DE3116067C2 - - Google Patents

Description

The invention relates to the use of adenosine derivatives for Anti-inflammatory and relieving the inflammation associated Pain and fever.

Adenosine preparations with antihypertensive activity are available from the GB PS 11 01 108 known, those with cytostatic efficacy Eur. J. Med. Chem., 12, 2 (1977), pp. 105 to 108.

The compounds having therapeutic activity according to the present invention Invention have the following general formula:

in which mean:
R is a straight-chain or branched alkyl radical having 1 to 18 carbon atoms, or a phenylalkylene radical in which the alkylene chain has 1 to 6 carbon atoms;
R₁ is hydrogen, an aliphatic acyl radical having 1 to 6 carbon atoms or an aromatic acyl radical;
R₂ is hydrogen, an aliphatic acyl radical having 1 to 6 carbon atoms or an aromatic acyl radical, or the radicals R₂ together form an isopropylidene chain,
n 0 or the number 1.

If R₁ is hydrogen, the invention also includes the acid addition salts the compounds of general formula (I).

Preferred meanings for R are the following radicals: methyl, Ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, Hexyl, heptyl, octyl, decyl, dodecyl, hexadecyl, octadecyl or Benzyl.  

Preferred meanings for the radical R₁ are the following radicals: Hydrogen, acetyl, propionyl, butyryl, benzoyl or tosyl.

Preferred meanings for the radical R₂ are the following radicals: Hydrogen, acetyl, propionyl, butyryl, benzoyl or tosyl.

Preferred acid addition salts of the compounds of the general Formula (I) are the chlorides, sulfates, phosphates, formates, Acetates, citrates, tartrates, methanesulfonates or p-toluenesulfonates.

The compounds of general formula (I) are in part new.

The compounds of the general formula (I) may vary the meanings of the various remains after different Process are produced.

For the preparation of the class of compounds of the following general formula

in which R has the abovementioned meaning, the Legraverand method (Legraverand, M., Ibanez, S., et al. [1977], Eur. J. Med. Chem., 12, 105-108), in which adenosine by reaction with thionyl chloride in hexamethylphosphoramide into which 5'-chloro-5'-deoxyadenosine is converted.  

The 5'-chloro-5'-deoxydenosine is then converted by reaction with the corresponding mercaptan in 2N sodium hydroxide solution at 80 ° C converted into the desired thioether.

The thioethers are then recrystallized from water or purified from lower aliphatic alcohols.

The compounds of formula (Ia) may then be stoichiometric Amount of the desired acid in the corresponding Salt are transferred.

The compounds of the following general formula

in which R, R₁ and R₂ have the above meaning, with the In that R₁ and R₂ are not hydrogen or an isopropylidene chain Mean, according to the method of Satom and Makono (Satom, K., Makino, K. [1951], Nature, 167, 238), in which the corresponding compounds of the general Formula (Ia) with the desired acyl chloride in anhydrous pyridine. The products are preferably recrystallized from a 1: 1 chloroform / petroleum ether mixture.

The compounds of the general formula

in which R and R₁ have the above meaning are prepared by implementation of the corresponding compounds of general formula (I) in which R₂ is hydrogen, with acetone in the presence of ZnCl₂, also by the method Satom and Makino (which already mentioned above has been). The products obtained are preferably by Recrystallization from a 1: 1 chloroform / petroleum ether mixture cleaned.

The compounds of the general formula

in which R, R₁ and R₂ have the above meaning be by oxidation of the corresponding thioether, which after the described above, with bromine or hydrogen peroxide in aqueous solution (Green Stein, J.P., Winitz, M. [1961], - Chemistry of the Amino Acids - John Wiley & Sons, Inc., 2146). The products obtained are purified by recrystallization from water.

Of all manufactured products has become for the purpose of present invention, a compound as particularly interesting proved, namely the 5'-deoxy-5'-methylthioadenosin (MTA) of the following formula:

which a physiological already present in the living organism Connection is.

For the production of this product, a method was found this is particularly simple and from the industrial point of view is economical. The process consists essentially in that the hydrolysis of S-adenosylmethionine (SAME) under strictly critical conditions, which gives a virtually complete hydrolysis and a can achieve complete crystallization of the MTA:

The controlled hydrolysis process can be applied to any SAME regardless of how it was produced.

However, the way of making the SAME solution also stands an influencing factor in the implementation of the new Process in an economically suitable way.

The following compiled process steps the economic execution of the method.

• a) Normal Brothefe is prepared according to the method of Schlenk (Schlenk, F. [1965], Enzymology, 29, 283) by treatment enriched with methionine to SAME.  
• b) The yeast cells suspended in water are lysed by treatment with ethyl or methyl acetate at ambient temperature (DE-OS 23 36 401).
  By adjusting the pH to a value between 4 and 6 and filtering, an aqueous solution containing virtually all of the present SAME in the original yeast is obtained.
• c) The solution is placed under vacuum at temperatures of 35-40 ° C concentrated to about one tenth of its original volume.
• d) The concentrate is refluxed for about 30 minutes heated, and then the pH is raised with soda 7 set.
• e) The solution is allowed to stand at 0-5 ° C, and the precipitated MTA is practically complete with good purity collected.

The stages c, d and e, which - as already mentioned - for the complete selective hydrolysis of SAME to MTA without formation of by-products are critically needed are new.

The preparation of some products used according to the invention will be described below.

Example 1 Preparation of 5'-deoxy-5'-methylthioadenosine (MTA)

11 liters of ethyl acetate and 11 liters of water are at ambient temperature given to 90 kg of bread yeast, by adding Methionine is enriched to SAME until the SAME content 6.88 g / kg. After 30minütigem, vigorous stirring is the pH adjusted to 4.5 with dilute H₂SO₄, the mixture filtered and the residue washed with water, so that a 140 liter solution with a SAME content of 4.40 g / liter receives, which equates to 99.5% of the original  Material present SAME is.

The lysate thus obtained is placed under vacuum (30 mm Hg, 35-40 ° C). concentrated to a volume of about 14 liters. The concentrated Solution is at atmospheric pressure for 30 minutes under reflux conditions heated. It is then cooled to 20 ° C, the pH is with 40% soda set to 7, and then you leave that Whole stand in a refrigerated container (+ 3 ° C) overnight.

A formed white precipitate is filtered off, in 10 liters boiling, distilled water dissolved and cooled crystallized out of the solution.

This gives 410 g of crystalline high purity MTA in a yield of 90%, based on the hydrolysed SAME. The properties of the product agree with those from an MTA obtained by other means.

example 2 Preparation of 5'-deoxy-5-ethylthioadenosine

1 kg of adenosine is placed under a nitrogen atmosphere in 10 liters Hexamethylphosphoramide dissolved, and under cooling Added 7.5 liters of thionyl chloride.

The reaction mixture is allowed to stand at ambient temperature for 20 hours react long. Then 10 liters of water are added and the mixture is neutralized with 2N NaOH.

The then forming 5'-deoxy-5'-chloradenosine is allowed on Crystallize at 3 ° C overnight. Then it is filtered off. This gives 0.950 kg of 5'-deoxy-5'-chloroadenosine (yield 89%). 0.950 kg of 5'-deoxy-5'-chloroadenosine are dissolved in 10 liters of 2N NaOH dissolved, and then 200 ml of ethanethiol are added. The mixture is heated to 80 ° C and reacted for 1 hour. Then is neutralized with glacial acetic acid. The then forming 5'-deoxy 5'-ethylthioadenosine is allowed to precipitate overnight at 3 ° C. It  is filtered off and recrystallized from water. You get 0.830 kg of product (yield 80%, based on the previous Step).

example 3 Preparation of other compounds of general formula (Ia)

The procedure of Example 2 is repeated, but with instead of ethanethiol propanethio, butanethiol, isobutanethiol, Pentanethiol, hexanethiol and benzylthiol can be used.

example 4 Preparation of N⁶, 2 ', 3'-triacetyl-5'-deoxy-5'-thioadenosine

1 kg of MTA is suspended in 10 liters of anhydrous pyridine, and then 3 l of acetic anhydride are added. One lets the Mix for 4 hours. There are then 20 liters of water added and the mixture concentrated under vacuum, taking one getting a pyridine-free, oily mass. This will be in a hot 1: 1 mixture of petroleum ether / chloroform (10 liters) dissolved, and then allowed to crystallize. The product is recrystallized from a 1: 1 petroleum ether / chloroform mixture. This gives 1.140 kg of product (yield 80%).

example 5 Preparation of other compounds of general formula (Ib)

The procedure described in Example 4 is repeated, wherein however, other thioether or propionic anhydride, butyric anhydride, Benzoyl chloride or tosyl chloride instead of MTA be used.

example 6 Preparation of 5'-deoxy-2 ', 3'-isopropylidene-5'-methylthioadenosine

1 kg of MTA are dissolved in 25 l of anhydrous acetone, and it  Then 2.5 kg of molten ZnCl₂ are added. The reaction is carried out under reflux for 5 hours. The mixture is then reduced to one third of its volume under vacuum The original volume is concentrated and then it is 7.5 kg Barium hydroxide octahydrate added in aqueous suspension. Until the neutral reaction is then carbon dioxide through the Mixture bubbled through. The mixture is filtered off and the residue washed with acetone. The filtrate is added Concentrated vacuum, and you get a syrupy residue. This is dissolved in a hot 1: 1 chloroform / petroleum ether Mixture (10 l), filtered, and then allowed to crystallize. The product is made from a 1: 1 chloroform / Petroleum ether mixture recrystallized to give 0.795 kg Product (yield 70%).

example 7 Preparation of other compounds of general formula (Ic)

The procedure of Example 6 is repeated, but instead of of MTA from the corresponding adenosine derivatives becomes.

example 8 Preparation of MTA sulfoxide

1 kg of MTA is suspended in 10 liters of water and cooled Bromine added.

The bromine-containing aqueous solution is by the oxidation of the MTA to sulfoxide instantly discolored.

The addition of bromine is continued until the solution does not discolored again.

By adding a small amount of MTA, the solution is decolorized.

The aqueous solution is then treated with Amberlite IRA-93 resin (registered  Trademark of Rohm & Haas for a weak basic ion exchange resin with a polystyrene matrix), until the reaction of bromide ions disappears.

The mixture is filtered off and the residue with water washed. The aqueous solution is concentrated to 10 liters, with Activated charcoal (100 g) treated and lyophilized. You get 0.950 kg of product (yield 90%).

example 9 Preparation of other compounds of the general formula (Id)

The procedure of Example 8 is repeated, but instead of of MTA from the corresponding adenosine derivatives becomes.

As already mentioned above, it was found that the compounds the general formula (I) a strong anti-inflammatory Have effectiveness accompanied by one analgesic and antipyretic efficacy.

The anti-inflammatory efficacy was originally for some representatives of the compounds by the experimental edema test in the rat, caused by Carragen, demonstrated by the percentage protection after the method of Winter (J. Pharm. exper. Therap., 141, 369, 1963). The values obtained are as follows Table 1 is compiled.

Table 1

From the table it can be seen that the ED₅₀ value of MTA 37 mg / kg is the lowest value for those Compounds that have been administered orally.

In the same test, the ED₅₀ value of indomethacin is 9 mg / kg. These doses, however, cause serious injury in the stomach area, while at ED₅₀ doses of MTA no Side effects occur in the gastrointestinal tract. It is still noteworthy that the LD value of indomethacin in the rat 12 mg / kg (Martelli, A., in Aspetti di farmacologia dell: infiammazione, page 73, published by Tamburini Milan, 1973), while the LD₅₀ value of MTA in the rat <200 mg / kg, administered orally.

Thus, the following therapeutic indices are obtained:

Indomethacin TI logo CNRS logo INIST 1.3 MTA TI = <54.05

The compounds were also a series of subjected to pharmacological tests with the aim of their anti-inflammatory Effectiveness to confirm and their anticatalytic and to demonstrate antipyretic activity.

The results obtained in some of these tests with MTA are given below, wherein this compound in all Cases where the administration was oral, was the most effective Product proved to be the safest Compound is because it is an existing physiological in the organism Connection is, as already mentioned above.

As mentioned above, that is Process for the preparation of SAME MTAs from From an industrial point of view the easiest and most economical methods, and it allows the connection to to produce a particularly low price and in the trade bring to.  

From the test results of Table 1 it can be seen that that methylthioadenosine sulfoxide is particularly effective when it administered intramuscularly. The greater effectiveness of this Compound at intramuscular administration was at all confirmed tests. For MTA sulfoxide are too given some significant values, but should be noted All compounds examined are in proved effective in all cases, albeit at different Levels.

• A. Anti-inflammatory activity:
  The products were tested in rat pleurisy induced by carrageenin according to the method of Velo (Velo, GP, Dunn, CJ, et al., [1973] J. Path., 111, 149).
  At an orally administered dose of 75 mg / kg, MTA gave a protection of 42.4%, calculated on the basis of exudate volume, and 48.8%, calculated on the basis of the number of all cells present in the exudate.
  Comparable protection was achieved with 10 mg / kg indomethacin, ie at a dose much closer to the LD₅₀ value. In the same test, the MTA-sulfoxide gave a protection of 75.8%, calculated on the basis of the exudate volume, or 76.4%, calculated on the said number of cells present in the exudate, the administration being intramuscular and the dose was 80 mg / kg.
• B. Anti-inflammatory activity:
  In the granuloma test induced in the rat by cotton pellets (Winter, CA, Riseley, EA, et al. (1963), J. Pharm. Exper. Ther., 141, 369), which is important for chronic inflammation, MTA gave a 30% protection with an oral dose of 9 mg / kg, with a TI of 222.
• C. The analgesic efficacy of the products was examined in two tests considered very significant.
  In the Roberts hot plate mouse test (Roberts, E., Simonsen, DG [1966], Biochem Pharmac., 15, 1875), the MTA provided 50% protection at an orally administered dose of 37 mg / kg. An approximately equivalent protection of 58% was achieved with an orally administered dose of 100 mg / kg amidopyrine.
  In the same test, MTA sulfoxide provided 50% protection at an intramuscular dose of 20 mg / kg and an orally administered dose of 100 mg / kg.
  In the stretch test caused by phenylquinone (Siegmund, E., Cadmus, R., GO-LU [1957], Proc. Soc. Exp. Biol. Med., 95, 729), the MTA afforded protection of 51% in one oral administered dose of 37 mg / kg. In the same test, an ED₅₀ of 10 mg / kg was determined for the MTA sulfoxide, provided that the administration is intramuscular.
• D. Antipyretic Activity:
  This activity was measured for the products of fever caused by brewer's yeast in rats (Winder, CV, et al., 1961, J. Pharmacol. Exp. Ther., 133, 117).
  The antipyretic activity obtained one hour after the oral administration of MTA at a dose of 300 mg / kg was demonstrated to be a 4.59% reduction in temperature for control animals treated with the yeast only. This percentage corresponds to a temperature reduction from 38.8 ° C to 37.4 ° C.
  In comparison, amidopyrin caused a 4.69% reduction in temperature at an orally administered dose of 200 mg / kg and an intramuscular dose of MTA at a dose of 80 mg / kg gave a 2.35% reduction in temperature.

Other positive features

• E. Efficacy against platelet aggregation:
  The compounds were also tested for a potential effect on platelet aggregation.
  Platelet aggregation is known as a complex phenomenon that can be divided into two stages, a first stage due to the direct action of a stimulant (for example adenosine diphosphate, ie ADP, or epinephrine), and a second stage, due to the aggregation itself, caused by the ADP released by the platelets. That is, when the platelets come into contact with subendothelial collagen, the collagen initiates a whole series of reactions leading to the release of ADP by the platelets. This ADP then causes the second wave of platelet aggregation. To measure the effectiveness of the compounds against this aggregation, the following tests were performed:
  • 1) "in vitro" anti-platelet aggregation tests by ADP and collagen in the presence of the products;
  • 2) "in vitro" tests against platelet aggregation caused by arachidonic acid (AA);
  • 3) "in vivo" tests against platelet aggregation caused by ADP and collagen in subjects treated with the products.
• In this case, the best results were obtained again with MTA and therefore the results obtained using this product are representative of the properties of all compounds.
  • 1) "in vitro" tests:
    Without stasis, blood was drawn and an anticoagulant (3.8% sodium citrate solution) was added to give a blood: citrate ratio of 9: 1. Platelet rich plasma and platelet poor plasma were obtained by centrifugation at ambient temperature. Platelet aggregation was determined by the method of Born & Cross (GVR Born and MJ Cross, J. Physiol., London, 168, 178, 1963) in the platelet-rich plasma fraction. The aggregating agents were used in the following concentrations: ADP (Sigma) 1 μM; Collagen (horn) 5 μg / ml; Arachidonic acid 4 × 10 ^-4 M.
    As a reference substance having an anti-aggregation activity, adenosine was used at a concentration of 1 × 10 ^-5 M.
    The results obtained with ADP are shown in Fig. 1, in which the abscissa indicates the time in minutes, and the ordinate the percentage aggregation.
    Curve 1 relates to the control samples, curve 2 to the 1 × 10 ^-5 M adenosine treated samples, and curve 3 to the 5 × 10 ^-4 M MTA treated samples. From the graph, it can be seen that the MTA causes a very large reduction in primary platelet aggregation due to the ADP, thus inhibiting the second wave of aggregation.
    The same tests performed on collagen gave negative results, that is, MTA did not show any inhibitory effects on platelet aggregation induced by collagen.
  • 2) Figure 2 shows the effects at various MTA concentrations on platelet aggregation elicited by AA at a concentration of ^{4x10 -4} M. Curve 1 relates to the control samples, curve 2 relates to the samples with MTA at a concentration of 2.5 × 10 ^-4 M, curve 3 relates to the samples with MTA at a concentration of 5 × 10 ^-4 M, and curve 4 finally, those samples containing MTA at a concentration of 10 ^-3 M are concerned. It is evident that the inhibitory effect of MTA on platelet aggregation is proportional to the concentration. The ability of the MTA to increase the inhibitory effect of prostacyclin (PGI₂) on AA-induced aggregation was also examined. In Fig. 3, the curve 1 relates to the control samples, the curve 2 samples with an MTA concentration of 5 × 10 ^-4 M, the curve 3 those samples having a PGI₂ concentration of 5 × 10 ^-9 M, and the curve 4 those samples containing a mixture of 5 x 10 ^-4 M MTA and 5 x 10 ^-9 M PGI₂. From Figure 3 it can be seen that, when used in admixture, there is a strong increase in the efficiency of aggregation at concentrations which are otherwise ineffective.
  • 3) "in vivo" tests:
    Three apparently healthy, volunteers aged 35, 42 and 48, respectively, who had not taken any medication for at least 15 days, were subjected to an aggregation test before and after taking the products at a dose of 100 mg every 8 hours for 3 days and these tests were then evaluated. The blood sample for the determination of platelet aggregation was taken 2 hours before the last dose of the product to be tested.
    Fig. 4 shows the results obtained with MTA.
    The solid curves refer to those obtained with blood samples from untreated patients, while the dotted curves refer to those obtained with the same but MTA-treated patients.
    It is evident that MTA strongly inhibits ADP (1 μM) induced platelet aggregation "in vivo".
    The same test was repeated with the addition of 5 μg / ml collagen to the blood, and it was found that MTA is not effective in inhibiting collagen-induced platelet aggregation but only prolongs the latent time of this phenomenon.
    The fact that MTA (and more or less similarly the other products of the same group) strongly inhibits ADP-induced platelet aggregation, while having virtually no effect on collagen-induced aggregation, indicates that MTA inhibits the first wave of aggregation, while having a negligible direct influence on the second aggregation wave.
    The use of the compounds in conjunction with other known anti-aggregation agents which are generally effective with respect to the second wave while having little efficacy against the first wave is therefore of particular interest.
    Due to the demonstrated efficacy, MTA (and the other compounds of this series, even if less effective) are not only useful as anti-platelet aggregation agents, but also as antithrombotic and arteriosclerotic agents because of the altered relationship between platelets and vessel walls in addition to the fact that this is the basis for the mechanism of thrombogenesis, also plays a primary role in atherosclerosis disease.
• F: sleep-inducing activity:
  For this, the Morris test was used (Morris, RW [1966], Arch. Int. Pharmacodyn, 161, No. 2, 380).
  In this test, MTA increased the duration of sleep induced pentobarbital in the mouse by 87% at an intramuscular dose of 20 mg / kg.
• G. Acute toxicity:
  The compounds have virtually no acute toxicity when administered orally. When using therapeutic doses, they have virtually no toxicity regardless of the mode of administration.

The following values were determined for MTA and MTA sulfoxide:

MTA-DL₅₀ in mouse, oral <2000 mg / kg MTA-DL₅₀ in the mouse, intravenously 360 mg / kg MTA sulfoxide DL₅₀ in mouse, oral <2000 mg / kg MTA sulfoxide-DL₅₀ in the mouse, intravenously 400 mg / kg

The adenosine derivatives of the general formula (I) can be diluted with any suitable pharmacologically acceptable Excipients, in any therapeutically applicable form be administered orally, parenterally, intravenously or rectal. For local treatment, they can also be used in products used for external application.  

Some examples of typical pharmaceutical compositions with MTA are exemplified below.

100 mg capsules MTA | 100.2 mg mannitol 195.0 mg magnesium stearate  5.0 mg 300.2 mg

50 mg capsules MTA | 50.1 mg mannitol 100.0 mg magnesium stearate  3.0 mg 153.0 mg

100-mg tablets MTA | 100.2 mg Strength 100.0 mg magnesium stearate 15.0 mg lactose  85.0 mg 300.2 mg

50 mg tablets MTA | 50.1 mg Strength 120.0 mg magnesium stearate 15.0 mg lactose 115.0 mg 300.1 mg

100 mg suppositories MTA | 100.2 mg suppository 1700.0 mg 1800.2 mg

50 mg suppositories MTA | 50.1 mg suppository 1450.0 mg 1500.1 mg

50 mg vial for injection MTA · HCl (equivalent to 56.15 mg) | 50 mg Lidocaine HCl 25 mg Water on 3 ml

25 mg vial for injection MTA · HCl (equivalent to 28.07 mg) | 25 mg Lidocaine HCl 20 mg Water on 2 ml

100 mg oral dose MTA · HCl (equivalent to 112.3 mg) | 100 mg Citrus flavor 0.25 mg sugar 1 g Antifermentierungsmittel 50 mg Water on 5 ml

50 mg oral dose MTA · HCl (equivalent to 56.15 mg) | 50 mg Citrus flavor 0.015 mg sugar 0.5 g Antifermentierungsmittel 30 mg Water on 5 ml

100 g of ointment MTA | 5 g Base for water-soluble ointment, on 100 g antioxidant 0.1 g

Claims (8)

1. Use of a compound of the general formula in which mean:
R is a straight-chain or branched alkyl radical having 1 to 18 carbon atoms or a phenylalkylene radical in which the alkylene chain has 1 to 6 carbon atoms;
R₁ is hydrogen, an aliphatic acyl radical having 1 to 6 carbon atoms or an aromatic acyl radical;
R₂ is hydrogen, an aliphatic acyl radical having 1 to 6 carbon atoms or an aromatic acyl radical, or the radicals R₂ together form an isopropylidene chain;
n 0 or the number 1,
for anti-inflammatory and relieving pain and fever associated with inflammation. 2. Use according to claim 1, characterized in that in the general formula (I) R₁ and R₂ are hydrogen, R is the methyl radical and n 0 mean. 3. Use according to claim 1, characterized in that in the general formula (I) R₁ and R₂ are hydrogen, R is the methyl radical and n is the number 1. 4. Use according to claim 1, characterized in that in the general formula (I) R₁ and R₂ is hydrogen, R is a straight-chain or branched alkyl radical having 1 to 12 carbon atoms and n 0 mean. 5. Use according to claim 1, characterized in that in the general formula (I) R₁ and R₂ is hydrogen, R is the benzyl radical and n 0 mean. 6. Use according to claim 1, characterized in that in the general formula (I) R₁ and R₂ the benzoyl radical, R is the methyl radical and n is 0. 7. Use according to claim 1, characterized in that in the general formula (I) R₁ and R₂ is the tosyl radical, R is the methyl radical and n is 0. 8. Use according to claim 1, characterized in that in the general formula (I) R₂-R₂ the isopropylene radical, R is the methyl radical and n is 0.