DE69330091T2 - IMIDAZOLINE DERIVATIVES, THEIR PRODUCTION AND BLOOD PRESSURE HOLDERS - Google Patents

Description

The present invention relates to new imidazoline derivatives, a process for their preparation and a very safe therapeutic agent for maintaining blood pressure. The agent contains an imidazoline derivative as the active ingredient and is used for maintaining blood pressure in humans in the treatment of shock by removing excess nitric oxide (hereinafter referred to as "NO"). This is precisely the substance that constitutes the endothelium-derived relaxation factor (hereinafter referred to as "EDRF"), which causes the relaxation of vascular smooth muscles.

In the past, it was well known that endothelial cells secrete a substance that contributes to the relaxation of vascular smooth muscle, i.e. EDRF (Nature, vol. 288 (1980), pp. 373-376). After some time, it was confirmed that NO produced by L-arginine is precisely the substance that constitutes EDRF (Nature, vol. 327 (1987), pp. 524-526; and Proc. Natl. Acad. Soc., USA, vol. 84 (1987), pp. 9265-9269).

As NO-synthesizing enzymes (NO synthases) produced by the endothelial cells, the following two types of enzymes exist: (1) the endogenous type, which is always present in the vascular endothelium (Pharm. Rev., vol. 43 (1991), pp. 109-142; The Lancet, vol. ii (1989), pp. 997-1000) and (2) the type derived from endotoxin or cytokines such as tumor necrosis factor (TNF) (Pharm. Rev., vol. 43 (1991), pp. 109-142). While the former type (1) is thought to contribute to the physiological adaptation of the vascular resistance, it is assumed that the sudden advance of NO synthesis due to the latter type (2) causes the decrease in vascular resistance and the accompanying fall in blood pressure, which presumably leads to shock. Most patients in such a syndrome do not respond to circulatory agents such as norepinephrine and dopamine and do not recover from the fatal state.

Various therapies have been investigated to combat shock such as that described above, caused by endotoxin or cytokines. One of these therapeutic attempts, which has been carried out experimentally, involved the intention of treating laboratory animals in endotoxin shock or patients in septic shock by the application of an L-arginine analogue, which is a NO synthase inhibitor (The Lancet, vol. 338 (1991), pp. 1555-1557; The Lancet, vol. 338 (1991), pp. 1557-1558). Since the increasing formation of EDRF (namely NO) is the consistent and universal phenomenon that occurs with the drop in blood pressure of a patient in endotoxic shock, the effort to stop the amount of NO is an excellent therapeutic way to treat endotoxic shock or similar cases. The advantage of the therapeutic agent capable of neutralizing even the action of NO is obvious when considering the complexity that lies in the therapy of neutralizing the action of endotoxin or individual cytokine.

Nevertheless, reports have been submitted regarding the fact that, since the effect of this NO synthase inhibitor in maintaining blood pressure when the inhibitor is administered to treat shock is highly dose dependent, caution should be exercised in determining the dose to be administered. This is true in the sense that if the dose is too low, the effectiveness of the inhibitor is completely lost, and conversely, in the case of an overdose, the patient is placed in an extremely dangerous state induced by a violent rise and fall in blood pressure (The Lancet, volume 338 (1991), pages 1555-1557; The Lancet, volume 338 (1991), pages 1557-1558). In view of the importance of the above reports, there are still unresolved issues related to the safety of the NO synthase inhibitor. Moreover, since it is an L-arginine derivative, there is the possibility that, conversely, this inhibitor may affect L-arginine-dependent protein-synthesizing systems other than NO synthase, as well as metabolic systems such as the urea cycle (Krebs-Henseleit). In this sense, it is feared that the administration of this NO synthase inhibitor will be accompanied by some side effects. In particular, it will understandably lead to many unknown questions if such an inhibitor is administered in an excessive amount over an extended period of time.

The present inventors have investigated a method for stably maintaining blood pressure by removing excess NO generated for the purpose of solving the above-mentioned problem without administering any NO synthase inhibitor.

Despite a report on a process for producing cigarette filters impregnated with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (hereinafter "PTIO") having the following general formula in which R2 represents a hydrogen atom, the purpose of which is to remove NO from cigarette smoke (British Patent 1235880), and on another process in which PTIO is used as the NO oxidizing agent in an apparatus for determining nitrogen oxides (NO and NO) in the atmosphere ("Kankyo To Sokutei Gijutsu" (Environment and Measurement Technology), Volume 12 (1985), pages 32-39), it is not known that compounds of general formula II, including PTIO, have ever shown any efficacy in maintaining blood pressure.

As a result of their investigations on the effectiveness of PTIO and its derivatives as a therapeutic agent for maintaining blood pressure, the present inventors have found the various novel PTIO derivatives represented by the following general formula I in which the phenyl group of PTIO is substituted by a specific substituent, and a process for producing these substances. Furthermore, the inventors have found that PTIO represented by the general formula II and a component containing the above-mentioned novel or known PTIO derivatives have an effectiveness as a therapeutic agent for maintaining blood pressure. Thus, the inventors have completed the present invention.

The object of the present invention is therefore to provide an effective and safe therapeutic agent for maintaining blood pressure, using new or known compounds with the effectiveness of a therapeutic agent for maintaining blood pressure, which have a new mechanism of NO elimination.

That is, the present invention relates to imidazoline derivatives of the general formula I

in which R¹ represents the carboxyl group or the carboxymethoxy group, or pharmaceutically acceptable salts thereof, and a therapeutic agent for maintaining blood pressure, which contains as active ingredient an imidazoline derivative of the following general formula II

in which R² represents a hydrogen atom, the carboxyl group or the carboxymethoxy group, or a pharmaceutically acceptable salt thereof.

The preferred pharmaceutically acceptable salt is sodium or potassium salt.

Regarding the synthesis of PTIO, which is represented by the general formula II in which R2 represents a hydrogen atom, it is well known that the compound can be obtained by reacting 2,3-bis(hydroxyamino)2,3-dimethylbutane with benzaldehyde (J. Am. Chem. Soc., Volume 90 (1968), pages 1078-1079).

On the other hand, the novel imidazoline derivatives of the general formula I can be obtained by reacting 2,3-bis(hydroxyamino)2,3-dimethylbutane with a benzaldehyde derivative. For example, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (hereinafter referred to as "C-PTIO") of the general formula I, in which R¹ represents a carboxyl group, or a pharmaceutically acceptable salt thereof is obtainable by reacting 2,3-bis(hydroxyamino)2,3-dimethylbutane with p-formylphenoxybenzoic acid.

Furthermore, 2-(4-carboxymethoxylphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (hereinafter referred to as "cm-PTIO") of the general formula I in which R¹ represents a carboxymethoxy group, or a pharmaceutically acceptable salt thereof, can be prepared by reacting 2,3-bis(hydroxyamino)2,3-dimethylbutane with p-formylphenoxyacetic acid.

The PTIO and the imidazoline derivatives having the substituent indicated in the general formula I (hereinafter collectively referred to as "PTIO and its derivatives") thus prepared can be analytically identified by 1H nuclear magnetic resonance (NMR), infrared absorption spectrometry, mass spectrometry or electron spin resonance (ESR) of the product obtained and the intermediates thereof.

The therapeutic agent for maintaining blood pressure according to the present invention can be used as a mixture containing active ingredients with low toxicity. The effective dose with low toxicity is 5 to 500 mg/kg weight/day.

The therapeutic agent for maintaining blood pressure according to the present invention can be applied in the form of an injection, an oral administration, a suppository or an aerosol spray.

In case of injection, any route of subcutaneous intramuscular, intravenous and intraarterial administration is applicable. It is also possible to administer the therapeutic agent by way of continuous intravenous infusion. Although there is no limitation on the concentrations of PTIO and its derivatives, in case of injection it is preferred to use concentrations of PTIO and its derivatives of 0.001 to 5% by weight. It is also possible that PTIO and its derivatives, both prepared in powder form, combined with a liquid solvent and administered by injection.

The type of preparation for oral administration can be selected as required. Some examples of this type are tablets, granules, pills, liquid medicines, syrup, lozenges and drops. All of the above preparations can be prepared in a conventional manner. Furthermore, PTIO and its derivatives can be suspended in oil so that enteral absorption can be improved.

PTIO and its derivatives can be converted into the form of a suppository for anal or vaginal administration in the usual way.

Preparations in the form of an aerosol spray can be prepared in a conventional manner using a suitable propellant.

PTIO and its derivatives react with NO in a molar ratio of 1:1 and are converted into nitrogen dioxide (hereinafter referred to as "NO2"), which is inactive toward vascular smooth muscle cells, by taking up an oxygen atom. Furthermore, NO2 is converted into nitrite and nitrate ions in aqueous solution. The present inventors have found that PTIO and its derivatives themselves are converted into substances which are inactive toward NO, that PTIO and its derivatives strongly suppress relaxation of NO-dependent vascular smooth muscle cells, and also that the blood pressure and pulse rate of animals administered PTIO and its derivatives remained at normal levels even when an aqueous solution saturated with NO was introduced into their blood vessels by infusion. As mentioned, PTIO and its derivatives do not interfere with NO synthesis, but rather eliminate excess NO produced.

Therefore, they serve as a very safe therapeutic agent for maintaining blood pressure.

The present invention is explained in detail with the help of the following examples.

EXAMPLE 1: Synthesis of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO).

24.6 g of 2,3-bis(hydroxyamino)2,3-dimethylbutane sulfate was dissolved in 100 ml of water. After neutralization with 1 m KHCO₃ under ice-cooling, the solution was mixed with 15.0 g of p-formylbenzoic acid and stirred overnight at room temperature. The precipitate was dried. 16.9 g of 1,3-dihydroxy- 4,4,5,5-tetramethyl-2-(4-carboxyphenyl)-imidazole (I) were obtained.

After this intermediate (I) was subjected to observation by ¹H-NMR and fast atom bombardment mass spectrometry (FAB-MS) for identification, the sample was verified as the above-mentioned intermediate (I) with the following test results:

(1) ¹H-NMR (DMSO -d₆)

δ: 7.73 (dd, 4 H, Ar-H)

4.57 (s, 1H, C-H)

4.10 (broad s, 2 H, NOH)

1.05 (s, 6H, CH3)

1.09 (s, 6H, CH3)

(2) FAB MS

m/z 279 (M-H)⊃min;

Then 14.0 g of (I) were dissolved in 100 ml of N,N-dimethylformaldehyde. The solution was mixed with 23.5 g of PbO₂ and left to react for three hours at room temperature. Then the PbO₂ was filtered off. After the concentrated residue of the filtrate had been dissolved in water The solution was freeze-dried to obtain 12.5 g of C-PTIO. The resulting product was identified by infrared absorption spectrometry, mass spectrometry, ¹H nuclear NMR and ESR as follows:

(1) IR (KBr disk: 1360 cm⊃min;¹ (N-O)

(2) FAB MS

m/z 276 (M-H)⊃min;

(3) ¹H-NMR (D₂O)

δ: 2.90 (d, 12H, CH3 ) 7.90 (s, 4H, Ar-H)

(4) ESR (0.25 m phosphoric acid buffer, pH 7.5)

aN1, ³ = 0.81 mT

Based on the above experimental results, it was confirmed that the product was 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO).

EXAMPLE 2: Synthesis of 2-(4-carboxymethoxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (CM-PTIO)

24.6 g of 2,3-bis(hydroxyamino)2,3-dimethylbutane sulfate was dissolved in 100 mL of water. After the solution was neutralized with 1 m KHCO3 under ice cooling, it was mixed with 18.0 g of p-phenoxyacetic acid and stirred overnight at room temperature. Then the precipitate formed was dried and dissolved in 100 mL of N,N-dimethylformaldehyde. After adding 45 g of PbO2, the liquid was allowed to react at room temperature for three hours. After filtering off PbO2, the filtrate was concentrated. The residue was dissolved in water and this aqueous solution was freeze-dried to obtain 15.1 g of CM-PTIO. The analytical data thus determined are as follows:

(1) IR (KBr disk: 1360 cm⊃min;¹ (N-O)

(5) FAB MS

m/z 306 (M-H)⊃min;

(3) ¹H-NMR (D₂O)

δ: 1.35 (d, 12 H, CH3 )

1.65 (s, 2H, CH2)

7.10 (d, 2 H, Ar-H)

8.05 (d, 2 H, Ar-H)

(4) ESR (0.25 m phosphoric acid buffer, pH 7.5)

aN1, ³ = 0.82 mT

Based on these test results, the product was identified as 2-(4-carboxymethoxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide (CM-PTIO).

EXAMPLE 3: Preparation of an injection with C-PTIO

C-PTIO was dissolved in 800 ml of distilled water for injection. After adding sodium chloride to this aqueous solution, an injection preparation with a total volume of 100 ml was obtained by adding distilled water as needed for injection. After sterilizing the solution by passing it through a bacterial filter, an injection preparation containing C-PTIO was obtained with the following ingredients:

C-PTIO 2.0 g

Sodium chloride 9.0 g

Distilled water for injection Appropriate volume

Total volume 1000 ml

EXAMPLE 4: Preparation of a PTIO preparation

PTIO, lactose and starch were mixed and converted into wet granules with an aqueous solution of polyvinylpyrrolidone. After the material was dried and sieved, the granules were kneaded with magnesium stearate and pressed into tablets, each weighing 500 mg and composed of the following ingredients:

EXAMPLE 5: Preparation of a suppository with CM-PTIO

The following ingredients were melt mixed and formed into 10 suppositories:

CM-PTIO 510 g

Macrogol 4000 (polyethylene glycol) 2.0 g

Macrogol 1500 (polyethylene glycol) 9.0 g

EXAMPLE 6: Suppressing EDRF-dependent relaxation (dilation) of vascular smooth muscle cells by a therapeutic agent to maintain blood pressure

A rabbit aorta with unremoved vascular endothelium was cut into a ring shape and suspended in an organ bath filled with Krebs-Ringer solution. Its tension was continuously recorded. After the rabbit aorta was contracted by contact with 0.15 µm phenylephrine, it was completely relaxed by adding 3 µm acetylcholine. It is well known that relaxation of vascular smooth muscle by acetylcholine from EDRF, namely NO. The degree of the ability of PTIO to suppress relaxation caused by acetylcholine was determined by adding C-PTIO or cm-PTIO, which had been prepared according to Examples 1 and 2. The determination was carried out by registering the concentration at 30, 100 and 300 µm. NG-methylarginine, which is a NO synthase inhibitor, was used as a reference at the corresponding concentrations. The test results are given in Table 1. Table 1: The ability of PTIO, C-PTIO, cm-PTIO and NG-methylarginine to suppress EDRF-dependent relaxation (dilation) of vascular smooth muscles

Note 1: Vascular smooth muscles contracted by 0.15 µm phenylephrine were relaxed (dilated) 100% by 3.0 µm acetylcholine. The percentage of suppression of relaxation was assigned a score of 0%.

Note 2: Each measurement represents the mean of 10 test results plus or minus the standard deviation. As can be clearly seen from Table 1, PTIO, C-PTIO and CM-PTIO suppressed EDRF-dependent relaxation of vascular smooth muscle in a concentration-dependent manner. The intensity of suppression caused by PTIO and CM-PTIO was either equivalent to or slightly more intense than that of NG-methylarginine. The intensity of suppression caused by C-PTIO was twice as strong as that of the above-mentioned substances.

EXAMPLE 7: The effect of PTIO on the suppression of a fall in blood pressure caused by intravenous infusion of an aqueous solution of NO

Tail veins of rats anesthetized with Nembutal (pentobarbital sodium) were cannulated to provide a route for intravenous administration of a therapeutic agent. The mean blood pressure and pulse rate were monitored based on the pulse pressure wave of the tail artery using an automatic blood pressure measuring device (PS-200) manufactured by Riken Kaihatsu Company Limited.

2.0 mg/ml of a PTIO injection prepared according to the method of Example 1 was administered to a first group of rats by continuous intravenous infusion at a rate of 6.0 ml/h for ten minutes. After 4 minutes had passed, 1.0 ml of a physiological sodium chloride solution saturated with NO was continuously administered intravenously by infusion. The second group of rats were not given PTIO, but were given NO in a similar manner instead. Except for the period when the said dose was administered, a physiological sodium chloride solution was administered by continuous intravenous infusion at a rate of 6.0 ml/h. During the entire period, blood pressure and pulse rate were continuously monitored. In addition, the ratio of the blood pressure and pulse rate recorded five minutes after the administration of the dose to those recorded five minutes before the administration of the dose was recorded. The test result is shown in Table 2. Table 2: The effect of PTIO on changes in blood pressure and pulse rate induced by NO administration.

LEGEND: "Group 1": A group of three rats administered 2 mg/ml PTIO at a rate of 6 ml/h for ten minutes.

"Test group 2": reference group of three rats that were not administered with PTIO.

The measurement represents the average plus minus the standard deviation.

Table 2 shows that despite the decrease in blood pressure by about 40% and the increase in pulse rate by about 20% following administration of a NO-saturated physiological sodium chloride solution (test group 2), there were almost no changes in blood pressure and pulse rate when PTIO had been administered beforehand (test group 1).

The present invention provides a therapeutic agent for maintaining blood pressure in the treatment of shock. A high level of safety is achieved in therapeutic use of this agent, intended for maintaining blood pressure in the treatment of shock resulting from a drop in blood pressure caused by burns, septicemia, stings by bees, wasps or hornets, snake bites, anaphylaxis, poisoning by organophosphorus compounds or side effects of various cytokine therapies. It is also possible to use the agent as an effective accelerator for cardiac drugs.

Claims (6)

1. Imidazoline derivatives of the following general formula (I) in which R¹ represents the carboxyl group or the carboxymethoxy group, and pharmaceutically acceptable salts thereof. 2. Process for preparing the imidazoline derivatives according to claim 1, characterized in that 2,3-bis-(hydroxyamino)-2,3-dimethylbutane is reacted with formylbenzoic acid. 3. Process for preparing the imidazoline derivatives according to claim 1, characterized in that 2,3-bis- (hydroxyamino)-2,3-dimethylbutane is reacted with formylphenoxyacetic acid. 4. Therapeutic agent for maintaining blood pressure, characterized in that it contains as active ingredient an imidazoline derivative of the general formula (II) in which R² represents a hydrogen atom, the carboxyl group or the carboxymethoxy group, and pharmaceutically acceptable salts thereof. 5. Therapeutic agent according to claim 4, characterized in that its active ingredient is 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. 6. Therapeutic agent according to claim 4 or 5, characterized in that the pharmaceutically acceptable salt is the potassium or sodium salt.