2,4-and 2,5-bistetrazolylpyridines and the use thereof as pharmaceuticals

The invention relates to 2,4- and 2,5-bistetrazolylpyridines. Said compounds inhibit the enzymes proline hydroxylase and lysine hydroxylase and bring about a selective inhibition of collagen biosynthesis. They are used as fibrosuppressants and immunosuppressants.

Compounds which inhibit the enzymes proline hydroxylase and lysine 
hydroxylase bring about very selective inhibition of collagen biosynthesis 
by influencing the collagen-specific hydroxylation reaction. In the course 
of this, protein-bound proline or lysine is hydroxylated by the enzymes 
proline hydroxylase or lysine hydroxylase respectively. If this reaction 
is suppressed by inhibitors, the resulting collagen molecule is 
underhydroxylated, is unable to function and can be released by the cells 
into the extracellular space only in small amount. The underhydroxylated 
collagen is additionally unable to be incorporated in the collagen matrix 
and very easily undergoes proteolytic degradation. The consequence of 
these effects is an overall reduction in the amount of collagen deposited 
outside the cells. 
It is known that the inhibition of proline hydroxylase by known inhibitors 
such as .alpha.,.alpha.-dipyridyl leads to inhibition of Cl.sub.q 
biosynthesis by macrophages (W. Muller et al., FEBS Lett. 90 (1978), 218; 
Immunobiology 155 (1978), 47). This results in the classical pathway of 
complement activation becoming inoperative. Inhibitors of proline 
hydroxylase therefore also act as immunosuppressants, for example in 
immune complex diseases. 
It is known that the enzyme proline hydroxylase is efficiently inhibited by 
pyridine-2,4- and -2,5-dicarboxylic acid (K. Majamaa et al., Eur. J. 
Biochem. 138 (1984)239-245). These compounds are, however, active as 
inhibitors in cell culture only in very high concentration (Tschank, G. et 
al., Biochem. J. 248 (1987) 625-633). 
DE-A 34 32 094 describes pyridine-2,4- and -2,5-dicarboxylic diesters with 
16 carbon atoms in the ester alkyl moiety as pharmaceuticals for 
inhibiting proline hydroxylase and lysine hydroxylase. 
However, these lower alkyl diesters have the disadvantage that they are too 
quickly cleaved to the acids in the body and do not reach their site of 
action in the cell in sufficiently high concentration and thus are less 
suitable for possible administration as pharmaceuticals. 
DE-A 37 03 959, DE-A 37 03 962 and DE-A 37 03 963 describe, in general 
form, mixed esters/amides, higher alkylated diesters and diamides of 
pyridine-2,4- and -2,5-dicarboxylic acid, which effectively inhibit 
collagen biosynthesis in an animal model. Thus, DE-A 37 03 959 describes, 
inter alia, the synthesis of 
N,N'-bis(2-methoxyethyl)pyridine-2,4-dicarboxamide and 
N,N'-bis(3-isopropoxypropyl)pyridine-2,4-dicarboxamide. 
German Patent Applications P 38 26 471.4 and P38 28 140.6 propose an 
improved process for the preparation of 
N,N'-bis(2-methoxyethyl)pyridine-2,4-dicarboxamides. 
German Patent Application P 39 24 093.2 proposes novel 
N,N'-bis(alkoxyalkyl)pyridine-2,4-dicarboxamides. 
German Patent Application P 40 01 002.3 describes the use of 
di(nitroxyalkyl) amides of pyridine-2,4- and -2,5-dicarboxylic acids for 
the preparation of pharmaceuticals inhibiting proline hydroxylase and 
lysine hydroxylase. 
Both pyridine-2,4 - and -2,5-dicarboxamide (Hirakata et al., J. Pharm. Soc. 
Japan 77 (1957) 219 and Haring et al., Helv. 37 (1954) 147, 153), and 
pyridine-2,4- and -2,5-dicarbohydrazide (Itai et al., Bl. Nation. Hyg. 
Labor. Tokyo, 74 (1956) 115, 117 and Shinohara et al., Chem. High 
Polymers, Japan, 15 (1958) 839) have already been disclosed as agents for 
tuberculosis. 
JP 53/28175 (78/28175) describes N,N'-bis(2-nitroxyethyl)pyridine-2,4-2,4- 
and -2,5-dicarboxamides as substances with a vasodilator effect. 
German Patent Application P 40 20 570.3 describes the use of 2,4- and 
2,5-substituted pyridine N-oxides for the preparation of pharmaceuticals 
inhibiting proline hydroxylase and lysine hydroxylase. 
It has now been found, surprisingly, that 2,4 - and 
2,5-bistetrazolylpyridines of the formula I indicated below, and the 
physiologically tolerated salts thereof, effectively inhibit lysine 
hydroxylase and proline hydroxylase in an animal model. 
Specific 2,4- and 2,5-bistetrazolylpyridines are compounds of the formula I 
##STR1## 
where A is 
##STR2## 
and where R is H,(C.sub.2 -C.sub.6)alkyl, (C.sub.2 -C.sub.6)alkenyl, 
(C.sub.2 -C.sub.6)alkynyl, but preferably in each case alkyl where 
appropriate, substituted by carboxyl or carboxy(C.sub.1 -C.sub.4)alkyl 
ester, by arylcarbonyl, especially phenylcarbonyl, by (C.sub.1 
-C.sub.4)alkylaminocarbonyl, where substitution by (C.sub.1 
-C.sub.6)alkoxy, especially (C.sub.1 -C.sub.4)alkoxy, is possible for 
alkyl in turn, by aminocarbonyl, where the nitrogen can be substituted 
once, but preferably twice, by alkyl, especially (C.sub.1 -C.sub.3 )alkyl, 
(C.sub.6 -C.sub.10)aryl, especially phenyl or naphthyl, where appropriate 
substituted by halogen (chlorine or bromine), (C.sub.1 -C.sub.6)alkyl or 
(C.sub.1 -C.sub.3)alkoxy, Ar(C.sub.2 -C.sub.6)alkyl, where one or more, 
but a maximum of 3, CH.sub.2 groups in the alkyl radical can, where 
appropriate, be replaced by hetero atoms, especially N, O or S, and Ar is, 
in particular, phenyl, cycloalkyl- or cycloalkenyl-(C.sub.0 
-C.sub.6)alkyl, especially (C.sub.5 -C.sub.6)cycloalkyl or -alkenyl, where 
appropriate with aryl ring, especially benzene ring, fused on, where 1 to 
3 CH.sub.2 groups in the cycle are replaced by hereto atoms such as O, S 
or N, especially O and/or N, and/or groups such as C.dbd.O, and the cycle 
can, where appropriate, also be substituted by alkyl, especially (C.sub.1 
-C.sub.4)alkyl. 
Particularly preferred are alkyl radicals with 1-4 carbon atoms, Ar(C.sub.1 
-C.sub.4)alkyl with one CH.sub.2 group in the alkyl radical replaced by a 
hetero atom, especially N or O, cycloalkyl or cycloalkenyl, where, if two 
CH.sub.2 a groups have each been replaced by a C.dbd.O group, a third 
CH.sub.2 group is replaced by N, or, if two CH.sub.2 groups have each been 
replaced by one O, a third group is replaced by C.dbd.O or N. The 
resulting compounds are, in particular, axially symmetrical, i.e. the 
third group in each case is bonded to the other two. 
Particularly preferred compounds of the formula I are those in which the 
radicals R are identical for both substituents A. To be understood as 
preferred among these are those compounds for which the substitution 
patterns for A (2,4 or 2,5 ) are identical. 
The invention also relates to a process for the preparation of compounds of 
the formula I, in which a compound of the formula II 
##STR3## 
is reacted with NaN.sub.3 and NH.sub.4 Cl in a suitable organic solvent. 
If the intention is to obtain compounds of the formula I in which R is not 
H, the said reaction is then followed by a substitution reaction in which 
R is replaced by the particular substituent. For this, a solution of 2,4- 
or 2,5-bis(5-tetrazolyl)pyridine in a suitable solvent, preferably DMF or 
acetone, is mixed at room temperature with an excess of a suitable base, 
for example triethylamine or NaOH. Subsequently, for example, an 
alkylating agent is added as such or dissolved in DMF, and the reaction 
mixture is left to stir or is heated to boiling under reflux until little 
or no precursor is still detectable by thin-layer chromatography (silica 
gel, mobile phase ethyl acetate). Excess alkylating agent is, where 
appropriate, decomposed by addition of concentrated ammonia. Working up is 
carried out by either 
a) evaporation of the mixture and purification by flash chromatography or 
recrystallization of the residue or 
b) partition between water and a suitable solvent, preferably ethyl acetate 
or CH.sub.2 Cl.sub.2, drying of the organic phases with Na.sub.2 SO.sub.4, 
evaporation of the solution and purification by flash chromatography or 
recrystallization of the residue. 
The compounds of the formula I according to the invention have valuable 
pharmacological properties and display, in particular, activity as 
inhibitors of proline hydroxylase and lysine hydroxylase, as 
fibrosuppressant and as immunosuppressant. 
Fibrogenase activity can be determined by radioimmunological determination 
of the N-terminal propeptide of collagen type III or of the N- or 
C-terminal crosslinking domain of collagen type IV (7s collagen or type IV 
collagen NC.sub.1) in serum. 
For this purpose, the hydroxyproline, procollagen III peptide, 7s collagen 
and type IV collagen NC.sub.1 concentrations were measured in the livers 
of 
a) untreated rats (control) 
b) rats to which tetrachloromethane had been administered (CCl.sub.4 
control) 
c) rats to which initially CCL.sub.4 and then a compound according to the 
invention had been administered (this test method is described by 
Roullier, C., experimental toxic injury of the liver; in The Liver C. 
Roullier, Vol. 2, pages 335-476, New York, Academic Press, 1964). 
The compounds of the formula I can be used as medicaments in the form of 
pharmaceutical products which contain them, where appropriate together 
with tolerated pharmaceutical vehicles. The compounds can be used as 
medicines, for example in the form of pharmaceutical products which 
contain these compounds mixed with an inorganic or organic pharmaceutical 
vehicle suitable for enteral, percutaneous or parenteral administration, 
such as, for example, water, gum arabic, gelatin, lactose, starch, 
magnesium stearate, talc, vegetable oils, polyalkylene glycols, vaseline 
etc. 
They can be administered for this purpose orally in doses of 0.01-25.0 
mg/kg/day, preferably 0.01-5.0 mg/kg/day, or parenterally in doses of 
0.001-5 mg/kg/day, preferably 0.001-2.5 mg/kg/day, especially 0.005-1.0 
mg/kg/day. The dosage can also be increased in severe cases. However, 
lower doses often suffice in many cases. These data are based on an adult 
weighing about 75 kg.