Amino acid and peptide esters of leuko-indoaniline compounds and compositions for the detection of proteolytic enzymes

This invention relates to new amino acid and peptide esters of leuko-indoaniline compounds and to a process for their preparation. In additional aspect, the invention relates to compositions containing such compounds for the detection of proteolytic enzymes and to methods for detecting such enzymes.

This invention relates to new amino acid and peptide esters of 
leuko-indoaniline compounds and to a process for their preparation. In 
additional aspect, the invention relates to compositions containing such 
compounds for the detection of proteolytic enzymes and to methods for 
detecting such enzymes. 
The detection of leukocytes in body fluids, especially in the urine, is 
extremely important in the diagnosis of diseases of kidney and of the 
urogenital tract. 
Hitherto, this detection has been carried out by the laborious counting of 
the leukocytes in non-centrifuged urine or in urine sediment. 
It is inherent in both methods that only intact leukocytes are detected. On 
the other hand, it is known that the rate of leukocyte lysis is subject to 
enormous variations, depending upon the urine medium; thus, for example, 
in strongly alkaline urines, the leukocyte half-lifetime can be only 60 
minutes. The result is leukocyte counts that are too low or, when urine 
samples have been left to stand for a rather long time, even falsely 
negative findings. 
Apart from lysis error, the quantitative microscopic determination of the 
leukocytes in non-centrifuged, homogenized urine in a counting chamber 
gives quite dependable values. However, in practice, this method is only 
rarely used, since it is laborious, tiring, and time-consuming, and 
requires the use of trained personnel. 
The overwhelming majority of the leukocyte determinations in urine are 
carried out in a medical practice by the so-called viewing field method on 
the urine sediment. For this purpose, the material to be investigated 
(sediment) must first be obtained by centrifuging. In this process, 
however, other components of the urine are also concentrated, such as, for 
example, salts and epthelial cells, which can make the microscopic 
counting of the leukocytes considerably more difficult. The varying 
content of sediment, inhomogeneities of the sediment, as well as possible 
differing microscopic magnifications or differing optical equipment for 
the microscope results in the fact that reports of this type on the number 
of leukocytes per microscopic viewing field may be affected by errors of 
several hundred percent. 
It was the object of the present invention, therefore, to provide a 
diagnostic agent with which leukocytes present in body fluids can be 
detected in a manner that is simple and easy to use, and as quickly and 
completely as possible. 
Since leukocytes possess a broad-spectrum enzyme activity, an enzymatic 
reaction suggests itself as a detection principle for such a leukocyte 
test. 
U.S. Pat. No. 3,087,794 has already described and claimed a leukocyte 
detection method which is carried out via the peroxidase activity present 
in the granular leukocytes (granulocytes). An absorbent carrier, 
impregnated with hydrogen peroxide and an organic indicator, for example, 
o-tolidine, indicates the presence of leukocytes by the formation of a 
colored oxidation product. Such a test, however, has serious 
disadvantages: for one thing, peroxidase reactions using o-tolidine quite 
generally have a considerable tendency to be disturbed by reducing 
substances in the urine, such as, for example, ascorbic acid. Furthermore, 
there are references in several places in the literature to the 
instability of leukocyte peroxidase in the urine medium, which gives rise 
to falsely negative findings (see L. Mettler, Med. Welt 23 (1972), 399). 
Even more serious is the unsatisfactory selectivity toward erythrocytes 
that can be expected. 
For some years, detection methods that depend on the esterolytic activity 
of the enzymes present in the systems to be determined have their fixed 
place in histo- and cyctochemical enzymology (cf., for example A. G. E. 
Pearse, Histochemistry: Theoretical and Applied, 3rd Ed. 
(Edinburgh/London/New York: Churchill Livingstone, 1968)). In principle, 
colorless or faintly colored esters are used, which mostly break down, 
through enzymatic splitting, into a colorless acid component and into an 
also colorless alcohol or phenol component. The latter is then converted 
into colored products, in a reaction that follows the enzymatic 
saponification (for example, by coupling with diazonium salts or oxidative 
reactions). 
Thus, for example, F. Schmalzl and H. Braunsteiner, in Klin. Wschr. 46 
(1968):642, describe a specific cytochemical leukocyte esterase detection 
with naphthol-AS-D-chloroacetate as substrate and a diazonium salt for the 
formation of a colored azo-compound. 
As an agent for the quick and simple detection of leukocytes, for example 
in urine, two-component systems of this type have not proved suitable, 
since as is known, many compounds occurring in urine, such as, 
urobilinogen, stercobilinogen, bilirubin, and others, react with diazonium 
salts. Furthermore, this detection method is much too insensitive. For 
example, samples containing 5,000 leukocytes/.mu.l do not show any 
reaction. 
Surprisingly, it was now found that stable agents, which quickly produce an 
indication, and with which leukocytes can easily be detected in body 
fluids, are obtained when amino acid esters or peptide esters of 
leuko-indoanilines are used as a substrate for the detection of the 
esterases (proteases) present in the neutrophilic leukocyte granulocytes. 
The subject of the present invention, therefore, is new amino acid and 
peptide esters of leuko-indoanilines of the general formula I 
##STR1## 
in which 
R.sub.1, R.sub.2, which may be the same or different, are hydrogen, a lower 
alkyl or a hydroxy-lower alkyl group or, together, an alkylene or 
alkylene-oxy-alkylene chain, 
R.sub.3, R.sub.4, R.sub.5, R.sub.6, which may be the same or different, are 
hydrogen or a halogen, a lower alkyl group, if necessary completely 
halogen substituted, a lower alkoxy, an aralkoxy, a hydroxy or a nitro 
group, 
R.sub.7, R.sub.8, R.sub.9, R.sub.10, which may be the same or different, 
are hydrogen or a halogen, a lower alkyl group, if necessary completely 
halogen substituted, a lower alkoxy, an aralkoxy, a lower acylamino, an 
acylalkenyl, a hydroxy, a lower alkylmercapto, a lower alkylsulfonyl, a 
carboxy, or a carbonyl group which, if necessary, is substituted by a 
lower alkoxy, aralkoxy, amino or lower alkylamino group or, at times, two 
adjacent substituents may represent a saturated or unsaturated hydrocarbon 
chain, in which a member of the chain may be replaced by a nitrogen atom, 
A is an amino acid or a peptide radical, and 
B is a nitrogen protective group customary in peptide chemistry or derived 
from it, as well as processes for the preparation of these compounds and 
their use for the preparation of agents for the detection of proteolytic 
enzymes. 
The preparation of the new amino acid and peptide esters from 
leuko-indoanilines of general formula I can take place according to 
methods that are themselves known from peptide chemistry. 
Preferably, the corresponding leuko-indoaniline compounds of general 
formula II 
##STR2## 
in which R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, 
R.sub.8, R.sub.9, and R.sub.10 have the meanings indicated above, are 
caused to react in a known manner with amino acids or peptides of the 
general formula III 
EQU HO--A--B (III) 
in which A and B have the meanings indicated above, or with appropriate 
reactive derivatives of them. 
The acid chlorides or active esters are used as reactive derivatives. 
The indoaniline compounds or the leuko-indoaniline substances of general 
formula II that are prepared from them by means of ordinary reducing 
agents (such as, for example, sodium sulfite or sodium dithionite) are 
known compounds; cf., for example, P. W. Vittum, G. H. Brown, J. Amer. 
Chem. Soc. 68 (1946): 2235 and J. Amer. Chem. Soc. 69 (1947): 152 and S 
Hunig, P. Richters, Liebigs Ann. 612 (1957): 282, or can be prepared 
analogously to known compounds. This is also true for amino acids and 
peptides of general formula III; cf., for example, Houben-Weyl, Methoden 
der organischen Chemie, Vol. 15/1/. 
The subject of the present invention is, furthermore, an agent for the 
detection of proteolytic enzymes, especially for the detection of the 
proteases present in the leukocytes, consisting of an absorbent carrier, a 
film layer, a lyophilisate, a solution or a reagent tablet containing one 
or more chromogens, and a suitable buffering substance, as well as the 
customarily used adjuvants, if necessary, characterized in that amino acid 
and/or peptide esters of leuko-indoanilines of general formula I, 
indicated above, are used as chromogens. 
The test principle of the agent according to the present invention is based 
on splitting the colorless amino acid and peptide esters of 
leuko-indoanilines of general formula I, which are claimed as chromogens, 
by means of esterases (proteases) into the practically colorless 
leuko-indoaniline compounds of general formula II, which are converted, in 
a secondary reaction, into the deep-blue colored indoanilines of general 
formula IIa, by means of atmospheric oxygen or an oxidizing agent. 
##STR3## 
The substrates according to the present invention, as well as the agents 
prepared with them according to the present invention, are especially 
suited for the general detection of proteolytic enzymes, especially for 
the detection of the proteases present in the leukocytes. Other 
proteolytic enzymes, however, can also be detected with them, such as, for 
example, elastase, chymotrypsin or trypsin in purely aqueous solutions or 
also in body fluids, such as, for example, plasma, serum, liquor, 
pancreatic secretions, or aqueous stool extracts. 
The lower alkyl radicals that appear in the definitions of R.sub.1, 
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, 
and R.sub.10 contain 1 to 6, preferably 1 to 4, carbon atoms, the methyl, 
ethyl, and n-butyl, as well as the methoxy, methylmercapto, and 
methylsulfonyl groups, being particularly preferred. 
Hydrocarbon radicals with 1 to 6, preferably 2 to 5, carbon atoms, which 
may contain oxygen or nitrogen atoms as members of the chain, are to be 
understood under an "alkylene" and "alkylene oxyalkylene chain" in the 
definition of R.sub.1 and R.sub.2, as well as under a "saturated or 
unsaturated hydrocarbon chain" in the definition of R.sub.7, R.sub.8, 
R.sub.9, and R.sub.10. The butylene, pentylene, ethylene oxyethylene, 
butadienylene, and azabutadienylene radicals are especially preferred. 
In the definition of R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, 
R.sub.9, and R.sub.10, "halogen" means fluorine, chlorine, bromine, or 
iodine, preferably fluorine, chlorine, or bromine, bromine or fluorine 
being very especially preferred. 
In the definition of R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, 
R.sub.9, and R.sub.10, phenyl and naphthyl radicals substituted by lower 
alkoxy groups are in question as the aralkoxy group, the alkyl radical 
containing 1 to 5, preferably 1 to 3, carbon atoms. The benzoyl radical is 
especially preferred. 
The amido groups of the lower aliphatic carboxylic acids with 1 to 5, 
preferably 1 to 3, carbon atoms, are to be understood under a "lower 
acylamino group" in the definition of R.sub.7, R.sub.8, R.sub.9, and 
R.sub.10. The acetylamino radical is especially preferred. 
The "acylalkenyl group" in the definition of R.sub.7, R.sub.8, R.sub.9, and 
R.sub.10 means an alkenyl group substituted by radicals of aromatic 
carboxylic acids, such as, for example, the benzoic or naphthoic acids, 
the alkenyl group containing 2 to 5, preferably 2 to 3, carbon atoms. The 
benzoylvinyl radical is especially preferred. 
The radicals of the natural .alpha.-amino acids in their L or D form, or 
even their racemic form, preferably come into question as the "amino acid 
radical" in the definition of A. Especially preferred are the radicals of 
glycine, alanine, valine, serine, leucine, methionine, phenylalanine, and 
tyrosine. Any hydroxyl group that may possibly be present can be protected 
in a familiar manner, preferably by means of an acetyl or benzyl radical. 
In the definition of A, di-, tri-, tetra-, and pentapeptides, preferably 
di- and tripeptides, are to be understood under a "peptide radical," the 
amino acids mentioned above preferably being used as amino acid 
components. 
Acyl, oxycarbonyl, and sulfonyl groups, for example, should be understood 
as a "nitrogen protective group customary in peptide chemistry" in the 
definition of B. 
The amino acid and peptide esters of leuko-indoanilines of the general 
formula I, which are used as chromogens according to the present 
invention, are employed in concentrations of 10.sup.-4 1 mol/liter, 
preferably 10.sup.-3 to 10.sup.-1 mol/liter of impregnating solution, 
coating mass, or liquid to be investigated. 
An additional component of the diagnostic agent for the detection of 
proteolytic enzymes and, especially, of leukocyte proteases is a suitable 
buffer system. For this purpose, for example, a phosphate, borate, 
barbiturate, tris-(hydroxymethyl)aminomethane (=tris), 
2-amino-2-methyl-propane-1,3 diol (=amediol) or amino acid buffer come 
under consideration. The pH value and capacity of the buffer are selected 
in such a way that a pH value of 6-10, and preferably of 7-9, appears in 
the measuring solution or on the test strip. 
Furthermore, in the production of the agent according to the present 
invention for the detection of proteolytic enzymes, especially of 
leukocyte proteases in body fluids, oxidizing agents may be used in 
addition, in order to convert the leuko-indoaniline compounds of the 
general formula II, initially formed in the enzymatic reaction, into the 
deep-blue indoanilines of the general formula IIa. 
These oxidizing agents, such as, for example, potassium 
hexacyanoferrate-III, potassium peroxodisulfate, potassium metaperiodate, 
sodium perborate, or potassium chromate are used in concentrations of 
10.sup.-4 to 10.sup.-1 mol/liter preferably 10.sup.-3 to 10.sup.-2 
mol/liter of impregnating solution coating mass, or fluid to be 
investigated. 
An additional component of the agent for the detection of proteolytic 
enzymes according to the invention can be a wetting agent, since a more 
homogeneous color distribution and, in some cases, more brilliant colors 
can be achieved by means of this. Cation-active and also anion-active, as 
well as amphoteric and non-iogenic wetting agents may be used, in 
concentrations of 0.05-2% (w/v), preferably 0.1-1% (w/v). 
In the production of the agent for the detection of proteolytic enzymes 
according to the present invention, furthermore, commonly used 
antioxidants (cf., for example, Ullmanns Enzyklopadie der technischen 
Chemie, Vol. 8), such as, for example, those of the phenol type, may be 
added, since, by so doing, the stability of the amino acid and peptide 
esters used according to the present invention is considerably improved 
with respect to oxidative decomposition processes. In the process, the 
antioxidants are used both as monocomponents and as synergistic systems 
(for example, hydroquinone plus hydroquinone monoalkyl ether). The 
antioxidants are used in concentrations of 10.sup.-4 to 10.sup.-2 
mol/liter, and in so doing, the concentration should be selected in such a 
way that the oxidative secondary step to the colored indoanilines, which 
follows the enzymatic splitting of the amino acid esters into the 
leuko-indoaniline compounds, is not disturbed. 
A phosphoric or phosphonic acid amide of general formula IV may serve as a 
stabilizer, as an additional component of the agent according to the 
present invention 
##STR4## 
in which R.sub.11 means a dialkylamino, an alkoxy, an aryloxy, an alkyl, 
or an aryl group, or an N-morpholine radical, and R.sub.12 and R.sub.13 
mean a dialkylamino group or an N-morpholine radical. 
In the definition of R.sub.11, hydrocarbon radicals with up to 10 carbon 
atoms come under consideration as the "alkoxy or alkyl group." 
In the definition of R.sub.11, phenyl or naphthyl radicals, if necessary 
substituted by halogen, lower alkyl, or alkoxy groups, should be 
understood under the definition of "aryl or aryloxy group." 
An astonishing stabilization of the preparations is achieved with the aid 
of compounds of general formula IV. 
Phosphoric and phosphonic acid amides of general formula IV are well known 
compounds. In German Pat. No. 2 235 127, for example, they are used as 
stabilizers of test strip preparations that operate on the basis of 
peroxidase detection. 
The stabilizers of the phosphoric and phosphonic acid amide type of general 
formula IV are added to the aqueous impregnating solution in 
concentrations of 1-20% (w/v), preferably 5-15% (w/v). 
Surprisingly, it was found that the reaction times of the diagnostic agent 
for the detection of proteolytic enzymes, especially of proteolytic 
leukocyte enzymes, according to the present invention, can be considerably 
shortened if one or more activators are used in addition to the previously 
mentioned chromogens and adjuvants. Suitable activators, for example, are 
(a) Alcohols of general formula V, 
EQU R.sub.14 --K--OH (V), 
in which R.sub.14 means hydrogen, a hydroxy or a lower alkoxy group, and K 
means a hydrocarbon radical; 
(b) Metallic complexes of general formula VI, 
EQU D.sub.m [M(CN).sub.n (NO).sub.p ] (VI) 
in which 
D means an alkali metal ion, 
M a heavy metal ion, 
m a whole number from 2 to 5, 
n a whole number from 4 to 8, and 
p 0 or 1. 
The "lower alkoxy group" in the definition of R.sub.14 contains 1 to 5, 
preferably 3 to 4, carbon atoms. The butyloxy group is especially 
preferred. 
The hydrocarbon radical K in the definition of R.sub.14 may be 
straight-chained or branched, saturated or unsaturated, cyclic or acylic, 
and contains 1 to 30, preferably 2 to 22, carbon atoms in the acyclic 
compounds, and 3 to 20, preferably 6 to 17, carbon atoms in the cyclic 
compounds. 
In the activators of general formula VI, sodium and potassium ions 
preferably come under consideration as alkali metal ions D, and ions of 
the metals iron, nickel, chromium, manganese, cobalt, molybdenum, and 
vanadium preferably come under consideration as heavy metal ions M. 
The following may be mentioned as examples of activators that can be used 
in the sense of the present invention: 
1. 1-Hexanol 
2. 1-Heptanol 
3. 1-Octanol 
4. 1-Nonanol 
5. 1-Decanol 
6. 1-Dodecanol 
7. 1-Tetradecanol 
8. 1-Pentadecanol 
9. 1-Hexadecanol 
10. 1-Heptadecanol 
11. 1-Octadecanol 
12. 1-Nonadecanol 
13. 1-Eicosanol 
14. 1-Docosanol 
15. Cyclohexanol 
16. 1-Cyclohexen-1-ol 
17. Cycloheptanol 
18. Cyclooctanol 
19. Cyclononanol 
20. Cyclodecanol 
21. Cyclododecanol 
22. Cycloheptadecanol 
23. 9-Cycloheptadecen-1-ol 
24. Citronellol 
25. Geraniol 
26. Nerol 
27. Linalool 
28. Farnesol 
29. Nerolidol 
30. cis-9-Octadecen-(1)-ol 
31. Phytol 
32. 1,5-Pentanediol 
33. 1,6-Hexanediol 
34. 1,7-Heptanediol 
35. 1,8-Octanediol 
36. 1,9-Nonanediol 
37. 1,10-Decandiol 
38. 1,12-Dodecandiol 
39. 2-Butyloxyethanol 
40. 2-(2-Butyloxyethyloxy)ethanol 
41. Tripotassium hexacyanoferrate-III 
42. Tetrapotassium hexacyanoferrate-II 
43. Dipotassium tetracyanonickelate-II 
44. Trisodium octacyanomolybdate-V 
45. Disodium pentacyanonitrosylferrate-II 
46. Tripotassium pentacyanonitrosylmanganate-I 
47. Tripotassium pentacyanonitrosylchromate-I 
48. Tripotassium pentacyanonitrosylcobaltate-I 
49. Pentapotassium pentacyanonitrosylvanadate-I 
All the compounds used as activators according to the present invention are 
familiar. 
The activators of general formula V are used in concentrations of 0.5-10%, 
preferably 1-5% (w/v), and the activators of general formula VI in 
concentrations of 10.sup.-4 to 1 mol/liter, preferably 10.sup.-3 to 
10.sup.-1 mol/liter of impregnating solution. 
For the production of the agent according to the present invention, for 
example, absorbent carriers, preferably filter paper, cellulose, or 
synthetic fiber fleeces, are impregnated with solutions of the necessary 
reagents usually used for the production of test strips (substrate, 
buffer, if necessary a wetting agent, an oxidizing agent, etc.) in readily 
volatile solvents, such as, for example, water, methanol, ethanol, or 
acetone. This is expediently done in two separate steps: First, 
impregnation is carried out with an aqueous solution containing the buffer 
and other water-soluble additives. After that, impregnation is carried out 
with a solution of the protease substrate of general formula I. In special 
cases, the impregnation sequence can also be reversed. 
The finished test papers may be used as such, or stuck on to handles, in a 
familiar manner, or expediently sealed between plastics and fine-meshed 
networks according to Federal Republic of Germany Pat. No. 21 18 455. 
For the production of film-coated test strips, all the reagents are 
introduced into a solution or dispersion of a film-forming substance, such 
as, for example, a polyvinyl ester or a polyamide, and homogeneously 
mixed. The mixture is spread in a thin layer on a plastic carrier and 
dried. After drying, the film-coated test strips according to the present 
invention are cut up, and may be used as such, or stuck on to handles, in 
a familiar manner, or, for example, sealed between plastics and 
fine-meshed networks according to Federal Republic of Germany Pat. No. 21 
18 455. 
The diagnostic agent, according to the present invention, for the detection 
of proteolytic enzymes, especially of leukocyte proteases, in the form of 
powder mixtures or reagent tablets, can be produced by treating and 
granulating the abovementioned components of the test with ordinary 
galenical additives. Additives of this kind are, for example, 
carbohydrates, such as, mono, oligo, or polysaccharides, or sugar 
alcohols, such as, mannitol, sorbitol, or xylitol, or other soluble inert 
compounds, such as polyethylene glycols or polyvinylpyrrolidone. In 
general, the powder mixtures or reagent tablets have a final weight of 
about 50-200 mg., preferably 50-80 mg. 
For the production of lyophilizates, with a total weight, in each case, of 
about 5-20 mg, preferably of about 10 mg, a solution is freeze-dried 
which, along with all the reagents needed for the test, contains ordinary 
structure builders, such as, for example, polyvinylpyrrolidone, and 
possibly additional filling materials, such as, for example, mannitol, 
sorbitol, or xylitol. 
The diagnostic agent according to the present invention, in the form of a 
solution, preferably contains all of the reagents needed for the test. As 
solvents, water or mixtures of water with a water-soluble organic solvent, 
such as, for example, methanol, ethanol, acetone, or dimethylformamide 
come under consideration. It may be advantageous, for reasons of storage 
stability, to divide the reagents needed for the test into two or more 
solutions which are not mixed together until the time of the actual 
investigation. 
The diagnostic agents produced in this way make it possible, after dipping 
them into the body fluid to be investigated or after adding them to the 
body fluid in question, to detect, rapidly and simply, the presence of 
proteolytic enzymes by means of color formation, which can be evaluated 
visually or photometrically, for example, by reflectance photometry or in 
a cuvette. Since the activity of the leukocyte proteases per cell may be 
regarded as an essentially constant magnitude, the leukocyte concentration 
of the body fluid under investigation can be determined from the intensity 
of the color formation. At the same time, with the diagnostic agent 
according to the present invention, both intact and lyzed leukocytes are 
detected, since the activity of the leukocyte proteases is fully 
maintained even after the lysis of the leukocytes. Consequently, an error 
due to lysis does not occur.