemical structure, enabling them to join together and form tough, dense layers of connective tissue, including scar tissue. Lysyl oxidase is the key to the cross¬ linking of collagen and subsequent formation of scar tissue.

Ironically, the very process designed to repair the body can lead to dangerous complications. Like epoxy, scar tissue serves only a structural role. It fills in the gaps, but cannot contribute to the function of the organ in which it appears. For example, as fibrotic scar tissue replaces heart muscle damaged by hypertension, the heart becomes less elastic and thus less able to do its job. Similarly, pulmonary fibrosis causes the lungs to stiffen and decrease in size, a condition that can become life-threatening. Fibrotic growth can also proliferate and invade the healthy tissue that surrounds it even after the original injury heals.

Too much scar tissue thus causes physiological roadblocks that disfigure, cripple or kill.

There are a number of disease conditions in which increased cardiovascular fibrosis appears to be important. The myocardium, or heart muscle, may be affected by an increase in fibrous tissue content diffusely, or in one of several locations: the endocardial lining (endomyocardial fibrosis), the pericardial lining (constrictive pericarditis), the cardiac valves or the vasculature. The specific consequences of the increased fibrosis depends to some extent on its location, e.g., fibrosis in the area of the conduction system can cause heart block and other cardiac arrhythmias. However, the general effect of myocardial fibrosis, relatively independent of location, is to increase myocardial passive stiffness, thereby decreasing the ability of the heart's chambers to relax adequately and fill with blood during the diastolic phase of the cardiac cycle. Such impaired relaxation is referred to as "diastolic dysfunction" and results in a decreased cardiac output.

The clinical picture of congestive heart failure with pulmonary congestion occurs if the left ventricle is primarily affected, or with systemic congestion if the right ventricle is primarily affected. Thus, such affected patients can have the clinical syndrome of congestive heart failure despite normal systolic or contractile functions, i.e., the myocardium contracts normally but its failure to relax normally causes the clinical heart failure syndrome. Approximately 35% of all cases of heart failure are due primarily to such diastolic dysfunction. In addition, increased fibrosis within the coronary arterial wall causes a reduced coronary vasodilator ability, i.e., the arterial fibrosis appears to impair the ability of the vessels to dilate and increase blood flow.

Despite its widespread occurrence and clinical importance, there is no accepted effective therapy to reverse myocardial fibrosis once it has become established. A therapeutic approach with the potential to decrease myocardial and vascular fibrosis, either decreasing the fibrous tissue content and/or decreasing its stiffness coefficient, could have important clinical benefits by improving cardiac distensibility and diastolic chamber filling, and also by improving coronary arterial vasodilator function. Lysyl oxidase inhibitor therapeutics could be important in reversing the pathophysiology of the "stiff heart" syndrome which is common to all conditions associated with increased myocardial fibrosis. Such anti-fibrotic therapeutics could comprise a new class of drugs useful in the treatment of heart failure.

Atherosclerotic plaque has elevated levels of lysyl oxidase indicating that there is a fibrotic component to atherosclerosis. Approximately 445,000 people are hospitalized for atherosclerosis in the United States each year. Another

804,000 undergo costly and risky treatment for stenosis of the carotid and other precerebral arteries (210,000), of the mitral and aortic valves (57,000), and of the coronary vessels (537,000). About 25% to 40%, or about 200,000 to 320,000, of the patients treated for stenosis experience a reclosing (or restenosis) after the initial blockage has been cleared away. Since patients who will have recurrent problems cannot be identified in advance, a therapeutic to prevent restenosis could be given to all patients treated for arterial stenosis.

Cirrhosis is a degenerative liver condition that is manifested by the increased and disordered synthesis and deposition of fibrous connective tissue. Initially, the liver becomes enlarged. As fibrous tissue proliferates, the texture of the liver becomes granular. Blood vessels become dysfunctional, causing greatly decreased hepatic blood circulation and gastrointestinal hemorrhage as portal hypertension develops. Eventually, the liver shrinks, its structure disintegrates, and scar tissue predominates. Cirrhosis impairs liver function because normal liver cells die and are replaced by non-functional fibrotic growth. In advanced stages of cirrhosis, the patient often dies of coma, jaundice, infection, high blood pressure, and hemorrhage. The primary causes of liver fibrosis are alcoholism and chronic hepatitis infections, but several other metabolic abnormalities and genetic mutations can lead to liver fibrosis and cirrhosis-even in the child or adolescent. The primary method for preventing liver fibrosis is to control its causes.

Although there are now vaccines against hepatitis B, hundreds of thousands of people worldwide are already infected, and vaccination programs are not yet well established. In addition, the incidence of hepatitis C is increasing, and there is no preventative treatment for this strain. There are few effective therapeutics for hepatitis once an individual is infected.

Corticosteroids have proven disappointingly ineffective and possibly even deleterious. Trials of interferon are being conducted with somewhat promising results, but the use of interferon as a proven therapeutic is still uncertain. In addition, hepatitis B patients often get a coinfection with delta agent. Such an infection usually causes further liver deterioration, and there is no effective treatment. The accepted regimen for uncomplicated alcohol-related liver disease is the complete avoidance of alcohol, adequate diet, and limited sodium intake.

The only available effective treatment for people with advanced liver cirrhosis is a costly liver transplant. An effective therapeutic treatment would thus reduce hospitalization costs, disability and the long-term care necessary for chronic advanced liver disease. A wide variety of acute or chronic conditions cause cellular injury and the subsequent accumulation of collagenous scar tissue (fibrosis) in the lung. These include: bacterial and viral infections; exposure to pulmonary toxins (asbestosis, silica, certain chemotherapy drugs); systemic disorders (scleroderma, rheumatoid arthritis); infectious disease (tuberculosis) and acute injuries that result in adult respiratory distress syndrome (ARDS). ARDS frequently occurs following multiple organ injury from motor vehicle accidents or severe infections, and its resolution may be accompanied by fibrosis. Sometimes the cause of pulmonary fibrosis is unknown. In such cases, the disease is termed idiopathic pulmonary fibrosis (IDP). The accumulation of excess fibrotic tissue renders the lung less compliant, and the lung decreases in size. The degree of symptomatology often relates to the extent of collagen deposition. Most patients experience a loss of exercise tolerance, and in some the disease is incapacitating or life threatening. Progressive pulmonary fibrosis can lead to cor pulmonale and respiratory failure.

Similar to lung cancer, pulmonary fibrosis has a very poor prognosis. Although numerous therapies have been tried, no proven therapy for pulmonary fibrosis is currently available. Anti-inflammatory drugs are currently used including corticosteroids, but the results are not encouraging.

As a visceral organ heals following surgery, fibrous scar tissue forms along the incision. This fibrotic tissue may invade the surface of adjoining organs and cause them to adhere to one another. While such adhesions are themselves painless and may cause no problems, they can produce obstruction and malfunction by distorting the organ or organs involved. Surgical adhesions occur commonly after abdominal surgery, in the pleura, the pericardium and around the pelvic organs. They can cause intestinal obstruction, infertility and complications after cardiac surgery, and there is currently no preventive treatment. Hypertrophic scars form over the original wound site much as normal scar tissue does. They correspond in shape and approximate size to the original wound, but they are more elevated, harder and often darker in color than normal scars. Hypertrophic scars may develop at any time until a wound matures, a process that can take up to several months to complete. Similar to burn victims, surgical patients may develop contractures due to hypertrophic scarring. Contractures may occur in soft tissue implants such as those in breast reconstruction; they may also arise after surgery to repair damaged tendons and joints.

A keloid represents an exaggeration of the normal fibroblastic response to injury. Keloids appear as thickened, irregular growths which extend beyond the site of the original wound. Although benign, keloids often cause itching and pain as well as an unacceptable level of disfigurement. The therapeutic approach to both keloids and hypertrophic scarring is generally limited, and steroids are the only treatment. Once a keloid has formed, corticosteroids may be injected into the lesion at four- week intervals to cause atrophy and/or to help diminish symptoms. In cases of severe hypertrophic scarring, surgical scar revision may be the only recourse.

Hypertrophic (very severe) scarring may represent the most challenging aspect of treating a burn patient. Hypertrophic scarring can cause severe contractures which inhibit function and create a major cosmetic problem.

Burn contractures are different than other types of contractures because of the extent of tissue damage usually seen. A burn scar will shorten until it meets an opposing force. Collagen fibers in the wound fuse together, particularly if the wound is in an area of loose tissue, as seen when a joint is flexed. This shortened fused mass of collagen tissue results in contracture formation that exerts forces strong enough to cause joint subluxation or to limit functional activity. Burn scars may cover an entire extremity, and as the scar tissue contracts, it may affect more than one joint, causing severe or total loss of mobility.

Bone and joint changes have been reported to occur in about 2% to 5% of severely burned patients. They are particularly devastating when they occur in children. Bone growth disturbances can occur in children if the epiphyses are damaged or if scar tissue crossing a joint prevents normal growth from occurring.

Joint dislocation or immobility is found in both adults and children and can result from scar tissue contracture even after wound closure.

Lysyl oxidase inhibitors have been promoted as effective in the inhibition of collagen cross-linking. U.S. Patent Nos. 5,182,297 (Palfreyman et al), 5,252,608 (Palfreyman et al.), and 4,997,854 (Kagan et al.) describe various lysyl oxidase inhibitors. Other inhibitors include penicillamine, homocysteine [Siegel,

J. Biol. Chem. 252:254 (1977)] and beta-aminopropionitrile (BAPN). Here we describe a new class of compounds useful in the inhibition of collagen cross¬ linking. These and other advantages of the present invention will become apparent from the following detailed description.

These and other advantages of the present invention will become apparent from the following detailed description.

SUMMARY OF THE INVENTION

In accordance with the present invention, compounds having anti-fibrotic effects are provided. Also provided is a method for treating disorders, diseases or conditions associated with pathological fibrotic states. The compounds useful in the present invention are homocysteine thiolactone and selected derivatives thereof.

One embodiment of the present invention is to provide a method of treating diseases and conditions associated with the abnormal deposition of collagen in a patient comprising administering to the patient a therapeutically effective amount of a compound of the formula:

X

and salts thereof, wherein R 1 and R 2 are the same or different, a hydrogen (-H), or an unsaturated or saturated aliphatic, alicyclic or aromatic hydrocarbon radical having from 1 to

50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherein said carbons are substituted with a substituent selected from the group consisting of hydrogen (-H), chloro, fluoro, bromo, iodo, hydroxyl

(-OH), alkoxyl (-OR'), acyl (-COR'), carboxyl (-CO₂H), carboxyl esters

(-CO₂R'), amido (CONR₂"), amino (-NR"₂), nitro (-NO₂), nitroso (-NO), aza

(-N=N-R), diazonium (-N₂ ⁺) azido (-N₃) hydrazino (-NR'-NR'₂), cyano (-CN), isocyano (-NC), cyanato (NCO-), isocyanato (OCN-), thiocyanato (NCS-), isothiocyanato (SCN-), thioamido (-C(S)NR'₂), thioether (-SR'), thiol (-SH), sulfoxide (-S(O)R'), sulfone (-S(O)₂R'), sulfoximino (-S(O)(NR")R'), sulfonic acid (-SO3H), sulfonyl esters (-SO3R'), sulfinic acid (-SO₂H), sulfinyl esters (-SO₂R'), sulfenic acid (-SOH), sulfenyl esters (-SOR'), phospho (-OP(O)(OR)₂), phosphono (-P(O)(OR)₂), urea (-NR'C(O)NR"₂), and silyl (-SiR'₃), and wherein R' is a hydrogen (-H) or is an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, and R" is a hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherem R 3 may be a hydrogen (-H) or R 5 -NH₂, and wherein R may be a hydrogen (-H) or R -NH₂, and where R or R is an unsaturated or saturated branched or unbranched aliphatic, alicyclic, heterocyclic, aromatic or heteroaromatic radical having from 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherein said carbons are substituted with a substituent selected from the group consisting of hydrogen (-H), chloro, fluoro, bromo, iodo, hydroxyl (-OH), alkoxyl (-OR'), acyl (-COR'), carboxyl (-CO₂H), carboxyl esters (-CO₂R'), amido (CONR₂"), amino (-NR"₂), nitro (-NO₂), nitroso (-NO), aza (-N=N-R), diazonium (-N₂ ⁺) azido (-N₃) hydrazino (-NR'-NR'₂), cyano (-CN), isocyano (-NC), cyanato (NCO-), isocyanato (OCN-), thiocyanato (NCS-), isothiocyanato (SCN-), thioamido (-C(S)NR'₂), thioether (-SR'), thiol (-SH), sulfoxide (-S(O)R'), sulfone (-S(O)₂R'), sulfoximino (-S(O)(NR")R'), sulfonic acid (-SO3H), sulfonyl esters (-SO3R'), sulfinic acid (-SO₂H), sulfinyl esters (-SO₂R'), sulfenic acid (-SOH), sulfenyl esters (-SOR'), phospho (-OP(O)(OR)₂), phosphono (-P(O)(OR)₂), urea

(-NR'C(O)NR"₂), and silyl (-SiR'₃), and wherein R' is an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, and wherein R" is a hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, and wherein n is integer from 1 to 3, X is S or O, Y is S or O, with the provision that X or Y must be S. Either the R or S chirality of the stereocenters, and thus any stereoisomer embodied by the formula, may be used.

In another embodiment, a method of treating diseases and conditions associated with the abnormal deposition of collagen in a patient comprising administering to the patient a therapeutically effective amount of homocysteine thiolactone.

In a preferred embodiment, provided is a compound of the structure:

where m is an integer from 1 to 50, preferably 1 to 25 and most preferably 1 to 5, wherein R , R , R , R , R , and R are in a cis or trans relationship and are the same or different, a hydrogen (-H) or an unsaturated or saturated aliphatic, alicyclic or aromatic hydrocarbon radical having from 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherein said carbons are substituted with a substituent selected from the group consisting of hydrogen (-H), chloro, fluoro, bromo, iodo, hydroxyl (-OH), alkoxyl (-OR'), acyl (-COR'), carboxyl (-CO₂H), carboxyl esters (-CO₂R'), amido (CONR₂"), amino (-NR"₂), nitro (-NO₂), nitroso (-NO), aza (-N=N-R'), diazonium (-N₂ ) azido (-N₃) hydrazino (-NR'-NR'₂), cyano (-CN), isocyano (-NC), cyanato (NCO-), isocyanato (OCN-), thiocyanato (NCS-), isothiocyanato

(SCN-), thioamido (-C(S)NR'₂), thioether (-SR'), thiol (-SH), sulfoxide (-S(O)R'), sulfone (-S(O)₂R'), sulfoximino (-S(O)(NR")R'), sulfonic acid (-SO₃H), sulfonyl esters (-SO3R'), sulfinic acid (-SO 2H), sulfinyl esters (-SO ), sulfenic acid (-SOH), sulfenyl esters (-SOR'), phospho (-OP(O)(OR)₂), phosphono (-P(O)(OR)₂), urea (-NR'C(O)NR"₂), and silyl (-SiR'₃), and wherein R' is hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, and R" is a hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherein n is integer from 1 to 3, X is S or O, Y is S or O, with the provision that X or Y must be S. Compounds having this structure may be likewise used in a method of treating diseases and conditions associated with the abnormal deposition of collagen in a patient comprising administering to the patient a therapeutically effective amount of said compound. Either the R or S chirality of the stereocenters, and thus any stereoisomer embodied by the formula, may be used.

Also provided is a compound of the structure: X

wherein Z is hydrogen (-H), -(CH₂)_m NR'R",or -(CH ^ _mOR', where m is an integer from 1 to 50, preferably 1 to 25 and most preferably 1 to 5, wherein R ,

2 3 4 5

R , R , R and R are in a cis or trans relationship and are the same or different, a hydrogen (-H) or an unsaturated or saturated aliphatic, alicyclic or aromatic hydrocarbon radical having from 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherein said carbons are substituted with a substituent selected from the group consisting of hydrogen (-H), chloro, fluoro, bromo, iodo, hydroxyl (-OH), alkoxyl (-OR'), acyl (-COR'), carboxyl (-CO₂H), carboxyl esters (-CO₂R'), amido (CONR₂ "), amino (-NR"₂), nitro (-NO₂), nitroso (-NO), aza (-N=N-R'), diazonium (-N₂ ) azido (-N ^ hydrazino (-NR'-NR'₂), cyano (-CN), isocyano (-NC), cyanato (NCO-), isocyanato (OCN-), thiocyanato (NCS-), isothiocyanato (SCN-), thioamido (-C(S)NR'₂), thioether (-SR'), thiol (-SH), sulfoxide (-S(O)R'), sulfone (-S(O)₂R'), sulfoximino (-S(O)(NR")R'), sulfonic acid (-SO₃H), sulfonyl esters

(-SO3R'), sulfinic acid (-SO₂H), sulfinyl esters (-SO₂R'), sulfenic acid (-SOH), sulfenyl esters (-SOR'), phospho (-OP(O)(OR)₂), phosphono (-P(O)(OR)₂), urea (-NR'C(O)NR"₂), and silyl (-SiR'₃), and wherein R' is hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, and R" is a hydrogen (- H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherein n is integer from 1 to 3, X is S or O, Y is S or O, with the provision that

X or Y must be S. Either the R or S chirality of the stereocenters, and thus any stereoisomer embodied by the formula, may be used.

Most preferably, the compounds are selected from the group consisting of glycylhomocysteine thiolactone, β-alanylhomocysteine thiolactone, γ-aminobutyrylhomocysteine thiolactone, lysylhomocysteine thiolactone, and ε-aminocaproylhomocysteine thiolactone. Either the D- or L-amino acids may be used.

Also provided are compounds useful for treating diseases and conditions associated with the abnormal deposition of collagen in a patient. An additional embodiment of the present invention is to provide compositions useful in treating diseases and conditions associated with the abnormal deposition of collagen in a patient in need thereof.

These and other objects and advantages are described in the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the inhibition of lysyl oxidase by homocysteine thiolactone using a diaminopentane substrate.

Figure 2 shows the inhibition of lysyl oxidase by homocysteine thiolactone using a tropoelastin substrate.

Figure 3 shows the inhibition of lysyl oxidase by homocysteine thiolactone using a collagen substrate.

Figure 4 shows the prevention of left ventricular stiffness by homocysteine thiolactone.

Figure 5 shows the reduction of histologically demonstrable collagen in isoproterenol + homocysteine thiolactone- treated animals. Figure 5(A) shows the area of section; 5(B) shows the fibrotic area, and 5(C) shows percent fibrosis.

DRTAILED DESCRIPTION OF THE INVENTION

and salts thereof,

1 2 wherein R and R are the same or different, a hydrogen (-H), or an unsaturated or saturated aliphatic, alicyclic or aromatic hydrocarbon radical having from 1 to

50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherein said carbons are substituted with a substituent selected from the group consisting of hydrogen (-H), chloro, fluoro, bromo, iodo, hydroxyl (-OH), alkoxyl (-OR'), acyl (-COR'), carboxyl (-CO₂H), carboxyl esters (-CO₂R'), amido (CONR₂"), amino (-NR"₂), nitro (-N0₂), nitroso (-NO), aza (-N=N-R), diazonium (-N₂ ⁺) azido (-N₃) hydrazino (-NR'-NR'₂), cyano (-CN), isocyano (-NC), cyanato (NCO-), isocyanato (OCN-), thiocyanato (NCS-), isothiocyanato (SCN-), thioamido (-C(S)NR'₂), thioether (-SR'), thiol (-SH), sulfoxide (-S(O)R'), sulfone (-S(O)₂R'), sulfoximino (-S(O)(NR")R'), sulfonic acid (-SO3H), sulfonyl esters (-SO3R'), sulfinic acid (-SO₂H), sulfinyl esters (-SO₂R')₅ sulfenic acid (-SOH), sulfenyl esters (-SOR'), phospho (-OP(O)(OR)₂), phosphono (-P(O)(OR)₂), urea (-NR'C(O)NR"₂), and silyl (-SiR'₃), and wherein

R' a is hydrogen (-H) or is an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, and R" is a hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most

3 5 preferably 1 to 15 carbon atoms, wherein R may be a hydrogen (-H) or R -NH₂, and wherein R may be a hydrogen (-H) or R -NH₂, and where R or R is an unsaturated or saturated branched or unbranched aliphatic, alicyclic, heterocyclic, aromatic or heteroaromatic radical having from 1 to 50 carbon atoms, preferably

1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherein said carbons are substituted with a substituent selected from the group consisting of hydrogen (-H), chloro, fluoro, bromo, iodo, hydroxyl (-OH), alkoxyl (-OR'), acyl (-COR'), carboxyl (-CO₂H), carboxyl esters (-CO₂R'), amido (CONR₂"), amino (-NR"₂), nitro (-NO₂), nitroso (-NO), aza (-N=N-R), diazonium (-N₂ ⁺) azido (-N₃) hydrazino (-NR'-NR'₂), cyano (-CN), isocyano (-NC), cyanato (NCO-), isocyanato (OCN-), thiocyanato (NCS-), isothiocyanato (SCN-), thioamido

(-C(S)NR'₂), thioether (-SR'), thiol (-SH), sulfoxide (-S(O)R'), sulfone (-S(O)₂R'), sulfoximino (-S(O)(NR")R'), sulfonic acid (-SO3H), sulfonyl esters (-SO3R'), sulfinic acid (-SO₂H), sulfinyl esters (-SO₂R'), sulfenic acid (-SOH), sulfenyl esters (-SOR'), phospho (-OP(O)(OR)₂), phosphono (-P(O)(OR)₂), urea (-NR'C(O)NR"₂), and silyl (-SiR'₃), and wherein R' is an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, and wherein R" is a hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, and wherein n is integer from 1 to 3, X is S or O, Y is S or O, with the provision that X or Y must be S. Either the R or S chirality of the stereocenters, and thus any stereoisomer embodied by the formula, may be used.

In a preferred embodiment, provided is a compound of the structure:

X

where m is an integer from 1 to 50, preferably 1 to 25 and most preferably 1 to 5, wherein R , R , R , R , R , and R are in a cis or trans relationship and are the same or different, a hydrogen (-H) or an unsaturated or saturated aliphatic, alicyclic or aromatic hydrocarbon radical having from 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherein said carbons are substituted with a substituent selected from the group consisting of hydrogen (-H), chloro, fluoro, bromo, iodo, hydroxyl (-OH), alkoxyl (-OR'), acyl (-COR'), carboxyl (-CO₂H), carboxyl esters (-CO₂R'), amido (CONR₂"), amino (-NR"₂), nitro (-NO₂), nitroso (-NO), aza (-N=N-R')₅ diazonium (-N₂ ) azido (-N3) hydrazino (-NR'-NR' , cyano (-CN), isocyano (-NC), cyanato (NCO-), isocyanato (OCN-), thiocyanato (NCS-), isothiocyanato (SCN-), thioamido (-C(S)NR'₂), thioether (-SR'), thiol (-SH), sulfoxide (-S(O)R'), sulfone (-S(O)₂R'), sulfoximino (-S(O)(NR")R'), sulfonic acid (-SO3H), sulfonyl esters (-SO3R'), sulfinic acid (-SO₂H), sulfinyl esters (-SO₂R'), sulfenic acid (-SOH), sulfenyl esters (-SOR'), phospho (-OP(O)(OR)₂), phosphono (-P(O)(OR)₂), urea (-NR'C(O)NR"₂), and silyl (-SiR' ^ and wherein R' is hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, and R" is a hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherein n is integer from 1 to 3, X is S or O, Y is S or O, with the provision that X or Y must be S. Compounds having this structure may be likewise used in a method of treating diseases and conditions associated with the abnormal deposition of collagen in a patient comprising administering to the patient a therapeutically effective amount of said compound. Either the R or S enantiomers may be used.

Also provided is a compound of the structure:

X

R wherein Z is hydrogen (-H), -(CH₂)_m NR'R",or -(CH₂)_m OR', where m is an integer from 1 to 50, preferably 1 to 25 and most preferably 1 to 5, wherein R ,

2 3 4 5

R , R , R and R are in a cis or trans relationship and are the same or different, a hydrogen (-H) or an unsaturated or saturated aliphatic, alicyclic or aromatic hydrocarbon radical having from 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherein said carbons are substituted with a substituent selected from the group consisting of hydrogen (-H), chloro, fluoro, bromo, iodo, hydroxyl (-OH), alkoxyl (-OR'), acyl (-COR'), carboxyl (-CO₂H), carboxyl esters (-CO₂R'), amido (CONR₂"), amino (-NR"₂), nitro (-NO₂), nitroso (-NO), aza (-N=N-R'), diazonium (-N₂ ) azido (-N3) hydrazino (-NR'-NR'₂), cyano (-CN), isocyano (-NC), cyanato (NCO-), isocyanato (OCN-), thiocyanato (NCS-), isothiocyanato (SCN-), thioamido (-C(S)NR'₂), thioether (-SR'), thiol (-SH), sulfoxide (-S(O)R'), sulfone (-S(O)₂R'), sulfoximino (-S(O)(NR")R'), sulfonic acid (-SO3H), sulfonyl esters (-SO3R'), sulfinic acid (-SO₂H), sulfinyl esters (-SO₂R'), sulfenic acid (-SOH), sulfenyl esters (-SOR'), phospho (-OP(O)(OR)₂), phosphono (-P(O)(OR)₂), urea (-NR'C(O)NR"₂), and silyl (-SiR'₃), and wherein R' is hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, and R" is a hydrogen (- H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, preferably 1 to 25 carbon atoms, and most preferably 1 to 15 carbon atoms, wherein n is integer from 1 to 3, m is an integer of from 1 to 25, preferably 1 to 10, X is S or O, Y is S or O, with the provision that X or Y must be S. Either the R or S chirality of the stereocenters, and thus any stereoisomer embodied by the formula, may be used.

Most preferably, the compounds are selected from the group consisting of glycylhomocysteine thiolactone, β-alanylhomocysteine thiolactone, γ-aminobutyrylhomocysteine thiolactone, lysylhomocysteine thiolactone, and ε-aminocaproylhomocysteine thiolactone. Either the D- or L-amino acids may be used. Additionally, mixtures of any of the above compounds may be used as well. Also provided are compounds useful for treating diseases and conditions associated with the abnormal deposition of collagen in a patient. An additional embodiment of the present invention is to provide compositions useful in treating diseases and conditions associated with the abnormal deposition of collagen in a patient in need thereof.

Provided herein is a method of preventing, treating, lessen and/or control fibroses in disorders, conditions or diseases associated with the abnormal deposition of collagen. The compounds of the present invention provide therapeutic effects when administered to human or animal subjects. Also provided herein is a method of using the compounds of the present invention to prevent, lessen treat and/or control fibrosis in disorders, conditions or diseases associated with the abnormal deposition of collagen conditions or diseases associated with the abnormal deposition of collagen including but are not limited to, cardiovascular fibrosis, arterialstenosis, restenosis, liver cirrhosis, liver fibrosis, pulmonary fibrosis, hypertrophic scar formation, keloid conditions, burn scarring, diabetes, surgical scarring, corneal scarring and tumor formation, fibrotic changes in blood vessels due to aging, inflammation and atherosclerosis or injury including medical intervention by surgery or catheterization.

Compounds of the present invention can be administered in any appropriate carrier for oral, topical, inhalatory, or parenteral administration. The compounds can be introduced by any means that provides treatment, therapy or amelioration of fibrotic conditions in living humans or animals. A therapeutically effective amount is the amount that provides treatment, therapy or amelioration of fibrotic conditions or abnormal deposition of collagen. It is well recognized in the art that inhibition of lysyl oxidase activity provides an effective means of assessing therapeutic effectiveness of an antifibrotic agent. The dosage administered will vary and be dependent upon the age, health, and weight of the recipient; the kind of concurrent treatment, if any; the frequency of treatment; and the nature of the therapeutic effect desired. Generally, the expected effective therapeutic daily dosage of the compound will be from about 0.005 mg/kg to 100 mg/kg, preferably about 0.01 mg/kg to about 50 mg/kg and most preferably about 0.5 to about 10 mg/kg and even more preferably about 0.6 to about 2 mg/kg. The true dosage and best route of administration may be established after thorough assessment of potential toxicity, inhibitory potency, and efficacy using techniques known to those skilled in the art.

If the compounds are to be applied topically, they can be admixed in a pharmacologically inert topical carrier such as a gel, an ointment, a lotion, or a cream; and include such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils. Other possible topical carriers are liquid petrolatum, isopropylpalmitate, polyethylene glycol ethanol 95%, polyoxyethylene monolaurate 5% in water, sodium lauryl sulfate 5% in water, and the like. Materials such as anti-oxidants, humectants, viscosity stabilizers, and the like may be added, if necessary. Administration by inhalation will utilize pressurized gases, propellants, and emulsifiers. Also, the compounds may be disposed within devices placed on, in, or under the skin; such devices include patches and implants which release the active material into the skin or body either by diffusion or by an active release mechanism. Similarly, if the compounds are administered parenterally, they will be prepared in sterile form; in multiple or single dose formats; and dispersed in a fluid carrier such as sterile physiological saline or 5% dextrose solutions commonly used with injectables.

The compounds of the present invention may be administered in combination with each other or separately, or with other anti-fibrotic agents. The following examples illustrate the teachings of the present invention and are not intended as limiting the scope of the invention. Example 1: Inhibition of Lysyl Oxidase Lysyl Oxidase Purification:

This example shows that homocysteine thiolactone inhibits lysyl oxidase. Fifty fresh bovine aorta (from 2-6-weeks-old calves; 562 grams) were obtained from the slaughterhouse and cleaned of the fat materials. They were ground coarsely using a chilled meat grinder, and homogenized in a Waring blender with 3 volumes of 16 mM phosphate buffer pH 7.8 containing 0.15 M NaCl. Phenylmethylsulfonyl (PMSF) and iodoacetamide were added to a final concentration of 1 mM before blending. The homogenate was centrifuged at 8000 rpm for 20 minutes. The pellet was against extracted with the same buffer and centrifuged. The resulting pellet was homogenized in 16 mM phosphate buffer pH 7.8 and centrifuged. The pellet was extracted with 4 M urea in 16 mM phosphate buffer pH 7.8 and stirred for an hour. After centrifugation, the pellet was again extracted with urea buffer as before. The supernatants from 3 urea extracts were pooled and filtered through a cheese cloth.

The pooled urea extracts were stirred with 500 grams of hydroxy apatite previously equilibrated with 16 mM phosphate buffer pH 7.8. After 1.30 hours, the contents were centrifuged at 4000 rpm for 10 minutes and concentrated to a final volume of 1 L in an SI Y 10 ultrafiltration cartridge. The concentrated sample was dialyzed against 72 L of 16 mM phosphate buffer pH 7.8 for 18 hours. After dialysis equal volumes of 1 M phosphate buffer pH 7.8 were added. The precipitate formed after 30 minutes and was collected after centrifiiging at 8000 rpm for 20 minutes.

The precipitate was dissolved in 15 ml of 6 M urea in 16 mM phosphate buffer pH 7.8 containing 30 mM NaCl and chromatographed on a Sephacryl-S-200 (120x5 cm) using 6 M urea buffer. Active fractions (from 1482-1632 ml elution) were pooled and concentrated to 20 ml using an Amicon YM-10 membrane. The concentrated sample was loaded on Sephacryl-S-100 (120x5 cm) and eluted with 6 M urea buffer. The active fractions from 481 ml to 520 ml elution were pooled and concentrated to 14 ml with an Amicon YM- 10 membrane and dialyzed against 12 L of 16 mM phosphate buffer pH 7.8. The purity of the enzyme was checked by 10% SDS polyacrylamide gel electrophoresis. Two Coomassie positive bands with the molecular weight of 32 Kd and 24 Kd were resolved. Western blot analysis revealed that both the bands were positive against lysyl oxidase antibody. Assay of Lysyl Oxidase: 1. Fluorometric assay:

The 2 ml assay mixture consists of 2.5 mM 1,5-diaminopentane, 0.75 mM homovannilic acid, 7.6 units of horse radish peroxidase, 1.5 M urea, and 50 mM borate buffer, pH 8.2. The reaction was started by adding 2.5-5 μg enzyme. The fluorescence of homovannilic acid dimer formed was measured at 315 nm excitation and 425 nm emission using a Perkin Elmer MPF-44B fluorescence spectrophotometer or an Aminco Bowman Series 2 Luminescence spectrometer. The assay was carried out at 55 °C. 2. Tritiated elastin assay:

The assay mixture (800 μl) consists of 125,000 CPM of tropoelastin labelled with tritiated lysine, 100 mM borate buffer pH 8.2 and 150 mM NaCl.

The reaction was started by adding 6-10 μg of enzyme and incubated at 37 °C for 2 hours. The tritiated water released by the action of lysyl oxidase was vacuum distilled. Four hundred μl of the labelled water was mixed with 5 ml of scintillation fluid and counted using LKB 1217 Rack Beta Liquid Scintillation Counter.

3. Tritiated collagen assay: About 300,000 to 500,000 CPM of tritiated lysine labelled collagen (12 to

18 μl) was preincubated at 37 °C for 1 hour to make the fibril. The buffer (100 mM phosphate buffer pH 7.8 containing 150 ml NaCl) and enzyme was added to labelled collagen fibril to a final volume of 750 μl. After 2 hour incubation at 37 °C, the assay mixture was vacuum distilled and 400 μl of labelled water was counted after mixing with 5 ml of scintillation fluid.

Tritiated lysine collagen was prepared as follows: The collagen substrate was prepared as detailed by Prokop and Tuderman (Methods in Enzymology, vol. 82, Part A. Edited by L. W. Cunningham and D. W. Frederiksen, eds., pp. 305-319, 1982). Calvarical parietal bones from 72 chick embryos (17 days old) were incubated at 37 °C in 50 ml flasks with lysine deficient medium (10 ml/ 12 calvaria) obtained from GIBCO-BRL (#32400-020) and supplemented with leucine, methionine, 2.5 mg BAPN, 2.5 mg ascorbic acid and penicillin. After 30 minutes incubation, the medium was changed and 4-5, H lysine (1 mCi/ml) was added. After incubation for 24 hours at 37 °C, the calvaria were removed and homogenized in 160 ml of 1 M NaCl in 0.05 M Tris HC1 pH 7.4 at 4 °C and centrifuged at 20,000 x g for 10 minutes. The collagen was precipitated by adding solid sodium chloride to 20% concentration. The precipitate was centrifuged at 10,000 rpm for 15 minutes and the pellet was dissolved in 30 ml of 0.15 M NaCl in 100 mM phosphate buffer pH 7.8 and dialyzed against the same buffer for 3 days. The dialysate was centrifuged at 30,000 x g for 15 minutes and the supernatant was used as substrate. Tritiated recombinant tropoelastin was prepared as follows: PAS - tropoelastin bacteria (E. coli) are inoculated on an LB plate [15 g bacto agar in 1 liter of L Broth (LB) liquid medium], autoclaved and 25 ml poured to sterile petri dish and solidified. LB medium was prepared by adding 10 g Bactotryptone, 5 g bacto yeast extract, 5 g NaCl, and water to 1 liter and autoclaved. 50 μg/ml ampicillin was added before use. After stripping the bacteria, the plate was incubated at 37 °C overnight. One colony was transferred to 50 ml LB medium containing ampicillin and incubated overnight at 37 °C. The cells were diluted to 1 liter by LB medium with the final optical density of 0.1 at 620 nm and cultured until the absorbance reached 0.3. The cells were centrifuged at 5000 rpm for 10 minutes. The cells were washed with lysine deficient RPM1-1640 medium (Sigma, St. Louis, MO). (The medium powder was dissolved in water and 3.5 g of sodium bicarbonate was added. After adjusting the pH 6.8, 10.25 ml of 200 mm glutamine, 5 ml of 20 mM methionine, 4.8 ml 79 mM leucine were added and made up to 1 liter with water and filtered.) The cells were suspended in 500 ml

RPMI -1640 medium and incubated for 10 minutes. Protein expression was stimulated by adding 500 ml of nalidixic acid (60 mg/ml in 0.1 N NaCl.)

3 After 2 hour post stimulation, 4,5-[ H]lysine was added to a final concentration of 2 μCi/ml and incubated 3 more hours at 38 °C. The cells were centrifuged at 5000 rpm for 10 minutes. The cells were washed twice in PBS [1 liter PBS consists of 0.2 g KC1, 0.2 g potassium phosphate (monobasic), 8 g sodium chloride, and 2.89 g of sodium phosphate • 12 H₂O (dibasic)], and autoclaved. The cells were suspended in 11 ml of 50 mM Tris buffer pH 8 containing EDTA, 1 mM DTT, 1 mM PMSF and 15% glycerol and centrifuged. The cells were weighed and 3 ml of buffer (50 mM Tris pH 8, 2 mM EDTA, 1 mM

DTT) was added per gram of cells and centrifuged at 10,000 rpm for 15 minutes.

To the cell pellet, 300 mg cyanogen bromide was added to each 500 ml of LB medium used and formic acid was added to a final concentration of 70% (w/v).

The content was stirred overnight at room temperature. 5 ml of water was added to reduce the viscosity and centrifuged at 1500 rpm for 15 minutes. The supernatant was dialysed against 3 changes of 6 liters of 0.2 N glacial acetic acid for 36 hours. The dialysate was centrifuged for 30 minutes at 1500 rpm and the supernatent was aliquated (400 μl) in Eppendorf tube. They were lyophilized and stored at -80 °C.

As shown Figures 1, 2 and 3, homocysteine thiolactone (FM4011) inhibits lysyl oxidase in its physiologic reactions with diaminopentane (Figure 1), tropoelastin (Figure 2) or collagen (Figure 3) as substrates. A number of other inhibitors such as certain vicinyl diamines do not act to inhibit the catalysis of the oxidation of all three of these substrates. The action on collagen is, of course, medically significant.

Example 2 : Beneficial effect of homocysteine thiolactone on corneal healing in the rabbit

This example shows that homocysteine thiolactone promotes corneal healing.

Homocysteine thiolactone was administered to both control and surgically wounded rabbit corneas. Homocysteine thiolactone was applied in physiological saline three times a day at a concentration of 10 mM, which is more than one thousand times the I50 of homocysteine thiolactone for lysyl oxidase with diaminopentane as substrate. There was no inflammation above control levels in either unwounded or wounded experimental animals.

After two weeks of post-surgical treatment with either saline or saline containing 10 mM homocysteine thiolactone, we noted that the extent of developing fibrosis was markedly less in the drug-treated animals. -28-

Example 3: Prevention of left-ventricular stiffness by homocysteine thiolactone

This example shows that homocysteine thiolactone (FM4011) prevents left-ventricular stiffness. Male Sprague Dawley rats weighing 350-470 g were randomly assigned to a control, or an isoproterenol group (isoproterenol) (500 μg/kg/24h for 10 days: i.p.), or an isoproterenol + homocysteine thiolactone (homocysteine thiolactone)(100 mg/kg/24h, i.v.) Two days prior to the first isoproterenol injection animals had an Alzet pump (2 ml) surgically placed in the subscapular region with a sterile piece of PE tubing connecting it to the jugular vein for drug delivery. Isoprotereonol + homocysteine thiolactone were infused for 10 consecutive days with isoproterenol at the previously listed dose.

Rats were anesthetized with 0.5 - 0.8 ml sodium pentabarbital and weighed. Once unresponsiveness was determined, hearts were extracted and placed in a tared beaker of chilled saline (0.9%) such that residual blood could be washed away and wet heart weight determined. Within 20 seconds of determining the heart weight, hearts were mounted on a short perfusion cannula in a Langendorff fashion. Hearts were retrogradely perfused with a red-blood cell perfusate. Briefly, fresh whole cow blood was collected at a local slaughterhouse in a vessel containing approximately 6,000 units of sodium heparin and 100,000 units of penicillin per liter. The containers of blood were immediately placed on ice to facilitate rapid cooling for transportation. The whole blood was then spun in a refrigerated centrifuge (5° C) at a rotor speed of 3,000 rpm for 15 minutes. The supernatant was aspirated and the resulting packed cells were mixed 1 : 1 with calcium-free Krebs-Henseleit buffer. The centrifugation and resuspension steps were repeated three times, resulting in packed red cells that were essentially free of white cells and platelets. The packed red-blood cells were mixed 1:1 with calcim free buffer and stored for future use at 4° C. Immediately prior to experimentation, blood was once again washed and mixed in a red-blood cell perfusate consisting of bovine red-blood cells at a final hematocrit of 40% suspended in a Krebs-Henseliet buffer containing, in mM/L: NaCl 118, KC1 4.7, KH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 25.5, glucose 5.5, lactate 1 , palmitic acid 0.4, and heparin 60 mU/ml, and 4g % bovine serum albumin (Sigma Chemical Co., St.

2+ Louis, MO). In order to mimic in situ physiological extracellular Ca

2+ concentrations as closely as possible, the perfusate ionized Ca concentration was

2+ adjusted to ImM Ca for rats by means of a calcium specific electrode. Gentamycin (0.2 mg/dl) was added to the red-blood cell perfusate to retard bacterial growth. The perfusate was gassed with 20% O₂, 3% CO₂, and 77% N , to achieve a PO₂ of 100-160 mmHg and a pH of 7.35 to 7.4.

Following initial perfusion of the heart, a small apical drain was inserted into the left ventricle for collection of Thebesian drainage. A second cannula was inserted into the pulmonary artery for collection of venous effluent. Pacing was established at 5.5 Hz for rats and 3 Hz for rabbits by placing an electrode (model 59, Grass Instrument Co., Quincy, Mass.) and a thermistor (model 400, Yellow Springs, Boulder, CO) into the right ventricle through the superior vena cavae and right atrium. A collapsed plastic balloon was inserted into the left ventricle via the left atrium. The balloon was connected to a pressure transducer (Gould Statham

P23dB) to measure left ventricular pressure and its first derivative. All hearts were loaded with an initial balloon volume set to yield an end-diastolic pressure of approximately 10 mmHg for equilibration.

Hearts were placed in a 0.9% saline bath and maintained at 37° C. All hearts were perfused at a constant level of coronary flow yielding a coronary perfusion pressure of 100 mmHg. Changes in coronary resistance were measured by alterations in coronary perfusion pressure via a pressure transducer (Gould- Statham P23dB. Gould Inc., Oxnard, Calif) attached to a side-arm of the aortic cannula by a short, inflexible tubing. Coronary flow was measured by timed collection of venous affluent. Coronary perfusion pressure (CPP), left ventricular pressure, and its first derivative were recorded continuously on a Gould physiological recorder.

All hearts were provided with an equilibration period of approximately 18 minutes. During this time, coronary flow was set to a level that elicited a coronary perfusion pressure of approximately 100 mmHg. Following equilibration, baseline myocardial performance was assessed. Following equilibration hearts underwent two pressure volume curves, such that the balloon volume was set to 0 and progressively infalted with 100 μl increments. The P/V relationship was terminated when end-diastolic pressure exceeded 50 mmHg. The second P/V curve was used for analysis. Following P/V relationship hearts were withdrawn and rinsed in saline. Hearts were dissected into left and right ventricle, atria and weighed. The left ventricle was sectioned, and a mid-portion was used for histological determination of fibrosis by Masson's trichrome staining and analyzed with planimetry.

Following the determination that catecholamine-induced fibrosis provided a convenient animal model for cardiac fibrosis, we infused animals intravenously with homocysteine thiolactone (85mg/kg/24h) concomitantly with the subcutaneous administration of isoproterenol (500μg/kg/24h for lOd). The three groups - untreated controls, isoproterenol only, and isoproterenol + homocysteine thiolactone - were compared. The data show that homocysteine thiolactone prevents the development of left- ventricular stiffness found in the isoproterenol- only group (Figure 4). In Figure 4, the open circles represent control (n=10), the open squares represent isoproterenol treated animals (n=8) and the closed squares represent isoproterenol + homocysteine thiolactone treated animals (n=l 1). "*" represents p<0.01 and "**" represents p<0.001. Significantly, the reduction of histologically demonstrable collagen (Figure 5) in isoproterenol + homocysteine thiolactone-treated animals validates the concept underlying the general approach described and claimed herein to the treatment and prevention of fibrosis, namely, that a reduction in crosslinking will result in lowered accumulation of collagen under fibrogenic conditions.

In Figure 5, fixed samples of tissue were embedded, sectioned and stained with Masson's trichrome stain. Serial sections from the left mid- ventricular region (3μM) were photographed. Images were digitized, stored on floppy disks and analyzed by computerized planimetry for specific staining. Data are expressed as mean ± SE. "*" indicates a significant difference from control values. "#" indicates a significant difference between isoproterenol-only and isoproterenol + homocysteine thiolactone values. Figure 5(A) shows the area of section; 5(B) shows the fibrotic area, and 5(C) shows percent fibrosis. Example 4: Inhibition of lysyl oxidase by homocysteine thiolactone analogs.

This example shows that homocysteine thiolactone analogs inhibit lysyl oxidase.

Lysyl oxidase was prepared and its activity measured as in Example 1 with the exception that homocysteine thiolactone analogs were substituted for homocysteine thiolactone. The Table below shows the inhibition (expressed as l5ø/diaminopentane) :

COMPOUND Iso/diaminopentane glycylhomocysteine thiolactone lOμM β-alanylhomocysteine thiolactone lOμM γ-aminobutyrylhomocysteine thiolactone 5μM e-aminocaproylhomocysteine thiolactone 25 μM lysylhomocysteine thiolactone 4μM Example 5: Synthesis of homocysteine thiolactone derivatives:

This example demonstrates the synthesis of the following homocysteine thiolactone derivatives: (I) glycylhomocysteine thiolactone; (II) β- L-alanylhomocysteine thiolactone; (III) γ-aminobutyrylhomocysteine thiolactone;

(IV) L-lysylhomocysteine thiolactone; and (V) ε-aminocaproylhomocysteine thiolactone. The above compounds were synthesized by peptide bond formation at the amino group of homocysteine thiolactone. All syntheses were carried out in solution in dichloromethane by well known procedures (e.g., Rich, D. and Singh, J. In The Peptides, vol. 1 , pp. 241 -261 , Academic Press, 1979; Bodanszky,

M. et al., J. Org. Chem., 38, 3565-3570, 1973). One to five millimoles of homocysteine thiolactone hydrochloride (Sigma Chemical Company) was dissolved in five to twenty-five milliliters of dichloromethane containing equimolar amounts of triethylamine (Sigma Chemical Company). In the case of compounds I, II, III and V, equimolar amounts of N-tertiary-butyloxycarbonyl (t-

BOC) derivatives of glycine, β-L-alanine, γ-aminobutyric acid, and ε- aminocaproic acid, respectively, dissolved in five to one hundred milliliters of dichloromethane were added to solutions of homocysteine thiolactone. All N- tBOC-protected derivatives were purchased from Sigma Chemical Company. To the mixtures were added equimolar amounts of dicyclohexylcarbondiimide (DCC;

Sigma Chemical Company) by addition of appropriate volumes of a 1M solution in dichloromethane. Reactions were incubated at ambient temperature and progress of reactions was monitored by thin-layer chromatography (TLC) on silica gel-G (E. Merck Darmstadt) in chloroform:methanol (4:1) followed by staining with 0.3% ninhydrin in ethanol. Due to ultraviolet light absorbance of homocysteine thiolactone (γ_max=238nm), it was possible to see the appearance of product by examination of the TLC strip under short-wave ultraviolet light. In this solvent system, homocysteine thiolactone has an R_f of about 0.68, while products have R_f's greater than 0.8. Homocysteine thiolactone gives a distinctly reddish spot with ninhydrin. Despite the blocked amino group of the products, they gave some color, which developed slowly upon heating the strip to about 110 °C; colors ranged from yellow to greenish purple. In general, reaction proceeded rapidly, and further processing was routinely carried out after two to three hours. However, reactions could be left for twenty-four hours if necessary. Conditions for the preparation of product IV were identical, except that N_ε, N_B-di-tBOC-L-lysine N- hydroxysuccinimide ester (Sigma Chemical Company) was reacted directly with equimolar homocysteine without the addition of DCC.

At the termination of reactions, the reaction mixes were placed on a rotary evaporator and dried under reduced pressure. The residues were dissolved in dichloromethane:ethyl acetate (1:1) and flash-chromatographed on columns of silica gel (10-50 grams; E. Merck Darmstadt). Elution was carried out under air pressure with 200-500 milliliters of the appropriate solvent. Fractions of 10-20 milliliters were collected. One to three μL samples of each fraction were spotted on a strip of backed silica gel and examined under short-wave ultraviolet light and then stained with ninhydrin to identify product-containing fractions. Purity was assessed by TLC in dichloromethane: ethyl acetate (1 :1), in which homocysteine thiolactone remains at the origin. Pure fractions were pooled and dried under reduced pressure by rotary evaporation. N-tBOC blocking groups were removed by treatment with trifluoroactic acid (TFA). To the dry residue were added one to two milliliters of neat (TFA) or 50% TFA in dichloromethane. Progress of the de¬ blocking reactions was followed by TLC on silica gel in chloroform:methanol (4:1) monitored by ultraviolet absorption and ninhydrin staining. After de¬ blocking, each product stains purple with ninhydrin and moves much more slowly in this solvent. TFA was removed by warming under reduced pressure. Hydrochloride salts (the dihydrochloride in the case of lysyl homocysteine thiolactone) were formed by dissolving the residues (trifluoroactetate salts) in one to two milliliters of 6N HCl at room temperature. After ten minutes HCl was removed and the solutions were dried by warming under reduced pressure. Dissolution in 6N HCl and drying was repeated twice more, and the residue was washed several times in diethyl ether, which was removed by decantation. Products were whitish crystalline powders whose absorption spectra showed maxima at about 210nm and 238nm. Treatment with base, which opens the thiolactone ring, destroys the peak at 238nm. Many other variations and modifications may be made in the techniques herein before described, by those having skill in this technology, without departing from the concept of the present invention. Accordingly, it should be clearly understood that the foregoing description is illustrative only, and not intended as a limitation on the scope of the invention.

Claims

CLAIMS What is claimed is:

1. A method of treating diseases and conditions associated with the abnormal deposition of collagen in a patient, which comprises administrating to the patient a therapeutically effective amount of a compound of the formula:

and salts thereof,

1 2 wherein R and R are the same or different, a hydrogen (-H), or an unsaturated or saturated aliphatic, alicyclic or aromatic hydrocarbon radical having from 1 to 50 carbon atoms, wherein said carbons are substituted with a substituent selected from the group consisting of hydrogen (-H), chloro, fluoro, bromo, iodo, hydroxyl (-OH), alkoxyl (-OR'), acyl (-COR'), carboxyl (-C0₂H), carboxyl esters (-CO₂R'), amido (CONR₂"), amino (-NR"₂), nitro

(-NO₂), nitroso (-NO), aza (-N=N-R'), diazonium (-N₂ ) azido

(-N₃) hydrazino (-NR'-NR' $, cyano (-CN), isocyano (-NC), cyanato (NCO-), isocyanato (OCN-), thiocyanato (NCS-), isothiocyanato (SCN-), thioamido (-C(S)NR'₂), thioether (-SR'), thiol (-SH), sulfoxide (-S(O)R'), sulfone (-S(O)₂R'), sulfoximino (-S(O)(NR")R'), sulfonic acid (-SO₃H), sulfonyl esters (-SO₃R'), sulfinic acid (-SO₂H), sulfinyl esters (-SO₂R'), sulfenic acid (-SOH), sulfenyl esters (-SOR'), phospho (-OP(O)(OR)₂), phosphono (-P(O)(OR)₂), urea (-NR'C(O)NR"₂), and silyl (-SiR'₃), and wherein R' a hydrogen (-H) or is an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, and R" is a hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms,

3 5 4 wherein R may be a hydrogen (-H) or R -NH₂, and wherein R may be a hydrogen (-H) or R -NH₂, and where R or R is an unsaturated or saturated branched or unbranched aliphatic, alicyclic, heterocyclic, aromatic or heteroaromatic radical having from 1 to 50 carbon atoms, wherein said carbons are substituted with a substituent selected from the group consisting of hydrogen (-H), chloro, fluoro, bromo, iodo, hydroxyl (-OH), alkoxyl (-OR'), acyl (-COR'), carboxyl (-CO₂H), carboxyl esters (-CO₂R'), amido (CONR₂"), amino (-NR"₂), nitro (-NO₂), nitroso (-NO), aza (-N=N-R'), diazonium (-N azido (-N₃) hydrazino (-NR'-NR'₂), cyano (-CN), isocyano (-NC), cyanato (NCO-), isocyanato (OCN-), thiocyanato (NCS-), isothiocyanato (SCN-), thioamido (-C(S)NR'₂), thioether (-SR'), thiol (-SH), sulfoxide (-S(O)R'), sulfone (-S(O)₂R'), sulfoximino (-S(O)(NR")R'), sulfonic acid (-SO₃H), sulfonyl esters (-SO₃R'), sulfinic acid (-SO₂H), sulfinyl esters (-SO 2 '), sulfenic acid (-SOH), sulfenyl esters (-SOR'), phospho (-OP(O)(OR)₂), phosphono (-P(O)(OR)₂), urea (-NR'C(O)NR"₂), and silyl (-SiR'₃), and wherein R' is an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, and wherein R" is a hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, and wherein n is integer from 1 to 3, X is S or O, Y is S or O, with the provision that X or Y must be S.

2. The method accordmg to claim 1 wherein R 1 or R 2 is an unsaturated or saturated aliphatic, alicyclic, or aromatic hydrocarbon radical having from 1 to 25 carbon atoms.

3. The method according to claim 1 wherein R 1 or R 2 is an unsaturated or saturated aliphatic, alicyclic, or aromatic hydrocarbon radical having from 1 to 15 carbon atoms.

4. The method according to claim 1 wherein R' is an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 25 carbon atoms.

5. The method according to claim 1 wherein R' is an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 15 carbon atoms.

6. The method according to claim 1 wherein R" is an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 25 carbon atoms.

7. The method according to claim 1 wherein R" is an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 15 carbon atoms.

8. The method according to claim 1 wherein R and R are the same or different, an unsaturated or saturated branched or unbranched aliphatic, alicyclic heterocyclic, aromatic or heteroaromatic hydrocarbon radical having from 1 to 25 carbon atoms.

9. The method according to claim 1 wherein R and R are the same or different, an unsaturated or saturated branched or unbranched aliphatic, alicyclic heterocyclic, or aromatic heteroaromatic hydrocarbon radical having from 1 to 15 carbon atoms.

10. The method according to claim 1 wherein the compound is selected from the group consisting of homocysteine thiolactone, glycylhomocysteine thiolactone, β-alanylhomocysteine thiolactone, γ-aminobutyrylhomocysteine thiolactone, lysylhomocysteine thiolactone, and ε-aminocaproylhomocysteine thiolactone, or mixtures thereof.

11. The method according to claim 1 wherein the disease or condition associated with the abnormal deposition of collagen is selected from the group consisting of atherosclerosis, arterial stenosis, restenosis, liver fibrosis, pulmonary fibrosis, surgical adhesions and burns.

12. A compound of the formula:

where m is an integer from 1 to 50, wherein R , R , R , R , R , and R are in a cis or trans relationship and are the same or different, a hydrogen (-H) or an unsaturated or saturated aliphatic, alicyclic or aromatic hydrocarbon radical having from 1 to 50 carbon atoms, wherein said carbons are substituted with a substituent selected from the group consisting of hydrogen (-H), chloro, fluoro, bromo, iodo, hydroxyl (-OH), alkoxyl (-OR'), acyl (-COR'), carboxyl (-CO₂H), carboxyl esters (-CO₂R'), amido (CONR₂ "), amino (-NR"₂), nitro (-NC^), nitroso (-NO), aza (-N=N-R'), diazonium (-N ) azido (-N3) hydrazino (-NR'-NR'₂), cyano (-CN), isocyano (-NC), cyanato (NCO-), isocyanato (OCN-), thiocyanato (NCS-), isothiocyanato (SCN-), thioamido (-C(S)NR'₂), thioether (-SR'), thiol (-SH), sulfoxide (-S(O)R'), sulfone (-S(O)₂R'), sulfoximino (-S(O)(NR")R'), sulfonic acid (-SO3H), sulfonyl esters (-SO3R'), sulfinic acid (-SO₂H), sulfinyl esters (-SO₂R'), sulfenic acid (-SOH), sulfenyl esters (-SOR'), phospho (-OP(O)(OR)₂), phosphono (-P(O)(OR)₂), urea (-NR'C(O)NR"₂), and silyl (-SiR'₃), and wherein R' is hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, and R" is a hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, wherein n is integer from 1 to 3, X is S or O, Y is S or O, with the provision that X or Y must be S.

13. The compound according to claim 12 wherein m is an integer of

14. The compound according to claim 12 wherein m is an integer of

15. A method of treating diseases and conditions associated with the abnormal deposition of collagen in a patient, which comprises administering to the patient a therapeutically effective amount of the compound of claim 12.

16. The method according to claim 15 wherein the disease or condition associated with the abnormal deposition of collagen is selected from the group consisting of atherosclerosis, arterial stenosis, restenosis, liver fibrosis, pulmonary fibrosis, surgical adhesions and burns.

17. The method according to claim 15 wherein said compound is selected from the group consisting of glycylhomocysteine thiolactone, β- alanylhomocysteine thiolactone, γ-aminobutyrylhomocysteine thiolactone, lysylhomocysteine thiolactone, and ε-aminocaproylhomocysteine thiolactone, or mixtures thereof.

18. A compound of the formula:

X

wherein Z is hydrogen (-H), -(CH₂)_m NR'R",or -(CH₂)_m OR', wherein m is an integer from 1 to 50,

1 2 3 4 5 wherein R , R , R , R and R are in a cis or trans relationship and are the same or different, a hydrogen (-H) or an unsaturated or saturated aliphatic, alicyclic or aromatic hydrocarbon radical having from 1 to 50 carbon atoms, wherein said carbons are substituted with a substituent selected from the group consisting of hydrogen (-H), chloro, fluoro, bromo, iodo, hydroxyl (-OH), alkoxyl (-OR'), acyl

(-COR'), carboxyl (-CO₂H), carboxyl esters (-CO₂R'), amido (CONR₂"), amino (-NR" nitro (-NO ),₂nitroso (-NO), aza (-N=N-R), diazonium (-N₂ ⁺) azido (-N₃) hydrazino (-NR'-NR'₂), cyano (-CN), isocyano (-NC), cyanato (NCO-), isocyanato (OCN-), thiocyanato (NCS-), isothiocyanato (SCN-), thioamido (-C(S)NR'₂), thioether (-SR'), thiol (-SH), sulfoxide (-S(O)R'), sulfone (-S(O)₂R')₅ sulfoximino (-S(O)(NR")R'), sulfonic acid (-SO₃H), sulfonyl esters (-SO₃R'), sulfinic acid (-SO₂H), sulfinyl esters (-SO₂R'), sulfenic acid (-SOH), sulfenyl esters (-SOR'), phospho (-OP(O)(OR)₂), phosphono (-P(O)(OR)₂), urea (-NR'C(O)NR"₂), and silyl (-SiR'₃), and wherein R' is hydrogen

(-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, and R" is a hydrogen (-H) or an alkyl, alkenyl, alkynyl, aryl or heterocyclic of 1 to 50 carbon atoms, wherein n is an integer from 1 to 3, X is S or O, Y is S or O, with the provision that X or Y must be S.

19. A method of treating diseases and conditions associated with the abnormal deposition of collagen in a patient, which comprises administering to the patient a therapeutically effective amount of the compound of claim 18.

20. The method according to claim 19 wherein the disease or condition associated with the abnormal deposition of collagen is selected from the group consisting of atherosclerosis, arterial stenosis, restenosis, liver fibrosis, pulmonary fibrosis, surgical adhesions and burns.

21. The method according to claim 19 wherein said compound is selected from the group consisting of glycylhomocysteine thiolactone, β- alanylhomocysteine thiolactone, γ-aminobutyrylhomocysteine thiolactone, lysylhomocysteine thiolactone, and ε-aminocaproylhomocysteine thiolactone, or mixtures thereof.