Process for the preparation of nitrosylated propylene glycol, compositions comprising the same and medical uses thereof

Abstract

The present invention relates to a novel method of synthesizing mononitrosylated and dinitrosylated propanediols and novel compositions and pharmaceutical formulations comprising said compounds. The method is carried out by reacting the corresponding non-nitrosylated propanediol with a source of nitrite, optionally in the presence of a suitable acid. Therein, when the source of nitrite is an organic nitrite, the reaction step is performed in a suitable organic solvent and when the source of nitrite is an inorganic nitrite, the reaction step is performed in a biphasic solvent mixture comprising an aqueous phase and a non-aqueous phase. The present invention further relates to methods of treating conditions in which the administration of nitric oxide (NO) has a beneficial effect, by administering said compounds, compositions or formulations.

Description

Technical Field

[0001] This invention relates to a novel method for synthesizing mononitrosylpropanediol and dinitrosylpropanediol, as well as novel compositions and pharmaceutical formulations comprising said compounds. The invention also relates to a method for treating conditions in which the administration of nitric oxide (NO) has a beneficial effect, said method being carried out by administering said compounds, compositions, or formulations. Background Technology

[0002] The enumeration or discussion of obviously previously published literature in this specification should not be construed as an admission that the literature is part of current advanced technology or common knowledge.

[0003] Until recently, pulmonary hypertension (PH) was defined as an increase in mean pulmonary artery pressure (mPAP) at rest of 25 mmHg or higher, and it could be divided into a slower-developing chronic form (PH) and acute pulmonary hypertension (aPH). This definition has recently been updated to an increase in mean pulmonary artery pressure (mPAP) at rest of 20 mmHg or higher combined with a Wood Unit value >3. In aPH, acutely induced pulmonary vasoconstriction rapidly increases mPAP; it can be triggered as a response to various conditions such as major surgery (e.g., cardiac surgery), pulmonary embolism, and sepsis. In aPH, right ventricular adaptation has not yet developed, increasing the risk of right ventricular failure. Furthermore, patients often develop severe illness due to the triggering of aPH and typically have very low systemic blood pressure. In patients with the more chronic form of PH, aPH can superimpose on chronic PH, resulting in detrimental hypertension leading to right ventricular failure and death. Acute pulmonary hypertension is a huge problem that causes chaotic deaths and suffering for millions of people worldwide, and because diagnosis often requires right heart catheterization and there is a lack of effective selective treatments for the lungs, this problem is not fully documented and understood.

[0004] Under normal circumstances, the right ventricle draws deoxygenated blood from the systemic circulation and pumps it through the lungs, where the cardiac output in pulmonary circulation is equal to the volume of blood circulating in all other organs of the body. Despite the high flow rate through the lungs, the blood pressure in pulmonary circulation is only one-fifth that in systemic circulation. The low resistance in pulmonary circulation is attributed to the large cross-sectional area of ​​the pulmonary arteries and the fact that pulmonary vessels are much shorter than systemic vessels. The left ventricle is a powerful pump that carries blood through the systemic circulation to organs such as the liver, stomach, kidneys, and the heart itself (resisting high pressure), and it is well known that high blood pressure in systemic circulation can cause a number of health problems, including heart failure, stroke, and kidney disease.

[0005] Many physiological factors influence the complex control of blood flow in the systemic and pulmonary circulations. Blood vessels in the systemic circulation are typically in a state of vasoconstriction (small muscles in the vessel walls cause the vessels to constrict), while blood vessels in the pulmonary circulation are in a state of constant vasodilation (i.e., relaxed, widened vessels), thus maintaining very low resistance to blood flow and resulting in very low blood pressure compared to the systemic circulation.

[0006] In many life-threatening illnesses and after major surgery, the pathophysiological response accompanying a strong inflammatory reaction alters the physiological state of the systemic and pulmonary vessels. These alterations often result in a sudden vasodilation of systemic blood vessels, leading to extremely low systemic blood pressure (systemic hypotension), which can reduce blood flow to vital organs such as the brain, heart, liver, and kidneys. Paradoxically, a sudden pulmonary vasoconstriction leads to acute pulmonary hypertension and right heart failure, which reduces cardiac output and further exacerbates systemic hypotension. These critically ill and hemodynamically unstable patients often require intensive care, where the challenging task is to balance pharmacological therapy with vasopressors and cardiotonics to restore systemic blood pressure and pulmonary diaphragmatic agents to reduce acute, life-threatening pulmonary hypertension.

[0007] Acute pulmonary hypertension (aPH) is frequently missed, and treatment is often delayed (Rosenkranz, Stephan, et al., *European Heart Journal*, 37(12), 942-954 (2016)). aPH is so deadly because the right ventricle is a weak pump that normally resists low pressure, and there is a risk of failure (right ventricle failure) if the mean pressure in the pulmonary circulation rapidly reaches >40 mmHg. Acute PH is a distinct critical condition and should not be confused with chronic pulmonary hypertension (Tiller, D, et al., *PLoS One*, 8(3), e59225 (2013)). In chronic diseases, as the pressure in the pulmonary circulation gradually increases over time, the right ventricle adapts and increases in size and strength, and can then maintain a much higher outflow pressure. Even healthy individuals with conditions such as infection, pulmonary embolism (a blood clot in the lungs), or major surgery can develop aPH and experience worsening complications.

[0008] Acute pulmonary hypertension is a huge problem that causes pain, premature birth, death and suffering to millions of people around the world, and is not fully documented and understood due to a lack of proper diagnosis and treatment.

[0009] Currently, treatment options for patients developing aPH are severely limited. This is because patients typically present with extremely low systemic blood pressure. Attempting to treat aPH with intravenous (IV) vasodilators often results in fatal systemic hypotension because currently available drugs pass through the lungs (usually <30 seconds) and 'spill over' into the systemic circulation. Therefore, the best intravenous medications for treating aPH only dilate pulmonary vessels and have no effect on systemic circulation. To date, there are no intravenous pulmonary selective vasodilators on the market.

[0010] To overcome the systemic side effects of intravenously administered vasodilators, administration via inhalation of nitric oxide or prostacyclin has been developed. Unfortunately, while these drugs are effective in some cases, they are often insufficient because they are typically inactivated before reaching the target pulmonary vessels. Another major drawback of inhaled medications currently in use is that inhalation administration is more complex than intravenous infusion. The complexity of administering inhaled nitric oxide is so considerable that healthcare professionals require specialized training, and consequently, many hospitals lack the equipment even due to the associated high cost.

[0011] Nitric oxide (NO) is an important molecule in several biological systems. It is continuously produced in the lungs and can be measured at ppb (parts per billion) levels in exhaled air. The discovery of endogenous NO in exhaled air and its use as a diagnostic marker of inflammation dates back to the early 1990s (e.g., see WO 93 / 05709 and WO 95 / 02181). Today, the importance of endogenous NO is widely recognized, as evidenced by its use in clinical NO analyzers (…). The first NO analyzer customized for routine clinical use by asthma patients, manufactured by AEROCRINE AB in Solna, Sweden, has been proven.

[0012] Since these early experiments, it has been widely recognized that endogenous nitric oxide (NO) is crucial as a mediator of vasodilation in blood vessels. Specifically, nitric oxide plays a vital role in regulating pulmonary vascular tone to optimize ventilation-perfusion matching in healthy adults (i.e., matching the air reaching the alveoli with the blood reaching the alveoli via capillaries), ensuring that the oxygen supplied by ventilation is just sufficient to fully saturate the blood; see, for example, Persson et al., Acta Physiol Scand., 1990, 140, 449-57. Measuring exhaled NO is a good method for monitoring changes in endogenous NO production or clearance in the lungs (Gustafsson et al., Biochem. Biophys. Res. Commun., 1991, 181, 852-7).

[0013] Because ventilation-perfusion mismatch and elevated pulmonary artery blood pressure are characteristic of pulmonary embolism, inhaled NO has been tested as a potential treatment. For example, US 5,670,177 describes a method for treating or preventing ischemia comprising administering a gaseous mixture containing NO and carbon dioxide to a patient via an intravascular route, wherein the NO is present in an amount effective in treating or preventing ischemia. US 6,103,769 discloses a similar method, except that it uses a NO-saturated saline solution.

[0014] In addition, nitric oxide / oxygen blends are used as a last resort gas mixture in intensive care to promote capillary and lung dilation in the treatment of primary pulmonary hypertension in neonates and post-meconium aspiration associated with birth defects (see Barrington et al., Cochrane Database Syst.Rev., 2001, 4, CD000399 and Chotigeat et al., Journal of the Thai Medical Association, 2007, 90, 266-71). Similarly, NO can be administered as a rescue therapy for patients with acute right ventricular failure secondary to pulmonary embolism (Summerfield et al., Respir.Care., 2011, 57, 444-8). In Europe and Japan, inhaled NO is also approved for the treatment of acute pulmonary hypertension in patients undergoing cardiac surgery.

[0015] As an alternative to providing NO in a gaseous or dissolved form, the use of NO delivery compounds has been investigated. For example, WO 94 / 16740 describes the use of NO delivery compounds such as S-nitrosothiols, thionitrites, thionitrates, sydnonimines, furazolidone oxides, organic nitrates, nitroprussides, nitroglycerin, iron-nitrosoyl compounds, etc., for the treatment or prevention of alcoholic liver injury.

[0016] Currently, nitrates are used to treat the symptoms of angina (chest pain). Nitrates work by relaxing blood vessels and increasing the supply of blood and oxygen to the heart while reducing its workload. Examples of currently available nitrate medications include:

[0017] a) Nitroglycerin (trinitroglycerin ester) (1,2,3-propanetriol nitrate), now mostly administered sublingually, is used to suppress acute attacks of angina. However, severe headache and dizziness due to its rapid and widespread vasodilation are frequent side effects. Nitroglycerin infusion concentrate, diluted in isotonic glucose or normal saline, can also be used for intravenous infusion.

[0018] b) Isosorbide mononitrate (1,4:3,6-didehydro-D-glucol-5-nitrate), which is considered a preventative agent for angina. Tolerance development is an issue in long-term treatment regimens. Common side effects of nitroglycerin include headache and dizziness.

[0019] c) Isosorbide dinitrate (1,4:3,6-didehydro-D-glucol-2,5-nitrate), which is used acutely and prophylactically for angina and heart failure.

[0020] d) Pentaerythritol nitrate, a group of organic nitrates, is known to exert long-term antioxidant and anti-atherosclerotic effects through currently unknown mechanisms. Pentaerythritol tetranitrate has been investigated for nitrate tolerance (an undesirable development in nitrate therapy) and has been experimentally tested in pulmonary hypertension.

[0021] Many of these nitrate compounds, as well as other nitrate and nitrite compounds, have been tested in vivo and found to produce NO. For example, trinitroglycerin, ethyl nitrite, isobutyl nitrate, isobutyl nitrite, isoamyl nitrite, and butyl nitrite have been tested in rabbit models and found to have a significant correlation between their NO production in vivo and their effects on blood pressure (Cederqvist et al., Biochem. Pharmacol., 1994, 47, 1047-53).

[0022] Therefore, certain organic nitrites have been shown to be effective in treating male impotence and erectile dysfunction by application to the penile surface or corpora cavernosa (see US 5,646,181).

[0023] Recently, the roles of dietary nitrates and nitrites have been reassessed, specifically as endogenous producers of NO in the arginine-nitric oxide system, and their role in host defense has been identified (Larsen et al., *The New England Journal of Medicine*, 2006, 355, 2792-3). Therefore, L-arginine and its esters, such as ethyl-L-arginine, methyl-L-arginine, and butyl-L-arginine, have been used to increase endogenous NO production.

[0024] WO 2006 / 031191 describes compositions and methods for the therapeutic delivery of gaseous nitric oxide. Such compositions for delivering gaseous NO contain compounds, such as alcohols, carbohydrates, and proteins, capable of forming reversible bonds or associating with NO.

[0025] WO 2007 / 106034 describes a method for producing organic nitrites from a compound, said compound being a mono- or polyol or an aldehyde or ketone derivative thereof. The method comprises degassing an aqueous solution of said compound and then purging it with gaseous nitric oxide (NO).

[0026] Nilsson, KF et al., Biochemical Pharmacology, 82(3), 248-259 (2011) discussed the formation and identification of novel bioactive organic nitrites.

[0027] Despite recent advancements, existing compounds, compositions, and preparation methods still have many drawbacks.

[0028] For example, many of the compounds and compositions currently available are associated with undesirable properties or side effects, such as toxicity issues, delayed action, irreversible effects, or prolonged action. One particular problem frequently encountered when NO-donor compounds are administered via infusion is the production of methemoglobin (metHb).

[0029] Furthermore, known organic nitrites and their therapeutic uses are frequently associated with problems that may be attributed to impurities and degradation products present in the composition. The preparation of pharmaceutical formulations containing organic nitrites is also challenging because the mixing steps and mediators used can trigger further degradation.

[0030] In addition, the use of inhaled nitric oxide and oxygen poses serious problems due to the generation of nitrogen dioxide, and must be continuously monitored during application.

[0031] Some existing preparation methods only provide relatively low concentrations of organic nitrites in aqueous solutions, which means that the storage and transport properties of such formulations are often unsatisfactory.

[0032] In addition to the desired organic nitrites, the existing preparation methods also result in the dissolution of large amounts of NO gas and inorganic nitrites in the solution. Due to the high reactivity of NO, the solution must be handled and stored carefully to avoid sudden and spontaneous decomposition. NO gas may also react with the plastic materials in the storage container.

[0033] In addition, the presence of inorganic nitrites can increase the proportion of metHb in the blood, which is a dose-limited side effect.

[0034] Therefore, there is an urgent need for a method for preparing NO-delivery compounds and compositions comprising them, which overcomes one or more drawbacks associated with existing preparation methods and compositions. There is also a need for a method that allows the use of compounds and compositions obtained from such methods. Detailed Implementation

[0035] The inventors have unexpectedly discovered a method for preparing NO-delivery compounds that overcomes one or more drawbacks associated with prior art preparation methods.

[0036] For example, the method of the present invention provides a relatively high concentration of the compound of the present invention in solution, thereby providing ease of handling and minimizing storage volume and transportation costs. Furthermore, the method of the present invention does not result in dissolved nitric oxide gas or inorganic nitrites, thereby minimizing the risk of sudden and spontaneous decomposition and reducing the likelihood of side effects when the product of the method is used for treatment. The method of the present invention also results in only very low levels of other impurities.

[0037] Furthermore, the inventors have discovered that such methods can deliver chemically stable non-aqueous compositions and formulations containing these compounds, which allows for convenient transport and storage prior to therapeutic use. Additionally, the inventors have developed convenient means of using such compositions and formulations by combining them with a suitable aqueous buffer solution.

[0038] method

[0039] In a first aspect of the invention, a method is provided for preparing a composition comprising one or more compounds of formula I.

[0040]

[0041] in:

[0042] R 1 R 2 and R 3 Each can be independently represented as H or -NO;

[0043] n is 0 or 1;

[0044] Where, when n is 0, then R 1 Let H be the sum of the values ​​of n and n, and when n is 1, R is the sum of the values ​​of n and n. 2 For H; and

[0045] The condition is R 1 R 2 and R 3 At least one of them represents -NO,

[0046] The method includes the following steps:

[0047] (i) Optionally, in the presence of a suitable acid, the corresponding R is such that 1 R 2 and R 3 This indicates that compound I of formula H reacts with nitrite / ester source.

[0048] in:

[0049] (a) When the nitrite / ester source is an organic nitrite, step (i) is performed in a suitable organic solvent; and

[0050] (b) When the nitrite / ester source is an inorganic nitrite, step (i) is performed in a two-phase solvent mixture comprising an aqueous phase and a non-aqueous phase.

[0051] The method described herein may be referred to as "the method of the present invention" or similar terms.

[0052] To avoid any doubt, the product of the method of the present invention (i.e., the compound of formula I) may also (or alternatively) be referred to as mono- and dinitrosylated 1,2-propanediol or 1,3-propanediol (or a mixture of such compounds, i.e. a composition containing one or more mono- or dinitrosylated 1,2- or 1,3-propanediol).

[0053] To avoid ambiguity, the corresponding R 1 R 2 and R 3 Compounds of formula I representing H can be referred to as the corresponding 1,2-propanediol and / or 1,3-propanediol (i.e., the structure corresponding to the desired product). In other words, the corresponding compound of formula I can be a compound according to formula (Ia) as defined below.

[0054]

[0055] To avoid ambiguity, if the integer (n or 1-n) related to the oxygen atom is 0, then there is no oxygen atom and the substituent R is not present. 1 and R 2 (and the corresponding H in the compound of formula (Ia)) is bonded to the corresponding carbon.

[0056] Those skilled in the art will understand that references to the methods of the present invention (or similarly, “methods of the present invention”, etc.) will include references to all embodiments and their specific features.

[0057] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0058] Without departing from the disclosure of this invention, all embodiments of the invention and specific features mentioned herein may be used alone or in combination with any other embodiments and / or specific features mentioned herein (therefore more specific embodiments and features as disclosed herein are described).

[0059] As used herein, the term "comprising" will have its usual meaning in the art, indicating that the component includes, but is not limited to, the relevant features (i.e., including, among other things). Thus, the term "comprising" will include components that refer to substances primarily composed of the relevant substances.

[0060] As used herein, unless otherwise stated, the terms “consists essentially of” and “consisting essentially of” will mean that the relevant component is formed of at least 80% (e.g., at least 85%, at least 90%, or at least 95%, such as at least 99%) of (one or more) of the specified substance, according to the relevant measure (e.g., by weight). The terms “consists essentially of” and “consisting essentially of” may be replaced by “consists of” and “consisting of”, respectively.

[0061] To avoid ambiguity, the term "comprising" will also include references to components consisting of (one or more) related substances.

[0062] Thus, those skilled in the art will understand that reference to the preparation of a composition comprising one or more compounds of formula (I) means the preparation of a composition comprising a certain amount of one or more compounds as components, optionally together with other compounds, the structures of which are defined as in formula I. The method of the present invention may also refer to a method for preparing compounds of formula I (i.e., a method for preparing one or more compounds of formula I).

[0063] Those skilled in the art will understand that references to methods for preparing compounds of formula I should be understood as indicating that the methods of the present invention can result in the preparation of one or more types of compounds, each as described by formula I as defined herein (e.g., as a mixture thereof in the presence of more than one such compound).

[0064] Thus, those skilled in the art will also understand that the compounds formed in the method of the present invention can take the form of a mixture of each mononitrite and dinitrite product, the relative amount of each product varying according to the concentration of the compound of Formula I.

[0065] Specifically, the method of the present invention allows for the preparation of compositions in which at least 50 wt%, 60 wt%, 70 wt%, or 80 wt% (e.g., at least 90 wt% or at least 99 wt%, for example, at least 99.9 wt%) of a compound of formula I is mononitrified, such that R 1 R2 and R 3 Each can be represented independently as H or -NO, provided that R is... 1 R 2 Or R 3 One of them represents -NO and the other groups represent H.

[0066] Specifically, the method of the present invention can lead to the preparation of compounds comprising one or more of formula I along with one or more corresponding R. 1 R 2 and R 3 A composition of a compound of formula I representing H (i.e., 1,2-propanediol and / or 1,3-propanediol, such as unreacted 1,2-propanediol and / or 1,3-propanediol starting materials) and optionally other compounds.

[0067] In some embodiments, the method of the present invention may be for preparing compounds mainly composed of one or more of formula I and one or more corresponding R. 1 R 2 and R 3 A method for representing a composition of a compound of formula I (i.e., 1,2-propanediol and / or 1,3-propanediol; for example, as a mixture thereof) of H.

[0068] Those skilled in the art will understand that the term "reaction" refers to bringing together the relevant components in a manner that causes a chemical reaction to occur (e.g., in suitable conditions and media). Specifically, reference to reacting the starting material (i.e., 1,2-propanediol and / or 1,3-propanediol) with a nitrite / ester source will refer to a chemical reaction between the starting material and the nitrite / ester (i.e., the nitrite / ester provided by the nitrite / ester source).

[0069] Those skilled in the art will understand that referring to "nitrite / ester source" can alternatively simply mean "nitrite / ester," since it is the nitrite / ester source that provides the nitrite / ester that undergoes the chemical reaction. Thus, referring to a nitrite / ester source will be understood as referring to a compound that provides the nitrite / ester moiety (depending on the nitrite / ester source present, which may exist in ionic or covalent form) for the reaction. Therefore, a nitrite / ester source can be referred to as a source of reactive (or reactivity-prone) nitrite / ester (or nitrite / ester moiety). To avoid ambiguity, a nitrite / ester source can be an inorganic nitrite or an organic nitrite ester.

[0070] As indicated herein, when the nitrite / ester source is an organic nitrite, step (i) is performed in a suitable organic solvent.

[0071] Those skilled in the art will understand that various organic nitrites, such as alkyl nitrites, can be used in the methods of this invention.

[0072] Specific alkyl nitrites that may be mentioned include ethyl nitrite, propyl nitrite, butyl nitrite, and amylene pentyl nitrite. In specific embodiments, the alkyl nitrite is n-butyl nitrite, isobutyl nitrite, or tert-butyl nitrite, such as tert-butyl nitrite.

[0073] When the nitrite / ester source is an organic nitrite ester, a person skilled in the art will be able to select a suitable solvent. For example, suitable solvents may include those suitable organic components, and mixtures thereof, referred to herein as biphasic solvent systems.

[0074] To avoid ambiguity, unless otherwise stated, references to the execution of the methods of the present invention in suitable organic solvents do not imply the presence of other non-organic solvents, such as water.

[0075] In certain embodiments, where the method of the invention is performed in a suitable organic solvent, the solvent may be substantially anhydrous (which may be referred to as “anhydrous” or “dry”), which may indicate that the solvent contains less than about 1% by weight (e.g., less than about 0.1% by weight, such as less than about 0.01% by weight) of water.

[0076] The term “about” is defined herein as meaning that the defined value may deviate from ±10%, such as ±5%, for example ±4%, ±3%, ±2%, or ±1%. The term “about” may be removed from the entire specification without departing from the teachings of the invention.

[0077] As indicated herein, when the nitrite / ester source is an inorganic nitrite, step (i) is performed in a two-phase solvent mixture comprising an aqueous phase and a non-aqueous phase.

[0078] Those skilled in the art will understand that, as used herein, the term "two-phase solvent mixture" will refer to a system consisting of two solvents or solvent mixtures that do not mix to form a single solvent phase, but rather exist as two distinct (i.e., non-mixable) phases.

[0079] When such solvent mixtures contain water and organic solvents (or mixtures of organic solvents), it can be said that such solvent systems contain both an "aqueous phase" and an "organic phase." To avoid ambiguity, the term biphase does not indicate the presence of substances that form other phases besides the solvent system, such as substances that form a solid phase (that is, other phases may also exist).

[0080] Specific inorganic nitrite / ester sources that can be mentioned include metal nitrites, such as alkali metal nitrites and alkaline earth metal nitrites. Ionic liquids can also be suitable inorganic nitrite / ester sources.

[0081] To avoid ambiguity, the term alkali metals will have its usual meaning in the art, referring to IUPAC Group 1 elements and cations, including lithium, sodium, potassium, rubidium, cesium, and francium.

[0082] To avoid ambiguity, the term alkaline earth metals will have its usual meaning in the art, referring to IUPAC Group 2 elements and cations, including beryllium, magnesium, calcium, strontium, barium, and radium.

[0083] More specific inorganic nitrites that may be mentioned include alkali metal nitrites such as lithium nitrite, sodium nitrite, and potassium nitrite. In a particular embodiment, the nitrite / ester source is sodium nitrite.

[0084] Alternatively, the metal nitrite can be an alkaline earth metal nitrite, such as lithium nitrite, magnesium nitrite, or calcium nitrite.

[0085] To avoid any ambiguity, those skilled in the art will understand that the non-aqueous phase in a two-phase solvent system can be an organic solvent, and therefore can be referred to as the organic phase.

[0086] Technicians will be able to select suitable non-aqueous (i.e., organic) solvents based on the properties of the aqueous phase. For example, in cases where a certain level of a substance (e.g., an ionic solid, such as a salt) is dissolved in the aqueous phase, various organic solvents can be selected to form a two-phase solvent system.

[0087] In certain embodiments, the non-aqueous phase consists of an organic solvent that is immiscible with water. In more specific embodiments, the organic solvent that is immiscible with water is an aprotic organic solvent.

[0088] Specific water-immiscible organic solvents that can be mentioned (i.e., specific solvents that form a non-aqueous phase) include ethers (e.g., tert-butyl methyl ether, cyclopentyl methyl ether, methyltetrahydrofuran, diethyl ether, diisopropyl ether) and dichloromethane (DCM).

[0089] More specific water-immiscible organic solvents that may be mentioned (i.e., specific solvents that form a non-aqueous phase) include dichloromethane, diethyl ether, and tert-butyl methyl ether. In a more specific embodiment, the water-immiscible organic solvent is tert-butyl methyl ether.

[0090] In some of the embodiments that may be mentioned, the solvent mixture may contain an excess of R. 1 R 2 and R 3 The compound of formula I represents H (i.e., 1,2-propanediol and / or 1,3-propanediol). To avoid ambiguity, in this case, 1,2-propanediol and / or 1,3-propanediol (i.e., where R...) 1 R 2 and R 3Compounds of formula I (representing H) can exist as both a solvent (e.g., a component of a solvent mixture) and a reactant. Thus, in specific embodiments, the method involves preparing a compound of formula I such that R is present in the corresponding... 1 R 2 and R 3 Methods representing solutions of compounds of formula I, i.e., 1,2-propanediol and / or 1,3-propanediol (e.g., in the form of a mixture comprising 1,2-propanediol and / or 1,3-propanediol, as applicable). In some embodiments, when the nitrite / ester source is an organic nitrite, the solvent may be primarily composed of R. 1 R 2 and R 3 The composition of compound I (i.e., 1,2-propanediol and / or 1,3-propanediol) represents H. That is, where R... 1 R 2 and R 3 Compounds of formula I representing H can act as both solvents and reactants.

[0091] In an alternative embodiment, when the nitrite / ester source is an inorganic nitrite, step (i) can be performed in a single solvent, wherein the solvent may consist primarily of R. 1 R 2 and R 3 The composition of compound I (i.e., 1,2-propanediol and / or 1,3-propanediol) represents H. That is, where R... 1 R 2 and R 3 Compounds of formula I representing H can act as both solvents and reactants.

[0092] In an alternative embodiment, relative to where R 1 R 2 and R 3 The starting materials of formula I representing H (i.e., 1,2-propanediol and / or 1,3-propanediol) can be used to carry out the method of the present invention with an excess of nitrite / ester.

[0093] As used herein, the term “excess” will have its usual meaning in the art, indicating that the component is present in an amount greater than the stoichiometric amount for the reaction in which it is a reactant.

[0094] As indicated herein, the method of the present invention (specifically, the reaction between components) is optionally carried out in the presence of a suitable acid.

[0095] Specific methods of the invention that may be mentioned include those in which the step of reacting the starting material (i.e., 1,2-propanediol and / or 1,3-propanediol) with a nitrite / ester source is carried out in the presence of a suitable acid.

[0096] Specific acids that can be mentioned as suitable acids include Brønsted acids (i.e., proton donor acids), and more specifically, these acids can be called strong acids.

[0097] For the avoidance of ambiguity, the term "strong acid" will have its usual meaning in the art, referring to a Brønsted acid that is substantially completely dissociated in aqueous solution under equilibrium conditions. Specifically, reference to a strong acid may refer to a Brønsted acid having a pKa (in water) of less than about 5 (e.g., less than about 4.8). For the avoidance of ambiguity, for polyprotic acids such as sulfuric acid, the term strong acid refers to the dissociation of the first proton.

[0098] Certain strong acids that may be mentioned include those with a pKa (in water) less than about 1, such as those less than about 0 (e.g., less than about -1 or -2). For example, strong acids that may be mentioned include those with a pKa (in water) of about -3. Those skilled in the art will understand that, as is known to those skilled in the art, suitable acids may include non-nucleotide-dependent nucleic acids.

[0099] Particularly suitable acids that can be mentioned include sulfuric acid, phosphoric acid, trifluoroacetic acid, and acetic acid.

[0100] More specific suitable acids that can be mentioned include inorganic acids (e.g., strong inorganic acids), such as sulfuric acid.

[0101] Within the teachings of this invention, those skilled in the art will be able to select appropriate amounts of reactants for use in the method of this invention. For example, corresponding to R 1 R 2 and R 3 The ratio (i.e., molar ratio) of the compound of formula I representing H to the nitrite / ester to the acid (in the presence of the nitrite) can be about 1:about 1 to about 5:about 0.5 to about 3.5, for example about 1:about 1 to about 3:about 0.5 to about 2 (e.g., about 1:4:2.7, or about 1:2:0.95, or about 1:2:1). To avoid ambiguity, in the absence of a suitable acid, the corresponding R... 1 R 2 and R 3 The ratio between the compound of formula I representing H and the nitrite / ester remains applicable.

[0102] In a particular embodiment, method step (i) is performed at a temperature of about -30°C to about 5°C, such as about -30°C to about 0°C, for example about -30°C to about -10°C, preferably about -25°C to about -15°C.

[0103] In certain embodiments, method step (i) is performed in an inert atmosphere such as nitrogen or argon, preferably argon. Furthermore, in certain embodiments, any step of the method may be performed in an inert atmosphere such as nitrogen or argon, preferably argon.

[0104] Specific methods of the present invention that may be mentioned, particularly those using a two-phase solvent system, include those that further comprise the following steps after step (i) (e.g., directly after step (i):

[0105] (ii) Remove substantially all of the aqueous phase from the solvent mixture (i.e., remove substantially all of the water).

[0106] Those skilled in the art will understand that the aqueous phase can be removed from the solvent mixture by any suitable method and using any suitable equipment known in the art (e.g., by using a separatory funnel or similar equipment).

[0107] As used herein, unless otherwise stated, the term “substantially all” will mean at least 80% (e.g., at least 85%, at least 90%, or at least 95%, such as at least 99%) of the specified substance, according to the relevant measure (e.g., by weight).

[0108] Technicians will also understand that the phrase “removing substantially all of the aqueous phase from the solvent mixture” can be replaced by the phrase “removing some or all of the aqueous phase from the solvent mixture” or simply “removing the aqueous phase from the solvent mixture”.

[0109] To avoid ambiguity, in the context removed, the term aqueous phase will refer to the (separated) phase formed by water and the components dissolved therein.

[0110] Specific methods of the present invention that may be mentioned, particularly those using a two-phase solvent system, include those methods that further comprise (in the order shown) the following steps after step (i) (e.g., directly after step (i)):

[0111] (ii) Remove some or all (e.g., virtually all) of the aqueous phase (i.e., water);

[0112] (iii) Wash the remaining organic phase with one or more other aqueous phases;

[0113] (iv) Optionally repeat steps (ii) and (iii) once or more.

[0114] Other methods of the invention that may be mentioned, particularly those using a two-phase solvent system, include those that further comprise (in the order shown) the following steps after step (i) (e.g., directly after step (i)):

[0115] (ii) Remove some or all (e.g., virtually all) of the aqueous phase (i.e., water);

[0116] (iii) Wash the remaining organic phase with one or more other aqueous phases;

[0117] (iv) Optionally repeat steps (ii) and (iii) once or more;

[0118] (v) Optionally reduce the organic phase (i.e., reduce the amount / volume of the organic phase), such as by removing some or substantially all of the water-immiscible organic solvents (e.g., organic solvents other than 1,2-propanediol and / or 1,3-propanediol), and

[0119] (vi) Optionally dry the product.

[0120] Steps (ii) to (vi) can be performed in any order, provided that steps (ii) to (iv) are performed before steps (v) and (vi).

[0121] In certain embodiments, method steps (ii) to (iv) can be performed at temperatures from about -20°C to about 5°C, such as from about -10°C to about 5°C.

[0122] In a particular embodiment, method step (v) can be performed at a temperature of about 0°C to about 30°C, such as about 10°C to about 30°C, for example about 15°C to about 30°C.

[0123] In a particular embodiment, method step (v) is performed for no more than 6 hours, for example, no more than 5 hours, preferably no more than 4 hours.

[0124] In a particular embodiment, each of steps (ii) through (vi) is performed, as indicated in the order indicated.

[0125] To avoid any ambiguity, those skilled in the art will understand that washing the remaining organic phase with one or more additional aqueous phases will refer to the following steps: adding another portion of an aqueous solvent (e.g., water); mixing with the (separated) organic phase (e.g., by stirring and / or shaking together); and removing substantially all of the aqueous phase, and optionally repeating the steps once or more.

[0126] Those skilled in the art will understand that step (iii) can be performed by any suitable method and using any suitable equipment known in the art (e.g., using a separatory funnel).

[0127] Those skilled in the art will understand that step (v) can be performed by any suitable method and using any suitable equipment known in the art (e.g., by evaporation under reduced pressure).

[0128] In the context of step (v), references to the removal of some organic phases can specifically refer to the removal of substantially all water-immiscible organic solvents as defined herein. More specifically, the removal of water-immiscible organic solvents can refer to the removal of at least 99% by weight (e.g., at least 99.5% by weight, 99.9% by weight, or specifically 99.99% by weight) of water-immiscible organic solvents.

[0129] This removal of water-immiscible organic solvents can also refer to removal in such a way that the product after such removal contains less than 1% by weight (e.g., less than 0.5% by weight, 0.1% by weight, for example less than 0.05% by weight, less than 0.01% by weight) of water-immiscible organic solvents.

[0130] To avoid ambiguity, in the context of step (v), reference to the removal of an organic phase, such as an organic solvent immiscible with water, will refer to the removal of any such solvent as defined herein (e.g., the removal of dichloromethane or tert-butyl methyl ether). In the presence of additional organic solvents (such as those immiscible with water, e.g., excess 1,2-propanediol and / or 1,3-propanediol acting as a solvent), a portion of such solvents may also be removed (e.g., along with the water-immiscible organic solvent).

[0131] In the context of step (vi), reference to dried product refers to the removal of water from the material remaining after the aforementioned steps. Such removal of water may refer to the removal such that the dried product contains less than 1% by weight (e.g., less than 0.5% by weight or less than 0.1% by weight, for example, less than 0.05% by weight or less than 0.01% by weight) of water.

[0132] Those skilled in the art will understand that step (vi) can be performed by any suitable method and using any suitable equipment known in the art (e.g., by contacting the remaining organic phase with a suitable drying agent such as anhydrous sodium sulfate, anhydrous magnesium sulfate and / or molecular sieve).

[0133] Specific methods of the invention that may be mentioned include those in which the method further comprises (e.g., after step (i), and if present, other steps as described herein) the following steps: adding an additional amount of the corresponding R 1 R 2 and R 3 Compounds of formula I representing H (i.e., 1,2-propanediol and / or 1,3-propanediol) such that one or more compounds of formula I and the corresponding R are... 1 R 2 and R 3The combined mixture of compounds of formula I representing H (i.e., 1,2-propanediol and / or 1,3-propanediol) contains about 0.01% to about 9% by weight (e.g., about 0.01% to about 5% by weight, such as about 3% to about 5% by weight, or about 5% to about 7% by weight) of one or more compounds of the present invention.

[0134] As outlined above, all embodiments of the invention and specific features mentioned herein may be used alone or in combination with any other embodiments and / or specific features mentioned herein without departing from the disclosure of the invention (therefore more specific embodiments and specific features as disclosed herein are described).

[0135] For example, method step (i) performed at a temperature of about -30°C to about 5°C can be combined with the following: method steps (ii) to (iv) features performed at a temperature of about -20°C to about 5°C; method step (v) features performed at a temperature of about 0°C to about 30°C; and / or method step (v) features performed for no more than 6 hours.

[0136] More specific methods that may be mentioned include those in which the parameters specified are based on the examples provided in this article.

[0137] The specific product of the method of the present invention is a compound according to formula (II).

[0138]

[0139] Where R 2 and R 3 Each can be represented independently as H or -NO, provided that R is... 2 and R 3 At least one of them represents -NO, wherein the method includes the step of reacting 1,2-propanediol (i.e. the starting material) with a nitrite / ester source under the conditions described herein (including all examples thereof).

[0140] There are two enantiomers of the compound according to formula (II), in R and S forms, as described below:

[0141]

[0142] Another specific product of the method of the present invention is a compound according to formula (III) as described below:

[0143]

[0144] Where R 1 and R 3 Each can be represented independently as H or -NO, provided that R is... 1 and R 3At least one of them represents -NO, wherein the method includes the step of reacting 1,3-propanediol with a nitrite / ester source.

[0145] The two specific methods described above for producing compounds according to formulas (II) and (III) can be carried out together or independently of each other.

[0146] Based on the biphasic nature of the reaction mixture, the optional addition of a phase-transfer catalyst (PTC) can support product formation. Common PTCs include, but are not limited to, tetraalkylammonium ions such as Me4N+, Et4N+, Bu4N+, or Bu3(N+)CH2PHCl, as well as counter ions such as =Cl-, Br-, HSO4-, or other types of alkylammonium PTCs, such as... 336, stoichiometry < 1 equivalent, for example, but not exclusively, in the range of about 0.05 to about 40 mol%, such as about 0.1 to about 30 mol%, such as about 0.1 to about 20 mol%.

[0147] Another specific product of the method of the present invention is a compound according to formula (IV) as described below.

[0148]

[0149] Where R 4 and R 5 Each can be represented independently as H or -NO, provided that R is... 4 and R 5 At least one of them represents -NO.

[0150] Therefore, a specific method of the present invention is used for preparing compositions comprising one or more compounds of formula (IV).

[0151]

[0152] Where R 4 and R 5 Each can be represented independently as H or -NO, provided that R is... 4 and R 5 At least one of them represents -NO,

[0153] The method includes the following steps:

[0154] (i) Optionally, in the presence of a suitable acid, 1,2-propanediol is reacted with a nitrite / ester source.

[0155] in:

[0156] (a) When the nitrite / ester source is an organic nitrite, step (i) is performed in a suitable organic solvent; and

[0157] (b) When the nitrite / ester source is an inorganic nitrite, step (i) is performed in a two-phase solvent mixture containing an aqueous phase and a non-aqueous phase.

[0158] Any method steps outlined herein can be combined with the specific method described above for formula (IV), and specific embodiments are outlined below.

[0159] In a particular method, the inorganic nitrite is a metal nitrite, optionally wherein the metal nitrite is an alkali metal nitrite or an alkaline earth metal nitrite, preferably an alkali metal nitrite.

[0160] In a specific embodiment, the alkali metal nitrite is sodium nitrite.

[0161] In another specific embodiment, the organic nitrite is an alkyl nitrite, such as tert-butyl nitrite.

[0162] In certain methods, the suitable acid is a strong acid, such as a strong inorganic acid (e.g., sulfuric acid).

[0163] In certain embodiments, the non-aqueous phase comprises an organic solvent that is immiscible with water, such as an aprotic organic solvent that is immiscible with water.

[0164] In the examples, the organic solvent that is immiscible with water is dichloromethane.

[0165] In certain methods, the solvent mixture further comprises an excess of 1,2-propanediol.

[0166] In another specific method, after step (i), the method further includes the following steps:

[0167] (ii) Remove substantially all of the aqueous phase from the solvent mixture.

[0168] In one embodiment, after step (i), the method further includes the following steps:

[0169] (ii) Remove some or all (e.g., virtually all) of the aqueous phase (i.e., water);

[0170] (iii) Wash the remaining organic phase with one or more other aqueous phases;

[0171] (iv) Optionally repeat steps (ii) and (iii) once or more;

[0172] (v) Optionally reduce the organic phase (i.e., reduce the amount / volume of the organic phase),

[0173] as well as

[0174] (vi) Optionally dry the product.

[0175] Steps (ii) to (vi) can be performed in any order, provided that steps (ii) to (iv) are performed before steps (v) and (vi).

[0176] In a particular embodiment, the method further comprises the step of adding an additional amount of 1,2-propanediol, such that the combined mixture of one or more Formula I compounds and 1,2-propanediol comprises about 0.01% by weight to about 9% by weight of one or more Formula IV compounds.

[0177] Products and compositions

[0178] In a second aspect of the invention, products prepared using the methods of the invention (such as products obtained by or obtainable by the methods of the invention) (i.e., products produced according to the first aspect of the invention, including all embodiments and specific features thereof), are provided, said products may be referred to as "compounds of the invention".

[0179] The compounds of this invention may contain asymmetric carbon atoms as outlined above, and thus will exhibit optical isomerism. Various stereoisomers can be eliminated by separating racemic or other mixtures of the compounds using conventional techniques, such as stepwise crystallization or HPLC. Alternatively, the desired optical isomers can be prepared by reacting a suitable optically active starting material with a chiral auxiliary under conditions that do not induce racemization (i.e., the “chiral pool” method), which can then be removed at an appropriate stage by derivatization (i.e., resolution, including dynamic resolution); for example, with a pure chiral acid, followed by conventional methods such as chromatography, or by reaction with a suitable chiral reagent or chiral catalyst, under conditions known to those skilled in the art. All stereoisomers and mixtures thereof are included within the scope of this invention.

[0180] The method of the present invention advantageously allows for the preparation of substantially non-aqueous compositions comprising one or more compounds of the present invention. Specifically, the method of the present invention allows for relatively high concentrations of one or more compounds of the present invention in the composition, thereby providing ease of handling and minimizing storage volume and transportation costs.

[0181] Therefore, in a third aspect of the invention, a substantially non-aqueous composition is provided, comprising:

[0182] (a) One or more compounds of formula I as defined herein; and

[0183] (b) One or more corresponding R 1 R 2 and R 3Compounds of formula I representing H (e.g., 1,2-propanediol and / or 1,3-propanediol),

[0184] The composition may be referred to below as "the substantially non-aqueous composition of the present invention".

[0185] Those skilled in the art will understand that the substantially non-aqueous compositions of the present invention mentioned herein will include all embodiments and their specific forms.

[0186] As used herein, reference to “substantially non-aqueous” will refer to a component containing less than 1% by weight (e.g., less than 0.5% by weight or less than 0.1% by weight, such as less than 0.05% by weight or less than 0.01% by weight).

[0187] Specific, substantially non-aqueous compositions of the present invention that may be mentioned include those in which the composition comprises about 0.01 wt% to about 9 wt% (e.g., about 0.01 wt% to about 5 wt%, such as about 3 wt% to about 5 wt%, or about 5 wt% to about 7 wt%) of one or more compounds of the present invention (i.e., compounds of formula I).

[0188] Specific, substantially non-aqueous compositions of the present invention that may be mentioned include those in which the composition comprises a compound according to formula (II). Preferably, the compound according to formula (II) is in the S form.

[0189] The S form of the compound according to formula (II) is preferred because it has a higher metabolic rate than the R form. Furthermore, the S form has a different metabolic degradation pathway, resulting in lower toxicity of its metabolites compared to those of the R form.

[0190] Specific, substantially non-aqueous compositions of the present invention that may be mentioned include those in which the composition comprises a compound according to formula (III).

[0191] Preferably, the compound according to formula (II) is in the S form, but it is conceivable that the product is a mixture of the S and R forms of formula (II), wherein the S form is preferably present in enantiomeric excess (ee).

[0192] In certain embodiments, when the product of the method is a compound according to formula (II), the compound according to formula (II) may be in excess of the enantiomer in the S form of the compound. That is, more than 50 ee% of the product is in the S form, such as more than or equal to 60 ee%, 70 ee%, 80 ee%, 90 ee%, 95 ee%, or 98 ee% of the product is in the S form.

[0193] To achieve an enantiomeric excess of the S form of compound (II), in the examples, the starting material (i.e., 1-2-propylene glycol) may be present in an enantiomeric excess in the S form. That is, 50 ee% or more of the starting material (i.e., 1-2-propylene glycol) is in the S form, such as 60 ee%, 70 ee%, 80 ee%, 90 ee%, 95 ee%, or 98 ee% of the starting material is in the S form.

[0194] In embodiments where the product is a mononitrosylated compound according to formula (II), greater than 50% by weight of the product is nitrosylated at position 2 (i.e., R). 2 (for -NO), such as being nitrosylated at the 2 position between about 55 wt% and about 80 wt%, for example between about 55 wt% and 75 wt%.

[0195] Specific, substantially non-aqueous compositions of the present invention may be mentioned, wherein the composition comprises primarily one or more compounds of formula I and corresponding compounds wherein R 1 R 2 and R 3 The composition of H represents a compound of formula I (i.e., 1,2-propanediol and / or 1,3-propanediol).

[0196] Specifically, when the starting material is 1,2-propanediol, the substantially non-aqueous composition of the present invention may comprise (or specifically consist of, or more specifically consist of) one or more compounds of formula II and 1,2-propanediol.

[0197] Similarly, when the starting material is 1,3-propanediol, the substantially non-aqueous composition of the present invention may comprise (or specifically consist of, or more specifically consist of) one or more compounds of formula III and 1,3-propanediol.

[0198] The term "mainly composed of" means that the defined features are present in at least 90% by weight, such as at least 95% by weight, 96% by weight, 97% by weight, 98% by weight, or 99% by weight.

[0199] Furthermore, specific, substantially non-aqueous compositions of the present invention may be mentioned, including those comprising (or specifically, mainly composed of) one or more compounds of formulas (II) and (III) and those of 1,2-propanediol and 1,3-propanediol.

[0200] Specific, substantially non-aqueous compositions of the present invention that may be mentioned include those in which the composition is substantially free of dissolved nitric oxide.

[0201] The term "substantially free of" means that the non-aqueous composition of the present invention contains less than 5% by weight, 4% by weight, 3% by weight, 2% by weight, or 1% by weight of dissolved nitric oxide, such as less than 0.5% by weight or 0.1% by weight.

[0202] Furthermore, specific, substantially non-aqueous compositions of the present invention may contain:

[0203] (a) One or more compounds of formula IV

[0204]

[0205] Where R 4 and R 5 Each can be represented independently as H or -NO, provided that R is... 4 and R 5 At least one of them represents -NO; and

[0206] (b) 1,2-Propanediol.

[0207] The compounds of this invention and the substantially non-aqueous compositions of this invention can be used as pharmaceuticals. These compounds can be administered alone or in the form of known pharmaceutical compositions / formulations.

[0208] Therefore, in a fourth aspect, a pharmaceutical formulation is provided comprising the substantially non-aqueous composition of the present invention and optionally one or more pharmaceutically acceptable excipients, the formulation being referred to below as "the pharmaceutical formulation of the present invention".

[0209] Those skilled in the art will understand that any mention of pharmaceutical formulations of the present invention herein includes references to all embodiments and their specific forms.

[0210] As used herein, pharmaceutically acceptable excipients include reference agents, adjuvants, carriers, diluents, pH adjusters and buffers, tension adjusters, stabilizers, wetting agents, etc. Specifically, such excipients may include adjuvants, diluents, or carriers.

[0211] Specific pharmaceutical formulations of the present invention may be mentioned, including those wherein the pharmaceutical formulation comprises at least one pharmaceutically acceptable excipient.

[0212] Specific pharmaceutical formulations of the present invention that may be mentioned include those in which one or more pharmaceutically acceptable excipients are substantially non-aqueous.

[0213] To avoid ambiguity, the compounds of the present invention mentioned herein for specific uses (and, similarly, the uses and methods of use associated with the compounds of the present invention) are also applicable to compositions and pharmaceutical formulations comprising the compounds of the present invention as described herein.

[0214] Surprisingly, it has been found that the compounds of the present invention can be administered to patients (i.e., subjects) with a suitable aqueous buffer. Specifically, it has been unexpectedly found that the compounds of the present invention are suitably stable in an aqueous buffer (e.g., present without significant degradation for at least 15 minutes), and more specifically, wherein the aqueous buffer is non-nucleophilic and weakly basic.

[0215] According to a fifth aspect of the present invention, a multi-part kit is provided, comprising:

[0216] (A) Pharmaceutical formulations of the present invention (i.e., in the fourth aspect of the invention, including all embodiments and specific features thereof); and

[0217] (B) A suitable aqueous buffer solution,

[0218] Components (A) and (B) are provided in a form suitable for application to each other.

[0219] The multipart kit is referred to below as the "multipart kit of the present invention".

[0220] The specific embodiments mentioned include those in which the buffer solution is non-nucleophilic and weakly basic.

[0221] More specific embodiments that may be mentioned include those in which the pH of the buffer solution is from about 7.1 to about 10 (e.g., about 8 or about 9.2), such as carbonate (e.g., NaHCO3, which may be at a pH of about 7.4 or about 8.0) buffer solutions or physiological phosphate buffer solutions (optionally at a pH of 8) or mixtures thereof. Physiological saline may also be used as a buffer solution.

[0222] Specifically, the buffer solution may be the buffer solution used in Example 5 as described below, such as a carbonate buffer solution with pH 9.2, a phosphate buffer solution with pH 8.0 (e.g., 0.154 mol buffer), or a NaHCO3 buffer solution with pH 8.0.

[0223] Therefore, according to a sixth aspect of the invention, a combined product is provided by mixing the following together:

[0224] (A) Pharmaceutical formulations of the present invention (i.e., in the fourth aspect of the invention, including all embodiments and specific features thereof); and

[0225] (B) A suitable aqueous buffer solution as defined in the fifth aspect of the invention (including all embodiments and specific features therein),

[0226] The combined product is referred to below as "the combined product of the present invention".

[0227] According to a seventh aspect of the invention, a method for preparing the combined product of the invention (i.e., in the sixth aspect of the invention, including all embodiments and specific features thereof) is provided, comprising the step of combining the following:

[0228] (A) Pharmaceutical formulations of the present invention (i.e., in the fourth aspect of the invention, including all embodiments and specific features thereof); and

[0229] (B) Suitable aqueous buffer solutions as defined in the fifth aspect of the invention (including all embodiments and specific features therein).

[0230] Specific multipart kits and combination products of the present invention that may be mentioned include those in which the ratio of the pharmaceutical formulation of the present invention to a suitable aqueous buffer is about 1:2 to 1:99 or about 3:7 to 1:99 (e.g., about 1:3 to 1:99) by volume.

[0231] Alternatively, the multipart kits of the present invention may include those in which the kit contains instructions for mixing the components (e.g., prior to administration, such as by using techniques as described herein) such that the ratio of the pharmaceutical formulation of the present invention to a suitable aqueous buffer is about 3:7 to 1:99 (e.g., about 1:3 to 1:99) by volume.

[0232] Medical use

[0233] As discussed above, the compounds of the present invention, the non-aqueous compositions of the present invention, the pharmaceutical formulations of the present invention, and therefore the multi-component kits and combination products comprising them can be used to treat conditions in which the administration of NO has a beneficial effect.

[0234] According to an eighth aspect of the invention, non-aqueous compositions as defined above (i.e., in the third aspect of the invention, including all embodiments and specific features thereof), pharmaceutical formulations as defined above (i.e., in the fourth aspect of the invention, including all embodiments and specific features thereof), multi-component kits as defined above (i.e., in the fifth aspect of the invention, including all embodiments and specific features thereof), or combination products as defined above (i.e., in the sixth aspect of the invention, including all embodiments and specific features thereof) are provided for treating conditions in which the administration of NO has a beneficial effect.

[0235] In an alternative eighth aspect of the invention, a method for treating a condition in which the administration of NO has a beneficial effect is provided, comprising administering to a patient in need a therapeutically effective amount of the pharmaceutical formulation of the invention or a combination product of the invention.

[0236] In another alternative eighth aspect of the invention, a method for treating a condition in which the administration of NO has a beneficial effect is provided, comprising administering a therapeutically effective amount of the component to a patient in need:

[0237] (A) Pharmaceutical formulations of the present invention (i.e., in the fourth aspect of the invention, including all embodiments and specific features thereof); and

[0238] (B) Suitable aqueous buffer solutions as defined in the fifth aspect of the invention (including all embodiments and specific features therein).

[0239] Specific methods that may be mentioned include the mixing of components (A) and (B) performed before administration to the patient (e.g., by co-administering components (A) and (B)).

[0240] The inventors have discovered that the application of the compounds of the present invention may damage blood cells via hemolysis due to osmotic pressure, and these effects can be controlled or avoided by applying the compounds of the present invention in a suitable aqueous buffer solution.

[0241] More specific methods that may be mentioned include those in which mixing is performed via a mixing flow process, for example, during administration to a patient. Even more specific methods that may be mentioned include those in which the mixing flow process is intravenous infusion using a Y-position connector.

[0242] Those skilled in the art will understand that references to treatment of a specific condition (or similarly, treatment of the condition) take on their normal meaning in the medical field. Specifically, the term may refer to the reduction of the severity of one or more clinical symptoms and / or signs associated with the condition. For example, in the case of pulmonary embolism, the term may refer to the reduction of the severity of chest pain, shortness of breath, and / or pulmonary hypertension achieved through vasodilation.

[0243] As used herein, reference to "patient" will refer to a living subject being treated, including mammalian (e.g., human) patients. Specifically, the term "patient" may refer to a human subject. The term "patient" may also refer to an animal (e.g., a mammal), such as a domestic pet (e.g., a cat, and specifically a dog), livestock, and horses.

[0244] As used herein, the term effective amount will refer to the amount of compound that imparts a therapeutic effect to the treated patient. The effect can be objective (i.e., measurable by some test or biomarker) or subjective (i.e., the subject gives indications of the effect and / or feels the effect).

[0245] As indicated herein, the pharmaceutical formulations of the present invention can be used to treat conditions in which the administration of NO has a beneficial effect.

[0246] Specific conditions that may be mentioned include those selected from the following groups: acute pulmonary vasoconstriction of different etiologies; pulmonary hypertension of different etiologies, including primary and secondary hypertension; conditions requiring vasodilation of different etiologies; systemic hypertension of different etiologies; regional vasoconstriction of different etiologies; localized vasoconstriction of different etiologies; acute heart failure (with or without preserved ejection fraction (HFpEF)); coronary artery disease; myocardial infarction; ischemic heart disease; angina pectoris; unstable angina pectoris; arrhythmia; acute pulmonary hypertension in patients undergoing cardiac surgery; acidosis; respiratory tract inflammation; cystic fibrosis; COPD; and immobile cilia. Syndrome; lung inflammation; pulmonary fibrosis; adult respiratory distress syndrome; acute pulmonary edema; acute mountain sickness; asthma; bronchitis; hypoxia of various causes; stroke; cerebral vasoconstriction; gastrointestinal inflammation; gastrointestinal dysfunction; gastrointestinal complications; IBD; Crohn's disease; ulcerative colitis; liver disease; pancreatic disease; urethritis and cystitis; skin inflammation; diabetic ulcers; diabetic neuropathy; psoriasis; inflammation of various causes; wound healing; organ protection in ischemia-reperfusion conditions; organ transplantation; tissue transplantation; cell transplantation; acute kidney disease; uterine insufficiency; cervical insufficiency; eye diseases, such as glaucoma and conditions requiring smooth muscle relaxation.

[0247] More specific conditions that can be mentioned are pulmonary hypertension of various causes, including primary and secondary hypertension, and acute heart failure (with or without preserved ejection fraction (HFpEF)). For example, the condition can be pulmonary hypertension caused by surgery.

[0248] Pulmonary hypertension is defined as an increase in mean pulmonary artery pressure (mPAP) at rest of 20 mmHg or higher, combined with a Wood Unit value >3.

[0249] Technicians will be able to determine how to administer the drug formulations described herein with appropriate buffer solutions in treatment. Specifically, such combinations of drug formulations and buffer solutions as described herein can be administered intravenously or intra-arterially.

[0250] Technicians will be able to determine the appropriate dose of the active ingredient to be used for treatment based on the properties of the formulation used (e.g., a combination of a pharmaceutical formulation as described herein and a suitable buffer solution), the condition to be treated, and the patient's condition (e.g., disease state). For example, when administered intravenously or intra-arterially to an adult, an appropriate dose may be about 0.5 to about 3,000 nmol / kg / min, such as about 1 to about 3,000 nmol / kg / min, such as about 5 to about 3,000 nmol / kg / min of one or more compounds of formula I. Such doses may be administered by infusion (continuous or pulsatile), such as over a long period of time (e.g., 1 to 2 hours or even up to a week), or as a single (pump) dose (e.g., a single dose or a single dose for each treatment intervention, such as a single dose as needed, or a single dose every 24 hours during treatment).

[0251] Those skilled in the art will understand that the temperature at which the formulations of the present invention (i.e., pharmaceutical compositions comprising a compound of formula I) are formed and / or administered (i.e., administered to a subject) in treatment can be the ambient temperature at which the administration occurs (i.e., room temperature) or can be controlled. For example, such formulations can be formed and / or administered at room temperature or at a reduced temperature (i.e., below room temperature), such as from about 0 to about 25°C.

[0252] In certain embodiments, the compound of formula (II) is particularly important for use in humans, and the compound of formula (III) is particularly important for use in veterinary applications.

[0253] Without being bound by theory, it is believed that when administered to patients, compounds of formula I are hydrolyzed to release nitric oxide, which provides the desired therapeutic effect. It is believed that the methods described herein unexpectedly allow for the preparation of suitable concentrated and stable compositions containing the desired active ingredient. Furthermore, it is believed that a certain type of buffer solution has been unexpectedly discovered that allows for the safe administration of such compositions without significant degradation of (one or more) the active ingredient. Attached Figure Description

[0254] Figure 1 The results of analysis on the stability of the compositions of the present invention, as described in Example 5 herein, in various buffer solutions are shown.

[0255] Figure 2The results of an in vivo study, as described in Example 6 of this document, are shown, comparing the therapeutic effects of the compositions of the present invention compared to inhaled nitric oxide. Changes in mean pulmonary artery pressure (MPAP, Fig. a) and pulmonary vascular resistance (PVR, Fig. c), mean arterial pressure (MAP, Fig. b), systemic vascular resistance (SVR, Fig. d), and methemoglobin concentration were observed in anesthetized and mechanically ventilated pigs receiving intravenous infusions of PDNO (15, 30, 45, and 60 nmol / kg). -1 minute -1 To sodium bicarbonate carrier fluid [50 mg ml] -1 [; Infusion rate is 10 times the PDNO infusion rate]; pulmonary vasoconstrictor U46619 (60-150 ng kg) is infused intravenously or intravenously in n=6) -1 minute -1 Inhaled NO (5, 10, 20, and 40 ppm; n = 7) during the period. The dose of inhaled NO is converted to nmol / kg. -1 minute -1 The x-axis is on a logarithmic scale. Data are presented as median and interquartile range. *Indicates statistical significance of the indicated dose of U46619 alone for each drug. φ indicates the effect at 45 nmol / kg. -1 minute -1 The statistical difference between PDNO at 5 ppm and inhaled NO at 5 ppm represents the equivalent amount of NO delivery.

[0256] Figure 3 Results of reducing mean systemic arterial pressure and pulmonary artery pressure using 1,2-PDNO-R, 1,2-PDNO-S and 1,3-PDNO are shown, and the experimental procedure is outlined in Example 13.

[0257] Example

[0258] The present invention is illustrated by the following examples, which are not intended to limit the overall scope of the invention.

[0259] abbreviation

[0260] aq water-based

[0261] conc concentration

[0262] GC gas chromatography

[0263] NMR (Nuclear Magnetic Resonance)

[0264] equiv. equivalent

[0265] rel.vol. Relative volume

[0266] General Procedure

[0267] The starting materials and chemical reagents specified in the synthesis described below are commercially available from many suppliers, such as Sigma Aldrich.

[0268] All NMR experiments were performed at 298 kHz using Bruker Topspin 2.1 software on a Bruker 500 MHz AVI instrument equipped with a QNP probe with Z-gradient. Unless otherwise stated, the signal was referenced at 7.27 ppm residual CHCl3.

[0269] Stability determination

[0270] Stability determination of the samples was performed by GC / FID under the following conditions. 1,4-Dioxane was used as an internal standard (IS; approximately 0.50 mg / ml in CH3CN).

[0271] GC column: Rxi-5Sil MS, 20m × 0.18mm, 0.72μm

[0272] Carrier gas: Helium

[0273] Inlet temperature: 200℃, split ratio: 30:1

[0274] Constant flow rate: 1.0 ml / min

[0275] Oven temperature distribution: 40℃ (3 minutes), 10℃ / minute, 250℃ (3 minutes)

[0276] FID: Temperature 300℃; H2 flow rate 30ml / min, air flow rate 400ml / min, supplemental flow rate (N2) 25ml / min

[0277] In vivo studies

[0278] Prior to the experiment, the Linköping Regional Animal Ethics Committee ( Regional Animalethics Committee (Linköping, Sweden) Sweden) (Approval No. 953) has received ethical approval. Anesthesia management, surgical instrument handling, and measurement methods have recently been described (Dogan et al. 2018, Sadeghi et al. 2018).

[0279] In summary, eight male and female pigs (Swedish rural breed, crossbred between Hampshire and Yorkshire; 3-4 months old; average weight 27 kg, range 21-34 kg) were pre-treated with azaperone on the farm and transported to the laboratory. In the laboratory, anesthesia was induced using a mixture of tiletamine, zolazepam, and azaperone (intramuscular injection). Propofol was administered via a peripheral venous catheter in the ear vein if necessary. A bolus dose of atropine and cefuroxime was administered intravenously. The animals were endotracheally intubated and mechanically ventilated (positive end-expiratory pressure 5 cm H2O, minute ventilation adjusted to normal ventilation). General anesthesia was maintained by continuous intravenous infusion of propofol and fentanyl, with additional bolus doses administered if necessary. Ringer's acetate and glucose solution was continuously administered intravenously to replace fluid loss. After surgical instrumentation, heparin was administered via intravenous bolus. Following the experiment, the animals were euthanized under general anesthesia with propofol injection, followed by a rapid intravenous injection of potassium chloride (40 mmol) to confirm cardiac arrest.

[0280] The animal was instrumented using a catheter in the right carotid artery to measure systemic arterial blood pressure and heart rate, and to sample arterial blood. A sheath was placed in the right external jugular vein to introduce a pulmonary artery catheter. This catheter was used to continuously measure pulmonary artery blood pressure, semi-continuous cardiac output, and intermittent pulmonary wedge pressure. A central venous catheter was inserted into the left external jugular vein for drug and fluid administration. All fluid and drug administrations were performed using a power injector or drip pump. A catheter was inserted into the bladder. Respiratory gases, pressures, and volumes were measured at the endotracheal intubation site. Respiratory and hemodynamic variables were measured using a Datex AS / 3 (Helsinki, Finland) and data were collected using a computer system (MP150 / Acknowledge 3.9.1, BIOPAC system, Goleta, CA, USA). Blood gas analysis and methemoglobin concentration were measured using a blood gas analyzer (GEM 4000, Instrumental Laboratory, Lexington, Massachusetts, USA). Pulmonary and systemic vascular resistance were calculated using standard formulas. A one-hour intervention-free period was followed after surgical instrumentation.

[0281] Due to the non-normal distribution, the data exist with median and interquartile range. Using the ideal gas law and minute ventilation, and assuming complete absorption of NO in the lungs, the ppm dose of inhaled NO is converted to nmol / kg. -1minute -1 Dosage was measured in units. Within this range, drug data were analyzed using the Friedman test and Wilcoxon's signed-rank test for post-hoc multiple comparisons. The Mann-Whitney U test was used to compare the maximum dose with similar NO delivery doses (45 nmol / kg). -1 minute -1 The drug was compared with PDNO (5 ppm inhaled NO). In multiple comparisons, the Benjamini-Hochberg boosting procedure was used and the critical P value was adjusted to 0.05.

[0282] Example 1 - Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1, using sodium nitrite 2-bis(nitroso)propane

[0283] Add 1,2-propanediol (15 ml, 205 mmol), water (100 ml), dichloromethane (200 ml), and sodium nitrite (57 g, 826 mmol) to a 500 ml three-necked round-bottom flask. Cool the mixture to 0°C in an ice bath. Add concentrated sulfuric acid (30 ml, 546 mmol) and water (30 ml) to a dropping funnel and cool to 5°C in a refrigerator. Adjust the funnel to fit the round-bottom flask and add the acid to the nitrite mixture over two hours. Stir the mixture magnetically for 20 minutes, then pour it into a separatory funnel along with more dichloromethane (100 ml) and water (100 ml). The organic phase was separated, dried with sodium sulfate, and reduced on a rotary evaporator to give a mixture of 1,2-propanediol (3 wt%), 1-(nitrosooxy)-prop-2-ol (23 wt%), 2-(nitrosooxy)-prop-1-ol (13 wt%) and 1,2-bis(nitrosooxy)propane (57 wt%).

[0284] Example 2 - Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1, using sodium nitrite 2-bis(nitroso)propane

[0285] 1,2-Propanediol (20 ml, 273.4 mmol), water (60 ml), dichloromethane (120 ml), and sodium nitrite (37.72 g, 546.7 mmol) were added to a 0.5-liter reactor equipped with a stirrer and purged with nitrogen, and maintained under nitrogen atmosphere throughout the following reaction. The mixture was cooled to below 5°C by cooling the hood to 0°C. Concentrated sulfuric acid (26.3 g, 260.1 mmol) and water were added to a dropping funnel. Connect the funnel to the reactor and add the acid to the nitrite mixture over 33 minutes. Stir the mixture for 54 minutes and then pour it into a flask containing saturated sodium bicarbonate aqueous solution (100 ml). Transfer the mixture to a separatory funnel and wash the organic phase. Discard the aqueous phase and wash the organic phase with an additional saturated sodium bicarbonate aqueous solution (100 ml). Dry the organic phase with magnesium sulfate and then transfer it to a 1 L round-bottom flask with 1,2-propanediol (120 ml, 1640 mmol). Reduce the solution under reduced pressure on a rotary evaporator until dichloromethane is removed. Monitor the removal of dichloromethane by NMR. A clear solution (134 g) containing 1,2-propanediol (82.8 wt%), 1-(nitrosooxy)-prop-2-ol (10.4 wt%), 2-nitrosooxy)-prop-1-ol (6 wt%), and 1,2-bis(nitrosooxy)propane (0.8 wt%) is obtained.

[0286] 1 ¹H-NMR, δppm: 5.61 (br s 1H), 4.75-5.58 (m, 2H), 4.11 (br s, 1H), 3.90-3.87 (m, 1H), 3.83-3.69 (m, 2H), 3.60 (dd, J = 3.0, 11.2 Hz, 1H), 3.38 (dd, J = 7.9, 11.2 Hz, 1H), 1.47 (d, J = 6.6 Hz, 3H), 1.39 (d, J = 6.4 Hz, 3H), 1.26 (d, J = 6.4 Hz, 3H), 1.15 (d, J = 6.3 Hz, 3H). The signals for CH and CH2 in 1,2-bis(nitrosooxy)propane were below the detection limit.

[0287] Example 3 - Preparation of 1-(nitrosooxy)-prop-2-ol and 2-(nitrosooxy)-prop-1-ol from tert-butyl nitrite and 1,2-bis(nitrosooxy)propane

[0288] 2 ml of tert-butyl nitrite (15.1 mmol) was added to a round-bottom flask containing 11 ml of 1,2-propanediol (150.3 mmol), and the resulting solution was stirred at ambient temperature. Then, 1 ml of the reaction solution was mixed with 7.5 ml of 1,2-propanediol.

[0289] Example 4 - Non-aqueous forms of 1-(nitrosooxy)-prop-2-ol, 2-(nitrosooxy)-prop-1-ol, and 1,2-propanediol Stability of the mixture

[0290] Three different concentrations of 1-(nitrosooxy)-propane-2-ol and 2-(nitrosooxy)-propane-1-ol in 1,2-propanediol were prepared and stored in a refrigerator (5°C) and a freezer (-20°C). Aliquots of each solution were periodically taken and analyzed by GC to determine the concentrations of 1-(nitrosooxy)-propane-2-ol and 2-(nitrosooxy)-propane-1-ol.

[0291] The results of the GC analysis are shown in the table below (column: Rxi-5Sil MS, 20m × 0.18mm, film thickness 0.36; support: He; injection port: 250℃, split ratio 100:1; constant flow rate: 1.0ml / min; oven temperature distribution: 40℃ (3min), 10℃ / min, 80℃ (0min), 30℃ / min, 250℃ (3min); FID: 300℃, H2 flow rate 30ml / min, air flow rate 400ml / min, makeup flow rate (N2) 25ml / min; internal standard: 1,1,1,3,5,5,5-heptamethyltrisiloxane):

[0292]

[0293] Note: No pressure increase was observed in any sample.

[0294] Example 5 - 1-(nitrosooxy)-prop-2-ol and 2-(nitrosooxy)-prop-1-ol (PDNO) and 1,2-propanediol Stability of buffered aqueous solutions of alcohols (PD)

[0295] Add 100 μl of the stability sample to a GC vial. Add 400 μl of PD / buffer solution (1:9) and 400 μl of CH3CN. Then add 500 μl of CH2Cl2 and extract the mixture by gently shaking for 1 minute. Transfer 500 μl of the organic phase (lower phase) to another GC vial and add 50 μl of IS. Analyze the extract by GC / FID under the conditions described above.

[0296] Calibration curves were constructed for 1-nitrite and 2-nitrite, respectively. Peak area ratio (nitrite / IS) and nitrite concentration were plotted. Standard solutions were prepared using high-concentration PDNO / PD stock solutions. Nitrite concentration was calculated in %w / w.

[0297] table below and Figure 1 The results obtained are shown in the figure.

[0298]

[0299] *Based on known values.

[0300] Example 6 - In vivo study

[0301] After collecting baseline data, thromboxane A2-mimicking 9,11-dideoxy-9α,11α-methylepoxyPGF was administered via continuous intravenous infusion. 2α (U46619, Cayman Chemical, Michigan, USA; provided with methyl acetate and diluted in 0.9% NaCl to a final concentration of 30 μg / ml) -1 60-150 ng kg -1 minute -1 Stable pulmonary hypertension was induced by gradually increasing the dose of PDNO (15, 30, 45, and 60 nmol / kg) to achieve a target mean pulmonary artery pressure of 35-45 mmHg. -1 minute -1 ) Carrier stream of sodium bicarbonate solution administered via continuous intravenous infusion (50 mg / ml) -1 pH approximately 8; Fresenius Kabi, Uppsala, Sweden; infusion rate 10 times that of PDNO infusion rate) or inhaled NO (5, 10, 20, and 40 ppm; delivered from a 1000 ppm nitrogen reservoir to the respiratory branch of a Servo 300 ventilator with an inhaled NO delivery unit [Siemens-Elema, Stockholm, Sweden], using a non-randomized crossover design with a 30-minute washout time between doses. Verify the correct dose of inhaled NO using an NO analyzer. Administer each dose 5–10 minutes apart. Extract hemodynamic and respiratory data and sample arterial blood at the last minute of each administration.

[0302] Intravenous infusion of U46619 induced stable pulmonary hypertension, with mean pulmonary artery pressure of 43 (37-48) mmHg and 43 (41-46) mmHg before NO inhalation and PDNO infusion, respectively, and pulmonary vascular resistance of 8.3 (6.7-11.7) mmHg / min. -1 and 9.8 (7.7-12.5) mmHg per minute -1 Both inhaled NO and intravenously infused PDNO significantly reduced pulmonary artery pressure and vascular resistance, but PDNO reduced mean pulmonary artery pressure more effectively (with a steeper slope) than inhaled NO, and at comparable doses, PDNO significantly reduced pulmonary vascular resistance (e.g., pulmonary artery pressure) compared to inhaled NO. Figure 2 (As shown). No drug significantly affected mean arterial pressure and systemic vascular resistance, but at comparable doses, the PDNO group had slightly lower systemic vascular resistance compared to the inhalable NO group. Figure 2Both drugs significantly reduced the ratio of pulmonary to systemic vascular resistance, and at the highest dose, the ratio was slightly lower in the inhaled NO group compared to the PDNO group (data not shown). U46619 slightly decreased cardiac output, but no drug significantly altered cardiac output (data not shown). No drug significantly affected methemoglobin concentration, but there was a trend towards an increase in the inhaled NO group, and methemoglobin concentration appeared to be correlated with the delivered NO dose. Figure 2 U46619 slightly lowers the arterial partial pressure of oxygen, and both drugs tend to normalize this variable (data not shown).

[0303] Example 7 - Solvent-free preparation of 1-(nitrosooxy)-propane-2-ol and 2-(nitrosooxy)-propane-2-ol using sodium nitrite 1-Alcohol and 1,2-bis(nitrosooxy)propane

[0304] Add water (30 ml) and sodium nitrite (19.01 g, 272.8 mmol) to a 100 ml three-necked round-bottom flask, rinse with nitrogen, and cool to 1 °C on a water bath cooled with an external cooler. Add 1,2-propanediol (10 ml, 136.7 mmol). Pre-cool concentrated sulfuric acid (7 ml, 127.4 mmol) and water (20 ml) to room temperature and add dropwise over one hour via a dropping funnel. During the addition, the aqueous layer forms a thick slurry, and a second green layer forms. Before the acid addition is complete (5 ml remaining), remove the flask from the cooling bath and pour the green layer into a separatory funnel, washing with a 2× saturated NaHCO3 aqueous solution. The green layer decolorizes to yellow and, after separation, is dried over Na2SO4 and filtered through a syringe filter. 13mm, 0.45μM The reaction yielded 1.1 g of a mixture of approximately 0.25 / 0.1 / 1 1-(nitrosooxy)-propane-2-ol / 2-(nitrosooxy)-propane-1-ol / 1,2-bis(nitrosooxy)propane. The starting material 1,2-propanediol was not detected within the NMR sensitivity limit.

[0305] 1 H-NMR, δppm:5.81-5.76(m,br,1.0H),5.63(br,0.1H),4.93(br,2.08H),4.73-4.65(br,m,0.47H),4.1 4(br,0.19H),3.84-3.77(br,m,0.22H),1.49-1.48(br,m,3.21H),1.43(br,0.51H),1.28(br,0.72H).

[0306] Example 8 - Preparation of (2S)-1-(nitrosooxy)-prop-2-ol, (2S)-2-(nitrosooxy)-prop-1-ol and (2S)-1,2-bis(nitrosooxy)propane

[0307] (S)-1,2-propanediol (5 ml, 66.97 mmol), water (15 ml), dichloromethane (30 ml), and sodium nitrite (9.34 g, 134 mmol) were added to a 100 ml three-necked round-bottom flask, rinsed with nitrogen, and cooled to 1 °C on a water bath cooled with an external cooler. Concentrated sulfuric acid (3.5 ml, 63.69 mmol) and water (10 ml) were pre-cooled to room temperature and added dropwise over 1 hour via a syringe pump. After the addition, the mixture was stirred for another 60 minutes. After separation of the two layers, the DCM layer was diluted with an additional 15 ml of DCM and washed with a saturated aqueous solution of NaHCO3 (15 ml), then with brine (15 ml), dried over Na2SO4, filtered through a sintered glass filter, and reduced under vacuum. The residue was redissolved in 30 ml of DCM, washed with 1.4% w / w saturated bicarbonate aqueous solution, dried over Na2SO4, filtered through a sintered glass filter and vacuum reduced to give 1 g of product mixture. Based on NMR, the mixture consisted of (2S)-1,2-propanediol (3%), (2S)-1-(nitrosooxy)-prop-2-ol (23%), (2S)-2-(nitrosooxy)-prop-1-ol (14%) and (2S)-1,2-bis(nitrosooxy)propane (60%).

[0308] 1 H-NMR, δppm:5.83-5.74(m,1.0H),5.66-5.57(br,0.22H),4.99-4.85(br,1.98H),4.76-4.59(br,0.77H),4.17-4.07(br,0.38 H), 3.86-3.73 (br, 0.40H), 1.8-1.6 (br, 0.97H), 1.48 (d, J = 6.7Hz, 3.12H), 1.40 (d, J = 6.6Hz, 0.63H), 1.28 (d, J = 6.5Hz, 1.15H).

[0309] Example 9 - Preparation of (2R)-1-(nitrosooxy)-prop-2-ol, (2R)-2-(nitrosooxy)-prop-1-ol and (2R)-1,2-bis(nitrosooxy)propane

[0310] (R)-1,2-propanediol (5 ml, 66.97 mmol), water (15 ml), dichloromethane (30 ml), and sodium nitrite (9.34 g, 134 mmol) were added to a 100 ml three-necked round-bottom flask, rinsed with nitrogen, and cooled to 1 °C on a water bath cooled with an external cooler. Concentrated sulfuric acid (3.5 ml, 63.69 mmol) and water (10 ml) were pre-cooled to room temperature and added dropwise over 1 hour via a syringe pump. After the addition, the mixture was stirred for another 55 minutes. After separation of the two layers, the DCM layer was diluted with an additional 10 ml of DCM, washed with a saturated aqueous solution of NaHCO3 (20 ml), dried over Na2SO4, filtered through a sintered glass filter, and reduced under vacuum. Based on NMR, the mixture consisted of (2R)-1,2-propanediol (17%), (2R)-1-(nitrosooxy)-prop-2-ol (16%), (2R)-2-(nitrosooxy)-prop-1-ol (7%) and (2R)-1,2-bis(nitrosooxy)propane (59%).

[0311] 1 H-NMR, δppm:5.83-5.74(m,1.0H),5.66-5.57(br,0.12H),4.99-4.85(br,2.10H),4.76-4.59(br,0.53H),4.17-4.07(br ,0.24H),3.86-3.73(br,0.28H),2.4-2.1(br,0.38H),1.48(d,J=6.8Hz,3.20H),1.40(br,0.56H),1.28(br(d),0.88H).

[0312] Example 10 - Preparation of 1-(nitrosooxy)prop-3-ol and 1,3-bis(nitrosooxy)propane

[0313] 1,3-Propanediol (2.5 g, 32.86 mmol), water (7 ml), dichloromethane (15 ml), and sodium nitrite (4.53 g, 65.7 mmol) were added to a 100 ml round-bottom flask, rinsed with nitrogen, and cooled to 0 °C for 15 minutes in a water bath cooled with an external cooler. Concentrated sulfuric acid (1.7 ml, 31.2 mmol) and water (5 ml) were pre-cooled to room temperature and added dropwise over 5 minutes. After addition, the mixture was stirred at 0 °C for another 60 minutes. The two layers were then separated, and the organic phase was diluted with an additional 10 ml of DCM, washed with saturated NaHCO3 aqueous solution (2 x 25 ml), dried over MgSO4, and filtered through a sintered glass filter. Finally, 1,3-Propanediol (16.4 g, 216 mmol) was added to the organic phase, and the DCM was removed under vacuum. Based on NMR, the mixture (18.1 g) contained 1,3-propanediol (86.9 wt%), 1-(nitrosooxy)-prop-3-ol (11.8 wt%) and 1,3-bis(nitrosooxy)propane (1.3 wt%).

[0314] ¹H-NMR, δ 4.76–4.88 (m, 2H), 3.83 (t, J = 5.7 Hz, 2H), 3.73 (t, J = 6.1 Hz, 2H), 2.79 (s, 1H), 2.18 (quintet, J = 6.3 Hz, 2H), 1.99 (quintet, J = 6.2 Hz, 2H), 1.80 (quintet, J = 5.7 Hz, 2H).

[0315] Example 11 - Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and sodium nitrite Scale-up method for 1,2-bis(nitrosooxy)propane

[0316] 11.1 Chemicals used

[0317] The starting materials were purchased from the supplier list in the table below. Unless otherwise stated, these chemicals should be used as is without further purification.

[0318]

[0319] 11.2 General Procedure for Synthesizing PDNO Using DCM as a Solvent (Original Method)

[0320] The round-bottom flask was equipped with a stirrer and a dropping funnel. Water (3.0 veq.) was added, and sodium nitrite (2.0 veq.) was added to the flask. The solution was cooled (0°C), and PD (1.0 veq.) and DCM (6 veq.) were also added. During further cooling, a sulfuric acid solution (1.0 veq. of H2SO4 and 2.0 veq. of water) was prepared. The sulfuric acid solution was then added dropwise to the reaction mixture while maintaining the reaction mixture between 0°C and 5°C. After the acid was completely added, the solution was stirred for another 1 hour to complete the reaction.

[0321] The reaction was then quenched with a saturated NaHCO3 solution (6.0 relative volume). The phases were separated, and the organic layer was further washed with NaHCO3 solution (6.0 relative volume). The organic phase was dried over MgSO4, filtered, diluted with PD, and concentrated under reduced pressure using a rotary evaporator (water bath temperature 40°C).

[0322] The product obtained is a pale yellow liquid.

[0323] 11.3 General Synthesis of PDNO Using TBME as a Solvent

[0324] The round-bottom flask is equipped with a stirrer and a dropping funnel. Purge with argon for several minutes. Prepare a dilute sulfuric acid solution (1.0 equivalent H₂SO₄, 2.0 relative volume of water) beforehand and pre-cool (-30°C). Add water to the flask (3.0 relative volume). Add sodium nitrite (2.0 equivalent) to the water. Add TBME (7.5 relative volume). Add propylene glycol (1.0 equivalent) and cool the reaction mixture (-20°C), purging continuously with argon. Stir the reaction mixture thoroughly while adding the pre-cooled sulfuric acid dropwise. Monitor the reaction temperature throughout the acid addition. After the addition, further stir the reaction mixture at a cool temperature (-20°C) for 30–60 minutes. Then, raise the temperature of the reaction mixture (-5°C). Terminate the reaction by quenching with a saturated NaHCO₃ solution (6.0 relative volume). Separate the phases. The organic layer is further washed with a saturated NaHCO₃ solution until the pH reaches 7–8. The organic phase is then dried over MgSO₄. The crude PDNO solution was diluted with PD (3 relative volume) and further concentrated under reduced pressure at ambient temperature (25°C).

[0325] The crude PDNO solution was further purified using a riser evaporator.

[0326] PDNO was obtained as a pale yellow liquid.

[0327] 11.4 Detailed Synthesis of PDNO Using TBME as Solvent

[0328] This method was designed to produce approximately 7.5 L of 7% PDNO solution in a single synthesis (one “run”). Several syntheses were performed to obtain the desired batch size. Purity was determined using GC analysis for each run. Runs within the organic-related compound specification range could be pooled to obtain a single batch. All crude PDNO batches were then purified. Following purification, the strong PDNO solution was further diluted with PD to obtain the desired concentration (typically 7% PDNO solution).

[0329] A suitable double-walled reactor (60L) is equipped with a specific "cup stirrer," a dropping funnel, and an accessory for argon. The reactor is flushed with a constant flow of argon for 5 to 10 minutes. Water (3.0L) is added to the reactor. Sodium nitrite (2.0 equivalent, 1886g) is added through the reactor. The reaction mixture is further stirred until all salts are dissolved. 1,2-Propane glycol (1.0 equivalent, 1040g, 1L) is added, followed by tert-butyl methyl ether (7.5 relative volume, 7.5L). The reaction mixture is then cooled to an internal reaction temperature of -20°C by continuous stirring and an argon flow. Sulfuric acid (1.0 equivalent, 1340g, 728ml) is simultaneously diluted with water (2.0L) and cooled to -30°C. Once the internal reaction temperature of -20°C is reached, the dilute acid is added dropwise to the reaction mixture while vigorous stirring.

[0330] The stirring speed was varied during the addition of acid, starting at a high speed of approximately 350 rpm and decreasing to a slower speed (approximately 180 rpm) until the reaction was complete. This variation in stirring speed was due to the two-phase reaction system and the slow precipitation of sodium sulfate as the reaction progressed (due to the addition of increasing amounts of sulfuric acid).

[0331] Monitor the reaction temperature throughout the sulfuric acid addition process. Ideally, the temperature should be within the range of (-20±3)℃. Furthermore, stir the reaction at (-20±3)℃ for 30-60 minutes.

[0332] The reaction was heated to -5°C to 0°C. The reaction was terminated by adding a saturated NaHCO3 solution (6.0 L relative volume), followed by water (10 L). The phases were separated, and the organic layer was transferred to a separate double-walled reactor and cooled to 0°C to -5°C. The organic layer was washed several times (approximately 2-3 times) with a saturated NaHCO3 solution (4.0 L relative volume). The pH of the aqueous phase was monitored after each washing step. The pH was approximately 7-8. The aqueous phase was discarded. The organic layer was dried over MgSO4 and filtered through Whatman filter paper.

[0333] The crude PDNO (solution in TBME) was diluted by adding additional PD (3.0 relative volume, 3.0 L). This crude PDNO was then transferred to a rotary evaporator and concentrated under reduced pressure. The water bath temperature was maintained at a maximum of 25°C during evaporation. Most of the TBME was removed by evaporation within a timeframe of 1.5 to 2.0 hours.

[0334] The organic solvent can then be evaporated using a high vacuum pump at a water bath temperature of (0±2) °C for several hours (PDNO purity was monitored under these conditions during formation, and the product purity was unaffected for 6 hours).

[0335] 11.5 Further purification of crude PDNO solution

[0336] Final purification of the PDNO solution was accomplished via riser evaporation. The PDNO solution was distilled under high vacuum at 0°C using a continuous stream of dilute PDNO vapor. The tank containing the “crude” PDNO solution was cooled to 0°C. The entire distillation process was carried out at 0°C. The tank containing the “purified” PDNO was also cooled from -10°C to 0°C. After each evaporation run of the entire batch of PDNO, residual organic solvents (TBME) could be checked via GC. The evaporation was continued until the desired limit for residual solvents was reached. For PDNO, the limit for residual solvents was 1000 ppm.

[0337] 11.6 Preparation of the final diluent

[0338] After purification, PDNO is further diluted to achieve the desired concentration. The first step is to filter the PDNO solution into a clean glass vial using a Waterman filter. Additionally, the PDNO solution is determined by q-NMR. The amount of PD to be diluted can be calculated. PD is first filtered through a Waterman filter. The final dilution can be performed at ambient temperature. The calculated amount of PD is added to the PDNO solution (or vice versa). The resulting mixture is shaken for several minutes to obtain a homogeneous solution. The final PDNO solution is then filled into a product vial.

[0339] PDNO (7.5 kg; 7% solution) was obtained as a pale yellow liquid.

[0340] Example 12 - Hemodynamic Effects of Intravenous PDNO: The Influence of Various Carrier Solutions on Anesthetized Pigs

[0341] The hemodynamic effects of various carrier solutions on intravenously administered 1,2-propanediol (PDNO) organic mononitrite in anesthetized pigs were investigated.

[0342] Ethical approval was obtained from the Linköping Regional Animal Ethics Committee (Linköping, Sweden; Approval No. 953) prior to the experiment. The study was conducted in accordance with Directive 2010 / 63 / EU on the protection of animals used for scientific purposes. The study included two healthy 3-month-old pigs (crossbred between Hampshire and Yorkshire, Swedish rural breeds; weighing 26 and 27 kg respectively).

[0343] Animals were pre-administered azapiridone at the farm and transported to the laboratory. In the laboratory, anesthesia was induced using a mixture of telexamine, zoprazepam, and azapiridone (intramuscular injection). Propofol was administered via a peripheral venous catheter in the ear vein if necessary. A bolus dose of atropine and cefuroxime was administered intravenously. Animals were endotracheally intubated and mechanically ventilated (positive end-expiratory pressure 5 cm H2O, minute ventilation adjusted to normal ventilation). General anesthesia was maintained by continuous intravenous infusion of propofol and fentanyl, with additional bolus doses administered if necessary. Ringer's acetate and glucose solution were continuously administered intravenously to replace fluid loss. Heparin was administered intravenously as a bolus dose after surgical instrumentation. Following the experiment, animals were euthanized under general anesthesia with propofol injection, followed by a rapid intravenous injection of potassium chloride (40 mmol) to confirm cardiac arrest.

[0344] Instrumentation was performed on the animal using an arterial catheter in the right carotid artery to measure systemic arterial blood pressure and heart rate. A sheath was placed in the right external jugular vein to introduce a pulmonary artery catheter. This catheter was used for continuous measurement of pulmonary artery blood pressure and semi-continuous cardiac output. A central venous catheter was inserted into the left external jugular vein for drug and fluid administration. All fluid and drug administrations were performed using a power injector or drip pump. A catheter was inserted into the bladder. Hemodynamic variables were measured using a Datex AS / 3 (Helsinki, Finland), and data were collected using a computer system (MP150 / Acknowledge 3.9.1, BIOPAC system, Goleta, California, USA). A no-intervention period of at least 1 hour was followed after surgical instrumentation.

[0345] At 30 nmol kg -1 minute -1 PDNO was administered via intravenous infusion over 15 minutes (Research Institutes of Sweden, Södertälje, Sweden). Sweden) to sodium bicarbonate (14 mg / ml) -1 The carrier solution was infused in physiological phosphate buffer or saline at pH 7.4 or 8.0, or pH 8, at a rate nine times that of PDNO infusion. The carrier solution was prepared using standard chemicals. Hemodynamic effects were measured at the end of each infusion.

[0346] The results are shown in the table below. For healthy, anesthetized pigs, baseline values ​​prior to each intravenous combination of PDNO and carrier solution were normal. Intravenous PDNO in a pH 8 bicarbonate-buffered carrier solution reduced mean systemic arterial pressure (MAP) and mean pulmonary artery pressure (MPAP) by -11 ± 1.2 mmHg and -2.4 ± 0.8 mmHg, respectively, while in a saline carrier solution, intravenous PDNO reduced MAP and MPAP by -6.9 ± 2.5 mmHg and -2.4 ± 0.1 mmHg, respectively. The effects of intravenous PDNO combined with pH 7.4 bicarbonate-buffered and pH 8 phosphate-buffered on MAP and MPAP were similar to those of saline. Heart rate and semicontinuous cardiac output were only slightly affected by infusion.

[0347] Accept 30 nmol kg -1 minute -1 Hemodynamic variables in anesthetized and mechanically ventilated pigs receiving repeated intravenous infusions of PDNO in combination with various carrier solutions (n=2 for each carrier solution).

[0348]

[0349] The data exists as the mean and standard deviation.

[0350] Mean systemic arterial pressure (MAP), mean pulmonary artery pressure (MPAP), heart rate (HR), and semi-continuous cardiac output (CCO).

[0351] Compared with physiological saline, physiological phosphate buffer, and bicarbonate buffer carrier solutions at pH 7.4, intravenous PDNO bound to bicarbonate buffer carrier solution at pH 8 produces a greater hemodynamic effect.

[0352] Example 13 - Pharmacological study of 1,2-PDNO-R, 1,2-PDNO-S and 1,3-PDNO in anesthetized pigs

[0353] Ethical approval was obtained from the Linköping Regional Animal Ethics Committee (Linköping, Sweden; Approval No. 953) prior to the experiment. In short, two male and female pigs (Swedish rural breed, crossbred between Hampshire and Yorkshire; 3-4 months old; 24-26 kg) were pre-administered with azapiridone on the farm and transported to the laboratory. In the laboratory, anesthesia was induced using a mixture of telexamine, zoprazepam, and azapiridone (intramuscular injection). Propofol was administered via a peripheral venous catheter in the ear vein if necessary. A bolus dose of atropine and cefuroxime was administered intravenously. The animals were endotracheally intubated and mechanically ventilated (positive end-expiratory pressure of 5 cm H2O, minute ventilation adjusted to normal ventilation). General anesthesia was maintained by continuous intravenous infusion of propofol and fentanyl, with additional bolus doses administered if necessary. Ringer's acetate and glucose solution were continuously administered intravenously to replace fluid loss. Heparin was administered by intravenous bolus dose after surgical instrumentation. Following the experiment, the animals were given general anesthesia with propofol injection, followed by a rapid intravenous injection of potassium chloride (40 mmol) to kill them and confirm cardiac arrest.

[0354] Instrumentation was performed on the animal using a catheter in the right carotid artery to measure systemic arterial blood pressure and heart rate. A sheath was placed in the right external jugular vein to introduce a pulmonary artery catheter. This catheter was used for continuous measurement of pulmonary artery blood pressure, semi-continuous cardiac output, and intermittent pulmonary wedge pressure. A central venous catheter was inserted into the left external jugular vein for drug and fluid administration. All fluid and drug administrations were performed using a power injector or drip pump. A catheter was inserted into the bladder. Respiratory gases, pressures, and volumes were measured at the endotracheal intubation site. Respiratory and hemodynamic variables were measured using a Datex AS / 3 (Helsinki, Finland), and data were collected using a computer system (MP100 or MP150 / Acknowledge 3.9.1, BIOPAC system, Goleta, California, USA). A one-hour intervention-free period was followed after surgical instrumentation.

[0355] After collecting baseline data, 1,2-PDNO-R (43 nmol kg) will be administered intravenously over 10–15 minutes. -1 minute -1 ), 1,2-PDNO-S (43 nmol kg) -1 minute -1 ) and 1,3-PDNO (30 nmol kg -1 minute -1 The carrier stream (14 mg / ml) was applied to the sodium bicarbonate solution. -1 The infusion rate was approximately 9 times that of PDNO (pH approximately 8). Hemodynamic and respiratory data were extracted at the end of each administration.

[0356] 1,2-PDNO-R, 1,2-PDNO-S, and 1,3-PDNO reduced mean systemic and pulmonary artery pressure, as shown in this diagram. Figure 3 The conclusion is that 1,2-PDNO-R, 1,2-PDNO-S, and 1,3-PDNO induce systemic and pulmonary vasodilation, thus demonstrating their vasodilatory capabilities.

Claims

1. A process for the preparation of a composition comprising one or more compounds of formula I ###0001### wherein n is 0 or 1 ; （I） wherein R 1 , R 2 and R 3 each independently represent H or -NO, said process comprising the steps of: wherein when n is 0, R 1 is H; and wherein when n is 1, R 2 is H, with the proviso that at least one of R 1 R 2 and R 3 represents -NO, wherein: (i) reacting a corresponding compound of formula I, wherein R 1 , R 2 and R 3 represent H, with a source of nitrite, and (ii) reacting the resulting compound of formula I, wherein R 1 , R 2 and R 3 represent H, with a source of nitrate. (a) when the source of nitrite is an organic nitrite ester, step (i) is performed in a suitable organic solvent; and (b) when the source of nitrite is an inorganic nitrite salt, step (i) is performed in a biphasic solvent mixture comprising an aqueous phase and a non-aqueous phase; and wherein, step (i) is performed in the presence of a suitable acid. The method further comprises the step of adding a further amount of a compound of formula I, wherein R 1 , R 2 and R 3 represent H, such that the combined mixture of the one or more compounds of formula I and the compound of formula I, wherein R 1 , R 2 and R 3 represent H, comprises 0.01 wt.% to 9 wt.% of the one or more compounds of formula I.

2. The method of claim 1, wherein, said inorganic nitrite salt is a metal nitrite salt.

3. The method of claim 1, wherein, said metal nitrite salt is an alkali metal nitrite salt or an alkaline earth metal nitrite salt.

4. The method of claim 3, wherein, said metal nitrite salt is an alkali metal nitrite salt.

5. The method of claim 4, wherein, said alkali metal nitrite salt is sodium nitrite.

6. The method of claim 5, wherein, said organic nitrite ester is an alkyl nitrite ester.

7. The method of claim 1, wherein, said alkyl nitrite ester is tert-butyl nitrite.

8. The method of claim 7, wherein, said suitable acid is a strong acid.

9. The method of claim 2, wherein, said strong acid is a strong inorganic acid.

10. The method of claim 9, wherein, said strong inorganic acid is sulfuric acid.

11. The method of claim 10, wherein, said non-aqueous phase comprises a water-immiscible organic solvent.

12. The method of claim 1, wherein, said water-immiscible organic solvent is a water-immiscible aprotic organic solvent.

13. The method of claim 12, wherein, said water-immiscible organic solvent is dichloromethane or tert-butyl methyl ether.

14. The method of claim 12, wherein, after step (i), the process further comprises the step of:

15. The method of any one of claims 1 to 14, wherein, The solvent mixture further comprises an excess of a solvent in which R 1 , R 2 and R 3 denote H.

16. The method of any one of claims 1 to 14, wherein, (ii) removing substantially all of the aqueous phase from the solvent mixture. after step (i), the process further comprises one or more of the following steps:

17. The method of any one of claims 1 to 14, wherein, (ii) removing some or all or substantially all of the aqueous phase; (iii) washing the remaining organic phase with one or more additional aqueous phases. after step (iii), the process comprises:

18. The method of claim 17, wherein, (iv) repeating steps (ii) and (iii) one or more times. after step (iv), the process comprises:

19. The method of claim 18, wherein, (v) reducing the amount / volume of the organic phase. after step (v), the process comprises:

20. The method of claim 19, wherein, (vi) drying the product, wherein steps (ii) to (vi) can be performed in any order, provided that steps (ii) to (iv) are performed prior to steps (v) and (vi).

21. A composition comprising: (a) one or more compounds of formula I ###0002### wherein n is 0 or 1 ; and wherein the composition comprises 0.01 to 9% by weight of the one or more compounds of formula I; （I） wherein R 1 , R 2 , and R 3 each independently represent H or -NO, wherein the composition comprises at least 80% by weight of components (a) and (b); wherein when n is 0, R 1 is H; and wherein when n is 1, R 2 is H, provided that at least one of R 1 R 2 and R 3 represents -NO; and (b) wherein R 1 , R 2 and R 3 represent H; and (b) wherein R 1 , R 2 and R 3 represent H; and wherein the composition comprises less than 5 wt.% of dissolved nitric oxide.

22. The composition according to claim 21, wherein the composition comprises less than 1% by weight of water.

23. A composition comprising: (a) one or more compounds of formula I ###0003### wherein n is 0 or 1 ; and wherein the composition comprises 0.01 to 9% by weight of the one or more compounds of formula I; wherein the composition comprises less than 1% by weight of water; and （I） wherein, R 1 , R 2 , and R 3 each independently represent H or -NO, wherein the composition comprises less than 5 wt.% of dissolved nitric oxide. wherein when n is 0, R 1 is H; and wherein when n is 1, R 2 is H, provided that at least one of R 1 , R 2 , and R 3 represents -NO; and (b) wherein R 1 , R 2 and R 3 represent H; and (b) wherein R 1 , R 2 and R 3 represent H; and 25. A pharmaceutical formulation comprising the composition according to any one of claims 21 to 24. ​ ​ 24. The composition of any one of claims 21-23, wherein the composition consists essentially of the one or more compounds of Formula I and compounds of Formula I wherein R 1 , R 2 , and R 3 represent H. ​ 26. The pharmaceutical formulation of claim 25, further comprising one or more pharmaceutically acceptable excipients; wherein, the one or more pharmaceutically acceptable excipients are non-aqueous.

27. A kit-of-parts comprising: (A) the pharmaceutical formulation of claim 25 or 26; and (B) a suitable aqueous buffer, wherein components (A) and (B) are provided in a form suitable for administration to each other.

28. A combination product formed by mixing together: (A) the pharmaceutical formulation of claim 25 or 26; and (B) a suitable aqueous buffer.

29. The kit-of-parts of claim 27 or the combination product of claim 28, wherein the ratio of the pharmaceutical formulation to the suitable buffer is 3:7 to 1 :99 by volume.

30. The kit of parts according to claim 27, or the combination product according to claim 28, wherein, the suitable aqueous buffer is non-nucleophilic and weakly basic.

31. The kit of parts according to any one of claims 27, 29-30, or the combination product according to any one of claims 28 to 30, wherein, the suitable aqueous buffer maintains a pH of 7.1 to 10.

32. The kit of parts according to any one of claims 27, 29-30, or the combination product according to any one of claims 28 to 30, wherein, the suitable aqueous buffer is a carbonate buffer or a phosphate buffer or a mixture thereof.

33. A method for preparing the combination product of any one of claims 28-32, comprising the step of mixing together: (A) the pharmaceutical formulation of claim 25 or 26; and (B) the suitable aqueous buffer of any one of claims 30 to 32.

34. The composition of any one of claims 21 to 24, the pharmaceutical formulation of any one of claims 25-26, the kit-of-parts of any one of claims 27, 29-32, or the combination product of any one of claims 28 to 32, for use in the manufacture of a medicament for the treatment of acute pulmonary vasoconstriction of different etiologies; pulmonary hypertension of different etiologies, including primary and secondary hypertension; conditions of different etiologies requiring vasodilation; systemic hypertension of different etiologies; regional vasoconstriction of different etiologies; local vasoconstriction of different etiologies; acute heart failure; and acute pulmonary hypertension in patients undergoing cardiac surgery.

35. The use of a therapeutically effective amount of the pharmaceutical formulation of claim 25 or 26 or the combination product of any one of claims 28 to 32, in the manufacture of a medicament for the treatment of acute pulmonary vasoconstriction of different etiologies; pulmonary hypertension of different etiologies, including primary and secondary hypertension; conditions of different etiologies requiring vasodilation; systemic hypertension of different etiologies; regional vasoconstriction of different etiologies; local vasoconstriction of different etiologies; acute heart failure; and acute pulmonary hypertension in patients undergoing cardiac surgery.

36. A therapeutically effective amount of a component for use in the preparation of a medicament for the treatment of acute pulmonary vasoconstriction of different etiologies; pulmonary hypertension of different etiologies, including essential hypertension and secondary hypertension; conditions of different etiologies requiring vasodilation; systemic hypertension of different etiologies; regional vasoconstriction of different etiologies; local vasoconstriction of different etiologies; acute heart failure; and acute pulmonary hypertension in patients undergoing cardiac surgery, wherein, the components comprise: (A) the pharmaceutical formulation of claim 25 or 26; and (B) the suitable aqueous buffer of any one of claims 29 to 32, and wherein the components (A) and (B) are mixed prior to administration.

37. The use according to any one of claims 34-36, wherein, the acute heart failure is with or without preserved ejection fraction.

38. The use of claim 36 or 37, wherein, The mixing of components (A) and (B) is performed immediately prior to administration to the patient.

39. The use of claim 38, wherein, The administration is achieved by co-administration of the components (A) and (B).

40. The use of any one of claims 36 to 39, wherein, The mixing is performed by a mixing stream method.

41. The use of claim 40, wherein, The mixing stream method occurs at the time of administration of the components to the patient.

42. The use of any one of claims 34-36, wherein, The condition is selected from the group consisting of pulmonary hypertension of different origin, including primary and secondary hypertension; and acute heart failure.

43. The use of claim 42, wherein, The acute heart failure is with or without preserved ejection fraction.

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