WO2009151744A1 - S-nitrosomercapto compounds and related derivatives - Google Patents

Abstract

The present invention is directed to mercapto-based and S- nitrosomercapto-based SNO compounds and their derivatives, and their use in treating a lack of normal breathing control, including the treatment of apnea and hypoventilation associated with sleep, obesity, certain medicines and other medical conditions.

Description

TITLE S-Nitrosomercapto Compounds and Related Derivatives

CROS S REFERENCE TO RELATED APPLICATIONS

Application claims priority under 35 U. S. C. § 119(e) to US Provisional Application No. 61/072,919, filed on April 2, 2008, which application is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

Normal control of breathing is a complex process that involves the body's interpretation and response to chemical stimuli such as carbon dioxide, pH and oxygen levels in blood, tissues and the brain. Breathing control is also affected by wakefulness (i.e., whether the patient is awake or sleeping). Within the brain medulla, there is a respiratory control center that interprets the various signals that affect respiration and issues commands to the muscles that perform the work of breathing. Key muscle groups are located in the abdomen, diaphragm, pharynx and thorax. Sensors located centrally and peripherally provide input to the brain's central respiration control areas that enables response to changing oxygen requirements. Normal respiratory rhythm is maintained primarily by the body's rapid response to changes in carbon dioxide levels (CO₂). Increased CO₂ levels signal the body to increase breathing rate and depth, resulting in higher oxygen levels and subsequent lower CO₂ levels. Conversely, low CO₂ levels can result in periods of apnea (no breathing) since the stimulation to breathe is absent; this is observed in hyperventilation events.

The ability of a mammal to breathe, and to modify breathing according to the amount of oxygen available and demands of the body, is essential for survival. There are a variety of conditions that are characterized by loss of respiratory rhythm due to either a primary or secondary cause. Examples of a primary loss of breathing rhythm control are apneas and congenital central hypoventilation syndrome.

Secondary loss of breathing rhythm may be due to chronic cardiopulmonary diseases, excessive weight, certain drugs and/or factors that affect the neurological system.

The most common primary loss of breathing rhythm control is sleep apnea, characterized by frequent periods of no or partial breathing during sleep. When a patient with sleep apnea goes to sleep, respiratory drive and the muscle tone in the airway decrease and the airway collapses during inspiration, causing an obstruction to normal breathing. Key factors that contribute to these apneas include decrease in CO₂ receptor sensitivity, decrease in hypoxic ventilatory response sensitivity (e.g., decreased response to low oxygen levels) and loss of "wakefulness". Normal breathing rhythm is disturbed, resulting in hypoxia (and the associated oxidative stress) and eventually severe cardiovascular consequences (high blood pressure, stroke and heart attack). Similarly to sleep apnea, individuals who snore also present loss in upper airway muscles tone, causing inefficient airflow, which may result in hypoxia.

During sleep, breathing changes with the stage or depth of sleep. When episodes of apnea become more frequent and last longer, they can cause the body's oxygen level to decrease, which can disrupt sleep. The patient may not fully awaken, but is aroused from the deep restful stages of sleep, and thus feels tired the next day. Commonly, these patients have exhibited loud snoring for many years and are generally male. The sleep problems are often aggravated by alcohol or sedative medications.

There are two main types of sleep apnea that may occur together. The most common is obstructive sleep apnea, during which breathing is blocked by a temporary obstruction of the main airway, usually in the back of the throat. This often occurs because the tongue and throat muscles relax, causing the main airway to close and stopping airflow. After a short interval lasting seconds to minutes, the oxygen level drops, causing breathing efforts to become more vigorous, which eventually opens the obstruction and allows airflow to resume. This often occurs with a loud snort and jerking of the body, causing the patient to arouse from deep sleep. After a few breaths, the oxygen level returns to normal, the patient falls back to sleep, the muscles of the main airway relax and the obstruction occurs again. This cycle is then repeated over and over during certain stages of sleep. Most people with obstructive sleep apnea snore, suggesting that their main airway is already partly obstructed during sleep, but not all people who snore have obstructive sleep apnea. A less common form of sleep apnea is central sleep apnea, so named because the central control of breathing is abnormal. This control center lies in the brain, and its function can be disrupted by a variety of factors. There is no obstruction to airflow. The patient with sleep apnea stops breathing because the brain suddenly fails to signal the muscles of the chest and diaphragm to keep breathing. These patients do not resume breathing with a snort and body jerk, but merely start and stop breathing at various intervals. Although the mechanism is different than obstructive sleep apnea, sleep is still disturbed by the periodic decreases in oxygen, and the patients suffer from the same daytime symptoms. Some patients may suffer from a combination of the two causes of apnea, a disorder which is called mixed-sleep apnea, also termed "complex sleep apnea".

Another example of primary loss of breathing rhythm control is classic congenital central hypoventilation syndrome (CCHS), characterized by adequate ventilation while the affected individual is awake and by hypoventilation with normal respiratory rates and shallow breathing during sleep; more severely affected individuals hypoventilate when both awake and asleep.

Secondary loss of breathing rhythm may be due to chronic cardiopulmonary diseases (e.g., heart failure, chronic bronchitis, emphysema, and impending respiratory failure), excessive weight (e.g., obesity-hypoventilation syndrome), certain drugs (e.g., anesthetics, sedatives, anxiolytics, hypnotics, alcohol and narcotic analgesics) and/or factors that affect the neurological system (e.g., stroke, tumor, trauma, radiation damage and ALS).

In general, chronic cardiopulmonary diseases cause hypoxia due to a reduction in the amount of gases exchanged by the lungs. The deficit in oxygen delivered to the blood ultimately puts a high burden on the heart to sustain increasing blood output, raising the probability of heart failure.

Obesity hypoventilation syndrome is a condition in which severely overweight people fail to breathe rapidly enough or deeply enough, resulting in low blood oxygen levels and high blood carbon dioxide (CO₂) levels. Affected individuals may also frequently stop breathing altogether for short periods of time during sleep (obstructive sleep apnea), resulting in many partial awakenings during the night. The disease strains the heart, eventually leading to symptoms of heart failure, such as leg swelling and various other related symptoms. The most effective treatment is weight loss, but it is often possible to relieve the symptoms by nocturnal ventilation with positive airway pressure (CPAP) or related methods. Obesity hypoventilation syndrome is defined as the combination of obesity (body mass index above 30 kg/m²), hypoxia (falling oxygen levels in blood) during sleep, and hypercapnia (increased blood carbon dioxide levels) during the day, resulting from hypoventilation (excessively slow or shallow breathing).

Drug-induced respiratory depression is a life-threatening condition caused by analgesic, hypnotic, and anesthesia medications. Although it is a leading cause of death from the overdose of some classes of abused drugs, respiratory depression also arises during normal, physician-supervised procedures such as surgical anesthesia, post-operative analgesia, and as a result of normal outpatient management of pain from cancer, accidents, or illnesses. The majority of adverse events occurring with these drugs take place during the dose adjustment period, when two or more central nervous depressants are taken together, or when patients take prescribed drugs in ways not intended by their physician.

Although only 0.5 % to 1.2 % of total adverse drug events caused by prescription medications are respiratory in nature, these serious side effects account for 25 % to 30 % of drug-induced deaths. Opiates and barbiturates are the primary drugs classes responsible for these effects. Opiates include the standard pain-killing drugs morphine, fentanyl, and codeine, as well as related products vicodin, hydrocodone, and oxycontin. Barbiturates comprise the sedative drugs amobarbital, aprobarbital, butabarbital, pentobarbital, and others. Sleeping disorders are another common predisposing factor for respiratory depression, in this case known as sleep apnea.

Currently, the only way to counter opiate-induced respiratory depression is to administer opiate receptor antagonists, which block the effectiveness of opiate analgesia. This approach may prevent a serious side effect or even death, but it dramatically reduces the effectiveness of drugs administered for management of severe pain.

Estimates for afflicted individuals for several of the most frequent conditions in the United States include, sleep apneas: 15-20 million; obesity- hypo ventilation syndrome: 5-10 million; chronic heart disease: 5 million; chronic obstructive pulmonary disease (COPD)/chronic bronchitis: 10 million; drug-induced hypoventilation: 2-5 million; and mechanical ventilation weaning: 0.5 million.

The definitive treatment for many breathing control disorders is either mechanical ventilation or positive airway pressure devices [e.g., continuous positive airway pressure device (CPAP device), bi-level positive airway pressure device (BiPAP device)]. Generally these mechanical ventilation devices have poor compliance. Pharmaceutical products that could be used either alone or as an adjuvant to positive airway pressure devices, thereby lowering the pressure required to maintain airway patency, would be an important advance to either improve compliance with these PAP devices or provide an alternative means of treatment. Several pharmacologic agents have been proposed as interventions to control respiration in sleep-related breathing disorders. Such agents cited in the literature include Progestin, Almitrine and Acetazolmide (DeBacker, 1995, Eur. Respir. J. 8: 1372-1383); medroxyprogesterone, thyroid replacement, acetazolamide, theophylline, tricyclic antidepressants, serotonin reuptake inhibitors and clonidine (Hudgel & Thanakitcharu, 1998, Am. J. Respir. Crit. Care Med. 158: 691-699). In 2005, a review on treatment of obstructive sleep apnea included benzodiazepines, narcotics, acetazolamide, serotonin agonists, serotonin re-uptake inhibitors, serotonin antagonists and antidepressants (Qureshi & Lee-Chiong, 2005, TL Sem. Resp. Crit. Care Med. 26: 96-108). In particular, low doses of the carbonic anhydrase inhibitor acetazolamide appeared to exert a beneficial effect not related to its traditional action of reducing pH as a mechanism of respiratory stimulation (DeBacker, 1995, Eur. Respir. J. 8: 1372-1383). In a small, uncontrolled clinical study in central apnea patients, low doses of acetazolamide were found to decrease apnea episodes from 25.5 pre-treatment to 6.8 after one month of treatment (73%). Smaller reductions (about 25%) were seen in patients that had predominantly obstructive sleep apnea.

More recently, a serotonin agonist/antagonist combination increased motor tone in the portion of the throat that collapses in obstructive sleep apnea (Carley & Radulovacki, 1999, Am. J. Respir. Crit. Care Med. 160: 1824-1829). This concept is currently in commercial development by a partnership comprised of Organon and Cypress Bioscience and a separate group, BTG pic (see, for example, US Patent Application Publication Nos. 2006/0039866, 2006/0039867 and 2006/0122127).

In a different approach, the S-nitrosomercapto signaling pathway may be used to exert control over respiration by increasing minute ventilation

(International Patent Application Publication No. WO2003/015605, the entirety of which is incorporated by reference herein). S-nitrosomercapto compounds have been identified as potentially having a wide range of therapeutic uses in medicine (Hogg, 2002, Annu. Rev. Pharmacol. Toxicol. 42: 585-600, Hogg, 2000, Free Rad. Biol. Med. 28: 1478-1486, Gaston et al., 2006, Am. J. Respir. Crit. Care Med. 173: 1186- 1193, Palmer, 2005, Proc. Am. Thorac. Soc. 3: 166-169). These compounds have a mercapto group (SH) that binds to nitric oxide (NO). Depending upon the structure, the mercapto groups may act as carriers of nitric oxide (NO) or covalently bond the NO. It is also possible that certain molecules have some characteristics of both NO carriers and covalently bound molecules.

Certain S-nitrosomercapto agents act as signaling agents critical to the control of the rate and depth of breathing (respiratory control), ventilation-perfusion matching, upper airway muscle tone and pulmonary vascular tone. In fact, the centrally-mediated hypoxic ventilatory response system was shown to be under the control of certain S-nitrosomercapto compounds, and these compounds may induce the body's typical response to low oxygen levels, triggering, among other reactions, increases in the rate and depth of breathing. The ability to restore respiratory drive in patients in whom it is impaired will open up a new era of therapeutics for the 50 million patients in the United States alone that have a condition associated with diminished respiratory drive (Am. J. Respir. Crit. Care Med. 2006, 173: 1186-1193).

There have been several recent breakthroughs in understanding the detailed mechanism of action of S-nitrosomercapto compounds. Chen et al. (J. Biol. Chem. 2006, 281: 9190-9199) recently reported that S-nitrosoglutathione, when used in cystic fibrosis, stimulates chloride secretion via both cGMP-dependent and cGMP- independent mechanisms. Whether S-nitrosomercapto compounds exert an all-or- nothing effect on cGMP has been an important issue in understanding the molecular biology of these compounds (Zhang and Hogg, 2004, Am. J. Physiol. - Lung Cell. MoI. Physiol. 287: 467-474). S-nitrosomercapto compounds activate HIF-I (hypoxia-inducible factor, 1) by an interesting pathway. Normoxia is sensed by proline hydroxylation at residues Pro564 and Pro402 of the α subunit of HIF-I . In hypoxia, hydroxylation does not occur, and HIF-I is stabilized, dimerizes with a β subunit, and alters the transcription of hypoxia-regulated genes. Activation of HIF-I has been proposed as a therapeutic treatment for tissue ischemia, ischemia/hypoxia disorders, myocardial ischemia, and peripheral ischemia, and activators of HIF-I have been proposed as chemopreventive agents to reduce reperfusion injury following heart attack and stroke (Nagle & Zhou, 2006, Curr. Pharm. Des. 12: 2673-2678). Conversely, inhibitors of HIF-I have been proposed for various types of adjunctive cancer treatment (Melillo, 2006, MoI. Cancer Res. 4: 601-605).

Much scientific effort has been centered on accurate and reproducible methods for detection of S-nitrosomercapto-containing proteins, a difficult challenge due to the liability of the nitrosomercapto group. S-nitrosated proteins have been identified with a biotin-switch assay (Zhang et al., 2005, Free Rad. Biol. Med. 38: 874-881) and labeled with affinity and radioactive tags. These are important steps that will ultimately lead to the identification of the specific target proteins that are responsible for the biological effects on breathing control. Criteria have been proposed to help establish that a specific bioactivity is associated with S-nitrosation or denitrosation of a specific protein (Am. J. Physiol. Lung Cell. MoI. Physiol. 2004, 287: 465-466).

The S-nitrosomercapto compounds previously shown to increase respiratory drive via the hypoxic ventilatory drive pathway are not ideal as modern day medicines. For example, N-acetyl-L-cysteine (which acts as a S-nitrosomercapto prodrug) is poorly orally absorbed and is subject to extensive first pass metabolism by the liver. The most common clinical use for N-acetyl-L-cysteine (e.g., Acetadote™, Cumberland Pharmaceuticals, Nashville, TN) is for acetaminophen overdose, where the intended therapeutic effect is to form cysteine as a metabolic breakdown product. In summary, poor or inefficient respiratory drive results in hypoventilation, which further results in hypoxia. A primary initial clinical manifestation of hypoxia is drowsiness or excessive daytime sleepiness. Accordingly, drugs that cause decreased respiratory drive and the resulting hypoxia are sometimes limited in their usefulness due to the fear of a life-threatening respiratory depression and/or the excessive daytime sleepiness that negatively impacts quality of life.

Another outcome of hypoxia from respiratory drive deficiency is oxidative stress that has been linked to longer term cardiovascular and/or metabolic outcomes. Accordingly, a single compound, or in the alternative, a combination pharmaceutical product, that could safely restore all or part of the body's normal breathing control system in response to changes in CO₂ and/or oxygen would be of benefit in decreasing the incidence and severity of breathing control disturbances. There is currently an unmet need for such a product that can be administered to a patient with minimal side effects. The present invention addresses and meets these needs. BRIEF SUMMARY OF THE INVENTION

The invention includes one or more S-nitrosomercapto-based SNO compounds selected from the group consisting of (S)-2-(3,3-dimethylureido)-3- (nitrosomercapto)propanoic acid, (R)-2-(3,3-dimethylureido)-3-(nitrosomercapto)- propanoic acid, (R)-3-nitrosomercapto-2-(methoxycarbonylarnino)propanoic acid, (S)-3-nitrosomercapto-2-(methoxycarbonylamino)propanoic acid, (S)-2-((S)-2,4- diaminobutanamido)-3-(nitrosomercapto)propanoic acid, (R)-2-((S)-2,4- diaminobutanamido)-3-(nitrosomercapto)propanoic acid, (R)-S-nitroso-N- acetylcysteine, (S)-S-nitroso-N-acetylcysteine, (R)-2-acetamido-3 -nitrosomercapto-N- (methylsulfonyl)propanamide, (R)-N-(2-nitrosomercapto- 1 -( 1 H-tetrazol-5- yl)ethyl)acetamide, (R)-3-nitrosomercapto-2-propionamidopropanoic acid, (R)-2- benzamido-3-(nitrosomercapto)propanoic acid, (S)-4-(2-acetamido-3- (nitrosomercapto)-propanamido)benzoic acid, (R)-4-(2-acetamido-3- (nitrosomercapto)propanamido)benzoic acid, (R)-2-acetamido-3 -nitrosomercapto-3 - methylbutanoic acid, (S)-2-acetamido-3 -nitrosomercapto-3 -methylbutanoic acid, (S)- 2-(benzo[d]oxazol-2-ylamino)-3-nitrosomercapto-3-methylbutanoic acid, (R)-2- (benzo[d]oxazol-2-ylamino)-3-nitrosomercapto-3-methylbutanoic acid, 2- (benzo[d]oxazol-2-ylamino)-3-(nitrosomercapto)propanoic acid, (R)-2- (benzo[d]oxazol-2-ylamino)-3-(nitrosomercapto)propanoic acid, (S)-methyl 2- (benzo[d]oxazol-2-ylamino)-4-(nitrosomercapto)butanoate, (S)-methyl 2-(5- fluorobenzo[d]oxazol-2-ylamino)-4-(nitrosomercapto)butanoate, (R)-2-(6- chlorobenzo[d]oxazol-2-ylamino)-3-nitrosomercapto-propanoic acid, (R)-3- nitrosomercapto-2-(4-methylbenzo[d]oxazol-2-ylamino) propanoic acid, S-nitroso- (R)-2-(benzo[d]oxazol-2-ylamino)-2-(lH-tetrazol-5-yl)-l-nitrosomercaptoethane, (R)- 2-(2-(benzo[d]oxazol-2-ylamino)-3-nitrosomercapto-propanamido)acetic acid, (R)- methyl 2-(2-(benzo[d]oxazol-2-ylamino)-3-(nitrosomercapto)propanamido)acetate, 2- (benzo [d]thiazol-2-ylamino)-3 -(nitrosomercapto)propanoic acid, (S)-2- (benzo[d]thiazol-2-ylamino)-4-(nitrosomercapto)butanoic acid, (S)-methyl 4- (nitrosomercapto)-2-(benzo[d]thiazol-2-ylamino)butanoate, (S)-2-(benzo[d]thiazol-2- ylamino)-3 -nitrosomercapto-3 -methylbutanoic acid, (S)-methyl 2-(benzo[d]thiazol-2- ylamino)-3-nitrosomercapto-3-methylbutanoate, (R)-3-nitrosomercapto-2-(thiazol-2- ylamino)propanoic acid, (S)-3-nitrosomercapto-2-(thiazol-2-ylamino)propanoic acid, 3 -nitrosomercapto-2-(thiazol-2-ylamino)propanoic acid, (R)-2-(5 -(ethoxycarbonyl)-4- methylthiazol-2-ylamino)-3-(nitrosomercapto)propanoic acid, (R)-3-nitrosomercapto- 2-(5-methyl-4-phenylthiazol-2-ylamino)propanoic acid, (S)-4-nitrosomercapto-2-(4- phenylthiazol-2-ylamino)butanoic acid, (S)-4-nitrosomercapto-2-(5-methyl-4- phenylthiazol-2-ylamino)butanoic acid, (S)-4-nitrosomercapto-2-(thiazol-2- ylamino)butanoic acid, (S)-4-nitrosomercapto-2-(methyl(thiazol-2-yl)amino)butanoic acid, (R)-2-(4-tert-butylthiazol-2-ylamino)-3-(nitrosomercaptopropanoic acid, (R)-2- (4,5-dimethylthiazol-2-ylamino)-3-(nitrosomercapto)propanoic acid, (R)-3- nitrosomercapto-2-(4,5,6,7-tetrahydrobenzo[d]thiazol-2-ylamino)propanoic acid, (R)- 3-nitrosomercapto-2-(5-nitropyridin-2-ylamino)propanoic acid, 3-nitrosomercapto-2- (5 -(trifluoromethyl)pyridin-2-ylamino)propanoic acid, (R)-2-(4,6-bis(4- methylpiperazin-l-yl)-l,3,5-triazin-2-ylamino)-3-(nitrosomercapto)propanoic acid, (R)-2-(4,6-bis(dimethylamino)- 1 ,3 ,5-triazin-2-ylamino)-3- (nitrosomercapto)propanoic acid, (R)-2-(l-(4,6-bis(allylamino)-l ,3,5-triazin-2- yl)piperidin-4-ylamino)-3 -(nitrosomercapto)propanoic acid, (S)-4-nitrosomercapto-2- (pyrimidin-2-ylamino)butanoic acid, (R)-2-(6-chloropyrimidin-4-ylamino)-3- nitrosomercapto-3-methylbutanoic acid, (R)-2-(3-nitrosomercapto-2-(thiazol-2- ylamino) propanamido)acetic acid, (R)-2-(lH-tetrazol-5-yl)-2-(thiazol-2-ylamino)-l- nitrosomercaptoethane, (S)-4-nitrosomercapto-2-(methyl(thiazol-2-yl)amino)butanoic acid, (R)-4-nitrosomercapto-2-(methyl(thiazol-2-yl)amino)butanoic acid, (R)-3- nitrosomercapto-3 -methyl-2-(thiazol-2-ylamino)butanoic acid, (S)-3 -nitrosomercapto- 3-methyl-2-(thiazol-2-ylamino)butanoic acid, (2R,3R)-ethyl l-acetyl-3- (nitrosomercapto)pyrrolidine-2-carboxylate, (2S,4S)-l-tert-butyl 2-ethyl 4- (nitrosomercapto)pyrrolidine-l ,2-dicarboxylate, (2S,4S)-ethyl 1 -acetyl-4- (nitrosomercapto)pyrrolidine-2-carboxylate, (2S,3S)-ethyl 1 -acetyl-3- (nitrosomercapto)pyrrolidine-2-carboxylate, (R)-2-acetamido-3-nitrosomercapto-N- phenylpropanamide, (R)-2-acetamido-N-(4-chlorophenyl)-3 -nitrosomercapto- propanamide, (R)-2-acetamido-N-(3-chlorophenyl)-3-nitrosomercapto-propanamide, (R)-2-acetamido-N-(2-chlorophenyl)-3-nitrosomercapto-propanamide, (R)-3-(2- acetamido-3-nitrosomercapto-propanamido)benzoic acid, (R)-ethyl 2-acetamido-3- (nitrosomercapto)propanoate, (R)-3-acetamido-2-(nitrosomercaptomethyl)propanoic acid, (S)-2-acetamido-4-(nitrosomercapto)-butanoic acid, (R)-3-nitrosomercapto-2-(5- (4-methylpiperazin-l-yl) pentanamido)propanoic acid, (R)-2-acetamido-3- nitrosomercapto-3-methylbutanoic acid, (R)-2-acetamido-2-(4-methylpiperazin- 1 - yl)carbonyl- 1 -nitrosomercapto-ethane, (R)-2-acetamido-2-morpholinocarbonyl- 1 - nitrosomercapto-ethane, (S)-ethyl 1 -((R)-2-acetamido-3-nitrosomercapto-propanoyl)- pyrrolidine-2-carboxylate, (S)- 1 -((R)-2-acetamido-3 -nitrosomercapto-propanoyl)- pyrrolidine-2-carboxylic acid, (S)-l-((R)-2-(benzo[d]oxazol-2-ylamino)-3- nitrosomercapto-propanoyl)pyrrolidine-2-carboxylic acid, (S)-3 -nitrosomercapto-2- (phenylsulfonamido)-propanoic acid, (R)-3-nitrosomercapto-2-(phenylsulfonamido)- propanoic acid, (R)-5-(l-carboxy-2-(nitrosomercapto)-ethylamino)pentanoic acid, (R)-3 -nitrosomercapto-2-(4-(4-methylpiperazin- 1 -yl) butanamido)propanoic acid, (R)-3-nitrosomercapto-2-(5-(4-methylpiperazin-l-yl) pentanamido)propanoic acid, S- nitroso-cysteine, (R)-3 -nitrosomercapto-2-(4-morpholinobutanamido)propanoic acid, (R)-2-(benzo[d]oxazol-2-ylamino)-3-nitrosomercapto-N- (methylsulfonyl)propanamide, (S)-2-(benzo[d]oxazol-2-ylamino)-3-nitrosomercapto- 3-methyl-N-(methylsulfonyl)butanamide, (S)-2-(benzo[d]oxazol-2-ylamino)-3- nitrosomercapto-3 -methyl-N-(phenylsulfonyl)butanamide, (R)-methyl 2-(2- (benzo[d]oxazol-2-ylthio)acetamido)-3-nitrosomercaptopropanoate, and (R)-methyl 2-(2-(benzo[d]oxazol-2-ylthio)acetamido)-3-nitrosomercapto-3-niethylbutanoate, a salt thereof and mixtures thereof.

The invention also includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and one or more S-nitrosomercapto-based SNO compounds, a pharmaceutically acceptable salt thereof and mixtures thereof. The invention further includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more mercapto-based SNO compounds selected from the group consisting of (S)-2-(3,3-dimethylureido)-3- mercaptopropanoic acid, (R)-2-(3,3-dimethylureido)-3-mercaptopropanoic acid, (R)- 3 -mercapto-2-(methoxycarbonylarnino)propanoic acid, (S)-3 -mercapto-2- (methoxycarbonylamino)-propanoic acid, (S)-2-((S)-2,4-diaminobutanamido)-3- mercaptopropanoic acid, (R)-2-((S)-2,4-diaminobutanamido)-3-mercaptopropanoic acid, (R)-2-acetamido-3 -mercapto-N-(methylsulfonyl)propanamide, (R)-N-(2- mercapto- 1 -( 1 H-tetrazol-5-yl)ethyl)acetamide, (S)-4-(2-acetamido-3- mercaptopropanamido)benzoic acid, (R)-4-(2-acetamido-3- mercaptopropanamido)benzoic acid, (S)-2-acetamido-3 -mercapto-3 -methylbutanoic acid, (S)-2-(benzo[d]oxazol-2-ylamino)-3-mercapto-3-methylbutanoic acid, (R)-2- (benzo[d]oxazol-2-ylamino)-3-mercapto-3-methylbutanoic acid, (R)-2- (benzo[d]oxazol-2-ylamino)-3 -mercaptopropanoic acid, (2S,2'S)-dimethyl 4,4'- disulfanediylbis(2-(benzo[d]oxazol-2-ylamino)butanoate), (S)-methyl 2- (benzo[d]oxazol-2-ylamino)-4-mercaptobutanoate, (S)-methyl 4-(acetylthio)-2- (benzo[d]oxazol-2-ylamino)butanoate, (S)-3-(benzo[d]oxazol-2- ylamino)dihydrothiophen-2(3H)-one, (2S,2'S)-dimethyl 4,4'-disulfanediylbis(2-(5- fluorobenzo[d]oxazol-2-ylamino)butanoate), (S)-methyl 4-(acetylthio)-2-(5- fluorobenzo[d]oxazol-2-ylamino)butanoate, (S)-3-(5-fluorobenzo[d]oxazol-2- ylamino)dihydrothiophen-2(3H)-one, (S)-2-(benzo[d]thiazol-2-ylamino)-4- mercaptobutanoic acid, (S)-4-(acetylthio)-2-(benzo[d]thiazol-2-ylamino)butanoic acid, (S)-methyl 4-(acetylthio)-2-(benzo[d]thiazol-2-ylamino)butanoate, (S)-3- (benzo[d]thiazol-2-ylamino) dihydrothiophen-2(3H)-one, (S)-2-(benzo[d]thiazol-2- ylamino)-3-mercapto-3-methylbutanoic acid, (S)-methyl 2-(benzo[d]thiazol-2- ylamino)-3-mercapto-3-methylbutanoate, (R)-3-mercapto-2-(thiazol-2- ylamino)propanoic acid, (S)-3-mercapto-2-(thiazol-2-ylamino)propanoic acid, (R)-3- mercapto-2-(5-methyl-4-phenylthiazol-2-ylamino)propanoic acid, (S)-4-mercapto-2- (4-phenylthiazol-2-ylamino)butanoic acid, sodium (S)-4-mercapto-2-(4- phenylthiazol-2-ylamino)butanoate, sodium (S)-4-mercapto-2-(5-methyl-4- phenylthiazol-2-ylamino)butanoate, (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid, sodium (S)-4-mercapto-2-(thiazol-2-ylamino)butanoate, sodium (S)-4-mercapto- 2-(methyl(thiazol-2-yl)amino)butanoate, (R)-2-(4-tert-butylthiazol-2-ylamino)-3- mercaptopropanoic acid, (R)-2-(4,5-dimethylthiazol-2-ylamino)-3-mercaptopropanoic acid, (R)-3-mercapto-2-(4,5,6,7-tetrahydrobenzo[d]thiazol-2-ylamino)propanoic acid, (R)-3-mercapto-2-(5-nitropyridin-2-ylamino)propanoic acid, 3-mercapto-2-(5- (trifluoromethyl)pyridin-2-ylamino)propanoic acid, (R)-2-(4,6-bis(4-methylpiperazin- 1 -yl)-l ,3,5-triazin-2-ylamino)-3-mercaptopropanoic acid, (R)-2-(4,6- bis(dimethylamino)-l,3,5-triazin-2-ylamino)-3-mercaptopropanoic acid, (R)-2-(l- (4,6-bis(allylamino)- 1 ,3,5-triazin-2-yl)piperidin-4-ylamino)-3-mercaptopropanoic acid, sodium (S)-4-mercapto-2-(pyrimidin-2-ylamino)butanoate, (R)-2-(6- chloropyrimidin-4-ylamino)-3-mercapto-3-methylbutanoic acid hydrochloride, (S)-4- mercapto-2-(methyl(thiazol-2-yl)amino)butanoic acid, (R)-4-mercapto-2- (methyl(thiazol-2-yl)amino)butanoic acid, sodium (S)-4-mercapto-2-(thiazol-2- ylamino)butanoate, (R)-3-mercapto-3-methyl-2-(thiazol-2-ylamino)butanoic acid, (S)- 3-mercapto-3-methyl-2-(tniazol-2-ylarnino)butanoic acid, (2R,3R)-ethyl l-acetyl-3- mercaptopyrrolidine-2-carboxylate, (2S,3 S)-ethyl 1 -acetyl-3 -mercaptopyrrolidine-2- carboxylate, (S)-I -((R)-2-(benzo[d]oxazol-2-ylamino)-3-mercapto-propanoyl)- pyrrolidine-2-carboxylic acid, (S)-3-mercapto-2-(phenylsulfonamido)propanoic acid, (R)-3-mercapto-2-(phenylsulfonatnido)propanoic acid, (R)-methyl 3-mercapto-2-(5- morpholinopentanamido)propanoate, (R)-3-mercapto-2-(5-morpholinopentanamido)- propanoic acid, (R)-2-(benzo[d]oxazol-2-ylamino)-3-mercapto-N-(methylsulfonyl)- propanamide, (S)-2-(benzo[d]oxazol-2-ylamino)-3-mercapto-3-methyl-N- (methylsulfonyl)butanamide, (S)-2-(benzo[d]oxazol-2-ylamino)-3-mercapto-3- methyl-N-(phenylsulfonyl)butanamide, (R)-methyl 2-(2-(benzo[d]oxazol-2- ylthio)acetamido)-3-mercaptopropanoate, and (R)-methyl 2-(2-(benzo[d]oxazol-2- ylthio)acetamido)-3-mercapto-3-methylbutanoate, a pharmaceutically acceptable salt thereof and mixtures thereof. The invention also includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier, a first composition comprising one or more SNO compounds, a pharmaceutically acceptable salt thereof and mixtures thereof, and a second composition. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, a first composition comprising one or more mercapto-based SNO compounds, a pharmaceutically acceptable salt thereof and mixtures thereof, and a second composition. In another embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, a first composition comprising one or more S-nitrosomercapto-based SNO compounds, a pharmaceutically acceptable salt thereof and mixtures thereof, and a second composition. The second composition may comprise a second compound with an activity selected from the group consisting of stabilizing breathing rhythm, increasing the patency of the upper airway, promoting wakefulness, decreasing the incidence and/or severity of seizures, decreasing inflammation, decreasing respiratory drive, and improving lung function. In one embodiment of the invention, the second compound is not a SNO compound, but has the activity of or ability of stabilizing breathing rhythm. The invention further includes methods of administering the composition of the invention in order to bring about the desired effect of the second compound.

The invention also encompasses a pharmaceutical composition optionally comprising a third or additional compound. The third or additional compound may be another compound similar to the first component, or a compound similar to the second component, or a compound different from both the first and second components. In some embodiments the third compound is a SNO compound. In other embodiments, the third compound is not a SNO compound. The invention also encompasses methods of therapeutic treatment of the breathing of a mammal by way of administering a composition of the invention. In one embodiment, the composition of the invention useful for treatment of the mammal comprises a pharmaceutically acceptable carrier, and one or more SNO compounds, a pharmaceutically acceptable salt thereof and mixtures thereof. In a preferred embodiment, the composition of the invention useful for treatment of the mammal comprises a pharmaceutically acceptable carrier, and one or more mercapto- based SNO compounds, a pharmaceutically acceptable salt thereof and mixtures thereof. In another preferred embodiment, the composition of the invention useful for treatment of the mammal comprises a pharmaceutically acceptable carrier, and one or more S-nitrosomercapto-based SNO compounds, a pharmaceutically acceptable salt thereof and mixtures thereof.

In yet another embodiment, the mammal is a human.

Methods of treatment of the invention include administration of a composition of the invention to stabilize the breathing rhythm of a mammal, and administration of a composition of the invention to increase minute ventilation in a mammal. The invention also includes a method of administration of one or more compounds of the invention in conjunction with the use of a mechanical ventilation device or positive airway pressure device. In one embodiment, the method of the invention further comprises administering a second compound, wherein the second compound is selected from the group consisting of a carbonic anhydrase inhibitor, a respiratory stimulant, a narcotic antagonist and a hormone. In another embodiment, the second compound is selected from the group consisting of a serotonin agonist, a serotonin antagonist, a tetracyclic antidepressant, a agent that acts on dopamine and an agent that acts on norepinephrine. In another embodiment, the second compound is selected from the group consisting of an antihistamine, a leukotriene antagonist, a 5-lipoxygenase inhibitor, a steroid and a COX-2 inhibitor. In another embodiment, the second compound is selected from the group consisting of an opioid analgesic, a sedative hypnotic and a general anesthetic. In yet another embodiment, the second compound is selected from the group consisting of a steroid, a bronchodilator and an anticholinergic.

The invention further comprises a method of stabilizing the breathing rhythm of a mammal, wherein the method comprises administering to the mammal a composition comprising one or more SNO compounds, a pharmaceutically acceptable salt thereof or mixtures thereof, the method further comprising treating the mammal with a ventilation assist device. In a preferred embodiment, the ventilation assist device is selected from the group consisting of a mechanical ventilator, a continuous positive airway pressure (CPAP) device and a bi-level positive airway pressure (BiPAP) device.

In one embodiment of the invention, a composition of the invention is administered via parenteral, oral, or buccal route. In another embodiment, the parenteral route of administration is selected from the group consisting of transdermal, intravenous, intramuscular and intradermal. In yet another embodiment, a composition of the invention is administered by at least two routes of administration.

The present invention further includes a method of increasing minute ventilation (V_E) at the level of the brainstem respiratory control centers in the nucleus tractus solitarius of a mammal, comprising the step of administering to the mammal one or more SNO compounds, a pharmaceutically acceptable salt thereof or mixtures thereof, wherein the one or more mercapto-based SNO compound have the activity of increasing minute ventilation (V_E) at the level of the brainstem respiratory control centers in the nucleus tractus solitarius. The invention further includes a method of increasing minute ventilation (V_E) at the level of the brainstem respiratory control centers in the nucleus tractus solitarius of a mammal. The method comprises the steps of administering to the mammal a therapeutic composition comprising a first composition comprising a first component that is a SNO compound, a pharmaceutically acceptable salt thereof or mixtures thereof, and a second component comprising a second compound that is not a SNO compound, wherein the second component has the activity of increasing minute ventilation (V_E) at the level of the brainstem respiratory control centers in the nucleus tractus solitarius.

The invention further relates to one of the aforementioned compounds for use in the treatment of a mammal in order to stabilize the breathing rhythm of the mammal, or increase minute ventilation at the level of the brainstem respiratory control centers in the nucleus tractus solitarius of the mammal.

In another embodiment, the aforementioned compounds are used for preparation of a medicament for stabilizing the breathing rhythm of a mammal, or increasing minute ventilation at the level of the brainstem respiratory control centers in the nucleus tractus solitarius of a mammal.

The invention further includes a process for producing a mercapto amino-derivatized compound of Formula (H),

wherein R₁ is H, alkyl, acyl, aryl or heteroaryl; R₂ is alkyl, acyl, aryl or heteroaryl; R' is H, alkyl, aryl or heteroaryl; and Y is C₁-C₆ alkylene; comprising the steps of: (i) converting a mercapto amino compound of Formula (E),

wherein R₁, R' and Y are as above, to a tritylsulfanyl amino compound of Formula

(F), wherein R₁, R' and Y are as above;

(ii) reacting the tritylsulfanyl amino compound of Formula (F) with R₂X, wherein X is halide, mesylate, tosylate, triflate, or carboxylate; and R₂ is alkyl, acyl, aryl or heteroaryl; to form a tritylsulfanyl amino-derivatized compound of Formula (G), wherein R₁, R₂, R' and Y are as above;

and (iii) treating the tritylsulfanyl amino-derivatized compound of Formula (G) with an acid-containing mixture to form the mercapto amino-derivatized compound of Formula (H). The invention also includes a method of producing a S- nitrosomercapto compound of Formula (S), wherein R₁ is H, alkyl, acyl, aryl or heteroaryl; R₂ is alkyl, acyl, aryl or heteroaryl; R' is H, alkyl, aryl or heteroaryl; Y is C₁-C₆ alkylene;

comprising the step of treating a compound of Formula (H), wherein R₁, R₂, R' and Y are as above,

H

Y^

^Ri ° (H) with a nitrite equivalent, optionally in the presence of a chelating agent. In one embodiment, the nitrite equivalent is organic. In another embodiment, the nitrite equivalent is inorganic.

As envisioned in the present invention with respect to the disclosed compositions of matter and methods, in one aspect the embodiments of the invention comprise the components and/or steps disclosed therein. In another aspect, the embodiments of the invention consist essentially of the components and/or steps disclosed therein. In yet another aspect, the embodiments of the invention consist of the components and/or steps disclosed therein. BRIEF DESCRIPTION OF THE DRAWINGS For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1 is a synthetic scheme that depicts the preparation of (R)-3- mercapto-2-(5-methyl-4-phenyl-thiazol-2-ylamino)-propionic acid, (R)-2-(4-tert- butyl-thiazol-2-ylamino)-3-mercapto-propionic acid, (R)-2-(4,5-dimethyl-thiazol-2- ylamino)-3-mercapto-propionic acid, and (R)-3-mercapto-2-(4,5,6,7-tetrahydro- benzothiazol-2-ylamino)-propionic acid.

Figure 2 is a synthetic scheme that depicts the preparation of (R)-2-(5- nitropyridin-2-ylamino)-3 -mercapto-propionic acid.

Figure 3 is a synthetic scheme that depicts the preparation of (R)-2- (benzoxazol-2-ylamino)-3-mercapto-propionic acid hydrochloride.

Figure 4 is a synthetic scheme that depicts the preparation of 3- mercapto-2-(5-trifluoromethyl-pyridin-2-ylamino)-propionic acid hydrochloride.

Figure 5 is a synthetic scheme that depicts the preparation of (R)-2- [4,6-bis-(4-methyl-piperazin- 1 -yl)-[ 1 ,3 ,5]triazin-2-ylamino]-3-mercapto-propionic acid and (R)-2-(4,6-bis-dimethylamino-[l,3,5]triazin-2-ylamino)-3-mercapto- propionic acid.

Figure 6 is a synthetic scheme that depicts the preparation of (R)-2-[l- (4,6-bis-allylamino- [1,3 ,5 ]triazin-2-yl)-piperidin-4-ylamino] -3 -mercapto-propionic acid, sodium salt. Figure 7 is a synthetic scheme that depicts the preparation of (S)-2-

(pyrimidin-2-ylamino)-4-mercapto-butyric acid, sodium salt.

Figure 8 is a synthetic scheme that depicts the preparation of sodium (S)-4-mercapto-2-(5-methyl-4-phenyl-thiazol-2-ylamino)-butyrate and sodium (S)-4- mercapto-2-(4-phenyl-thiazol-2-ylamino)-butyrate. Figure 9 is a synthetic scheme that depicts the preparation of (S)-4- mercapto-2-(thiazol-2-ylamino)-butyric acid and sodium (S)-4-mercapto-2-(thiazol-2- ylamino)-butyrate.

Figure 10 is a synthetic scheme that depicts the preparation of sodium (S)-4-mercapto-2-(methyl-thiazol-2-yl-amino)-butyrate. Figure 11 is a synthetic scheme that depicts the preparation of (R)-2- (6-chloro-pyrimidin-4-ylamino)-3-mercapto-3 -methyl-butyric acid hydrochloride.

Figure 12 is a synthetic scheme that depicts the preparation of (S)-2- (benzothiazol-2-yl)amino-3-rnercapto-3 -methyl-butyric acid and methyl (S)-2- (benzothiazol-2-yl)amino-3 -mercapto-3 -methyl-butyrate.

Figure 13 is a synthetic scheme that depicts the preparation of (S)-2- (benzothiazol-2-ylamino)-4-mercapto-butyric acid, (S)-2-(benzothiazol-2-ylamino)- (4-acetylthio)-butyric acid, methyl (S)-2-(benzothiazol-2-ylamino)-(4-acetylthio)- butyrate and (S)-3-(benzo[d]thiazol-2-ylamino)dihydrothiophen-2(3H)-one. Figure 14 is a synthetic scheme that depicts the preparation of methyl

(S)-2-(benzothiazol-2-yl)amino-4-acetylthio-butyrate and (S)-2-(benzothiazol-2- yl)amino-4-acetylthio-butyric acid.

Figure 15 is a synthetic scheme that depicts the preparation of dimethyl L-4,4'-disulfanediylbis(2-(benzoxazol-2-yl)amino)-butyrate, methyl (S)-2- (benzoxazol-2-ylamino)-4-mercapto-butyrate, (S)-methyl 4-(acetylthio)-2- (benzo[d]oxazol-2-ylamino)butanoate, and (S)-3-(benκo[d]oxazol-2- ylamino)dihydrothiophen-2(3H)-one.

Figure 16 is a synthetic scheme that depicts the preparation of methyl (R)-3-mercapto-2-(5-moφholinopentanamido)propanoate, and (R)-3-mercapto-2-(5- morpholinopentanamido)propanoic acid.

Figure 17 is a synthetic scheme that depicts the preparation of (2S,2'S)- dimethyl 4,4'-disulfanediyl-bis(2-(5-fluorobenzo[d]oxazol-2-ylamino) butanoate), (S)- methyl 4-(acetylthio)-2-(5-fluorobenzo[d]oxazol-2-ylamino)butanoate, and (S)-3-(5- fluorobenzo[d]oxazol-2-ylamino)dihydrothiophen-2(3H)-one. Figure 18 is a synthetic scheme that depicts the preparation of (R)-2- acetamido-3-mercapto-N-(methylsulfonyl)propanamide.

Figure 19 is a synthetic scheme that depicts the preparation of (R)-N- (2-mercapto- 1 -(I H-tetrazol-5-yl)ethyl)acetamide.

Figure 20 is a synthetic scheme that depicts the preparation of 4-S- (nitrosomercapto)-2-(thiazol-2-yl)amino-butyric acid.

Figure 21 depicts the ¹H NMR spectra of 4-S-nitrosomercapto-2- (thiazol-2-yl)amino-butyric acid and (2S,2'S)-4,4'-disulfanediyl-bis(2-(thiazol-2- ylamino) butanoic acid), in CD₃OD. Figure 22 is a graph that summarizes the time-dependent stability of 4- (S-nitroso)mercapto-2-(thiazol-2-yl)amino-butyric acid in different aqueous buffers.

Figure 23 depicts the results of an example experiment demonstrating that (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid accelerated the normalization of blood pH after opioid analgesic administration.

Figure 24 depicts the results of an example experiment demonstrating that (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid accelerated the normalization of blood pCO₂ after opioid analgesic administration.

Figure 25 depicts the results of an example experiment demonstrating that (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid accelerated the normalization of blood pθ₂ after opioid analgesic administration.

Figure 26 depicts the results of an example experiment demonstrating that (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid accelerated the normalization OfFO₂Hb after opioid analgesic administration. Figure 27 depicts the results of an example experiment demonstrating that (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid accelerated the normalization of CHCO₃-(P) after opioid analgesic administration.

Figure 28 depicts the results of an example experiment demonstrating that (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid accelerated the normalization of cGLU after opioid analgesic administration.

Figure 29 depicts the results of an example experiment demonstrating that (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid immediately and completely reversed opioid analgesic-associated suppression of Minute Ventilation and decreased the time to arousal. Figure 30 depicts the results of an example experiment demonstrating that (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid immediately and completely reversed opioid analgesic-associated suppression of Tidal Volume and decreased the time to arousal.

Figure 31 depicts the results of an example experiment demonstrating that (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid anesthetic-associated suppression of Minute Ventilation.

Figure 32 depicts the results of an example experiment demonstrating that (S)-4-mercapto-2-(4-phenylthiazol-2-ylamino)butanoic acid reverses fentanyl- induced depression of tidal volume in rat. Figure 33 depicts the results of an example experiment demonstrating that (S)- l-((R)-2-(benzoxazol-2-ylamino)-3-mercaptopropanoyl)-pyrrolidine-2- carboxylic acid protects against fentanyl-induced depression of tidal volume in rat.

Figure 34 depicts the results of an example experiment demonstrating that (S)- 1 -((R)-2-(benzoxazol-2-ylamino)-3-mercaptopropanoyl)-pyrrolidine-2- carboxylic acid protects against fentanyl-induced depression of minute volume in rat.

Figure 35 depicts the results of an example experiment demonstrating that (S)-4-nitrosomercapto-2-(thiazol-2-ylamino)butanoic acid protects against fentanyl-induced depression of tidal volume in rat. Figure 36 depicts the results of an example experiment demonstrating that (S)-4-nitrosomercapto-2-(thiazol-2-ylamino)butanoic acid protects against fentanyl-induced depression of tidal volume in rat in a dose-dependent manner.

Figure 37 depicts a series of HPLC traces showing the stability of 4- nitrosomercapto-2-(thiazol-2-ylamino)butanoic acid in solution at various time points over 24 hours and at various time points as solid.

Figure 38 depicts mass spectral data corresponding to the various time points set forth in the stability study shown in figure 37.

DETAILED DESCRIPTION The invention relates to the discovery that monotherapy using an S- nitrosomercapto compound (or SNO compound), or a derivative or modification thereof, as well as combination products including a SNO compound that combine a compound to restore respiratory rhythm with an agent that helps reduce oxidative stress, provides an important dual mode of action to alleviate short and long-term consequences of hypoxia.

The compositions and methods of the invention can be used to prevent a loss of normal breathing, or to restore normal breathing after a loss occurs. One non-limiting example of a loss of normal breathing is respiratory depression. Respiratory depression results in hypoventilation, which further results in hypoxia. A primary initial clinical manifestation of hypoxia is drowsiness or excessive daytime sleepiness. Accordingly, drugs that cause decreased respiratory drive and the resulting hypoxia are sometimes limited in their usefulness due to the fear of a life- threatening respiratory depression and/or the excessive daytime sleepiness that negatively impacts quality of life. There are a wide variety of disorders that have loss of normal breathing or respiratory depression as a primary or secondary feature of the disorder, which can be treated using the compositions and methods of the present invention. Examples of a primary loss of normal breathing include: apneas (central, mixed and obstructive) and congenital central hypoventilation syndrome. Secondary loss of normal breathing may be due to certain drugs (e.g., anesthetics, sedatives, anxiolytics, hypnotics, alcohol, opioid analgesics), chronic cardio-pulmonary diseases (e.g., heart failure, chronic bronchitis, emphysema, and impending respiratory failure), excessive weight (e.g., obesity-hypoventilation syndrome) and/or factors that affect the neurological system (e.g., stroke, tumor, trauma, radiation damage, ALS).

Generally, patients in need of analgesia or anesthesia may receive one agent, or a combination of multiple agents, to create a state of partial or full unconsciousness to allow for medical procedures, such as surgery, to be performed. A common undesirable action of many agents used for analgesia and anesthesia (e.g., opioid analgesics, barbiturates, benzodiazepines, inhaled anesthetics, propofol) is respiratory depression. Examples of opioid analgesics include morphine, codeine, fentanyl, buprenorphine, meperidine, methadone, sufentanil, alfentanil, and the like. Examples of barbiturates include allobarbital, alphenal, amobarbital, aprobarbital, barbexaclone, barbital, brallobarbital, butabarbital, butalbital, butobarbital, butallylonal, crotylbarbital, cyclobarbital, cyclopal, ethallobarbital, febarbamate, heptabarbital, hexethal, hexobarbital, mephobarbital, metharbital, methohexital, methylphenobarbital, narcobarbital, nealbarbital, pentobarbital, phenobarbital, probarbital, propallylonal, proxibarbal, proxibarbital, reposal, secbutabarbital, secobarbital, sigmodal, talbutal, thialbarbital, thiamylal, thiobarbital, thiobutabarbital, thiopental, valofane, vinbarbital, and vinylbital. Examples of benzodiazepines include midazolam, clonazepam, diazepam, alprazolam and the like. Examples of inhaled anesthetics included halothane, enflurane, isoflurane, sevoflurane, desflurane, and the like. Not only can the respiratory depressant effect occur soon after administration of the agent, but the effects of the anesthetic and/or analgesic agent can linger for hours or days after the procedure. The compositions and methods of the invention can be used to diminish, prevent or reverse drug-induced respiratory depression.

Although not wishing to be bound to any particular theory, the observed protection may be due to an inherent respiratory stimulation via a hypoxia- mimetic activity and/or the restoration of the normal ability of chemoreceptors (e.g., carotid body) to respond to changes in carbon dioxide and/or oxygen. The present invention can be used to utilize this pathway and to signal to the ventilatory control centers in the brain that a hypoxic ventilatory response should occur, thereby resulting, at least in part, in an increase in breathing rate and efficiency (e.g., tidal volume).

By way of another non-limiting example, excessive weight can decrease respiratory drive resulting in hypoventilation and hypoxia. This condition is called obesity-hypoventilation syndrome. Excessive weight is also a risk factor in sleep related breathing disorders. A mono- or combination-composition comprising an SNO compound, or derivative or modification thereof, is therefore useful to counteract the respiratory depressant effects of obesity.

Mono- or combination-compositions of the invention are also useful for increasing the muscle tone of the upper airway, improving ventilatory/perfusion match and increasing erythropoietin production, among other things, as set forth in detail herein.

In one aspect, the present invention relates to a single-drug approach to the treatment of sleep apnea by using hypoxic ventilatory response control, by way of administration of an SNO compound, or derivative or modification thereof. The present invention also relates to a combination, or "multi-drug", approach to the treatment of sleep apnea by combining hypoxic ventilatory response control, by way of administration of one or more SNO compounds, or derivatives or modifications thereof, with other drugs or devices that provide a complimentary or enhancing activity. The invention provides that a composition comprising a combination of two or more compounds may provide enhanced effectiveness in the treatment of disorders of breathing control by acting upon on two or more physiological pathways, wherein one of the pathways is affected by S-nitrosomercapto treatment for restoration of respiratory rhythm. In another aspect of the invention, a composition comprising a combination of two or more compounds may provide enhanced effectiveness in the treatment of disorders of breathing control by acting upon the same physiological pathway. When more than one compound is used as a combination therapy, the order of administration of each compound with respect to the other will be apparent to the skilled artisan and the invention should not be limited to any particular order of administration of the compounds.

Definitions As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. As used herein, the term "about" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used.

As used herein, the term "modulate" is meant to refer to any change in biological state, i.e. increasing, decreasing, and the like.

As used herein, the term "depressed control of breathing" refers to a condition, a disease or a state in a mammal that causes or triggers lack of normal breathing control. Examples of conditions associated with depressed control of breathing are sleep apnea (central, mixed and obstructive, including but not limited to co-existing conditions of heart failure, kidney disease and stroke), sleep-disordered breathing (especially with snoring and arousals), chronic bronchitis, COPD, asthma, allergy, neurological diseases (e.g., stroke and amyotrophic lateral sclerosis, also known as ALS), snoring, obesity-hypoventilation syndrome, apnea of prematurity, respiratory depression due to drugs (e.g., narcotic analgesics, sedatives, alcohol, sleeping pills and anesthetics), central congenital hypoventilation syndrome, hypoventilation due to stroke, trauma, surgery and/or radiation, and acclimatization to high altitude.

As used herein, the term "apnea" means the absence of normal breathing resulting in intermittent stoppages of breathing.

"Hypopnea" is similar in many respects to apnea; however, breathing does not fully stop but is partially stopped (i.e., less than 100% of normal breathing, but more than 0% of normal breathing). Hypopnea is also referred to herein as "partial apnea" and can be subdivided into obstructive, central or mixed types.

As used herein, "Cheyne-Stokes respiration" refers to a specific pattern of breathing characterized by a crescendo pattern of breathing that results in apneas and/or hypopneas. A hallmark of this condition is that breathing becomes out of phase with blood oxygen levels.

As used herein, the term "patency" refers to the state or condition of an airway being open or unblocked. As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

The term "expression", as used herein, is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

As used herein, an "isolated nucleic acid" refers to a nucleic acid segment or fragment that has been separated from sequences that flank it in a naturally occurring state, i.e., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence. As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence that is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements that are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one that expresses the gene product in a tissue specific manner.

As used herein, the term "hypoxia" refers to a deficiency in the amount of oxygen being taken in by an organism, as well as to a deficiency in the amount of oxygen that is transported to tissues in a organism. It should be understood that the term hypoxia is meant to be a general term that denotes the diminution of oxygen, and as used herein also includes ischemia, which diminishes oxygen levels though partial or complete interruption of blood supply.

As used herein, the term "normoxia" refers to a homoeostasis or "normal condition" regarding the amount of oxygen being taken in by an organism, as well as to a homeostasis or "normal condition" with respect to the amount of oxygen transported to tissues in a organism.

"S-Nitrosomercapto pathway," as the term is used herein, refers to the signaling pathway and the signaling mechanisms that occur as the information pertaining to blood levels of oxygen is transmitted to the brain through S- nitrosomercapto signaling. While not wishing to be bound by any particular theory, examples of S-nitrosomercapto compounds or "SNO compounds" according to the invention include compounds having an activity or effect mediated at least in part by S-nitrosomercapto compounds and their ability to act as hypoxia-mimetics, agents that increase upper airway muscle tone or increase expression of hypoxia inducible factor- 1 (HIF-I).

As used herein, a "therapeutically effective amount" is the amount of a therapeutic composition sufficient to provide a beneficial effect to a mammal to which the composition is administered. As used herein, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl and the like. Preferred alkyl groups herein contain 1 to 6 carbon atoms. Alkyl groups may be optionally substituted with one to three groups selected from halo, amino, methoxy, ethoxy, hydroxyl, methylthio, methylsulfonyl, nitro, aryl, heterocyclyl and heteroaryl. The term "alkyl" as used herein also refers to ring-containing alkyl radicals, such as cyclohexyl, cyclopentyl, cyclopropyl, cyclopropylmethyl and norbornyl, optionally substituted with one to three groups chosen from halo, amino, methoxy, ethoxy, hydroxyl, methylthio, methylsulfonyl, nitro, aryl, heterocyclyl and heteroaryl. As used herein, the term "alkylene" refers to a divalent branched or unbranched saturated hydrocarbon group, such as methylene (-CH₂-), 1 ,2-ethylene (-CH₂CH₂-), 1,3-propylene (-CH₂CH₂CH₂-), 1 ,2-propylene (-CH₂CH(CH₃)-) and the like. Preferred alkylene groups herein contain 1 to 6 carbon atoms (C₁-C₆), optionally substituted with one to three groups selected from alkyl, halo, methoxy, ethoxy, aryl, heterocyclyl and heteroaryl. .

As used herein, the term "aryl," employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic group containing one or more rings (typically one, two or three rings). Multiple rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include, but are not limited to, phenyl, anthracyl and naphthyl. Preferred aryl groups are phenyl and naphthyl, most preferred is phenyl. Aryl groups may be optionally substituted with one to three groups selected from halo, amino, methoxy, ethoxy, hydroxyl, methylthio, methylsulfonyl, nitro, aryl, heterocyclyl and heteroaryl.

As used herein, the term "heterocycle", "heterocyclyl" or "heterocyclic" by itself or as part of another substituent means, unless otherwise stated, an unsubstituted or substituted, stable, mono- or multicyclic heterocyclic ring system consisting of carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quaternized. The heterocycle may be attached to the compound of which it is a component, unless otherwise stated, at any heteroatom or carbon atom in the heterocycle that affords a stable structure. Heterocyclic groups may be optionally substituted with one to three groups selected from halo, amino, methoxy, ethoxy, hydroxyl, methylthio, methylsulfonyl, nitro, aryl, heterocyclyl and heteroaryl.

Examples of non-aromatic heterocycles include monocyclic groups such as: aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrrolinyl, imidazolinyl, pyrazolidinyl, dioxolanyl, sulfolanyl, 2,3-dihydrofuranyl, 2,5-dihydrofuranyl, tetrahydrofuranyl, thiophanyl, piperidinyl, 1,2,3,6- tetrahydropyridinyl, 1 ,4-dihydropyridinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyranyl, 2,3-dihydropyranyl, tetrahydropyranyl, 1,4-dioxanyl, 1,3-dioxanyl, homopiperazinyl, homopiperidinyl, 1,3-dioxepinyl, 4,7-dihydro-l,3-dioxepinyl and hexamethyleneoxide.

As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heterocycle having aromatic character. A monocyclic heteroaryl group is preferably a 5-, 6-, or 7-membered ring, examples of which are pyrrolyl, furyl, thienyl, pyridyl, pyrimidinyl and pyrazinyl. A polycyclic heteroaryl may comprise multiple aromatic rings or may include one or more partially saturated rings. Heteroaryl groups may be optionally substituted with one to three groups selected from halo, amino, methoxy, ethoxy, hydroxyl, methylthio, methylsulfonyl, nitro, aryl, heterocyclyl and heteroaryl.

Examples of monocyclic heteroaryl groups include, for example, six- membered monocyclic aromatic rings such as, for example, pyridyl, pyrazinyl, pyrimidinyl and pyridazinyl; and fϊve-membered monocyclic aromatic rings such as, for example, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3- thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.

Examples of polycyclic heteroaryl groups containing a partially saturated ring include tetrahydroquinolyl and 2,3-dihydrobenzofuryl.

Examples of polycyclic heteroaryls include indolyl, indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl, 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl, quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, chromene-2-one-yl (coumarinyl), dihydrocoumarin, chromene-4-one-yl, benzofuryl, 1,5-naphthyridinyl, 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl, benzoxazolyl, benzothiazolyl, purinyl, benzimidazolyl, benzotriazolyl, thioxanthinyl, benzazepinyl, benzodiazepinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl and quinolizidinyl.

As used herein, the term "acyl" refers to a group of formula R-C(=O)-, wherein R is H, alkyl, aryl or heteroaryl. Examples of acyl groups are formyl, acetyl, propionyl, butyryl, benzoyl, nicotinoyl and the like. As used herein, a "nitrite equivalent" is a chemical reagent that behaves like a nitrite, allowing the conversion of a mercapto compound to a S- nitrosomercapto compound. Nitrite equivalents may be inorganic or organic. Non- limiting examples of inorganic nitrite equivalents are sodium nitrite, potassium nitrite, cesium nitrite and calcium nitrate. Non-limiting examples of organic nitrites are ethyl nitrite, butyl nitrite and t-butyl nitrite, or any other commercially available or synthetically prepared organic nitrite. As used herein, a "chelating agent" represents a chemical compound capable of complexing ions in solution. Non-limiting examples of chelating agents are EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriamine pentaacetic acid), and EGTA (ethylene glycol tetraacetic acid).

Description

There exists a need in the art to identify safe and effective S- nitrosomercapto compounds (SNO compounds). There is also a need for pharmaceutical compositions containing such agents, as well as methods relating to the use thereof to treat, for example, sleep apnea, chronic obstructive pulmonary disease, obesity-hypoventilation syndrome, drug-induced respiratory depression and apnea of prematurity. SNO compounds that promote the expression of hypoxia- inducible factor- 1 may also be useful in treating reperfusion injury and certain cancers. The preferred embodiments of the present invention fulfill these needs, and provide other advantages, as set forth herein.

In an embodiment, SNO compounds are provided that have one of the following general structures, including stereoisomers, prodrugs, and pharmaceutically acceptable salts thereof: (i) mercapto compounds, characterized by the presence of a - SH group in their structures, and herein referred to as "mercapto-based SNO compounds"; (ii) S-nitrosomercapto compounds, characterized by the presence of a - SNO group in their structures, and herein referred to as "S-nitrosomercapto-based SNO compounds".

As used herein, the term "SNO compound" refers to a mercapto-based SNO compound, a S-nitrosomercapto-based SNO compound, and mixtures thereof. SNO compounds of the invention have utility over a wide range of therapeutic applications, and may be used to treat a variety of disorders, illnesses, and pathological conditions including, but not limited to, a variety of apneas, hypopneas, hypoventilation conditions, reperfusion/ischemia conditions.

Compositions of the Invention and Uses Thereof

The present invention includes compositions and methods for treating disordered control of breathing. In one embodiment, the invention provides methods and compositions for treating sleep apnea. In another embodiment, the invention provides methods and compositions for treating drug-related disordered control of breathing.

The compounds and methods of the present invention should be understood to be applicable to any other respiratory control that is associated with an S-nitrosomercapto signaling pathway. That is, the present invention provides that a composition comprising a combination of two or more compounds may provide enhanced effectiveness in the treatment of disorders of breathing control by acting upon on two or more physiological pathways, wherein one of the pathways is affected by S-nitrosomercapto treatment for restoration of respiratory rhythm. The compounds of the invention comprise mercaptans, mercaptan derivatives and S-nitrosomercaptans, as listed in Tables 1 and 2. As defined herein, the mercaptans are characterized by the presence of a -SH group, and the S- nitrosomercaptans are characterized by the presence of a -SNO group. Useful mercaptan derivatives within the invention include protected mercaptans, which may be converted to the corresponding mercaptans in vivo. Examples of protected mercaptan include, but are not limited to, thioesters, thiolactones, symmetrical disulfides (R₃-S-S-R₃) and non-symmetrical disulfides (R₃-S-S-Rb, wherein R₃ is different from R_b). The compounds of the present invention include the symmetrical disulfides and non-symmetrical disulfides derived from any of the mercaptans depicted in Table 1 or any of the S-nitrosomercaptans depicted in Table 2.

In an aspect of the invention, any one of the compounds disclosed in Tables 1 and 2 may be used to treat any of the above described conditions according to the methodologies set forth herein. It will be understood that the compounds disclosed in Tables 1 and 2, or derivatives or modifications thereof, may be administered to a patient. Such a compound can be administered alone, as a pharmaceutical composition, or in combination with one or more other compounds, as described in detail elsewhere herein. Table 1. Mercapto-based SNO compounds.

Table 2. S-nitrosomercapto-based SNO compounds.

General Synthesis of The Compounds of The Invention

Compounds of the invention may be prepared according to the procedures set forth herein. These synthetic routes are considered to be exemplary and non-limiting as the skilled artisan knows how to select and implement alternative synthetic strategies to arrive at the desired compounds. Suitable alternative synthetic methods may be identified by reference to the literature describing synthesis of analogous compounds, and then performing the synthesis of the desired compound following the route used for the analogous compounds, modifying the starting materials, reagents, and reaction conditions as appropriate to synthesize a desired compound.

Reference may be made to sources such as "Comprehensive Organic Synthesis", Eds. Trost & Fleming (Pergamon Press 1991), "Comprehensive Organic Functional Group Transformations", Eds. Katritzky, Cohn & Rees (Pergamon Press, 1996), "Comprehensive Organic Functional Group Transformations II", Eds. Katritzky & Taylor (Editor) (Elsevier, 2nd Edition, 2004), "Comprehensive Heterocyclic Chemistry", Eds. Katritzky & Rees (Pergamon Press, 1984), and "Comprehensive Heterocyclic Chemistry II", Eds. Katritzky, Rees, & Scriven (Pergamon Press, 1996), the entire disclosures of which are incorporated herein by reference.

As used herein, the group Ri is H, alkyl, acyl, aryl or heteroaryl; the group R₂ is alkyl, acyl, aryl or heteroaryl; the group R' is H, alkyl, aryl or heteroaryl; the group P is a protective group of the mercapto group; the group X is halide, mesylate, tosylate, triflate or carboxylate; and the group Y is C₁-C₆ alkylene. The key synthetic transformations are outlined herein and depicted in Scheme 1. As shown in Scheme 1, a mercapto-containing amino acid (Compound A) may be converted to Compound D using the following steps: protection of the mercapto group with group "P" to afford Compound B; derivatization of Compound B at the amino center with group R₂ to afford Compound C; and removal of the "P" protective group at the mercapto group to afford Compound D.

Scheme 1.

Compounds such as cysteine, homocysteine, penicillamine, and related mercapto-containing amino acids, including their respective salts, esters, enantiomers and enantiomeric mixtures thereof, are applicable to this general synthetic pathway. Solubilization of the starting material may be ensured by use of co-solvents and/or adjustment of the pH of the solution. Separately, depending upon the presence of additional functional groups or reactive centers within a given molecule, additional synthetic steps may be needed. Additional synthetic steps that may alter the general scheme described above are shown in the specific examples that follow.

Different protective groups "P" may be used within the present invention. Non-limiting examples of "P groups are trityl, benzyl, substituted benzyl, acyl, Cbz (benzyloxycarbonyl) and alkylsulfanyl. These groups may be introduced using chemistry that is consistent with their chemical structure. For example, introduction of a benzyl protective group may be achieved by treating the corresponding compound with benzyl chloride in the presence of base, and introduction of a Cbz protective group may be achieved by treating the corresponding compound with CbzCl (benzyloxycarbonyl chloride) in the presence of base. By the same token, removal of such protective groups may be achieved based on the specific chemical properties of the protected mercapto group. For example, removal of the benzyl protective group may be achieved by treating the benzylsulfanyl compound with sodium metal in liquid ammonia at low temperature (such as -30 ⁰C). Removal of the Cbz group protective group may be achieved by treating the Cbz-protected mercapto compound with lithium hydroxide in a solvent such as a THF-water mixture.

A preferred protective group within the invention is triphenylmethyl (trityl, Trt). The mercapto group may be protected with a trityl group by reaction with triphenylmethanol in the presence of acid, such as, but not limited to, trifluoroacetic acid or hydrochloric acid, or by reaction with triphenylmethyl chloride in the presence of a tertiary organic amine, such as, but not limited to, pyridine or triethylamine. The trityl group is sensitive to acid and may be removed by treating the tritylsulfanyl protected compound with an acid, such as dilute hydrochloric acid or an aqueous solution of trifluoroacetic acid. One skilled in the art should appreciate that the choice of protective group, protection method and deprotection method is a function of the molecule under preparation.

More specifically, as shown in Scheme 2, a mercapto-containing compound (Compound E) may be reacted with triphenylmethanol in the presence of acid, such as trifluoroacetic acid, to afford the corresponding tritylsulfanyl-protected amino acid (compound F). Alternatively, Compound E may be reacted with triphenylmethyl chloride in the presence of a tertiary amine, such as pyridine or triethylamine, to afford compound F.

The nitrogen center may be further derivatized with reagent R₂-X, wherein R₂ is alkyl, acyl, aryl or heteroaryl, and X is a leaving group, such as halide, mesylate, tosylate, triflate or carboxylate, to afford Compound G. For example, alkylation of the nitrogen center may be accomplished with a suitably activated alkyl halide, mesylate, tosylate or triflate. Acylation of the nitrogen center may be accomplished with an acyl halide or an acid anhydride, for example. Heteroarylation of the nitrogen center may be accomplished by reaction with a suitably activated heteroaryl halide, mesylate, tosylate or triflate in the presence of a base, for example. Suitable heteroaryl halides include compounds such as, but not limted to, 2- bromopyridine, 2-chloropyridine, 2-chloropyrimidine, 2-bromothiazole, 2- chlorobenzoxazole, 2-bromobenzothiazole and substituted variations thereof, for example. Bases that may be used for this transformation include, but are not limited to, sodium and potassium hydroxide, sodium hydride and potassium tert-butoxide.

Removal of the trityl protective group of compound G to afford Compound H may be achieved by treatment with an acid-containing mixture, such as trifluoroacetic acid/triethylsilane, an aqueous solution of trifluoroacetic acid, or an aqueous solution of hydrochloric acid, or by use of any of the reported methods in Wuts & Greene, 2007, "Protective Groups in Organic Synthesis", 4^th Edition, John Wiley & Sons Inc., New York, New York.

Scheme 2.

Alternatively, the compounds of the invention may be prepared by a synthetic route where intermediate mercapto compounds may be oxidized to the corresponding disulfide compounds, and then derivatized as appropriate, as exemplified in Scheme 3. Treatment of the disulfide compound with a reducing agent such as DTT (dithiothreitol) may convert the disulfide back to the corresponding mercapto compound. A homocystine derivative (Compound I) may be reacted with an activated heterocyclic halide in the presence of base to afford the N-substituted derivative (Compound J). Reductive cleavage of the disulfide linkage to the mercapto Compound K may be performed by reacting Compound J with DTT (dithiothreitol) or with zinc metal dust (Zn). The reduction of the disulfide to the mercapto compound may be performed using other reagents known in the literature, as long as such reagents are compatible with the compounds under consideration, as should be apparent to those skilled in the art. Scheme 3.

Alternatively, the compounds of this invention may be prepared using hydroxyl amino acids as starting materials, as shown in Scheme 4. In this methodology, a hydroxyl amino acid, such as Compound L, may be reacted with an acylating, alkylating, arylating or heteroarylating agent, typically in the presence of base, to afford Compound M. The hydroxyl group may then be converted to a leaving group such as halide, triflate, mesylate, tosylate or carboxylate, affording Compound N, which may be then treated with a sulfur nucleophile, such as a thiocarboxylate (e.g., thioacetate or thiopropionate). The resultant thioester (Compound O) may be a suitable mercapto compound prodrug or it may be subjected to hydrolysis to yield the desired free mercapto compound (Compound P).

Scheme 4

The compounds of the invention also include thioesters and thiolactones, which may be converted to the corresponding mercapto compounds in vivo. Thioesters may be prepared by acylation of mercapto compounds, whereby the mercapto compound is treated with at least one equivalent of an acylating reagent, such as an acyl halide or an acid anhydride, in a solvent such as tetrahydrofuran or dioxane, in the presence of a base such as pyridine, triethylamine, or diisopropylethylamine. A thiolactone such as Compound R may be prepared by the cyclization of a mercapto compound such as Compound Q, as shown in Scheme 5. Scheme 5

S-nitrosomercapto compounds may be prepared from the corresponding mercapto compounds. In order to perform such transformation, the mercapto compound may be dissolved in an organic solvent, such as methanol, ethanol, tetrahydrofuran or dioxane, in the presence of an inorganic acid, such as hydrochloric acid or sulfuric acid. The mercapto solution may then be treated with a solution of an organic nitrite, such as ethyl nitrite, butyl nitrite, tert-butyl nitrite, or any other commercially available or synthetically prepared organic nitrite. Alternatively, the mercapto compound may be treated with a solution of sodium nitrite, optionally containing a chelating agent, such as EDTA (ethylenediaminetetraacetic acid), DTPA (diethylene triamine pentaacetic acid), or EGTA (ethylene glycol tetraacetic acid). The molar ratio of nitrite source to compound of the invention may vary from 1 :1 to 10:1, depending on the reactivity of the nitrite source. The reaction may be run at 0 ⁰C to room temperature. The conversion is preferentially performed in the absence of direct light, as to minimize any possible decomposition of the reagents or products.

Completion of the nitrosylation reaction may be determined by the Saville assay (Saville, 1958, Analyst 83: 670-672). The method uses mercury (II) chloride to convert the S-nitrosomercaptocompound to the corresponding mercapto compound, releasing nitrous acid, which reacts with sulfanilamide and N-I- naphthylethylenediamine dihydrochloride to form a colored azo compound. The concentration of the azo compound may be determined based on its absorbance at 540 nm (-50,000 M^"1 cm^"1). It will be understood that when any of the compounds disclosed in

Table 1 or Table 2 contains one or more chiral centers, the compounds may exist in, and may be isolated in pure enantiomeric or diastereomeric forms or as mixtures of enantiomers, mixtures of diasteromers, or mixtures of diastereomers and enantiomers. For each of the compounds identified herein, the present invention therefore includes the compounds' enantiomers, all possible diastereomers, if any, racemates, and mixtures thereof.

Diastereomers may be resolved by known separation techniques including silica gel chromatography, normal and reversed phase HPLC, and crystallization.

An "isolated optical isomer" is a compound that has been substantially purified from the corresponding optical isomer(s) of the same formula. Preferably, the isolated isomer is at least about 80%, more preferably at least 90% pure, even more preferably at least 98% pure, most preferably at least about 99% pure, by weight, as quantitated by chiral HPLC.

Mixtures containing optical isomers of a given compound may be purified to provide isolated optical isomers by well known chiral separation techniques. According to one such method, a racemic mixture of a compound found in Table 1 or Table 2, or a chiral intermediate thereof, is separated into 99% wt.% pure optical isomers by HPLC or SFC using a suitable chiral column, such as a member of the series of DAICEL® CHIRALPAK® family of columns (Daicel Chemical Industries, Ltd., Tokyo, Japan). It is within the ability of one ordinary skill in the art to determine the appropriate operating parameters.

Combination with Other Therapeutic Agents

In another aspect of the invention, a composition comprising a combination of two or more compounds may provide enhanced effectiveness in the treatment of disorders of breathing control by acting upon the same physiological pathway. In one aspect of the invention, a composition is used to treat depressed control of breathing. In another aspect, a composition is used to treat sleep apnea. In yet another aspect, a composition is used to treat drug-induced depressed control of breathing. In yet another aspect, a composition is used to treat obesity-associated depressed control of breathing.

According to the present invention, the second compound used in conjunction with an SNO compound can be selected for a specific property or activity, as described in detail herein. In one aspect of the invention, the third, fourth, or additional compound can similarly be a non-SNO compound, selected for a specific property or activity, as described in detail herein. Below are listed non- limiting examples of such compounds, which should not be considered in any way to be the only such compounds useful in the present invention. The skilled artisan, when armed with the present disclosure, will understand how to identify a second (or third, fourth, fifth, and so on) compound useful in combination with an SNO compound according to the present invention.

Table 1. Examples of compounds useful in combination with a compound according to the present invention a. A compound with the activity of stabilizing breathing rhythm: i. Carbonic anhydrase inhibitor (e.g., acetazolamide, topiramate) ii. Respiratory stimulation (e.g., caffeine, theophylline, doxapram) iii. Narcotic antagonists (e.g., naloxone) iv. Hormones (e.g., medroxyprogesterone) b. A compound with the activity of increasing the patency of the upper airway by activity on serotonin, dopamine, norepinephrine or GABA: i. Serotonin agents (e.g., 5HTl A agonist buspirone, serotonin reuptake inhibitors, 5HT3 receptor antagonists such as ondansetron) ii. Dopamine and/or norepinephrine agents (e.g., ropinerole, milnacipran) iii. Tetracyclic antidepressants (e.g., mirtazipine, setiptiline) c. A compound with the activity of promoting wakefulness: i. Modafinil, r-modafinil, amphetamine d. A compound with the activity of decreasing seizures: i. Zonisamide e. A compound with the activity of increasing the patency of the upper airway by decreasing inflammation: i. Antihistamines (e.g., cetirizine, azelastine, desloratidine, fexofenadine) ii. Leukotriene antagonists (e.g., montelukast) iii. 5-lipoxygenase inhibitors (e.g., zileuton) iv. Steroids (e.g., fluticasone) v. COX-2 inhibitors f. A compound with the activity of decreasing respiratory drive as a side effect to its primary therapeutic effect: i. Opoid analgesics (e.g., morphine, meperidine, fentanyl, oxycodone, buprenorphine) ii. Sedative hypnotics (e.g., lorazepam, Zolpidem, zaleplon) iii. General anesthetics (e.g., halothane, enflurane, thiopental) iv. Ethyl alcohol g. A compound with the activity of improving lung function in diseases such as asthma and/or chronic obstructive pulmonary disease: i. Steroids (e.g., budesonide, fluticasone, salmeterol/fluticasone combinations) ii. Bronchodilators (e.g., salbutamol, salmeterol) iii. Anticholinergic (e.g., tiotropium, ipatropium) h. A device use to assist breathing through mechanical ventilation or positive airway pressure: i. Mechanical ventilators ii. CPAP iii. BiPAP

Combination therapeutics in the pharmaceutical industry are common, and the preparation and use of such combination therapeutics will be understood by the skilled artisan. For example, Advair™ (GlaxoSmithKline) is a combination of a steroid compound and a bronchodilating compound, and is used for treatment of asthma. Combinations comprising two or more compounds according to the present invention include, but are not limited to, SNO compounds + acetazolamide (and other carbonic anhydrase inhibitors including topiramate), SNO compounds + serotonin agonist agents (e.g., 5HTl A agonist buspirone; serotonin re-uptake inhibitors), SNO compounds + serotonin antagonist agents (e.g., 5HT3 receptor antagonists, such as ondansetron), SNO compounds + tetracyclic antidepressants (e.g., mirtazipine, setiptiline), SNO compounds + modafinil, SNO compounds + r- modafmil, SNO compounds + compounds that effect the neuronal uptake of norepinephrine and/or dopamine (e.g., ropinerole, milnacipran), SNO compounds + zonisamide, SNO compounds + agents that stimulate brain activity and/or are opoid antagonists (e.g., doxapram, naloxone, caffeine), SNO compounds + narcotic analgesics that cause respiratory depression (e.g., morphine, meperidine, fentanyl, oxycodone, buprenorphine), SNO compounds + general anesthesics that cause respiratory depression (halothane, enflurane, thiopental), SNO compounds + theophylline SNO compounds + steroid and/or bronchodilator agents commonly used to treat asthma or chronic obstructive pulmonary disease (e.g., budesonide, fluticasone, salbutamol, formoterol, salmeterol/fluticasone combinations, tiotropium, ipatropium), SNO + antihistamines (e.g., cetirizine, azelastine, desloratidine, fexofenadine), SNO compounds + sedative/hypnotics (e.g., lorazepam, Zolpidem, zaleplon), and SNO compounds in combination with positive airway pressure breathing devices (including CPAP and BiPAP). Other compounds useful in combination with SNO compounds, as set forth herein, are described in US Patent Application Publication No. 20060039866, which is incorporated by reference herein in its entirety. It will be understood that any of the compounds of Table 1 or Table 2 can be substituted for the generic "SNO" compound as described in any aspect of the present invention.

In one embodiment of the invention, a combination of two or more compounds, wherein at least one compound acts through the S-nitrosomercapto pathway, would provide an additive or synergistic effect to restore normal breathing rhythm. In another embodiment of the invention, a combination of two or more compounds, wherein at least one compound acts through the S-nitrosomercapto pathway, provides an effect to counteract the respiratory depressant effect of another drug that may or may not be administered at the same time. SNO compounds have been described to have various clinical benefits.

These include, but are not limited to, increase in respiratory drive, increase in muscle tone in the upper airway, improvement of oxygen exchange in the lungs ("ventilatory perfusion matching"), and increased production of erythropoietin (EPO), a natural hormone that increases red blood cell production. Increased EPO production may be especially useful in patients that have breathing problems (with the accompanying hypoxia) and anemia. Such conditions result in a "double negative effect" of low oxygen levels and a low count of cells that carry the oxygen (e.g., apnea of prematurity, kidney dialysis patients). In one aspect of the invention, a compound of the invention is useful in the form in which the compound is administered. That is, the chemical structure and formula of the compound that is administered to the patient is the compound that is active according to a method of the invention. In another aspect, a compound of the invention is active in a form other than that structure or formula that is administered to a patient. In this aspect of the invention, a compound must first be altered, added to, broken down, metabolized or otherwise modified from the form in which the compound is administered to the patient. See, for example, International Patent Application Publication No. WO 03/015605, the entirety of which is incorporated by reference herein.

By way of several non-limiting examples, an SNO compound encompassed by the present invention includes an analog of N-acetylcysteine, a derivative of N-acetylcysteine, a modification of N-acetylcysteine, and a metabolite of N-acetylcysteine. It will be understood by the skilled artisan, when armed with the disclosure set forth herein, that analogs and derivatives of SNO compounds can be prepared and used according to the invention set forth herein. The skilled artisan will understand how to identify which portion or portions of an SNO compound to modify, and further, how to make such modifications, in accordance with the present invention. Additionally, based on the detailed description set forth herein, the skilled artisan will know how to assay such compounds to identify analogs or derivatives that have the activity of a compound of the invention, namely, the ability to control breathing in accordance with the present invention, when used in combination with one or more additional compounds. Acetazolamide has been used for many years as a mild diuretic (i.e., to increase urine output or to help treat mountain sickness). Acetazolamide is also believed to work through the carbon dioxide based respiratory drive pathway. It is proposed to work by lowering the pH of the blood, but this may not be the only way it affects respiratory drive. Decreases in respiratory drive may be caused by poor function of the carbon dioxide component, the oxygen component, or both components together. These components are, in fact, interrelated and causing an effect on one may affect the other and the overall respiratory drive.

Therefore, in one embodiment of the invention, in cases such as sleep apnea where both CO₂ and O₂ drive is diminished, a combination composition is used to provide a clinical benefit and/or treatment of the patient. That is, in one embodiment, the invention provides a method of treating sleep apnea.

Traditional thinking was previously that the doses of acetazolamide needed for treatment are too toxic for long-term use in a large number of patients. However, lower doses of acetazolamide may be sufficient to produce the desired effects on respiratory drive, particularly in combination with one or more other components according to the present invention. Other compounds that may be more effective at lower doses, due to the prevalence of side effects when used at higher doses, include, but are not limited to theophylline. A combination composition according to the invention is useful to treat any condition characterized by lack of normal breathing control. By way of a non- limiting example, such conditions include sleep apnea (central, mixed and obstructive including but not limited to co-existing conditions of heart failure, kidney disease and stroke), sleep-disordered breathing (especially with snoring and arousals), chronic bronchitis, COPD, asthma, allergy and neurological diseases (e.g., stroke, and amyotrophic lateral sclerosis, also known as ALS). Other conditions that may be treated with the methods and compositions of the present invention include, but should not be limited to, snoring, obesity-hypoventilation syndrome, apnea of prematurity, respiratory depression due to drugs (e.g., narcotic analgesics, sedatives, alcohol, sleeping pills, anesthetics), central congenital hypoventilation syndrome, hypoventilation due to stroke, trauma, surgery and/or radiation, and acclimatization to high altitude.

A combination composition according to the invention is also useful to assist in the treatment of any condition that is treatable using a positive airway pressure (PAP) device, as described elsewhere herein.

By way of a non-limiting example, the present invention may also be used to treat and/or alleviate symptoms of, or to facilitate, acclimatization to high altititude. Genetic diversity plays a role in how people respond to low oxygen levels. Some respond quickly by increasing the rate and depth of breathing (the hypoxic ventilatory response) while some others are slower. There are some cases where the ability to adapt quickly is important. For example, soldiers quickly inserted into a battle situation at high altitude (e.g., 12,000 feet in Afghanistan) need to operate at peak performance. A slow response to hypoxia will result in excessive tiredness and poor work performance. For soldiers this may be life-threatening. For the extreme altitude mentioned the case is fairly clear-cut. There also may be application at lesser altitudes such as the transition from New York to Denver (5,000 ft) or the jet lag from a long airplane flight (cabin pressure of 6,000 feet).

Serotonin agonist or re-uptake inhibitor compounds (e.g., Mirtazapine) have been demonstrated in animals to help restore the tone of the upper airway to prevent collapse. In an aspect of the invention, an SNO/serotonin agonist combination composition is used, whereby the SNO compound is used to improve respiratory drive, and the serotonin agonist improves the upper airway tone to help air flow and help prevent obstruction. In another embodiment, the invention includes a combination of a

SNO compound with an agent intended to reduce oxidative stress. When the body stops breathing and oxygen levels drop, there are a series of reactions leading to oxidative stress that is believed to be directly causative of the cardiovascular complications associated with sleep apnea and other conditions. The cardiovascular complications are the main cause of death.

In an aspect of the invention, a combination composition comprises N- acetylcysteine, which is used to reduce oxidative stress through a metabolic pathway unrelated to SNO production. That is, the invention also includes methods and combination compositions in which N-acetylcysteine or another SNO compound reduces oxidative stress in combination with another drug, either a second SNO compound, or a non-SNO compound such as, but not limited to, acetazolamide, wherein the second compound acts to increase respiratory drive. Other combinations useful in the methods and compositions of the invention will be understood by the skilled artisan, when armed with the disclosure set forth in the present disclosure. In another aspect, the invention includes a combination composition comprising an SNO compound and a compound that treats and/or prevents oxidative stress in a mammal. In one embodiment, the invention includes a method of treating a patient lacking normal breathing by administering a compound of the invention.

Frequent hypoxia/reoxygenation events, which replicate oxygenation patterns in sleep apnea, induce in one embodiment NADPH oxidase and proinflammatory gene expression in select brain regions, including in another embodiment, in wake-active neurons. In one embodiment, lack of a functional NADPH oxidase and pharmacological inhibition of NADPH oxidase is determined to confer resistance to intermittent hypoxia-induced neurobehavioral, redox and pro- inflammatory changes, thereby emphasizing a potential target to prevent oxidative morbidities in persons with obstructive sleep apnea (OSA).

US Patent Application Publication No. 20060154856 (incorporated herein by reference in its entirety) identifies NADPH oxidase as an important source of intermittent hypoxia-induced injury in the brain. In another embodiment, NADPH oxidase activation in persons with OSA contributes to the cardiovascular morbidities associated with this disease. The NADPH oxidase pathway is therefore a valuable pharmacotherapeutic target for both neurobehavioral and cardiovascular morbidities of the prevalent disorder, sleep apnea. According to one aspect of the present invention, the invention provides a method for treating a cardiovascular morbidity, a neurobehavioral morbidity or a combination thereof, resulting from sleep apnea hypopnea syndrome in a subject, comprising administering to said subject a therapeutically effective amount of a composition comprising an NADPH oxidase inhibitor, and at least one other compound. In one embodiment, the at least one other compound is an inhibitor of the S-nitrosomercapto signaling pathway. NADPH oxidase inhibitors include, but are not limited to, apocynin, or 4-hydroxy-3'- methoxyacetophenon, N-vanillylnonanamide, and staurosporine.

In another embodiment, the invention includes a combination of a SNO compound with an agent intended to reduce inflammation. Examples include a leukotriene receptor antagonist (or 5 -lipoxygenase inhibitor), antihistamine or antiinflammatory agent (e.g., COX-2 inhibitor or steroid). In one aspect, the invention includes a method of using such a combination composition to treat a patient lacking normal breathing.

Patients with sleep disordered breathing have turbulent airflow that causes inflammation and reduces their ability to efficiently get air. As discussed elsewhere herein, SNO compounds increase respiratory drive and may increase the diameter of the upper airway passages. Therefore, according to the invention, a combination composition comprising a SNO compound plus an anti-inflammatory compound is useful to provide a complimentary therapeutic benefit (Goldbart et al, 2005, Am. J. Respir. Crit. Care. Med. 172: 364-370).

By way of a non-limiting example, leukotriene antagonist therapy, using compositions and methods of the present invention, will decrease inflammation that results from turbulent airflow, ordering the breathing of a patient suffering from lack of normal breathing. This is because disturbed airflow causes inflammation that further restricts airflow, since the inflammation decreases the size of the airway passages. According to the present invention, combination composition products that include an anti-inflammatory agent are useful to provide an additional benefit for both adult and pediatric patients with various forms of sleep disordered breathing. In one aspect of the invention, a combination product of a SNO compound prodrug or a SNO compound in combination with a leukotriene antagonist (or a 5-lipoxygenaseoxidase inhibitor) are useful to treat disordered control of breathing, while at the same time, minimizing the inflammation associated with such breathing disorders. In another aspect of the invention, the invention includes a combination composition comprising three or more compounds for the treatment of a disease or disorder involving a lack of normal breathing control. The invention also includes methods for treating a mammal, wherein the method uses a combination composition comprising three or more compounds for the treatment of a disease or disorder involving a lack of normal breathing control. A composition according to the invention may comprise one or more SNO compounds. In another embodiment, a composition according to the invention may comprise three or more non-SNO compounds. Compounds useful in a combination composition of the invention are described in detail elsewhere herein. In another aspect of the invention, a method of treating a patient lacking normal breathing comprises administering a compound of the invention, as described herein, and additionally treating the patient using a device for treatment of a lack of normal breathing. As described in detail elsewhere herein, such devices include, but are not limited to, ventilation devices, CPAP and BiPAP devices. A mechanical ventilation is a method to mechanically assist or replace spontaneous breathing. Mechanical ventilation is typically used after an invasive intubation, a procedure wherein an endotracheal or tracheostomy tube is inserted into the airway. It is normally used in acute settings, such as in the ICU, for a short period of time during a serious illness. It may also be used at home or in a nursing or rehabilitation institution, if patients have chronic illnesses that require long-term ventilation assistance. The main form of mechanical ventilation is positive pressure ventilation, which works by increasing the pressure in the patient's airway and thus forcing air into the lungs. Less common today are negative pressure ventilators (for example, the "iron lung") that create a negative pressure environment around the patient's chest, thus sucking air into the lungs. Mechanical ventilation is often a life- saving intervention, but carries many potential complications including pneumothorax, airway injury, alveolar damage, and ventilator-associated pneumonia. For this reason the pressure and volume of gas used is strictly controlled, and reduced as soon as possible. Types of mechanical ventilation are: conventional ventilation, high frequency ventilation, non-invasive ventilation (non-invasive positive pressure pentilation or NIPPV), proportional assist aentilation (PAV), adaptive support ventilation (ASV) and neurally adjusted ventilatory assist (NAVA).

Non-invasive ventilation refers to all modalities that assist ventilation without the use of an endotracheal tube. Non-invasive ventilation is primarily aimed at minimizing patient discomfort and the complications associated with invasive ventilation, and is often used in cardiac disease, exacerbations of chronic pulmonary disease, sleep apnea, and neuromuscular diseases. Non-invasive ventilation refers only to the patient interface and not the mode of ventilation used; modes may include spontaneous or control modes and may be either pressure or volume modes. Some commonly used modes of NIPPV include:

(a) Continuous positive airway pressure (CPAP). This kind of machine has been used mainly by patients for the treatment of sleep apnea at home, but now is in widespread use across intensive care units as a form of ventilation. The CPAP machine stops upper airway obstruction by delivering a stream of compressed air via a hose to a nasal pillow, nose mask or full-face mask, splinting the airway (keeping it open under air pressure) so that unobstructed breathing becomes possible, reducing and/or preventing apneas and hypopneas. When the machine is turned on, but prior to the mask being placed on the head, a flow of air comes through the mask. After the mask is placed on the head, it is sealed to the face and the air stops flowing. At this point, it is only the air pressure that accomplishes the desired result. This has the additional benefit of reducing or eliminating the extremely loud snoring that sometimes accompanies sleep apnea.

(b) Bi-level positive airway pressure (BIPAP). Pressures alternate between inspiratory positive airway pressure (IPAP) and a lower expiratory positive airway pressure (EPAP), triggered by patient effort. On many such devices, backup rates may be set, which deliver IPAP pressures even if patients fail to initiate a breath.

(c) Intermittent positive pressure ventilation (IPPV), via mouthpiece or mask. Methods

As set forth in detail elsewhere herein, any composition, or combination composition of the invention is useful in a therapeutic treatment of the breathing of a mammal. In an aspect, the mammal is a human. A therapeutic treatment of the breathing of a mammal includes, but is not limited to improvement or correction of a non-normal breathing condition in a mammal.

A composition of the invention is administered to a mammal in any manner known in the art, and further, in any manner known or determined to be a beneficial manner for administration of the composition for obtaining a therapeutic effect according to the invention. Methods and manufactures for administration of a composition of the invention are set forth in greater detail elsewhere herein. In an embodiment, a composition is administered to the brainstem of a mammal. In another embodiment, a composition is administered to the respiratory center of the brainstem of a mammal. In yet another embodiment, a composition is administered to the nucleus tractus solitarius of the brainstem of a mammal.

In an embodiment, a method of the invention includes administration of a composition of the invention to stabilize the breathing rhythm of a mammal. In another embodiment, a method of the invention includes administration of a composition of the invention to increase minute ventilation of a mammal. In an aspect, minute ventilation of a mammal is increased at the level of the brainstem respiratory control center in the nucleus tractus solitarius.

In an aspect, a method of the invention includes administration of a combination composition of the invention, in which a first compound is useful for stabilizing the breathing rhythm of a mammal, and a second compound has a distinct effect on the mammal. In an embodiment, a method of the invention includes administration of a combination composition of the invention, in which a second compound can increase the patency of the upper airway in a mammal. In another embodiment, a method of the invention includes administration of a combination composition of the invention, in which a second compound promotes wakefulness in a mammal. In another embodiment, a method of the invention includes administration of a combination composition of the invention, in which a second compound decreases frequency and/or intensity of seizures in a mammal. In another embodiment, a method of the invention includes administration of a combination composition of the invention, in which a second compound decreases inflammation in a mammal. In another embodiment, a method of the invention includes administration of a combination composition of the invention, in which a second compound decreases respiratory drive in a mammal. In another embodiment, a method of the invention includes administration of a combination composition of the invention, in which a second compound improves lung function in a mammal.

In one embodiment of the inevntion, when two compounds are to be administered to a subject, the two compounds may be administered concomitantly or nearly concomitantly, wherein the term "nearly concomitantly" indicates that the compounds are administered at the same time or as close in time as physically and medically possible based on the forms of administration used and general set-up for administration. In another embodiment, when two compounds are to be administered to the subject, the two compounds may be administered sequentially. In one preferred embodiment, the second compound is administered 5 minutes or less after the first compound. In another preferred embodiment, the second compound is administered 5 to 10 minutes after the first compound. In yet another preferred embodiment, the second compound is administered 10 to 20 minutes after the first compound. In yet another preferred embodiment, the second compound is administered 20 to 40 minutes after the first compound. In yet another preferred embodiment, the second compound is administered 40 minutes to 2 hours after the first compound. In yet another preferred embodiment, the second compound is administered 2 hours to 4 hours after the first compound. In yet another preferred embodiment, the second compound is administered 4 hours to 8 hours after the first compound. In yet another preferred embodiment, the second compound is administered 8 hours to 16 hours after the first compound. In yet another preferred embodiment, the second compound is administered 16 hours to 1 day after the first compound. In yet another preferred embodiment, the second compound is administered 1 day to 2 days after the first compound. In yet another preferred embodiment, the second compound is administered 2 days to 4 days after the first compound. One skilled in the art should be able to identify the best sequence and interval for administration, based on the activity(ies) of the two compounds to be administered.

In one embodiment of the invention, when three or more compounds are to be administered to a subject, the three or more compounds may be administered concomitantly or nearly concomitantly, wherein the term "nearly concomitantly" indicates that the three or more compounds are administered at the same time or as close in time as physically and medically possible based on the forms of administration used and general set-up for administration. In another embodiment, the three or more compounds are not administered concomitantly. In a preferred embodiment, the three or more compounds are administered sequentially, with intervals between each administration that are independently 5 minutes or less, 5 to 10 minutes, 10 to 20 minutes, 20 to 40 minutes, 40 minutes to 2 hours, 2 hours to 4 hours, 4 hours to 8 hours, 8 hours to 16 hours, 16 hours to 1 day, 1 day to 2 days, 2 days to 4 days, or any fractions or multiples thereof. In another preferred embodiment, the three or more compounds are combined in one or more groups of compounds, wherein each one or more groups of compounds are administered concomitantly or nearly concomitantly. In another preferred embodiment, the three or more compounds are combined in one or more groups of compounds, wherein each one or more groups of compounds are administered sequentially. One skilled in the art should be able to identify the best sequence, combination of compounds and interval for administration, based on the activity(ies) of the three or more compounds to be administered.

Pharmaceutical Compositions & Formulations The invention also encompasses the use of pharmaceutical compositions of an appropriate protein or peptide and/or isolated nucleic acid to practice the methods of the invention. The compositions and combinations of compounds set forth herein can be used alone or in combination with additional compounds to produce additive, complementary or synergistic effects in the treatment of disordered breathing, and in the treatment of sleep-related breathing disorders.

In an embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In another embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.

Pharmaceutically acceptable carriers that are useful include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Pharmaceutical compositions that are useful in the methods of the invention may be administered, prepared, packaged, and/or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and imrnunologically-based formulations.

The compositions of the invention may be administered via numerous routes, including, but not limited to, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, or ophthalmic administration routes. A composition of the invention may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

Compounds identified using any of the methods described herein, and combinations of such compounds, may be formulated and administered to a mammal for treatment of disordered control of breathing.

Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

An obstacle for topical administration of pharmaceuticals is the stratum corneum layer of the epidermis. The stratum corneum is a highly resistant layer comprised of protein, cholesterol, sphingolipids, free fatty acids and various other lipids, and includes cornified and living cells. One of the factors that limit the penetration rate (flux) of a compound through the stratum corneum is the amount of the active substance that can be loaded or applied onto the skin surface. The greater the amount of active substance which is applied per unit of area of the skin, the greater the concentration gradient between the skin surface and the lower layers of the skin, and in turn the greater the diffusion force of the active substance through the skin. Therefore, a formulation containing a greater concentration of the active substance is more likely to result in penetration of the active substance through the skin, and more of it, and at a more consistent rate, than a formulation having a lesser concentration, all other things being equal.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.

Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs. Pharmaceutical compositions that are useful in the methods of the invention may be administered systemically in oral solid formulations, ophthalmic, suppository, aerosol, topical or other similar formulations. In addition to the compound such as heparin sulfate, or a biological equivalent thereof, such pharmaceutical compositions may contain pharmaceutically acceptable carriers and other ingredients known to enhance and facilitate drug administration. Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer compounds according to the methods of the invention. A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

Enhancers of permeation may be used. These materials increase the rate of penetration of drugs across the skin. Typical enhancers in the art include ethanol, glycerol monolaurate, PGML (polyethylene glycol monolaurate), dimethylsulfoxide, and the like. Other enhancers include oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositions of the invention may contain liposomes. The composition of the liposomes and their use are known in the art (for example, see US Patent No. 6,323,219).

The source of active compound to be formulated will generally depend upon the particular form of the compound. Small organic molecules and peptidyl or oligo fragments can be chemically synthesized and provided in a pure form suitable for pharmaceutical usage. Products of natural extracts can be purified according to techniques known in the art. Recombinant sources of compounds are also available to those of ordinary skill in the art.

In alternative embodiments, the topically active pharmaceutical composition may be optionally combined with other ingredients such as adjuvants, anti-oxidants, chelating agents, surfactants, foaming agents, wetting agents, emulsifying agents, viscosifiers, buffering agents, preservatives, and the like. In another embodiment, a permeation or penetration enhancer is included in the composition and is effective in improving the percutaneous penetration of the active ingredient into and through the stratum corneum with respect to a composition lacking the permeation enhancer. Various permeation enhancers, including oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known to those of skill in the art. In another aspect, the composition may further comprise a hydrotropic agent, which functions to increase disorder in the structure of the stratum corneum, and thus allows increased transport across the stratum corneum. Various hydrotropic agents such as isopropyl alcohol, propylene glycol, or sodium xylene sulfonate, are known to those of skill in the art.

The topically active pharmaceutical composition should be applied in an amount effective to affect desired changes. As used herein "amount effective" shall mean an amount sufficient to cover the region of skin surface where a change is desired. An active compound should be present in the amount of from about 0.0001% to about 15% by weight volume of the composition. More preferable, it should be present in an amount from about 0.0005% to about 5% of the composition; most preferably, it should be present in an amount of from about 0.001% to about 1% of the composition. Such compounds may be synthetically-or naturally derived.

Liquid derivatives and natural extracts made directly from biological sources may be employed in the compositions of this invention in a concentration (w/v) from about 1 to about 99%. Fractions of natural extracts and protease inhibitors may have a different preferred rage, from about 0.01% to about 20% and, more preferably, from about 1% to about 10% of the composition. Of course, mixtures of the active agents of this invention may be combined and used together in the same formulation, or in serial applications of different formulations. The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of an aqueous gel because of repeated patient use when it is exposed to contaminants in the environment from, for example, exposure to air or the patient's skin, including contact with the fingers used for applying a composition of the invention such as a therapeutic gel or cream. Examples of preservatives useful in accordance with the invention include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid. The composition preferably includes an antioxidant and a chelating agent that inhibits the degradation of the compound for use in the invention in the aqueous gel formulation. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the preferred range of about 0.01% to 0.3% and more preferably BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. Preferably, the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Particularly preferred chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20% and more preferably in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition which may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the particularly preferred antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art. Controlled-release preparations may also be used and the methods for the use of such preparations are known to those of skill in the art. In some cases, the dosage forms to be used can be provided as slow or controlled-release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the invention. Thus, single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps, and caplets, that are adapted for controlled-release are encompassed by the present invention.

Most controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood level of the drug, and thus can affect the occurrence of side effects.

Most controlled-release formulations are designed to initially release an amount of drug that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds. The term "controlled-release component" in the context of the present invention is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres or a combination thereof that facilitates the controlled-release of the active ingredient. Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., poly oxy ethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol. Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in- water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents, such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as poly oxy ethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents. As used herein, an "oily" liquid is one which comprises a carbon- containing liquid molecule and which exhibits a less polar character than water. A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, a paste, a gel, toothpaste, a mouthwash, a coating, an oral rinse, or an emulsion. The terms oral rinse and mouthwash are used interchangeably herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for oral or buccal administration. Such a formulation may comprise, but is not limited to, a gel, a liquid, a suspension, a paste, toothpaste, a mouthwash or oral rinse, and a coating. For example, an oral rinse of the invention may comprise a compound of the invention at about 1.4 %, chlorhexidine gluconate (0.12%), ethanol (11.2%), sodium saccharin (0.15%), FD&C Blue No. 1 (0.001%), peppermint oil (0.5%), glycerine (10.0%), Tween 60 (0.3%), and water to 100%. In another embodiment, a toothpaste of the invention may comprise a compound of the invention at about 5.5%, sorbitol, 70% in water (25.0%), sodium saccharin (0.15%), sodium lauryl sulfate (1.75%), carbopol 934, 6% dispersion in (15%), oil of spearmint (1.0%), sodium hydroxide, 50% in water (0.76%), dibasic calcium phosphate dihydrate (45%), and water to 100%. The examples of formulations described herein are not exhaustive and it is understood that the invention includes additional modifications of these and other formulations not described herein, but which are known to those of skill in the art.

A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form, such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycollate. Known surface-active agents include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in US Patents Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use. A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.

Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient that is solid at ordinary room temperature (i.e., about 20°C) and that is liquid at the rectal temperature of the subject (i.e., about 37°C in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives.

Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives. Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations that are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other

"additional ingredients" that may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985,

Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

Typically, dosages of the compound of the invention that may be administered to an animal, preferably a human, will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration.

The compound can be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, and the type and age of the animal.

EXAMPLES

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

Example 1 : Characterization of a compound having "S-nitrosomercapto-like" activity according to the invention

One embodiment of the invention includes characterization of one or more molecules that interact with the hemoglobin contained in red blood cells so as to induce a reaction between the SNO or SNO prodrug to create SNOHb (Doctor et al., 2005, PNAS 102: 5709-5714). This change, when identified by screening, identifies small molecules that can act as SNO signaling agents. Example 2: Characterization of compounds of the invention using a method of evaluating breathing control

An established method for evaluating the effects of drugs that act on breathing control is to create closed systems where the key factors that affect breathing can be tightly controlled and monitored. For example, control systems are established for oxygen concentration, carbon dioxide concentration and atmospheric pressure.

For animal based evaluations, systems are available that allow for either whole body or nose-only evaluation of multiple respiratory function measurements. There are also established animal models (e.g., guinea pig, dog, rodent) of respiration in combination with allergy, inflammation, COPD and narcotic analgesic use. By way of a non-limiting example, Lovelace Respiratory Research Institute (Albuquerque, NM) has extensive experience in establishing such models as part of evaluation for new drugs and environmental exposure purposes. In another non-limiting example, Onal et al. described a method in humans of evaluating various treatments on the genioglossal muscle (Onal et al., 1981, Am. Rev. Respir. Dis. 124: 215-7). Muscle tone in the upper airway is a critical component in condtions such as sleep apnea and snoring and this publication suggests that there is an intimate relationship between central respiratory control and airway muscle tone. Accordingly, agents such as SNO compounds that affect the control of breathing may also improve the patency of the upper airway by incrasing muscle tone in this region. Horner and Bradley recently provided a review on clinical and animal model data regarding sleep and the control of ventilation (Am. J. Respir. Crit. Care Med. 2006, 173: 827-832). They repeated Onal's earlier call for evaluation of agents that affect the upper airway musculature and reference a variety of animal and human models that may be helpful. This reference is incorporated herein in its entirety.

Example 3: Novel model to assess the biological activity and potency of compounds used to restore ventilatory control For the experimental model test species, Sprague-Dawley male rats

(Charles River) were used, at a weight of 250-300 grams. Fentanyl was used at doses ranging from 75 to 150 micrograms per kilogram to induce respiratory depression ranging from 25% to 50%. The effect of fentanyl and other narcotic analgesics in inducing respiratory depression in rats and humans is well documented in the literature (Dahan et al, 2005, British Journal of Anaesthesia 94 (6): 825-34). Such models of induced respiratory depression are accepted in the art as representative of respiratory depression occurring in nature.

Test compounds according to the inevntion may be given before, simultaneous or after administration of fentanyl in this model. The order of administration is critical, since some compounds require biological activation to be fully effective. The skilled artisan would be aware of, or be able to determine, compounds that require biological activation to become fully effective.

The speed of administration via intravenous infusion is also important to elicit two distinct phases of respiratory depression. Within the first few minutes there is an initial deep respiratory depression effect (0 to 5 minutes) often accompanied by apnea (breathing stoppage). This is followed by a recovery of breathing and a prolonged respiratory depression lasting 45-60 minutes. Proper administration of fentanyl is critical to obtain both phases of the respiratory depression curve since the compounds of the invention may affect either one or both parts of the curve with differing clinical uses, depending upon the activity.

Example 4 : Characterization of compounds of the invention using human models Hildebrandt et al. (Blood 2002, 99: 1552-1555) described a protocol that was used for evaluation of N-acetylcysteine under varying conditions of oxygen and carbon dioxide concentrations. In addition, the United States military (Naval Aerospace Medical Research Command, Pensacola FL, US Army Research Institute of Environmental Medicine, Natick, MA) has developed methods that include both whole body and face only exposure/monitoring systems (Sausen et al., 2003, Aviat. Space Environ. Med. 74: 1190-7).

In another embodiment, hospitalized patients who are connected to mechanical ventilation devices represent an opportunity to closely evaluate the effects of drugs on respiration. Levels of oxygen and carbon dioxide can be controlled in an environment where respiration parameters are measured on a minute-by-minute basis. In addition to the animal and human-based systems described herein, there is an emerging field in which certain biochemical markers are used to indicate chronic oxidative stress resulting from hypoxia. One such example is the use of various isoprostanes to indicate oxidative stress (Cracowski & Durand, 2006, Fundam. Clin. Pharmacol. 20: 417-27). Example 5 : (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid accelerated the normalization of blood pH after opioid analgesic administration To assess its effect on blood pH, (S)-4-mercapto-2-(thiazol-2- ylamino)butanoic acid (100 μmol/kg) was administered intravenously to Sprague

Dawley rats 15 minutes after an intravenous dose of fentanyl (75 μg/kg) (Figure 23).

Blood pH is a parameter commonly used in clinical medicine to evaluate a patient's health.

Example 6: (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid accelerated the normalization of blood pCO? after opioid analgesic administration To measure its effect on the partial pressure of carbon dioxide (pCO₂) in the blood, (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid (100 μmol/kg) was administered intravenously to Sprague Dawley rats 15 minutes after an intravenous dose of fentanyl (75 μg/kg) (Figure 24). pCO₂ is a parameter commonly used in clinical medicine to evaluate a patient's health.

Example 7: (S)-4-mercapto-2-('thiazol-2-ylamino^*)butanoic acid accelerated the normalization of blood pO? after opioid analgesic administration To measure its effect on the partial pressure of oxygen (pθ₂) in the blood, (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid (100 μmol/kg) was administered intravenously to Sprague Dawley rats 15 minutes after an intravenous dose of fentanyl (75 μg/kg) (Figure 25). pθ₂ is a parameter commonly used in clinical medicine to evaluate a patient's health.

Example 8: (S)-4-mercapto-2-rthiazol-2-ylamino)butanoic acid accelerated the normalization of FChHb after opioid analgesic administration To measure its effect on the fraction of total hemoglobin that is oxyhemoglobin (FO₂Hb) in the blood, (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid (100 μmol/kg) was administered intravenously to Sprague Dawley rats 15 minutes after an intravenous dose of fentanyl (75 μg/kg) (Figure 26). FO₂Hb is a parameter commonly used in clinical medicine to evaluate a patient's health. Example 9: (SV4-mercapto-2-(tMazol-2-ylamino)butanoic acid accelerated the normalization of cHCO_rfP) after opioid analgesic administration To measure its effect on the concentration of bicarbonate ions (mM) in the plasma (cHCO₃-(P)), (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid (100 μmol/kg) was administered intravenously to Sprague Dawley rats 15 minutes after an intravenous dose of fentanyl (75 μg/kg) (Figure 27). CHCO₃-(P) is a parameter commonly used in clinical medicine to evaluate a patient's health.

Example 10: (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid accelerated the normalization of cGLU after opioid analgesic administration

To measure its effect on the concentration of D-glucose (cGLU) in the blood, (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid (100 μmol/kg) was administered intravenously to Sprague Dawley rats 15 minutes after an intravenous dose of fentanyl (75 μg/kg) (Figure 28). cGLU is a parameter commonly used in clinical medicine to evaluate a patient's health.

Example 11 : (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid immediately and completely reversed opioid analgesic-associated suppression of Minute Ventilation and decreased the time to arousal To measure its effect on Minute Ventilation (i.e., the amount of air breathed per minute) and the time to arousal, (S)-4-mercapto-2-(thiazol-2- ylamino)butanoic acid (100 μmol/kg) was administered intravenously to Sprague Dawley rats 15 minutes after an intravenous dose of fentanyl (75 μg/kg). (S)-4- mercapto-2-(thiazol-2-ylamino)butanoic acid immediately and completely reversed fentanyl-associated suppression of Minute Ventilation and decreased the time to arousal (i.e., "woke" the rats up) (Figure 29).

Example 12: (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid immediately and completely reversed opioid analgesic-associated suppression of Tidal Volume and decreased the time to arousal

To measure its effect on Tidal Volume (i.e., the depth of respiration) and the time to arousal, (S)-4-mercapto-2-(thiazol-2-ylarnino)butanoic acid (100 μmol/kg) was administered intravenously to Sprague Dawley rats 15 minutes after an intravenous dose of fentanyl (75 μg/kg). (S)-4-mercapto-2-(thiazol-2- ylamino)butanoic acid immediately and completely reversed fentanyl-associated suppression of Tidal Volume and decreased the time to arousal (i.e., "woke" the rats up) (Figure 30). The data disclosed in Figure 30, taken in light of the data disclosed in Figure 29, suggest the majority of the effect of (S)-4-mercapto-2-(thiazol-2- ylamino)butanoic acid was due to increases in Tidal Volume rather than to increases in the respiratory rate.

Example 13: (S>4-mercapto-2-(thiazol-2-ylamino)butanoic acid diminished anesthetic-associated suppression of Minute Ventilation To measure its effect on preventing anesthetic-associated suppression of Minute Ventilation (i.e., the amount of air breathed per minute), (S)-4-mercapto-2- (thiazol-2-ylamino)butanoic acid (100 μmol/kg) was administered intravenously to Sprague Dawley rats 15 minutes before an intravenous dose of sodium pentobarbital (30 mg/kg). (S)-4-mercapto-2-(thiazol-2-ylamino)butanoic acid diminished pentobarbital-associated suppression of Minute Ventilation (Figure 31). The data disclosed in Figure 30 demonstrate that the effect (S)-4-mercapto-2-(thiazol-2- ylamino)butanoic acid has on Minute Ventilation is not limited to fentanyl, which exerts its effects through opioid receptors, but also counters the respiratory depression effects of pentobarbital, which is an anesthetic agent that works through a mechanism not involving opioid receptors.

Example 14: (S>4-mercapto-2-(4-phenylthiazol-2-ylamino)butanoic acid reverses fentanyl-induced depression of tidal volume in rat To measure its effect on Tidal Volume (i.e., the depth of respiration), (S)-4-mercapto-2-(4-phenylthiazol-2-ylamino)butanoic acid (100 μmol/kg) was administered intravenously to Sprague Dawley rats 5 minutes before an intravenous dose of fentanyl (75 μg/kg). (S)-4-mercapto-2-(4-phenylthiazol-2-ylamino)butanoic acid reversed fentanyl-associated suppression of Tidal Volume (Figure 32).

Example 15 : (S)- 1 -((R)-2-(benzoxazol-2-ylamino )-3 -mercaptopropanoylV pyrrolidine-2-carboxylic acid protects against fentanyl-induced depression of tidal volume in rat

To measure its effect on Tidal Volume (i.e., the depth of respiration), (S)-I -((R)-2-(benzoxazol-2-ylamino)-3 -mercaptopropanoyl)-pyrrolidine-2-carboxylic acid (100 μmol/kg) was administered intravenously to Sprague Dawley rats 15 minutes before an intravenous dose of fentanyl (75 μg/kg). (S)-l-((R)-2-(benzoxazol- 2-ylamino)-3-mercaptopropanoyl)-pyrrolidine-2-carboxylic acid protected against fentanyl-associated suppression of Tidal Volume (Figure 33).

Example 16: fSVl-(YRV2-(benzoxazol-2-ylamino)-3-mercaptopropanovD- pyrrolidine-2-carboxylic acid protects against fentanyl-induced depression of minute volume in rat

To measure its effect on Minute Volume (i.e., the amount of air breathed per minute), (S)- 1 -((R)-2-(benzoxazol-2-ylamino)-3 -mercaptopropanoyl)- pyrrolidine-2-carboxylic acid (100 μmol/kg) was administered intravenously to Sprague Dawley rats 15 minutes before an intravenous dose of fentanyl (75 μg/kg). (S)- 1 -((R)-2-(benzoxazol-2-y lamino)-3 -mercaptopropanoyl)-pyrrolidine-2-carboxylic acid_protected against fentanyl-associated suppression of Minute Volume (Figure 34).

Example 17: (SV4-nitrosomercapto-2-fthiazol-2-ylamino)butanoic acid protects against fentanyl-induced depression of tidal volume in rat

To measure its effect on Tidal Volume (i.e., the depth of respiration), (S)-4-nitrosomercapto-2-(thiazol-2-ylamino)butanoic acid (1 μmol/kg/minute) was administered by intravenous infusion to Sprague Dawley rats. After 40 minutes the rats received fentanyl (15 μg/kg) as a bolus IV injection, and 35 minutes later, the rats received a second IV bolus dose of fentanyl (25 μg/kg). Approximately 30 minutes later, the rats received naloxone (1.5 mg/kg) to demonstrate reversal of opioid- induced effects on breathing. (S)-4-nitrosomercapto-2-(thiazol-2-ylamino)butanoic acid protected against drug-associated suppression of Tidal Volume (Figure 35). Animals that received IV-infused (S)-4-nitrosomercapto-2-(thiazol-2- ylamino)butanoic acid exhibited markedly higher tidal volume versus animals that did not receive the compound but were challenged with fentanyl.

Example 18: (SV4-nitrosomercapto-2-(^'thiazol-2-ylamino)butanoic acid protects against fentanyl-induced depression of tidal volume in rat in a dose- dependent manner

To measure its effect on Tidal Volume (i.e., the depth of respiration), (S)-4-nitrosomercapto-2-(thiazol-2-ylamino)butanoic acid (1 μmol/kg/minute) was administered by intravenous infusion to Sprague Dawley rats. After approximately 40 minutes, the rats received an IV bolus dose of fentanyl (15 μg/kg) and a second IV bolus dose of fentanyl (25 μg/kg) 35 minutes later. Approximately 30 minutes later, the rats received naloxone (1.5 mg/kg) to demonstrate reversal of opioid-induced effects on breathing. (S)-4-nitrosomercapto-2-(thiazol-2-ylamino)butanoic acid protected against drug-associated suppression of Tidal Volume (Figure 36), as compared to a control group of animals that did not receive the compound but were challenged with fentanyl.

As will be understood by the skilled artisan when armed with the present disclosure, any of the above-described methods can be used to evaluate compounds and/or methods of the present invention.

Preparation of Compounds According to The Present Invention Synthetic methods for compounds according to the present invention are presented herein. These methods are considered to be exemplary methods only as a chemist of ordinary skill in the art will understand that there may be alternative methods to arrive at the described compounds. An alternative method may comprise of, but is not limited to, the use of alternative protecting groups as example (see Wuts & Greene, 2007, "Protective Groups in Organic Synthesis", 4^th Edition, John Wiley & Sons Inc., New York, New York). For example, and as described below, an appropriately substituted benzyl group may be used as an alternative to a triphenylmethyl-derived protecting group when masking of a mercapto sulfur center is necessary. All reactions were monitored via TLC or HPLC. It is within the ability of the ordinarily skilled artisan to determine appropriate TLC or HPLC conditions for a given compound/reaction.

Where it is stated that compounds were prepared in the manner described for an earlier Preparation or Example, the skilled artisan will appreciate that reaction times, number of equivalents of reagents and reaction temperatures may be modified for each specific reaction, and that it may nevertheless be necessary or desirable to employ different workup or purification conditions. The structures of all final products were verified by ¹H NMR and mass spec analysis. Unless otherwise reported, compounds containing a chiral center were greater than 95% of the desired enantiomer.

The abbreviations used in the present application are listed below:

Ar argon aq. aqueous

CHCl₃ chlorofrom

CH₂Cl₂ or DCM dichloromethane

Cs₂CO₃ cesium carbonate

DIPEA N,N-diisopropylethylamine

DTT dithiothreitol

EtOH ethanol

EtOAc ethyl acetate

Et₃SiH triethylsilane

HOAc acetic acid

KHSO₄ potassium bisulfate

K₂CO₃ potassium carbonate

MeOH methanol

NaHCO₃ sodium bicarbonate

Na₂SO₄ sodium sulfate

Pd(OAc)₂ palladium (II) acetate

PE petroleum ether

PMB para-methoxybenzyl sat. saturated t-Bu (tert-butyl) tertiary butyl

TEA triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethyl xanthene

EDCI 1 -ethyl-3-(3 '-dimethylaminopropyl) carbodiimide

HOBt N-hydroxybenzotriazole

KI potassium iodide

TsCl tosyl chloride

BuONO butyl nitrite DTPA diethylenetriamine pentaacetic acid

EtONO ethyl nitrite

NNED naphthyl (N-l-ethylene)diamine

SAA sulfanilamide

Although the preparations, below, are directed to a single enantiomer (unless otherwise stated) each of the procedures below may be modified to prepare the "other" enantiomer or desired diastereomer of the title compound. For example, a synthesis using D-penicillamine may be modified to use L-penicillamine to produce the desired optical isomer of the title compound of the synthetic route. Similarly, a procedure utilizing (S)-cysteine may be modified to use (R)-cysteine to obtain the desired optical isomer.

Synthetic Example 1 (R)-3-Mercapto-2-(5-methyl-4-phenyl-thiazoI-2-yIamino)-propionic acid

Step 1 : S-Trityl-L-cysteine falso known as (R)-2-amino-3- (tritylsulfanyl)propanoic acid)

L-Cysteine hydrochloride (5.0 g, 31.7 mmol) and trityl chloride (13.5 g, 48.4 mmol) were stirred in DMF (20 ml) for 48 h at room temperature. A 10% NaOAc solution (175 mL) was then added, and the precipitate was filtered and washed with water. Afterward, the residue was suspended in acetone and stirred at 50 °C for 30 min, then cooled and filtered. The residue was washed with acetone (cold) and diethyl ether. After drying 8.86 g (77%) S-trityl-L-cysteine was obtained. Step 2: (R)-2-(^'5-Methyl-4-phenyl-thiazol-2-ylamino)-3-tritylsulfanyl-propionic acid

2-Bromo-l-phenyl-propan-l-one (2.13 g, 10.0 mmol) and potassium thiocyanate (1.04 g, 10.7 mmol) were stirred in EtOH (35 mL) for 3h at 5O⁰C. S- Trityl-L-cysteine (3.64 g, 10.0 mmol), triethylamine (1.5 mL, 11.0 mmol) and EtOH (15 mL) were added. The reaction mixture was stirred at 5O⁰C for 16 h. The ethanol was removed in vacuum and the residue was partitioned between water and EtOAc. The water phase was twice extracted with EtOAc. The combined organic extracts were washed with water, brine, dried over anhydrous Na₂SO₄ and concentrated. The crude product was purified by flash column chromatography using gradient elution from CH₂Cl₂/Et0H (9:1) to CH₂Cl₂/Et0H (7:1) to give 3.37 g (63%) of (R)-2-(5- methyl-4-phenyl-thiazol-2-ylamino)-3-tritylsulfanyl-propionic acid.

Step 3: (R)-3-Mercapto-2-(5-methyl-4-phenyl-thiazol-2-ylamino)-propionic acid

To a solution of (R)-2-(5-methyl-4-phenyl-thiazol-2-ylamino)-3- tritylsulfanyl-propionic acid (1.24 g, 2.31 mmol) in CH₂Cl₂ (16 mL), triethylsilane (Et₃SiH) (0.88 g, 1.20 mL, 7.59 mmol) was added followed by trifluoroacetic acid (TFA) (17 mL). The reaction mixture was stirred under Ar atmosphere at O⁰C for 40 minutes. The volatiles were removed in vacuum. The residue was purified by flash column chromatography using gradient elution from CH₂Cl₂/Et0H (98:2) to CH₂CVEtOH (1 : 1) to give 0.33 g (49%) of (R)-3-mercapto-2-(5-methyl-4-phenyl- thiazol-2-ylamino)-propionic acid. 400 MHz ¹H-NMR (DMSO-d₆, ppm): 13.2-12.7 (IH, br s) 8.0-7.8 (IH, br s) 7.59-7.54 (2H, m) 7.43-7.36 (2H, m) 7.32-7.27 (IH, m) 4.53.4.47 (IH, m) 3.06-2.99 (IH, m) 2.92-2.83 (IH, m) 2.46 (IH, t, J=8.1 Hz) 2.34 (3H, s). ESI-MS (m/z): 295 [M+H]⁺.

Synthetic Example 2

(R)-2-(4-tert-Butyl-thiazol-2-ylamino)-3-mercapto-propionic acid Step 1 : (RV2-r4-tert-Butyl-thiazol-2-ylamino)-3-tritylsulfanyl-propionic acid The title compound was prepared in 56% yield from S-trityl-L-cysteine and 1 -bromo-3,3-dimethyl-butan-2-one following the procedure used for preparing (R)-2-(5-methyl-4-phenyl-thiazol-2-ylamino)-3-tritylsulfanyl-propionic acid. Step 2: (R)-2-(^"4-tert-Butyl-thiazol-2-ylamino)-3-mercapto-propionic acid

The title compound was prepared in 49% yield from (R)-2-(4-tert-butyl- thiazol-2-ylamino)-3 -tritylsulfanyl-propionic acid following the procedure used for preparing (R)-3-mercapto-2-(5-methyl-4-phenyl-thiazol-2-ylamino)-propionic acid. 200 MHz ¹H-NMR (DMSO-d₆, ppm): 8.3-8.0 (IH, br s) 6.24 (IH, s) 4.51-4.36 (IH, m) 3.08-2.76 (2H, m) 2.56-2.42 (IH, m, overlapped with DMSO) 1.18 (9H, s). ESI- MS (m/z): 261 [M+H]⁺. Synthetic Example 3 (R)-2-(4,5-DimethyI-thiazoI-2-yIamino)-3-mercapto-propionic acid

Step 1 : CR^')-2-(4,5-Dimethyl-thiazol-2-ylamino)-3-tritylsulfajiyl-propionic acid The title compound was prepared in 40% yield from S-trityl-L-cysteine and 3-bromo-2-butanone following the procedure used for preparing (R)-2-(5-methyl-

4-phenyl-thiazol-2-y lamino)-3 -trity lsulfanyl-propionic acid.

Step 2: fRV2-(4,5-Dimethyl-thiazol-2-ylaminoV3-mercapto-propionic acid

The title compound was prepared in 93% yield from (R)-2-(4,5-dimethyl- thiazol-2-ylamino)-3-tritylsulfanyl-propionic acid following the procedure used for preparing (R)-3-mercapto-2-(5-methyl-4-phenyl-thiazol-2-ylamino)-propionic acid.

200 MHz ¹H-NMR (DMSO-d₆, ppm): 7.7-7.6 (IH, br s) 4.43-4.30 (IH, m) 3.00-2.72

(2H, m) 2.55-2.34 (IH, m, overlapped with DMSO) 2.08 (3H, s) 1.96 (3H, s). ESI-MS

(m/z): 233 [MH-H]⁺.

Synthetic Example 4

(R)-3-Mercapto-2-(4,5,6,7-tetrahydro-benzothiazoI-2-yIamino)-propionic acid

Step 1 : (RV2-C4.5.6 J-Tetrahvdro-benzothiazol-2-ylaminoV3-tritylsulfanyl- propionic acid 2-Chloro-cyclohexanone (1.06 g, 8.00 mmol) and potassium thiocyanate (1.04 g, 9.06 mmol) in MeCN (24 mL) are heated at 120 ⁰C for 80 min under microwave conditions. The reaction mixture was filtered and the solvent was removed in vacuum. The resultant residue was dissolved in EtOH (40 mL). S-Trityl- L-cysteine (2.91 g, 8.00 mmol) and triethylamine (1.2 mL, 8.61 mmol) were added. The reaction mixture was stirred at 5O⁰C for 6.5 h, after which time the solvents were removed in vacuum. The residue was dissolved in CH₂Cl₂, washed with 0.1N HCl solution, then with brine, dried over anhydrous Na₂SO₄ and concentrated. The crude product was purified by flash column chromatography using gradient elution from CH₂Cl₂MeOH (50:1) to CH₂Cl₂-MeOH (3:1) to give 1.72 g (43%) of (R)-2-(4,5,6,7- tetrahydro-benzothiazol-2-ylamino)-3 -tritylsulfanyl-propionic acid. Step 2: (R)-3-Mercapto-2-r4.5,6J-tetrahydro-benzothiazol-2-ylamino)-propionic acid

The title compound was prepared in 42% yield from (R)-2-(4,5,6,7- tetrahydro-benzothiazol-2-ylamino)-3 -tritylsulfanyl-propionic acid following the procedure used for preparing (R)-3-mercapto-2-(5-methyl-4-phenyl-thiazol-2- ylamino)-propionic acid. 400 MHz ¹H-NMR (CDCl₃, ppm): 10.23 (IH, s) 9.7-7.4 (2H, br s) 4.10-4.00 (IH, m) 3.15-3.00 (2H, m) 2.61-2.50 (4H, m) 1.86-1.76 (4H, m). ESI-MS (m/z): 259 [M+H]⁺.

Synthetic Example 5

(R)-2-(5-Nitro-pyridin-2-ylamino)-3-mercapto-propionic acid

Step 1: (R)-2-(5-Nitro-pyridin-2-ylamino)-3-tritylsulfanyl-propionic acid

A pressure tube was charged with S-trityl-L-cysteine (2.00 g, 5.50 mmol), 2-chloro-5-nitro-pyridine (0.88 g, 5.55 mmol), K₂CO₃ (1.90 g, 13.75 mmol) and EtOH (50 mL). The mixture was heated at 90 ⁰C for 16 h, after which time the mixture was filtered, and the ethanol was removed in vacuum. The residue was purified by flash column chromatography using gradient elution from CH₂Cl₂ZMeOH (20:1) to CH₂Cl₂ZMeOH (5:1) to give 0.80 g (30%) of (R)-2-(5-nitro-pyridin-2- ylamino)-3 -tritylsulfanyl-propionic acid. Step 2: (R)-2-(5-Nitro-pyridin-2-ylamino)-3-mercapto-propionic acid

The title compound was prepared in 49% yield from (R)-2-(5-nitro- pyridin-2-ylamino)-3 -tritylsulfanyl-propionic acid following the procedure used for preparing (R)-2-(5-methyl-4-phenyl-thiazol-2-ylamino)-3-tritylsulfanyl-propionic acid. 200 MHz ¹H-NMR (DMSO-d₆, ppm): 8.88 (lH,d, J=2.7 Hz) 8.34 (IH, d, J=7.6 Hz) 8.15 (IH, dd, J=9.2 Hz, 2.7 Hz) 6.76 (IH, d, J=9.2 Hz) 4.84-4.70 (IH, m) 3.09- 2.78 (2H, m) 2.51 (IH, t, J=8.8 Hz, overlapped with DMSO). ESI-MS (m/z): 244 [M+H]⁺.

Synthetic Example 6 (R)-2-(Benzoxazol-2-ylamino)-3-mercapto-propionic acid hydrochloride

Step 1: (R>2-(Benzoxazol-2-ylaminoV3 -tritylsulfanyl-propionic acid

The mixture of S-trityl-L-cysteine (5.0 g, 13.76 mmol), 2-chloro-l,3- benzoxazole (2.32 g, 15.11 mmol) and diisopropylethylamine (DIPEA) (9.1 mL, 55.01 mmol) in DMF (20 mL) was heated at 80 ⁰C for 1 h under Ar atmosphere. The mixture was poured into water (100 mL) and acidified by addition of 5% aqueous KHSO₄ to reach pH~3. The product was extracted into EtOAc (2 x 150 mL) and the combined organic phases were washed with water, brine and dried over Na₂SO₄. The solvent was evaporated to give crude (R)-2-(benzoxazol-2-ylamino)-3-tritylsulfanyl- propionic acid (6.60 g, 100%), which was used in the next step without purification. Step 2: (R>2-(Benzoxazol-2-ylamino>3-mercapto-propionic acid hydrochloride To a solution of (R)-2-(benzoxazol-2-ylamino)-3-tritylsulfanyl-propionic acid (6.60 g, 13.73 mmol) in CH₂Cl₂ (15 mL), Et₃SiH (6.03 g, 8.4 niL, 51.86 mmol) was added followed by TFA (15 mL). The reaction mixture was stirred under Ar atmosphere at O⁰C for 1 h. Volatile materials were then removed in vacuum. The resultant residue was purified by flash column chromatography using gradient elution from CH₂Cl₂/Me0H (98:2) to CH₂Cl₂/Me0H (85:15) to give 1.95 g (60%) of (R)-2- (benzoxazol-2-y lamino)-3 -mercapto-propionic acid.

The obtained free amino acid (1.45 g; 6.09 mmol) was suspended in THF (110 mL) and 4 M HCl in dioxane (1.5 mL, 6.09 mmol) was added. The volatiles were removed in vacuum to give the title compound. 200 MHz ¹H-NMR (DMSO-d₆, ppm): 12.0-11.5 (IH, br s) 10.8-9.8 (2H, m) 7.29-7.16 (2H, m) 7.12-7.03 (IH, m) 6.93-6.81 (IH, m) 5.11-4.79 (IH, m) 4.01-3.81 (IH, m, overlapped with water) 3.75- 3.58 (IH, m). ESI-MS (m/z): 239 [M+H]⁺.

Synthetic Example 7

3-Mercapto-2-(5-trifluoromethyI-pyridin-2-yIamino)-propionic acid hydrochloride

Step 1 : l-(5-Trifluoromethyl-pyridin-2-yl)-aziridine-2-carboxylic acid methyl ester

An oven dried pressure tube was charged with aziridine-2-carboxylic acid methyl ester (1.50 g, 6.64 mmol), 2-bromo-5-trifluoromethyl-pyridine (2.01 g, 19.88 mmol), Pd(OAc)₂ (45 mg, 0.20 mmol), Xantphos (156 mg, 0.27 mmol) and Cs₂CO₃ (4.30 g, 13.20 mmol). The vial was purged with argon, toluene (7 mL) was added, and the mixture was heated at 9O⁰C for 48 h. The reaction mixture was filtered through celite. The celite pad was washed with EtOAc (2 x 30 mL). Volatiles were removed in vacuum, and the residue was purified by flash column chromatography using PE/EtOAc (4/1) as eluent to give 0.95 g (58%) of l-(5-trifluoromethyl-pyridin- 2-yl)-aziridine-2-carboxylic acid methyl ester. Step 2: 2-(5-Trifluoromethyl-pyridin-2-ylaminoV3-tritylsulfanyl-propionic acid methyl ester

An oven dried flask was charged with triphenylmercaptomethane (2.02 g, 7.31 mmol), BF₃OEt₂ (463 μL, 3.66 mmol) and CH₂Cl₂ (10 mL). The mixture was cooled to 0 ⁰C (NaCl-ice bath), and l-(5-trifluoromethyl-pyridin-2-yl)-aziridine-2- carboxylic acid methyl ester (0.60 g, 2.44 mmol) in CH₂Cl₂ (15 mL) was added dropwise. The reaction mixture was stirred at room temperature for 20 h. An additional portion of BF₃OEt₂ (200 μl, 1.58 mmol) was added and the reaction mixture was stirred for 2 h. The mixture was poured into saturated NaHCO₃ solution (50 mL), extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with water, brine and dried over Na₂SO₄. The solvent was removed in vacuum and residue was purified using preparative LC to give 0.92 g (72%) of the title compound. Step 3 : 2-(5 -Trifluor omethyl-pyr idin-2- ylamino)-3 -trity lsulfanyl-propionic acid The mixture of 2-(5-trifluoromethyl-pyridin-2-ylamino)-3-tritylsulfanyl- propionic acid methyl ester (1.2 g, 2.30 mmol) and 2N NaOH solution (6 mL) in acetonitrile (12 mL) was stirred at room temperature for 4 h. The mixture was acidified by addition of 5% KHSO₄ to reach pH~3. The product was extracted into EtOAc (2 x 100 mL) and the combined organic phases were washed with water, brine and dried over Na₂SO₄. The solvent was evaporated and the crude product filtered through silica gel using PE/EtOAc to give the title compound in 75% yield (0.88 g). Step 4: 3-Mercapto-2-(5-trifluoromethyl-pyridin-2-ylamino)-propionic acid hydrochloride The title compound was prepared in 45% yield from 2-(5-trifluoromethyl- pyridin-2-ylamino)-3-tritylsulfanyl-propionic acid following the procedure used for preparing (R)-2-(5-methyl-4-phenyl-thiazol-2-ylamino)-3-tritylsulfanyl-propionic acid. 40O MHz ¹H-NMR (DMSO-(I₆₅ PPm): 8.29-8.25 (IH, m) 7.81-7.72 (IH, m) 7.69 (IH, dd, J=9.0, 2.2 Hz) 6.81 (IH, d, J=9.0 Hz) 4.72-4.65 (IH, m) 3.01-2.92 (IH, m) 2.90-2.80 (IH, m) 2.51 (IH, t, J=8.4 Hz). ESI-MS (m/z): 267 [M+H]⁺.

Synthetic Example 8

(R)-2-[4,6-Bis-(4-methyl-piperazin-l-yl)-[l,3,5]triazin-2-ylamino]-3-mercapto- propionic acid

Step 1 : (R)-2-r4,6-Dichloro-[l,3,5]triazin-2-ylamino)-3-tritylsulfanyl-propionic acid t-butyl ester

To a solution of cyanuric chloride (2.43 g, 13.16 mmol) in THF (160 mL) at 0 ⁰C was added (R)-2-amino-3-tritylsulfanyl-propionic acid tert-butyl ester hydrochloride (6.00 g, 13.16 mmol) in THF (60 mL), followed by DIPEA (5.7 mL, 33.06 mmol). The reaction mixture was stirred at 0 ⁰C for Ih, then at room temperature for 1 h. Volatile materials were removed in vacuum, the residue was taken up in EtOAc (300 mL), washed with water, brine and dried over Na₂SO₄. The solvent was evaporated to give crude (R)-2-(4,6-dichloro-[l,3,5]triazin-2-ylamino)-3- tritylsulfanyl-propionic acid tert-butyl ester (7.04 g, 94%), which was used in the next step without purification.

Step 2: rRV2-r4.6-Bis-r4-methyl-piperazin-l-ylVπ 3.51triazin-2-ylaminol-3- tritylsulfanyl-propionic acid t-butyl ester A mixture of (R)-2-(4,6-dichloro-[l,3,5]triazin-2-ylamino)-3- tritylsulfanyl-propionic acid tert-butyl ester (2.70 g, 4.76 mmol), 1-methyl-piperazine (2.1 mL, 19.05 mmol) and DIPEA (3.3 mL, 16.71 mmol) in THF (120 mL) was heated at reflux for 48 h. The solvent was removed in vacuum and the residue was partitioned between brine (100 mL) and EtOAc (200 mL). The organic phase was dried over anhydrous Na₂SO₄ and concentrated. The crude product was purified by flash column chromatography using CH₂Cl₂/Et0H (50:1) to give the title compound (1.88 g, 57%).

Step 3: rRV2-F4.6-Bis-(^'4-methyl-piperazin-l-ylV[1.3.51triazin-2-ylaminol-3- mercapto-propionic acid

To a solution of (R)-2-[4,6-bis-(4-methyl-piperazin-l-yl)-[l,3,5]triazin-2- ylamino]-3-tritylsulfanyl-propionic acid tert-butyl ester (1.66 g, 2.39 mmol) in CH₂Cl₂ (18 mL), Et₃SiH (3.00 mL, 18.78 mmol) was added followed by TFA (19 mL). The reaction mixture was stirred under argon atmosphere at room temperature for 24 h. Volatile materials were removed in vacuum and the residue was purified by reverse phase chromatography using gradient elution from MeOH/0.1% CF₃COOH (5: 1) to MeOH) to give 0.37 g (39%) of (R)-2-[4,6-bis-(4-methyl-piperazin-l-yl)- [l,3,5]triazin-2-ylamino]-3-mercapto-propionic acid. 200 MHz ¹H-NMR (DMSO-d₆, ppm): 10.5-10.1 (IH, br s) 7.28 (IH, d, J = 7.5 Hz) 4.80-4.52 (4H, m) 4.50-4.35 (IH, m) 4.1-3.6 (2H, m, overlapped with water) 3.54-3.31 (4H, m) 3.27-2.80 (8H, m,) 2.79 (6H, s) 2.56-2.44 (IH, m, overlapped with DMSO). ESI-MS (m/z): 397 [M+H]⁺.

Synthetic Example 9

(R)-2-(4,6-Bis-dimethyIamino-[l,3,5]triazin-2-yIamino)-3-mercapto-propionic acid

Step 1 : (R)-2-(4,6-Bis-dimethylamino-|^'l,3,51triazin-2-ylaminoV3-tritylsulfanyl- propionic acid tert-butyl ester A pressure tube was charged with (R)-2-(4,6-dichloro-[l,3,5]triazin-2- ylamino)-3-tritylsulfanyl-propionic acid tert-butyl ester (2.70 g, 4.76 mmol), N₅N- dimethylamine hydrochloride (1.55 g, 19.01 mmol) and DIPEA (6.60 mL, 37.91 mmol) and THF (120 mL). The reaction mixture was heated at 70 ⁰C for 48 h. Volatile materials were removed in vacuum and the residue was partitioned between brine (100 mL) and EtOAc (200 mL). The organic phase was dried over anhydrous Na₂SO₄ and concentrated. The crude product was purified by flash column chromatography using as eluent CH₂Cl₂-EtOH (50:1) to give the title compound (1.75 g, 63%). Step 2: (R)-2-(4,6-Bis-dimethylamino-[l ,3,51triazin-2-ylamino>3-mercapto- propionic acid

The title compound was prepared in 59% yield from ((R)-2-(4,6-bis- dimethylamino-[l ,3,5]triazin-2-ylamino)-3-tritylsulfanyl-propionic acid tert-butyl ester using the procedure outlined for (R)-2-[4,6-bis-(4-methyl-piperazin-l-yl)- [1 ,3,5]triazin-2-ylamino]-3-mercapto-propionic acid. 200 MHz ¹H-NMR (DMSO-d₆, ppm): 12.8-12.4 (IH, br s) 6.74 (IH, d, J = 7.4 Hz) 4.47-4.33 (IH, m) 2.98 (12H, s) 2.98-2.78 (2H, m) 2.55-2.45 (IH, m, overlapped with DMSO). ESI-MS (m/z): 287 [M+H]⁺.

Synthetic Example 10

(R)-2-[l-(4,6-Bis-allylamino-[l,3,5]triazin-2-yl)-piperidin-4-ylamino]-3- mercapto-propionic acid, sodium salt

Step 1 : 8-(4,6-Dichloro-[l ,3,51triazin-2-yl)-l ,4-dioxa-8-aza-spiro[4.51decane

To a solution of cyanuric chloride (12.88 g, 69.84 mmol) in THF (65 mL), 1 ,4-dioxa-8-aza-spiro[4.5]decane (10.00 g, 8.95 mL, 69.84 mmol) and DIPEA (11.5 mL, 69.84 mmol) in THF (50 mL) were added dropwise at 0 ⁰C. The reaction mixture was stirred at 0 ⁰C for 45 min. Volatile materials were removed in vacuum, water (100 mL) was added and the resulting suspension was extracted with EtOAc (2 x 200 mL). The combined organics were washed with water, brine and dried over Na₂SO₄. The solvent was evaporated and the residue crystallized from acetonitrile to yield 15.47 g (76%) of the title compound.

Step 2: N.N'-DiaUyl-6-α .4-dioxa-8-aza-spiror4.51dec-8-ylVπ ,3,51triazine-2,4- diamine A pressure tube was charged with 8-(4,6-dichloro-[l,3,5]triazin-2-yl)-l,4- dioxa-8-aza-spiro[4.5]decane (15.47 g, 53.10 mmol), allylamine (23.4 mL, 312.20 mmol), DIPEA (26.0 mL, 159.30 mmol) and THF (100 mL). The reaction mixture was heated at 70 ⁰C for 48 h. Volatiles were removed in vacuum and the residue was partitioned between water (100 mL) and EtOAc (2 x 200 mL). The organic phase was dried over anhydrous Na₂SO₄ and concentrated to yield 17.60 g (100%) of N₅N'- diallyl-6-(l ,4-dioxa-8-aza-spiro[4.5]dec-8-yl)-[l ,3,5]triazine-2,4-diamine, which was used in the next step without purification. Step 3: l-(4,6-Bis-allylamino-ri,3,51triazin-2-yl)-piperidin-4-one To a solution of N,N'-diallyl-6-(l,4-dioxa-8-aza-spiro[4.5]dec-8-yl)-

[l,3,5]triazine-2,4-diamine (9.01 g, 27.11 mmol) in THF (270 mL), 6N HCl (270 mL) was added. The reaction mixture was stirred at room temperature for 16 h. THF was evaporated and residue was neutralized by addition OfNH₄OHtO reach pH~6, then extracted with EtOAc. The combined organic extracts were washed with water, brine, dried over anhydrous Na₂SO₄ and purified by flash column chromatography using eluent PE/EtOAc (3:1) to give the desired product (6.54 g, 84%). Step 4: CR)-2-| ^"1 -f4,6-Bis-allylammo-ri 3,51triazin-2-yl)-piperidin-4-ylammo]-3- tritylsulfanyl-propionic acid tert-butyl ester A mixture of l-(4,6-bis-allylamino-[l,3,5]triazin-2-yl)-piperidin-4-one (600 mg, 2.08 mmol), (R)-2-arnino-3-tritylsulfanyl-propionic acid tert-butyl ester hydrochloride (949 mg, 2.08 mmol) and NaBH(OAc)₃ (555 mg, 2.62 mmol) in THF (25 mL) was stirred at room temperature for 16 h. The volatile materials were evaporated, NaHCO₃ (saturated solution) was added, and the resulting suspension was extracted with EtOAc (2 x 100 mL). The combined organic extracts were washed with water, brine, dried over anhydrous Na₂SO₄, and purified by preparative LC using PE/EtOAc as eluent to give the title compound (852 mg, 59%). Step 5: CRV2-ri-(^'4.6-Bis-allylamino-rL3,51triazin-2-ylVpiperidin-4-ylamino1-3- mercapto-propionic acid, sodium salt To a solution of (R)-2-[l-(4,6-bis-allylamino-[l,3,5]triazin-2-yl)-piperidin- 4-ylamino]-3-tritylsulfanyl-propionic acid tert-butyl ester (842 mg, 1.22 mmol) in CH₂Cl₂ (9 mL), Et₃SiH (2.00 mL, 12.21 mmol) was added, followed by TFA (9 mL). The reaction mixture was stirred under argon atmosphere at room temperature for 24 h. The volatiles were removed in vacuum and the residue was purified by flash column chromatography using gradient elution from CH₂Cl₂/Me0H (99:1) to CH₂Cl₂/Et0H (6:1) to give the title compound (358 mg, 39%) as the free amino acid.

The obtained product was dissolved in acetonitrile and NaOH (36 mg, 0.90 mmol, leq) in water (0.5 mL) was added. The volatiles were removed in vacuum and the resultant product was dried under high vacuum to give the sodium carboxylate salt of the title compound. 400 MHz ¹H-NMR (DMSOd₆, ppm): 6.90-6.77 (IH, br s) 6.77-6.10 (IH, br s) 5.90-5.79 (2H, m) 5.14-4.98 (4H, m) 4.56-4.46 (2H, m) 3.88-3.79 (4H, m) 3.73-3.66 (IH, m) 3.5-3.1 (IH, m, overlapped with water) 3.03-2.90 (2H, m) 2.87-2.75 (2H, m) 1.92-1.78 (2H, m) 1.36-1.21 (2H, m). ESI-MS (m/z): 394 [M+H]⁺.

Synthetic Example 11

(S)-2-(Pyrimidin-2-ylamino)-4-mercapto-butyric acid, sodium salt Step 1. (S)-2-Amino-4-mercapto-butyric acid

A 1.5-liter round bottom flask, equipped with a mechanical stirrer, was charged with L-methionine (20 g, 134 mmol). The reaction flask was flushed with argon and cooled to -78⁰C with a dry ice-acetone bath. Anhydrous ammonia gas was condensed in the flask until the starting material was completely dissolved. The dry ice bath was removed, and sodium metal (11 g, 483 mmol) was added in pieces to the refluxing ammonia solution. The progress of the reaction was monitored by quenching a small aliquot of the reaction and analyzing via TLC and HPLC. After the reaction was complete, any visible remaining pieces of sodium metal were carefully removed and ammonium acetate (9.46 g, 130 mmol) was added in portions to quench the reaction mixture. The ammonia was evaporated overnight under a stream of argon. The resulting solid of (S)-2-amino-4-mercapto-butyric acid was ground with a mortar and pestle, and used in the next step without purification. Step 2. (S>2-Amino-4-tritylsulfanyl-butyric acid (also known as S-trityl-L- cysteine)

A 1.5-liter round bottom flask, equipped with a mechanical stirrer, was charged with crude (S)-2-amino-4-mercapto-butyric acid and triphenylmethanol (36.2 g, 139 mmol). The reaction flask was flushed with argon and cooled with ice-water bath. Chloroform (300 mL) was added, followed by the addition of trifluoroacetic acid (130 mL) in portions. The reaction mixture was stirred at room temperature for 3 h, and the solvents were removed in vacuum. The final traces of TFA were removed under high vacuum. Water (1 liter) was added, the resulting suspension was cooled to 10 ⁰C and sodium hydroxide (42 g) was added in portions until pH of 13 was achieved. The resulting precipitate was filtered off and washed with water. The filtrate was suspended in water (800 mL) and the pH was adjusted to 4 by the addition of citric acid. tert-Butyl methyl ether (300 mL) was added, and the solid was filtered, washed with water and tert-butyl methyl ether. After drying at high vacuum at 4O⁰C, 34.2 g (70%) of (S)-2-amino-4-tritylsulfanyl-butyric acid was obtained. 200 MHz ¹H NMR (DMSOd₆, ppm): 7.38-7.09 (15H, m) 3.03 (IH, t, J=6.2 Hz) 2.25 (2H, t, J=7.9 Hz) 1.90-1.50 (2H, m). ESI-MS (m/z): 378 [M+H]+. Step 3: (S)-2-(Pyrimidin-2-ylamino>4-tritylsulfanyl-butγric acid A mixture of (S)-2-amino-4-tritylsulfanyl-butyric acid (3.00 g, 7.95 mmol), 2-chloropyrimidine (0.91 g, 7.95 mmol) and NaOH (0.70 g, 17.50 mmol) was heated at 50 ⁰C for 16 h. Water was added (50 mL), and the resulting suspension was extracted with EtOAc (4 x 50 mL). The combined organic extracts were washed with water, brine, dried over anhydrous Na₂SO₄. The product was purified by preparative LC using PE/EtOAc as eluent to give (S)-2-(pyrimidin-2-ylamino)-4-tritylsulfanyl- butyric acid (1.09 g, 30%). Step 4. fS)-2-(Pyrimidin-2-ylamino)-4-sulfanyl-butyric acid, sodium salt

To a solution of (S)-2-(pyrimidin-2-ylamino)-4-tritylsulfanyl-butyric acid (1.09 g, 2.39 mmol) in CH₂Cl₂ (10 mL), Et₃SiH (1.3 mL, 7.89 mmol) was added followed by TFA (0.9 mL, 11.95 mmol). The reaction mixture was stirred under argon atmosphere at 0 ⁰C for 40 min. The volatiles were removed in vacuum and the residue was purified by flash column chromatography using gradient elution from CH₂Cl₂MeOH (100:1) to CH₂Cl₂/Et0H (5:1) to give (S)-2-(pyrimidin-2-ylamino)-4- tritylsulfanyl-butyric acid (432 mg, 85%). The obtained product was dissolved in EtOH, and NaOAc (166 mg, 2.02 mmol, 1 eq) in EtOH was added. The volatiles were removed in vacuum to give the title compound as the sodium carboxylate salt. 200 MHz ¹H-NMR (DMSO-d₆, ppm): 8.31 (2H, d, J=4.8 Hz) 7.59-7.44 (IH, m) 6.65 (IH, dd, J=4.8, 4.8 Hz) 4.54-4.39 (IH, m) 2.68-2.50 (2H, m, overlapped with DMSO) 2.35 (IH, t, J=8.0 Hz) 2.11-1.95 (2H, m). ESI-MS (m/z): 214 [M+H]⁺.

Synthetic Example 12

Sodium (S)-4-mercapto-2-(5-methyl-4-phenyl-thiazol-2-ylamino)-butyrate

Step 1 : (S)-2-f5-Methyl-4-phenyl-thiazol-2-ylammo^')-4-tritylsulfanyl-butyric acid The title compound was prepared in 58% yield from (S)-2-amino-4- tritylsulfanyl-butyric acid and 2-bromo-l-phenyl-propan-l-one using the procedure outlined for (R)-2-(5-methyl-4-phenyl-thiazol-2-ylamino)-3-tritylsulfanyl-propionic acid. Step 2: Sodium (SV4-mercapto-2-(^'5-methyl-4-phenyl-thiazol-2-ylaminoVbutyrate

The title compound was prepared in 32% yield from (S)-2-(5-methyl-4- phenyl-thiazol-2-ylamino)-4-tritylsulfanyl-butyric acid using the procedure outlined for (S)-2-(pyrimidin-2-ylamino)-4-mercapto-butyric acid, sodium salt. 400 MHz ¹H- NMR (D₂O, ppm): 7.41-7.21 (5H, m) 3.89 (IH, dd, J=7.7; 2.7 Hz) 2.74-2.61 (IH, m) 2.58-2.39 (2H, m) 2.15 (3H, s) 2.04-1.82 (2H, m). ESI-MS (m/z): 309 [M+H]⁺.

Synthetic Example 13

Sodium (S)-4-mercapto-2-(4-phenyl-thiazol-2-ylamino)-butyrate

Step 1 : (SV2-(4-Phenyl-thiazol-2-ylamino)-4-tritylsulfanyl-butyric acid The title compound was prepared in 51% yield from (S)-2-amino-4- tritylsulfanyl-butyric acid and 2-bromo-l-phenyl-ethanone using the procedure outlined for (R)-2-(5-methyl-4-phenyl-thiazol-2-ylamino)-3-tritylsulfanyl-propionic acid. Step 2: Sodium fS)-4-mercapto-2-f4-phenyl-thiazol-2-ylaminoVbutyrate The title compound was prepared in 56% yield from (S)-2-(4-phenyl- thiazol-2-ylamino)-4-tritylsulfanyl-butyric acid using the procedure outlined for (S)- 2-(pyrimidin-2-ylamino)-4-mercapto-butyric acid, sodium salt. 400 MHz ¹H-NMR (D₂O, ppm): 8.31 (IH, s) 7.69-7.58 (2H, m) 7.38-7.19 (3H, m) 6.79 (IH, s) 4,06 (IH, m) 2.66-2.42 (2H, m) 2.06-1.88 (2H, m). ESI-MS (m/z): 295 [M+H]⁺.

Synthetic Example 14

(S)-4-Mercapto-2-(thiazol-2-ylamino)-butyric acid

Step 1 : CS)-2-(3-Benzoyl-thioureido>4-tritylsulfanyl-butyric acid

To a solution of (S)-2-amino-4-tritylsulfanyl-butyric acid (8.00 g, 21.20 mmol) in acetone (300 mL), benzoyl isothiocyanate (3.46 g, 2.86 mL, 21.20 mmol) was added, and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed in vacuum to give a crude (S)-2-(3-benzoyl-thioureido)-4- tritylsulfanyl-butyric acid, which was used in the next step without purification. 400 MHz ¹H-NMR (DMSO-d₆, ppm): 13.4-12.9 (IH, br s), 11.48 (IH, s), 11.07 (IH, d, J=7.4 Hz), 7.94-7.88 (2H, m), 7.66-7.60 (IH, m), 7.54-7.48 (2H, m), 7.31-7.19 (12H, m), 7.18-7.12 (3H, m), 4.87-4.80 (IH, m), 2.21-2.01 (2H, m), 2.01-1.78 (2H, m). Step 2: (S)-2-Thioureido-4-tritylsulfanyl-butyric acid

Crude (S)-2-(3-benzoyl-thioureido)-4-tritylsulfanyl-butyric acid was suspended in 1 ,4-dioxane (75 mL), and IN NaOH solution (150 mL) was added. The reaction mixture was stirred at 7O⁰C for 2 h, cooled to room temperature; 5% KHSO₄ solution was added until pH of 3 was achieved. The resultant suspension was extracted with EtOAc (3x75 mL). The combined organic extracts were washed with water, brine, and dried over anhydrous Na₂SO₄. The solvent was removed in vacuum to give a crude (S)-2-thioureido-4-tritylsulfanyl-butyric acid, which was used in the next step without purification. 400 MHz ¹H-NMR (DMSO-d₆, ppm): 13.1-12.3 (IH, br s), 7.65 (IH, d, J=8.1 Hz), 7.34-7.14 (15H, m), 4.72-7.64 (IH, m), 2.20-1.99 (2H, m), 1.75-1.57 (2H, m). Step 3: (SV2-rThiazol-2-ylaminoV4-tritylsulfanyl-butyric acid To a solution of (S)-2-thioureido-4-tritylsulfanyl-butyric acid (21.20 mmol) in ethanol (50 mL), chloroacetaldehyde (15 mL, 50% water content) was added; the reaction mixture was heated at 6O⁰C for 16 h. The volatiles were removed in vacuum; the residue was partitioned between water and EtOAc. The organic phase was washed with water, brine, dried over anhydrous Na₂SO₄ and concentrated. The crude product was purified by flash column chromatography using gradient elution from CH₂Cl₂-EtOH (99:1) to CH₂Cl₂-EtOH (1 :1) to give (S)-2-(thiazol-2-ylamino)-4- tritylsulfanyl-butyric acid (5.04 g), 52 % in 3 steps from (S)-2-amino-4-tritylsulfanyl- butyric acid. 400 MHz ¹H-NMR (DMSO-d₆, ppm): 13.2-11.7 (IH, br s), 7.65 (IH, d, J-8.2 Hz), 7.28-7.13 (15H, m), 6.95 (IH, d, J=3.6 Hz), 6.61 (IH, d, J=3.6 Hz), 4.31- 4.22 (IH, m), 2.26-2.17 (IH, m), 2.13-2.05 (IH, m), 1.73-1.64 (2H, m). ESI-MS (m/z) 461 [M+H]+. Step 4. (S)-4-Mercapto-2-(thiazol-2-ylamino^')-butyric acid

To a solution of (S)-2-(thiazol-2-ylamino)-4-tritylsulfanyl-butyric acid (3.95 g, 8.58 mmol) in CH₂Cl₂ (10 mL), Et₃SiH (3.99 g, 6.37 mL, 34.32 mmol) was added followed by TFA (6.37 mL, 85.80 mmol). The reaction mixture was stirred under Ar atmosphere at O⁰C for 40 minutes. The volatiles were removed in vacuo; the residue was purified by flash column chromatography using gradient elution from CH₂Cl₂/Et0H (98:2) to CH₂Cl₂/Et0H (1 :1) to give (S)-4-mercapto-2-(thiazol-2- ylamino)-butyric acid (1.38 g, 72%). 400 MHz ¹H-NMR (DMSO-d₆, ppm): 12.9- 12.4 (IH, br s), 7.85 (IH, d, J=7.9 Hz), 6.99 (IH, d, J=3.6 Hz), 6.64 (IH, d, J=3.6 Hz), 4.41-4.33 (IH, m), 2.62-2.51 (2H, m), 2.43 (IH, t, J=8.0 Hz), 2.03-1.93 (2H, m). ESI-MS (m/z) 219 [M+H]+.

Synthetic Example 15

(S)-4-Mercapto-2-(thiazol-2-ylamino)-butyric acid, sodium salt

(S)-4-Mercapto-2-(thiazol-2-ylamino)-butyric acid (1.35 g, 6.18 mmol) was suspended in THF (20 mL), and NaOH (245 mg, 6.12 mmol) in water was added. Reaction mixture was stirred for 5 minutes under Ar atmosphere. The volatiles were removed in vacuum, and the residue was dried under high vacuum to give (S)-4- mercapto-2-(thiazol-2-ylamino)-butyric acid, sodium salt. 400 MHz ¹H-NMR (D₂O, ppm): 6.91 (IH, d, J=3.6 Hz), 6.50 (IH, d, J=3.6 Hz), 3.97 (IH, dd, J=8.5, 5.0 Hz), 2.57-2.42 (2H, m), 2.02-1.85 (2H₅ m).

Synthetic Example 16

Sodium (S)-4-mercapto-2-(methyl-thiazol-2-yl-amino)-butyrate

Step 1 : (S)-2-(Thiazol-2-ylamino)-4-tritylsulfanyl-butyric acid

An oven dried vial was charged with (S)-2-amino-4-tritylsulfanyl-butyric acid (200 mg, 0.53 mmol), CuI (10 mg, 0.05 mmol), N,N'-dimethylethylene-diamine (14 μL, 0.12 mmol), K₂CO₃ (220 mg, 1.59 mmol) and 2-bromothiazole (174 mg, 1.06 mmol). The vial was flushed with argon, toluene (3 mL) was added, and the reaction mixture was heated at 9O⁰C for 24 h. The reaction mixture was filtered through Celite, concentrated and purified by flash column chromatography using gradient elution from CH₂Cl₂/Et0H (20:1) to CH₂Cl₂/Et0H (1 :1) to give (S)-4-mercapto-2- (thiazol-2-ylamino)-butyric acid (88 mg, 36%). 400 MHz ¹H-NMR (DMSO-d₆, ppm): 13.2-11.7 (IH, br s), 7.65 (IH, d, J-8.2 Hz), 7.28-7.13 (15H, m), 6.95 (IH, d, J=3.6 Hz), 6.61 (IH, d, J=3.6 Hz), 4.31-4.22 (IH, m), 2.26-2.17 (IH, m), 2.13-2.05 (IH, m), 1.73-1.64 (2H, m). ESI-MS (m/z) 461 [M+H]+. Step 2: (S)-2-(Methyl-thiazol-2-yl-amino)-4-tritylsulfanyl-butyric acid, and rSV2-r3-Methyl-3H-thiazol-r2E)-ylideneaminol-4-tritylsulfanyl-butyric acid

A solution of (S)-2-(thiazol-2-ylamino)-4-tritylsulfanyl -butyric acid (3.34 g, 7.25 mmol) in THF (80 mL) was added to a stirred suspension of NaH (60% suspension in mineral oil) (580 mg, 14.50 mmol) in THF (40 mL) at 0 ⁰C. The mixture was stirred at 0 ⁰C for 30 min. After this time, MeI (4.20 g, 1.8 mL, 29.00 mmol) was added in portions and the mixture was stirred at room temperature for 24 h. The mixture was poured into water (100 mL), acidified by addition of 5% KHSO₄ to reach pH~3 and extracted with EtOAc (2 x 150 mL). The combined organic extracts were washed with water, brine and dried over Na₂SO₄. The product was purified by flash column chromatography using gradient elution from CH₂Cl₂-EtOH (99:1) to CH₂Cl₂-EtOH (1:1) to give (S)-2-(methyl-thiazol-2-yl-amino)-4- tritylsulfanyl-butyric acid (1.93 g, 56%) and (S)-2-[3-methyl-3H-thiazol-(2E)- ylideneamino]-4-tritylsulfanyl-butyric acid (690 mg, 20%). Step 3: Sodium (S)-4-mercapto-2-fmethyl-thiazol-2-yl-aminoybutyrate

The title compound was prepared in 65% yield from (S)-2-(methyl-thiazol- 2-yl-amino)-4-tritylsulfanyl-butyric acid using the procedure outlined for Example 11. 400 MHz ¹H-NMR (D₂O, ppm): 7.01 (IH, d, J=3.9 Hz), 6.59 (IH, d, J=3.9 Hz), 4.42 (IH, dd, 3=9.0, 6.2 Hz), 2.89 (3H, s), 2.58-2.56 (IH, m), 2.54-2.46 (IH, m), 2.36-2.27 (IH, m), 2.14-2.07 (2H, m). ESI-MS (m/z): 233 [M+H]⁺.

Synthetic Example 17

(R)-2-(6-Chloro-pyrimidin-4-ylamino)-3-mercapto-3-methyl-butyric acid hydrochloride Step 1. rR)-2-Amino-3-methyl-3-r2.4.6-trimethoxy-thiobenzylVbutyric acid trifluoroacetate

A suspension of (R)-2-amino-3-mercapto-3-methyl-butyric acid (10 g, 67.02 mmol) in CH₂Cl₂ (300 mL) was cooled to 0 ⁰C. Trifluoroacetic acid (110 mL) was added in portions followed by (2,4,6-trimethoxyphenyl)methanol (13.28 g, 67.02 mmol) in CH₂Cl₂ (280 mL). The reaction mixture was stirred at 0 ⁰C for 1 h and then at room temperature for 16 h. Volatile materials were removed in vacuum. Diethyl ether (300 mL) was added to the residue and the solid was filtered to give the title compound (19.15 g, 75%). Step 2. (RV2-(6-Chloro-pyrimidin-4-ylamino)-3-methyl-3-(2,4,6-trimethoxy- benzylsulfanylVbutyric acid

A mixture of (R)-2-amino-3-methyl-3-(2,4,6-trimethoxy- thiobenzyl)butyric acid trifluoroacetate (2.Og, 4.51 mmol), 4,6-dichloropyrimidine (949 mg, 6.37 mmol) and TEA (2.1 mL, 15.2 mmol) in DMF (20 mL) was stirred at room temperature for 24h, then poured into water (100 mL) and extracted with EtOAc (2 x 200 mL). The combined organic extracts were washed with water, brine and dried over Na₂SO₄. The product was purified by flash column chromatography using gradient elution from CH₂Cl₂-EtOH (99:1) to CH₂Cl₂-EtOH (97:3) to give the title compound (1.46 g, 73%) Step 3: (R)-2-(6-Chloro-pyrimidin-4-ylaminoV3-mercapto-3-methyl-butyric acid hydrochloride

To a solution of (R)-2-(6-chloro-pyrimidin-4-ylamino)-3-methyl-3-(2,4,6- trimethoxy-thiobenzyl)-butyric acid (1.46 g, 3.30 mmol) in CH₂Cl₂ (10 mL), Et₃SiH (1.8 mL, 10.89 mmol) was added followed by TFA (3 mL). The reaction mixture was stirred under an argon atmosphere at 0 ⁰C for 15 min and then at room temperature for 15 min. The volatile materials were removed in vacuum and the residue was purified by flash column chromatography using gradient elution from CH₂Cl₂-MeOH (99:1) to CH₂Cl₂-EtOH (50:1) to give the title compound (836 mg, 97%) as free amino acid.

The obtained product (500 mg, 1.91 mmol) was dissolved in THF, 2 M HCl in Et₂O (0.96 mL, 1.92 mmol, leq) was added. The precipitate was filtered to give the title product. 400 MHz ¹H-NMR (D₂O, ppm): 8.29 (IH, s) 6.82 (IH, s) 4.46 (IH, s) 1.42 (3H, s) 1.34 (3H, s). ESI-MS (m/z): 262, 264 [M+H]⁺.

Synthetic Example 18 (S)-2-(Benzothiazol-2-yl)amino-3-mercapto-3-methyl-butyric acid

Step 1. (SV2-Amino-3-methyl-3-triphenylmethylsulfanyl)-butyric acid

To a stirred solution of (S)-penicillamine (1.492 g, 10 mmol) and triphenylmethanol (3.12 g, 12 mmol) in AcOH (10 mL), was added BF₃-Et₂O (2.2 mL) dropwise at room temperature. The reaction was stirred for 2 hours. The mixture was then poured into aqueous sodium acetate (15 mL) and the white solid that separated was collected by filtration. The solid was washed with Et₂O (3 x 10 mL), and dried in vacuum to afford S-trityl-protected penicillamine as a white powder (2.1g, 53.6%). ¹H-NMR (DMSO-d₆, ppm, 500 MHz): δ = 7.22-7.57 (m, 15H), 1.94 (s, IH), 1.16 (s, 3H), 1.11 (s, 3H). MS (ESI): 414(M+23) ⁺. Step 2: fSV2-(Benzothiazol-2-vDamino-3-methyl-3- ftriphenylmethylsulfanyDbutyric acid

To a stirred solution of S-trityl-penicillamine (8.0 g, 20.4 mmol), K₂CO₃ (5.6 g, 41 mmol), and KOH (2.0 g, 35.6 mmol) in anhydrous DMF (60 mL), was added 2-chlorobenzothiazole (5.1 g, 30.6 mmol). The reaction solution was stirred at 80 ⁰C for 3 days under nitrogen. The solution was removed by distillation in vacuum.

To the resultant residue was added water (20 mL) and the pH was adjusted to pH 7.

This material was then extracted with EtOAc (5 x 100 mL), the combined organic layers were dried over Na₂SO₄, evaporated, and purified by silica gel column chromatography (DCM and methanol) to afford (S)-2-(benzthiazol-2-yl)amino-3- methyl-3-(triphenylmethylsulfanyl)-butyric acid (4.1g, 38.2%). MS (ESI):

243[Ph3C]⁺.

Step 3: (S)-2-(Benzothiazol-2-yl)arnino-3-mercapto-3 -methyl-butyric acid

To a solution of (S)-2-(benzothiazol-2-yl)amino-3-methyl-3- (triphenylmethylsulfanyl)butyric acid (550 mg, 1.0 mmol) in TFA (5 mL), triethylsilane (1 mL) was added dropwise at room temperature. The reaction mixture was stirred for 2 hours at room temperature. The solvent was removed by distillation in vacuum, and the residue purified by preparative HPLC to afford (S)-2-

(benzothiazol-2-yl)amino-3-mercapto-3 -methyl-butyric acid (205mg, 72%). ¹H-NMR (DMSO-d₆, 500 MHz): δ = 8.43 (d, J= 8.5 Hz, IH), 7.69 (d, J= 7.5Hz, IH), 7.39 (d,

J= 8.0 Hz, IH), 7.23 (t, J= 7.5 Hz, IH), 7.05 (t, J= 7.0 Hz, IH), 4.70 (d, J= 9.5 Hz,

IH), 2.99 (s, IH), 1.48 (s, 6H). MS (ESI): 283 [M+l] ⁺.

Synthetic Example 19 Methyl (S)-2-(benzothiazol-2-yl)amino-3-mercapto-3-methyl-butyrate

A solution of (S)-2-(benzothiazol-2-yl)amino-3-methyl-3-

(triphenylmethylsulfanyl)-butyric acid (1.1 g) and SOCl₂ (4 mL) in methanol (20 mL) was stirred at reflux for 4 hours. The volatile materials were removed by evaporation in vacuum and the residue was purified by preparative HPLC to afford methyl (S)-2- (benzothiazol-2-yl)amino-3-mercapto-3-methyl-butyrate (pale yellow oil, 188 mg, 30%). ¹H-NMR (CDCl₃, ppm, 500 MHz): δ = 8.41 (d, J= 8.5 Hz, IH), 7.59 (t, J= 8.0 Hz, IH), 7.35 (t, J= 7.0 Hz, IH), 7.18 (t, J= 7.5 Hz, IH), 4.53 (s, IH), 3.80 (s, 3H), 2.22 (s, IH), 1.59 (s, 3H), 1.52 (s, 3H). MS (ESI): 297[M+1]⁺. HPLC purity: 98% (254 nm). Synthetic Examples 20 and 21

(S)-2-(Benzothiazol-2-ylamino)-4-mercapto-butyric acid (Synthetic Example 20) (S)-3-(Benzo[d]thiazol-2-ylamino)dihydrothiophen-2(3H)-one (Synthetic Example 21) Step 1 : S-trityl-homocysteine.

Methionine (20.0 g, 134 mmol) was placed in a 1 -liter three-necked round bottom flask equipped with a cold finger condenser cooled to -78°C with dry ice/acetone. The reaction was flushed with argon and cooled to -78°C. Anhydrous ammonia was condensed in the flask until the methionine was completely dissolved. The dry ice bath was removed and sodium metal (1 1.1 g, 484 mmol) was added portion wise to the refluxing ammonia solution until the reaction mixture maintained its blue color. Ammonium acetate (NH₄OAc) (9.4 g, 122 mmol) was added in portions to quench the reaction and then the ammonia evaporated overnight under a stream of nitrogen gas. The resulting thick solid was broken up and combined with triphenylmethanol (36.6 g, 140 mmol). The mixture was placed under nitrogen atmosphere. With cooling by a water bath, chloroform (100 mL) was added, followed by the addition of TFA (200.0 g, 130.3 mL). The mixture was stirred for 2.5 h, and the volatile materials were removed in vacuum. Water (400 mL) was added and the solution was cooled in a water bath at 10^QC. Sodium hydroxide (42.3 g) was added in portions until a pH of 13 was achieved. The resulting white precipitate was suspended; filtered off and washed with distilled water. The solid was suspended in water (400 mL) and the pH was adjusted to 4 by the addition of solid citric acid, ether was added and the solid was filtered and washed with water followed by ether. The resultant solid was dried under high vacuum to afford the desired S-trityl- homocysteine (27.0 g, 55%). ¹H-NMR (DMSO-d₆, ppm, 500 MHz): δ = 7.23-7.35 (m, 15H), 2.96 (s, IH), 2.21 (s, 2H), 1.75 (s, IH), 1.53 (s, IH). Step 2: (S)-2-(Benzothiazol-2-ylamino)-4-tritylsulfanyl-butyric acid

A mixture of the S-trityl-homocysteine (5.0 g, 13.5 mmol), NaOH (1.6 g, 39.3 mmol) and 2-chlorobenzothiazole (6.6 g, 39.2 mmol) in DMSO (200 mL) was heated to 80⁰C. The mixture was stirred under N₂ for over Ih and monitored by LCMS and TLC. The mixture was cooled and adjusted pH to 5-6 by the addition of IN HCl and then extracted with ethyl acetate (3 x 20OmL). The combined organic layer was washed with brine and evaporated in vacuum to afford a brown oil from which the desired product (S)-2-(benzothiazol-2-ylamino)-4-tritylsulfanyl-butyric acid was isolated via silica gel chromatography. (3.5 g, 45%). ¹H-NMR (CDCl₃, ppm, 500 MHz): δ = 8.24 (d, J= 10.0 Hz, IH), 7.70 (d, J= 10.0 Hz, IH), 7.40 (d, J = 10.0 Hz, IH), 7.17-7.30 (m, 16H), 7.06 (t, J= 9.5 Hz, IH), 4.51 (dd, J= 9.0 Hz, 18.5 Hz, IH), 3.36 (br s, J= 12.0 Hz, IH), 2.15 - 2.34 (m, 2H), 1.81 (dd, J= 9.0 Hz and 17.0Hz, 2H). ¹³C NMR (CDCl₃, 125 MHz): δ (ppm) = 14.0, 20.6, 27.8, 30.0, 55.2, 59.6, 65.9, 118.2, 120.9, 121.1, 125.4, 126.5, 127.8, 128.9, 130.4, 144.2, 152.0, 165.5, 170.2, 173.1. ESI-MS: 511.2 (M⁺+H). HPLC purity: 87%. Step 3 : (S)-2-(Benzothiazol-2-ylamino)-4-mercapto-butyric acid, and (S)-3-(Benzo[dlthiazol-2-ylamino)dihvdrothiophen-2(3H)-one To a solution of (S)-2-(benzothiazol-2-ylamino)-4-tritylsulfanyl-butyric acid (1.5 g, 2.9 mmol) in dichloromethane (50 mL) at O⁰C, TFA (1 mL) was added and the mixture became yellow in color. Et₃SiH (1.5 mL) was added in a dropwise manner until the yellow colored disappeared. The reaction was then concentrated via rotary evaporation and then via vacuum pump to afford (S)-2-(benzothiazol-2- ylamino)-4-mercapto-butyric acid as yellow crystals. This material slowly converted to the corresponding mercaptoactone (S)-3-(benzo[d]thiazol-2- ylamino)dihydrothiophen-2(3H)-one under these acidic conditions (140 mg, 21%). ¹H-NMR (CDCl₃, ppm, 500 MHz): δ = 7.61 (t, J= 8.5 Hz, 2H), 7.48 (t, J= 7.0 Hz, IH), 7.34 (t, J= 8.0 Hz, IH), 4.07^1.10 (m, IH), 3.46-3.51 (m, 2H), 2.81-2.85 (m, IH), 2.55-2.63 (m, IH). ESI-MS: 251.1 (M⁺+H). HPLC purity: 100%.

Synthetic Example 22 (S)-2-(Benzothiazol-2-ylamino)-(4-acetylthio)-butyric acid

The mixture of (S)-2-(benzothiazol-2-ylamino)-4-mercapto-butyric acid (70 mg, 0.3 mmol), Ac₂O (3 mL) and KHCO₃ was stirred for Ih at O⁰C. The mixture was extracted with ethyl acetate (3 x 50 mL), washed with NH₄Cl (50 mL) and the aqueous layer extracted with additional ethyl acetate (3 x 50 mL). The combined organic layers were dried over Na₂SO₄ and evaporated in vacuum. The crude product was purified by preparative HPLC to afford (S)-2-(benzothiazol-2-ylamino)-(4- acetylthio)-butyric acid as white crystals (150 mg, 19%). ¹H-NMR (CDCl₃, ppm, 500 MHz): δ = 7.60 (d, J= 8.0 Hz, IH), 7.52 (t, J= 5.5 Hz, IH), 7.37 (t, J= 8.5 Hz, IH), 7.18 (t, J= 7.0 Hz, IH), 4.18-4.22 (m, IH), 3.10-3.18 (m, 2H), 2.30-2.43 (m, 5H). ESI-MS: 311.1 (M⁺+H). HPLC purity: 96%.

Synthetic Example 23 Methyl (S)-2-(benzothiazol-2-ylamino)-(4-acetylthio)-butyrate

To a solution of (S)-2-(benzothiazol-2-ylamino)-(4-acetylthio)-butyric acid (200 mg, 0.6 mmol) in Et₂O (2 niL) and MeOH (2 mL), 0°C, TMS-CHN₂ (10 mL) was added in a dropwise manner. The mixture was stirred cold for 10 min. The reaction was quenched with HOAc (1 mL) and the product extracted into ethyl acetate (2 x 100 mL), washed with brine, dried over Na₂SO₄ and evaporated in vacuum. The desired product was isolated by preparative HPLC (150 mg, 72%). ¹H-NMR (DMSO-d₆, 500 MHz): δ = 8.53 (d, J= 8.0 Hz, IH), 7.70 (d, J= 7.5 Hz, IH), 7.4 (d, J= 8.0 Hz, IH), 7.24 (t, J= 7.0 Hz, IH), 7.06 (t, J= 7.5 Hz, IH), 4.60 (q, J= 5.5 Hz and 16.0Hz, IH), 3.67 (s, 3H)₅ 2.96 (t, J= 8.0 Hz, 2H), 1.97-2.10 (m, 2H). ESI-MS: 325.1 (M⁺+H). HPLC purity: 100%.

Synthetic Example 24

Methyl (S)-2-(benzothiazol-2-yl)amino-4-acetylthio-butyrate

Step 1 : (S)-2-fBenzo[dlthiazol-2-ylamino)-4-hydroxybutanoic acid, and (RV3-rBenzordlthiazol-2-ylamino)dihvdrofuran-2r3HVone

To a mixture of L-homoserine (6.0 g, 50.5 mmol) and K₂CO₃ (27.9 g, 0.2 mol) in DMSO (30 mL), was added 2-chlorobenzothiazole (17.1 g, 101 mmol). After stirring for 19 h at 90⁰C, the reaction mixture was poured into saturated aqueous NaHCO₃ (150 mL) and extracted with ethyl acetate (3 x 150 mL). The aqueous layer was acidified to pH 5 using 6N HCl and then NaCl solution was added and the product extracted into ethyl acetate/THF (1 :1) (3 x 200 mL). The combined organic extracts were dried over Na₂SO₄ and evaporated to give the two title compounds as brown oil (14.4 g, crude), which were used in the next step directly.

(S)-2-(benzo[d]thiazol-2-ylamino)-4-hydroxybutanoic acid: ¹H-NMR (CDCl₃, ppm, 500 MHz): δ = 7.58 (d, J= 7.5 Hz, IH), 7.49 (d, J= 8.0 Hz₅ IH), 7.36 (t, J= 7.0 Hz, IH), 7.19 (t, J= 7.0 Hz, IH), 4.35 (br s, IH), 3.93-3.98 (m, IH), 3.84- 3.89 (m, IH), 2.28 (br s, 2H). ESI-MS: 253.0(M⁺+H). HPLC purity: 78.4 % (214 nm). (R)-3-(berizo[d]thiazol-2-ylamino)dihydrofuran-2(3H)-one: ¹H-NMR (CDCl₃, ppm, 500 MHz): δ = 7.58 (dd, J= 7.5 and 3.5 Hz, 2H), 7.31 (t, J= 8.0 Hz, IH), 7.13 (t, J= 8.0 Hz, IH), 4.71-4.76 (m, IH), 4.54 (t, J= 8.5 Hz, IH), 4.34-4.39 (m, IH), 3.08-3.13 (m, IH), 2.27-2.36 (m, IH). ESI-MS: 235.1(MVH). HPLC purity: 7.4 % (214 nm).

Step 2: (SV2-fBenzothiazol-2-yl)amino-4-bromo-butyric acid

45% HBr in acetic acid (30 mL) was added to a mixture of compounds (S)-2-(benzo[d]thiazol-2-ylamino)-4-hydroxybutanoic acid, and (R)-3- (benzo[d]thiazol-2-ylamino)dihydrofuran-2(3H)-one (7.0 g, crude) and the mixture was stirred for 2 h at 80 °C. The mixture was evaporated to afford (S)-2-

(benzothiazol-2-yl)amino-4-bromo-butyric acid as a brown oil (7.7 g, crude), which was used directly in the next step. ¹H-NMR (DMSOd₆, ppm, 500 MHz): δ = 8.03 (d, J= 7.5 Hz, IH), 7.66 (d, J= 8.0 Hz, IH), 7.59 (t, J= 8.0 Hz, IH), 7.44 (t, J= 7.5 Hz, IH), 4.66 (q, J= 7.5 Hz, IH), 4.25-4.30 (m, IH), 4.18-4.23 (m, IH), 2.52-2.56 (m, IH), 2.32-2.39 (m, IH). ESI-MS: 316.0 (M⁺-HH). HPLC purity: 53.4 % (214 nm). Step 3: Methyl (SV2-fbenzothiazol-2-vDamino-4-bromo-butyrate

To a solution of (S)-2-(benzothiazol-2-yl)amino-4-bromo-butyric acid (7.7 g, crude) in MeOH (100 mL) was added SOCl₂ (30 mL) at 0°C, and the mixture was allowed to warm to room temperature and then stirred for 15h. The mixture was evaporated to afford methyl (S)-2-(benzothiazol-2-yl)arnino-4-bromo-butyrate as a brown oil (7.3 g, crude), which was used in the next step directly. ¹H-NMR (DMSO- d₆, ppm, 500 MHz): δ = 8.06 (d, J= 8.0 Hz, IH), 7.67 (d, J= 8.5 Hz, IH), 7.59 (d, J = 7.5 Hz, IH), 7.45 (t, J= 8.0 Hz, IH), 4.83 (t, J= 5.0 Hz, IH), 4.31-4.36 (m, IH), 4.14-4.20 (m, IH), 3.79 (s, 3H), 2.49-2.55 (m, IH), 2.38-2.44 (m, IH). ESI-MS: 329.0(M⁺). HPLC purity: 55.3 % (214 nm).

Step 4: Methyl (SV2-fbenzothiazol-2-yl)amino-4-acetylthio-butyrate

To a stirred solution of methyl (S)-2-(benzothiazol-2-yl)amino-4-bromo- butyrate (7.7 g, crude) in DMF (300 mL), was added potassium thioacetate (5.0 g, 44.3 mmol) at room temperature. After stirring for 15 min, the mixture was diluted with ethyl acetate (300 mL), and washed with water (2 x 200 mL) and then with brine (1 x 200 mL). The mixture was evaporated to afford a brown oil, which was subjected to silica gel chromatography (ethyl acetate/PE=l :5-l :3). The resultant brown oil Ywas then purified via another silica gel chromatography (Eluent: i- PrOH/PE=l :50-l :20) to yield compound methyl (S)-2-(benzothiazol-2-yl)amino-4- acetylthio-butyrate as brown oil (3.91 g, 51.5%). ¹H-NMR (CDCl₃, ppm, 500 MHz): δ = 7.57 (q, J= 8.0 Hz, 2H), 7.30 (t, J= 7.5 Hz, IH), 7.12 (t, J= 7.5 Hz, IH), 4.79 (q, J= 7.0 Hz, IH), 3.81 (s, 3H), 3.00-3.06 (m, IH), 2.91-2.96 (m, IH), 2.32-2.36 (m, 4H), 2.08-2.15 (m, IH). ESI-MS: 325.0 (M⁺+H). HPLC purity: 100 % (214 nm).

Synthetic Example 25 (S)-2-(Benzothiazol-2-yl)amino-4-acetylthio-butyric acid

To a solution of methyl (S)-2-(benzothiazol-2-yl)amino-4-acetylthio- butyrate in THF (30 mL) was added 6N HCl (60 mL), and the mixture was stirred for 2O h. After this time, volatile materials were evaporated, and the aqueous layer was basified to pH 5 using sat. aq. NaHCO₃. The product was extracted into ethyl acetate (3 x 150 mL). The combined organic extracts were dried over Na₂SO₄ and evaporated to afford oil, which was purified by silica gel chromatography (ethyl acetate/PE=l : 3 -ethyl acetate) to yield a solid. The solid was purified via preparative HPLC to obtain (S)-2-(benzothiazol-2-yl)amino-4-acetylthio-butyric acid (1.9 g, 51%). ¹H-NMR (MeOD, ppm, 500 MHz): S = 7.70 (d, J= 8.0 Hz, IH), 7.50 (d, J= 8.0 Hz, IH), 7.38 (t, J= 7.5 Hz, IH), 7.22 (t, J= 7.5 Hz, IH), 4.63-4.66 (q, IH), 3.07 (t, J= 7.0 Hz, 2H), 2.28-2.35 (m, 4H), 2.11-2.18 (m, IH). ESI-MS: 311.0(M⁺+H). HPLC purity: 100 % (214 nm).

Synthetic Example 26

Methyl (L)-4,4'-disulfanediyl-bis(2-(benzoxazol-2-yl)amino)-buty rate

Step 1 : f2S.2'SVDimethyl 4.4'-disulfanediyl-bisf2-aminobutanoate)

Thionyl chloride (SOCl₂) (11.9 g, 100 mmol) was added slowly to MeOH (250 mL) in an ice-bath. L-Homocystine (13.4 g, 50 mmol) was added to the solution. The resulting mixture was continuously stirred overnight at 8O⁰C. The reaction mixture was concentrated in vacuum, and CHCl₃ (250 mL) was added. This mixture was then concentrated and dried under high vacuum to afford the desired homocystine bis-ester as a solid (14.2 g, 96 %). ESI-MS: 297.1(M+H)⁺. HPLC purity: 100% (254 nm).

Step 2: Dimethyl L-4,4'-disulfanediyl-bis[(2-(benzoxazol-2-vDamino>butyratel

To a solution of (2S,2'S)-dimethyl 4,4'-disulfanediyl-bis(2- aminobutanoate) (4.0 g, 13.6 mmol) in DMF (32 mL) cooled in an ice bath, was added DIPEA (7.0 g, 54.3 mmol). 2-Chloro-benzoxazole (5.0 g, 32.6 mmol) was added to the solution and the mixture was stirred overnight at room temperature. The reaction was diluted with water (20 mL) and extracted with ether acetate (3 x 60 mL) to remove excess benzoxazole. The crude product was purified by column chromatography with PE:ethyl acetate =1 :1 to afford the desired product, dimethyl L- 4,4 ' -disulfanediyl-bis [(2-(benzoxazol-2-yl)amino)-butyrate as a solid (3.8 g, 53.1 % yield). ¹H-NMR (CDCl₃, ppm, 500 MHz): δ = 7.36 (d, J= 7.5 Hz, IH), 7.21 (d, J= 8.0 Hz, IH), 7.14 (t, J= 8.0 Hz, IH), 7.01 (t, J= 8.0 Hz, IH), 4.71-4.74 (m, 2H), 3.74 (s, 6H), 2.76 (t, J = 7.5 Hz, 4H), 2.37-2.44 (m, 2H), 2.17-2.24 (m, 2H). ESI-MS: 530.6(M⁺+H). HPLC purity: 100% (254 nm).

Synthetic Example 27

Methyl (S)-2-(benzoxazol-2-ylamino)-4-mercapto-butyrate

To a solution of methyl L-4,4'-disulfanediyl-bis(2-(benzoxazol-2- yl)amino) butyrate (1.0 g, 2 mmol) in THF (15 mL), was added DTT (600 mg, 2 mmol) and the reaction was stirred overnight at room temperature. The reaction mixture was washed with ethyl acetate, concentrated and the crude product, methyl (S)-2-(benzoxazol-2-ylamino)-4-mercapto-butyrate, was used for next step directly.

Synthetic Examples 28 and 29 (S)-Methyl 4-(acetylthio)-2-(benzo[d]oxazol-2-ylamino)butanoate, Synthetic

Example 28,

(S)-3-(Benzo[d]oxazol-2-ylamino)dihydrothiophen-2(3H)-one, Synthetic Example

29

Crude methyl (S)-2-(benzoxazol-2-ylamino)-4-mercapto-butyrate was dissolved in THF (5 mL), and KHCO₃ (5 mL) and Ac₂O (5 mL) was added. The mixture was stirred 30 min and washed with water, extracted with ethyl acetate (2 x

30 mL) and purified by preparative TLC and preparative HPLC to afford the title compounds.

(S)-methyl 4-(acetylthio)-2-(benzo[d]oxazol-2-ylamino)butanoate: 150 mg, 15%; ¹H-NMR(CDCl₃, ppm, 500 MHz): δ = 9.01 (br s, IH), 7.40 (d, J= 7.5 Hz,

IH), 7.31 (d, J= 8.0 Hz, IH), 7.25 (t, J= 8.0 Hz, IH), 7.14 (t, J= 7.5 Hz, IH), 4.65

(dd, J= 4.5 and 8.0 Hz, IH), 3.80 (s, 3H), 2.97-3.07 (m, 2H), 2.31 (s, 3H), 2.30-2.35

(m, IH), 2.18-2.24 (m, IH). ESI-MS: 309.1(M⁺-HH). HPLC purity: 100% (254 nm). (S)-3-(benzo[d]oxazol-2-ylamino)dihydrothiophen-2(3H)-one: 150 mg, 15%; ¹H-NMR(CDCl₃, ppm, 500 MHz): δ = 7.38 (d, J= 7.5 Hz, IH), 7.26 (d, J= 8.0 Hz, IH), 7.18 (t, J= 7.5 Hz, IH), 7.07 (t, J= 7.0 Hz, IH), 4.65 (dd, J= 6.5 and 12.5 Hz, IH), 3.39-3.45 (m, IH), 3.28-3.32 (m, IH), 3.13-3.18 (m, 1H),2.O8-2.17 (m, IH). ¹³C NMR (CDCl₃, ppm, 125 MHz): <S = 27.3, 32.0, 62.3, 109.1, 116.7, 121.5, 124.1, 142.3, 148.6, 161.2, 204.6. ESI-MS: 235.1 (M⁺+H). Purity: 100% (214 nm).

Synthetic Example 29 (S)-3-(Benzo[d]oxazoI-2-ylammo)dihydrothiophen-2(3H)-one To a solution of methyl L-4,4'-disulfanediyl-bis(2-(benzoxazol-2- yl)amino)-butyrate (2.1 g, 4.0 mmol) in THF (25 mL) was added DTT (1.2 g, 7.9 mmol) and aqueous saturated KHCO₃ (10 mL). The resulting mixture was stirred at room temperature and monitored by TLC until the reaction was complete. The mixture was then concentrated and the residue was dissolved in 6N HCl. The resulting solution was heated at 60 °C for 3 hours and then concentrated by reduced pressure. The residue was purified by column chromatography with PE: ethyl acetate = 3:1 to afford the title compound as a solid (1.2 g, 68%). ¹H-NMR (CDCl₃, ppm, 500 MHz): δ = 7.39 (d, J= 7.5 Hz, IH), 7.28 (d, J= 8.0 Hz, IH), 7.19 (t, J= 7.5 Hz, IH), 7.08 (t, J= 7.5 Hz, IH), 4.56 (dd, J= 7.0, 13.0 Hz, IH), 3.40-3.46 (m, IH), 3.30- 3.34 (m, IH)₅. 3.17-3.22 (m, IH), 2.07-2.16 (m, IH).

Synthetic Example 30 (R)-Methyl 3-mercapto-2-(5-morphoIinopentanamido)propanoate

Step 1 : (RVmethyl 2-(5-bromopentanamido)-3-(tritylsulfanyDpropanoate Thionyl chloride (8.2 g, 68.9 mmol) was added to MeOH (500 mL) slowly with ice-cooling, and then S-trityl cysteine (25.0 g, 68.9 mmol) was added to the solution. The resulting mixture was continuously stirred overnight at 80°C. The solvent was concentrated by reduced pressure and purified by column chromatography with DCM:MeOH = 10:1 to afford S-trityl cysteine methyl ester as a white solid. (18.6 g, 72% yield).

To a solution of S-trityl cysteine methyl ester (7.5 g, 19.3 mmol) in DCM (50 mL) was added HOBt (5.2 g, 38.6 mL), EDCl (7.4 g, 38.6 mmol) and Et₃N (4.0 g, 38.6 mmol). After approximately 30 min, 5-bromovaleric acid (3.6 g, 20 mmol) was added. The resulting mixture was continuously stirred at room temperature for 4 hours. The mixture was then concentrated, water (100 mL) was added and the product was extracted into ethyl acetate (3 x 150 mL). The combined organics were washed with saturated NaCl solution, and purified by column chromatography with PE: ethyl acetate = 3:1 to afford the desired compound (6.0 g, 60%). Step 2: (R)-Methyl 2-(3-moφholinopentanamido>3-(tritylsulfanyl)propanoate

To a solution of (R)-methyl 2-(5-bromopentanamido)-3-

(tritylsulfanyl)propanoate (6.0 g, 11.1 mmol) in MeOH (20 mL), was added KI (3.7 g, 22.2 mmol). After 15 min, morpholine (9.7 g, 111 mmol) was added and then the mixture was stirred for four hrs at 65 °C. The mixture was concentrated under reduced pressure, diluted with water (150 mL) and extracted with ethyl acetate (2 x 150 mL), concentrated to afford the crude title compound (4.5 g, 74%). ESI-MS: 547.3 (M+H), Purity: 94% (214 nm), 100% (254 nm). Step 3: (R)-Methyl 3-mercapto-2-(5-morpholinopentanamido)propanoate To a solution of (R)-methyl 2-(5-morpholinopentanamido)-3- (tritylsulfanyl)propanoate (1.0 g, 1.8 mmol) in DCM (5 mL) was added TFA (5 mL) slowly. After ten minutes, Et₃SiH (5 mL) was added to the solution. The mixture was stirred at room temperature for an additional ten minutes. The mixture was then concentrated under reduced pressure, purified by column chromatography with DCM:MeOH =10:1 to afford the crude product which was purified by preparative HPLC to afford the title product (450 mg, 80%). ¹H-NMR (CDCl₃, ppm, 500 MHz): δ = 4.82-4.84 (m, IH), 3.99 (t, J= 4.5 Hz, 4H), 3.79 (s, 3H), 3.54 (d, J= 9.5 Hz, 2H), 2.97-3.08 (m, 4H), 2.84 (s, 2H), 2.35 (t, J= 7.0 Hz, 2H), 1.83-1.88 (m, 2H), 1.73-1.78 (m, 2H), 1.46 (t, J= 8.5 Hz, IH), 1.25 (s, IH). ¹³C NMR (CDCl₃, 125 MHz): 5(ppm) = 22.1, 22.7, 26.4, 34.9, 51.8, 52.7, 54.1, 57.0, 63.8, 77.2, 170.8, 172.6. ESI-MS: 305.2 (M⁺+H), HPLC purity: 93% (214 nm).

Synthetic Example 31 (R)-3-Mercapto-2-(5-morpholinopentanamido)propanoic acid

Step 1 : (R)-2-(5-Morpholinopentanamido)-3-(tritylsulfanyl)propanoic acid To a solution of (R)-methyl 2-(5-morpholinopentanamido)-3-

(tritylsulfanyl)propanoate (1.1 g, 2.0 mmol) in THF (6 mL) and MeOH (2 mL), was added aqueous LiOH (2.0 mL of a saturated solution). The mixture was stirred at room temperature for one hour. The reaction was then concentrated and acidified with HCl 1 N to pH 3, washed and filtered off to afford the desired product (1.0 g,

97%).

Step 2: (R)-3-mercapto-2-(5-morpholinopentanamido)propanoic acid

To a solution of (R)-2-(5-morpholinopentanamido)-3- (tritylsulfanyl)propanoic acid (1.0 g, 2.0 mmol) in DCM (5 mL) was added TFA (2 mL) slowly. After ten minutes, Et₃SiH (2 mL) was added and the mixture was stirred at room temperature for two hours. The mixture was then concentrated, dissolved in water and washed with petroleum ether. The water phase was concentrated to afford the title compound (162 mg, 30%). ¹H-NMR (D₂O, ppm, 500 MHz): δ = 4.50 (dd, J = 4.5 and 7.0 Hz, IH), 4.02 (dd, J= 3.5 and 8.0 Hz, 4H), 3.68-3.74 (m, 2H), 3.42-3.48 (m, 3H), 3.05-3.12 (m, 4H), 2.84-2.94 (m, 2H),1.57-1.72 (m, 4H). ¹³C NMR (D₂O, 125 MHz): (5 = 22.0, 22.4, 25.1, 34.4, 51.6, 54.9, 56.7, 63.8, 173.7, 176.1. ESI-MS: 291.1 (M⁺+H). HPLC purity: 73% (214 nm).

Example 32

(2S,2'S)-Dimethyl 4,4'-disulfanediyl-bis(2-(5-fluorobenzo[d]oxazol-2-ylamino) butanoate)

Step 1. 5-Fluoro-2-mercaptobenzoxazole

A mixture of 2-amine-4-fluorophenol (5.0 g) and potassium ethyl xanthate (1.0 eq), dissolved in 100 mL ethanol was heated under reflux for 7 hrs. The solvent was removed in vacuum and the residue was dissolved in water. The solution was acidified to pH 5 with glacial acetic acid. The product was filtered and crystallized to obtain 5-fluoro-2-mercaptobenzoxazole (5.O g, 75%).

Step 2. 2-Chloro-5-fluoro-benzoxazole To a suspension of 5-fluoro-2-mercapto-benzoxazole (5.0 g) in POCl₃ (8.8 eq) at room temperature, was added PCl₅ (1.2 eq) along with 20 mL DCM. After 24 hr of stirring at room temperature, the mixture was concentrated to remove excess

POCl₃. The residue was neutralized with cool aqueous NaOH mixed with CH₂Cl₂.

The organic layer was washed with brine and the crude product was purified by silica gel chromatography to obtain 2-chloro-5-fluoro-benzoxazole (4.1 g, 81%).

Step 3. (2S,2'SVDimethyl 4,4'-disulfanediyl-bis(2-(^'5-fluorobenzordloxazol-2- ylamino) butanoate) To a solution of 2-chloro-5-fluoro-benzoxazole in DMF (25 mL) was added DIPEA (1.0 eq) along with methyl homocystine (0.5 eq) at room temperature. The mixture was stirred at room temperature and monitored via TLC. After the reaction was complete, the crude product was extracted with ethyl acetate (3 x 100 mL) and washed with aqueous NH₄Cl. The combined organic layers were washed with a saturated solution of aqueous NaCl and dried over anhydrous sodium sulfate and then evaporated in vacuo. The resultant residue was purified via silica gel chromatography (eluent: from ethyl acetate/PE = 1 : 5 to 1 : 2) to give (2S,2'S)- dimethyl 4,4'-disulfanediyl-bis(2-(5-fluorobenzo[d]oxazol-2-ylamino) butanoate). ¹H-NMR (CDCl₃, ppm, 500 MHz): δ = 7.25-7.28 (m, 2H), 7.00-7.02 (m, 2H), 6.88- 6.93 (m, 2H), 4.69-4.71 (m, 2H), 3.79 (s, 6H), 2.76-2.82 (m, 4H), 2.41-2.48 (m, 2H), 2.19-2.26 (m, 2H). ESI-MS: 567 (M+H)⁺, Purity: 97% (254 nm).

Synthetic Example 33 (S)-MethyI 4-(acetyIthio)-2-(5-fluorobenzo[d]oxazol-2-ylamino)butanoate

To a solution of (2S,2'S)-dimethyl 4,4'-disulfanediyl-bis(2-(5- fluorobenzo[d]oxazol-2-ylamino) butanoate) in THF was added DTT (2.0 eq). The mixture was stirred at room temperature under nitrogen atmosphere and monitored by TLC. The crude product was extracted with ethyl acetate and washed with aqueous NaCl and dried over anhydrous sodium sulfate and then evaporated in vacuo. To the residue dissolved in THF were added IN aqueous K₂CO₃ (1.2 eq) and acetic anhydride (3.3 eq). The mixture was stirred at room temperature under nitrogen atmosphere and TLC monitored it. The crude product was extracted with ethyl acetate and the residue was purified by silica gel chromatography (eluent: from EA/PE = 1 :5 to 1 : 2.5) to give the title compound. ¹H-NMR (CDCl₃, ppm, 500 MHz): δ = 7.25-7.28 (m, IH), 6.98-7.00 (m, IH), 6.88-6.92 (m, IH), 4.65-4.68 (m, IH), 3.80 (s,3H), 2.93-3.09 (m, 2H), 2.32 (s, 3H), 2.27-2.35 (m, IH), 2.10-2.16 (m, IH). ESI- MS: 327.1 (M+H), Purity: 97% (254 nm).

Synthetic Example 34 (S)-3-(5-Fluorobenzo[d]oxazol-2-ylamino)dihydrothiophen-2(3H)-one (S)-methyl 4-(acetylthio)-2-(5-fluorobenzo[d]oxazol-2-ylamino)butanoate was placed in 6N aqueous HCl, stirred at 80°C and monitored via TLC. Once the reaction was complete, the solution was adjusted to pH 8 with sodium hydroxide and the product was extracted into ethyl acetate (2 x 100 mL). The residue was purified by silica gel chromatography to obtain the title compound as a white solid. ¹H-NMR (CDCl₃, 500 MHz): δ (ppmj = 7.27-7.30 (m, IH), 7.03-7.05 (m, IH), 6.91-6.95 (m, IH), 4.49-4.53 (m, IH), 3.41-3.46 (m, IH), 3.32-3.35 (m, IH), 3.17-3.22 (m, IH), 2.08-2.17 (m, IH). ESI-MS: 253.1 (M+H)⁺, Purity: 94% (214 nm).

Synthetic Example 35:

(R)-2-Acetamido-3-mercapto-N-(methylsulfonyl)propanamide

Step 1 : (R)-2-Acetamido-3-r4-methoxybenzylsulfanyl)propanoic acid

To a solution of N-acetyl-L-cysteine (10.0 g, 61.3 mmol) in 4N sodium hydroxide (100 mL), PMBCl (p-methoxybenzyl chloride, 15 mL) was added slowly at 0°C. The mixture was stirred at room temperature for 18 h. The resulting reaction solution was acidified with 2N HCl to pH 1 and extracted with ethyl acetate (4 x 150 mL). The combined organic layers was washed with brine, dried and concentrated. The crude product was crystallized with ethyl acetate to give S-PMB-protected N- acetyl cysteine. Step 2: (R)-2-Acetamido-3-(^'4-methoxybenzylsulfanyl)-N-(methylsulfonyl^') propanamide

To a stirred solution of S-PMB-protected N-acetyl cysteine (20.7 g, 73.1 mmol) in dry CH₂Cl₂ under nitrogen atmosphere, was added methanesulfonamide (34.7 g, 365.7 mmol), DMAP (4.4 g, 36.5 mmol) followed by EDCI (15.3 g, 80.4 mmol). The resulting mixture was stirred at room temperature for 24 h. The mixture was concentrated in vacuum and the residue was extracted with ethyl acetate (2 x 100 mL) and washed with brine. The crude product was purified by flash chromatography and further purification by preparative HPLC to obtain the desired (R)-2-acetamido-3- (4-methoxybenzylsulfanyl)-N-(methylsulfonyl)propanamide (3.0 g, 11%). ¹H-NMR (CD₃OD, ppm, 500 MHz): δ = 7.27 (d, J= 8.5 Hz, 2H), 6.87 (d, J- 8.5 Hz, 2H), 4.51 (t, J= 7.0 Hz, IH), 3.78 (s, 3H), 3.74 (d, J= 2.5 Hz, 2H), 3.24 (s, 3H), 2.79-2.84 (m, IH), 2.64-2.69 (m, IH), 1.99 (s, 3H). ESI-MS: 382.9 (M+Na)⁺, Purity: 100% (214 nm) Step 3: (Ry2-Acetamido-3-mercapto-N-(methylsulfonvDpropanamide A mixture of solution of TFA (20 mL) and anisole (5 mL) was added to

(R)-2-acetamido-3 -(4-methoxybenzylsulfanyl)-N-(methylsulfonyl)propanamide (1.5 g, 4.1 mmol) and the mixture was stirred at 52 ^ΩC for 3 h. After the starting material was consumed, the mixture was concentrated to remove excess TFA and the residue was purified by silica column and further purified by crystallization with ethyl acetate to obtain the title compound as a white solid. ¹H-NMR (CD₃OD, ppm, 500 MHz): δ = 4.45-4.48 (m, IH), 3.26 (s, 3H),. 2.90-2.93 (m, IH), 2.79-2.84 (m, IH), 2.03 (s, 3H). ESI-MS: 263.0 (M+Na)⁺, Purity: 38.7% (214 nm); 500.8 (2M+Na)⁺, Purity: 58.7% (214 nm).

Synthetic Example 36 (R)-N-(2-Mercapto-l-(lH-tetrazol-5-yl)ethyl)acetamide

Step 1 : (^"RVN-π-cvano-2-r4-methoxybenzylsulfanyl)ethvπacetamide A stirred solution of S-para-methoxybenzyl-N-acetylcysteine carboxamide

(22.5 g, 79.6 mmol) in anhydrous pyridine (150 mL) and anhydrous CH₂Cl₂ (75 mL) was cooled in an ice bath and TsCl (37.5 g, 196.6 mmol) was added. The resulting yellow solution was stirred at 0 ⁰C for 30 min and then the ice bath was removed, and stirring was continued for 3 hrs. The reaction mixture was evaporated in vacuum and the residue was dissolved in EtOAc (30 mL) and washed with aq. NaHCO₃ until pH~7. The organic phase was dried over MgSO₄, evaporated, and the residue was purified by silica gel column chromatography (petroleum ether and ethyl acetate) to get the desired compound (white solid, 15.4 g, 73%). ¹H-NMR (DMSO- d₆, ppm, 500 MHz): δ = 8.78 (d, J= 7.0 Hz, IH), 7.25 (d, J= 7.5 Hz, 2H), 6.89 (d, J= 7.5 Hz, 2H), 4.85 (d, J= 7.0 Hz₅ IH), 3.80 (s, 2H), 3.74 (s, 3H), 2.83-2.71 (m, 2H), 1.89 (s, 3H). MS (ESI): 265[M+1]⁺, 287[M+23]⁺.

Step 2: rRVN-(2-(^'4-MethoxybenzylsulfanylVl-πH-tetrazol-5-yl^N)ethvDacetamide To a stirred solution of (R)-N-(I -cyano-2-(4-methoxybenzylsulfanyl)ethyl) acetamide (15.0 g, 56.7 mmol) in anhydrous DMF (150 mL) under nitrogen, was added NH₄Cl (14.0 g, 261.8 mmol ) followed by NaN₃ (4.5 g, 69.2 mmol). The reaction mixture was heated at 105 ⁰C overnight under nitrogen. After this time, the solvent was removed by distillation in vacuum. The residue was purified by recrystallization in methanol to afford (R)-N-(2-(4-methoxybenzylsulfanyl)-l-(lH- tetrazol-5-yl)ethyl)acetamide (white solid, 11.2 g, 64 %). ¹H-NMR (DMSO-^, ppm, 500 MHz): (5 = 8.67 (d, J= 7.0 Hz, IH), 7.22 (d, J= 7.0 Hz, 2H), 6.88 (d, J= 8.0 Hz, 2H), 5.34 (d, J= 7.5 Hz, IH), 3.73 (s, 3H), 3.69 (s, 2H), 2.84-3.00 (m, 2H),1.89 (s, 3H). MS (ESI): 308[M+l]⁺, 330[M+23]⁺. Step 3 : f RVN-f 2-Mercapto- 1 -( 1 H-tetrazol-5-vDethvOacetamide A solution of TFA (30 mL) and anisole (7.5 mL) was added to (R)-N-(2- (4-methoxybenzylsulfanyl)-l-(lH-tetrazol-5-yl)ethyl)acetamide (4.5 g, 14.6 mmol), and the mixture was stirred at 55 ⁰C overnight. The solvent of the reaction mixture was removed by extraction of hexane (30 mL x 3) and the residue was evaporated in vacuum and purified by preparative HPLC to (R)-N-(2-mercapto- 1 -(I H-tetrazol-5- yl)ethyl)acetamide as a white solid, 1.89 g, 69%. ¹H-NMR (DMSO-d₆, ppm, 500 MHz): δ = 5.33 (t, J= 6.5 Hz, IH), 3.00-3.12 (m, 2H), 1.99 (s, 3H). MS (ESI): 188[M+1]⁺.

Synthetic Example 37

4-S-Nitrosomercapto-2-(thiazol-2-yl)amino-butyric acid

Procedure A:

A 0.1 M solution of 4-mercapto-2-(thiazol-2-yl)amino-butyric acid in methanol was prepared in an amber vial to protect against light. To a 1.0 mL aliquot was added 10 μL of concentrated HCl. The mixture was agitated using a vortex apparatus until complete dissolution was observed. At this time, 100 μL of the 10- 20% EtONO solution was added and the mixture was again mixed by using a vortex apparatus. After 5 min at room temperature, conversion to 4-(S-nitrosomercapto)-2- (thiazol-2-yl)amino-butyric acid was complete as determined by the Saville assay. Analysis of the LCMS trace of the reaction mixture indicated a new peak with m/z value of mz/ 248, uniquely attributed to the [M+H]+ molecular ion of Synthetic Example 37 (data not shown). The ¹H NMR of the reaction product was also consistent with the S-nitrosomercapto compound (Figure 21) and distinct from the disulfide (2S,2'S)-4,4'-disulfanediyl-bis(2-(thiazol-2-ylamino) butanoic acid). Butylnitrite, isobutyl nitrite and other organic nitrites can be used, by one skilled in the art, to produce nitrosated mercaptos following the procedure outlined in Example 37. Procedure B:

A 100 μmol quantity of mercapto (22 mg of 4-mercapto-2-(thiazol-2- yl)amino-butyric acid) was dissolved in 1.0 mL of 0.1 N HCl, to which 100 μmoles DTPA (chelating agent) was added. To this solution was added 1.0 mL of 0.1 M NaNO₂ in H₂O. The mixture was agitated using a vortex apparatus for approximately 30 seconds, then place directly onto ice. Saville assay - Quantification of nitrosation of mercaptos

The following stock reagents were prepared by one skilled in the art or purchased from commercial sources: (a) 10-20% ethylnitrite (EtONO) in ethanol; (b) 1 % Sulfanilamide (SAA) + 0.2% HgCl₂ in 0.1 N HCl solution (w/v);

(c) 0.02% Naphthyl (N- 1 -ethylene)diamine (NNED) in 0.1 N HCl (w/v), kept at 4⁰C in light sealed bottle.

The Saville procedure involved the following steps. To each well (triplicate) was added 50 μL of 50 μM solution of nitrosated mercapto Example 37 or 50 μL of 0.1 N HCl + 100 μM DTPA to serve as a blank. At this time, wells containing nitrosated mercapto and those that serve as blanks were chosen to either receive 50 μL of 1% SAA or 50 μL of 1% SAA + HgCl₂ as prepared above. All wells were incubated for 5 min at room temperature in the dark. After this time, each well received 50 μL of 0.02% NNED solution as prepared above, and incubation was continued for 5 minutes at room temperature in the dark. Each well was then analyzed on a Flex Station 3 (Molecular Devices, Sunnyvale, CA) plate reader at OD at 540 run. The determination of nitrosated mercapto was determined by subtracting the average background OD from each +/- Hg values and then calculating the percentage of nitrosated mercapto according to the equation:

% nitrosation = [("Hg" - "No Hg")/"Hg"] x 100

Reaction of 4-mercapto-2-(thiazol-2-yl)amino-butyric acid to produce 4- (S-nitrosomercapto)-2-(thiazol-2-yl)amino-butyric acid, using the nitrosation conditions described above, gives >95% nitrosation as determined by the Saville assay as described herein.

Stability studies of nitrosated mercaptos

Nitrosated mercaptos such the solution of 4-(S-nitrosomercapto)-2- (thiazol-2-yl)amino-butyric acid were diluted 1 :100 with the three separate pH 3.0, 5.0 and 7.0 PBS buffers (10 μL of compound into 990 μL of buffer; titrated with HCl if necessary). Aliquots were drawn for analysis for nitrosated mercapto content using the Saville assay described above. The stability of the 4-(S-nitrosomercapto)-2- (thiazol-2-yl)amino-butyric acid was measured at four different pH values over a period of up to 5 hours (Figure 22). The compound was found to be remarkably stable at acidic pH values (pH 1, pH 3), with slightly lower stability at pH 7 (-75% remaining compound after 5 hours of incubation) and at pH 5 (-50% remaining compound after 5 hours of incubation).

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed:

1. A composition comprising one or more mercapto-based

SNO compounds selected from the group consisting of (S)-2-(3,3- dimethylureido)-3 -mercaptopropanoic acid, (R)-2-(3 ,3 -dimethylureido)-3 - mercaptopropanoic acid, (R)-3-mercapto-2- (methoxycarbonylarnino)propanoic acid, (S)-3-mercapto-2- (methoxycarbonylamino)-propanoic acid, (S)-2-((S)-2,4-diaminobutanarnido)-

3 -mercaptopropanoic acid, (R)-2-((S)-2,4-diaminobutanarnido)-3- mercaptopropanoic acid, (R)-2-acetamido-3-mercapto-N- (methylsulfonyl)propanamide, (R)-N-(2-mercapto-l -(I H-tetrazol-5- yl)ethyl)acetamide, (S)-4-(2-acetamido-3-mercaptopropanamido)benzoic acid, (R)-4-(2-acetamido-3-mercaptopropanamido)benzoic acid, (S)-2-acetamido-3- mercapto-3-methylbutanoic acid, (S)-2-(benzo[d]oxazol-2-ylamino)-3- mercapto-3-methylbutanoic acid, (R)-2-(benzo[d]oxazol-2-ylamino)-3- mercapto-3-methylbutanoic acid, (R)-2-(benzo[d]oxazol-2-ylamino)-3- mercaptopropanoic acid, (2S,2'S)-dimethyl 4,4'-disulfanediylbis(2- (benzo[d]oxazol-2-ylamino)butanoate), (S)-methyl 2-(benzo[d]oxazol-2- ylamino)-4-mercaptobutanoate, (S)-methyl 4-(acetylthio)-2-(benzo[d]oxazol- 2-ylamino)butanoate, (S)-3-(benzo[d]oxazol-2-ylamino)dihydrothiophen- 2(3H)-one, (2S,2'S)-dimethyl 4,4'-disulfanediylbis(2-(5-fluorobenzo[d]oxazol- 2-ylamino)butanoate), (S)-methyl 4-(acetylthio)-2-(5-fluorobenzo[d]oxazol-2- ylamino)butanoate, (S)-3-(5-fluorobenzo[d]oxazol-2- ylamino)dihydrothiophen-2(3H)-one, (S)-2-(benzo[d]thiazol-2-ylamino)-4- mercaptobutanoic acid, (S)-4-(acetylthio)-2-(benzo[d]thiazol-2- ylamino)butanoic acid, (S)-methyl 4-(acetylthio)-2-(benzo[d]thiazol-2- ylamino)butanoate, (S)-3-(benzo[d]thiazol-2-ylamino) dihydrothiophen- 2(3H)-one, (S)-2-(benzo[d]thiazol-2-ylamino)-3-mercapto-3-methylbutanoic acid, (S)-methyl 2-(benzo[d]thiazol-2-ylamino)-3-mercapto-3- methylbutanoate, (R)-3-mercapto-2-(thiazol-2-ylamino)propanoic acid, (S)-3- mercapto-2-(thiazol-2-ylamino)propanoic acid, (R)-3-mercapto-2-(5-methyl- 4-phenylthiazol-2-ylamino)propanoic acid, (S)-4-mercapto-2-(4- phenylthiazol-2-ylamino)butanoic acid, sodium (S)-4-mercapto-2-(4- phenylthiazol-2-ylamino)butanoate, sodium (S)-4-mercapto-2-(5-methyl-4- phenylthiazol-2-ylamino)butanoate, (S)-4-mercapto-2-(thiazol-2- ylamino)butanoic acid, sodium (S)-4-mercapto-2-(thiazol-2- ylamino)butanoate, sodium (S)-4-mercapto-2-(methyl(thiazol-2- yl)amino)butanoate, (R)-2-(4-tert-butylthiazol-2-ylamino)-3 - mercaptopropanoic acid, (R)-2-(4,5-dimethylthiazol-2-ylamino)-3- mercaptopropanoic acid, (R)-3-mercapto-2-(4,5,6,7- tetrahydrobenzo [d]thiazol-2-ylamino)propanoic acid, (R)-3 -mercapto-2-(5- nitropyridin-2-ylamino)propanoic acid, 3-mercapto-2-(5-

(trifluoromethyl)pyridin-2-ylamino)propanoic acid, (R)-2-(4,6-bis(4- methylpiperazin- 1 -y I)- 1 , 3 , 5 -triazin-2-y lamino)-3 -mercaptopropanoic acid, (R)-2-(4,6-bis(dimethylamino)-l,3,5-triazin-2-ylamino)-3-mercaptopropanoic acid, (R)-2-( 1 -(4,6-bis(allylamino)- 1 ,3 ,5 -triazin-2-yl)piperidin-4-ylamino)-3 - mercaptopropanoic acid, sodium (S)-4-mercapto-2-(pyrimidin-2- ylamino)butanoate, (R)-2-(6-chloropyrimidin-4-ylamino)-3-mercapto-3- methylbutanoic acid hydrochloride, (S)-4-mercapto-2-(methyl(thiazol-2- yl)amino)butanoic acid, (R)-4-mercapto-2-(methyl(thiazol-2- yl)amino)butanoic acid, sodium (S)-4-mercapto-2-(thiazol-2- ylamino)butanoate, (R)-3 -mercapto-3 -methyl-2-(thiazol-2-ylamino)butanoic acid, (S)-3 -mercapto-3 -methyl-2-(thiazol-2-ylamino)butanoic acid, (2R,3R)- ethyl l-acetyl-3-mercaptopyrrolidine-2-carboxylate, (2S,3S)-ethyl l-acetyl-3- mercaptopyrrolidine-2-carboxylate, (S)- 1 -((R)-2-(benzo[d]oxazol-2-ylamino)- 3-mercapto-propanoyl)-pyrrolidine-2-carboxylic acid, (S)-3-mercapto-2- (phenylsulfonamido)propanoic acid, (R)-3-mercapto-2-

(phenylsulfonamido)propanoic acid, (R)-methyl 3-mercapto-2-(5- morpholinopentanamido)propanoate, (R)-3-mercapto-2-(5- morpholinopentanamido)-propanoic acid, (R)-2-(benzo[d]oxazol-2-ylamino)- 3-mercapto-N-(methylsulfonyl)propanamide, (S)-2-(benzo[d]oxazol-2- ylamino)-3 -mercapto-3 -methyl -N-(methylsulfonyl)butanamide, (S)-2-

(benzo[d]oxazol-2-ylamino)-3-mercapto-3-methyl-N- (phenylsulfonyl)butanamide, (R)-methyl 2-(2-(benzo[d]oxazol-2- ylthio)acetamido)-3-mercaptopropanoate, and (R)-methyl 2-(2- (benzo[d]oxazol-2-ylthio)acetamido)-3-mercapto-3-methylbutanoate; a pharmaceutically acceptable salt thereof, and mixtures thereof.

2. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more mercapto-based SNO compounds selected from the group consisting of (S)-2-(3,3-dimethylureido)- 3-mercaptopropanoic acid, (R)-2-(3,3-dimethylureido)-3-mercaptopropanoic acid, (R)-3-mercapto-2-(methoxycarbonylamino)propanoic acid, (S)-3- mercapto-2-(methoxycarbonylamino)-propanoic acid, (S)-2-((S)-2,4- diaminobutanamido)-3-mercaptopropanoic acid, (R)-2-((S)-2,4- diaminobutanamido)-3 -mercaptopropanoic acid, (R)-2-acetamido-3 -mercapto- N-(methylsulfonyl)propanamide, (R)-N-(2-mercapto- 1 -( 1 H-tetrazol-5- yl)ethyl)acetamide, (S)-4-(2-acetamido-3-mercaptopropanamido)benzoic acid, (R)-4-(2-acetamido-3-mercaptopropanamido)benzoic acid, (S)-2-acetamido-3- mercapto-3 -methylbutanoic acid, (S)-2-(benzo [d] oxazol-2-ylamino)-3 - mercapto-3-methylbutanoic acid, (R)-2-(benzo[d]oxazol-2-ylamino)-3- mercapto-3 -methylbutanoic acid, (R)-2-(benzo[d]oxazol-2-ylamino)-3- mercaptopropanoic acid, (2S,2'S)-dimethyl 4,4'-disulfanediylbis(2- (benzo[d]oxazol-2-ylamino)butanoate), (S)-methyl 2-(benzo[d]oxazol-2- ylamino)-4-mercaptobutanoate, (S)-methyl 4-(acetylthio)-2-(benzo[d]oxazol-

2-ylamino)butanoate, (S)-3 -(benzo [d]oxazol-2-ylamino)dihydrothiophen- 2(3H)-one, (2S,2'S)-dimethyl 4,4'-disulfanediylbis(2-(5-fluorobenzo[d]oxazol- 2-ylamino)butanoate), (S)-methyl 4-(acetylthio)-2-(5-fluorobenzo[d]oxazol-2- ylamino)butanoate, (S)-3-(5-fluorobenzo[d]oxazol-2- ylamino)dihydrothiophen-2(3H)-one, (S)-2-(benzo [d]thiazol-2-ylamino)-4- mercaptobutanoic acid, (S)-4-(acetylthio)-2-(benzo[d]thiazol-2- ylamino)butanoic acid, (S)-methyl 4-(acetylthio)-2-(benzo[d]thiazol-2- ylamino)butanoate, (S)-3 -(benzo [d]thiazol-2-ylamino) dihydrothiophen- 2(3H)-one, (S)-2-(benzo[d]thiazol-2-ylamino)-3-mercapto-3-methylbutanoic acid, (S)-methyl 2-(benzo[d]thiazol-2-ylamino)-3-mercapto-3- methylbutanoate, (R)-3-mercapto-2-(thiazol-2-ylamino)propanoic acid, (S)-3- mercapto-2-(thiazol-2-ylamino)propanoic acid, (R)-3-mercapto-2-(5-methyl- 4-phenylthiazol-2-ylamino)propanoic acid, (S)-4-mercapto-2-(4- phenylthiazol-2-ylamino)butanoic acid, sodium (S)-4-mercapto-2-(4- phenylthiazol-2-ylamino)butanoate, sodium (S)-4-mercapto-2-(5-methyl-4- phenylthiazol-2-ylamino)butanoate, (S)-4-mercapto-2-(thiazol-2- ylamino)butanoic acid, sodium (S)-4-mercapto-2-(thiazol-2- ylamino)butanoate, sodium (S)-4-mercapto-2-(methyl(thiazol-2- yl)amino)butanoate, (R)-2-(4-tert-butylthiazol-2-ylamino)-3 - mercaptopropanoic acid, (R)-2-(4,5-dimethylthiazol-2-ylamino)-3- mercaptopropanoic acid, (R)-3-mercapto-2-(4,5,6,7- tetrahydrobenzo[d]thiazol-2-ylamino)propanoic acid, (R)-3-mercapto-2-(5- nitropyridin-2-ylamino)propanoic acid, 3-mercapto-2-(5-

(trifluoromethyl)pyridin-2-ylamino)propanoic acid, (R)-2-(4,6-bis(4- methylpiperazin-l-yl)-l,3,5-triazin-2-ylamino)-3-mercaptopropanoic acid, (R)-2-(4,6-bis(dimethylamino)-l,3,5-triazin-2-ylamino)-3-mercaptopropanoic acid, (R)-2-(l-(4,6-bis(allylamino)-l,3,5-triazin-2-yl)piperidin-4-ylamino)-3- mercaptopropanoic acid, sodium (S)-4-mercapto-2-(pyrimidin-2- ylamino)butanoate, (R)-2-(6-chloropyrimidin-4-ylamino)-3 -mercapto-3 - methylbutanoic acid hydrochloride, (S)-4-mercapto-2-(methyl(thiazol-2- yl)amino)butanoic acid, (R)-4-mercapto-2-(methyl(thiazol-2- yl)amino)butanoic acid, sodium (S)-4-mercapto-2-(thiazol-2- ylamino)butanoate, (R)-3-mercapto-3-methyl-2-(thiazol-2-ylamino)butanoic acid, (S)-3-mercapto-3-methyl-2-(thiazol-2-ylamino)butanoic acid, (2R,3R)- ethyl l-acetyl-3-mercaptopyrrolidine-2-carboxylate, (2S,3S)-ethyl l-acetyl-3- mercaptopyrrolidine-2-carboxylate, (S)- 1 -((R)-2-(benzo [d]oxazol-2-ylamino)- 3-mercapto-propanoyl)-pyrrolidine-2-carboxylic acid, (S)-3-mercapto-2- (phenylsulfonamido)propanoic acid, (R)-3-mercapto-2-

(phenylsulfonamido)propanoic acid, (R)-methyl 3-mercapto-2-(5- morpholinopentanamido)propanoate, (R)-3-mercapto-2-(5- morpholinopentanamido)-propanoic acid, (R)-2-(benzo[d]oxazol-2-ylamino)- 3-mercapto-N-(methylsulfonyl)propanamide, (S)-2-(benzo[d]oxazol-2- ylamino)-3 -mercapto-3 -methyl-N-(methylsulfonyl)butanamide, (S)-2-

(benzo [d]oxazol-2-ylamino)-3 -mercapto-3 -methyl -N- (phenylsulfonyl)butanamide, (R)-methyl 2-(2-(benzo[d]oxazol-2- ylthio)acetamido)-3-mercaptopropanoate, and (R)-methyl 2-(2- (benzo[d]oxazol-2-ylthio)acetamido)-3-mercapto-3-methylbutanoate; a pharmaceutically acceptable salt thereof, and mixtures thereof.

3. A composition comprising one or more S- nitrosomercapto based SNO compounds, selected from the group consisting of

(S)-2-(3 ,3-dimethylureido)-3-(nitrosomercapto)propanoic acid, (R)-2-(3 ,3 - dimethylureido)-3-(nitrosomercapto)-propanoic acid, (R)-3-nitrosomercapto- 2-(methoxycarbonylamino)propanoic acid, (S)-3-nitrosomercapto-2- (methoxycarbonylamino)propanoic acid, (S)-2-((S)-2,4-diaminobutanamido)- 3-(nitrosomercapto)propanoic acid, (R)-2-((S)-2,4-diaminobutanamido)-3-

(nitrosomercapto)propanoic acid, (R)-S-nitroso-N-acetylcysteine, (S)-S- nitroso-N-acetylcysteine, (R)-2-acetamido-3 -nitrosomercapto-N- (methylsulfonyl) propanamide, (R)-N-(2-nitrosomercapto-l-(lH-tetrazol-5- yl)ethyl)acetamide, (R)-3 -nitrosomercapto-2-propionamidopropanoic acid, (R)-2-benzamido-3-(nitrosomercapto)propanoic acid, (S)-4-(2-acetamido-3-

(nitrosomercapto)-propanamido)benzoic acid, (R)-4-(2-acetamido-3- (nitrosomercapto) propanamido)benzoic acid, (R)-2-acetamido-3- nitrosomercapto-3 -methylbutanoic acid, (S)-2-acetamido-3 -nitrosomercapto- 3-methylbutanoic acid, (S)-2-(benzo[d]oxazol-2-ylamino)-3-nitrosomercapto- 3 -methylbutanoic acid, (R)-2-(benzo[d]oxazol-2-ylamino)-3-nitrosomercapto-

3 -methylbutanoic acid, 2-(benzo[d]oxazol-2-ylamino)-3- (nitrosomercapto)propanoic acid, (R)-2-(benzo [d] oxazol-2-ylamino)-3 - (nitrosomercapto)propanoic acid, (S)-methyl 2-(benzo[d]oxazol-2-ylamino)-4- (nitrosomercapto)butanoate, (S)-methyl 2-(5-fluorobenzo[d]oxazol-2- ylamino)-4-(nitrosomercapto)butanoate, (R)-2-(6-chlorobenzo[d]oxazol-2- ylamino)-3-nitrosomercapto-propanoic acid, (R)-3-nitrosomercapto-2-(4- methylbenzo[d]oxazol-2-ylamino) propanoic acid, S-nitroso-(R)-2- (benzo [d]oxazol-2-ylamino)-2-( 1 H-tetrazol-5-yl)- 1 -nitrosomercaptoethane, (R)-2-(2-(benzo[d]oxazol-2-ylamino)-3-nitrosomercapto-propanamido)acetic acid, (R)-methyl 2-(2-(benzo[d]oxazol-2-ylamino)-3-

(nitrosomercapto)propanamido)acetate, 2-(benzo[d]thiazol-2-ylamino)-3- (nitrosomercapto)propanoic acid, (S)-2-(benzo[d]thiazol-2-ylamino)-4- (nitrosomercapto)butanoic acid, (S)-methyl 4-(nitrosomercapto)-2- (benzo[d]thiazol-2-ylamino)butanoate, (S)-2-(benzo[d]thiazol-2-ylamino)-3- nitrosomercapto-3-methylbutanoic acid, (S)-methyl 2-(benzo[d]thiazol-2- ylamino)-3 -nitrosomercapto-3 -methylbutanoate, (R)-3 -nitrosomercapto-2- (thiazol-2-ylamino)propanoic acid, (S)-3 -nitrosomercapto-2-(thiazol-2- ylamino)propanoic acid, 3 -nitrosomercapto-2-(thiazol-2-ylamino)propanoic acid, (R)-2-(5-(ethoxycarbonyl)-4-methylthiazol-2-ylamino)-3-

(nitrosomercapto) propanoic acid, (R)-3-nitrosomercapto-2-(5-methyl-4- phenylthiazol-2-ylamino)propanoic acid, (S)-4-nitrosomercapto-2-(4- phenylthiazol-2-ylamino)butanoic acid, (S)-4-nitrosomercapto-2-(5-methyl-4- phenylthiazol-2-ylamino)butanoic acid, (S)-4-nitrosomercapto-2-(thiazol-2- ylamino)butanoic acid, (S)-4-nitrosomercapto-2-(methyl(thiazol-2- yl)amino)butanoic acid, (R)-2-(4-tert-butylthiazol-2-ylamino)-3- (nitrosomercaptopropanoic acid, (R)-2-(4,5-dimethylthiazol-2-ylamino)-3- (nitrosomercapto)propanoic acid, (R)-3-nitrosomercapto-2-(4,5,6,7- tetrahydrobenzo[d]thiazol-2-ylamino)propanoic acid, (R)-3-nitrosomercapto- 2-(5-nitropyridin-2-ylamino)propanoic acid, 3-nitrosomercapto-2-(5-

(trifluoromethyl)pyridin-2-ylamino)propanoic acid, (R)-2-(4,6-bis(4- methylpiperazin-l-yl)-l, 3, 5-triazin-2-ylamino)-3-(nitrosomercapto) propanoic acid, (R)-2-(4,6-bis(dimethylamino)-l ,3,5-triazin-2-ylamino)-3- (nitrosomercapto)propanoic acid, (R)-2-(l-(4,6-bis(allylamino)-l,3,5-triazin- 2-yl)piperidin-4-ylamino)-3-(nitrosomercapto)propanoic acid, (S)-4- nitrosomercapto-2-(pyrimidin-2-ylamino)butanoic acid, (R)-2-(6- chloropyrimidin-4-ylamino)-3 -nitrosomercapto-3 -methylbutanoic acid, (R)-2- (3-nitrosomercapto-2-(thiazol-2-ylamino) propanamido)acetic acid, (R)-2- ( 1 H-tetrazol-5-yl)-2-(thiazol-2-ylamino)- 1 -nitrosomercaptoethane, (S)-4- nitrosomercapto-2-(methyl(thiazol-2-yl)amino)butanoic acid, (R)-4- nitrosomercapto-2-(methyl(thiazol-2-yl)amino)butanoic acid, (R)-3- nitrosomercapto-3-methyl-2-(thiazol-2-ylamino)butanoic acid, (S)-3- nitrosomercapto-3 -methyl-2-(thiazol-2-ylamino)butanoic acid, (2R,3R)-ethyl 1 -acetyl-3 -(nitrosomercapto)pyrrolidine-2-carboxylate, (2S,4S)- 1 -tert-butyl 2- ethyl 4-(nitrosomercapto)pyrrolidine-l,2-dicarboxylate, (2S,4S)-ethyl 1- acetyl-4-(nitrosomercapto)pyrrolidine-2-carboxylate, (2S,3S)-ethyl l-acetyl-3- (nitrosomercapto)pyrrolidine-2-carboxylate, (R)-2-acetamido-3- nitrosomercapto-N-phenylpropanamide, (R)-2-acetamido-N-(4-chlorophenyl)- 3-nitrosomercapto-propanamide, (R)-2-acetamido-N-(3-chlorophenyl)-3- nitrosomercapto-propanamide, (R)-2-acetamido-N-(2-chlorophenyl)-3- nitrosomercapto-propanamide, (R)-3-(2-acetamido-3-nitrosomercapto- propanamido)benzoic acid, (R)-ethyl 2-acetamido-3- (nitrosomercapto)propanoate, (R)-3 -acetamido-2- (nitrosomercaptomethyl)propanoic acid, (S)-2-acetamido-4-(nitrosomercapto)- butanoic acid, (R)-3-nitrosomercapto-2-(5-(4-methylpiperazin-l-yl) pentanamido)propanoic acid, (R)-2-acetamido-3 -nitrosomercapto-3 - methylbutanoic acid, (R)-2-acetamido-2-(4-methylpiperazin- 1 -yl)carbonyl- 1 - nitrosomercapto-ethane, (R)-2-acetamido-2-morpholinocarbonyl- 1 - nitrosomercapto-ethane, (S)-ethyl 1 -((R)-2-acetamido-3 -nitrosomercapto- propanoyl)-pyrrolidine-2-carboxylate, (S)-l-((R)-2-acetamido-3- nitrosomercapto-propanoyl)-pyrrolidine-2-carboxylic acid, (S)- 1 -((R)-2- (benzo[d]oxazol-2-ylamino)-3-nitrosomercapto-propanoyl)pyrrolidine-2- carboxylic acid, (S)-3-nitrosomercapto-2-(phenylsulfonamido)-propanoic acid, (R)-3-nitrosomercapto-2-(phenylsulfonamido)-propanoic acid, (R)-5-(l- carboxy-2-(nitrosomercapto)-ethylamino)pentanoic acid, (R)-3- nitrosomercapto-2-(4-(4-methylpiperazin- 1 -yl) butanamido)propanoic acid, (R)-3-nitrosomercapto-2-(5-(4-methylpiperazin-l-yl) pentanamido)propanoic acid, S-nitroso-cysteine, (R)-3-nitrosomercapto-2-(4- morpholinobutanamido)propanoic acid, (R)-2-(benzo[d]oxazol-2-ylamino)-3- nitrosomercapto-N-(methylsulfonyl)propanamide, (S)-2-(benzo[d]oxazol-2- ylamino)-3 -nitrosomercapto-3 -methyl-N-(methylsulfonyl)butanamide, (S)-2- (benzo [d]oxazol-2-ylamino)-3 -nitrosomercapto-3 -methyl-N-(phenylsulfonyl) butanamide, (R)-methyl 2-(2-(benzo[d]oxazol-2-ylthio)acetamido)-3- nitrosomercaptopropanoate, and (R)-methyl 2-(2-(benzo[d]oxazol-2-ylthio) acetamido)-3-nitrosomercapto-3-methylbutanoate, a salt thereof, and mixtures thereof.

4. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, and one or more S-nitrosomercapto-based

SNO compounds, selected from the group consisting of (S)-2-(3,3- dimethylureido)-3-(nitrosomercapto)propanoic acid, (R)-2-(3,3- dimethylureido)-3 -(nitrosomercapto)-propanoic acid, (R)-3 -nitrosomercapto- 2-(methoxycarbonylamino)propanoic acid, (S)-3-nitrosomercapto-2- (methoxycarbonylamino)propanoic acid, (S)-2-((S)-2,4-diaminobutanamido)- 3-(nitrosomercapto)propanoic acid, (R)-2-((S)-2,4-diaminobutanamido)-3- (nitrosomercapto)propanoic acid, (R)-S-nitroso-N-acetylcysteine, (S)-S- nitroso-N-acetylcysteine, (R)-2-acetamido-3 -nitrosomercapto-N- (methylsulfonyl) propanamide, (R)-N-(2-nitrosomercapto- 1 -( 1 H-tetrazol-5- yl)ethyl)acetamide, (R)-3 -nitrosomercapto-2-propionamidopropanoic acid, (R)-2-benzamido-3-(nitrosomercapto)propanoic acid, (S)-4-(2-acetamido-3- (nitrosomercapto)-propanamido)benzoic acid, (R)-4-(2-acetamido-3- (nitrosomercapto)propanamido) benzoic acid, (R)-2-acetamido-3- nitrosomercapto-3-methylbutanoic acid, (S)-2-acetamido-3-nitrosomercapto-

3-methylbutanoic acid, (S)-2-(benzo[d]oxazol-2-ylamino)-3-nitrosomercapto- 3 -methylbutanoic acid, (R)-2-(benzo [d]oxazol-2-ylamino)-3 -nitrosomercapto- 3-methylbutanoic acid, 2-(benzo[d]oxazol-2-ylamino)-3- (nitrosomercapto)propanoic acid, (R)-2-(benzo[d]oxazol-2-ylamino)-3- (nitrosomercapto)propanoic acid, (S)-methyl 2-(benzo[d]oxazol-2-ylamino)-4-

(nitrosomercapto)butanoate, (S)-methyl 2-(5-fluorobenzo[d]oxazol-2- ylamino)-4-(nitrosomercapto)butanoate, (R)-2-(6-chlorobenzo[d]oxazol-2- ylamino)-3 -nitrosomercapto-propanoic acid, (R)-3 -nitrosomercapto-2-(4- methylbenzo[d]oxazol-2-ylamino) propanoic acid, S-nitroso-(R)-2- (benzo[d]oxazol-2-ylamino)-2-(lH-tetrazol-5-yl)-l-nitrosomercaptoethane,

(R)-2-(2-(benzo[d]oxazol-2-ylamino)-3-nitrosomercapto-propanamido)acetic acid, (R)-methyl 2-(2-(benzo[d]oxazol-2-ylamino)-3- (nitrosomercapto)propanamido) acetate, 2-(benzo [d]thiazol-2-ylamino)-3 - (nitrosomercapto)propanoic acid, (S)-2-(benzo[d]thiazol-2-ylamino)-4- (nitrosomercapto)butanoic acid, (S)-methyl 4-(nitrosomercapto)-2-

(benzo[d]thiazol-2-ylamino)butanoate, (S)-2-(benzo[d]thiazol-2-ylamino)-3- nitrosomercapto-3 -methylbutanoic acid, (S)-methyl 2-(benzo[d]thiazol-2- ylamino)-3 -nitrosomercapto-3 -methylbutanoate, (R)-3 -nitrosomercapto-2- (thiazol-2-ylamino)propanoic acid, (S)-3 -nitrosomercapto-2-(thiazol-2- ylamino)propanoic acid, 3-nitrosomercapto-2-(thiazol-2-ylamino)propanoic acid, (R)-2-(5-(ethoxycarbonyl)-4-methylthiazol-2-ylamino)-3- (nitrosomercapto)propanoic acid, (R)-3-nitrosomercapto-2-(5-methyl-4- phenylthiazol-2-ylamino)propanoic acid, (S)-4-nitrosomercapto-2-(4- phenylthiazol-2-ylamino)butanoic acid, (S)-4-nitrosomercapto-2-(5-methyl-4- phenylthiazol-2-ylamino)butanoic acid, (S)-4-nitrosomercapto-2-(thiazol-2- ylamino)butanoic acid, (S)-4-nitrosomercapto-2-(methyl(thiazol-2- yl)amino)butanoic acid, (R)-2-(4-tert-butylthiazol-2-ylamino)-3- (nitrosomercaptopropanoic acid, (R)-2-(4,5-dimethylthiazol-2-ylamino)-3- (nitrosomercapto)propanoic acid, (R)-3-nitrosomercapto-2-(4,5,6,7- tetrahydrobenzo[d]thiazol-2-ylamino)propanoic acid, (R)-3-nitrosomercapto- 2-(5-nitropyridin-2-ylamino)propanoic acid, 3-nitrosomercapto-2-(5- (trifluoromethyl)pyridin-2-ylamino)propanoic acid, (R)-2-(4,6-bis(4- methylpiperazin- 1 -yl)- 1 ,3 ,5-triazin-2-ylamino)-3-(nitrosomercapto)propanoic acid, (R)-2-(4,6-bis(dimethylamino)-l,3,5-triazin-2-ylamino)-3-

(nitrosomercapto)propanoic acid, (R)-2-(l-(4,6-bis(allylamino)-l,3,5-triazin- 2-yl)piperidin-4-ylamino)-3 -(nitrosomercapto)propanoic acid, (S)-4- nitrosomercapto-2-(pyrimidin-2-ylamino)butanoic acid, (R)-2-(6- chloropyrimidin-4-ylamino)-3-nitrosomercapto-3-methylbutanoic acid, (R)-2- (3-nitrosomercapto-2-(thiazol-2-ylamino) propanamido)acetic acid, (R)-2-

( 1 H-tetrazol-5 -yl)-2-(thiazol-2-ylamino)- 1 -nitrosomercaptoethane, (S)-4- nitrosomercapto-2-(methyl(thiazol-2-yl)amino)butanoic acid, (R)-4- nitrosomercapto-2-(methyl(thiazol-2-yl)amino)butanoic acid, (R)-3- nitrosomercapto-3 -methyl-2-(thiazol-2-ylamino)butanoic acid, (S)-3 - nitrosomercapto-3-methyl-2-(thiazol-2-ylamino)butanoic acid, (2R,3R)-ethyl

1 -acetyl-3-(nitrosomercapto)pyrrolidine-2-carboxylate, (2S,4S)-1 -tert-butyl 2- ethyl 4-(nitrosomercapto)pyrrolidine-l,2-dicarboxylate, (2S,4S)-ethyl 1- acetyl-4-(nitrosomercapto)pyrrolidine-2-carboxylate, (2S,3S)-ethyl 1 -acetyl-3- (nitrosomercapto)pyrrolidine-2-carboxylate, (R)-2-acetamido-3 - nitrosomercapto-N-phenylpropanamide, (R)-2-acetamido-N-(4-chlorophenyl)-

3-nitrosomercapto-propanamide, (R)-2-acetamido-N-(3-chlorophenyl)-3- nitrosomercapto-propanamide, (R)-2-acetamido-N-(2-chlorophenyl)-3- nitrosomercapto-propanamide, (R)-3-(2-acetamido-3-nitrosomercapto- propanamido)benzoic acid, (R)-ethyl 2-acetamido-3- (nitrosomercapto)propanoate, (R)-3-acetamido-2-

(nitrosomercaptomethyl)propanoic acid, (S)-2-acetamido-4-(nitrosomercapto)- butanoic acid, (R)-3-nitrosomercapto-2-(5-(4-methylpiperazin-l -yl) pentanamido)propanoic acid, (R)-2-acetamido-3-nitrosomercapto-3- methylbutanoic acid, (R)-2-acetamido-2-(4-methylpiperazin- 1 -yl)carbonyl- 1 - nitrosomercapto-ethane, (R)-2-acetamido-2-moφholinocarbonyl- 1 - nitrosomercapto-ethane, (S)-ethyl 1 -((R)-2-acetamido-3-nitrosomercapto- propanoyl)-pyrrolidine-2-carboxylate, (S)- 1 -((R)-2-acetamido-3 - nitrosomercapto-propanoyl)-pyrrolidine-2-carboxylic acid, (S)- 1 -((R)-2- (benzo[d]oxazol-2-ylamino)-3-nitrosomercapto-propanoyl)pyrrolidine-2- carboxylic acid, (S)-3-nitrosomercapto-2-(phenylsulfonamido)-propanoic acid, (R)-3-nitrosomercapto-2-(phenylsulfonamido)-propanoic acid, (R)-5-(l- carboxy-2-(nitrosomercapto)-ethylamino)pentanoic acid, (R)-3- nitrosomercapto-2-(4-(4-methylpiperazin- 1 -yl) butanamido)propanoic acid, (R)-3-nitrosomercapto-2-(5-(4-methylpiperazin-l-yl) pentanamido)propanoic acid, S-nitroso-cysteine, (R)-3-nitrosomercapto-2-(4- morpholinobutanamido)propanoic acid, (R)-2-(benzo[d]oxazol-2-ylamino)-3- nitrosomercapto-N-(methylsulfonyl)propanamide, (S)-2-(benzo[d]oxazol-2- ylamino)-3 -nitrosomercapto-3 -methyl -N-(methylsulfonyl)butanamide, (S)-2- (benzo[d]oxazol-2-ylamino)-3-nitrosomercapto-3-methyl-N-(phenylsulfonyl) butanamide, (R)-methyl 2-(2-(benzo[d]oxazol-2-ylthio)acetamido)-3- nitrosomercaptopropanoate, and (R)-methyl 2-(2-(benzo[d]oxazol-2- ylthio)acetamido)-3-nitrosomercapto-3-methylbutanoate, a pharmaceutically acceptable salt thereof and mixtures thereof.

5. A pharmaceutical composition for stabilizing breathing rhythm in a mammal, said pharmaceutical composition comprising:

(a) A first composition comprising one or more SNO compounds, a pharmaceutically acceptable salt thereof and mixtures thereof; and, (b) A second composition comprising a second compound, wherein said second compound has one or more biological activities selected from the group consisting of stabilization of breathing rhythm, increase of the patency of the upper airway, promotion of wakefulness, decrease in incidence and/or severity of seizures, decrease in inflammation, decrease in respiratory drive, and improvement in lung function.

6. The pharmaceutical composition of claim 5, wherein said second compound is a SNO compound.

7. The pharmaceutical composition of claim 5, wherein said second compound is not a SNO compound.

8. The pharmaceutical composition of claim 5, further comprising a third compound, wherein said third compound is a SNO compound.

9. The pharmaceutical composition of claim 5, further comprising a third compound, wherein said third compound is not a SNO compound.

10. A method of stabilizing the breathing rhythm of a mammal, said method comprising administering to said mammal a therapeutically effective amount of a composition comprising one or more SNO compounds, pharmaceutically acceptable salts thereof and mixtures thereof.

11. The method of claim 10, further comprising administering a composition comprising a second compound, wherein said second compound is selected from the group consisting of a carbonic anhydrase inhibitor, a respiratory stimulant, a narcotic antagonist and a hormone.

12. The method of claim 10, further comprising administering a composition comprising a second compound, wherein said second compound is selected from the group consisting of a serotonin agonist, a serotonin antagonist, a tetracyclic antidepressant, a agent that acts on dopamine and an agent which acts on norepinephrine.

13. The method of claim 10, further comprising administering a composition comprising a second compound, wherein said second compound is selected from the group consisting of an antihistamine, a leukotriene antagonist, a 5-lipoxygenase inhibitor, a steroid, and a COX-2 inhibitor.

14. The method of claim 10, further comprising administering a composition comprising a second compound, wherein said second compound is selected from the group consisting of an opioid analgesic, a sedative hypnotic and a general anesthetic.

15. The method of claim 10, further comprising administering a composition comprising a second compound, wherein said second compound is selected from the group consisting of a steroid, a bronchodilator and an anticholinergic.

16. A method stabilizing the breathing rhythm of a mammal, said method comprising administering to a mammal a composition comprising one or more SNO compounds, pharmaceutically acceptable salts thereof and mixtures thereof, said method further comprising treating said mammal with a ventilation assist device.

17. The method of claim 16, wherein said ventilation assist device is selected from the group consisting of a mechanical ventilator, a continuous positive airway pressure (CPAP) device and a bi-level positive airway pressure (BiPAP) device.

18. The method of claim 10, wherein said composition is administered by an administration mode selected from the group consisting of parenteral, oral and buccal.

19. The method of claim 18, wherein said parenteral administration mode is selected from the group consisting of transdermal, intravenous, intramuscular and intradermal.

20. The method of claim 18, wherein said composition is administered by at least two administration modes.

21. A method of increasing minute ventilation (V_E) at the level of the brainstem respiratory control centers in the nucleus tractus solitarius of a mammmal, said method comprising the step of administering to said mammal one or more SNO compounds, a pharmaceutically acceptable salt thereof and mixtures thereof, wherein said compound has the activity of increasing minute ventilation (V_E) at the level of the brainstem respiratory control centers in the nucleus tractus solitarius.

22. A method of increasing minute ventilation (V_E) at the level of the brainstem respiratory control centers in the nucleus tractus solitarius of a mammal, said method comprising the step of administering to said mammal a therapeutic composition comprising:

(a) A first composition comprising a first compound that is a SNO compound, a pharmaceutically acceptable salt thereof and mixtures thereof; and,

(b) A second composition comprising a second compound that is not an SNO compound, wherein said second compound has the activity of increasing minute ventilation (V_E) at the level of the brainstem respiratory control centers in the nucleus tractus solitarius.

23. A method of increasing minute ventilation (V_E) at the level of the brainstem respiratory control centers in the nucleus tractus solitarius of a mammal, said method comprising the step of administering to said mammal a therapeutic composition comprising: (a) A first composition comprising a first compound that is a SNO compound; and,

(b) A second composition comprising a second compound that is not a SNO compound, wherein said second compound has the activity of increasing minute ventilation (V_E) at the level of the brainstem respiratory control centers in the nucleus tractus solitarius.

24. A method for producing a mercapto amino-derivatized compound of Formula (H),

wherein

R₁ is H, alkyl, acyl, aryl or heteroaryl;

R₂ is alkyl, acyl, aryl or heteroaryl;

R' is H, alkyl, aryl or heteroaryl; and,

Y is C₁-C₆ alkylene; comprising the steps of:

(a) converting a mercapto amino compound of Formula (E),

wherein

R₁, R' and Y are as above, to a tritylsulfanyl amino compound of Formula (F),

wherein R₁, R' and Y are as above;

(b) reacting said tritylsulfanyl amino compound of Formula (F) with R₂X, wherein

X is halide, mesylate, tosylate, triflate or carboxylate; and, R₂ is alkyl, acyl, aryl or heteroaryl, to form atritylsulfanyl amino-derivatized compound of Formula (G)

wherein

R₁, R₂, R' and Y are as above; and,

(c) treating said tritylsulfanyl amino-derivatized compound of Formula (G) with an acid-containing mixture to form said mercapto amino-derivatized compound of Formula (H).

25. The method of claim 24, wherein said mercapto amino- derivatized compound of Formula (H) is a mercapto-based SNO compound.

26. A method of producing a S-nitrosomercapto compound of

Formula (S),

wherein R₁ is H, alkyl, acyl, aryl or heteroaryl;

R₂ is alkyl, acyl, aryl or heteroaryl; R' is H, alkyl, aryl or heteroaryl; and Y is C₁-C₆ alkylene; comprising the step of treating a compound of Formula (H),

wherein

R₁, R₂, R' and Y are as above, with a nitrite equivalent, which is selected from the group consisting of organic nitrite and inorganic nitrite, optionally in the presence of a chelating agent.

27. The method of claim 26, wherein said S-nitrosomercapto compound of Formula (S) is a S-nitrosomercapto-based SNO compound.