NO177187B - Analogous Process for Preparing Therapeutically Active N, N'-Bis (Alkoxy-alkyl) -pyridine-2,4-dicarboxylic Acid Diamides - Google Patents

Abstract

N,N'-bis(alkoxyalkyl)pyridine-2,4-dicarboxylic acid diamides of the formula I <IMAGE> where R<1> is linear or branched C1-C4-alkanediyl, R<2> is unbranched C1-C4-alkyl or hydrogen and n is 1 or 2 and R<1'>, R<2'> and n' have the same meanings as R<1>, R<2> and n, where R<1> and R<1'>, R<2> and R<2'>, and n and n' are identical or different, and their physiologically acceptable salts with the exception of N,N'-bis(2-methoxyethyl)pyridine-2,4-dicarboxylic acid diamide and N,N'-bis(2-hydroxyethyl)pyridine-2,4-dicarboxylic acid diamide. The compounds of the formula I inhibit prolin hydroxylase and lysin hydroxylase and are suitable as fibrosuppressants and immunosuppressants.

Description

The present invention relates to the preparation of new, therapeutically active N,N'-bis(alkoxy-alkyl)-pyridine-2,4-dicarboxylic acid diamides and physiologically acceptable salts thereof.

Compounds that inhibit proline and lysine hydroxylase cause a highly selective inhibition of collagen biosynthesis by influencing collagen-specific hydroxylation reactions. In their course, protein-bound proline or lysine is hydroxylated by the enzymes proline and lysine hydroxylase, respectively. If this reaction is prevented by means of inhibitors, a non-functional, underhydroxylated collagen molecule is formed which can only be released from the cells into the extracellular space in small quantities. Furthermore, the underhydroxylated collagen cannot be incorporated into the collagen matrix and is very easily degraded proteolytically. As a result of these effects, the total amount of extracellular storage collagen is reduced.

It is known that the inhibition of proline hydroxylase by means of known inhibitors, such as oc,a'-dipyridyl leads to an inhibition of Clq biosynthesis by macrophages (W. Muller et al., FEBS Lett. 90 (1978), 218; Immunbiology 155 (1978) 47). This results in an outcome of the classic method for complement activation. Inhibitors of proline hydroxylase therefore act as immunosuppressants, e.g. in immune complex diseases.

It is known that proline hydroxylase can be effectively inhibited by pyridine-2,4- and -2,5-dicarboxylic acid (K. Majamaa et al., Eur. J. Biochem. 138 (1984) 239-245). However, these compounds are only effective in cell culture as inhibitors in very high concentrations (Tschank, G. et al., Biochem. J. 238, 625-633, 1987).

DE-A 34 32 094 describes pyridine-2,4- and -2,5-dicarboxylic acid diesters with 1-6 C atoms in the ester alkyl part as drugs for inhibiting proline and lysine hydroxylase. . However, these low-alkylated diestes have the disadvantage that they break down too quickly to the acids in the organism and do not reach the site of action in the cell in a sufficiently high concentration and are thus less suitable for possible administration as medicine.

DE-A 37 03 959, DE-A 37 03 962 and DE-A 37 03 963 describe in general form mixed ester/amides, higher alkylated diesters and diamides of pyridine-2,4- and -2,5-dicarboxylic acids as effective inhibits collagen biosynthesis in an animal model.

Accordingly, DE-A 37 03 959 describes, among other things, the synthesis of N,N'-bis(2-methoxyethyl)-pyridine-2,4-dicarboxylic acid diamide

(III)

and N,N'-bis(3-isopropoxypropyl)-pyridine-2,4-dicarboxylic acid diamide (IV)

In the German patent publications P 38 26 471.4 and P 38 28 140.6, an improved process for the production of N,N'-bis(2-methoxyethyl)-pyridine-2,4-dicarboxylic acid diamide (III) is proposed.

However, the enteral resorbability of many of the compounds mentioned in DE-A 37 03 959 is still unsatisfactory, so that there is a need to provide compounds which, after oral administration, already at low dosages effectively inhibit the proline and lysine hydroxylase.

It has now been found that compounds of formula (I)

in which

R<1> is linear or branched C1-C4~alkanediyl,

R<2> is unbranched C₁-C₄ alkyl or hydrogen,

n means 1 or 2 and

R1', R<2>' and n' have the same meanings as R<1>, R<2> and n,

whereby R<1> and R<1>', R<2> and R<2>' and n and n' are identical

or different,

as well as their physiologically acceptable salts, except N,N'-bis(2-methoxyethyl)-pyridine-2,4-dicarboxylic acid diamide and N,N<*->bis(2-hydroxyethyl)-pyridine-2,4-dicarboxylic acid diamide, meet the above-mentioned task.

Compared to the compounds N,N'-bis(2-methoxyethyl)-pyridine-2,4-dicarboxylic acid diamide and N,N*-bis(3-isopropoxypropyl)-pyridine-2,4- dicarboxylic acid diamide exhibits the compounds of formula (I) both a better pharmacological activity and also a better enteral resorbability.

The above-mentioned compounds of formula (I) are produced according to the invention by a method characterized by the fact that

1. mixing pyridine-2,4-dicarboxylic acid with at least two equivalents of a halogenating agent, and that 2. dissolving at least two equivalents of hydroxyalkylamine or Alkoxyalkylamine of formula (II) or (II')

in which

R<1> and R<1>' mean linear or branched C₁-C₄alkanediyl, R<2> and R<2>' mean unbranched C₁-C₄alkyl or hydrogen, and n and n' mean 1 or 2 and

R1 and R*' , R<2> and R<2>' and n and n' are identical or different, whereby (II) and (II') are however different,

in a solvent and that the solution prepared according to 1. is brought into reaction with the solution prepared according to 2. and that the resulting compound of formula (I) is optionally then transferred to its physiologically acceptable salt.

In the process for the preparation of compounds of formula (I), the starting material commercially available pyridine-2,4-dicarboxylic acid is suspended in a solvent such as toluene and mixed at room temperature with a halogenating agent, preferably a chlorinating agent such as e.g. S0C12- With regard to the molar amount of pyridine-2,4-dicarboxylic acid used, 2-3 equivalents of a halogenating agent are used, preferably 2.5 equivalents. The obtained reaction mixture is heated to 90-110°C, preferably to 100°C, until no more gas evolution is observed and a clear solution is formed. Then, 10Sé is evaporated from the solution, preferably in a high vacuum (up to 10<3> torr), and the obtained carboxylic acid halide is reacted further.

With regard to the molar amount of pyridine-2,4-dicarboxylic acid used, the 2-4 times molar amount of commercial alkoxyalkylamine or hydroxyalkylamine is dissolved in a solvent such as toluene and preferably the 2-4 times molar amount of a base, such as triethylamine, is added . The carboxylic acid halide is brought into reaction with the alkoxyalkylamine, respectively the hydroxylalkylamine. This preferably takes place by adding the solution of the aforementioned alkylamine dropwise to the dissolved pyridine-2,4-dicarboxylic acid halide. However, it is also possible to add dropwise the solution of the carboxylic acid halide to the solution of the alkoxyalkylamine or the hydroxyalkylamine. The addition takes place at a temperature of -5 to +5°C, preferably at 0°C. The reaction mixture is then allowed to react by e.g. warm to room temperature and stir for a further 2-5 hours, preferably 3 hours. The product obtained is then acidified in order to separate excess hydroxy or alkoxyalkylamine from the desired product. The acidification can, for example, take place with 0.2 molar citric acid. The organic phases are then separated and washed with water. The organic phase is then dried, preferably over magnesium sulphate, and finally freed of solvent. On removal of the solvent, the product is formed as a white solid or as an oil.

For the preparation of the N,N'-bis(hydroxyalkyl)-pyridine-2,4-dicarboxylic acid diamide, the best way to proceed is to transfer a corresponding bis(alkoxyalkyl)diamide, preferably bis(methoxyalkyl)diamide, by methods known from the literature , for example with boron tribromide, to the corresponding bis(hydroxyalkyljdiamide.

Unsymmetrically substituted compounds of formula (I) can for example be synthesized in the following way: Reaction of a pyridine-2,4-dicarboxylic acid halide, preferably the -chloride, with substituted or unsubstituted benzyl alcohol to pyridine-2,4-dicarboxylic acid benzyl ester, subsequent selective saponification of the ester in the 2-position (e.g. in the presence of a copper catalyst, Acta Heiv. 44, 1963, p. 637), transfer of the free acid in the 2-position to the acid halide, reaction with a compound of formula (II<*>) to pyridine-4-carboxylic acid benzyl ester-2-carboxylic acid amide, hydrogenolytic cleavage of remaining benzyl protecting group (e.g. with B^/Pd, see Houben-Weyl, vol. IV/lc (1980), pp. 381-82) and subsequent transfer of the free acid at the 4-position of the pyridine ring to the acid halide. The acid halide can then be transferred with an amine (II) to the desired diamine (I) (see reaction core).

Optionally, the preparation of the products can take place, for example, by extraction or by chromatography, e.g. over silica gel. The isolated product can be recrystallized and optionally reacted with a suitable acid to a physiologically acceptable salt. As suitable acids, for example: mineral acids such as hydrogen chloride and hydrogen bromide as well as sulphurous, phosphoric, nitric or perchloric acid or organic acids such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, maleic acid, fumaric acid, phenylacetic acid, benzoic acid, methanesulfonic acid, toluenesulfonic acid, oxalic acid, 4-aminobenzoic acid, naphthalene-1,5-disulfonic acid or ascorbic acid.

The compounds of formula (I) can find use as drugs in the form of pharmaceutical preparations containing these together with acceptable pharmaceutical carriers. The compounds may find application as healing agents, e.g. In the form of pharmaceutical preparations containing these compounds in admixture with a pharmaceutical, organic or inorganic carrier suitable for enteral, percutaneous or parenteral administration, such as e.g. water, gum arabic, gelatin, milk sugar, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycol, petroleum jelly, etc.

The pharmaceutical preparations can be in solid form, e.g. as tablets, dragees, suppositories or capsules; in semi-solid form, e.g. as ointments, or in liquid form, e.g. as solutions, suspensions or emulsions. If necessary, they are sterilized and/or contain auxiliary substances, such as preservatives, stabilisers, wetting or emulsifying agents, salts for changing the osmotic pressure or buffers. They may also contain other therapeutically active substances.

It was found that the compounds of formula (I) exhibit unusually good enteral resorbability. The resorbability was examined in Wistar rats which were given the compounds prepared according to the invention intragastrically. The serum level decreased in the first hours after administration of the drug and reached after approx. 5 hours a still slightly decreasing plateau. From the first very high serum level directly after administration of the substances, one can conclude that the substances are well resorbable.

In the following, the invention will be described by means of examples.

Example 1

Pyridine- 2, 4- dicarboxylic acid- N, N'-( methoxypropyl) amld

3 6 pyridine-2,4-dicarboxylic acid is placed in 50 ml of toluene and 1 ml of DMF and added dropwise to a solution of 2.7 ml of thionyl chloride. It is heated until no more gas evolution is observed (approx. 2.5 hours). It is cooled, 5 ml of toluene is distilled off and added dropwise to a solution of 4.6 ml of 3-methoxypropylamine and 5 ml of triethylamine. After stirring the solution at room temperature for 4 hours, it is evaporated, the residue is taken up with water and extracted four times with methylene chloride. The combined organic phases are dried over magnesium sulfate and evaporated. The crude product is chromatographed with silica gel (solvent ethyl acetate).

Yield: 4.3 g; oil

Example 2

Pyridine-2. 4-dicarboxylic acid-bis-N,N'-(ethoxypropyl)amld Method, see example 1; amine component ethoxypropylamine;

Yield: 4.5 g, freezing point: 46-48°C.

<*>H-NMR (CDCl 3 ): S = 1.3 (6H, tr); 1.7-2.1 (4H, m);

3.3-3.8 (12H, m); 7.8-8.0 (1H, m);

8.4-8.5 (IB, m); 8.5-8.8 (1H, m).

Example 3

Pyridine-2.4-dicarboxylic acid-bis-N.N'-(2-dimethoxyethyl)amide Method, see example 1; amine component 2-dimethoxyethylamine;

Yield: 1.6 g (from 3 g of pyridine-2,4-dicarboxylic acid), oil.

<i>H-NMR (CDCl 3 ): S = 3.4 (12H, s); 3.7 (4H, m);

4.5 (2H, m); 7.9-8.0 (1H, m);

8.4-8.5 (1H, m); 8.7-8.8 (1H, m).

Example 4

Pyridine-2.4-dicarboxylic acid-bis-N,N,-(2-methoxyisopropyl)amide Method, see example 1; amine component 2-methoxypropylamine;

Yield: 3.3 g (from 3 g of pyridine-2,4-dicarboxylic acid), oil;

<i>H-NMR (CDCl 3 ): S = 1.3 (6H, d); 3.2 (6H, s); 3.5 (4H, d);

4.4 (2H, m); 7.9-8.0 (1H, m);

8.4-8.5 (1H, m); 8.7-8.8 (1H, m).

Example 5

Pyridine-2,4-dicarboxylic acid-bis-N.N'-(2-ethoxyethyl)amld Method, see example 1; amine component ethoxyethylamine;

Yield: 7.8 g (from 10 g of pyridine-2,4-dicarboxylic acid), Freezing point: 42-44°C.

<i>H-NMR (CDCl 3 ): S = 1.2 (3H, tr); 3.3-3.8 (12H, qu. and m);

7.9 (12, m); 8.4-8.5 (1H, m);

8.7-8.8 (1H, m).

Example 6

Pyridine-2.4-dicarboxylic acid-bis-N,N'-(3-hydroxyethyl)aroid °.5 g pyridine-2,4-dicarboxylic acid-bis-N,N'-(3-methoxyethyl)-amide dissolve in 10 ml of dichloromethane and add boron tribromide (11 ml, 1 molar solution in dichloromethane) dropwise at -78°C. After the addition is complete, the mixture is allowed to come to room temperature and stirred for 3 hours. It is poured onto 100 ml of saturated bicarbonate solution and extracted three times with ethyl acetate. The combined organic solvent is dried with magnesium sulfate and evaporated. The crude product is chromatographed on silica gel.

Yield: 0.45 g; oil

1 H-NMR (CDCl 3 ) S 1.5-2.2 (4H, m); 3.4 (4H, m);

3.6 (4H, m); 7.9-8.0 (1H, m);

8.4-8.5 (1H, m); 8.7-8.8 (1H, m).

Example 7a

Pyrldln-2.4-dicarboxylic acid dibenzyl ester 30 g of pyridine-2,4-dicarboxylic acid is transferred analogously to example 1 with 30 ml of thionyl chloride to acid chloride and reacted with 43.8 g of benzyl alcohol. The product is recrystallized from diisopropyl ether.

Yield: 42.1 g. Melting point: 63-65"C.

Example 7b

Pyridln-2-carboxylic acid-4-carboxylic acid benzyl ester

40 g of pyridine-2,4-dicarboxylic acid dibenzyl ester from example 7a is added to a suspension of 27.8 g of copper(II) nitrate in 700 ml of methanol. It is boiled for one hour under reflux and after cooling the copper complex is filtered off. The complex is suspended in dioxane and carbon sulphide is introduced. The precipitated copper sulphide is filtered off and the organic phase is evaporated. The product is stirred with petroleum ether.

Yield: 25.3 g Melting point: 113-115°C

Example 7c

Pvridin- 2-( 3- methoxypropyl ^- carboxylic acid ainide- 4- carboxyl acid benzvlester

3.9 g of pyridine-2-carboxylic acid-4-carboxylic acid benzyl ester from example 7b is transferred analogously to example 1 with 1.2 ml of thionyl chloride to the acid chloride and reacted with 3-methoxypropylamine to amide. The product is chromatographed for purification over silica gel with a mixture of cyclohexane/ethyl acetate (1:1).

Yield: 4.3 g Oil

Example 7d

Pyridine-4-carboxylic acid-2-(3-methoxypropyl)-carboxylic acid 4.3 g of the compound from example 7c are dissolved in 100 ml of dioxane and stirred with 500 mg of palladium/coal (105t) catalyst under normal pressure for 4 hours. After completion of the hydrogen absorption, it is sucked off from the catalyst and the solvent is drawn off.

Yield: 3.5 g Melting point: 124-126'C

Example 7e

Pyridin- 4- carboxylic acid-(2- methoxyethyl)- 2- carboxylic acid-(3- methoxypropyl)- diamide

Corresponding to example 1, 1.8 g of the compound from example 7d is transferred with 0.6 ml of thionyl chloride to the acid chloride and then reacted with 2-methoxyethylamine. The product is chromatographed for purification over silica gel with a mixture of dichloromethane/methanol (20:1).

Yield: 1.0 g Oil

1-H-NMR (CDCl 3 ): S = 1.9-2.0 (2H, qui); 3.4 (6H, s);

3.5-3.7 (8H, m); 6.9 (1H, s, br);

8.0 (1H, dd); 8.4 (1H, s, br );

8.5 (1H, s); 8.7 (1H, d).

Example 8

Pyr Idi n- 2- carboxylic acid - ( 2- methoxyethyl)- 4- carboxylic acid-( 3- methoxypropyl)- diamide

Analogous to examples 7a-e, the compound according to example 8 is prepared by using 2-methoxyethylamine in reaction step example 7c and 3-methoxypropylamine in reaction step example 7e.

Melting point: 69-72"C

<i>H-NMR (CDCl 3 ): S = 1.9-2.0 (2H, qui); 3.4 (3H, s);

3.45 (3H, s); 3.6-3.7 (8H, m);

7.4 (1H, br); 7.9 (1H, dd);

8.3 (1H, br); 8.4 (1H, d); 8.7 (1H, d).

Example 9

Enteral resorbability

Female Wistar rats with a body weight of approx. 150 g, received 50 mg/kg of the substance to be examined administered intragastrically by means of a pharyngeal tube. After 5; 10; 15; 30; 60; 120; 180 and 240 minutes each time 4 rats are anesthetized and the blood is removed via the vena cava. The blood is immediately centrifuged and the administered compound is extracted with ether from the serum. After evaporation of the ether, the residue is taken up in 100 ml of liquid. The fluid consists of 0.05 M phosphoric acid and acetonitrile (4:1). 50 µl of this sample are injected into an HPLC column. The detection takes place at TJV 200 nm and a retention time of 2.2 minutes. The results are documented in table 1.

Example 10

Pharmacological business

To demonstrate the effective inhibition of the proline and lysine hydroxylase by means of the compound produced according to the invention, the hydroxyproline concentration in the liver and the 7s-(IV)-collagen concentration in serum from

a) untreated rats (control)

b) rats that were administered carbon tetrachloride (CCI4 control) c) rats that were first given CCI4 and then a compound prepared according to the invention,

(this experimental method is described by Rouiller, C, Experimental toxic injury of the liver; in The Liver, C. Rouiller, volume 2, pp. 335-476, New York, Academic Press, 1964).

The potency of the compound produced according to the invention was determined as percentage inhibition of liver hydroxyproline and serum 7s-(IV) collagen synthesis after oral administration compared to control animals that were only administered carbon tetrachloride (CCl4 control). The results are shown in table 2. As a comparison substance, the compounds from examples 2 and 3 in DE-A 37 03 959 (N,N'-bis(2-methoxyethyl)-pyridine-2,4-dicarboxylic acid diamide and N,N<* ->bis(3-isopropoxypropyl)-pyridine-2,4-dicarboxylic acid diamide) listed. Surprisingly, the compounds produced according to the invention already show better activity after oral administration than the i.p.-administered compound from example 2 in DE-A 37 03 959.

Claims (12)

1. Analogous process for the preparation of therapeutically active N , N * -bis-(alkoxyalkyl)-pyridine-2,4-dicarboxylic acid diamides of formula (I) wherein R<*> is linear or branched C1-C4-alkanediyl, R<2> means unbranched C1-C4~alkyl or hydrogen, n means 1 or 2 and R<i>', R<2>' and n' have the same meanings as Ri, R<2> and n, whereby R<1> and R1', R2 and R<2>' and n and n' are identical or different and their physiologically acceptable salts, except N,N'-bis(2-methoxyethyl)-pyridine-2,4- dicarboxylic acid diamide and N,N'-bis(2-hydroxyethyl)-pyridine-2,4-dicarboxylic acid diamide, characterized by the fact that one 1. mixes pyridine-2,4-dicarboxylic acid with at least two equivalents of a halogenating agent, and that one 2. dissolves at least two equivalents of hydroxyalkylamine or Alkoxyalkylamine of formula (II) or (II') in which R<1> and R<1>' mean linear or branched C-j—C4 alkanediyl, R<2> and R<2>' mean unbranched C1-C4 alkyl or hydrogen, and n and n<*> mean 1 or 2 and R1 and R<1>', R<2> and R<2>' and n and n' are identical or different, whereby (II) and (II') are however different, in a solvent and that the solution prepared according to 1. is brought into reaction with the solution prepared according to 2. and that the resulting compound of formula (I) is optionally then transferred to its physiologically acceptable salt. 2. Process according to claim 1 for the preparation of compounds of formula (I) in which R<*> and R<1>', R<2> and R<2>' and n and n' have the same meaning, characterized in that correspondingly, optionally substituted starting materials are used. 3. Process according to claim 1 for the preparation of compounds of formula (I) in which the substituents -(R<1>)-(OR<2>)n and -(R1' )-(OR2' )n" are different, characterized in that corresponding , optionally substituted, starting materials are used. 4. Process according to claim 1 for the preparation of compounds of formula (I) in which R<1> means linear or branched C]-C3-alkyl and R<2> means unbranched C^-C2-alkyl or hydrogen, characterized in that correspondingly, optionally substituted starting materials are used. 5. Process according to claim 1 for the preparation of a compound with the formula or physiologically acceptable salts thereof, characterized in that corresponding, possibly substituted, starting materials are used. 6. Process according to claim 1 for the preparation of a compound with the formula as well as physiologically acceptable salts thereof, characterized in that corresponding, possibly substituted, starting materials are used. 7. Process according to claim 1 for the preparation of a compound with the formula as well as physiologically acceptable salts thereof, characterized in that corresponding, possibly substituted, starting materials are used. 8. Process according to claim 1 for the preparation of a compound with the formula as well as physiologically acceptable salts thereof, characterized in that corresponding, possibly substituted, starting materials are used. 9. Process according to claim 1 for the preparation of a compound with the formula as well as physiologically acceptable salts thereof, characterized in that corresponding, possibly substituted, starting materials are used. 10. Process according to claim 1 for the preparation of a compound with the formula as well as physiologically acceptable salts thereof, characterized in that corresponding, possibly substituted, starting materials are used. 11. Process according to claim 1 for the preparation of a compound with the formula as well as physiologically acceptable salts thereof, characterized in that corresponding, possibly substituted, starting materials are used. 12. Process according to claim 1 for the preparation of a compound with the formula as well as physiologically acceptable salts thereof, characterized in that corresponding, possibly substituted, starting materials are used.