JPH0524134B2 - - Google Patents

Description

[Detailed description of the invention]

This invention has a calcium antagonistic effect () The present invention relates to a therapeutic agent for cardiovascular and cerebral circulation disorders containing N-methyl-N-bis-(3,4-dimethoxy-phenyl-ethyl)amine or a salt thereof as an active ingredient. N-methyl-N used in the therapeutic agents of the invention
-Bis-(3,4-dimethoxy-phenyl-ethyl)amine (hereinafter referred to as "compound of the invention") is a compound of bis-(2-(3,4-dimethoxy-phenyl)ethyl)amine in a reducing medium. Produced by reaction with formaldehyde. US
Patent No. 2006114, Berichte 72B18−281939, and
In DE 617647 Rosemund, Kürz and Batt describe a series of bis-phenethylethylamines with papaverine-like properties and antispasmodic action, such as N-methyl-N-bis-(3,4-dimethoxy-
phenyl-ethyl)amine. Although Rosemund et al. did not show any data to support these structures, these compounds should originally have a structure different from that of the compounds of the present invention. This is because they reported a melting point of 230°C or 242°C for those hydrochlorides, but the hydrochloride of the compound obtained in the present invention has a melting point of 180-185°C even after recrystallization. This is because it melts. The structural formula of the compound of the present invention has also been revealed by NMR data. Moreover, as will be made clear later, it has been found that the compounds of the present invention and those of Rosemund et al. are completely different in pharmaceutical properties. Perhaps the different nature of the latter compound is due to the very unusual reaction of the N-unmethylated secondary amine to (3,4-dimethoxy-phenethyl)-amine with hydrogen over palladium/barium sulfate at elevated temperature by the following equation: This seems to be related to the fact that it is obtained by hydrogen cracking. Furthermore, DE 617 647 describes a method for the methylation of the above secondary amines with formaldehyde in the absence of reducing agents. And in US2006114, the methylation is carried out in the presence of formic acid. Nevertheless, as mentioned above, the structural formula was correctly confirmed.
-Methyl-N-bis-(3,4-dimethoxy-phenyl-ethyl)amine has a higher melting point of 242°C than that of amine hydrochloride. That is, according to the teachings of the above cited examples, compounds different from those obtained by repeating the experimental method described in the present invention are obtained. The pharmaceutical properties of the compound of formula () are also not foreseeable from its structure in relation to papaverine. In fact, it is known that the therapeutic action of papaverine is questionable. In this regard, see, for example, Gudeman and Gilman, ``Pharmaceutical Basis of Therapeutic Agents'', Matsukumilan Publishing, New York. It states that papaverine has not been shown to have therapeutic value for any condition. A similar conclusion was made by an expert panel of the US Food and Drug Administration (FDA). In other words, it is stated that there is no element proving the efficacy of papaverine in any of the indications indicated by its manufacturer. Based on these conclusions, the FDA has recommended that papaverine be withdrawn from the market (FDA Drug Bull 9, 26, 1979; Fed.Reg. 44.30443, 1979). Therefore, the compounds described in the above patents and literature have never been used therapeutically and no therapeutic indications can be derived from the teachings of the state of the art. It has now surprisingly been revealed that the compound of formula () has a stronger calcium antagonistic effect compared to the known new calcium antagonist nifedipine and is not associated with papaverine-like properties. In fact, the action of papaverine on calcium ions is complex, and stimulation of calcium ion flux at low concentrations (Carpeneda et al. J. Pharm.
Pharmacol.1971, 23, 502-505) and approx.
Antagonism at a concentration of 10 ^-4 M (Sanguinetti and West, J.Pharm.Exp.Ther., 219, 715,
(1981). The authors also show that the known calcium-antagonists papaverine and valapamil are mutually antagonistic in the slow infiltration with calcium ions into the isolated guinea pig atrium. Therefore, papaverine does not have a calcium antagonistic effect after all, and such properties should be expected from structurally related compounds. Furthermore, the biochemical action of papaverine and some of its derivatives is the inhibition of phosphodiesterases (Kuyubets and Pech, Nauyn
Schmiedebergs Arch.Pharm.267, 189−194,
(see 1970). On the contrary, surprisingly, N-methyl-N-bis-
It has been found that (3,4-dimethoxy-phenyl-ethyl)-amine not only has a strong calcium antagonistic effect, but also has no inhibitory effect on phosphodiesterase. Therefore, the therapeutic and pharmaceutical properties of the compounds of the present invention, despite their structural similarities, should be regarded as unforeseeable based on the above-mentioned literature and the pharmacology of papaverine. The compound of the present invention is therefore a novel drug that blocks calcium entry, and is a useful drug for cerebrovascular disease caused by cerebral anemia and myocarditis ischemia. The compounds of this invention are prepared using bis-(2-(3,4-dimethoxyphenyl)ethyl)amine under reductive amination conditions, i.e., treatment with formaldehyde in a reducing medium. The following examples are intended to illustrate the invention. Example (a) 3,4-dimethoxyphenylacetyl chloride. 29.5 g of 3,4-dimethoxyphenyl-acetic acid was added to anhydrous chloroform without ethanol.
Dissolve in 200c.c. Then 23.8 g of thionyl chloride are added and the mixture is heated under reflux for 4 hours. Excess solvent and reactants are removed under reduced pressure. The oily residue was distilled off under reduced pressure (10 mmHg),
Collect the fraction distilled out at 170-172°C. 26 g of pure acid chloride are thus obtained. Yield 80%. (b) N-(2-(3,4-dimethoxyphenyl)ethyl)-3,4-dimethoxyphenylacetamide. 21.7g of 2-(3,4-dimethoxyphenyl)
Ethylamine is dissolved in 150 c.c. of anhydrous chloroform and this solution is added to 15.2 g of anhydrous triethylamine. This solution was cooled to a temperature between 5-10°C and stirred to produce the 3,4-
Add a solution of 26 g of dimethoxyphenylacetyl chloride in 80 c.c. of anhydrous chloroform. The temperature is allowed to rise to room temperature and then heated under reflux for 8 hours. After cooling the mixture, chloroform
Add 200 c.c. and wash the organic layer with water, 5% HCl and water, and then 5% sodium hydroxide and water. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The solid thus obtained is recrystallized from pure ethanol.
30 g of pure amide are obtained. Yield 84%. Melting point 128
℃. This gives a single spot on chromatography using ethanol as the eluent. (c) Bis-(2-(3,4-dimethoxy-phenyl)ethyl)amine. 37.7 g of sodium borohydride are suspended in 1500 c.c. of anhydrous tetrahydrofuran under a stream of inert nitrogen. Add the amide 36 prepared in section (b) to this suspension.
While stirring and cooling the mixture to 10° C., 58 c.c. of glacial acetic acid are added and the mixture is heated under reflux for 4 hours. At the end, the solvent is removed under reduced pressure and the residue is treated with water and then with dilute hydrochloric acid to make it fully acidic. The mixture is then made alkaline by adding a solution of sodium hydroxide and extracted with 300 c.c. of dichloromethane each time. The organic layer is extracted with dilute hydrochloric acid, the acid solution is washed with dichloromethane, then cooled, made alkaline with potassium carbonate, and finally extracted four times with 200 c.c. of dichloromethane each time. The organic layer is dried over anhydrous sodium sulfate, filtered and stripped. Recrystallize the residual crude amine with ethanol to obtain 28.5g of purified product.
get it. Yield 75% melting point 56-58℃. A single spot is obtained on TLC (eluent n-butanol: ethanol: acetic acid: water = 60:20:40:10) Elemental analysis value Calculated value % C = 69.54; H = 7.88; N = 4.05; Experimental value % C=69.77; H=8.01; N=4.16. (d) N-methyl-N-bis-(3,4-dimethoxy-phenyl-ethyl)amine. Dissolve 10.4 g of the compound prepared in section (c) in 100 c.c. of methanol and add 300 c.c. of 37% formaldehyde.
Add. The mixture was boiled for 40 minutes while stirring, then cooled to 0°C in an ice bath, and 4 g
Add NaBH ₄ little by little. Stirring is continued for 1.5 hours at room temperature and methanol is evaporated under reduced pressure. Dissolve the residue in water and acidify with hydrochloric acid. After stirring for several minutes, it is cooled and made completely alkaline with sodium hydroxide. Extract this alkaline solution with dichloromethane and wash the organic layer with separated water.
Dry _{over Na2SO4} . Then filter and evaporate the solvent under reduced pressure until dry. Remove the residue twice each time.
-Crystallize using 100 c.c. of hexane. In this way, 6.7g of pure product is obtained. Yield 62%. melting point 67
−69℃. This gives a chromatographic spot on TLC using the same eluent as for the amine in section (c). Analytical value: C ₂₁ H ₂₉ NO ₄ (molecular weight = 359.47) Calculated value % C = 70.16; H = 8.13; N = 3.89; Experimental value % C = 70.23; H = 8.17; N = 3.85 The compounds thus obtained are as follows. Call it YS035.
The structural formula of this compound was confirmed by spectrocopy data. Spectrum H ¹ NMR (registered in CDCl ₃ with TMS as internal reference) The chemical displacements of promene, expressed in δ, are 2.35 (s, 3H N-CH ₃ ); 2.7 (s, 8H, N- _CH2 - _CH2 ) ₂ ); 3.8 (s, 12H, _4OCH3 )); 6.7 (s, 6H, aromatic compound). The hydrochloride salt of the compound YS035 has a melting point of 180-185°C. Elemental analysis C ₂₁ H ₃₀ NO ₄ Cl (molecular weight = 359.93) Calculated value % C = 63.70; H = 7.63; N = 3.54 Experimental value % C = 63.42; H = 7.45, N = 3.39 Calcium antagonistic effect of compound YS035 A series of pharmaceutical toxicity tests were conducted to determine its efficacy in comparison with the known drugs nifedipine and nicardipine. The results obtained are shown below. Acute toxicity of YS035. Acute toxicity was measured by the Richfield and Wilcoxon method (J. Pharm Therapy (1949), pages 96 and 99) by oral and intravenous injection into male rats. YS035 showed toxicity with an oral DL ₅₀ of 177.9 mg/Kg and an intravenous injection of 19 mg/Kg. Pharmacology Calcium antagonistic activity Inhibition of Ca ²⁺ uptake in rat brain synaptosomes Synaptosomes were prepared from rat brain homogenate by centrifugation at 23,500 rpm for 30 minutes in a filter gradient to 1.5 mg/cc in a solvent with the following composition: Diluted. NaCl 120mM, KCl 30mM, MgSO ₄ 1.2mM,
KH ₂ PO ₄ 0.4mM, N ₉ HCO ₃ 5mM, HES 20m
M, Glucose 10mM. Y035 was added to the medium at a final concentration of 250μM for comparison with nifedipine and papaverine.
After 15 min pre-incubation ⁴⁵ _CaCl2 final concentration 1mM
(0.2 μCi/cc was added to the medium). The first measurement was taken 2 minutes after addition. The concentration found was set as the 0 hour value. Other controls were performed after 5, 10 and 15 minutes. After each minute the reaction was stopped by centrifugation and radioactivity was measured in the supernatant and pellet. The latter was previously denatured and neutralized with PCT and then centrifuged. The results are shown in Table 1 and are expressed as the amount of calcium transferred per mg of protein after a maximum incubation time of 15 minutes.

[Table] Based on the data in the table, it is clear that the inhibitory activity of calcium uptake (uptake) exhibited by YS035 in rat brain synaptosomes is comparable to the activity inhibited by nifedipine. On the other hand, papaverine does not have calcium antagonistic effects. Blockade of Ca ²⁺ -uptake in neonatal hamster kidney cells. Neonatal hamster kidney cells were washed and suspended several times at a concentration of 5.10 ⁶ /cc in a medium having the following composition. NaCl 120mM, KCl 5.5mM Curcose 5.5mM, Na ₂ HPO ₄ 0.7mM NaHCO ₃ 25mM, MgCl ₂ 1.3mM Tris.HCl (PH 7.4) 10mM YS035 and nifedipine were compared and added at a final concentration of 230 μM. After 15 min preincubation at 30 °C, ⁴⁵ _CaCl2 1mM
(0.1 μCi/cc) was added to the medium to start the reaction.
The concentration at 0 hours was the concentration measured 2 minutes after addition. Other controls were performed every 5 minutes, 10 minutes, and 20 minutes. [Ca ²⁺ ] measurements were performed in the same manner as in the previous experiment. Results are protein at maximum period of 15 minute incubation
Moved per mg (1 mg protein = 5.10 ⁶ cells)
The amount of Ca is reported in Table 2. The obtained results demonstrate the inhibitory effect of both YS035 and nifedipine on Ca uptake in this experimental model. The action of the new compound is quantitatively superior to that of nifedipine.

[Table] In rat liver granules (mitochondria)
Blocking Ca ²⁺ − uptake. The granules prepared by the classical method were diluted to a concentration of 1.5 mg/cc in an incubation medium having the following composition. Satsuka Rose 200mM, KCl 20mM Tris/HClPH7.4 10mM, Succinate 2m
M rotenone 1 μM YS035 and nifedipine were compared and added to the medium to a final concentration of 250 μM. After 15 minutes of preincubation at 20°C, ⁴⁵ CaCl ₂ (50 nmol/mg proto) was added. In a similar manner to the previous experiment, the concentration measured after 2 minutes was taken as the 0 hour concentration. Other controls were established after 5, 10 and 15 minutes.
Ca ²⁺ measurements were performed in a similar manner to the previous experiment. The results are reported in Table 3 as the amount of Ca transferred per mg of protein.

[Table] Regarding Ca uptake in rat grains
The inhibitory effect of YS035 is evident from these results.
However, under these experimental conditions, the effect is somewhat inferior to that of nifedipine. Study of Ca ²⁺ -effrax in rat hepatic granules. Rat liver granules prepared in the same manner as in the previous experiment were charged with Ca and then placed in a medium from which this ion had been removed. Ca ²⁺ from the system granules to the media in the presence of a non-coupling agent or ruthenium lead
There is a flow of ions. YS035 and nifedipine were added at a concentration of 250 μM. In each case, two types of Ca flow activators were present or absent. The results reported in Table 4 are expressed as the amount of Ca transferred from the system granules per mg of protein per minute. Based on these data, it is clear that nifedipine has a non-coupling effect.
RYS035 does not show that effect.

[Table] Antagonistic effect on arachidonic acid-induced contraction of the aorta of domestic rabbits This study was conducted using autologous (autologous) blood as the perfusion solution and compared with nicardipine. Addition of 0.1 mg of arachidonic acid salt to the liquid causes isovolumic contraction of the aorta. Display this on a converter. The results, shown in Table 5, show that the inhibition of arachidonic acid contraction by the two products is comparable. It is because both molecules are involved in the synthesis of tramboxane A ₂ .
This may be because it causes a bypass during the synthesis of PGI ₂ . This phenomenon is demonstrated by the relaxation following the addition of arachidate to perfused blood. This result can be recorded at 0.1 mg/Kg of YS035 and 1 mg/Kg of nicardipine.

[Table] Inhibition of phosphodiesterase activity The phosphodiesterase activity of YS035 and papaverine was determined by the KG Nile method (Biochemistry. 5 , 150,
(1966). The following media were used: 2.5ml buffer tri-HCl 0.1MPH7.4; 50μ MgSO _{4.7H 2} O (10μg/ml); 50μ CAMP “Sigma” (1 mgg/ml); 5μ adenosine deaminase (2 mgg/ml); 20μ alkaline phosphatase “ sigma"
(2.6mg/ml) 15μ Phosphodiesterase (10mg/ml) YS035 was added to a final concentration of 100mM. And papaverine was adjusted to a concentration of 10mM. _ID50 value (50% inhibitory concentration) was 0.04mM for papaverine, whereas YS035 was found to be completely inactive. In vivo study YS035 and nicardipine were orally administered to rabbits for 3 consecutive days. The final administration of each sample was made one hour before killing the animals. Sections of the artery were incubated in PRP and Krebs-Henseleit solution. Table 6 shows the results obtained by adding arachidonic acid.
Shown below.

[Table] This study showed that YS035 and nicardipine directly stimulated endothelial cells in arteries to induce prostabrangins, especially prostabrangins.
This shows that it acts on the synthesis of PGI ₂ and is not due to the vasoconstrictive effect of thromboxane A ₂ on platelets. Effect on various types of cerebral infarction in rats Intramuscular injection of sodium arachidonate (0.1 mg)
causes an edematous effect in rats due to calcium accumulation and decreased cholesterin levels in brain tissue.
YS035 moderately reduced calcium entry at the 1 mg/Kg level when administered intravenously at doses of 1, 3.3, and 10 mg/Kg 15 minutes before induration, but had no effect on other biochemical changes. It has no effect. Antagonistic effects on neurological deficits and biochemical abnormalities during the post-anemia period in rats Anemia in rats is associated with a slight decrease in blood pressure (8-
9KPa) caused by bilateral occlusion of the carotid arteries.
Maintained for 60 minutes. After removing the bite, the post-anemic period lasts for several days. In this case, observations were continued until the third day, when the brain accumulation of Ca ⁺⁺ ions reached its maximum. YS035 or nicardipine was administered to rats at 1, 5, 18, 24, 42, and 71 hours after removal of the arterial occlusion, respectively. After 72 hours, the rats were sacrificed and the tissues were immediately removed to determine the water, calcium and potassium contents. The results are shown in Table 7.

【table】

[Table] The above results show that YS035 reduces the severity of brain edema, in particular it substantially reduces the intracellular accumulation of Ca ⁺⁺ ions. Nicardipine appears to have a similar effect, but its effect is less distinctive with respect to Ca ion accumulation. Anti-arrhythmia effect and anti-ischiemic effect in rats The left coronary artery is tied to induce early arrhythmia (30 minutes), especially lctopic beats, ventricular tachycardia (VT), and ventricular fibrillation in anesthetized rats. (Sely 1960,
(according to the methods of Clark 1980 and Paratz 1983). 0.156 YS035 15 minutes prior to coronary tying
Administer intravenously at doses between mg/Kg and 20 mg/Kg. The results obtained are shown below.

[Table] As can be seen from the table, injection of YS035 at doses between 0.625 and 10 mg/Kg prevented critical arrhythmia symptoms such as VF, VT and death. And there was no relationship between dose and observed effects. This is because there is a regression in results at doses higher than 10 mg/Kg. Effect on myocardial infarction in the subacute phase in dogs By tying the coronary arteries of the heart using the Harris method, the following effects can be obtained. (a) Decrease in coronary blood flow (b) Increase in coronary vascular resistance (c) Decrease in left ventricular working index (d) Increase in DPTI/TTI ratio (e) Decrease in aortic flow index (f) Glucose, oxygen and lactate Reduction in myocardial partial salt consumption (g) Reduction in myocardial free fatty acid uptake Intravenous administration of YS035 at a dose of 0.1 mg/Kg
The same dose was administered three times after ligating the coronary artery and within 48 hours, with the following results. (a) Coronary blood flow is similar to the control group or at most tends to rise slightly during the experiment. (b) Coronary vascular resistance is not only reduced but also lower compared to that of the control group. (c) Left ventricular work index is significantly higher than in dogs with induration. (d) DPTI/TTI ratio remains unchanged (e) Aortic flow index (f) Glucose consumption in the myocardium remains unchanged, while oxygen consumption increases substantially and lactate consumption tends to normalize ( g) Free fatty acid uptake increased to a value twice the basal flux measured in healthy control animals. This invention therefore shows all possibilities for using compounds of formula () in cardiovascular therapy. The invention therefore consists in pharmaceutical compositions containing a compound of the formula () as active ingredient, with which the customary auxiliaries commonly used in formulations can be used. The compounds of the invention can be administered orally or parenterally. The average daily dose of YS035 is 20 to 150 mg administered orally in 2 to 3 divided doses. Administration can be continued for a long period of time. YS035 for acute symptoms
can also be administered slowly intravascularly at a dose of 10 to 20 μg/Kg. Examples of the dosage form of the therapeutic agent of this invention include the following. (1) Gelatin capsule containing 10mg of YS035 (2) YS035 as an auxiliary agent commonly used in pharmaceutical formulations
Tablets (3) of 20 mg YS035HCl in sterile pyrogen-free distilled water.
A sterile liquid formulation suitable for parenteral administration containing 1 mg/cc of

Claims (1)

[Claims] 1 N-methyl-N with calcium antagonistic effect
-Bis-(3,4-dimethoxy-phenyl-ethyl)amine or a salt thereof as an active ingredient, together with an inert auxiliary agent, in a dosage unit form for the treatment of cardiovascular diseases and cerebral circulation disorders.