Compound, diagnostic marker for gastric cancer, method for detecting enzyme activity, method for diagnosing gastric cancer, kit containing the compound, use of the compound, and method for treating gastric cancer

Abstract

The present invention relates to a novel compound, a diagnostic marker, for use in medical treatment, more particularly in cancer diagnosis, particularly in the diagnosis of gastric cancer. The present invention also relates to an in vitro method for detecting enzyme activity present in a subject's body fluid, particularly derived from gastric cancer cells, using the compound. The present invention further relates to an in vitro method for diagnosing gastric cancer using the compound, a kit containing the compound, and the use of the compound for detecting enzyme activity specific to gastric cancer, as well as the use of the compound for diagnosing gastric cancer. The present invention also relates to the compound for use as a diagnostic marker for gastric cancer, and to a method for treating gastric cancer, including the procedure of carrying out the above-mentioned gastric cancer diagnostic method using the compound.

Description

[Technical Field]

[0001] The present invention relates to a novel compound and diagnostic marker for use in medical treatment, more particularly in cancer diagnosis, particularly in the diagnosis of gastric cancer. The present invention also relates to an in vitro detection method for detecting enzyme activity present in a subject's body fluid, particularly derived from gastric cancer cells, using the compound, an in vitro method for diagnosing gastric cancer using the compound, a kit containing the compound, the use of the compound for detecting enzyme activity specific to gastric cancer, the use of the compound for diagnosing gastric cancer, and the compound for use as a diagnostic marker for gastric cancer. The present invention also relates to a method for treating gastric cancer, comprising the procedure of carrying out the above-mentioned method for diagnosing gastric cancer. [Background technology]

[0002] Gastric cancer is the fifth most common cancer worldwide, with over 1.4 million new cases reported in 2020. Environmental conditions, including a diet consisting of artificially preserved, salted, and smoked foods, as well as foods containing nitrosamines, significantly contribute to an increased risk of gastric cancer. As with most cancers, smoking and excessive alcohol consumption, especially spirits, can increase the risk of developing the disease. Substances contained in tobacco smoke disrupt the peristaltic control mechanism through direct irritation of the gastric musculature and submucosal nerve plexus. This can result in reflux of duodenal contents into the stomach, gastric mucositis, and bile duct inflammation, which can influence the formation of neoplastic changes in glandular cells, ranging from inflammatory mucosal atrophy to metaplasia, dysplasia, and malignant transformation. In addition, infections, including bacterial infections, have received increasing attention. Helicobacter pylori infection is a risk factor for intestinal gastric cancer.

[0003] Symptoms caused by gastric cancer are often non-specific and may resemble those seen in other diseases, such as mucositis or peptic ulcer disease. For this reason, warning signs of the disease are often underestimated by both patients and physicians. Early symptoms of gastric cancer are nonspecific and include abdominal pain, weight loss, vomiting, loss of appetite, early satiety, frequent belching, dysphagia, weakness, and dyspepsia. Physical examination usually reveals abnormalities very slowly. Signs of advanced gastric cancer include a tumor palpable through the abdominal wall, ascites, enlarged lymph nodes in the left supraclavicular fossa (Virchow's), hepatomegaly, jaundice, pleural effusion, and palpable infiltrates rectally. The presence or complaint of any of these symptoms usually indicates that gastric cancer is already in an advanced stage, where curative treatment is no longer possible. A method that allows early detection of gastric cancer and differentiation from precancerous lesions is gastroscopy, which allows the collection of specimens for histopathological examination. Endoscopy for gastric cancer has a sensitivity of over 90%. Ultrasound and x-rays of the stomach using contrast agents are also used to diagnose stomach cancer.

[0004] CEA, CA 19-9, and CA 72-4 are the main cancer markers used in the diagnosis of gastric cancer. CEA is a nonspecific marker. Elevated levels are found in patients with colon and rectal cancer, gastric cancer, pancreatic cancer, cervical cancer, and lung cancer. In contrast, the glycoprotein antigen CA 19-9 is a marker released by proliferating cells of pancreatic, gastric, and colon cancer. The diagnostic utility of CEA and CA 19-9 is limited by their low sensitivity in gastric cancer, meaning that normal levels cannot exclude the presence of this cancer.

[0005] Although attempts have been made to use CA 72-4 as a predictive marker, its use in diagnostic tests for the detection of, including early-stage, gastric cancer is not recommended due to the low selectivity of this test. The diagnostic use of CA 72-4 together with other markers, namely CEA and CA 19-9 measured in gastric cancer patients, allows for very accurate monitoring and correct prognosis after surgery performed as a curative operation.

[0006] The processes of cancer cell initiation, proliferation, and dissemination are known to involve numerous factors, including numerous enzymes, particularly hydrolases, especially proteolytic enzymes. These enzymes catalyze the enzymatic (hydrolytic or proteolytic) cleavage of proteins and peptides into smaller fragments. This process allows cancer cells to colonize new tissues and expand by enhancing the process of blood vessel formation (angiogenesis), which allows for effective nutrient delivery to tumors. Furthermore, these enzymes exist as a result of the death of healthy cells during the tumor growth process. All of these processes contribute to the distinctive and specific profile of enzymatic (proteolytic) activity of cancer cells, which is characteristic of tumors.

[0007] Chromogenic peptide molecules are known in the art that change or increase the color of a test solution upon enzymatic degradation into smaller fragments, resulting in the release of a chromophore (e.g., 4-nitroanilide or 2-aminobenzoic acid) from the chromogenic peptide molecule.

[0008] Chromogenic molecules of this type and their uses are known, for example, from the publications of Erlanger BF, Kokowsky N, Cohen W., "The preparation and properties of two new chromogenic substrates of trypsin", Arch Biochem Biophys., November 1961; 95: 271-8, and Hojo K, Maeda M, Iguchi S, Smith T, Okamoto H, Kawasaki K., Amino acids and peptides. XXXV. "Facile preparation of p-nitroanilide analogs by the solid-phase method", Chem Pharm Bull (Tokyo), November 2000; 48(11): 1740-4.

[0009] However, the use of this class of compounds for the diagnosis of gastric cancer has not been reported to date. A method for obtaining chromogenic peptides is also known in the prior art, which involves coupling the individual components under appropriate time and stoichiometric conditions. The coupling method consists of successive steps: coupling the individual components (amino acid derivatives), washing the residues, sequentially removing the protecting groups, and washing again. This cycle is repeated for each amino acid residue. The resulting peptide is separated from the resin by reaction under acidic conditions. The solution is then separated from the resin in a filtration step, after which the peptide is precipitated from the solution using a non-polar solvent.

[0010] However, no chromogenic peptide compounds suitable for specific and early diagnosis of gastric cancer or methods for obtaining them are known in the prior art. Therefore, there is an urgent need in the art for "cancer markers" for gastric cancer that enable early, sensitive, and specific diagnosis of gastric cancer in a non-invasive and reliable manner, and for diagnostic and therapeutic methods using such diagnostic markers.

[0011] The object of the present invention is to provide novel and specific diagnostic markers for gastric cancer, diagnostic methods using such markers for non-invasive, rapid, highly sensitive, and specific early detection of gastric cancer, which may also be suitable as screening tests, and therapeutic methods using such markers.

[0012] These objects are achieved by the invention as defined in the appended claims, while preferred modifications thereof are defined in the dependent claims. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Erlanger BF, Kokowsky N, Cohen W., “The preparation and properties of two new chromogenic substrates of trypsin”, Arch Biochem Biophys., November 1961; 95:271-8 [Non-patent document 2] Hojo K, Maeda M, Iguchi S, Smith T, Okamoto H, Kawasaki K. Amino acids and peptides. XXXV. “Facile preparation of p-nitroanilide analogs by the solid-phase method”, Chem Pharm Bull (Tokyo), November 2000; 48(11):1740-4 Summary of the Invention

[0014] The present invention relates to a compound of formula 1: X1 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -X2 6 (Formula 1) The present invention provides a compound having the formula:

[0015] wherein X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, The compound undergoes enzymatic cleavage into fragments X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2), generating a measurable light signal due to the spatial separation of molecules C1 and C2.

[0016] The compounds according to the invention preferably undergo hydrolytic cleavage, more preferably proteolytic cleavage. Preferably, in the compound according to the invention, the pair of molecules C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO2), more preferably the pair of C1 and C2 is (ABZ) / pNA or ABZ / ANB-NH2.

[0017] Preferably, the compound according to the present invention is a compound having the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

[0018] More preferably, the compounds according to the invention undergo hydrolytic cleavage to produce the following fragment 1: ABZ-Gly-Thr-Thr-Gly-OH and fragment 2: ANB-NH2.

[0019] The present invention further provides an in vitro method for detecting enzyme activity, particularly derived from stomach cancer cells, present in a body fluid of a subject, the method comprising: a) A body fluid sample is subjected to a method according to Equation 1: X1 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -X2 6 (Formula 1) contacting the compound having the formula: [wherein X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, the compound undergoes enzymatic cleavage to give fragments X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2), and b) detecting a measurable light signal generated by the spatial separation of molecules C1 and C2; This includes:

[0020] In the method for detecting enzymatic activity according to the present invention, the enzymatic activity is preferably hydrolytic activity, more preferably proteolytic activity. In the method for detecting enzyme activity according to the present invention, the compound preferably has the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

[0021] In the method for detecting enzyme activity according to the present invention, the body fluid used is preferably urine, more preferably human urine. The present invention also relates to an in vitro method for diagnosing gastric cancer, which detects the presence or absence of gastric cancer in a subject by measuring gastric cancer-specific enzyme activity in a body fluid sample of the subject, the absence of said enzyme activity indicating the absence of gastric cancer, while the presence of said enzyme activity indicates the presence of gastric cancer.

[0022] In the method for detecting / diagnosing gastric cancer according to the present invention, the detection of enzyme activity is carried out by the method for detecting enzyme activity defined above. In the method for detecting / diagnosing stomach cancer according to the present invention, the enzyme activity is measured by the method according to Formula 1: X1 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -X2 6 (Formula 1) The reaction is carried out using a compound having the formula:

[0023] In the above formula, X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, The compound undergoes enzymatic cleavage into fragments X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2), generating a measurable light signal due to the spatial separation of molecules C1 and C2.

[0024] In the method for detecting / diagnosing gastric cancer according to the present invention, the body fluid sample is incubated with the compound in an assay buffer, preferably having a neutral or alkaline pH, more preferably a physiological pH, at a sample to assay buffer ratio ranging from 1:2 to 1:10, preferably 1:5.

[0025] In the method for detecting / diagnosing stomach cancer according to the present invention, the compound is preferably used at a concentration of 0.1 to 10 mg / mL, particularly 0.25 to 7.5 mg / mL. In the method for detecting / diagnosing gastric cancer according to the present invention, the compound preferably has the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

[0026] In the method for detecting / diagnosing gastric cancer according to the present invention, the sample used is preferably a urine sample, more preferably human urine. In the method for detecting / diagnosing gastric cancer according to the present invention, measuring the enzyme activity preferably comprises measuring the absorbance intensity within a range of 300 to 500 nm, more preferably within a range of 380 to 430 nm, particularly within a range of 405 nm, for 40 to 60 minutes at a temperature within a range of 25 to 40°C, more preferably within a range of 36 to 38°C.

[0027] The present invention further provides a kit comprising any compound according to the invention as defined above and an assay buffer. In the kit according to the present invention, the compound is preferably a compound having the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA.

[0028] The present invention also provides the use of any compound according to the invention as defined above for detecting an enzyme activity specific for gastric cancer. The present invention also provides the use of any compound according to the invention as defined above for the diagnosis of gastric cancer.

[0029] Preferably, in such uses, diagnosing gastric cancer includes detecting primary gastric cancer, detecting minimal residual disease after surgical resection of gastric cancer, and / or detecting recurrence of gastric cancer. Preferably, the compound in use according to the invention is a compound having the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA.

[0030] The present invention further provides any of the compounds according to the invention as defined above for use as a diagnostic marker for the detection of gastric cancer. Preferably, the compound for use as a diagnostic marker according to the present invention is a compound having the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA.

[0031] The present invention further comprises: a) detecting the presence of gastric cancer-specific enzyme activity in the body fluid sample to be tested by any of the methods defined above; and b) administering to the subject a treatment for gastric cancer if the presence of said enzymatic activity is found in said sample; It also offers treatments for stomach cancer.

[0032] Preferably, in the method of treatment according to the invention, after the end of the treatment according to point b), the activity of said enzyme specific for gastric cancer is monitored at defined time intervals. Preferably, in the treatment method according to the present invention, a urine sample, preferably human urine, is used as the sample.

[0033] Preferably, in the method of treatment according to the present invention, a compound having the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA is used as the compound. [Brief explanation of the drawings]

[0034] [Figure 1] FIG. 1 shows the results of chromatographic analysis of the substrate cleavage, i.e., ABZ-Gly-Thr-Thr-Gly-ANB-NH2, in urine samples from subjects with gastric cancer. [Figure 2] Figure 2 shows the hydrolysis rate of the substrate ABZ-Gly-Thr-Thr-Gly-ANB-NH2 in urine samples from subjects diagnosed with gastric cancer (samples 1–20) and urine collected from healthy subjects (samples 21–40). Arabic numerals indicate the numbers of the selected urine samples. [Figure 3]Figure 3 shows the hydrolysis selectivity of the substrate ABZ1-Gly2-Thr3-Thr4-Gly5-NH2 (i.e., the compound of Formula 2) in a urine sample collected from a subject diagnosed with gastric cancer (Sample 1) and urine samples collected from subjects diagnosed with different neoplastic diseases (cancers) (Samples 2-9). Arabic numerals indicate the individual cancer types. Samples tested for each cancer type were obtained from 20 different patients for each cancer tested. Results are average values ​​for a given cancer type. The results demonstrate selectivity of substrate cleavage in urine from gastric cancer patients compared to urine samples from other cancer patients. [Figure 4] Figure 4 shows the dependence of the hydrolysis level of the substrate ABZ1-Gly2-Thr3-Thr4-Gly5-NH2 on pH conditions. The values ​​shown on the x-axis indicate the pH of the environment. DETAILED DESCRIPTION OF THE INVENTION

[0035] It should be understood that the present invention is defined in the appended claims. This description illustrates various non-limiting aspects and embodiments of the present invention. The present invention is not limited to any particular methodology, protocols, or reagents used to carry it out, unless otherwise specified. The terms and scientific and technical expressions used herein have the meanings commonly known and used by those skilled in the art of the present invention. However, for the sake of clarity, the following expressions / terms and acronyms used in this patent shall be understood as follows:

[0036] Chromogenic compound or molecule means a compound that has chromogenic properties, which refers to the ability of a compound to form a colored product. A fluorescent compound or molecule refers to a compound that has fluorescent properties, which refers to the ability of a compound to form a product that fluoresces.

[0037] NMP represents N-methylpyrrolidone; DMF represents dimethylformamide; DCM represents methylene chloride or dichloromethane; pNA represents 4-nitroaniline or para-nitroaniline; ABZ represents 2-aminobenzoic acid, ANB-NH2 represents the amide of 5-amino-2-nitrobenzoic acid; Boc represents the tert-butyloxycarbonyl group; Fmoc represents the 9-fluorenylmethoxycarbonyl group; and TFA represents trifluoroacetic acid.

[0038] In the context of the present invention, the term gastric cancer should be understood as a primary cancer (malignant neoplasm) of the stomach that arises from tissues within the stomach. The most common type of gastric cancer is endometrial adenocarcinoma (approximately 90%), with the others being undifferentiated carcinoma, adenosquamous carcinoma, and other rarer variants. The term gastric cancer as used herein therefore includes any malignant neoplasm of the stomach that arises from tissues within the stomach.

[0039] In the context of the present invention, the term treatment of gastric cancer shall be understood to mean treatment at an early stage of the progression of the disease, making it possible to significantly extend the survival time of affected individuals and improve their quality of life.

[0040] In the context of the present invention, the term monitoring shall be understood to mean diagnosing the presence of minimal residual disease (MRD), i.e., a small number of cancer cells surviving in the body (during treatment or remission) in amounts that cannot be detected by standard diagnostic methods.

[0041] In the context of the present invention, the term subject is to be understood to mean a human subject or mammal suspected of having gastric cancer or belonging to a group at high risk of gastric cancer, or a human subject or mammal after resection of gastric cancer or after completion of treatment for gastric cancer. The subject is preferably a human subject.

[0042] The compounds of the present invention possess color-producing and fluorescent properties due to the presence of a chromophore, i.e., they contain a fluorescent donor and a fluorescent acceptor molecule. Specifically, their structure was developed so that upon contact with a test body fluid sample from a subject with gastric cancer, an increase in color is observed in the wavelength range of 380 to 440 nm, while no such effect is observed upon reaction with body fluid samples from healthy subjects or subjects diagnosed with another type of cancer. These compounds detect enzyme activity specific to gastric cancer, enabling specific and sensitive diagnosis of gastric cancer, especially at an early stage of its progression. The subject to be tested is preferably a human subject. The body fluid is preferably urine, more preferably human urine.

[0043] In a first aspect of the present invention, there is provided a novel compound having the formula 1: X1 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -X2 6 (Formula 1) wherein X1 is an amino acid derivative or peptide fragment containing molecule C1 or X1 consists of such molecule C1, X2 is an amino acid derivative or peptide fragment containing molecule C2 or X2 consists of such molecule C2, and the pair of molecules C1 and C2 is a fluorescent donor-acceptor pair. Superscripts indicate the position of the subsequent residue in the compound according to the invention and the order of attachment of the residues during synthesis. According to the invention, in this context, chemical formula 1 can alternatively be written without indicating the numbering of the residues. The core of all compounds according to the invention is a tetrapeptide with the four amino acid sequence shown, i.e., Gly-Thr-Thr-Gly (the notation in the three-letter amino acid abbreviation format is equivalent to the notation in the one-letter amino acid abbreviation format: GTTG). This sequence is also shown in the sequence listing as SEQ ID NO: 1.

[0044] The compounds of the present invention undergo enzymatic cleavage into fragments: X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2), generating a measurable optical signal due to the spatial separation of molecules C1 and C2. The measurable optical signal is measured by a method for measuring the change in absorbance / fluorescence after enzymatic cleavage of the compound. Preferably, molecules C1 and C2 are separated from each other by 10 or fewer amino acid residues to ensure efficient quenching of the fluorescent donor by the fluorescent acceptor. It will be apparent to those skilled in the art that the key factor is the distance between the fluorescent donor and acceptor. Therefore, the distance between molecules C1 and C2 may be greater than 10 amino acid residues if the amino acid sequence separating them folds into a twisted or condensed secondary structure, resulting in closer proximity of molecules C1 and C2 compared to the primary structure.

[0045] Due to its chromogenic properties and the presence of a reactive site at position 5 that allows enzymatic (preferably proteolytic) cleavage into smaller fragments, the compound is particularly suitable for use as a diagnostic marker, in particular a specific diagnostic biomarker for gastric cancer, in particular for the early diagnosis of gastric cancer.

[0046] In a preferred embodiment, the compounds according to the invention undergo hydrolytic cleavage, more preferably proteolytic cleavage. In a preferred embodiment, the pair of molecules C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, and ABZ / Tyr(3-NO2), and more preferably, the pair of molecules C1 and C2 is ABZ / pNA or ABZ / ANB-NH2.

[0047] In a preferred embodiment, the compound according to the present invention has formula 2: ABZ 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5-ANB 6 -NH2 (Formula 2) or a compound having formula 3: ABZ 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -pNA 6 (Formula 3) In the formula, ABZ represents 2-aminobenzoic acid, ANB-NH2 represents an amide of 5-amino-2-nitrobenzoic acid, and pNA represents 4-nitroaniline.

[0048] The compounds undergo hydrolytic cleavage to generate the following fragment 1: ABZ-Gly-Thr-Thr-Gly-OH and fragment 2: ANB-NH2 for compounds having formula 2, while the compounds having formula 3 generate the following fragment 1: ABZ-Gly-Thr-Thr-Gly-OH and fragment 2: pNA. Thus, fragment 2 is the free chromophore.

[0049] Upon enzymatic cleavage of the compound according to the invention, the molecules C1 and C2 are spatially separated, and the fluorescence emitted by the fluorescent donor is no longer quenched by the fluorescent acceptor, resulting in the generation of a measurable optical signal, which can be detected at wavelengths preferably between 300 and 500 nm, more preferably between 380 and 430 nm.

[0050] The compounds according to the invention can be obtained by known methods. For example, they can be obtained using a method for obtaining chromogenic peptides, which consists in carrying out the process on a solid support in the form of a resin bearing an Fmoc group (which is removed during the reaction). For example, it can be an amide resin, such as Teenage S RAM or RinkAmide, but any other commercially available resin can also be used. The resin used to carry out the method should be appropriately prepared. The preparation of the resin consists in increasing its volume by repeated washing with a hydrophobic solvent. Preferably, a resin with a substitution rate of 0.23 mmol / g is used. The Fmoc protecting group must be removed from the resin by washing with a 20% solvent solution.

[0051] Next, known methods for obtaining chromogenic peptides involve coupling the individual components under appropriate time and stoichiometric conditions. The coupling method consists of successive steps of coupling the individual components (amino acid derivatives), washing the residues, sequentially removing the protecting groups, and washing again. This cycle is repeated for each amino acid residue. The resulting peptide is separated from the resin by reaction under acidic conditions. Next, the solution is separated from the resin in a filtration step, and the peptide is precipitated from the resulting solution using a non-polar solvent. The peptide precipitate thus obtained is then centrifuged.

[0052] Exemplary detailed, but non-limiting, syntheses of compounds according to the invention are set forth below and in Example 1 below. The synthesis of the compounds according to the invention preferably involves carrying out the process on a solid support, preferably in the form of a resin bearing an Fmoc group. Prior to starting the process, the solid support is prepared by increasing its volume by repeated washing with a hydrophobic solvent, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and removing the Fmoc protecting group, preferably by washing with a 10-30% solution of piperidine in a solvent such as dimethylformamide, methylene chloride or N-methylpyrrolidone.

[0053] The method is then carried out in the following steps: a) Prior to coupling 5-amino-2-nitrobenzoic acid (ANB) (or another chromophore suitable for use in accordance with the claimed invention) to the resin, the solid support is washed with a 3-6% solution of N-methylmorpholine (NMM) in DMF, followed by DMF washing. A solution of ANB in ​​DMF is then prepared, to which TBTU, DMAP, and finally diisopropylethylamine (DIPEA) are added in the following order in excess relative to polymer substitution: ANB / TBTU / DMAP / DIPEA 3:3:2:6. The mixture thus prepared is added to the resin and mixed until uniform. The resin is then filtered under reduced pressure and washed with solvents such as DMF, DCM, and isopropanol. The conjugation of ANB to the resin is then continued using an excess of hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HATU) followed by hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU). After completion, the solid support is washed sequentially with DMF, DCM, and isopropanol and gently dried.

[0054] b) The attachment of an amino acid residue to ANB is carried out by reaction with the amino acid derivative Fmoc-Gly-OH. Here, at least a 5-fold molar excess of the amino acid derivative relative to the resin is dissolved in anhydrous pyridine and contacted with the resin with ANB attached. The whole is then cooled to a temperature not lower than -20°C, and POCl3 is added in a 1:1 ratio relative to the amount of amino acid derivative used. The whole is mixed, and the mixing steps are then carried out at room temperature and then at elevated temperature. Upon completion of the reaction, the resin is filtered under reduced pressure, washed with DMF and MeOH, and gently dried. The resulting intermediate compound is then subjected to the acylation step, followed by the attachment of the Gly-Thr-Thr fragment.

[0055] c) Acylation of the resulting intermediate compound is carried out with an amino acid derivative, preferably Fmoc-Thr(tBu)-OH, followed by Fmoc-Thr(tBu)-OH, then Fmoc-Gly-OH, and in the final step of the synthesis, Boc-Abz-OH. The acylation is carried out stepwise from residue 6 to 1 using an excess of diisopropylcarbodiimide as a coupling agent. After each step, the resin is washed with DMF and preferably subjected to the chloranil test (a test for the presence of a free amino group) to monitor the coupling of the amino acid derivative.

[0056] d) Removal of the Fmoc protecting group is carried out by washing with a 10-30% solution of piperidine in DMF, followed by washing with each solvent, namely DMF, isopropanol and methylene chloride.

[0057] e) Separation of the peptide from the resin is carried out using a mixture of TFA:phenol:water:TIPS, maintaining the ratio of 88:5:5:2 v / v / v / v, respectively. The mixture is stirred for at least 1 hour, preferably 3 hours, and the resulting precipitate is filtered under reduced pressure, then washed with diethyl ether, and the resulting peptide is centrifuged.

[0058] f) The preparation of the final product is carried out by dissolving the peptide in water by ultrasound and then subjecting it to lyophilization. In a second aspect of the present invention, there is provided an in vitro detection method for detecting enzymatic activity, preferably proteolytic activity, present in a body fluid of a subject, particularly from gastric cancer cells, the method comprising: a) contacting a body fluid sample with a compound according to the present invention; and b) detecting a measurable optical signal generated by spatial separation of molecules C1 and C2 present in the compound according to the present invention. In a preferred embodiment of this aspect, the test subject in this case is a human subject. In another preferred embodiment of this aspect, the body fluid is urine, particularly human urine.

[0059] In a preferred embodiment of this aspect, a compound having the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA is used.

[0060] In a third aspect of the present invention, there is provided an in vitro method for diagnosing gastric cancer. This method detects the presence or absence of gastric cancer in a subject by measuring gastric cancer-specific enzyme activity in a body fluid sample from the subject, where the absence of the enzyme activity indicates the absence of gastric cancer, while the presence of the enzyme activity indicates the presence of gastric cancer. Detection of such enzyme activity is preferably carried out using the method for detecting enzyme activity described above. In a preferred embodiment of this aspect, the subject is a human subject. In a preferred embodiment of this aspect, the body fluid is urine, particularly human urine. In a preferred embodiment of this aspect, the gastric cancer-specific enzyme activity is proteolytic activity. In a preferred embodiment of this aspect, a compound having the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA is used.

[0061] Furthermore, in a preferred embodiment of this aspect, the measurement of the enzyme activity in the method according to the present invention comprises measuring the absorbance intensity at 300 to 500 nm, preferably at 380 to 430 nm, and particularly at 405 nm, for 40 to 60 minutes at a temperature within a range of 25 to 40° C., preferably at 36 to 38° C. This makes it possible to obtain the strongest possible measurable optical signal resulting from an increase in absorbance or fluorescence.

[0062] Furthermore, in a preferred embodiment of the method according to the present invention, the measurement of the enzyme activity is carried out using the compound according to the present invention at a concentration ranging from 0.1 to 10 mg / mL, more preferably at a concentration of 1 mg / mL. In a preferred embodiment of the method according to the present invention, the test sample is incubated with the compound according to the present invention in an assay buffer having a neutral or alkaline pH, preferably physiological pH. The body fluid sample is preferably human urine, and the ratio of sample (e.g., urine sample) to assay buffer is in the range of 1:2 to 1:10, preferably 1:5. The sample is preferably collected from a subject referred for a diagnosis of gastric cancer. Preferably, the absorbance intensity is measured at 300 to 500 nm, preferably 380 to 430 nm, particularly 405 nm, for 40 to 60 minutes at a temperature ranging from 25 to 40°C, preferably 36 to 38°C. Under the above conditions, a maximally intense measurable light signal is obtained as a result of the increase in absorbance or fluorescence.

[0063] In a fourth aspect, the present invention provides a kit comprising any compound according to the present invention and an assay buffer. Assay buffers are known in the art, and a suitable buffer for use in the kit according to the present invention is, for example, but not limited to, Tris-HCl buffer. In a preferred embodiment, in the kit according to the present invention, the compound is a compound having the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA.

[0064] In a fifth aspect, the present invention provides the use of a compound according to the present invention for detecting enzyme activity specific to gastric cancer. In a sixth aspect, the present invention provides the use of a compound according to the present invention for diagnosing gastric cancer. Preferably, diagnosing gastric cancer according to the present invention includes detecting primary gastric cancer, detecting minimal residual disease after surgical resection of gastric cancer, and / or detecting recurrence of gastric cancer after previously completed gastric cancer treatment.

[0065] In a seventh aspect, the present invention provides a compound according to the present invention for use as a diagnostic marker for detecting gastric cancer. In a preferred embodiment of this aspect, the compound has the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA.

[0066] In an eighth aspect, the present invention provides a method for producing a medicament for a medicament comprising: a) detecting the presence of an enzymatic activity specific for gastric cancer in a body fluid sample to be examined by any of the methods according to the invention as defined above, and b) administering to the subject a treatment for gastric cancer if said enzymatic activity is found to be present in said sample; Provides treatment for stomach cancer.

[0067] In a preferred embodiment of the method, after the completion of treatment according to point b), the activity of the enzyme specific for gastric cancer is monitored at predetermined time intervals as known in the art, for example, weekly, every few weeks, monthly, every few months, yearly, or any other interval deemed appropriate by a person skilled in the art, in order to detect minimal residual disease or recurrence of gastric cancer after surgical resection. Furthermore, in a preferred embodiment of the method, a urine sample, preferably human urine, is used as the test sample. In a preferred embodiment of the method, a compound having the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3) is used as the compound.

[0068] An advantage of the present invention is that it provides novel compounds with suitable properties for use in the specific and sensitive detection of gastric cancer-specific enzyme activity, for use as diagnostic biomarkers for gastric cancer detection, and for use in the rapid, non-invasive diagnosis of gastric cancer, while enabling the detection of gastric cancer at an early stage of its progression. Another advantage is that the diagnostic method according to the present invention can also be successfully used as a screening test. This allows for a complete diagnosis at an early stage of cancer progression and, consequently, more effective treatment. Early diagnosis allows for surgical treatment, which significantly extends patient survival. It is also important for monitoring the effectiveness of applied surgical and / or chemotherapy treatments for gastric cancer, since it allows for the detection of any minimal residual disease or recurrence.

[0069] The present invention will now be described in the following figures and examples which are not intended in any way to limit the scope of the invention as defined in the claims. [Example]

[0070] The present invention is illustrated by the following non-limiting examples. Unless otherwise indicated, the following examples use known and / or commercially available equipment, methods, reaction conditions, reactants, and kits that are commonly used in the field to which the present invention pertains and that are recommended by the manufacturers of the respective reactants and kits.

[0071] Example 1: Synthesis of compounds according to the invention In this example, one representative compound according to the present invention, namely, compound ABZ 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5The synthesis of -NH2 is provided. The remaining peptides of the present invention can be synthesized in a similar manner. The superscripts indicate the position of the subsequent residue in the compound of the present invention and the order of attachment of the residues during synthesis. Alternatively, the compound of the present invention can be represented by a similar formula in which the positions of the residues are not indicated. This does not change the sequence of the residues in the compound of the present invention, which remains unchanged.

[0072] 1. Obtaining Chromogenic Peptides a) The first step of the synthesis was to obtain the chromogenic peptide, which was obtained by solid phase synthesis on a solid support using Fmoc / tBu chemistry, i.e., protection.

[0073] Array ABZ 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 Compounds with -NH2, where ABZ is 2-aminobenzoic acid, ANB-NH2 is the amide of 5-amino-2-benzoic acid, and ANB is 5-amino-2-benzoic acid, were obtained by solid-phase chemical synthesis using the following amino acid derivatives: Boc-ABZ, Fmoc-Gly, Fmoc-Thr(tBu), Fmoc-Thr(tBu), and Fmoc-Gly.

[0074] The synthesis of the compound according to the present invention, which can be used as a diagnostic marker for detecting gastric cancer, was carried out on a solid support that allows the conversion of 5-amino-2-benzoic acid to ANB-NH2 amide, namely the amide resin TentaGel S RAM (with a substitution rate of 0.23 mmol / g) from RAPP Polymere (Germany). However, it is also possible to use any other amide resin, such as Rink Amide (Germany).

[0075] The synthesis of the compounds was carried out manually on a laboratory shaker. For most steps, 25 mL sintered syringes for solid-phase synthesis were used as reactors. All final compounds obtained contained a 2-aminobenzoic acid (ABZ) molecule at position 1, i.e., the N-terminus, of their sequence, and a 5-amino-2-nitrobenzoic acid (ANB) molecule at position 6, i.e., the C-terminus. ABZ acts here as a fluorescent donor, while ANB (5-amino-2-nitrobenzoic acid) acts as a fluorescent quencher and simultaneously as a chromophore. The peptides contained at least, and preferably one, reactive site in their sequence (located between the amino acid residues Gly-ANB-NH2, i.e., at position 5 of the compound). The synthesis of the amino acid derivatives was carried out from residue 6 to 1, i.e., from the C-terminus to the N-terminus.

[0076] b) Binding of ANB to TentaGel S RAM resin: The synthesis of the peptide was carried out on TentaGel S RAM resin (Rapp Polymere) with a substitution rate of 0.23 mmol / g. In the first step, the resin was prepared, including loosening it through a washing cycle. Then, the Fmoc amino group protection was removed from the solid support with a 20% piperidine solution in NMP. A solvent washing cycle was then performed. A chloranil test was performed to confirm the presence of free amino groups.

[0077] Solvent wash cycle: DMF 1x10min; IsOH 1x10min; DCM 1x10min Removal of Fmoc protecting group: DMF 1x5 min; 20% piperidine in NMP 1x3 min; 20% piperidine in NMP 1x8 min Solvent wash cycle: DMF 3x2min; IsOH 3x2min; DCM 3x2min c) Chloranil test: The chloranil test consisted of transferring a few particles of resin from the reactor (syringe) with a spatula into a glass ampoule, after which 100 μL of a saturated solution of p-chloranil in toluene and 50 μL of fresh acetaldehyde were added to it. After 10 minutes, the color of the particles was controlled.

[0078] At this stage of the test, green particles were obtained, which is evidence of the presence of free amino groups. After confirming the removal of the 9-fluorenylmethoxycarbonyl protecting group from the resin, it was possible to proceed to the next step, i.e., the coupling of the ANB derivative (5-amino-2-nitrobenzoic acid).

[0079] d) Attachment of 5-amino-2-nitrobenzoic acid to a solid support The first step in the synthesis of the peptide was the placement of ANB onto 1 g of resin. Prior to the attachment of the chromophore, the resin used in the reaction was washed with the following solvents: DMF, DCM, and then DMF again, after which the Fmoc protecting group was removed from the functional groups of the solid support. One cycle of Fmoc protecting group removal included the following steps:

[0080] Removal of Fmoc protecting group: 20% piperidine in NMP 1x3 min; 20% piperidine in NMP 1x8 min e) Washing: DMF 3x2min; IsOH 3x2min; DCM 3x2min f) Chloranil test: The resin bearing free amino groups was washed with a 5% solution of N-methylmorpholine (NMM) in DMF, followed by DMF. The Fmoc protecting group removal procedure and washing cycle were carried out in a Merrifield vessel. In a separate flask, ANB was dissolved in DMF, followed by TBTU, DMAP, and finally diisopropylethylamine (DIPEA) in the following excess amounts relative to polymer substitution: ANB / TBTU / DMAP / DIPEA 3:3:2:6 v / v / v / v. The mixture thus prepared was added to the resin and stirred for 3 hours. The resin was filtered under reduced pressure, washed with DMF, DCM, and isopropanol, and the entire acylation procedure was repeated twice. To carry out the subsequent reaction of attaching ANB to the resin, hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HATU) and then hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU) were used. In the last step, the resin was washed successively with DMF, DCM, and isopropanol and air-dried.

[0081] g) Conjugation of the C-terminal amino acid residue (Fmoc-Gly-OH) to ANB: The corresponding amino acid derivative (9-fold molar excess relative to resin substitution) was dissolved in pyridine and transferred to the flask containing the ANB-attached resin. The whole was cooled (ice bath: 1 part by weight of NH4Cl, 1 part by weight of NaNO3, 1 part by weight of ice) until the temperature reached -15 °C. Once the desired temperature was reached, POCl3 was added (1:1 ratio relative to the amount of amino acid derivative used), and the whole was stirred on a magnetic stirrer for 20 minutes at -15 °C, 30 minutes at room temperature, and 6 hours at 40 °C (oil bath). Upon completion of the reaction, the resin was filtered under reduced pressure, washed with DMF and MeOH, and allowed to dry.

[0082] In the next step, a residue was attached to the P2 position (Fmoc-Thr(tBu)). After each conjugation of an amino acid residue, the resin was first washed with DMF for 5 minutes. Diisopropylcarbodiimide was used as a coupling agent for subsequent conjugations. The procedure was repeated twice.

[0083] After each acylation, a resin washing cycle was initiated and then a chloranil test was performed to monitor the binding of the amino acid derivative to the free amino acid groups of the resin. Solvent wash cycle: DMF 3x2min; IsOH 3x2min; DCM 3x2min Chloranil test: After the first two coupling steps, the particles were initially green and then gray, necessitating another acylation step. The resulting resin particles, tested by the chloranil test, became colorless, confirming the binding of ANB to the TentaGel S RAM resin, allowing the next step in peptide synthesis.

[0084] h) Attachment of subsequent protected amino acid residues: After washing both the resin and the binding fragment ANB-Gly in the reaction vessel with DMF, Fmoc was deprotected from the amino group to allow the binding of a protected amino acid derivative Thr.

[0085] Removal of Fmoc protecting group: DMF 1x5 min; 20% piperidine in NMP 1x3 min; 20% piperidine in NMP 1x8 min Solvent wash cycle: DMF 3x2min; IsOH 3x2min; DCM 3x2min Chloranil test: The chloranil test was successful, as evidenced by the green color of the resin particles, making it possible to proceed to the next step, ie the coupling of the amino acid residue Fmoc-Thr(tBu)-OH.

[0086] Binding of amino acid derivatives The resin was washed with DMF before the coupling step. The composition of the coupling mixture remained unchanged during attachment of the protected serine residue.

[0087] After each acylation, a solvent washing cycle was performed according to a specific procedure, followed by a chloranil test to check for the presence of free amino acid groups in the solution. Solvent wash cycle: DMF 3x2min; IsOH 3x2min; DCM 3x2min Chloranil test: During the test carried out after the second acylation, the resin particles remained colorless, making it possible to proceed to the next synthetic step, i.e., the introduction of another protected amino acid derivative, Fmoc-Gly, and a 2-aminobenzoic acid molecule. The coupling step was carried out according to the procedure discussed previously.

[0088] Tests carried out after attachment of the aforementioned residues showed good results, with the resin particles being colorless. 2. Removal of peptides from solid support After synthesis, the amide of the ABZ-Gly-Thr-Thr-Gly-ANB-NH2 peptide was removed from the solid support, and the side chain protecting groups were simultaneously removed using a mixture of TFA:phenol:water:TIPS (88:5:5:2, v / v / v / v) in a round-bottom flask on a magnetic stirrer.

[0089] After 3 hours, the contents of the flask were filtered under reduced pressure through a Schott funnel and washed with diethyl ether. The resulting precipitate was centrifuged for 20 minutes in a SIGMA 2K30 Laboratory Centrifuges. The precipitate obtained after centrifugation was dissolved in water by ultrasound and then freeze-dried. The remaining compounds according to the present invention can be obtained in a similar manner.

[0090] The identification / characterization of the novel compounds according to the present invention was confirmed using HPLC analysis, which was carried out under the following conditions: RP Bio Wide Pore Supelco C8 column, 250 mm x 4 mm, phase system A: 0.1% TFA in water, B: 80% acetonitrile in A, flow rate 1 mL / min, UV detection: 226 nm.

[0091] The analyses carried out confirmed that the compound according to the invention had been obtained. Example 2: Testing the properties of compounds according to the invention as cancer markers The activity of the novel compounds according to the present invention was investigated in a group of 20 subjects diagnosed with gastric cancer using a representative compound according to the present invention. The mechanism of action of the compounds according to the present invention (including the representative compound having formula 2) is a specific enzymatic cleavage, more specifically enzymatic hydrolysis, occurring at a position that results in the release of a free molecule of the respective chromophore, ANB-NH2 (amide of 5-amino-2-nitrobenzoic acid) in the case of the compound having formula 2, or pNA (para-nitroaniline) in the case of the compound having formula 3, which exhibits absorbance at wavelengths between 320 and 480 nm, particularly between 380 and 430 nm, and especially at 405 nm. The remaining compounds according to the present invention are characterized by a similar mechanism of action. For this purpose, the representative compound according to the present invention, ABZ 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -NH2 was dissolved in dimethyl sulfoxide (at a concentration of 0.5 mg / mL), and 50 μL of this solution was mixed with 120 μL of buffer solution (200 mM Tris-HCl, pH 8.0) and 80 μL of urine from a subject with gastric cancer. Measurements were performed in a 96-well plate designed for absorbance measurements, and each sample was analyzed in triplicate at a temperature of 37 °C. The measurement time was 60 min. During the measurement, the wavelength characteristic of the released chromophore (ANB-NH2) was monitored at a wavelength of 405 nm (range 380–430 nm).

[0092] As shown in Figure 1, RP HPLC analysis of a randomly selected series containing urine collected from individuals diagnosed with gastric cancer showed that the compound of the present invention was cleaved into the peptide fragment ABZ-Gly-Thr-Thr-Gly-OH and the chromophore group of the compound (ANB-NH2).

[0093] Measurements showed that the color intensity of the solution increased over time in all urine samples collected from individuals diagnosed with gastric cancer. The magnitude of the observed increase in absorbance over time varied for each sample tested. Different results were obtained for the 20 samples collected from healthy subjects, as no increase in absorbance within the diagnostic range was observed in any of the 20 urine samples tested.

[0094] The tests performed showed that all gastric cancer patient samples 1 to 20 underwent cleavage, but in samples 4 and 9, the substrate, i.e., ABZ-Gly-Thr-Thr-Gly-ANB-NH2, was cleaved less efficiently than in samples 6 and 9 (Table 1, Figure 2). This result may be due to differences in the activity and amount of enzymes responsible for enzymatic cleavage (proteolysis). Furthermore, the results shown in Table 1 below indicate that incubation of the substrate solution (compounds according to the present invention) with urine samples collected from healthy individuals (not diagnosed with cancer, marked sequentially with Arabic numerals 21 to 40) did not result in an increase in absorbance, thus indicating no hydrolysis of the test compound. This result indicates the absence of proteolytic enzymes specific / characteristic for gastric cancer.

[0095] Table 1. Results of absorbance analysis

[0096] [Table 1-1]

[0097] [Table 1-2]

[0098] Furthermore, the cleavage selectivity of the substrate, i.e., the compound according to the present invention, was also investigated depending on the type of cancer tested. The results of the tests carried out are shown in Figure 3. According to the results, the test substrate, i.e., ABZ, was incubated with samples collected from patients diagnosed with the following cancers: kidney cancer, lung cancer, intestinal cancer, prostate cancer, stomach cancer, pancreatic cancer, uterine cancer, and liver cancer. 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -ANB 6 It has been shown that -NH2 does not undergo cleavage and does not cause an increase in absorbance within a specific range. The test samples were in each case a mixture of 20 samples obtained from each of the cancers investigated. This demonstrates the cleavage selectivity of the compounds according to the present invention, making them suitable for the specific detection of enzyme activity specific to gastric cancer and the specific diagnosis of gastric cancer.

[0099] Table 2 below shows the results of three measurements taken for each sample. Table 2. Analysis results of cleavage selectivity

[0100] [Table 2]

[0101] Furthermore, the dependence of the proteolytic activity of representative compounds according to the present invention on reaction pH was also determined, and the experiments showed that the investigated materials possess at least one enzyme that exhibits maximum activity at alkaline pH (Figure 4).

[0102] The analyses carried out have confirmed that the compounds according to the invention are suitable for the sensitive and specific detection of enzyme activity specific for gastric cancer, as well as for the specific diagnosis of gastric cancer and as diagnostic markers for gastric cancer. The mechanism of action of the compounds according to the invention consists in their specific enzymatic cleavage at a position which leads to the release of free chromophore molecules, which generate a measurable light signal which can be used for diagnostic purposes, in particular for the diagnosis of gastric cancer according to the invention.

Claims

1. Formula 1: X1 １ -Gly ２ -Thr ３ -Thr ４ -Gly ５ -X2 ６ (Formula 1) A compound having the formula: wherein X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, The compound undergoes enzymatic cleavage into fragments X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2), generating a measurable optical signal due to the spatial separation of molecules C1 and C2. compound.

2. The compound of claim 1 , wherein the compound undergoes hydrolytic cleavage, preferably proteolytic cleavage.

3. In the compound, the pair of molecules C1 and C2 is 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH ２ , ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr (3-NO ２ ) Preferably, the pair of C1 and C2 is selected from the group consisting of ABZ / pNA or ABZ / ANB-NH ２ 3. The compound according to claim 1 or 2, wherein

4. The compound has the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH ２ The compound according to any one of claims 1 to 3, which is a compound having the formula: (Formula 2) or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

5. The compound undergoes hydrolytic cleavage to give the following fragment 1: ABZ-Gly-Thr-Thr-Gly-OH and fragment 2: ANB-NH ２ The compound of claim 4, which produces

6. 1. An in vitro method for detecting enzyme activity, particularly from stomach cancer cells, present in a body fluid of a subject, comprising: a) A body fluid sample is subjected to a quantification of the following: X1 １ -Gly ２ -Thr ３ -Thr ４ -Gly ５ -X2 ６ (Formula 1) contacting the compound having the formula: [wherein X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, the compound undergoes enzymatic cleavage to fragments X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2); and b) detecting a measurable light signal generated by the spatial separation of molecules C1 and C2; An in vitro method comprising:

7. 7. The in vitro method according to claim 6, wherein the enzymatic activity is a hydrolytic activity, preferably a proteolytic activity.

8. The compound may be a compound of Formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH ２ 8. The in vitro method according to claim 6 or 7, wherein a compound having the formula 2 or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (formula 3) is used.

9. The method according to any one of claims 6 to 8, wherein urine, preferably human urine, is used as the body fluid.

10. 1. An in vitro method for diagnosing gastric cancer, wherein the presence or absence of gastric cancer in a subject is detected by measuring gastric cancer-specific enzyme activity in a body fluid sample of the subject, wherein the absence of said enzyme activity indicates the absence of gastric cancer, while the presence of said enzyme activity indicates the presence of gastric cancer.

11. The method according to claim 10, wherein the detection of the enzymatic activity is carried out by the method defined in any one of claims 6 to 9.

12. The measurement of the enzyme activity is carried out according to Formula 1: X1 １ -Gly ２ -Thr ３ -Thr ４ -Gly ５ -X2 ６ (Formula 1) The method is carried out using a compound having the formula In the above formula, X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, 12. The method of claim 10 or 11, wherein the compound undergoes enzymatic cleavage into fragments X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2), generating a measurable optical signal due to the spatial separation of molecules C1 and C2.

13. 13. The method according to any one of claims 10 to 12, wherein the body fluid sample is incubated with the compound in an assay buffer having a neutral or alkaline pH, preferably a physiological pH, in a sample to assay buffer ratio ranging from 1:2 to 1:10, preferably 1:

5.

14. The method according to any one of claims 10 to 13, wherein the compound is used at a concentration of 0.1 to 10 mg / mL, in particular 0.25 to 7.5 mg / mL.

15. The compound may be a compound of Formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH ２ The method according to any one of claims 10 to 14, wherein a compound having the formula (2) or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (formula 3) is used.

16. The method according to any one of claims 10 to 15, wherein a urine sample, preferably human urine, is used as the sample.

17. 17. The method according to any one of claims 10 to 16, wherein the measurement of the enzyme activity comprises measuring the absorbance intensity in the range of 300 to 500 nm, preferably 380 to 430 nm, in particular 405 nm, for 40 to 60 minutes at a temperature in the range of 25 to 40°C, preferably 36 to 38°C.

18. A kit comprising a compound as defined in any one of claims 1 to 5 and an assay buffer.

19. The compound has the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH ２ 19. The kit of claim 18, wherein the compound is a compound having the formula: (Formula 2) or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

20. Use of a compound as defined in any one of claims 1 to 5 for detecting an enzyme activity specific for stomach cancer.

21. Use of a compound as defined in any one of claims 1 to 5 for the diagnosis of gastric cancer.

22. 22. The use according to claim 21, wherein diagnosing gastric cancer comprises detecting primary gastric cancer, detecting minimal residual disease after surgical resection of gastric cancer, and / or detecting recurrence of gastric cancer.

23. The compound has the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH ２ The use according to any one of claims 21 to 22, wherein the compound is a compound having the formula: (Formula 2) or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

24. A compound as defined in any one of claims 1 to 5 for use as a diagnostic marker for the detection of gastric cancer.

25. The compound has the formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH ２ 25. The compound for use according to claim 24, which is a compound having the formula: (Formula 2) or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

26. 1. A method of treating stomach cancer, comprising: a) detecting the presence of a gastric cancer-specific enzymatic activity in a body fluid sample to be examined by a method as defined in any one of claims 6 to 9, and b) administering to the subject a treatment for stomach cancer if the presence of said enzymatic activity is found in said sample. Treatment.

27. 27. The method according to claim 26, wherein after the end of the treatment according to point b), the activity of the enzyme specific for gastric cancer is monitored at predetermined time intervals.

28. 28. The method according to claim 26 or 27, characterized in that as sample a urine sample, preferably human urine, is used.

29. The compound may be a compound of Formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH ２ The method according to any one of claims 26 to 28, wherein a compound having the formula (2) or a compound having the formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (formula 3) is used.

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