JPS5833223B2 - Novel dipeptide derivatives and their salts - Google Patents

Description

[Detailed description of the invention] The present invention relates to novel dipeptide derivatives and salts thereof.

The relationship between the activities of various enzymes in the living body and diseases has been studied for some time, and the correlation between the activities of certain enzymes and diseases has been known and is widely used for diagnosis.

For example, activities such as leucine aminopeptidase activity, glutamate oxaloacetate transaminase, glutamate transaminase, and lactate dehydrogenase have been measured to distinguish between obstructive yellow scars and parenchymal yellow scars.

The activities of these enzymes are usually increased in patients suffering from certain diseases compared to normal people, and therefore accurate diagnosis cannot be made unless combined with other diagnostic methods such as measuring various enzyme activities. There wasn't.

Therefore, the present inventors have been conducting intensive research on enzymes and their activities in body fluids, which have the potential to more reliably, safely, and easily diagnose various diseases. -We synthesized a dipeptide derivative represented by proline p-nitroanilide (where X is an amino acid residue) and its salt, and first investigated the presence or absence of an enzyme that acts on the dipeptide derivative in the body. It has been discovered that there exists an enzyme, X-prolyl dipeptidyl aminopeptidase, which is a type of dipeptidyl aminopeptase that acts on the novel dipeptide derivative and decomposes it into X-L-florin and p-nitroaniline.

Next, the present inventors measured the enzyme activity in body fluids, particularly in serum, using the above-mentioned XL proline p-nitroanilide or its salt as a substrate, and found that it was normal and in patients suffering from various diseases. In humans, although there are some significant differences between men and women, especially between men and women in the young age group, the difference is almost constant. It has been found that the activity of the enzyme is increased in cancer, etc., and more surprisingly, the activity is decreased in gastrointestinal cancer, such as gastric cancer, pancreatic cancer, etc., and the present invention has been made. Measuring the activity of this enzyme using novel dipeptide derivatives and their salts as substrates will greatly contribute to disease diagnosis, especially gastrointestinal cancer diagnosis.In particular, it will be extremely effective in diagnosing liver metastasis of gastric cancer, for which there is no conventional diagnostic method. method, and recognized that this dipeptide derivative and its salts are effective as diagnostic agents.

Furthermore, the present inventors believe that the new substance XL-proline p-nitroanilide or its salt is a safe substance that does not cause problems such as carcinogenicity as a substrate for measuring this enzyme;
We also confirmed that it satisfies all the essential conditions for a substrate, including ease of measurement of the amount of substrate subjected to enzyme action, stability, linearity with respect to enzyme amount and contact reaction time, etc. The inventors have now completed the present invention, which is a novel dipeptide derivative and a salt thereof, which have important significance, and a diagnostic agent comprising the same.

The X constituting the new substance XL-proline p-nitroanilide according to the present invention may be any amino acid residue, including various amino acids widely found in proteins,
It is usually an α-amino acid residue having 15 or fewer carbon atoms,
It does not matter whether the body is D or DL.

For example, neutral amino acids such as glycine, alanine, valine, leucine, serine, threonine, phenylalanine, tyrosine, and driftfan; acidic amino acids such as aspartic acid and glutamic acid; and basic amino acids such as lysine and arginine. Even groups can be used.

Among them, glycine, alanine, aspartic acid, glutamic acid, lysine and arginine are more preferred.

In addition, the dipeptide derivative as a substrate can be used not only as a free form, but also in the form of a salt with an acid that does not inhibit the enzyme reaction, such as a salt with p-toluenesulfonic acid, hydrochloric acid, hydrobromic acid, etc., in the measurement system. It can also be introduced into

Among these various substrates, dipeptide derivatives in which X is a neutral amino acid residue and dipeptide derivatives in which X is a basic amino acid residue are the most susceptible to hydrolysis by this enzyme; Certain dipeptide derivatives are relatively resistant to degradation.

Therefore, dipeptide derivatives in which X is a basic amino acid residue or salts with various acids that do not inhibit the enzyme reaction, such as tosylate (p-)luenesulfonate), are effective as substrates, that is, as diagnostic agents. However, it has the disadvantage of being hygroscopic.

On the other hand, among dipeptide derivatives in which X is a neutral amino acid residue, glycyl-L-proline p-nitroanilide is hydrolyzed by this enzyme at an optimum pH of 8.7.
Contrary to the case of H7,0, X is larger than dipeptide derivatives in which X is a basic amino acid residue, and in particular, its various salts, such as hydrochloride and tosylate, are not hygroscopic and easy to handle, so this derivative or Salts thereof are preferred, especially the tosylate of glycyl-L-proline p-nitroanilide.

The novel substance XL-proline p-nitroanilide according to the present invention can be synthesized by methods well known in peptide chemistry.

That is, it is obtained by deprotecting N-m-protected-X-(,-proline p-nitroanilide).

Specifically, L-proline p-nitroanilide and the N-protected amino acid responsible for the N-protected N-protected-X-L prolinto-nitroanilide may be synthesized by the most advantageous method depending on the type of amino acid used, such as active esterification of the carboxyl group of the amino acid.

In this case, the N-protecting group may be appropriately selected from known protecting groups depending on the amino acid used, such as be/zyloxycarbonyl group, t-butyloxycarbonyl group, t-
An amyloxycarbonyl group or the like is used.

Furthermore, when the amino acid used has a side chain functional group, it is preferably protected in advance by a conventional method before being subjected to the reaction.

For example, in the case of acidic amino acids, the side chain carboxyl group is converted into an ester such as benzyl ester.

Also, commonly used protective groups for the guanidino group of arginine, such as evatosyl group, nitro group, p-nitrobenzyloxycarbonyl group, 2-(isolopyloxycarbonyl)
It is preferable to protect with a 3,4,5,6-titrachlorobenzoyl group, etc., especially the former two, and the Nε-amino group of lysine is preferably protected with an ordinary amino protecting group, preferably Nα- It may be used by protecting with the same protecting group as the amino protecting group.

The thus obtained N-protected-XL-proline p-nitroanilide is then deprotected, and the method for removing the protecting group is based on the presence of the N-protecting group and the side chain functional group. In this case, it may be carried out by appropriately selecting and combining known methods depending on the type of the protecting group.

At this time, simultaneous removal or change of other constituents such as p-nitroaniline residues is not allowed, and therefore it is important to select a method that selectively removes only the protecting group.

On the other hand, after first synthesizing N-protected-X-L-proline in the same manner, it was subjected to a condensation reaction with p-nitroaniline.
The desired dipeptide derivative can also be obtained by obtaining XL-proline p-nitroanilide and then deprotecting it as described above.

Enzyme activity can be measured without particular difficulty in diagnosing the presence or absence of a disease using the dipeptide derivative according to the present invention.

That is, first of all, a substrate solution is prepared, and as mentioned above, the novel dipeptide derivative XL-proline p-
two).

Prepare a solution of an appropriate concentration using anilide or its salt.

Since the present dipeptide derivatives have low solubility in water, it is advantageous to use suitable solubility-promoting substances, such as surfactants.

The pH of this substrate solution varies depending on the type of substrate used, but the optimum value can be easily determined experimentally by taking into consideration the rate of hydrolysis by this enzyme and the decomposition of the substrate by factors other than the enzyme (i.e., substrate stability). be able to.

Usually, the pH is selected to be around neutrality, more specifically around 6 to 9.

For measurement, the substrate solution and a sample containing X-prolyl dipeptidyl aminopeptidase, such as body fluid such as serum, are mixed and reacted at 30°C to 45°C for an appropriate time depending on the amount of substrate and enzyme, and then the enzyme activity is determined. The activity of this enzyme can be determined by inactivating it by a conventional method and quantifying free p-nitroaniline.

As is well known, p-nitroaniline is a compound with a pale yellow color, so it can be quantified with high accuracy by directly determining the absorbance of the reaction solution at 385 nm, which is the simplest method.

Of course, it is also possible to react with excess sodium nitrite under acidic conditions and quantify it more precisely as a diazo compound.

In this case, even if unreacted nitrite is quantified, the target free p-
The amount of nitroaniline can be calculated, and unreacted nitrous acid can be decomposed using sulfamino acid, ammonium sulfaminate, urea, etc., and then a coupling component such as naphthylethylenediamine can be reacted to form an azo compound, which can be converted into a product. It can also be quantified by the so-called azo-coupling method, which quantifies by measuring absorbance at a suitable wavelength.

In any case, this direct method is usually sufficient because the desired enzyme activity can be determined with high accuracy simply by directly measuring the absorbance of the enzyme reaction solution.

As is clear from the above explanation, the present invention provides useful** novel dipeptide derivatives and salts thereof, and can greatly contribute to medical diagnosis, especially gastrointestinal cancer diagnosis, in a safe and easy-to-operate manner. there is.

Hereinafter, the present invention will be explained in more detail with reference to Examples and Reference Examples.

Example 1 (N'-benzyloxycarbonyl-L-proline p-
Production of nitroanilide) N-benzyloxycarbonyl-L-florin (74
, 8fI, 0.3 mol) and p-nitroaniline (4
1,4F, 0.3 mol) in tetrahydrofuran (400
ml) and cooled to -10°C with stirring, then 20 ml of phosphorus oxychloride (50.6f, 0.33 mol)
It was dripped in minutes.

Subsequently, triethylamine (
92.4ml.

0.66 mol) was added dropwise over 40 minutes.

After completing the dropwise addition, add triethylamine to the reaction mixture,
The pH was maintained at 7-8 and stirred for an additional 3 hours at room temperature.

The reaction mixture was concentrated under reduced pressure to remove tetrahydrofuran, and the residue was dissolved in ethyl acetate (1,21%) and water (400%
r110, IN-hydrochloric acid (300ml x S times), water (4o
omz), 5% sodium bicarbonate aqueous solution (3007/
dX (3 times) and saturated saline (300rrLl), and then dried by adding magnesium sulfate.

After removing the desiccant, the residue was concentrated under reduced pressure and ethyl ether was added to obtain crude crystals (82ff).

This crude crystal is made of ethyl acetate (300 m0) and methanol (5 m0).
When recrystallized from a mixed solvent of 561
A pure product was obtained.

m, p, 159-161℃, Ca)'B'' -107,
6° (C=1.0, methanol), and the main island was subjected to thin layer chromatography (CHC13:CH30H:CH3CO
0H=95:5:3), a 100γ single spot was given.

Elemental analysis example (manufacture of L-proline p-nitroanilide hydrobromide) 26% hydrogen bromide acetic acid solution in N-benzyloxycarbonyl-L-proline p-nitroanilide (55,4P, 0.15 mol) (200 TLl) was added under cooling and stirred for 5 minutes.

Thereafter, after stirring at room temperature for 120 minutes, the reaction mixture was poured into dry ethyl ether (21) to precipitate crystals.

The solvent was removed by decantation, new ethyl ether was added, the crystals were collected by filtration, and thoroughly washed with ethyl ether.

Yield: 47 grams (quantitative). Example 3 (Production of N-benzyloxycarbonylglucyl-L-proline p-nitroanilide) L-proline p-nitroanilide hydrobromide (47
f? , 0.15 mol) in dimethylformamide (100
The mixture was dissolved in rrLO, cooled to 0° C. with stirring, and neutralized by adding triethylamine (22 ml).

Next, N-hydroxybenzotriazole (1f?)
and N-benzyloxycarbonylglycine droxysuccinimide ester (48f, 0.16 mol) were dissolved and stirred for 1 hour.

During stirring, triethylamine was added to adjust the pH of the reaction mixture to around 8.

Thereafter, the reaction was further stirred at room temperature for 19 hours, and then concentrated to dryness under reduced pressure.

Water (350ml) was added to the oily residue, and ethyl acetate (50ml) was added to the oily residue.
0TLl, 200TLl), and the extract was extracted with water (2001rLO1IN-hydrochloric acid (200TLl×2 times).
, water (2001'to, 5% hydrogen carbonate), IJum aqueous solution (200ml x 4 times), and water (200TLl) in this order.

After drying with magnesium sulfate, it was concentrated to dryness under reduced pressure.
- Hexane was added to obtain an amorphous powder.

Yield 67f? .

In addition, the main island has thin layer chromatography (CuCl2:C
H30H: CH3CO0H=95:5:3)
A 100γ single spot was given at .

※※Example 4 (Production of Chrysyl-L-proline p-nitroanilide) N-benzyloxycarbonylglycyl-L-proline p-nitroanilide (61P) was cooled with 26% hydrogen bromide acetic acid solution (250rILl). Add a few drops and stir for 5 minutes,
After stirring at room temperature for 2 hours, dry ethyl ether (
1.5 J) to precipitate crystals.

After exchanging ethyl ether twice by decantation, the crystals were collected by filtration, thoroughly washed with ethyl ether, and then dried under reduced pressure in a desiccator containing caustic soda.

Yield of glycyl-L-proline p-nitroanilide hydrobromide: 591° The above hydrobromide (20F) was dissolved in IM-sodium carbonate aqueous solution (i'o'oTLl), salt was added, and chloroform (100TLlX8 extraction).

The extract was washed with saturated brine (ioml), dried over magnesium sulfate, concentrated to dryness under reduced pressure, and crystallized from ethyl acetate-ethyl ether.

Yield 12.7? .

m, p, 118-120℃, @)b'-115,8゜(
C=1.0, methanol) ε-11600 (0, IN hydrochloric acid) Example 5 (Production of glycyl-L-proline p-nitroanilide tosylate) Crysyl-I, -7'loline p-nitro Anilide (58
47Q) and p-) luenesulfonic acid monoaqueous phase (380
After dissolving 9) in ethanol (lOmJ), the solution was left in a cold place to precipitate crystals.

The obtained crude crystals were recrystallized from ethanol-ethyl ether.

Yield 31211I9゜, p, 223-225℃ (decomposition), 0] is -81,0゜ (C = 1.0, methanol) Elemental analysis example 6 (t Butyloxycarbonylabutyl L-proline p-nitro Production of anilide) L-proline p-nitroanilide hydrobromide (9, Fl, 0.03
mol) was suspended in chloroform (40 mO), and neutralized by adding triacylamine (4.2 mO) while stirring while cooling.

Then, t-butyl ox'/force 11y ho = ru L-'y
Rani 7 (5,7F, 0.03 mol) was added, and a chloroform solution (5 mA) of dicyclohexycarbodiimide (6,2r, 0.03 mol) was added dropwise.

After the reaction was stirred at room temperature for 44 hours, a few drops of acetic acid was added and the mixture was further stirred for 2 hours. Insoluble dicyclohexylurea was removed by filtration, and the filtrate was concentrated under reduced pressure.

After adding water, extraction was performed with ethyl acetate, and the organic layer was washed successively with IN hydrochloric acid, water, 5% aqueous sodium bicarbonate solution, water, and saturated brine, and dried over sodium sulfate.

The mixture was concentrated to dryness under reduced pressure, and the residual oil was dissolved in ethyl ether and allowed to stand.

After removing a trace amount of insoluble matter, the mixture was again concentrated to dryness under reduced pressure, and n-hexane was added to obtain a powdery solid.

Yield 12.9fo ※※ Main island is Example 1
A single spot was obtained using thin layer chromatography as described above.

Example 7 (Production of L-alanyl-L-7'loline p-nitroanilide hydrochloride) t-7'Tyloxycarbonyl-L-alanyl-L-
Proline p-nitroanilide (12,2f) was dissolved in dioxane (20m0) and 6N-hydrochloric acid in dioxane solution (4
After stirring for 55 minutes, the mixture was concentrated to dryness under reduced pressure.

Dry ethyl ether was added to the residual oil, and the resulting powder solid was collected by filtration.

The powdered solid was dissolved in ethanol, crystallized by rubbing with a glass rod, and then recrystallized from hot ethanol.

Yield 6. C.I. m, p, 130-140℃, (cx)p
-103,4° (C=1.0, methanol) Elemental analysis example 8 (Production of t-4 t-4 tyloxycarbonyl-β-benzyl-L-aspartyl-L-7'loline p-nitroanilide) L-proline p-nitroanilide hydrobromide (9,
5f, 0.03 mol) and t-butyloxycarbonyl-β-benzyl-L-aspartic acid (9,7?, 0
．． 03 mol) in the same manner as in Example 6.

The crude product was purified by silica gel column chromatography (solvent system: chloroform:ethyl acetate=5:l).

Yield 10.3P.

Moreover, a single spot was obtained for the main island by thin layer chromatography similar to that in Example 1.

Example 9 (Manufacture of L-aspartyl-L-proline p-nitroanilide) *t-7"t-7" thyloxycarbonyl-β-benzyl-L-asharathyl-L-florin p-nitroanilide (to, 3ff)
Add anisole (8m) to it, add anhydrous hydrogen fluoride (approx. 50m)
The sample was treated at 0°C for 1 hour.

Hydrogen fluoride was distilled off under reduced pressure, and ethyl ether was added to the residue to form a precipitate, which was washed twice by decantation and then dried to obtain a powdery solid.

This crude product was dissolved in 0.1M acetic acid, washed with ethyl ether, and then dissolved in a strongly basic ion exchange resin (Amberl).
ite I R-400, acetate type, 200771
The effluent was concentrated to dryness under reduced pressure and crystallized.

A small amount of water was added and the crystals were collected by filtration and washed.

Yield 5.2'? . m, p, 144-145°C (decomposition),
■More 2100, 3° (C=1.0, N hydrochloric acid) ε幇翳-12t7o (0, IN hydrochloric acid) Elemental analysis example 10 (t-)-y-yloxycarbonyl-γ-benzyl L-
Production of glutamyl-L-proline p-nitroanilide) L-proline p-nitroanilide hydrobromide (9,5
f, 0.03 mol) and t-butyloxycarbonyl-
r-benzyl-L-J' rutamic acid fudicyclohexylamine salt 17. l? , 0.033 mol) in the same manner as in Example 6.

The crude product was purified by silica gel column chromatography (solvent system: chloroform:acetic acid=1:1).

Yield 12.7f'. The main island was subjected to thin layer chromatography similar to Example 1, giving a single spot.

Example 11 *
* (Production of L-glutamyl-L-7'loline p-nitroanilide) t-7' thyloxycarbonyl-γ-benzyl L-glutamyl-L-proline p-nitroanilide (ion, 0
．． 018 mol) was treated with 501 rLl of hydrogen fluoride for 1 hour while cooling with water.

After distilling off unreacted hydrogen fluoride under reduced pressure, the residue was washed with ether and treated with a strongly basic ion exchange resin (Amberlite).
It was passed through an IR-400 acetate type 15 QmA filter and eluted with a 0.1M acetic acid aqueous solution.

The effluent was concentrated to dryness under reduced pressure, and ethanol was added to the residue to isolate crystals.

Yield 4.7f? . m, p, 117-126°C (decomposition),
(. p-nitroanilide hydrobromide (9,5
? , 0.03 mo/l/) Oyohi Nct-N'-shihenzyloxycarbonyl-L-lysine (12,4f?
0.03 mol) in the same manner as in Example 6.

Yield 15.8f. The main island was subjected to thin layer chromatography as in Example 1 to give a single spot.

Example 13 (Production of L-IJ sil-L-proline p-nitroanilide cytosylate) Nα·Nε-shihenzyloxycarbonyl-L-lysyl-L-proline p-nitroanilide** (9,5f ,0
．． 015 mol) was dissolved in a 26% hydrogen bromide acetic acid solution (35 rft
was treated in the same manner as in Example 4, and L-lysyl-L
-7'loline p-nitroanilide dihydrobromide was obtained.

Yield: 7.8 ri. The hydrobromide salt was then passed through a strongly basic ion exchange resin (Amberlite I R-400, acetate type, 2001rLl) and eluted with 0.1 M acetic acid aqueous solution.

The effluent was concentrated to dryness under reduced pressure, and 1007 d of ethanol was added to the residue to dissolve it, followed by further dissolving an aqueous phase of p-)luenesulfonic acid (5,5y, 0.029 mol).

The mixture was concentrated to dryness under reduced pressure, and ether was added to the residue to isolate crystals.

Yield 10.1S'. m, p, 92-130°C (decomposition),
Phrase 5' = -32,6° (C = 1.1, water) Elemental analysis example 4 (Manufacture of Nα benzyloxycarbonyl G tosyl-L-arginyl-L-proline p-nitroanilide) Nα-benzyloxycarbonyl-NG -Tosyl-L-
Arginine cyclohexylamine salt (23,05',
0.041 mo#) Free N-benzyloxycarbonyl-NG-tosyl-L-arginine was derived, and 601 rL of chloroform was added and dissolved, followed by L-proline p-nitroanilide hydrobromide (12, Of, 0.041 mo#). 038 mol) was added.

While cooling, N-ethyl-N'-dimethylaminopropylcarpodiimide (6,8 TL, 110.038 mol) was added dropwise, stirred for 1 hour, and further stirred at room temperature for 17 hours.

The reaction solution was concentrated under reduced pressure, water was added to the residue, and the mixture was extracted with ethyl acetate.

The extract was washed successively with 1N hydrochloric acid, water, 5% aqueous sodium bicarbonate solution, water and saturated brine, and dried over sodium sulfate.

Then, the mixture was concentrated to dryness under reduced pressure, and ether was added to obtain a powdery solid.

Yield 22.3S'. A single spot of the crystal was obtained by thin layer chromatography as in Example 1.

Example 15 (Production of L-arginyl-L-7'' loline p-nitroanilide cytosylate) Nα-benzyloxycarbonyl-NG-tosyl-L-
Fulginyl-L-florin p-nitroanilide (8,2
y, 0.012 mol) with 30 ml of hydrogen fluoride under water cooling.
Time processed.

After distilling off unreacted hydrogen fluoride under reduced pressure and dissolving the residue in water,
Extracted with ether.

The aqueous layer was treated with a strongly basic ion exchange resin (Amberlite).
IR-400, acetate type, passed through 180 m0, 0
．． Eluted with 1M aqueous acetic acid.

The effluent contains p-toluenesulfonic acid l aqueous phase (4,6y,
After adding and dissolving 0.024 mol), the mixture was concentrated to dryness under reduced pressure.

Ether was added to the residue to obtain a powdered solid, which was recrystallized from hot water.

Yield 7.3? . m, p, 125-138℃ (decomposition), (
==E) b5-31.4° (C=1.0, water) Elemental analysis reference example 1 Glycyl-L-7'' loline p-nitroanilide tosylate in a 2% surfactant-containing aqueous solution (Nitukol) NP-1
01 Nikko Chemicals ■) to 3 mmol (rrtM,
The substrate solution was dissolved to give a concentration of rIlmol/73) and used as a substrate solution.

The following operations were performed in sequence using human serum as the enzyme solution, and then similar operations were performed while changing the incubation time.

The relationship between the reaction time and the amount of p-nitroaniline produced was determined.

The p-nitroaniline (nmol) produced by the enzyme through the above operations was determined by the formula, where E, C1, and S are the respective absorbances.

The results are shown in the table below. As is clear from the above results, it was confirmed that the present substrate was affected by the present enzyme in a linear relationship with the reaction time.

Reference Example 2 A substrate solution was prepared in the same manner as in Reference Example 1 using glycyl-L-proline p-nitroanilide tosylate.

Using human serum as the enzyme solution, the following operations using the azo coupling method were carried out in sequence, and then the same operations were carried out with increasing amounts of the enzyme solution.

The p-nitroaniline (n m
ol) is the absorbance of E, C and S, respectively.
It was determined by the formula.

The results are shown in the table below. *As is clear from the above results,
It was confirmed that this substrate was degraded in proportion to the amount of enzyme used.

Reference Example 3 A substrate solution with a concentration of 3 mmol was prepared in the same manner as in Reference Example 1 using various dipeptide derivatives and their salts.

In the method of Reference Example 1, using 0.511'Ll of 0.15 M) lis maleate buffer (pH = 7.0) in place of the glycine/sodium hydroxide buffer, the amount of p-nitroaniline produced was determined. , the activity of each substrate for the enzyme was calculated based on glycyl-L-proline p-nitroanilide.

However, cytosylate salts were used as substrates for lysine and arginine derivatives, tosylate salts were used for glycine derivatives, and hydrochloride salts were used for alanine derivatives.

Next, in the same manner, the optimum pH of the substrate was adjusted using Tris-maleic acid buffer for pH = 5.6 to 8.8 and glycine/sodium hydroxide buffer for pH = 8.2 to 9.4. was determined, and the rate constant km was determined at pH 7.0 by varying the substrate concentration.

At pH 8.7, L-proline p It showed high activity.

Reference Example 4 Compared to pH 7.0, glycyl-nitroanilide is 1.53 times higher. In the same manner as Reference Example 1, p-nitroaniline produced in 88 cases of normal human serum was determined, and the enzyme activity (I, U, /
1 rnl serum, ie μmol/mm-m0, was determined.

The results are shown in the table below. As is clear from the above results, although there are some significant differences between men and women, especially in young people (under 40 years old), it was confirmed that the activity of this enzyme in the serum of normal humans remains constant. It was done.

Example 16 **Using glycyl-L-proline p-nitroanilide tosylate as a diagnostic agent, three serum samples from hepatitis patients and three serum samples from gastric cancer patients were treated in the same manner as in Reference Example 1. Enzyme activity was determined.

The results are shown in the table below.

Example 17 Enzyme activities in serum from hepatitis patients and gastric cancer patients were determined using the various diagnostic agents listed in the following table and in the same manner as in Reference Example 1.

The results are shown in the table below. From the above results, it was confirmed that the dipeptide derivative and its salt according to the present invention are effective as a diagnostic agent, particularly as a diagnostic agent for liver and adjacent organ diseases and fire extinguisher cancer.

Claims (1)

[Claims] I X-L-proline p-nitroanilide (X is glycine, alanine, aspartic acid, glutamic acid,
A novel dipeptide derivative represented by an amino acid residue selected from the group consisting of lysine and arginine, and a salt thereof.