Compound-uterine body cancer diagnostic marker, method for detecting enzymatic activity, method for diagnosing uterine body cancer, kit containing compound, application of compound and method for treating uterine body cancer

Abstract

The present invention relates to a novel compound, a diagnostic marker, for use in medicine, more particularly in the diagnosis of cancer, in particular in the diagnosis of uterine body cancer. The invention also relates to an in vitro method for detecting the activity of an enzyme, in particular the activity of an enzyme derived from uterine cancer cells, present in a bodily fluid of a subject using the compound. The invention also relates to an in-vitro method for diagnosing the uterine body cancer by using the compound, a kit containing the compound, application of the compound to detection of specific enzyme activity of the uterine body cancer and application of the compound to diagnosis of the uterine body cancer. The invention also relates to a compound used as a diagnostic marker of uterine body cancer and a method for treating uterine body cancer. The method comprises a step of carrying out the diagnostic method of uterine body cancer as defined above by using the compound.

Description

[0001] The present invention relates to a novel compound, a diagnostic marker, for use in medicine, more specifically for cancer diagnosis, especially for the diagnosis of uterine end cancer. The present invention also relates to an in vitro method for detecting enzyme activity present in a subject's body fluid using the compound, especially enzyme activity derived from uterine end cancer cells, an in vitro method for diagnosing uterine end cancer using the compound, a kit containing the compound, the use of the compound for detecting uterine end cancer-specific enzyme activity, the use of the compound in diagnosing uterine end cancer, and the compound is used as a diagnostic marker for uterine end cancer. The present invention also relates to a method for treating uterine end cancer, comprising the steps of implementing the method for diagnosing uterine end cancer as described above. Background Art

[0002] In 2020, 417,000 patients worldwide will develop uterine corpus cancer (also known as endometrial cancer or cancer of the endometrium), making it the sixth most common malignancy in women. 75% of uterine corpus cancers occur after menopause. The most important influence on the development of uterine corpus cancer is the long-term stimulation of the endometrium by estrogen, accompanied by a lack of progesterone.

[0003] Risk factors for uterine corpus cancer include obesity, infertility, infertility, lack of physical activity, use of drugs that have an agonist effect on estrogen receptors, early first menstruation, late last menstruation, occurrence of anovulatory cycles, diabetes, endometrial hyperplasia, family burden, and Lynch syndrome. Symptoms of uterine corpus cancer are nonspecific and usually present as irregular and copious purulent discharge, spotting, and bleeding. Bleeding can be associated with uterine corpus cancer or with pathological endometrial hyperplasia.

[0004] Gynecological examination and rectal examination reveal features of genital tract bleeding. The main imaging test is transvaginal ultrasonography. The indication for curettage is when the endometrial thickness exceeds 12 cm, because in this case, a tumor or precancerous lesion is suspected. The results of histopathological examination of the material obtained after curettage confirm the diagnosis.

[0005] The prognosis for uterine corpus cancer is quite good, depending mainly on the stage of the disease, as well as the patient's age and general health. The earlier this cancer is detected, the greater the chance of a permanent cure. The five-year survival rate for cases caught early is over 90%, compared to only 20% for cases caught in later stages.

[0006] There are no laboratory tests, no "cancer markers," and no set of tests that can reliably diagnose uterine corpus cancer at an early stage. Attempts are being made to use CA-125 and HE4 as predictive markers; however, due to their low levels of specificity and sensitivity, they are not recommended for use as diagnostic tests for the detection of uterine corpus cancer, including early detection.

[0007] It is well known that the process of initiation, growth and spread of cancer cells involves many factors, including many enzymes, especially hydrolases and especially proteolytic enzymes. Such enzymes catalyze the enzymatic cleavage (hydrolysis or proteolysis) of proteins and peptides into smaller fragments. This process enables cancer cells to expand by colonizing new tissues and enhances the process of blood vessel formation (angiogenesis), which allows for the efficient delivery of nutrients to the tumor. In addition, these enzymes are the result of the death of healthy cells due to the tumor growth process. All these processes form a characteristic and specific profile of the enzymatic (proteolytic) activity of cancer cells, which is characteristic of tumors.

[0008] In this field, it is known that chromogenic peptide molecules are broken down into smaller fragments by enzymes, resulting in a change or increase in the color of the tested solution. This color development effect is the result of the release of chromophores (such as 4-nitroaniline or 2-aminobenzoic acid) from the chromogenic peptide molecules.

[0009] Chromophore molecules of this type and their uses are known, for example, from Erlanger BF, Kokowsky N, Cohen W., "The preparation and properties of two new chromogenic substrate softrypsin", 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.

[0010] However, the use of this class of compounds in the diagnosis of uterine corpus cancer has not been described so far.

[0011] Methods for obtaining chromogenic peptides are also known in the prior art, which methods include connecting individual components under appropriate time and stoichiometric conditions. The connection process includes subsequent steps, in which individual elements (amino acid derivatives) are connected, the residues are washed off, the protecting groups are removed in turn and washed again. This cycle is repeated for each amino acid residue. The obtained peptide is separated from the resin by reaction under acidic conditions. Subsequently, the solution is separated from the resin in a filtering process, and the peptide is then precipitated from the solution by a non-polar solvent.

[0012] However, chromogenic peptide compounds suitable for specific and early diagnosis of uterine corpus cancer or methods for obtaining them are not known in the prior art.

[0013] Therefore, in this field, there is an urgent need for a "cancer marker" for uterine corpus cancer, which will enable early, sensitive and specific diagnosis of uterine corpus cancer in a non-invasive and reliable manner, and a diagnostic method and a therapeutic method using such a diagnostic marker.

[0014] The object of the present invention is to provide a new, specific diagnostic marker for uterine end cancer and a diagnostic method using such a marker for non-invasive, rapid, sensitive and specific early detection of uterine end cancer, which is also suitable for screening tests, and a therapeutic method using such a marker.

[0015] These objects have been achieved by the invention defined in the attached patent claims, while preferred variants thereof are defined in the dependent claims. Summary of the invention

[0016] The present invention provides a compound having formula 1:

[0017] X1 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -X2 6 (Formula 1),

[0018] Wherein X1 comprises or consists of molecule C1, and X2 comprises or consists of molecule C2.

[0019] The molecular pair C1 and C2 are a pair of fluorescence donor and fluorescence acceptor, and the compound undergoes enzymatic cleavage into fragments X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2) when the molecules C1 and C2 are spatially separated, and generates a measurable light signal.

[0020] The compounds of the invention preferably undergo hydrolytic cleavage, more preferably proteolytic cleavage.

[0021] Preferably, in the compounds according to the present invention, the molecular pair C1 and C2 is selected from: 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 C1 and C2 is (ABZ / pNA or ABZ / ANB-NH2.

[0022] Preferably, the compound of the present invention is a compound having Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-ProArg-Thr-Ile-pNA (Formula 3).

[0023] More preferably, the compounds of the invention undergo hydrolytic cleavage to produce the following fragment 1: ABZ-Pro-Arg-Thr-Ile-OH and fragment 2: ANB-NH2.

[0024] The present invention also provides an in vitro method for detecting enzyme activity present in a subject's body fluid, in particular enzyme activity derived from uterine cancer cells, the method comprising:

[0025] a) contacting a body fluid sample with a compound of formula 1:

[0026] X1 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -X2 6 (Formula 1),

[0027] wherein X1 comprises or consists of molecule C1, X2 comprises or consists of molecule C2,

[0028] wherein the pair of molecules C1 and C2 are a pair of fluorescence donor and fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into fragments X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2), and b) a measurable light signal generated when the detection molecules C1 and C1 are spatially separated.

[0029] In the method for detecting enzyme activity according to the present invention, the enzyme activity is preferably hydrolytic activity, more preferably proteolytic activity.

[0030] In the method for detecting enzyme activity according to the present invention, it is preferred to use a compound of Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound of Formula 3: ABZ-ProArg-Thr-Ile-pNA (Formula 3).

[0031] In the method for detecting enzyme activity according to the present invention, urine is preferably used as the body fluid, and human urine is more preferably used.

[0032] The present invention also relates to an in vitro method for diagnosing uterine end cancer, wherein the presence or absence of uterine end cancer in a subject is detected by measuring the activity of an enzyme specific for uterine end cancer in a body fluid sample from the subject, and wherein the absence of the enzyme activity indicates the absence of uterine end cancer, while the presence of the enzyme activity indicates the presence of uterine end cancer.

[0033] In the method for detecting / diagnosing uterine corpus cancer according to the present invention, the detection of the enzyme activity is performed by the method for detecting the enzyme activity as defined above.

[0034] In the detection / diagnosis method of uterine end cancer according to the present invention, the enzyme activity is measured using a compound having Formula 1:

[0035] X1 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -X2 6 (Formula 1),

[0036] wherein X1 comprises or consists of molecule C1, X2 comprises or consists of molecule C2,

[0037] The molecular pair C1 and C2 are a pair of fluorescence donor and fluorescence acceptor, and the compound is enzymatically cleaved into fragments X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2) when the molecules C1 and C2 are spatially separated, and a measurable light signal is generated.

[0038] In the method for detecting / diagnosing uterine endometrial 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, in a sample to measurement buffer ratio in the range of 1:2 to 1:10, preferably 1:5.

[0039] In the method for detecting / diagnosing uterine end cancer according to the present invention, the compound is preferably used at a concentration of 0.1-10 mg / mL, particularly 0.25-7.5 mg / mL.

[0040] In the detection / diagnosis method of uterine end cancer according to the present invention, as the compound, it is preferred to use a compound having Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Pro-Aarg-Thr-Ile-pNA (Formula 3).

[0041] In the method for detecting / diagnosing endometrial cancer according to the present invention, as the sample, a urine sample is preferably used, and human urine is more preferably used.

[0042] In the method for detecting / diagnosing uterine body cancer according to the present invention, the measurement of the enzyme activity preferably includes measuring the absorption intensity in the range of 300-500nm, more preferably 380-430nm, and especially 405nm at a temperature in the range of 25-40°C, more preferably 36-38°C for 40-60 minutes.

[0043] The present invention also provides a kit comprising any compound of the present invention as described above and a measurement buffer.

[0044] In the kit of the present invention, the compound is preferably a compound having Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 or a compound having Formula 3: ABZ-ProArg-Thr-Ile-pNA.

[0045] The present invention also provides the use of any compound of the present invention as defined above for detecting uterine endometrial cancer-specific enzyme activity.

[0046] The present invention also provides the use of any compound of the present invention as defined above for the diagnosis of uterine corpus cancer.

[0047] Preferably, in such applications, the diagnosis of uterine corpus cancer includes the detection of primary uterine corpus cancer, the detection of minimal residual disease after surgical resection of uterine corpus cancer, and / or the detection of recurrence of uterine corpus cancer.

[0048] Preferably, the compound in the use of the present invention is a compound having formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 or a compound having formula 3: ABZ-ProArg-Thr-Ile-pNA.

[0049] The present invention also provides any compound of the present invention as defined above for use as a diagnostic marker for detecting uterine corpus cancer.

[0050] Preferably, the compound used as a diagnostic marker of the present invention is a compound having Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 or a compound having Formula 3: ABZ-ProArg-Thr-Ile-pNA.

[0051] The present invention also provides a method for treating endometrial cancer, wherein a) the presence of endometrial cancer-specific enzyme activity is detected in a body fluid sample from a subject by any method as defined above, and b) if the presence of said enzyme activity is found in said sample, a treatment for endometrial cancer is applied in the subject.

[0052] Preferably, in the treatment method according to the present invention, the uterine corpus cancer-specific enzyme activity is monitored at predetermined time intervals after the treatment according to point b) is completed.

[0053] Preferably, in the method of treatment according to the invention, a urine sample, preferably human urine, is used as sample.

[0054] Preferably, in the treatment method of the present invention, a compound having Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 or a compound having Formula 3: ABZ-ProArg-Thr-Ile-pNA is used as the compound. DETAILED DESCRIPTION

[0055] It should be understood that the invention is defined in the appended claims. This specification illustrates various non-limiting embodiments and examples of the invention. Unless otherwise indicated, the invention is not limited to any particular method, protocol or reagent for practicing the invention. The terms and scientific and technical expressions used herein have the meanings commonly known and used by those skilled in the art of the invention. However, for the sake of clarity, the following expressions / terms and abbreviations used in the patent should be understood as follows:

[0056] A chromogenic compound or chromogenic molecule is a compound that has the property of chromogenicity. Chromogenicity refers to the ability of a compound to form a colored product.

[0057] A fluorescent compound or fluorescent molecule is a compound that has the property of fluorescence. The fluorescent property refers to the ability of a compound to form products that emit fluorescence.

[0058] NMP represents N-methylpyrrolidone; DMF represents dimethylformamide; DCM represents methylene chloride or dichloromethane; pNA represents 4-nitroaniline or p-nitroaniline; ABZ represents 2-aminobenzoic acid, and ANB-NH2 represents the amide of 5-amino-2-nitrobenzoic acid; Boc represents tert-butyloxycarbonyl; Fmoc represents 9-fluorenyloxycarbonyl; and TFA represents trifluoroacetic acid.

[0059] In the context of the present invention, the term uterine corpus cancer is understood to refer to primary uterine corpus cancer (malignant tumor) formed by tissue located in the uterus. The most common uterine corpus cancer is endometrial adenocarcinoma (about 90%), and less common is serous uterine clear cell carcinoma. The term uterine corpus cancer used herein therefore includes all malignant uterine corpus tumors formed by tissue located in the uterus.

[0060] In the context of the present invention, the term "diagnosis of uterine corpus cancer" should be understood to refer to the identification of the disease, especially in its early stages, when other diagnostic methods are not sensitive and / or specific enough. As used herein, diagnosis of uterine corpus cancer also includes detection of minimal residual disease (MRD) after surgical resection of uterine corpus cancer and detection of uterine corpus cancer recurrence after previously completed treatment of uterine corpus cancer.

[0061] In the context of the present invention, the term "treatment of uterine corpus cancer" is understood to mean a treatment at an early stage of disease progression, which can significantly prolong survival time and improve the quality of life of the affected subject.

[0062] In the context of the present invention, the term "monitoring" is to be understood as referring to the diagnosis of minimal residual disease (MRD), i.e. the presence of a small number of cancer cells surviving in the organism (during treatment or remission) in an amount that cannot be detected by standard diagnostic methods.

[0063] In the context of the present invention, the term "subject" is understood to refer to a human subject or mammal suspected of having endometrial cancer, or alternatively, a human subject or mammal belonging to a group with increased risk of endometrial cancer, or a human subject or mammal after excision of endometrial cancer or completion of endometrial cancer treatment. The subject is preferably a human subject.

[0064] Due to the presence of chromophores, the compounds of the present invention have colorimetric and fluorescence properties, i.e., they contain molecules of fluorescence donors and acceptors. Due to their structure, an increase in color is observed in the wavelength range of 380-440 nm, particularly due to contact with a test body fluid sample from a subject suffering from endometrial cancer, while this effect is not observed in the reaction with a body fluid sample from a healthy subject or a subject suffering from another type of cancer, these compounds make it possible to detect endometrial cancer-specific enzymatic activity, particularly also to diagnose endometrial cancer in a specific and sensitive manner in the early stages of the progression of this cancer. The subject is preferably a human subject. The body fluid is preferably urine, more preferably human urine.

[0065] In the first aspect of the present invention, a novel compound is provided, which has formula 1:

[0066] X1 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -X2 6 (Formula 1),

[0067] Wherein X1 is an amino acid derivative or peptide containing molecule C1, or X1 consists of such molecule C1, X2 is an amino acid derivative or peptide containing molecule C2, or X2 consists of such molecule C2, wherein the pair of molecules C1 and C2 is a fluorescent donor and a fluorescent acceptor pair. The superscript indicates the subsequent position of the residues in the compound of the present invention and the order in which the residues are connected during the synthesis process. According to the present invention, in this case, Chemical Formula 1 can be written alternatively without indicating the number of the residues. The core of all compounds of the present invention is a tetrapeptide (Pro-Arg-Thr-Ile) with a specified sequence of 4 amino acids, which is also represented by SEQ ID No.1 in the sequence table.

[0068] The compound of the present invention is enzymatically cleaved into fragments: X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2), and a measurable light signal is generated when the molecules C1 and C2 are spatially separated. The measurable light signal is measured by measuring the absorbance / fluorescence change after the compound is cleaved. Preferably, the molecules C1 and C2 are separated from each other by no more than 10 amino acid residues, which ensures the effective quenching of the fluorescent donor by the fluorescent acceptor. For those skilled in the art, the key factor is obviously the distance between the fluorescent donor and the acceptor. Therefore, when the amino acid sequence separating the molecules C1 and C2 is folded into a twisted or condensed secondary structure, resulting in the proximity of the molecules C1 and C1 relative to the primary structure, the distance between the molecules C1 and C2 can be greater than 10 amino acid residues.

[0069] Due to the chromogenic properties and the presence of a reactive site at position 5, which 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 uterine endometrial cancer, especially for early diagnosis of uterine endometrial cancer.

[0070] In a preferred embodiment, the compounds of the invention undergo hydrolytic cleavage, more preferably proteolytic cleavage.

[0071] In a preferred embodiment, the molecular pair C1 and C2 are selected from: 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 molecular pair C1 and C2 are ABZ / pNA or ABZ / ANB-NH2.

[0072] In a preferred embodiment, the compounds of the present invention are:

[0073] The compound has Formula 2:

[0074] ABZ 1-Pro 2 -Arg 3 -Thr 4 -Ile 5 -ANB6-NH2 (Formula 2) or

[0075] Compound of formula 3:

[0076] ABZ 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -pNA6 (Formula 3),

[0077] Wherein ABZ represents 2-aminobenzoic acid, ANB-NH2 represents the amide of 5-amino-2-nitrobenzoic acid, and pNA represents 4-nitroaniline.

[0078] In the case of the compound of formula 2, the compound undergoes hydrolytic cleavage to produce the following fragment 1: ABZ-Pro-Arg-Thr-Ile-OH and fragment 2: ANB-NH2, while in the case of the compound of formula 3, the following fragment 1: ABZ-ProArg-Thr-Ile-OH and fragment 2: pNA are produced. Therefore, fragment 2 is a free chromophore.

[0079] The spatial separation of molecules C1 and C2 is the result of enzymatic cleavage of the compound of the present invention, which leads to the generation of a measurable light signal because the fluorescence emitted by the fluorescence donor is no longer quenched by the fluorescence acceptor. This measurable light signal can be preferably detected at a wavelength of 300-500nm, more preferably at 380-430nm.

[0080] The compounds of the invention can be obtained by known methods. For example, they can be obtained using a method for obtaining chromogenic peptides, which comprises carrying out the process on a solid support in the form of a resin having 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 process should be appropriately prepared. The preparation of the resin consists in increasing its volume by repeated washing with a hydrophobic solvent. It is preferred to use a resin with a deposition amount of 0.23 mmol / g. The resin must be washed with a 20% solvent solution to remove the Fmoc protecting group.

[0081] Then, the known method of obtaining chromogenic peptides comprises connecting the individual components under appropriate time and stoichiometric conditions. The connection process comprises subsequent steps, in which the individual elements (amino acid derivatives) are connected, the residues are washed off, the protecting groups are removed in turn and washed again. This cycle is repeated for each amino acid residue. The peptide obtained is separated from the resin by reaction under acidic conditions. Then, the solution is separated from the resin in a filtering process, and the peptide is then precipitated from the obtained solution by a non-polar solvent. The peptide precipitate obtained in this way is centrifuged.

[0082] Exemplary, detailed but non-limiting synthetic methods for compounds of the invention are described below and in Example 1 below.

[0083] The method for synthesizing the compounds of the present invention is that the method is carried out on a solid support in the form of a resin, preferably a solid support having an Fmoc group, wherein before the start of the method, the solid support is prepared by repeatedly washing with a hydrophobic solvent (preferably dimethylformamide, dichloromethane or N-methylpyrrolidone) to increase its volume and remove the Fmoc protecting group, preferably by washing with a 10-30% piperidine solution in a solvent (such as dimethylformamide, methylene chloride or N-methylpyridone).

[0084] The method then proceeds in the following steps:

[0085] a) before depositing 5-amino-2-nitrobenzoic acid ANB (or another chromophore suitable for the present invention as defined in the claims) on the resin, washing the solid support with a 3-6% solution of N-methylmorpholine (NMM) in DMF and then with DMF, thereafter preparing a solution of ANB in ​​DMF to which TBTU, DMAP and finally diisopropylethylamine (DIPEA) are added in sequence in the following excess relative to the polymer deposited: ANB / TBTU / DMAP / DIPEA, 3:3:2:6; The prepared mixture is added to the resin and mixed until uniform, and then the resin is filtered under reduced pressure and washed with solvents such as DMF, DCM and isopropanol, and then hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HATU) is used, followed by excess hexafluorophosphate-O-(7-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU) to continue to connect ANB to the resin. After completion, the solid support is washed successively with DMF, DCM and isopropanol, and then gently dried;

[0086] b) the connection of the amino acid residue to ANB is carried out by reaction with an amino acid derivative - Fmoc-Ile-OH, wherein at least five times molar excess of the amino acid derivative relative to the resin is dissolved in anhydrous pyridine and contacted with the deposited ANB, and then the whole is cooled to a temperature not lower than -20°C, and then POCl3 is added in a ratio of 1:1 relative to the amount of the amino acid derivative used and the whole is mixed, and then the mixing process is carried out at room temperature, and then the mixing is increased, and after the reaction is completed, the resin is filtered under reduced pressure, washed with DMF and MeOH and gently dried, and then the intermediate compound obtained is subjected to acylation treatment, and the Pro-Arg-Thr fragment is connected in sequence;

[0087] c) acylation of the intermediate compound obtained with an amino acid derivative, preferably Fmoc-Thr(tBu)-OH, followed by Fmoc-Arg(Pbf)-OH, then Fmoc-Pro-OH and in the last step of the synthesis with Boc-Abz-OH, the acylation being carried out in steps from residues 6 to 1, using diisopropylcarbodiimide used in excess as coupling agent, washing the resin with DMF after each step, preferably carrying out a chloranil test (a test for the presence of free amino groups), wherein the attachment of the amino acid derivative is monitored;

[0088] d) Removal of the Fmoc protecting group was performed by washing with 10-30% piperidine in DMF, followed by washing with each of DMF, isopropanol, and dichloromethane;

[0089] e) separating the peptide from the resin using a mixture: TFA:phenol:water:TIPS, while maintaining a ratio of 88:5:5:2 v / v / v / v, respectively, stirring the mixture for at least one hour, preferably three hours, filtering off the precipitate obtained under reduced pressure, then washing with diethyl ether, and centrifuging the peptide obtained;

[0090] f) The peptide is dissolved in water by ultrasonication and then freeze-dried to prepare a finished product.

[0091] A second aspect of the invention provides an in vitro method for detecting enzyme activity, preferably proteolytic activity, present in a subject's body fluid, in particular enzyme activity derived from uterine body cancer cells, the method comprising a) contacting a body fluid sample with a compound of the invention, and b) detecting a measurable light signal generated upon spatial separation of the molecules C1 and C2 present in the compound of the invention. In a preferred embodiment of this aspect, in this case, the subject being examined is a human subject. In another preferred embodiment of this aspect, the body fluid is urine, in particular human urine.

[0092] In a preferred embodiment of this aspect, a compound having the formula 2: ABZ-Pro-Arg-Thr-Le-ANB-NH2 or a compound having the formula 3: ABZ-Pro-Arg-Thr-Ile-pNA is used.

[0093] The third aspect of the present invention provides an in vitro method for diagnosing uterine end cancer, wherein the presence or absence of uterine end cancer in a subject is detected by measuring the uterine end cancer-specific enzyme activity in a body fluid sample from the subject, wherein the absence of the enzyme activity indicates the absence of uterine end cancer, and the presence of the enzyme activity indicates the presence of uterine end cancer. The detection of this enzyme activity is preferably carried out using the method for detecting enzyme activity as 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 enzyme activity specific to uterine end cancer is proteolytic activity. In a preferred embodiment of this aspect, a compound having formula 2: ABZ-Pro-Arg-Thr-Le-ANB-NH2 or a compound having formula 3: ABZ-Pro-Arg-Thr-Ile-pNA is used.

[0094] Furthermore, in a preferred embodiment of this aspect, the measurement of the enzyme activity in the method of the present invention comprises measuring the absorbance intensity within the range of 300-500 nm, preferably 380-430 nm, and particularly 405 nm at a temperature of 25-40° C., preferably 36-38° C., within 40-60 minutes. This enables the acquisition of a measurable light signal of maximum intensity caused by an increase in absorbance or fluorescence.

[0095] Furthermore, in a preferred embodiment of the method of the present invention, the enzyme activity is measured using a compound of the present invention at a concentration in the range of 0.1-10 mg / mL, more preferably at a concentration of 1 mg / mL. The sample is preferably taken from a subject referred for diagnosis of uterine body cancer. Preferably, the absorbance intensity is measured at a temperature of 25-40° C., preferably 36-38° C., within 40-60 minutes, within a range of 300-500 nm, preferably 380-430 nm, and in particular 405 nm. Under the above conditions, a measurable light signal of maximum intensity is obtained due to an increase in absorbance or fluorescence.

[0096] In a fourth aspect, the present invention provides a kit comprising any compound of the present invention and a measurement buffer. The measurement buffer is known in the art, and a buffer suitable for the kit of the present invention is, for example, but not limited to, a Tris-HCl buffer. In a preferred embodiment, in the kit according to the present invention, the compound is a compound having Formula 2: ABZ-Pro-Arg-Thr-Ile-Thr-ANB-NH2 or a compound having Formula 3: ABZ-Pro-Arg-Thr-Ile-pNA.

[0097] In a fifth aspect, the present invention provides the use of the compounds of the present invention for detecting uterine endometrial cancer-specific enzyme activity. In a sixth aspect, the present invention provides the use of the compounds according to the present invention for diagnosing uterine endometrial cancer. Preferably, according to the present invention, the diagnosis of uterine endometrial cancer includes the detection of primary uterine endometrial cancer, the detection of minimal residual disease after surgical resection of uterine endometrial cancer, and / or the detection of uterine endometrial cancer recurrence after previously completing uterine endometrial cancer treatment.

[0098] In a seventh aspect, the present invention provides a compound of the present invention for use as a diagnostic marker for the detection of uterine corpus cancer. 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-ProArg-Thr-Ile-pNA.

[0099] In an eighth aspect, the present invention provides a method for treating uterine corpus cancer, wherein

[0100] a) detecting the presence of endometrial cancer-specific enzyme activity in a body fluid sample from a subject by any of the methods of the present invention as defined above, and

[0101] b) applying a treatment for uterine corpus cancer in the subject when said enzyme activity is found to be present in said sample.

[0102] In a preferred embodiment of the method for treatment, after completion of the treatment according to point b), the uterine end cancer-specific enzyme activity is monitored at predetermined time intervals known in the art, such as weekly, weekly, weekly, monthly, monthly, annually, or any other interval deemed appropriate by a person skilled in the art to detect minimal residual disease after surgical resection or recurrence of uterine end cancer. In addition, in a preferred embodiment of the method, a urine sample, preferably human urine, is used as a test sample. In a preferred embodiment of the method of treatment, a compound having Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-ProArg-Thr-Ile-pNA (Formula 3) is used as the compound.

[0103] The advantage of the present invention is that a new compound is provided, which has an enzyme activity that makes it suitable for specific and sensitive detection of uterine body cancer, used as a diagnostic biomarker for detecting uterine body cancer, for rapid and non-invasive diagnosis of uterine body cancer, while being able to detect the properties of the uterine body at an early stage of uterine body cancer formation. Another advantage is that the diagnostic method according to the present invention can be successfully used for screening tests. This enables comprehensive diagnosis at an early stage of cancer formation, thereby obtaining more effective treatment. Early diagnosis allows surgical treatment, thereby significantly prolonging the patient's survival time. This is also important when monitoring the effectiveness of surgical treatment and / or chemotherapy for uterine body cancer, because minimal residual disease or recurrence (if any) can be detected.

[0104] The invention will now be illustrated in the following figures and examples which, however, are not intended in any way to limit the scope of the invention as defined in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 Shown are the results of chromatographic analysis of the cleavage of the substrate, ABZ-Pro-Arg-Thr-Ile-ANB-NH2 in a urine sample from a subject with uterine corpus cancer.

[0106] Figure 2 The hydrolysis rates of the matrix ABZ-Pro-Arg-Thr-Ile-ANB-NH2 in urine samples of subjects diagnosed with uterine corpus cancer (samples T1-T20) and urine samples of healthy subjects (samples 21-40) are shown. Arabic numerals indicate the number of the selected urine sample.

[0107] Figure 3 The substrate-ABZ is shown 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -ANB6-NH2 (i.e., compound of formula 2) in urine samples of subjects diagnosed with uterine body cancer (sample 1) and urine samples of subjects diagnosed with another neoplastic disease (samples 2-9). Arabic numerals represent the numbers of given cancer types. The test samples of each cancer are from 20 different patients of each test cancer. The results are the average values ​​of given cancer types. The results show that the selectivity of substrate cleavage in the urine of patients with uterine body cancer is compared with urine samples of patients with other tumors.

[0108] Figure 4 The substrate-ABZ is shown 1 -Pro 2 -Arg 3 -Thr4 -Ile 5 -Dependence of the hydrolysis level of ANB6-NH2 on pH conditions.

[0109] Example

[0110] The present invention is illustrated by the following non-limiting examples. Unless otherwise specified, the following examples use known and / or commercially available devices, methods, reaction conditions, reactants and kits, which are commonly used in the art to which the present invention belongs and are recommended by the manufacturers of the corresponding reactants and kits.

[0111] Example 1: Synthesis of the compounds of the present invention

[0112] This example describes the synthesis of a representative compound of the present invention, namely compound: ABZ 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -ANB6-NH2. The remaining peptides of the present invention can be synthesized in a similar manner. The superscripts indicate the subsequent position of the residues in the compounds of the present invention and the order in which the residues are connected during the synthesis. The compounds of the present invention can alternatively be represented by a similar formula that does not indicate the position of the residues, which does not change the order of the residues in the compounds of the present invention because it remains unchanged.

[0113] 1. Obtaining chromogenic peptides

[0114] a) The first step of the synthesis is to obtain the chromogenic peptide obtained by solid phase synthesis on a solid support using Fmoc / tBu chemistry, ie with protection.

[0115] In the solid phase chemical synthesis, the following amino acid derivatives were used to obtain the sequence ABZ 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -ANB6-NH2 compound (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):

[0116] Boc-ABZ, Fmoc-Pro, Fmoc-Arg(Pbf), Fmoc-Thr(tBu), Fmoc-Ile.

[0117] The synthesis of the compounds of the invention which can be used as diagnostic markers for the detection of uterine corpus cancer was carried out on a solid support capable of converting 5-amino-2-benzoic acid into ANB-NH2 amide, namely the amide resin TentaGel S RAM from RAPP Polymer (Germany) with a deposition of 0.23 mmol / g. However, any other amide resin can be used, for example Rinkamide (Germany).

[0118] The synthesis of this compound was performed manually using a laboratory shaker. In most steps, a 25 mL fritted syringe for solid phase synthesis was used as the reactor.

[0119] All the final compounds obtained contain 2-aminobenzoic acid (ABZ) 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 fluorescence donor, while ANB-5-amino-2-nitrobenzoic acid acts as a fluorescence quencher and at the same time as a chromophore. The peptide contains at least one and preferably one reactive site in its sequence, located between the amino acid residues Pro-ANB-NH2, i.e. position 5 of the compound. The synthesis consisting of the linked amino acid derivatives is carried out from residues 6 to 1, i.e. from the C-terminus to the N-terminus.

[0120] b) Deposition of ANB on TentaGel S RAM resin:

[0121] The peptide synthesis was carried out on TentaGel S RAM resin from Rapp Polymere with a deposit of 0.23 mmol / g. In the first step, the resin was prepared, including loosening it by washing cycles. Subsequently, the protection of the Fmoc-amino group was removed from the solid support with a 20% piperidine solution in NMP. Then, solvent washing cycles were carried out. In order to confirm the presence of free amino groups, a chloran test was performed.

[0122] Solvent Wash Cycle:

[0123] DMF 1×10 min; IsOH 1×10 min; DCM 1×10 min.

[0124] Removal of Fmoc protection:

[0125] DMF 1 x 5 min; 20% piperidine in NMP 1 x 3 min; 20% piperidine in NMP 1 x 8 min.

[0126] Solvent Wash Cycle:

[0127] DMF 3×2 min; IsOH 3×2 min; DCM 3×2 min.

[0128] c) Chloran test:

[0129] The chloran test consists of transferring a few pellets of resin from the reactor (syringe) into a glass ampoule with a spatula, to which 100 μL of a saturated solution of p-chloran in toluene and 50 μL of fresh acetaldehyde are then added. After 10 minutes, the pellet color is controlled.

[0130] At this stage, after testing, green particles were obtained, which confirmed the presence of free amino groups. After confirming the removal of the 9-Fmoc protection from the resin, the next step can be carried out, namely the attachment of the ANB derivative (5-amino-2-nitrobenzoic acid).

[0131] d) Deposition of 5-amino-2-nitrobenzoic acid on a solid support:

[0132] The first step of peptide synthesis is to deposit ANB on 1 g of resin. Before attaching the chromophore, the resin used for the reaction is washed with the following solvents: DMF, DCM and DMF, and then the Fmoc-protection is removed from the functional groups of the solid support. One cycle of removing the Fmoc protection consists of the following steps:

[0133] Removal of Fmoc protection:

[0134] 20% piperidine in NMP 1 x 3 min; 20% piperidine in NMP 1 x 8 min.

[0135] e) Washing:

[0136] DMF 3×2 min; IsOH 3×2 min; DCM 3×2 min.

[0137] f) Chlorine test:

[0138] The resin with free amino groups was washed with 5% DMF solution of N-methylmorpholine (NMM), then washed with DMF. The procedure and washing cycle for removing Fmoc protection were carried out in a Merrifield container. In another flask, ANB was dissolved in DMF, followed by addition of TBTU, DMAP and last diisopropylethylamine (DIPEA) with respect to the following excess of polymer deposition: ANB / TBTU / DMAP / DIPEA, 3:3:2:6 v / v / v / v. The mixture prepared in this way 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 whole acylation process was repeated twice. For the subsequent reaction of attaching ANB to the resin, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was used, followed by O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU). In the last step, the resin was washed with DMF, DCM, and isopropanol, and air-dried.

[0139] g) Connection of C-terminal amino acid residue (Fmoc-Ile-OH) to ANB:

[0140] The corresponding amino acid derivative (9-fold molar excess relative to the resin deposition) was dissolved in pyridine and transferred to the flask containing the resin deposited with ANB. The whole was cooled to a temperature of -15°C (ice bath: 1 part by weight of NH4Cl, 1 part by weight of NaNO3, 1 part by weight of ice). After reaching the desired temperature, POCl3 (in a 1:1 ratio with the amount of amino acid derivative used) was added and stirred on a magnetic stirrer: 20 minutes at -15°C, 30 minutes at room temperature, and 6 hours at 40°C (oil bath). After the reaction was complete, the resin was filtered under reduced pressure, washed with DMF and MeOH and dried.

[0141] In the next stage, the residue was attached at the P2 position (Fmoc-Thr(tBu)).

[0142] Before each attachment of amino acid residues, the resin was washed with DMF for 5 minutes. Diisopropylcarbodiimide was used as coupling agent in subsequent attachments. This procedure was repeated twice.

[0143] After each acylation, a resin wash cycle was initiated and then a chloran test was performed to monitor the attachment of the amino acid derivative to the free amino acid groups on the resin.

[0144] Solvent Wash Cycle:

[0145] DMF 3×2 min; IsOH 3×2 min; DCM 3×2 min.

[0146] Chloran test:

[0147] As a result of the tests performed, after the first two coupling procedures, the color of the particles was first green and then gray, so it was necessary to perform another acylation, resulting in the resin particles being colorless by the chloran test. This proved that the ANB was attached to the TentaGel S RAM resin and that the next step of peptide synthesis could be performed.

[0148] h) Subsequent attachment of protected amino acid residues:

[0149] The resin was washed with DMF together with the linker fragment ANB-Ile in the reactor, and then Fmoc was deprotected from the amino group to link the protected amino acid derivative Thr.

[0150] Removal of Fmoc protection:

[0151] DMF 1 x 5 min; 20% piperidine in NMP 1 x 3 min; 20% piperidine in NMP 1 x 8 min.

[0152] Solvent Wash Cycle:

[0153] DMF 3×2 min; IsOH 3×2 min; DCM 3×2 min.

[0154] Chloran test:

[0155] The Chloran test yielded a positive result, as evidenced by the green color of the resin particles. Therefore, the next step - attachment of the amino acid residue Fmoc-Arg)Pbf)-OH - could be carried out.

[0156] Linking of amino acid derivatives

[0157] Prior to the coupling process, the resin was washed with DMF. When attaching protected serine residues, the composition of the coupling mixture remained unchanged.

[0158] After each acylation, solvent wash cycles were performed according to the specified procedure, followed by a chloran test for the presence of free amino acid groups in solution.

[0159] Solvent Wash Cycle

[0160] DMF 3×2 min; IsOH 3×2 min; DCM 3×2 min.

[0161] Chloran test:

[0162] In tests performed after the second acylation, the resin particles were colorless and thus could proceed to the next step of the synthesis, which was the introduction of another protected amino acid derivative, Fmoc-Pro, and the 2-aminobenzoic acid molecule. The coupling process was performed according to the procedure discussed previously.

[0163] Tests performed after connecting the above residue showed positive results - the resin particles were colorless.

[0164] 2. Removal of peptides from solid supports

[0165] After synthesis, the amide of the ABZ-Pro-Arg-Thr-Le-ANB-NH2 peptide was removed from the solid support 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, while removing the side chain protection.

[0166] After 3 hours, the contents in the flask were filtered under reduced pressure in a Schott funnel and washed with ether. The precipitate obtained was centrifuged for 20 minutes on a SIGMA2K30 centrifuge (laboratory centrifuge). The precipitate obtained after centrifugation was dissolved in water by ultrasound and then freeze-dried. The remaining compounds of the present invention can be obtained in a similar manner.

[0167] The identity / characteristics of the novel compounds of the present invention were confirmed by HPLC analysis. The conditions of HPLC analysis were as follows: RPBio Wide Pore Supelco C8 column, 250mm 4mm, phase system A: 0.1% TFA aqueous solution, B: 80% acetonitrile solution A, flow rate 1mL / min, UV detection wavelength 226nm.

[0168] The analyses performed confirmed that the compound of the present invention was obtained.

[0169] Example 2: Testing the properties of the compounds of the invention as cancer markers

[0170] The activity of the novel compounds of the present invention was studied in 20 subjects diagnosed with uterine corpus cancer using representative compounds of the present invention. The mechanism of action of the compounds of the present invention, including the representative compounds of formula 2, is that a specific enzymatic cleavage, more specifically enzymatic hydrolysis, is carried out at the position leading to the release of the free molecule of the respective chromophore: ANB-NH2 (amide of 5-amino-2-nitrobenzoic acid) in the case of the compound of formula 2, and pNA (p-nitroaniline) in the case of the compound of formula 3, which exhibits absorbance at a wavelength of 320-480nm, in particular 380-430nm, in particular 405nm. The remaining compounds of the present invention are characterized by a similar mechanism of action. For this purpose, the representative compound ABZ of the present invention was used. 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5-ANB6-NH2 was dissolved in dimethyl sulfoxide (concentration of 0.5 mg / mL), and then 50 μL of the solution was mixed with 120 μL of buffer (200 mM Tris-HCl, pH 8.0) and 80 μL of urine from a subject with uterine corpus cancer. The measurements were performed on a 96-well plate designed for measuring absorbance, and each sample was analyzed three times at a temperature of 37 ° C. The measurement duration was 60 minutes. During the measurement, the wavelength characteristics of the released chromophore (ANB-NH2) were monitored at a wavelength of 405 nm (range 380-430 nm).

[0171] like Figure 1 As shown, RP HPLC analysis of a randomly selected system containing urine from a person diagnosed with uterine corpus cancer showed that the compound according to the invention was fragmented into the peptide fragment ABZ-Pro-Arg-Thr-Ile-OH and the chromophore (ANB-NH2) of the compound.

[0172] The measurements showed that the color intensity of the solution increased over time in all urine samples collected from people diagnosed with uterine corpus cancer. The magnitude of the observed increase in absorbance over time was different for each sample examined. A different effect was obtained for the 20 samples from healthy subjects, as no increase in absorbance within the diagnostic range was observed in any of the 20 urine samples tested.

[0173] The tests performed showed that cleavage occurred in all samples T1-T20 from humans with uterine corpus cancer, but in the case of samples T1 and T15, the substrate (i.e., ABZ-Pro-Arg-Thr-Ile-ANB-NH2) was not cleaved as efficiently as in the case of samples T2 or T19 (Table 1, Figure 2 ). Such results may be due to differences in the activity and quantity of the enzymes responsible for enzymatic cleavage (proteolysis). In addition, the results shown in Table 1 below show that incubation of the matrix solution - the compound according to the present invention - with urine samples extracted from healthy people (no cancer diagnosis, marked sequentially with Arabic numerals from 21 to 40) does not lead to an increase in absorbance, and therefore no hydrolysis of the test compound occurs. The results indicate the lack of proteolytic enzymes specific / characteristic of uterine corpus cancer.

[0174] Table 1. Absorbance analysis results

[0175]

[0176]

[0177] Furthermore, the dependence of the cleavage selectivity of the substrate (i.e. the compounds of the invention) on the cancer being tested was investigated. Figure 3As shown, they indicate that the substrate tested, namely ABZ 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -ANB6-NH2, incubated with samples extracted from patients with the following cancers: gastric cancer, cancer, cancer, prostate cancer, bladder cancer, pancreatic cancer, ovarian cancer and cancer, was not cleaved within a specific range and did not cause an increase in absorbance. In each case, the sample tested was a mixture of 20 samples from each cancer studied. This shows the cleavage selectivity of the compounds of the invention, which makes them suitable for the specific detection of uterine body cancer-specific enzymatic activity and the specific diagnosis of uterine body cancer.

[0178] Table 2 below shows the measurement results of each sample (triplicate).

[0179] Table 2. Results of lysis selectivity analysis

[0180] Uterine body 1 0.0141605416 0.0147606476 0.018740487 Kidney 2 0 0 0 Lung 3 0 0 0 Intestine 4 0 0 0 Prostate 5 0 0 0 Stomach 6 0 0 0 Pancreas 7 0 0 0 Ovaries 8 0 0 0 Liver 9 0 0 0

[0181] In addition, the dependence of the proteolytic activity of representative compounds of the invention on the reaction pH was measured. The experiments showed that at least one enzyme of the studied materials exhibited maximum activity at alkaline pH values ​​( Figure 4 ).

[0182] The assay performed demonstrates the suitability of the compounds of the invention for the sensitive and specific detection of uterine corpus cancer-specific enzymatic activity and, by this assay, they are also suitable for the specific diagnosis of uterine corpus cancer and as diagnostic markers for uterine corpus cancer. The mechanism of action of the compounds of the invention consists in their specific enzymatic cleavage at the site leading to the release of free chromophore molecules, which generates a measurable optical signal that can be used for diagnostic purposes, in particular for the diagnosis of uterine corpus cancer according to the invention.

[0183]

[0184]

[0185]

[0186]

Claims

1. A compound having formula 1: X1 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -X2 6 (Formula 1), wherein X1 comprises or consists of molecule C1, X2 comprises or consists of molecule C2, The molecular pair C1 and C2 are a pair of fluorescence donor and fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into fragments X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2) when the molecules C1 and C2 are spatially separated, and generates a measurable light signal.

2. The compound according to claim 1, which undergoes hydrolytic cleavage, preferably proteolysis.

3. The compound according to claim 1 or 2, wherein the molecular pair C1 and C2 are selected from: 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), preferably, the C1 and C2 pair is ABZ / pNA or ABZ / ANB-NH2.

4. The compound according to any one of claims 1 to 3, which is a compound having Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-ProArg-Thr-Ile-pNA (Formula 3).

5. The compound according to claim 4, which undergoes hydrolytic cleavage to produce the following fragment 1: ABZ-Pro-Arg-Thr-Ile-OH and fragment 2: ANB-NH2.

6. An in vitro method for detecting enzyme activity present in a subject's body fluid, in particular enzyme activity derived from uterine cancer cells, comprising: a) contacting a body fluid sample with a compound of formula 1: x1 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -X2 6 (1) wherein 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 fluorescence donor and fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into fragments X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2), and b) A measurable light signal is generated when the detection molecules C1 and C2 are spatially separated.

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

8. The in vitro method according to claim 6 or 7, wherein As the compound, a compound of Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound of Formula 3: ABZ-ProArg-Thr-Ile-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. An in vitro method for diagnosing endometrial cancer, wherein the presence or absence of endometrial cancer in a subject is detected by measuring an enzyme activity specific for endometrial cancer in a body fluid sample from the subject being examined, and wherein the absence of said enzyme activity indicates the absence of endometrial cancer, and the presence of said enzyme activity indicates the presence of endometrial cancer.

11. The method according to claim 10, wherein the detection of enzyme activity is performed by the method according to any one of claims 6 to 9.

12. The method according to claim 10 or 11, wherein the enzyme activity is measured using a compound of formula 1: x1 1 -Pro 2 -Arg 3 -Thr 4 -Ile 5 -X2 6 (1) wherein X1 comprises or consists of molecule C1, X2 comprises or consists of molecule C2, The molecular pair C1 and C2 are a pair of fluorescence donor and fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into fragments X1-Pro-Arg-Thr-Ile-OH (fragment 1) and X2 (fragment 2), generating a measurable light signal when the molecules C1 and C2 are spatially separated.

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

5.

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

15. The method according to claims 10 to 14, wherein: As the compound, a compound having Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-ProArg-Thr-Ile-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. 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 300-500 nm, preferably 380-430 nm, in particular 405 nm at a temperature of 25-40°C, preferably 36-38°C, within 40-60 minutes.

18. A kit comprising the compound according to any one of claims 1 to 5 and a measurement buffer.

19. The kit according to claim 18, wherein the compound is a compound having Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-ProArg-Thr-Ile-pNA (Formula 3).

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

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

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

23. The use according to any one of claims 21 to 22, wherein the compound is a compound having Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-ProArg-Thr-Ile-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 uterine corpus cancer.

25. The compound according to claim 24, which is a compound having Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-ProArg-Thr-Ile-pNA (Formula 3).

26. A method for treating uterine corpus cancer, wherein a) detecting the presence of uterine corpus cancer-specific enzyme activity in a body fluid sample from the subject by the method defined in any one of claims 6 to 9, and b) applying a treatment for uterine corpus cancer in said subject if the presence of said enzyme activity is found in said sample.

27. The method according to claim 26, wherein the uterine corpus cancer-specific enzyme activity is monitored at predetermined time intervals after the treatment according to point b) is completed.

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

29. The method according to any one of claims 26 to 28, wherein: As the compound, a compound of Formula 2: ABZ-Pro-Arg-Thr-Ile-ANB-NH2 (Formula 2) or a compound of Formula 3: ABZ-ProArg-Thr-Ile-pNA (Formula 3) is used.

Images