Compound - Diagnostic marker for endometrial cancer, method for detecting enzyme activity, method for diagnosing endometrial cancer, kit containing this compound, use of this compound, and method for treating endometrial cancer

Abstract

The present invention relates to novel compounds, diagnostic markers for use in medicine, more particularly in the diagnosis of cancer, especially endometrial cancer. The present invention also relates to an in vitro method for detecting, using the compounds, the enzymatic activity present in a biological fluid of a subject, especially originating from endometrial cancer cells. The present invention further relates to an in vitro diagnostic method for endometrial cancer using the compounds, a kit comprising the compounds, and the use of the compounds for detecting enzymatic activity specific for endometrial cancer, as well as the use of the compounds for the diagnosis of endometrial cancer. The present invention also relates to a method for treating endometrial cancer, which comprises using the compounds as a diagnostic marker for endometrial cancer and a procedure for carrying out the diagnostic method for endometrial cancer as described above using the compounds.

Description

Technical Field

[0001] The present invention relates to novel compounds and diagnostic markers for use in medicine, and more particularly for the diagnosis of cancer, especially endometrial cancer. The present invention also relates to an in vitro detection method for detecting the enzyme activity present in a subject's body fluid, especially derived from endometrial cancer cells, using the present compound, an in vitro diagnostic method for endometrial cancer using the present compound, a kit containing the present compound, the use of the present compound for detecting enzyme activity specific to endometrial cancer, the use of the present compound for diagnosing endometrial cancer, and the present compound for use as a diagnostic marker for endometrial cancer. The present invention further relates to a method for treating endometrial cancer, including the procedure for performing the diagnostic method for endometrial cancer as described above.

Background Art

[0002] In 2020, 417,000 patients worldwide developed endometrial cancer (also known as uterine corpus cancer), and endometrial cancer became the sixth most common cancer in women. Endometrial cancer occurs after menopause in 75% of cases. The most important influence on the development of endometrial cancer is the long-term stimulation of the endometrium by estrogen accompanied by a lack of progesterone.

[0003] Risk factors for endometrial cancer include obesity, infertility, nulliparity, lack of exercise, use of drugs with agonist action on estrogen receptors, early menarche, late menopause, occurrence of anovulatory cycles, diabetes, endometrial hyperplasia, family burden, Lynch syndrome, and the like. The symptoms of endometrial cancer are non-specific and often take the form of abnormal and excessive purulent discharge, spotting, and bleeding. Bleeding may be associated with endometrial cancer, but may also be associated with pathological endometrial hyperplasia.

[0004] When performing a transrectal examination in conjunction with a gynecological examination, the characteristics of bleeding from the reproductive tract become clear. The primary imaging examination is a transvaginal ultrasound examination. If the endometrial thickness exceeds 12 cm, in such cases, a neoplasm or pre-cancerous condition is suspected, which indicates the indication for curettage. The diagnosis is determined by the results of histopathological examination of the material obtained after curettage of the uterine cavity.

[0005] The prognosis of endometrial cancer is quite good, which mainly depends on the stage of the disease, as well as the patient's age and general health status. The earlier this cancer is detected, the higher the possibility of radical cure. The 5-year survival rate for early detection cases exceeds 90%, but in advanced stages, it only remains at 20%.

[0006] There is no clinical examination, "cancer marker", or test set that can enable an early and reliable diagnosis of endometrial cancer. Attempts have been made to use CA-125 and HE4 as predictive markers, but due to their low specificity and sensitivity, their use in diagnostic tests for detection, including early detection of endometrial cancer, is not recommended.

[0007] It is known that the processes of cancer cell generation, proliferation, and seeding involve a number of enzymes, especially hydrolases, and in particular, a number of factors including proteolytic enzymes. Such enzymes catalyze the process of enzymatically (hydrolytically or proteolytically) cleaving proteins and peptides into their small fragments. By this process, cancer cells can colonize new tissues and are able to proliferate and expand by enhancing the angiogenesis (neovascularization) process that enables effective nutrient delivery to the tumor. Moreover, these enzymes exist as a result of the death of healthy cells due to the tumor growth process. All these processes form a characteristic and specific profile of cancer cell enzyme (proteolytic) activity that is characteristic of tumors.

[0008] In this field, chromogenic peptide molecules that undergo enzymatic degradation into smaller fragments and cause a change or increase in the color of a test solution are known. This chromogenic effect is the result of the release of a chromophore (e.g., 4-nitroanilide or 2-aminobenzoic acid) from the chromogenic peptide molecule.

[0009] This type of chromogenic molecule and their use are known, for example, from the publications of "The preparation and properties of two new chromogenic substrates of trypsin" by Erlanger BF, Kokowsky N, Cohen W., Arch Biochem Biophys., November 1961; 95: 271-8 and "Amino acids and peptides. XXXV. 'Facile preparation of p-nitroanilide analogs by the solid-phase method'" by Hojo K, Maeda M, Iguchi S, Smith T, Okamoto H, Kawasaki K., Chem Pharm Bull (Tokyo), November 2000; 48(11): 1740-4.

[0010] However, there has been no report on the use of this class of compounds for the diagnosis of endometrial cancer. Methods for obtaining chromogenic peptides, which consist of binding individual components under appropriate time and stoichiometric conditions, are also known in the prior art. The binding method consists of a series of steps of binding individual components (amino acid derivatives), washing away residues, sequentially removing 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, after separating the solution from the resin in a filtration step, the peptide is precipitated from the solution with a non-polar solvent.

[0011] However, chromogenic peptide compounds suitable for specific and early diagnosis of endometrial cancer and methods for obtaining them are not known in the prior art. Therefore, there is an urgent need in the art for "cancer markers" for endometrial cancer that enable early, highly sensitive, and specific diagnosis of endometrial cancer in a non-invasive and reliable manner, as well as diagnostic and treatment methods using such diagnostic markers.

[0012] The object of the present invention is to provide a novel and specific diagnostic marker for endometrial cancer, a diagnostic method (suitable for screening tests) for non-invasive, rapid, highly sensitive, and specific early detection of endometrial cancer using such a marker, and a treatment method using such a marker.

[0013] These objects are achieved by the present invention as defined in the appended claims. On the other hand, preferred modifications thereof are defined in the dependent claims.

Prior Art Documents

Non-Patent Documents

[0014]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0015] The present invention provides a compound having the formula 1: X1 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -X2 6 (Formula 1) The present invention provides a compound having the formula (1).

[0016] In the formula, X1 comprises or consists of molecular C1, and X2 comprises or consists of molecular C2, wherein the pair of molecules C1 and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and the compound undergoes enzymatic cleavage to form a fragment X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2), generating an optical signal measurable by the spatial separation of molecules C1 and C2.

[0017] The compound according to the present invention preferably undergoes hydrolytic cleavage, more preferably proteolytic cleavage. Preferably, in the compound according to the present 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), and more preferably, the pair of C1 and C2 is (ABZ) / pNA or ABZ / ANB-NH2.

[0018] Preferably, the compound according to the present invention is a compound having the formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2), or a compound having the formula 3: ABZ-Pro-Arg-Thr-Ile-pNA (Formula 3).

[0019] More preferably, the compound according to the present invention undergoes hydrolytic cleavage to produce the following fragment 1: ABZ-Pro-Arg-Thr-Ile-OH and fragment 2: ANB-NH2.

[0020] The present invention further provides an in vitro detection method for detecting the enzyme activity present in a body fluid of a subject, particularly derived from endometrial cancer cells, the method comprising a) contacting a body fluid sample with a compound having the formula 1: X1 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -X2 6 (Formula 1) wherein the compound undergoes enzymatic cleavage to become fragment X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2), and [In the above formula, X1 comprises or consists of molecule C1, X2 comprises or consists of molecule C2, wherein the pair of molecules C1 and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and the compound undergoes enzymatic cleavage to become fragment X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2)], and then b) detecting a measurable optical signal generated by the spatial separation of molecule C1 and C2 including that.

[0021] In the method for detecting enzyme activity according to the present invention, the enzyme activity is preferably hydrolytic activity, and more preferably proteolytic activity. In the method for detecting enzyme activity according to the present invention, as the compound, preferably, a compound having the formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound having the formula 3: ABZ-Pro-Arg-Thr-Ile-pNA (Formula 3) is used.

[0022] In the method for detecting enzyme activity according to the present invention, as the body fluid, preferably, urine, more preferably, human urine is used. The present invention also relates to an in vitro diagnostic method for detecting the presence or absence of endometrial cancer in a subject by measuring the enzyme activity specific to endometrial cancer in a body fluid sample of the subject to be examined. The absence of the enzyme activity indicates the absence of endometrial cancer, while the presence of the enzyme activity indicates the presence of endometrial cancer.

[0023] In the method for detecting / diagnosing endometrial 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 endometrial cancer according to the present invention, the measurement of the enzyme activity is carried out using a compound having the formula 1: X1 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -X2 6 (Formula 1) and is carried out using a compound having the same.

[0024] In the above formula, X1 contains or consists of molecule C1, and X2 contains or consists of molecule C2, where the pair of molecules C1 and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and the compound undergoes enzymatic cleavage to become a fragment X1-Pro-Arg-Thr-Ile-OH (Fragment 1) and X2 (Fragment 2), generating a light signal measurable by the spatial separation of molecules C1 and C2.

[0025] In the method for detecting / diagnosing uterine corpus cancer according to the present invention, the body fluid sample is preferably incubated with the compound in a measurement buffer having a neutral or alkaline pH, more preferably a physiological pH, and in the range of a sample-to-measurement buffer ratio of 1:2 to 1:10, preferably 1:5.

[0026] In the method for detecting / diagnosing uterine corpus 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 uterine corpus cancer according to the present invention, as the compound, preferably, a compound having the formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound having the formula 3: ABZ-Pro-Arg-Thr-Ile-pNA (Formula 3) is used.

[0027] In the method for detecting / diagnosing uterine corpus cancer according to the present invention, as the sample, preferably, a urine sample, more preferably human urine is used. In the method for detecting / diagnosing uterine corpus cancer according to the present invention, the measurement of the enzyme activity preferably includes measuring the absorbance intensity at 405 nm within the range of 300 to 500 nm, more preferably 380 to 430 nm, for 40 to 60 minutes at a temperature within the range of 25 to 40 °C, more preferably 36 to 38 °C.

[0028] The present invention further provides a kit comprising any compound according to the present invention as defined above and a measurement buffer. In the kit according to the present invention, the compound is preferably a compound having the formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 or a compound having the formula 3: ABZ-Pro-Arg-Thr-Ile-pNA.

[0029] The present invention also provides the use of any compound according to the present invention as defined above for detecting an enzyme activity specific to uterine corpus cancer. The present invention also provides the use of any compound according to the present invention as defined above for diagnosing uterine corpus cancer.

[0030] Preferably, in such use, the diagnosis of endometrial cancer includes the detection of primary endometrial cancer, the detection of minimal residual disease after surgical resection of endometrial cancer, and / or the detection of recurrence of endometrial cancer.

[0031] Preferably, the compound in the use according to the present invention is a compound having the formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 or a compound having the formula 3: ABZ-Pro-Arg-Thr-Ile-pNA.

[0032] The present invention further provides any of the compounds according to the present invention as defined above for use as a diagnostic marker for the detection of endometrial cancer. Preferably, the compound for use as a diagnostic marker according to the present invention is a compound having the formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 or a compound having the formula 3: ABZ-Pro-Arg-Thr-Ile-pNA.

[0033] The present invention further provides a) detecting the presence of an enzyme activity specific to endometrial cancer in a body fluid sample to be examined by any of the methods defined above, and b) when the presence of said enzyme activity is found in said sample, applying it to the treatment of endometrial cancer, a method of treating endometrial cancer.

[0034] Preferably, in the treatment method according to the present invention, after the end of the treatment according to point b), the enzyme activity specific to endometrial cancer is monitored at predetermined time intervals. Preferably, in the treatment method according to the present invention, a urine sample, preferably human urine, is used as the sample.

[0035] Preferably, in the treatment method according to the present invention, a compound having the formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 or a compound having the formula 3: ABZ-Pro-Arg-Thr-Ile-pNA is used as the compound.

Brief Description of the Drawings

[0036]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0037] The present invention should be understood as being defined in the appended claims. In this description, various non-limiting aspects and examples of the present invention are illustrated. The present invention is not limited to any particular methodology, protocol or reagent used to practice it, unless otherwise specified. The terms as well as scientific and technical expressions used herein have the meanings commonly known and used by those skilled in the technical field of the present invention. However, for the sake of clarity, the following expressions / terms and acronyms used in this patent are to be understood as follows.

[0038] A chromogenic compound or chromogenic molecule means a compound having chromogenic properties. Chromogenic properties mean the ability of a compound to form a colored product. A fluorescent compound or fluorescent molecule means a compound having fluorescent properties. Fluorescent properties mean the ability of a compound to form a product that emits fluorescence.

[0039] 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, and ANB-NH2 represents the amide of 5-amino-2-nitrobenzoic acid; Boc represents a tert-butyloxycarbonyl group; Fmoc represents a 9-fluorenylmethoxycarbonyl group; and TFA represents trifluoroacetic acid.

[0040] In the context of the present invention, the term endometrial cancer is to be understood as meaning a primary endometrial cancer (malignant neoplasm) arising from tissues located within the uterus. The most frequent endometrial cancer is endometrial adenocarcinoma (about 90%), and less frequent is serous endometrial clear cell carcinoma. The term endometrial cancer as used herein thus includes all malignant endometrial neoplasms arising from tissues located within the uterus.

[0041] In the context of the present invention, the term "diagnosis of endometrial cancer" is to be understood to mean the identification of this disease, in particular at an early stage where other diagnostic methods are not sufficiently sensitive and / or specific. As used herein, the diagnosis of endometrial cancer also includes the detection of minimal residual disease (MRD) after surgical resection of endometrial cancer and the detection of recurrence of endometrial cancer after previously completed treatment of endometrial cancer.

[0042] In the context of the present invention, the term "treatment of endometrial cancer" is to be understood to mean treatment at an early stage of the progression of the disease that makes it possible to significantly prolong the survival time of the affected patient and improve the quality of life.

[0043] In the context of the present invention, the term "monitor" is to be understood to mean diagnosing the presence of minimal residual disease (MRD), i.e., a small number of cancer cells that have survived in the body in an amount that cannot be detected by standard diagnostic methods (during treatment or in remission).

[0044] In the context of the present invention, the term "subject" is to be understood to mean a human subject or mammal suspected of having endometrial cancer, or a human subject or mammal belonging to a group at high risk of endometrial cancer, or a human subject or mammal after resection of endometrial cancer or after completion of treatment of endometrial cancer. The subject is preferably a human subject.

[0045] The compounds according to the present invention have chromogenic properties and fluorescence properties due to the presence of a chromophore. That is, they contain molecules of a fluorescence donor and a fluorescence acceptor. Specifically, as a result of contact with a test body fluid sample of a test subject having endometrial cancer, an increase in color is observed in the wavelength range of 380 to 440 nm, while such an effect is not observed in the reaction with a body fluid sample of a healthy subject or a subject diagnosed with another type of cancer. Due to their structures developed in this way, these compounds detect enzyme activity specific to endometrial cancer and make it possible to diagnose endometrial cancer particularly specifically and sensitively, even at an early stage of the progression of this cancer. The test subject is preferably a human subject. The body fluid is preferably urine, more preferably human urine.

[0046] In a first aspect of the present invention, a novel compound is provided. This compound has the formula 1: X1 1 -Pro 2 -Arg 3 -Thr 4 -Ile 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 pair of a fluorescence donor and a fluorescence acceptor. The superscript letters indicate the position of the residues following in the compound according to the present invention and the binding order of the residues during synthesis. According to the present invention, in this context, Chemical Formula 1 can alternatively be represented without indicating the residue numbering. The core of all compounds according to the present invention is a tetrapeptide having the shown 4 amino acid sequence (Pro-Arg-Thr-Ile). This is also shown as SEQ ID NO: 1 in the sequence listing.

[0047] The compounds according to the present invention are subjected to enzymatic cleavage to become fragment: X1-Pro-Arg-Thr-Ile-OH (Fragment 1) and X2 (Fragment 2), and generate a measurable optical signal by 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, ensuring efficient quenching of the fluorescence donor by the fluorescence acceptor. It is clear to those skilled in the art that the key factor is the distance between the fluorescence donor and the acceptor. Therefore, if the amino acid sequence separating molecules C1 and C2 folds into a twisted or condensed secondary structure, resulting in molecules C1 and C2 being closer to each other compared to the primary structure, the distance between molecules C1 and C2 may be more than 10 amino acid residues.

[0048] This compound is particularly suitable for use in the early diagnosis of endometrial cancer, especially as a diagnostic marker, particularly a specific diagnostic biomarker for endometrial cancer, due to its coloring properties and the presence of a reactive site at the 5-position that allows for enzymatic (preferably proteolytic) cleavage into smaller fragments.

[0049] In a preferred embodiment, the compound according to the invention undergoes 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, ABZ / Tyr(3-NO2), and more preferably, the pair of molecules C1 and C2 is ABZ / pNA or ABZ / ANB-NH2.

[0050] In a preferred embodiment, the compound according to the invention Formula 2: ABZ 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -ANB 6 -NH2 (Formula 2) is a compound having, or Formula 3: ABZ 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -pNA 6 (Formula 3) is a compound having. In the formula, ABZ represents 2-aminobenzoic acid, ANB-NH2 represents the amide of 5-amino-2-nitrobenzoic acid, and pNA represents 4-nitroaniline.

[0051] This compound undergoes hydrolytic cleavage to produce, in the case of the compound having Formula 2, the following Fragment 1: ABZ-Pro-Arg-Thr-Ile-OH and Fragment 2: ANB-NH2. On the other hand, in the case of the compound having Formula 3, the following Fragment 1: ABZ-Pro-Arg-Thr-Ile-OH and Fragment 2: pNA are produced. Therefore, Fragment 2 is the free chromophore.

[0052] As a result of the enzymatic cleavage of the compound according to the present invention, when molecules C1 and C2 are spatially separated, a measurable optical signal is generated. This is because the fluorescence emitted from the fluorescence donor is no longer quenched by the fluorescence acceptor. Such a measurable optical signal can preferably be detected at a wavelength of 300 to 500 nm, more preferably 380 to 430 nm.

[0053] The compounds according to the present invention can be obtained by known methods. For example, it can be obtained using a method for obtaining a chromogenic peptide consisting of performing the process on a solid support in the form of a resin having an Fmoc group (removed during the reaction). For example, it can be an amide resin. For example, Teenage S RAM or RinkAmide, etc., but any other commercially available resin can also be used. The resin used to perform the method should be appropriately prepared. The preparation of the resin consists of increasing its volume by repeatedly washing with a hydrophobic solvent. Preferably, a resin having a loading amount of 0.23 mmol / g is used. The Fmoc protecting group must be removed from the resin by washing with a 20% solvent solution.

[0054] Next, known methods for obtaining chromogenic peptides involve combining the individual components under appropriate time and stoichiometric conditions. The coupling method consists of a series of steps of combining the individual constituents (amino acid derivatives), washing away 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, after separating the solution from the resin in a filtration step, the peptide is precipitated from the resulting solution with a non-polar solvent. The peptide precipitate thus obtained is centrifuged.

[0055] Exemplary and detailed, but non-limiting, synthetic methods of the compounds according to the present invention are described below and in Example 1 below. The synthetic method of the compounds according to the present invention preferably consists of carrying out the method on a solid support in the form of a resin having an Fmoc group. Before starting the method, the solid support is prepared by increasing its volume by repeatedly washing it with a hydrophobic solvent, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and removing the Fmoc protecting group, preferably by washing with a 10-30% piperidine solution in a solvent such as dimethylformamide, methylene chloride or N-methylpyrrolidone.

[0056] Next, the method is carried out in the following steps. a) Before the attachment of 5-amino-2-nitrobenzoic acid (ANB) (or another chromophore suitable for use according to the present invention as defined in the claims) to the resin, the solid support is washed with a 3 - 6% solution of N-methylmorpholine (NMM) in DMF, then washed with DMF. After that, a solution of ANB in DMF is prepared, and to this, TBTU, DMAP, and finally diisopropylethylamine (DIPEA) are added in turn, with the following excess amounts for polymer attachment, i.e., ANB / TBTU / DMAP / DIPEA are added in a ratio of 3:3:2:6. The mixture thus prepared is added to the resin and mixed until homogeneous. Then, the resin is filtered under reduced pressure, washed with solvents such as DMF, DCM, and isopropanol, and after that, the binding of ANB to the resin is continued using an excess of O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU), and then O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU). After completion, the solid support is washed with DMF, DCM, and isopropanol in turn and gently dried.

[0057] b) The attachment of the amino acid residue to ANB is carried out by reaction with the amino acid derivative Fmoc-Ile-OH. Here, at least a 5-fold molar excess of the amino acid derivative is dissolved in anhydrous pyridine and brought into contact with the resin to which ANB is attached. After that, the whole is cooled to a temperature not below -20°C, then POCl3 is added in a ratio of 1:1 with respect to the amount of the amino acid derivative used, the whole is mixed, then the mixing step is carried out at room temperature, then at an elevated temperature. When the reaction is complete, the resin is filtered under reduced pressure, washed with DMF and MeOH, gently dried, and then the resulting intermediate compound is subjected to an acylation step, and subsequently the fragment of Pro-Arg-Thr is attached.

[0058] c) Acylation of the resulting intermediate compound is carried out using amino acid derivatives, preferably Fmoc-Thr(tBu)-OH, then Fmoc-Arg(Pbf)-OH, then Fmoc-Pro-OH, and finally Boc-Abz-OH at the final stage of the synthesis. Acylation is carried out stepwise from residue 6 to 1 using diisopropylcarbodiimide as a coupling agent (used in excess). After each step, the resin is washed with DMF and preferably subjected to a chloranil test (a test to examine the presence of free amino groups) to monitor the binding of the amino acid derivative.

[0059] d) Removal of the Fmoc protecting group is carried out by washing with a 10 - 30% piperidine solution in DMF, followed by washing with each solvent, namely DMF, isopropanol, and methylene chloride.

[0060] e) Separation of the peptide from the resin is carried out using a mixture, namely TFA:phenol:water:TIPS, while 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, the resulting precipitate is filtered under reduced pressure, then washed with diethyl ether, and the resulting peptide is centrifuged.

[0061] f) Preparation of the final product is carried out by dissolving the peptide in water by sonication and then subjecting it to lyophilization. In a second aspect of the present invention, there is provided an in vitro detection method for detecting enzyme activity, preferably proteolytic activity, present in a body fluid of a subject, particularly derived from endometrial cancer cells. The method comprises a) contacting a body fluid sample with a compound according to the present invention, and b) detecting a measurable optical signal generated by the spatial separation of molecules C1 and C2 present in the compound according to the present invention. In a preferred embodiment of this aspect, the subject to be examined is a human subject in this case. In another preferred embodiment of this aspect, the body fluid is urine, particularly human urine.

[0062] In a preferred embodiment of this aspect, a compound having formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 or a compound having formula 3: ABZ-Pro-Arg-Thr-Ile-pNA is used.

[0063] In a third aspect of the present invention, an in vitro diagnostic method for endometrial cancer is provided. This detects the presence or absence of endometrial cancer in a subject by measuring the enzyme activity specific to endometrial cancer in a body fluid sample to be tested. The absence of said enzyme activity indicates the absence of endometrial cancer, while the presence of said enzyme activity indicates the presence of endometrial cancer. Detection of such enzyme activity is preferably carried out using a method for detecting the above enzyme activity. 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 enzyme activity specific to endometrial cancer is proteolytic activity. In a preferred embodiment of this aspect, a compound having formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 or a compound having formula 3: ABZ-Pro-Arg-Thr-Ile-pNA is used.

[0064] 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 - 500 nm, preferably in the range of 380 - 430 nm, particularly 405 nm, for 40 - 60 minutes at a temperature within the range of 25 - 40 °C, preferably 36 - 38 °C. This makes it possible to obtain the strongest measurable optical signal resulting from an increase in absorbance or fluorescence.

[0065] 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 in the range of 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 a measurement buffer having a neutral or alkaline pH, preferably physiological pH. The body fluid sample is preferably human urine, and the ratio of the sample (e.g., urine sample) to the measurement buffer is in the range of 1:2 to 1:10, preferably 1:5. The sample is preferably collected from a subject who has been consulted for the diagnosis of endometrial cancer. Preferably, the absorbance intensity is measured at a temperature in the range of 25 to 40 °C, preferably 36 to 38 °C, for 40 to 60 minutes, at 405 nm within the range of 300 to 500 nm, preferably 380 to 430 nm. Under the above conditions, the strongest measurable optical signal is obtained as a result of an increase in absorbance or fluorescence.

[0066] In a fourth aspect, the present invention provides a kit comprising any compound according to the present invention and a measurement buffer. The measurement buffer is known in the art, and a buffer suitable 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-Pro-Arg-Thr-Ile-Thr-ANB-NH2 or a compound having the formula 3: ABZ-Pro-Arg-Thr-Ile-pNA.

[0067] In a fifth aspect, the present invention provides the use of a compound according to the present invention for detecting enzyme activity specific to endometrial cancer. In a sixth aspect, the present invention provides the use of a compound according to the present invention for the diagnosis of endometrial cancer. Preferably, the diagnosis of endometrial cancer includes, according to the present invention, the detection of primary endometrial cancer, the detection of minimal residual disease after surgical resection of endometrial cancer, and / or the detection of recurrence of endometrial cancer after a previously completed treatment for endometrial cancer.

[0068] In a seventh aspect, the present invention provides a compound according to the present invention for use as a diagnostic marker for endometrial cancer detection. In a preferred embodiment of this aspect, the compound is a compound having formula 2: ABZ - Pro - Arg - Thr - Ile - ANB - NH2 or a compound having formula 3: ABZ - Pro - Arg - Thr - Ile - pNA.

[0069] In an eighth aspect, the present invention a) detecting the presence of an enzyme activity specific to endometrial cancer in a body fluid sample to be tested by any of the methods according to the present invention defined above, and b) when it is found that the enzyme activity is present in the sample, applying it to the treatment of endometrial cancer, provides a method for treating endometrial cancer.

[0070] In a preferred embodiment of the treatment method, after completion of the treatment according to point b), in order to detect minimal residual disease or recurrence after surgical resection of endometrial cancer, the enzyme activity specific to endometrial cancer is monitored at predetermined time intervals as known in the art, for example, weekly, every few weeks, monthly, every few months, annually, or any other interval that a person skilled in the art deems appropriate. Further, 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 treatment method, a compound having formula 2: ABZ - Pro - Arg - Thr - Ile - ANB - NH2 (formula 2) or a compound having formula 3: ABZ - Pro - Arg - Thr - Ile - pNA (formula 3) is used as the compound.

[0071] The advantages of the present invention are to provide a novel compound having appropriate characteristics for use as a diagnostic biomarker for endometrial cancer detection, for specifically and sensitively detecting enzyme activity specific to endometrial cancer while enabling detection of endometrial cancer at an early stage of its progression, for use in rapid and non-invasive diagnosis of endometrial cancer. Another advantage is that the diagnostic method according to the present invention can be successfully used for screening tests. This enables a complete diagnosis at an early stage of cancer progression and, as a result, more effective treatment. Early diagnosis enables surgical treatment that significantly extends the patient's survival time. It is also important when monitoring the effectiveness of applied surgical treatment and / or chemotherapy for endometrial cancer because it is possible to detect any minimal residual disease or recurrence.

[0072] Next, the present invention will be described with reference to the following drawings and the following examples, which are not intended to limit the scope of the present invention as defined in the claims in any way.

Example

[0073] The present invention is illustrated by the following non-limiting examples. Unless otherwise indicated, the following examples use known and / or commercially available devices, methods, reaction conditions, reactants, and sets, which are generally used in the field to which the present invention pertains and are recommended by the manufacturers of the respective reactants and kits.

[0074] Example 1: Synthesis of the compound according to the present invention In this example, one representative compound according to the present invention, namely compound: ABZ 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -ANB 6-NH2 synthesis is provided. The remaining peptides according to the present invention can be synthesized in the same manner. The superscript characters indicate the position of the residues following in the compounds according to the present invention and the binding order of the residues during synthesis. The compounds according to the present invention can alternatively be represented by a similar formula that does not indicate the position of the residues. By this, the sequence of the residues in the compounds according to the present invention is not changed and remains invariant.

[0075] 1. Obtaining a chromogenic peptide a) The first step of the synthesis was to obtain a chromogenic peptide. This was obtained by using Fmoc / tBu chemistry, i.e., solid-phase synthesis on a solid support using protection.

[0076] Sequence ABZ 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -ANB 6 A compound having -NH2 [wherein ABZ is 2-aminobenzoic acid, ANB-NH2 is the amide of 5-amino-2-benzoic acid, and ANB is 5-amino-2-benzoic acid] was obtained by the method of solid-phase chemical synthesis using the following amino acid derivatives: Boc-ABZ, Fmoc-Pro, Fmoc-Arg(Pbf), Fmoc-Thr(tBu), Fmoc-Ile.

[0077] The synthesis of the compounds according to the present invention that can be used as diagnostic markers for endometrial cancer detection was carried out on a solid support that enables the conversion of 5-amino-2-benzoic acid to the ANB-NH2 amide, i.e., amide resin TentaGel S RAM (having an attachment amount of 0.23 mmol / g) manufactured by RAPP Polymere GmbH. However, it is also possible to use any other amide resin, for example, Rink Amide.

[0078] The synthesis of the compound was carried out manually using a laboratory shaker. In most steps, a 25 mL sintered syringe for solid-phase synthesis was used as the reactor. All of the final compounds obtained contained 2-aminobenzoic acid (ABZ) at the first position, i.e., the N-terminus, of their sequences and 5-amino-2-nitrobenzoic acid (ANB) molecules at the sixth position, i.e., the C-terminus. Here, ABZ acts as a fluorescence donor, while ANB (5-amino-2-nitrobenzoic acid) acts as a fluorescence quencher and, at the same time, as a chromophore. The peptides contained at least and preferably one reactive site in their sequences (located between the amino acid residues Pro-ANB-NH2, i.e., at the 5th position of the compound). The synthesis for attaching the amino acid derivatives was carried out from residue 6 to 1, i.e., from the C-terminus to the N-terminus.

[0079] b) Attachment of ANB to TentaGel S RAM resin: The synthesis of the peptides was carried out on TentaGel S RAM resin from Rapp Polymere with a loading of 0.23 mmol / g. In the first step, the resin was prepared, including loosening the resin by a washing cycle. Subsequently, the protection of the Fmoc amino group was removed from the solid support with a 20% piperidine solution in NMP. Then a solvent washing cycle was carried out. A chloranil test was performed to confirm the presence of free amino groups.

[0080] Solvent washing cycle: DMF 1 x 10 min; IsOH 1 x 10 min; DCM 1 x 10 min Removal of the Fmoc protecting group: DMF 1 x 5 min; 20% piperidine in NMP 1 x 3 min; 20% piperidine in NMP 1 x 8 min Solvent washing cycle: DMF 3 x 2 min; IsOH 3 x 2 min; DCM 3 x 2 min c) Chloranil test: The chloranil test consisted of transferring a few grains of resin from the reactor (syringe) to a glass ampoule with a spatula and then adding 100 μL of a saturated solution of p-chloranil in toluene and 50 μL of fresh acetaldehyde to it. After 10 minutes, the color of the particles was checked.

[0081] At this stage after the test was carried out, green particles were obtained. This is evidence of the presence of free amino groups. After confirming the removal of the 9-fluorenylmethoxycarbonyl protecting group from the resin, it became possible to proceed to the next step, namely the attachment of the ANB derivative (5-amino-2-nitrobenzoic acid).

[0082] d) Attachment of 5-amino-2-nitrobenzoic acid to the solid support The first step in the synthesis of the peptide was the attachment of ANB onto 1 g of the resin. Before the attachment of the chromophore, the resin used in the reaction was washed with the following solvents, namely DMF, DCM, and then again with DMF, and then the Fmoc protecting group was removed from the functional group of the solid support. One cycle of removing the Fmoc protecting group included the following steps.

[0083] Removal of the Fmoc protecting group: 20% piperidine in NMP 1 x 3 min; 20% piperidine in NMP 1 x 8 min e) Washing: DMF 3 x 2 min; IsOH 3 x 2 min; DCM 3 x 2 min f) Chloranil test: The resin having free amino groups was washed with a 5% solution of N-methylmorpholine (NMM) in DMF and then washed with DMF. The Fmoc protecting group removal procedure and washing cycles were carried out in a Merrifield vessel. In a separate flask, ANB was dissolved in DMF, and subsequently TBTU, DMAP, and finally diisopropylethylamine (DIPEA) were added in the following excess amounts with respect to polymer attachment, i.e., ANB / TBTU / DMAP / DIPEA at 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 next reaction of binding ANB to the resin, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was used, followed by O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU). In the final step, the resin was washed successively with DMF, DCM, and isopropanol and air-dried.

[0084] g) Binding of the C-terminal amino acid residue (Fmoc-Ile-OH) to ANB: The corresponding amino acid derivative (9-fold molar excess with respect to resin attachment) was dissolved in pyridine and transferred to a flask containing the ANB-attached resin. The whole was cooled until the temperature reached -15 °C (ice bath: 1 part by weight of NH4Cl, 1 part by weight of NaNO3, 1 part by weight of ice). Once the desired temperature was reached, POCl3 was added (in a 1:1 ratio with respect to the amount of amino acid derivative used), and the whole was stirred on a magnetic stirrer at -15 °C for 20 minutes, at room temperature for 30 minutes, and at 40 °C (oil bath) for 6 hours. When the reaction was complete, the resin was filtered under reduced pressure, washed with DMF and MeOH, and left to dry.

[0085] In the next step, the residue was attached to the P2 position (Fmoc-Thr(tBu)). Before each binding of an amino acid residue, the resin was first washed with DMF for 5 minutes. Diisopropylcarbodiimide was used as the coupling agent for subsequent bindings. The procedure was repeated twice.

[0086] After each acylation, a resin washing cycle was initiated, followed by a chloranil test to monitor the attachment of the amino acid derivative to the free amino acid groups of the resin. Solvent washing cycle: DMF 3 x 2 min; IsOH 3 x 2 min; DCM 3 x 2 min Chloranil test: After the first two coupling procedures, as a result of the tests carried out, the color of the particles was initially green and then gray, so another acylation had to be carried out. As a result, the resin particles tested in the chloranil test became colorless. Since this is evidence that ANB was attached to the TentaGel S RAM resin, it became possible to proceed to the next peptide synthesis step.

[0087] h) Attachment of subsequent protected amino acid residues: After washing both the resin in the reactor and the coupling fragment ANB-Ile with DMF, the Fmoc was deprotected from the amino group to attach the protected amino acid derivative Thr.

[0088] Removal of the Fmoc protecting group: DMF 1 x 5 min; 20% piperidine in NMP 1 x 3 min; 20% piperidine in NMP 1 x 8 min Solvent washing cycle: DMF 3 x 2 min; IsOH 3 x 2 min; DCM 3 x 2 min Chloranil test: The chloranil test gave good results, as evident from the green color of the resin particles, so it became possible to proceed to the next step, namely the attachment of the amino acid residue Fmoc-Arg(Pbf)-OH.

[0089] Attachment of amino acid derivatives The resin was washed with DMF before the coupling step. The composition of the coupling mixture remained unchanged also when attaching the protected serine residue.

[0090] After each acylation, a solvent washing cycle was carried out according to a specific procedure, and then a chloranil test was performed to examine the presence of free amino acid groups in the solution. Solvent washing cycle: DMF 3 x 2 min; IsOH 3 x 2 min; DCM 3 x 2 min Chloranil test: During the test performed after the second acylation, since the resin particles were colorless, it was possible to proceed to the next synthesis step, namely the introduction of another protected amino acid derivative, Fmoc-Pro, and 2-aminobenzoic acid molecule. The coupling step was carried out according to the procedure discussed above.

[0091] The test performed after the attachment of the aforementioned residues showed that the resin particles were colorless and gave good results. 2. Removal of the peptide from the solid support After synthesis, the amide of the ABZ-Pro-Arg-Thr-Ile-ANB-NH2 peptide was removed from the solid support, and at the same time, the side chain protecting groups were 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.

[0092] After 3 hours, the contents of the flask were filtered under reduced pressure through a sintered (Schott) funnel and washed with diethyl ether. The resulting precipitate was centrifuged for 20 minutes in a SIGMA 2K30 centrifuge (Laboratory Centrifuges). The precipitate obtained after centrifugation was dissolved in water by sonication and then subjected to lyophilization. The remaining compounds according to the present invention can also be obtained in the same manner.

[0093] The identification / characterization of the novel compounds according to the present invention was confirmed using HPLC analysis. The conditions for HPLC analysis were as follows. RP Bio Wide Pore Supelco C8 column, 250 mm 4 mm, phase system A: 0.1% TFA in water, B: 80% acetonitrile in A, flow rate 1 mL / min, UV detection: 226 nm.

[0094] The analysis performed confirmed the obtainment of the compounds according to the present invention. Example 2: Test of the characteristics of the compound according to the present invention as a cancer marker The activity of the novel compounds according to the present invention was examined using representative compounds according to the present invention in a group of 20 subjects diagnosed with uterine corpus cancer. The mechanism of action of the compounds according to the present invention (including the representative compound having formula 2) is, in the case of the compound having formula 2, specific enzymatic cleavage occurring at a position that results in the release of the free molecule of the respective chromophore, ANB-NH2 (amide of 5-amino-2-nitrobenzoic acid), or in the case of the compound having formula 3, pNA (para-nitroanilide), more specifically enzymatic hydrolysis, which exhibit absorbance at wavelengths of 320 - 480 nm, especially 380 - 430 nm, particularly 405 nm. The remaining compounds according to the present invention are also characterized by a similar mechanism of action. For this purpose, a representative compound according to the present invention, ABZ 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -ANB 6 -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 (200 mM Tris-HCl, pH 8.0) and 80 μL of urine from a subject suffering from uterine corpus cancer. The measurement was carried out on a 96-well plate designed for absorbance measurement, and each sample was analyzed 3 times at a temperature of 37 °C. The measurement time was 60 minutes. During the measurement, the wavelength characteristic of the released chromophore (ANB-NH2) was monitored at a wavelength of 405 nm (range 380 - 430 nm).

[0095] As shown in Figure 1, according to RP HPLC analysis of a random selection system containing urine collected from a person diagnosed with uterine corpus cancer, the compounds according to the present invention were shown to be cleaved into the peptide fragment ABZ-Pro-Arg-Thr-Ile-OH and the chromophore group (ANB-NH2) of the compound.

[0096] According to the measurement, it was shown that the color intensity of the solution increased with time in all urine samples collected from people diagnosed with endometrial cancer. The scale of the observed increase in absorbance over time varied for each test sample. Different results were obtained because no increase in absorbance within the diagnostic range was observed in any of the 20 urine samples tested from healthy subjects.

[0097] According to the tests conducted, all samples T1 - T20 from endometrial cancer patients were subjected to cleavage. However, for samples T1 and T15, it was shown that the cleavage of the substrate, namely ABZ - Pro - Arg - Thr - Ile - ANB - NH2, proceeded less efficiently than in the case of samples T2 or T19 (Table 1, Figure 2). Such results would be due to differences in the activity and amount of the enzyme responsible for enzymatic cleavage (proteolysis). Furthermore, the results shown in Table 1 below indicate that when the substrate solution (the compound according to the present invention) was incubated with urine samples collected from healthy subjects (marked with Arabic numerals 21 - 40 in sequence, not diagnosed with cancer), the absorbance did not increase, and thus hydrolysis of the test compound did not occur. This result indicates the absence of proteolytic enzymes specific / characteristic to endometrial cancer.

[0098] Table 1. Results of absorbance analysis

[0099]

Table 1 - 1

[0100]

Table 1 - 2

[0101] Furthermore, the cleavage selectivity of the substrate, i.e., the compound according to the present invention, was also investigated with respect to its dependence on the type of cancer being examined. The results of the tests conducted are shown in FIG. 3. According to this, the test substrate, i.e., ABZ 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -ANB 6 -NH2 was shown not to undergo cleavage and not to 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 indicates the cleavage selectivity of the compound according to the present invention and makes this compound suitable for the specific detection of enzyme activity specific to endometrial cancer and the specific diagnosis of endometrial cancer.

[0102] The following Table 2 shows the results of three measurements obtained for each sample. Table 2. Analysis results of cleavage selectivity

[0103]

Table 2

[0104] Furthermore, measurements were also carried out on the dependence of the proteolytic activity of the representative compound according to the present invention on the reaction pH. The experiment showed that the material under investigation has at least one enzyme that exhibits maximum activity at alkaline pH (FIG. 4).

[0105] From the analysis conducted, it was confirmed that the compounds according to the present invention are suitable for highly sensitive and specific detection of enzyme activity specific to endometrial cancer, are likewise suitable for specific diagnosis of endometrial cancer, and are also suitable as diagnostic markers for endometrial cancer. The mechanism of action of the compounds according to the present invention lies in their specific enzymatic cleavage at positions that result in the release of free chromophore molecules, thereby generating a measurable optical signal that can be used for diagnostic purposes, particularly for the diagnosis of endometrial cancer according to the present invention.

Claims

1. Formula 1: X1 １ -Pro ２ -Arg ３ -Thr ４ -Ile ５ -X2 ６ (Formula 1) A compound having, In the formula, X1 contains or consists of molecule C1, and X2 contains or consists of molecule C2. Here, the pairs of molecules C1 and C2 are a fluorescent donor and a fluorescent receptor. The compound undergoes enzymatic cleavage to form fragments X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2), and the spatial separation of molecules C1 and C2 generates a measurable optical signal. compound.

2. The compound according to 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 ), and is preferably selected from the group consisting of, and more preferably, the pair of C1 and C2 is ABZ / pNA or ABZ / ANB-NH ２ The compound according to claim 1 or 2, which is

4. The compound has the formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH ２ The compound according to claim 1 or 2, which is a compound having (Formula 2) or a compound having Formula 3: ABZ-Pro-Arg-Thr-Ile-pNA (Formula 3).

5. The aforementioned compound undergoes hydrolytic cleavage to obtain the following fragments: Fragment 1: ABZ-Pro-Arg-Thr-Ile-OH and Fragment 2: ANB-NH ２ The compound according to claim 4, which produces

6. An in vitro method for detecting enzyme activity, particularly that derived from endometrial cancer cells, present in the body fluids of a target, a) Prepare a body fluid sample using formula 1: X1 １ -Pro ２ -Arg ３ -Thr ４ -Ile ５ -X2 ６ (Formula 1) By contacting a compound having the following properties, [In the above formula, X1 contains or consists of molecule C1, and X2 contains or consists of molecule C2, Here, the pairs of molecules C1 and C2 are a fluorescent donor and a fluorescent receptor. The compound undergoes enzymatic cleavage to form fragment X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2), and b) Detect a measurable optical signal generated by the spatial separation of molecules C1 and C2. An in vitro method that includes this.

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

8. As the aforementioned compound, Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH ２ The in vitro method according to claim 6 or 7, wherein a compound having (Formula 2) or a compound having Formula 3: ABZ-Pro-Arg-Thr-Ile-pNA (Formula 3) is used.

9. The method according to claim 6 or 7, wherein urine, preferably human urine, is used as the body fluid.

10. An in vitro method for diagnosing endometrial cancer in which the presence or absence of endometrial cancer in a subject is detected by measuring the enzyme activity specific to endometrial cancer in a body fluid sample of the subject, wherein the absence of the enzyme activity indicates the absence of endometrial cancer, and the presence of the enzyme activity indicates the presence of endometrial cancer, wherein the measurement of the enzyme activity is given by Equation 1: X1 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -X2 6 (Formula 1) This is carried out using a compound having the following properties: In the above formula, X1 contains or consists of molecule C1, and X2 contains or consists of molecule C2. Here, the pairs of molecules C1 and C2 are a fluorescent donor and a fluorescent receptor. The compound undergoes enzymatic cleavage to form fragments X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2), and the spatial separation of molecules C1 and C2 generates a measurable optical signal. In vitro method.

11. The method according to claim 10, wherein the detection of enzyme activity is carried out by the method defined in claim 6.

12. The method according to claim 10 or 11, wherein the body fluid sample is incubated with the compound in a measurement buffer having a neutral or alkaline pH, preferably physiological pH, in a sample-to-measurement buffer ratio within the range of 1:2 to 1:10, preferably 1:

5.

13. The method according to claim 10 or 11, wherein the compound is used at a concentration of 0.1 to 10 mg / mL, particularly 0.25 to 7.5 mg / mL.

14. As the aforementioned compound, Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH ２ The method according to claim 10 or 11, wherein a compound having (Formula 2) or a compound having Formula 3: ABZ-Pro-Arg-Thr-Ile-pNA (Formula 3) is used.

15. The method according to claim 10 or 11, wherein a urine sample, preferably human urine, is used as the aforementioned sample.

16. The method according to claim 10 or 11, wherein the measurement of the enzyme activity includes measuring the absorbance in the range of 300 to 500 nm, preferably 380 to 430 nm, particularly at 405 nm, for 40 to 60 minutes at a temperature in the range of 25 to 40°C, preferably 36 to 38°C.

17. A kit comprising the compound defined in claim 1 or 2 and a measurement buffer.

18. The compound has the formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH ２ The kit according to claim 17, wherein the compound is having (Formula 2) or is having Formula 3: ABZ-Pro-Arg-Thr-Ile-pNA (Formula 3).

19. Use of a compound as defined in claim 1 or 2 for detecting enzyme activity specific to endometrial cancer.

20. A kit as defined in claim 17 for the diagnosis of endometrial cancer.

21. The kit according to claim 20, wherein the diagnosis of endometrial cancer includes the detection of primary endometrial cancer, the detection of minimal residual disease after surgical resection of endometrial cancer, and / or the detection of recurrence of endometrial cancer.

22. The compound has the formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH ２ The kit according to claim 20, wherein the compound is having (Formula 2) or is a compound having Formula 3: ABZ-Pro-Arg-Thr-Ile-pNA (Formula 3).

23. A diagnostic marker for detecting endometrial cancer, comprising the compound defined in claim 1 or 2.

24. The compound has the formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH ２ The diagnostic marker according to claim 23, which is a compound having (Formula 2) or a compound having Formula 3: ABZ-Pro-Arg-Thr-Ile-pNA (Formula 3).

25. a) The presence of enzyme activity specific to endometrial cancer is detected in the body fluid sample to be tested by the method defined in claim 6, and b) If the presence of the enzyme activity is found in the sample, it shall be applied to the treatment of endometrial cancer. The kit according to claim 17 for use in the treatment of endometrial cancer.

26. The kit for use according to claim 25, wherein, after the completion of treatment in accordance with point b), the enzyme activity specific to endometrial cancer is monitored at predetermined time intervals.

27. The kit for use according to claim 25, characterized in that a urine sample, preferably human urine, is used as the sample.

28. As the aforementioned compound, Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH ２ A kit for use according to claim 25, wherein a compound having (Formula 2) or a compound having Formula 3: ABZ-Pro-Arg-Thr-Ile-pNA (Formula 3) is used.

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