SE466238B - THERAPEUTIC COMPOSITION CONTAINING ADENOSIN DERIVATIVES - Google Patents

Description

Preferred values for R 1 are: acetyl, propionyl, butyryl, benzoyl or tosyl.

Preferred values for R 2 are: acetyl, propionyl, butyryl, benzoyl or tosyl.

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

The compounds of formula (I) are prepared by various methods in accordance with the meanings of the various substituents. For the preparation of the group of compounds of the formula: t - fa: ff 2 15 'fi ". -_ In (IS) for which R has the meanings given above, the Legrazgverand method (Legraverand M. Ibanez S., et al. (1977) ÉEur. J. Med. Chem. 12, 105-108), in which adenosine is converted to 5 * -chloro-5'-deoxyadenosine by reaction with thionyl chloride in hexamethylphosphoramide.

A 5'-chloro-5'-deoxyadenosine is then converted to the required thioether by reaction with the corresponding mercaptan in a 2N sodium hydroxide solution at 80 ° C.

The thioethers obtained are purified by recrystallization from water or from lower aliphatic alcohols. 466 238 ~ ___.__._.____.__.__, _ __ The compounds of the general formula "5 ara-a, (Ib) m cae-sa 15 OR GR in which R, R1 and R2 are the same as above defined, provided that R 1 and R 2 are other than hydrogen or an isopropylidene chain, have been prepared by the Satom and Makino method (Satom K, Makino K (1951) Nature 167, 238) by substituting the corresponding compounds of formula (Ia) reacted with the required acyl chloride in anhydrous pyridine The products are preferably recrystallized from a 1: 1 mixture of chloroform / petroleum ether.

The compounds of formula 466 238 10 15 20 25 30 35 f * (Is) G '///' \\\\ cu 's in which R and R1 are the same as defined above are prepared by substituting the corresponding compounds of formula (I ), where R 2 is H, is reacted with acetone in the presence of ZnCl 2, again according to the Satom and Makino method (referred to above). The products obtained are preferably purified by crystallization from a 1: 1 mixture of chloroform / petroleum ether.

The compounds of the formula Fish, (Ia): Ä fa 10 15 20 25 30 466 238 5 Stein J-PH Winitz M. (leen-cnemiscry of the amino acids - John Wiley & Sons Inc; 2146). The products obtained are purified by recrystallization from water.

A product of interest in connection with the invention is 5'-deoxy-5'-methylthioadenosine (MTA) of formula (II) which is a physiological compound already present in living organisms.

Below is a method of producing this product, which is particularly simple and economical from an industrial point of view. This process essentially consists of hydrolysis of S-adenosylmethionine (SAME) under strictly controlled critical conditions, leading to virtually total hydrolysis and complete crystallization of MTA. 466 238 10 15 20 25 30 35 "z. Sslma H2 ° ca -š-cx -ca -ca-cocn -_--) 2 | 2 2“ s son ox CH2-S-C33 ï fl z + ao-cnz-caz -cn-coon + a * The controlled hydrolysis procedure can be applied to SAME prepared by any means.

The process for preparing the SAME solution is also a influencing factor in carrying out the described process in an economically appropriate manner.

The following work steps provide the most economical embodiment of the process: a) Normal bread yeast is enriched with SAME by treatment with methionine according to the Schlenk method (Schlenk F. (1965) Enzymologie 29, 283). b) The yeast cells suspended in water are subjected to lysis by treatment with ethyl or methyl acetate at ambient temperature (DT-OS P23 3640l.4).

By adjusting the pH to between 4 and 6 and filtering, an aqueous solution is obtained, which contains practically all the SAME present in the original yeast. c) The solution is concentrated in vacuo at 35-40 ° C to about 1/10 of its initial volume. d) The concentrate is boiled under reflux for about 30 minutes and the pH is adjusted to 7 with soda. e) The solution must be left at 0-5 ° C and precipitated MTAs are collected practically completely and in good purity.

Steps c, d and e, which as indicated are critically necessary for obtaining completely selective hydrolysis of SAME to MTA without the formation of by-products, are new.

The preparation of certain products used in the present invention is described below.

EXAMPLE 1 Preparation of 5'-deoxy-5'-methylthioadenosine (MTA) 11 liters of ethyl acetate and 11 liters of water at ambient temperature were added to 90 kg of bread yeast, which had been enriched with SAME by adding methionine until the SAME content was 6. 88 g / kg.

After careful stirring for 30 minutes, the pH was adjusted to 4.5 with dilute H 2 SO 4. The mixture was filtered and the residue was washed with water to give 140 liters of solution with a SAME content of 4.40 g / liter, which corresponds to 99.5% of the SAME present in the starting material.

The lysate thus obtained was concentrated in vacuo (30 mm Hg; 35-40 ° C) to a volume of about 14 liters.

The concentrated solution was refluxed at normal pressure for 30 minutes. It was cooled to 20 ° C, the pH was adjusted to 7 with 40% soda overnight in a cooling chamber (+ 3 ° c).

A white precipitate formed, and it was left over which was filtered, dissolved in 10 liters of boiling distilled water and crystallized by cooling this solution. 410 g of crystalline MTA of high purity were obtained, which corresponds to a yield of 90% with respect to SAME 466 258 10 15 20 25 30 so 8 which was subjected to hydrolysis.

The characteristics of the product obtained coincide with those of pure MTA obtained in another way.

EXAMPLE 2 Preparation of 5'-deoxy-5'-ethylthioadenosine 1 kg of adenosine was dissolved under a nitrogen atmosphere in 10 liters of hexamethylphosphoramide and 7.5 liters of thionyl chloride was added under cooling. The mixture was allowed to react at ambient temperature for 20 hours. 10 liters of water were added. and the mixture was neutralized with 2N NaOH. The 5'-deoxy-5'-chloroadenosine thus formed was allowed to crystallize overnight at 3 ° C. It was then filtered. 0.950 kg of 5'-deoxy-5'-chloroadenosine were obtained (yield 89%). 0.950 kg of 5'-deoxy-5'-chloroadenosine was dissolved in 10 liters of 2N NaOH, and 200 ml of ethanethiol was added. The mixture was heated to 80 ° C and allowed to react for 1 hour. It was neutralized with glacial acetic acid. The 5'-deoxy-5'-ethylthioadenosine thus formed was allowed to precipitate overnight at 3 ° C. The precipitate was filtered off and recrystallized from water. 0.830 kg of product was obtained (yield 80% with respect to the previous step).

EXAMPLE 3 Preparation of other compounds of formula Ia The procedure described in Example 2 was carried out, except that propanethiol, butanethiol, isobutanethiol, pentanethiol, hexanethiol and benzylthiol were used instead of the ethanethiol.

EXAMPLE 4 Preparation of N6, 2 ', 3'-triacetyl-5'-deoxy-5'-thioadenosine. 1 kg of MTA was suspended in 10 liters of anhydrous pyridine and 3 liters of acetic anhydride were added. The mixture. was allowed to react for 4 hours. 2 liters of water were added and the mixture was concentrated in vacuo to give an oily mass which was free of pyridine. This was dissolved in a hot 1: 1 mixture of petroleum ether / chloroform (10 liters) and left to crystallize. The product was recrystallized from a 1: 1 mixture of petroleum ether / chloroform. 1,140 kg of product were obtained (yield 80%).

EXAMPLE 5 Preparation of other compounds of formula Ib.

The procedure described in Example 4 was repeated, but other thioethers or propionic anhydride, butyric anhydride, benzoyl chloride or tosyl chloride were used in place of MTA.

EXAMPLE 6 Preparation of 5'-deoxy-2 ', 3'-isopropylidene-5'-methylthioadenosine. 1 kg of MTA was suspended in 25 liters of anhydrous acetone, and 2.5 kg of molten Znclz was added. The reaction was allowed to proceed at reflux for 5 hours. The mixture was then concentrated in vacuo to 1/3 of its initial volume, and 7.5 kg of barium hydroxide octahydrate in aqueous suspension was added. Carbon dioxide was then bubbled through the mixture to neutrality. The mixture was filtered and the residue was washed with acetone. The filtrate was concentrated in vacuo to give a syrupy residue. This was taken up in a hot 1: 1 mixture of chloroform / petroleum ether (10 liters), filtered and left to crystallize.

The product was recrystallized from 1: 1 chloroform / petroleum ether to give 0.795 kg of product (yield 70%).

EXAMPLE 7 Preparation of other compounds of formula Ic.

The procedure described in Example 6 was performed, but starting from the corresponding adenosine derivatives instead of MTA.

EXAMPLE 8 Preparation of MTA sulfoxide. 1 kg of MTA was suspended in 10 liters, and bromine was added while cooling.

The aqueous solution containing bromine of: was immediately colored by oxidation of MTA to sulfoxide. 466 238 10 15 20 25 10 Addition of bromine was continued until the solution no longer decolorized.

The solution was decolorized by further adding small amounts of MTA.

The aqueous solution was treated with Amberlite IRA 93 resin (registered trademark of Rohm and Haas for a weak base ion exchange resin with a polystyrene matrix) until the bromine ion reaction disappeared.

The mixture was filtered and the residue was washed with water. The aqueous solution was concentrated to 10 liters, then treated with activated carbon (100 g) and lyophilized. 0.950 kg of product was obtained (yield 90%).

EXAMPLE 9 Preparation of other compounds of formula Id.

The procedure described in Example 8 was followed, but starting from the corresponding adenosine derivatives instead of MTA.

As initially stated, it has been found that the compounds of formula I possess strong anti-inflammatory activity, followed by analgesic and antipyretic action.

The anti-inflammatory activity was initially shown for certain compounds in this class by testing experimental edema caused by carrageenan in rats, by determining the percentage protection according to the Winter method (J. Pharm. Exper. Therap. 141, 369 1963).

The values obtained are shown in Table 1. 466 238 fi, 11 TABLE 1 Compound of formula (I) Oral ad- Z Protection, administered based on dose, Egg / kg edema development line no a = -cu.n1 = n2 = 1i 37 50 n = on - ca3, n1 = n2 = x 23 (;) 50 n = o zw-c fl z-cón, n1 = n2 = H u? 1o_ cx n = on = -cH2-cH .a1 = n2 = 11 85 62 \ G33 n = on = - (cH2) ¿-c1i, 121 = R2 = H _ 95 20 n = oa - - (cH _, _) u-ca3. al = 122 = H 112 10 n = o x = - (cxz) u-cx, nl = 52 = H 90 á 25 n = o n = -c fl a-cx3. ni = az = x 80 un n = oa = - (ca) -cx.a = a = H '80 53 2 2 1 z / ° "3 n = oa = -ca .ai = a2 = u Bø # 5 \ CH3 n = oa = - (cn2) 3-cx3. RI = az = H 85 39 n = 0 R = (šH-CHZ-Cl-íj. H1 = H2 = H 85 35 cxß n = o = z = - ( cH2) 7-c: -z3. al = n: = H zoo h? n = oaf - (cH2) 9-c: 3, 31 = a2 = n 106 33 n = 1a = -cx3. a1 = a2 = ä 156 '50 n = 1 a = -cx3, a1 = a2 = as, s (a) 50 n = 0 3 = -cuy 31 = H2 = -cc-cl-Ij 114 h? N = 0g = .cu3, a1 = a = w = y1 201 »15 n = on = -cxy al; az = -co-coxs 161 * 10 n = 0 3 = 4:33, 31 = H, 82-31 = isopropyl 91 '20 Indomethacin 9 50 (a) indicates that the product was administered intranuscularly 466 238 10 15 20 25 30 35 12 As shown in Table 1, the ED50 for MTA is 37 mg / kg, and is thus the lowest for the compounds tested, vidoral administration.

In the same test, the ED50 for indomethacin was 9 mg / kg.

At these doses, severe gastric lesions occur, whereas at the ED50 doses MMA does not give rise to any secondary effects on the gastrointestinal system. It should also be noted that the LD for indomethacin in rats is 12 mg / kg (Martelli A. in Aspetti di Pharmacologia dell'-infiammazione, page 73, published by Tamburini-Milan 1973), whereas the LD50 for MTA in rats is> 200 mg / kg when administered orally.

The following therapeutic indices were thus obtained: Indomethacin TI = 1.3 MTA TI => 54.05 The compounds of the invention were also subjected to a series of pharmacological tests in order to confirm their anti-inflammatory activity and to show their analgesic and antipyretic activity.

The results obtained with some of these tests with MTA are given below, since MTA is a product which in all cases proved to be the most active in oral administration, and it is certainly the safest because, as already stated, it is a compound which is physiologically present in the organism.

From an industrial manufacturing point of view, the process for producing MTA from SAME, as discovered, is by far the simplest and most economical, and makes it possible to market it at an extremely low price.

As can be seen from the data in Table 1, methylthioadenosine sulfoxide is particularly active when administered intramuscularly. The greater activity of said compound in incremuecular administration was confirmed in all tests performed. Some significant data regarding MTA sulfoxide are also given, but it should be noted, however, that all of the compounds tested were found to be active in all cases, albeit at different levels.

A - The anti-inflammatory activity.

The products were tested by pleurisy induced in rat by the carrageenan according to the Velo method (Velo G.P., DUNN C.J. et al. (1973) J. Path. Lll, 149).

MTA at a dose of 75 mg / kg provided by oral administration a protection of 42.4%, calculated on the volume of the exudate, and 48.8%, calculated on the total number of cells present in the exudate.

A comparable protection was obtained with 10 mg / kg indomethas - ie at a dose much closer to LD50. In the same test, the MTA sulfoxide gave a protection of 75.8%, calculated on the volume of the exudate, and 76.4%, calculated on the total number of cells present in the exudate, when the compound was administered intramuscularly at a dose of 80 mg / kg.

B - Anti-inflammatory activity In the granuloma test in rats by cotton pellets (Winter C.A., Riseley E.A. et al (1963) J. Pharm. Exper.

Ther. 141, 369), which is significant for chronic inflammation, gave MTA 30% protection at an oral dose of 9 mg / kg, with a therapeutic index of 222.

C - The analgesic activity of the products was determined by two tests, edge.

- When testing mice on hot plate according to Roberts (Roberts E. Simonsen D.G. (1966) Biochem. Pharmac. 15, 1875), MTA gave 50% protection at an oral dose of 37 mg / kg. An approximately equivalent protection of 58% was obtained with 100 mg / kg amidopyrine when administered orally.

In the same test, MTA sulfoxide gave 50% protection at a dose of 20 mg / kg when administered intramuscularly, and at a dose of 100 mg / kg, cin, which were considered very significant when the compound was administered orally.

- In the tensile test with phenylquinone (Siegmund E., Cadmus R., GO-LU (1957) Proc. Soc. Exp. Bio. Med. 95, 729), MTA gave a protection of 51% at an oral dose of 37 mg / kg. 466 238 _ lo 15 .zyo in 25. 3Q 35 a secondary stage due to aggregation, 14 In the same test, MTA sulfoxide has an ED50 of 10 mg / kg when administered intramuscularly.

D - Antipyretic activity This was measured for the new products by fever, which was induced in rat with brewer's yeast (Winter C.V. et al (1961) J. Pnarmacoi. Exp. That. 133, 117).

The antipyretic effect, which was evaluated 1 hour after oral administration of MTA at a dose of 300 mg / kg, resulted in a temperature reduction of 4.59% with respect to the controls treated only with yeast. This percentage corresponds to a temperature decrease from 38 ° C: iii 37.4 ° C.

In comparison, amidopyrine administered orally at a dose of 200 mg / kg gave a temperature decrease of 4.69%, and intramuscular administration of MTA at a dose of 80 mg / kg gave a temperature decrease of 2.35%. 'E - Platelet anti-aggregation activity.

The compounds of the invention were also evaluated in view of their possible antiplatelet capacity.

It is known that platelet aggregation is a complex phenomenon, which can be divided into a primary stage 'depending on the direct action of a stimulus (eg adenosine diphosphate, ie ADP, or epinephrine) and which is induced by platelet release of ADP. In this regard, when the platelets come in contact with subendothelial collagen, the collagen initiates a whole series of reactions, which lead to the platelets' release of ADP. It is this ADP that causes the second wave of platelet aggregation. The following tests were performed to evaluate the anti-aggregation effect of the new compounds: l) "in vitro" test for platelet aggregation, induced by ADP and collagen, in the presence of the new products; 2) "in vitro" test for platelet aggregation, induced by arachindonic acid (AA); 10 15 20 25 30 35 466 238 15 3) "in vivo" test for platelet aggregation, induced by ADP and collagen in persons treated with the new products.

In this case, the most significant results were again obtained with MTA, so the results obtained using this product are given as an indication of the behavior of the whole class. 1) "in vitro" test Blood was drained, and an anticoagulant (3.8% sodium citrate) 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 estimated using the method of Born & Cross (G.V.R. Born and M.J. Coss, J. Physiol., Lond. 168, 178, 1963) on the plasma fraction as on platelets.

The aggregation agent was used in the following concentrations: ADP (Sigma) 1 PM; collagen (Horn) 5 pg / ml; arachidonic acid 4x10-4M. was rich Adenosine at a concentration of lx10-SM was used as a reference substance for anti-aggregation activity.

The results obtained with ADP are shown in the diagram in Fig. 1, where the abscissa indicates the time in minutes and the ordinate percent aggregation.

Curve 1 relates to controls, curve 2 to the samples treated with 1x10-5M adenosine and curve 3 to the samples treated with 5x10 -4M MTA.

The curve pattern shows that MTA greatly reduces primary platelet aggregation due to ADP and, as a result, inhibits the second wave of aggregation.

The same test performed with collagen gave negative results. That is, MTA showed no inhibitory effect against platelet aggregation induced by collagen, which was worth noting. 466 238 10 15 20 25 30 35 16 2) Fig. 2 shows the effects of different MTA concentrations on platelet aggregation induced by AA at a concentration of 4x10 -4 M. Curve 1 refers to controls, curve 2 to MTA at a concentration of 2.5x10 4 M, curve 3 to MTA at a concentration of 5x10 4 M and curve 4 to MTA at a concentration of 10-3M. As can be seen, the platelet aggregation inhibitory effect of MTA is proportional to its concentration. The ability of MTA to increase the inhibitory effect of prostacyclin (PGI2) in AA-induced aggregation was also examined. In Fig. 3, curve 1 refers to controls, curve 2 to MTA at a concentration of 5x10 -4 M, curve 3 to PGI2 at a concentration of 5x10-9M, and curve 4 to a mixture consisting of 5x10 -4 MTA and 5x1Q'9M PGI2. It can be seen from Figure 3 that when the compounds are used in admixture, a strong increase in anti-aggregation effect occurs at concentrations which are in themselves ineffective. 3) "in vivo" test.

Three apparently healthy subjects 35, 48 years old, 42 and those who had not taken any drug for at least 15 days were subjected to aggregation tests before and after taking the new products at a dose of 100 mg every 8 hours for 3 days, after which these tests were evaluated.

The platelet aggregation blood sample was taken 2 hours before taking the last dose of the product tested.

Fig. 4 shows the results obtained with MTA.

More precisely, the solid curves refer to values obtained with blood samples from untreated patients, whereas the dashed curves refer to values obtained for the same patient which were treated with MTA. U It is clear that MTA strongly inhibits platelet aggregation induced by ADP (1 pM) "in vivo".

The same test repeated with the addition of 5 pg / ml 'of collagen to the blood showed that MTA is not effective in inhibiting platelet aggregation induced by collagen, but only prolongs the latency of the phenomenon.

The fact that MTA (and in a more or less comparable way the other products in the same class) strongly inhibits platelet aggregation induced by ADP, whereas it has virtually no effect on aggregation induced by collagen, indicates that MTA inhibits the first wave of aggregation, as it has a negligible direct effect on the second wave of action.

Its use in association with other known anti-aggregation drugs, which are generally active against the second wave and only to a small extent, is therefore particularly aggregate against the first wave, is interesting.

The activity shown suggests that the use of MTA (and the other compounds in this series, although less effective) is not only as a platelet anti-aggregation drug, but also as an antithrombotic and anti-atherosclerotic drug, for that. Altered ratios of platelets to vessel walls also play a primary role in atherosclerotic disease in addition to being a basis for thrombogenesis mechanisms.

F - Sleep-inducing activity The Morris test was used (Morris R.W.

Pharmacodyn 161, No. 2, 380) In this test, MTA increased by 87% the duration of sleep, induced by pentobarbital in a mouse, at an intramuscular dose of 20 mg / kg.

G - Acute Toxicity I (1966) Arch. int.

The compositions of the present invention are practically free from acute toxicity when administered orally. They are practically free from toxicity at the therapeutic doses for all modes of administration. 466 238 with MTA are given below in illustrative - 100 mg capsules 15 20 25 30 35 The following values were obtained for MTA and MTA sulfoxide: MTA - LDSO in mouse oral intravenous> 2000 mg / kg 360 mg / kg MTA sulfoxide - LDSO in mouse orally intravenously> 2000 mg / kg 400 mg / kg The adenosine derivatives of formula (I) may be administered diluted with suitable pharmacologically acceptable additives, in any therapeutically useful form, such as orally, parenterally or by venous or rectal route. They can also be used in products for external use through topical application.

Some examples of typical pharmaceutical compositions MTA Mannitol Magnesium Stearate 50 mg capsules MTÅ Mannitol Magnesium Stearate 100 mg tablets MTA Starch Magnesium Stearate Lactose 50 mg tablets MTA Starch Magnesium Stearate Lactose Purpose: 100.2 mg 195.0 mg 5.0 mg 300.2 mg 50.1 mg 100.0 mg 3.0 mg 153.1 mg 100.2 mg 100.0 mg 15.0 mg 85.0 mg 300.2 mg 50.1 mg 120.0 mg 15.0 mg 115.0 mg 300 , 1 mg 10 15 20 25 30 35 466 19 100 mg suppository MTA 100.2 mg Suppository mass l700.0 mg _ 1800.2 mg SO mg suppository MTA '50.1 mg Suppository mass l450.0 mg l500, l mg 50 mg vial MTA. HCl (56.15 mg mainly equivalent) 50 mg Lidocaine. HCl 25 mg Water up to 3 ml 25 mg vial MTA. HCl (28.07 mg mainly equivalent) 25 mg Lidocaine. HCl 20 mg Water up to 2 ml 100 mg oral dose MTA. HCl (II2.3 mg mainly equivalent) 100 mg Citrus flavor 0.025 mg Sugar 1 g Antifertilizer 50 mg Water up to 5 ml 50 mg oral dose MTA. HCl (56, 1.5 mg mainly equivalent) 50 mg Citrus flavor 0.015 mg Sugar 0.5 9 Antifermenting agent 30 mg Water up to 5 ml 238 466 238 20 - 100 g ointment MTA 5 g Base for water-soluble ointment, up to 100 g Antioxidant 0.1 g.

Claims (4)

5 10 15 20 25 30 466 238 21 PATENT REQUIREMENTS 1. A therapeutic composition having anti-inflammatory, analgesic and antipyretic activity, characterized in that it comprises as its active ingredient at least one compound of the general formula NB-R 'aliphatic acyl group having 1 to 6 carbon atoms or one or tosyl group, aliphatic acyl group having 1-6 carbon atoms or one or tosyl group, benzoyl-R 2 is a benzoyl- or alternatively the groups R 2 together form an isopropylidene chain, and n is 0 or 1. A therapeutic composition according to claim 1, characterized in that it comprises as its active ingredient a compound of formula (I) wherein R 1 = R - benzoyl, R = methyl and n = 0. Therapeutic composition according to claim 1, characterized in that it comprises as its active ingredient a compound of formula (I), wherein R 1 = R 2 = tosyl, R = methyl and n = 0. 466 238 22 Therapeutic composition according to claim 1, characterized in that it comprises as its active ingredient a compound of formula (I), wherein R 2 - R 2 = isopropylene, R = methyl and n = 0.