PROCESSES FOR PREPARING NITROSYLATED PROPANEDIOLS, COMPOSITIONS COMPRISING THE SAME, AND MEDICAL USES THEREOF
20220002223 · 2022-01-06
Inventors
- Per Håkan Agvald (Stockholm, SE)
- Leif Christofer Adding (Åkersberga, SE)
- Kristofer Bo Ingemar Nilsson (Örebro, SE)
- Anna Lena Elisabeth Minidis (Järna, SE)
- Johan Salman Malmberg (Stockholm, SE)
- Alexander Bogdan Emil Minidis (Järna, SE)
Cpc classification
A61K31/047
HUMAN NECESSITIES
International classification
Abstract
Disclosed is a process for the synthesis of mono- and bis-nitrosylated propanediols, as well as compositions and pharmaceutical formulations that includes the compounds. The process proceeds by reacting a corresponding propanediol that is not nitrosylated with a source of nitrite, optionally in the presence of a suitable acid. When the source of nitrite is an organic nitrite, reacting step is performed in a suitable organic solvent, and when the source of nitrite is an inorganic nitrite, the reacting step is performed in a bi-phasic solvent mixture comprising an aqueous phase and a non-aqueous phase. Also disclosed are methods of treating a condition wherein administration of nitric oxide (NO) has a beneficial effect by administering said compounds, compositions or formulations.
Claims
1. A process for the preparation of a composition comprising one or more compounds of formula I ##STR00009## wherein R.sup.1, R.sup.2 and R.sup.3 each independently represent H or —NO, wherein n is 0 or 1; wherein when n is 0, R.sup.1 is H; and wherein when n is 1, R.sup.2 is H provided that at least one of R.sup.1 R.sup.2 and R.sup.3 represents —NO, said process comprising the step of: (i) reacting a corresponding compound of formula I but wherein R.sup.1, R.sup.2 and R.sup.3 represent H with a source of nitrite, optionally in the presence of a suitable acid, wherein: (a) when the source of nitrite is an organic nitrite, step (i) is performed in a suitable organic solvent; and (b) when the source of nitrite is an inorganic nitrite, step (i) is performed in a bi-phasic solvent mixture comprising an aqueous phase and a non-aqueous phase.
2. A process as claimed in claim 1, wherein inorganic nitrite is a metal nitrite.
3. A process as claimed in claim 2, wherein the metal nitrite is an alkali metal nitrite or an alkaline earth metal nitrite.
4. A process as claimed in claim 3, wherein the metal nitrite is an alkali metal nitrite.
5. A process as claimed in claim 4, wherein the alkali metal nitrite is sodium nitrite.
6. A process as claimed in claim 2, wherein the organic nitrite is an alkyl nitrite.
7. A process as claimed in claim 6, wherein the alkyl nitrite is tert-butyl nitrite.
8. A process as claimed in any one of claims 1 to 7, wherein the suitable acid is a strong acid, such as a strong mineral acid.
9. A process as claimed in claim 8, wherein the strong mineral acid is sulphuric acid.
10. A process as claimed in any one of claims 1 to 9, wherein the non-aqueous phase comprises a water immiscible organic solvent, such as a water immiscible aprotic organic solvent.
11. A process as claimed in claim 10, wherein the water immiscible organic solvent is dichloromethane or tert-butylmethyl ether.
12. A process as claimed in any one of claims 1 to 11, wherein the solvent mixture further comprises excess of a compound of formula I but wherein R.sup.1, R.sup.2 and R.sup.3 represent H.
13. A process as claimed in any one of claims 1 to 12, wherein after step (i) the process further comprises the step of: (ii) removing substantially all of the aqueous phase from the solvent mixture.
14. A process as claimed any one of claims 1 to 12, wherein after step (i) the process further comprises the step(s) of: (ii) removing some or all (e.g. substantially all) of the aqueous phase (i.e. of water); (iii) washing the remaining organic phase with one or more further aqueous phase; (iv) optionally repeating steps (ii) and (iii) one or more times; (v) optionally reducing (i.e. reducing the amount/volume of) the organic phase, and (vi) optionally drying the product, wherein steps (ii) to (vi) may be performed in any order provided that steps (ii) to (iv) are performed before steps (v) and (vi).
15. A process as claimed in any one of claims 1 to 12, wherein the process further comprises the step of adding a further amount of a compound of formula I but wherein R.sup.1, R.sup.2 and R.sup.3 represent H, such that the combined mixture of the one or more compounds of formula I and a compound of formula I but wherein R.sup.1, R.sup.2 and R.sup.3 represent H comprises from about 0.01% to about 9% by weight of the one or more compounds of formula I.
16. A product obtained or obtainable by a process as claimed in any one of claims 1 to 15.
17. A substantially non-aqueous composition comprising: (a) one or more compounds of formula I ##STR00010## wherein R.sup.1, R.sup.2 and R.sup.3 each independently represent H or —NO, wherein n is 0 or 1; and wherein when n is 0, R.sup.1 is H; and wherein when n is 1, R.sup.2 is H provided that at least one of R.sup.1 R.sup.2 and R.sup.3 represents —NO; and (b) a compound of formula I but wherein R.sup.1, R.sup.2 and R.sup.3 represent H.
18. A composition as claimed in claim 17, wherein the composition comprises from about 0.01% to about 9% by weight of the one or more compounds of formula I.
19. A composition as claimed in any one of claims 17 and 18, wherein the composition is substantially free of dissolved nitric oxide.
20. A composition as claimed in any one of claims 17 to 19, wherein the composition consists essentially of the one or more compounds of formula I and a compound of formula I but wherein R.sup.1, R.sup.2 and R.sup.3 represent H.
21. A pharmaceutical formulation comprising a composition as claimed in any one of claims 17 to 20, and optionally one or more pharmaceutically acceptable excipients.
22. A pharmaceutical formulation as claimed in claim 21, wherein the one or more pharmaceutically acceptable excipients are non-aqueous.
23. A kit-of-parts comprising: (A) a pharmaceutical formulation as claimed in any one of claims 21 and 22; and (B) a suitable aqueous buffer, wherein components (A) and (B) are provided in a form suitable for administration with each other.
24. A combination product formed by mixing together: (A) a pharmaceutical formulation as claimed in any one of claims 21 and 22; and (B) a suitable aqueous buffer.
25. A kit-of-parts as claimed in claim 23 or a combination product as claimed in claim 24, wherein the ratio of the pharmaceutical formulation and the suitable buffer is from about 3:7 to about 1:99 by volume.
26. A kit-of-parts as claimed in any one of claims 23 and 25, or a combination product as claimed in any one of claims 24 and 25, wherein the buffer is non-nucleophilic and weakly basic.
27. A kit-of-parts as claimed in any one of claims 23, 25 and 26, or a combination product as claimed in any one of claims 24 to 26, wherein the buffer maintains a pH of from about 7.1 to about 10.
28. A kit-of-parts as claimed in any one of claims 23 and 25 to 27, or a combination product as claimed in any one of claims 24 to 27, wherein the buffer is a carbonate buffer or a phosphate buffer, or a mixture thereof.
29. A process for preparing a combination product as claimed in any one of claims 24 and 26 to 28, comprising the step of mixing together: (A) a pharmaceutical formulation as claimed in any one of claims 21 and 22; and (B) a suitable aqueous buffer as defined in any one of claims 26 to 28.
30. A composition as claimed in any one of claims 17 to 20, a pharmaceutical formulation as claimed in any one of claims 21 and 22, a kit-of-parts as claimed in any one of claims 23 and 25 to 28, or a combination product as claimed in any one of claims 24 to 28, for use in the treatment of a condition wherein administration of NO has a beneficial effect.
31. A method of treating a condition wherein administration of NO has a beneficial effect comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical formulation as claimed in any one of claims 21 and 22 or a combination product as claimed in any one of claims 24 to 28.
32. A method of treating a condition wherein administration of NO has a beneficial effect comprising administering to a patient in need thereof a therapeutically effective amount of components: (A) a pharmaceutical formulation as claimed in any one of claims 21 and 22; and (B) a suitable aqueous buffer as defined in any one of claims 25 to 28, wherein the components (A) and (B) are mixed prior to administration.
33. The use as claimed in claim 30 or the method as claimed in any one of claims 31 and 32, wherein the condition is selected from the group consisting of: acute pulmonary vasoconstriction of different genesis; pulmonary hypertension of different genesis, including primary hypertension and secondary hypertension; conditions of different genesis in need of vasodilation; systemic hypertension of different genesis; regional vasoconstriction of different genesis; local vasoconstriction of different genesis; acute heart failure (with or without preserved ejection fraction (HFpEF)); coronary heart disease; myocardial infarction; ischemic heart disease; angina pectoris; instable angina; cardiac arrhythmia; acute pulmonary hypertension in cardiac surgery patients; acidosis; inflammation of the airways; cystic fibrosis; COPD; immotile cilia syndrome; inflammation of the lung; pulmonary fibrosis; adult respiratory distress syndrome; acute pulmonary oedema; acute mountain sickness; asthma; bronchitis; hypoxia of different genesis; stroke; cerebral vasoconstriction; inflammation of the gastrointestinal tract; gastrointestinal dysfunction; gastrointestinal complication; IBD; Crohn's disease; ulcerous colitis; liver disease; pancreas disease; inflammation of the bladder of the urethral tract; inflammation of the skin; diabetic ulcers; diabetic neuropathy; psoriasis; inflammation of different genesis; wound healing; organ protection in ischemia-reperfusion conditions; organ transplantation; tissue transplantation; cell transplantation; acute kidney disease; uterus relaxation; cervix relaxation; and conditions where smooth muscle relaxation is needed.
34. The method as claimed in any one of claims 32 and 33, wherein the mixing of components (A) and (B) is performed immediately prior to administration to the patient, such as by co-administration thereof.
35. The method as claimed in any one of claims 32 to 34, wherein the mixing is performed by a mixed flow process.
36. The method as claimed in claim 35, wherein the mixed flow process occurs at the point of administration of the components to the patient.
37. The use according to claim 33, wherein the condition is selected from the group consisting of pulmonary hypertension of different genesis, including primary hypertension and secondary hypertension; and acute heart failure (with or without preserved ejection fraction (HFpEF)).
Description
BRIEF DESCRIPTION OF THE FIGURES
[0198]
[0199]
[0200]
EXAMPLES
[0201] The invention is illustrated by way of the following examples, which are not intended to be limiting on the general scope of the invention.
Abbreviations
[0202] aq aqueous
conc concentration
GC gas chromatography
NMR nuclear magnetic resonance
equiv. equivalent(s)
rel. vol. relative volume(s)
General Procedures
[0203] Starting materials and chemical reagents specified in the preparations described below are commercially available from a number of suppliers, such as Sigma Aldrich.
[0204] All NMR experiments were performed at 298K on a Bruker 500 MHz AVI instrument equipped with a QNP probe-head with Z-gradients using the Bruker Topspin 2.1 software. Signals were referenced to residual CHCl.sub.3 at 7.27 ppm, unless stated otherwise.
Stability Assays
[0205] Assays of the stability samples were performed by GC/FID, under the following conditions. 1,4-Dioxane was used as the Internal Standard (IS; approximately 0.50 mg/ml in CH.sub.3CN).
GC column: Rxi-5Sil MS, 20 m×0.18 mm, 0.72 μm
Carrier gas: Helium
[0206] Inlet: 200° C., split ratio 30:1
Constant flow: 1.0 ml/min
Oven temperature profile: 40° C. (3 min), 10° C./min, 250° C. (3 min)
FID: temp 300° C.; H.sub.2 flow 30 ml/min, Air flow 400 ml/min, make-up flow (N.sub.2) 25 ml/min
In Vivo Studies
[0207] Prior to experimentation, ethical approval was received from Linköping's regional animal ethics committee (Linköping, Sweden; approval number 953). Anaesthetic management, surgical instrumentation and methods for measurements were recently described (Dogan et al. 2018, Sadeghi et al. 2018).
[0208] In brief, 8 male and female pigs (a crossbreed between Swedish country breed, Hampshire and Yorkshire; 3-4 months old; mean body weight 27 kg, range 21-34 kg) were premedicated with azaperone at the farm and transported to the laboratory. At the laboratory, anaesthesia was induced with a mixture of tiletamine, zolazepam and azaperone (intramuscular injection). Propofol was given in a peripheral venous catheter in an ear vein, if needed. Bolus doses of atropine and cefuroxime were administered intravenously. The animals were endotracheally intubated and mechanically ventilated (5 cm H.sub.2O in positive end-expiratory pressure, minute ventilation was adjusted to normoventilation). General anaesthesia was maintained with propofol and fentanyl via continuous intravenous infusions, and additional bolus doses were given if needed. Ringer's acetate and glucose solutions were continuously administered intravenously to substitute for fluid loss. Heparin was given as an intravenous bolus dose after the surgical instrumentation. After the experiments the animals were killed in general anaesthesia with a propofol injection followed by a rapid intravenous injection of potassium chloride (40 mmol), and asystolia was confirmed.
[0209] The animals were instrumented with an arterial catheter in the right carotid artery for measurement of systemic arterial blood pressure and heart rate, and for arterial blood sampling. A sheath was placed in the right external jugular vein for introduction of a pulmonary-arterial catheter. This catheter was used for continuous measurement of pulmonary arterial blood pressure, semi-continuous cardiac output and intermittent pulmonary wedge pressure. A central venous catheter was inserted in the left external jugular vein for drug and fluid administration. All fluid and drug administrations were done by motorised syringe or drip pumps. The urinary bladder was catheterized. Respiratory gases, pressures and volumes were measured at the endotracheal tube. Respiratory and hemodynamic variables were measured by a Datex AS/3 (Helsinki, Finland) and data were collected by a computerised system (MP150/Acknowledge 3.9.1, BIOPAC systems, Goleta, Calif., USA). Blood gases and methemoglobin concentration were measured by a blood gas machine (GEM 4000, Instrumental Laboratory, Lexington, Mass., USA). Pulmonary and systemic vascular resistances were calculated with standard formula. After surgical instrumentation, a 1 h intervention-free period followed.
[0210] Data were presented as median and interquartile range due to non-normal distribution. The ppm dose of inhaled NO was converted to dose in nmol kg.sup.−1 min.sup.−1 using the ideal gas law and minute ventilation, and assuming full uptake of NO in the lung. Within drug data were analysed with Friedman's test with Wilcoxon's signed rank test for post-hoc multiple comparisons. Mann Whitney U tests were used to compare the drugs at the maximal dose and at the dose of similar NO delivery (45 nmol kg.sup.−1 min.sup.−1 of PDNO and 5 ppm of inhaled NO). A critical P-value of 0.05 was used and adjusted by Benjamini-Hochberg's step-up procedure in multiple comparisons.
Example 1—Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane with sodium nitrite
[0211] 1,2-propanediol (15 mL, 205 mmol), water (100 mL), dichloromethane (200 mL) and sodium nitrite (57 g, 826 mmol) were added to a 500 mL three-necked round bottom flask. The mixture was cooled down to 0° C. with an ice bath. Concentrated sulphuric acid (30 mL, 546 mmol) and water (30 mL) were added to a dropping funnel and cooled to 5° C. in a refrigerator. The funnel was adapted to the round bottom flask and the acid added to the nitrite mixture during two hours. The mixture was stirred with a magnet for 20 minutes and then poured into a separation funnel together with more dichloromethane (100 mL) and water (100 mL). The organic phase was separated and dried with sodium sulphate, and reduced on a rotavapor to yield a mixture of 1,2-propanediol (3 wt. %), 1-(nitrosooxy)-propan-2-ol (23 wt. %) 2-(nitrosooxy)-propan-1-ol (13 wt. %) and 1,2-bis(nitrosooxy)propane (57 wt. %).
Example 2—Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane with sodium nitrite
[0212] 1,2-propandiol (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 reactor fitted with a stirrer and flushed with nitrogen and kept during the course of the following reaction under nitrogen. The mixture was cooled down to below 5° C. by cooling the mantle to 0° C. Concentrated sulphuric acid (26.3 g, 260.1 mmol) and water were added to a dropping funnel. The funnel was attached (to the reactor and the acid was added to the nitrite mixture during 33 minutes. The mixture was stirred for 54 minutes and then poured into a flask containing an aqueous saturated sodium bicarbonate solution (100 mL). The mixture was transferred to a separation funnel and the organic phase was washed. The aqueous phase was discarded, and the organic phase was washed with additional aqueous saturated sodium bicarbonate solution (100 mL). The organic phase was dried with magnesium sulphate and then transferred to a 1 L round bottom flask together with 1,2-propandiol (120 ml, 1640 mmol). The solution was reduced on a rotavapor under reduced pressure until the dichloromethane was removed. The removal of dichloromethane was monitored by NMR. A clear solution (134 g) containing 1,2-propandiol (82.8 wt. %), 1-(nitrosooxy)-propan-2-ol (10.4 wt. %), 2-nitrosooxy)-propan-1-ol (6 wt. %) and 1,2-bis(nitrosooxy)propane (0.8 wt. %) was obtained.
[0213] .sup.1H-NMR, δ ppm: 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), Signals for CH and CH.sub.2 of the 1,2-bis(nitrosooxy)propane were below the detection limit.
Example 3—Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane with tert-butyl nitrite
[0214] Tert-butyl nitrite (2 mL, 15.1 mmol) was added to a round bottom flask with 1,2-propanediol (11 mL, 150.3 mmol) and the obtained solution was stirred at ambient temperature. 1 mL of the reaction solution was then mixed with 7.5 mL 1,2-propanediol.
Example 4—Stability of Non-Aqueous Mixtures of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-propanediol
[0215] Three different concentrations of 1-(nitrosooxy)-propan-2-ol and 2-(nitrosooxy)-propan-1-ol in 1,2-propanediol were prepared and stored in both a refrigerator (5° C.) and freezer (−20° C.). Aliquots of each solution were taken periodically and analysed by GC to determine the concentration of 1-(nitrosooxy)-propan-2-ol and 2-(nitrosooxy)-propan-1-ol.
[0216] The results of the GC analysis are shown in the table below (column: Rxi-5Sil MS, 20 m×0.18 mm, 0.36 film thickness; carrier: He; Inlet: 250° C., split ratio 100:1; constant flow: 1.0 mL/min; oven temperature profile: 40° C. (3 min), 10° C./min, 80° C. (0 min), 30° C./min, 250° C. (3 min); FID: 300° C., H.sub.2 flow 30 mL/min, air flow 400 mL/min, make-up flow (N.sub.2) 25 mL/min; internal standard: 1,1,1,3,5,5,5-heptamethyl trisiloxane):
TABLE-US-00001 Refrigerator (5° C.) Freezer (−20° C.) Concentration Concentration (% w/w) (% w/w) 1- 2- 1- 2- Stability Sample Nitrite Nitrite Total Nitrite Nitrite Total Start High conc. 3.75 2.94 6.69 3.75 2.94 6.69 Start Medium 0.81 0.61 1.42 0.81 0.61 1.42 conc. Start Low conc. 0.08 0.06 0.14 0.08 0.06 0.14 14 days High 3.72 2.91 6.63 3.76 2.89 6.65 conc. 10 days Medium 0.86 0.67 1.53 0.81 0.63 1.44 conc. 10 days Low 0.08 0.06 0.14 0.08 0.06 0.14 conc. 28 days High 3.67 2.90 6.57 3.72 2.93 6.65 conc. 27 days Medium 0.81 0.63 1.44 0.74 0.57 1.31 conc. 27 days Low 0.09 0.07 0.16 0.07 0.06 0.13 conc. 56 days High 3.47 2.69 6.16 3.55 2.74 6.29 conc. 64 days Medium 0.73 0.57 1.30 0.74 0.58 1.32 conc. 64 days Low 0.07 0.06 0.13 0.07 0.06 0.13 conc. 84 days High 3.33 2.59 5.92 3.50 2.71 6.21 conc. 84 days Medium 0.77 0.60 1.37 0.78 0.62 1.40 conc. 84 days Low 0.07 0.06 0.13 0.08 0.06 0.14 conc. Note: no build-up of pressure was observed for any of the samples.
Example 5—Stability of Aqueous Buffered Solutions of 1-(nitrosooxy)-propan-2-ol and 2-(nitrosooxy)-propan-1-ol (PDNO), and 1,2-propanediol (PD)
[0217] 100 μl of the stability sample was added to a GC vial. 400 μl of a solution PD/Buffer (1:9) and 400 μl CH.sub.3CN were added. Then 500 μl CH.sub.2Cl.sub.2 was added and the mixture extracted by gentle shaking for 1 min. 500 μl of the organic phase (lower phase) was transferred to another GC vial and 50 μl IS added. The extract was analysed by GC/FID according to the conditions above.
[0218] Calibration curves for 1-nitrite and 2-nitrite, respectively, were constructed. The peak area ratio (Nitrite/IS) vs. amount of nitrite was plotted. Stock solutions of PDNO/PD at high concentration were used for preparing the standards. Concentrations of nitrites in % w/w were calculated.
[0219] The results obtained are shown in the table below and in
TABLE-US-00002 Change in concentration PDNO/Buffer or Saline (1:9) % of the original concentration, i.e. 0.02% w/w 5% 0.154M 0.154M NaHCO.sub.3 Carbonate Phosphate Sample (pH 8)* (pH 9.2)* (pH 8.0)* Saline 0 min 100.0 100.0 100.0 45.3 15 min 91.1 95.8 97.4 43.0 30 min 85.1 92.5 93.1 42.9 45 min 75.7 85.5 86.4 40.9 60 min 72.5 79.6 84.1 38.8 *Based on known values.
Example 6—In Vivo Studies
[0220] After collecting baseline data, stable pulmonary hypertension was induced by continuous intravenous infusion of the thromboxane A2-mimetic 9,11-dideoxy-9α, 11α-methanoepoxy PGF.sub.2α (U46619, Cayman Chemical, Michigan, USA; supplied in methyl acetate and diluted in NaCl 0.9% to a final concentration of 30 μg ml.sup.−1; 60-150 ng kg.sup.−1 min.sup.−1 to a target mean pulmonary arterial pressure of 35-45 mmHg). Thereafter either a continuous intravenous infusion of PDNO in increasing doses (15, 30, 45 and 60 nmol kg.sup.−1 min.sup.−1) into a carrier flow of a solution of sodium bicarbonate (50 mg ml.sup.−1; pH approximately 8; Fresenius Kabi, Uppsala, Sweden; infusion rate 10 times of the PDNO infusion rate) or inhalation of NO (5, 10, 20 and 40 ppm; delivered into the inspiratory limb of a Servo 300 ventilator with a inhaled NO dosing unit [Siemens-Elema, Stockholm, Sweden] from a tank of 1000 ppm in nitrogen) in non-randomised cross-over design with a wash-out period of 30 min between the drugs. Correct dosage of inhaled NO was checked with a NO analyser. Each dose was administered for 5-10 min. Hemodynamic and respiratory data were extracted, and arterial blood was sampled, at the last minute of each dose.
[0221] Intravenous infusion of U46619 induced stable pulmonary hypertension with mean pulmonary arterial pressure of 43 (37-48) mmHg and 43 (41-46) mmHg, and pulmonary vascular resistance of 8.3 (6.7-11.7) mmHg min l.sup.−1 and 9.8 (7.7-12.5) mmHg min l.sup.−1, before NO inhalation and PDNO infusion, respectively. Both inhaled NO and intravenously infused PDNO significantly reduced pulmonary arterial pressure and vascular resistance, but PDNO decreased mean pulmonary arterial pressure more efficiently (steeper slope) than inhaled NO, and PDNO decreased pulmonary vascular resistance significantly more compared to inhaled NO at a comparable dosage (as shown in
Example 7—Solvent Free Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol, and 1,2-bis(nitrosooxy)propane with sodium nitrite
[0222] Water (30 mL) and sodium nitrite (19.01 g, 272.8 mmol) were added to a 100 mL three-necked round bottom flask, flushed with nitrogen and cooled down to 1° C. on a water bath cooled with an external cooler. 1,2-Propanediol (10 mL, 136.7 mmol) was added. Concentrated sulphuric acid (7 mL, 127.4 mmol) and water (20 mL) were pre-cooled to room temperature and added dropwise during one hour via a dropping funnel. During the addition, the water layer formed a thick slurry and a green second layer was formed. Before completion of acid addition (5 mL remaining) the flask was removed from the cooling bath and the green layer was decanted into a separation funnel and washed with 2× saturated aqueous NaHCO.sub.3 solution. The green layer faded to yellow and after separation was dried over Na.sub.2SO.sub.4 and filtered through a syringe filter (Acrodisc® 13 mm, 0.45 μM SUPOR®) to yield 1.1 g mixture of approximately 0.25/0.1/1 of 1-(nitrosooxy)-propan-2-ol/2-(nitrosooxy)-propan-1-ol/1,2-bis(nitrosooxy)propane. No starting-material 1,2-propanediol could be detected within the limits of NMR sensitivity.
[0223] .sup.1H-NMR, δ ppm: 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), 4.14 (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).
Example 8—Preparation of (2S)-1-(nitrosooxy)-propan-2-ol, (2S)-2-(nitrosooxy)-propan-1-ol and (2S)-1,2-bis(nitrosooxy)propane
[0224] (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, flushed with nitrogen and cooled down to 1° C. on a water bath cooled with an external cooler. Concentrated sulphuric acid (3.5 mL, 63.69 mmol) and water (10 mL) were pre-cooled to room temperature and added dropwise via a syringe-pump during 1 h. After addition the mixture was stirred for additional 60 minutes. After separation of the two layers, the DCM layer was diluted with additional DCM (15 mL) and washed with sat. aq. NaHCO.sub.3 (15 mL), followed by brine (15 mL), then dried over Na.sub.2SO.sub.4, filtered over a sintered glass filter and reduced in vacuo. The residue was taken up again in 30 mL DCM, washed with 1.4% w/w aq. bicarbonate solution, then dried over Na.sub.2SO.sub.4, filtered over a sintered glass filter and reduced in vacuo to yield 1 g of product mixture. The mixture of consisted of (2S)-1,2-propanediol (3%), (2S)-1-(nitrosooxy)-propan-2-ol (23%), (2S)-2-(nitrosooxy)-propan-1-ol (14%) and (2S)-1,2-bis(nitrosooxy)propane (60%) based on NMR.
[0225] .sup.1H-NMR, δ ppm: 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, 0.38H), 3.86-3.73 (br, 0.40H), 1.8-1.6 (br, 0.97H), 1.48 (d, J=6.7 Hz, 3.12H), 1.40 (d, J=6.6 Hz, 0.63H), 1.28 (d, J=6.5 Hz, 1.15H).
Example 9—Preparation of (2R)-1-(nitrosooxy)-propan-2-ol, (2R)-2-(nitrosooxy)-propan-1-ol and (2R)-1,2-bis(nitrosooxy)propane
[0226] (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, flushed with nitrogen and cooled down to 1° C. on a water bath cooled with an external cooler. Concentrated sulphuric acid (3.5 mL, 63.69 mmol) and water (10 mL) were pre-cooled to room temperature and added dropwise via a syringe-pump during 1 h. After addition the mixture was stirred for additional 55 minutes. After separation of the two layers, the DCM layer was diluted with additional DCM (10 mL) and washed with saturated aqueous NaHCO.sub.3 (20 mL), then dried over Na.sub.2SO.sub.4, filtered over a sintered glass filter and reduced in vacuo. The mixture of consisted of (2R)-1,2-propanediol (17%), (2R)-1-(nitrosooxy)-propan-2-ol (16%), (2R)-2-(nitrosooxy)-propan-1-ol (7%) and (2R)-1,2-bis(nitrosooxy)propane (59%) based on NMR.
[0227] .sup.1H-NMR, δ ppm: 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=6.8 Hz, 3.20H), 1.40 (br, 0.56H), 1.28 (br(d), 0.88H).
Example 10—Preparation of 1-(nitrosooxy)propan-3-ol and 1,3-bis(nitrosooxy)propane
[0228] 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, flushed with nitrogen and cooled down to 0° C. for 15 min on a water bath cooled with an external cooler. Concentrated sulphuric acid (1.7 mL, 31.2 mmol) and water (5 mL) were pre-cooled to room temperature and added dropwise for 5 minutes. After addition the mixture was stirred for additional 60 minutes at 0° C. The two layers was then separated, and the organic phase was diluted with additional DCM (10 mL), washed with saturated aqueous NaHCO.sub.3 (2×25 mL), dried over MgSO.sub.4, filtered over a sintered glass filter. Finally, 1,3-propanediol (16.4 g 216 mmol) was added to the organic phase followed by removal of DCM in vacuo. Based on NMR the mixture (18.1 g) contained 1,3-propandiol (86.9 wt. %), 1-(nitrosooxy)-propan-3-ol (11.8 wt. %), and 1,3-bis(nitrosooxy)propane (1.3 wt. %).
[0229] 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).
Example 11—Scaled Up Process for the Preparation of 1-(nitrosooxy)-propan-2-ol, 2-(nitrosooxy)-propan-1-ol and 1,2-bis(nitrosooxy)propane with sodium nitrite
11.1 Chemicals Used
[0230] Starting materials were purchased from the list of suppliers in the table below. Unless otherwise noted the chemicals were used as received without further purification.
TABLE-US-00003 List of used chemicals and solvents Chemical/ Solvent Grade Supplier 1,2-Propanediol EMPROVE ® ESSENTIAL Merck Ph. Eur. or BP or USP, ≥99% Sodium nitrite Conforms to current ACS, VWR, Acros USP or Ph. Eur., ≥97% Sulfuric acid ≥95.0, Conforms to VWR, Acros current ACS, USP or Ph. Eur. TBME Conforms to current ACS, VWR, Acros USP or Ph. Eur., ≥99% Sodium bicarbonate Conforms to current ACS, VWR, Acros USP or Ph. Eur. Magnesium sulfate USP, dried VWR, Acros Arqon 4.8 or higher Linde AG, Westfalen AG
11.2 General Procedure for the Synthesis of PDNO Using DCM as Solvent (Origin Process)
[0231] A round bottom flask was equipped with a stirrer and dropping funnel. Water (3.0 veq.) was added and sodium nitrite (2.0 equiv.) was charged to the flask. The solution was cooled (0° C.) and PD (1.0 equiv.) and DCM (6 rel. vol.) were also added. During further cooling, a sulfuric acid solution (1.0 eq. H.sub.2SO.sub.4, 2.0 rel. vol. water) was prepared. The sulfuric acid solution was further added dropwise to the reaction mixture while keeping the reaction mixture between 0° C. and 5° C. After complete addition of the acid, the solution was further stirred for 1 h to complete reaction.
[0232] Then, the reaction was quenched with saturated NaHCO.sub.3 solution (6.0 rel. vol.). The phases were separated, and the organic layer was further washed with NaHCO.sub.3 solution (6.0 rel. vol.). The organic phase was dried over MgSO.sub.4, filtered, diluted with PD, and concentrated under reduced pressure using a rotary evaporator (water bath temperature 40° C.).
[0233] The product was obtained as a slightly yellowish liquid.
11.3 General Synthesis of PDNO Using TBME as Solvent
[0234] A round bottom flask was equipped with stirrer and dropping funnel. Argon was flushed through for several minutes. A diluted sulfuric acid solution (1.0 eq. H.sub.2SO.sub.4, 2.0 rel. vol. water) was prepared in advanced and precooled (−30° C.). Water was added to the flask (3.0 rel. vol.). Sodium nitrite (2.0 equiv.) was added into the water. TBME (7.5 rel. vol.) was added. Propanediol (1.0 equiv.) was added and the reaction mixture was cooled (−20° C.) flushing constantly with argon. The reaction mixture was stirred well while adding dropwise the precooled sulfuric acid. The reaction temperature was monitored during the entire addition of the acid. After addition, the reaction mixture was further stirred (30-60 min) at cold temperature (−20° C.). Afterwards, the reaction mixture was allowed to warm up (−5° C.). The reaction was stopped by quenching with saturated NaHCO.sub.3 solution (6.0 rel. vol.). The phases were separated. The organic layer was further washed with saturated NaHCO.sub.3 solution until a pH value of 7-8 was obtained. The organic phase was then dried over MgSO.sub.4. The crude PDNO solution was diluted with PD (3 rel. vol.) and further concentrated under reduced pressure at ambient temperature (25° C.).
[0235] The crude PDNO solution was further purified using a vertical tube evaporation apparatus.
[0236] PDNO was obtained as a slightly yellowish liquid.
11.4 Detailed Synthesis of PDNO Using TBME as Solvent
[0237] The process was designed to produce approx. 7.5 L of 7% PDNO solution with one synthesis (one “run”). The synthesis was performed several times, to give the desired batch size. GC analysis was used each single run for purity determination. The runs which are within the specifications for the organic related compounds can be blended together to yield one batch. The entire crude PDNO batch was then purified. After purification, the strong PDNO solution was then further diluted with PD to yield the desired concentration (usually 7% PDNO solution).
[0238] A suitable double wall reactor (60 L) was equipped with specific “cup-stirrer”, dropping funnel and attachment for argon. The reactor was flushed for 5 min to 10 min with a constant argon stream. Water (3.0 L) was added to the reactor. Sodium nitrite (2.0 equiv., 1886 g) was added through the reactor. The reaction was further stirred until all of the salt was dissolved. 1,2-propanediol (1.0 equiv., 1040 g, 1 L) was added, followed by tert-butylmethyl ether (7.5 rel. vol., 7.5 L). The reaction mixture was then cooled by continuous stirring and argon flow at an inner reaction temperature of −20° C. Meanwhile sulfuric acid (1.0 equiv., 1340 g, 728 mL) was diluted with water (2.0 L) and cooled at −30° C. After reaching an inner reaction temperature of −20° C., the diluted acid was added dropwise to the reaction mixture while vigorous stirring.
[0239] The stirring speed was varied during the addition of the acid. Starting with approx. 350 rpm to a slower stirring speed by the end of the reaction (approx. 180 rpm). This variation of the stirring speed is due the two-phase reaction system and the slowly precipitation of sodium sulfate by further progress of the reaction (due to the addition of more and more sulfuric acid).
[0240] During the entire addition of the sulfuric acid, the reaction temperature was monitored. The temperature should ideally be in range of (−20±3) ° C. In addition, the reaction was stirred for 30-60 min at (−20±3) ° C.
[0241] The reaction was allowed to warm up to −5° C. to 0° C. The reaction was stopped by the addition of saturated NaHCO.sub.3 solution (6.0 rel. vol 6.0 L) followed by the addition of water (10 L). The phases were separated and the organic layer was transferred into a separate double wall reactor and chilled at 0° C. to −5° C. The organic layer was washed several times (approx. 2-3 times) with saturated NaHCO.sub.3 solution (4.0 rel. vol., 4.0 L). The pH value of the water phase was monitored after each washing step. The pH value was about 7-8. The water phases were discarded. The organic layer was dried over MgSO.sub.4 and filtered over a Whatman filter paper.
[0242] The crude PDNO (solution in TBME) was diluted by the addition of further PD (3.0 rel. vol., 3.0 L). This crude PDNO was transferred to a rotary evaporator and concentrated under reduced pressure. The water bath temperature during the evaporation was maintained at a maximum temperature of 25° C. The evaporation of the main amount of TBME was removed in a time range between 1.5 h and 2.0 h.
[0243] The evaporation of the organic solvents could then be continued at a water bath temperature at (0±2) ° C. for several hours using a high vacuum pump (during the development the PDNO purity was monitored at these conditions, and over a period of 6 h the product purity was not affected).
11.5 Further Purification of the Crude PDNO Solution
[0244] The final purification of the PDNO solution was done by vertical tube evaporation. The PDNO solution was distilled under high vacuum with a continuous thin steam of PDNO at 0° C. The storing tank for the “crude” PDNO solution was chilled at 0° C. The entire distillation was performed at 0° C. The storage tank for the “purified” PDNO was also chilled at −10° C. to 0° C. After each run of the evaporation of the entire batch PDNO, the residual organic solvent (TBME) can be checked via GC. This evaporation was continued until the desired limit for the residual solvents was achieved. In the case of PDNO the limit for the residual solvent is 1000 ppm.
11.6 Preparation of the Final Dilution
[0245] After purification, PDNO was further diluted to reach the favoured concentration. The first step was to filter the PDNO solution into a clean glass bottle via Whatman filter. In addition, the assay of the PDNO solution was determined via q-NMR. The amount of PD for dilution can be calculated. The PD was filtered first over a Whatman filter. The final dilution can be done at ambient temperatures. The calculated amount of PD was added to the PDNO solution (or the other way around). The resulting mixture was shaken for several minutes to obtain a homogeneous solution. The final PDNO solution was filled into the product bottles.
[0246] PDNO (7.5 kg; 7% solution) was yielded as a slightly yellowish liquid.
Example 12—Hemodynamic Effects of Intravenous PDNO: Influence of Various Carrier Solutions in Anesthetized Pigs
[0247] The influence of various carrier solutions on the hemodynamic effects of the organic mononitrites of 1,2-propanediol (PDNO), administered intravenously in anesthetized pigs, was studied.
[0248] Prior to experimentation, ethical approval was received from Linköping's regional animal ethics committee (Linköping, Sweden; approval number 953). The study was conducted in accordance with the Directive 2010/63/EU on the protection of animals used for scientific purposes. Two healthy domestic 3-month-old pigs (a crossbreed between Swedish country breed, Hampshire and Yorkshire; a body weight of 26 and 27 kg) were included in the study.
[0249] The animals were premedicated with azaperone at the farm and transported to the laboratory. At the laboratory, anesthesia was induced with a mixture of tiletamine, zolazepam and azaperone (intramuscular injection). Propofol was given in a peripheral venous catheter in an ear vein, if needed. Bolus doses of atropine and cefuroxime were administered intravenously. The animals were endotracheally intubated and mechanically ventilated (5 cm H.sub.2O in positive end-expiratory pressure, minute ventilation was adjusted to normoventilation). General anesthesia was maintained with propofol and fentanyl via continuous intravenous infusions, and additional bolus doses were given if needed. Ringer's acetate and glucose solutions were continuously administered intravenously to substitute for fluid loss. Heparin was given as an intravenous bolus dose after the surgical instrumentation. After the experiments the animals were killed in general anesthesia with a propofol injection followed by a rapid intravenous injection of potassium chloride (40 mmol), and asystole was confirmed.
[0250] The animals were instrumented with an arterial catheter in the right carotid artery for measurement of systemic arterial blood pressure and heart rate. A sheath was placed in the right external jugular vein for introduction of a pulmonary-arterial catheter. This catheter was used for continuous measurement of pulmonary arterial blood pressure and semi-continuous cardiac output. A central venous catheter was inserted in the left external jugular vein for drug and fluid administration. All fluid and drug administrations were done by motorized syringe or drip pumps. The urinary bladder was catheterized. Hemodynamic variables were measured by a Datex AS/3 (Helsinki, Finland) and data were collected by a computerized system (MP150/Acknowledge 3.9.1, BIOPAC systems, Goleta, Calif., USA). After surgical instrumentation, at least an 1 h intervention-free period followed.
[0251] Intravenous infusions of PDNO were administered (Research Institutes of Sweden, Södertälje, Sweden) at 30 nmol kg.sup.−1 min.sup.−1 for 15 min into a carrier flow, at an infusion rate 9 times of the PDNO infusion rate, of sodium bicarbonate (14 mg mL.sup.−1; pH 7.4 or 8.0), physiological phosphate buffer at pH 8 or physiological saline. Standard chemicals were used to produce the carrier solutions. The hemodynamic effect was measured at the end of each infusion.
[0252] The results are shown in the table below. Baseline values before each intravenous combination of PDNO and carrier solution were normal for healthy, anesthetized pigs. Intravenous PDNO in a carrier solution of bicarbonate buffer at pH 8 decreased mean systemic arterial pressure (MAP) and mean pulmonary arterial pressure (MPAP) by −11±1.2 mmHg and −2.4±0.8 mmHg, respectively, whereas intravenous PDNO in a carrier solution of physiological saline decreased MAP and MPAP by −6.9±2.5 mmHg and −2.4±0.1 mmHg. Intravenous PDNO in combination with bicarbonate buffer at pH 7.4 and physiological phosphate buffer at pH 8 had similar effects on MAP and MPAP as physiological saline. Heart rate and semi-continuous cardiac output were only slightly affected by the infusions.
[0253] Hemodynamic variables in anesthetized and mechanically ventilated pigs subjected to repeated intravenous infusions PDNO at 30 nmol kg.sup.−′min.sup.−1 in combination with various carrier solutions (n=2 per carrier solution).
TABLE-US-00004 HR CCO PDNO at 30 nmol kg.sup.−1 MAP MPAP (beats (L min.sup.−1 in carrier solutions (mmHg) (mmHg) min.sup.−1) min.sup.−1) Baseline 66.5 ± 1.0 18.9 ± 2.8 86 ± 1 4.5 ± 0.6 Bicarbonate buffer 55.5 ± 2.2 16.5 ± 1.9 93 ± 11 4.4 ± 0.1 at pH 8.0 Baseline 67.5 ± 2.8 20.7 ± 1.7 81 ± 6 3.8 ± 0.5 Physiological saline 60.6 ± 0.3 18.3 ± 1.8 83 ± 8 3.9 ± 0.4 Baseline 65.6 ± 1.1 20.6 ± 2.0 76 ± 1 3.6 ± 0.1 Phosphate buffer at pH 8 59.0 ± 0.7 18.0 ± 1.6 82 ± 7 3.8 ± 0.2 Baseline 68.3 ± 4.0 20.8 ± 1.8 77 ± 1 3.4 ± 0.1 Bicarbonate buffer at 61.0 ± 5.9 17.2 ± 1.6 81 ± 5 3.6 ± 0.1 pH 7.4
[0254] Data are presented as means and standard deviations.
[0255] Mean systemic arterial pressure (MAP), mean pulmonary arterial pressure (MPAP), heart rate (HR), semi-continuous cardiac output (CCO).
[0256] Intravenous PDNO in combination with a carrier solution of bicarbonate buffer at pH 8 produced larger hemodynamic effects compared to carrier solutions of physiological saline, physiological phosphate buffer and bicarbonate buffer at pH 7.4.
Example 13—Pharmacological Investigation of 1,2-PDNO—R, 1,2-PDNO—S and 1,3-PDNO in Anesthetised Pigs
[0257] Prior to experimentation, ethical approval was received from Linköping's regional animal ethics committee (Linköping, Sweden; approval number 953). In brief, 2 male and female pigs (a crossbreed between Swedish country breed, Hampshire and Yorkshire; 3-4 months old; 24-26 kg) were premedicated with azaperone at the farm and transported to the laboratory. At the laboratory, anaesthesia was induced with a mixture of tiletamine, zolazepam and azaperone (intramuscular injection). Propofol was given in a peripheral venous catheter in an ear vein, if needed. Bolus doses of atropine and cefuroxime were administered intravenously. The animals were endotracheally intubated and mechanically ventilated (5 cm H.sub.2O in positive end-expiratory pressure, minute ventilation was adjusted to normoventilation). General anaesthesia was maintained with propofol and fentanyl via continuous intravenous infusions, and additional bolus doses were given if needed. Ringer's acetate and glucose solutions were continuously administered intravenously to substitute for fluid loss. Heparin was given as an intravenous bolus dose after the surgical instrumentation. After the experiments the animals were killed in general anaesthesia with a propofol injection followed by a rapid intravenous injection of potassium chloride (40 mmol), and asystolia was confirmed.
[0258] The animals were instrumented with an arterial catheter in the right carotid artery for measurement of systemic arterial blood pressure and heart rate. A sheath was placed in the right external jugular vein for introduction of a pulmonary-arterial catheter. This catheter was used for continuous measurement of pulmonary arterial blood pressure, semi-continuous cardiac output and intermittent pulmonary wedge pressure. A central venous catheter was inserted in the left external jugular vein for drug and fluid administration. All fluid and drug administrations were done by motorised syringe or drip pumps. The urinary bladder was catheterized. Respiratory gases, pressures and volumes were measured at the endotracheal tube. Respiratory and hemodynamic variables were measured by a Datex AS/3 (Helsinki, Finland) and data were collected by a computerised system (MP100 or MP150/Acknowledge 3.9.1, BIOPAC systems, Goleta, Calif., USA). After surgical instrumentation, a 1 h intervention-free period followed.
[0259] After collecting baseline data, a 10-15 min intravenous infusion of 1,2-PDNO—R (43 nmol kg.sup.−1 min.sup.−1), 1,2-PDNO—S (43 nmol kg.sup.−1 min) and 1,3-PDNO (30 nmol kg.sup.−1 min.sup.−1) was administered into a carrier flow of a solution of sodium bicarbonate (14 mg mL.sup.−1; pH approximately 8; infusion rate 9 times of the PDNO infusion rate). Hemodynamic and respiratory data were extracted at the end of each dose.
[0260] 1,2-PDNO—R, 1,2-PDNO—S and 1,3-PDNO decreased mean systemic and pulmonary arterial pressure, which are shown in