NOVEL DIAGNOSTIC MARKER FOR PROSTATE CANCER
20230204587 · 2023-06-29
Assignee
Inventors
Cpc classification
International classification
Abstract
The invention provides a compound characterized by formula 1: X1-Trp-Glu-Gly-Asn-X2, wherein cleavage of the compound into a fragment 1 comprising X1 and a fragment 2 comprising X2 generates a detectable signal. The invention further provides an in vitro method for detecting protease activity in a subject's body fluid, comprising contacting the body fluid with the compound of the invention and detecting a signal, wherein the body fluid may comprise a hydrolytic enzyme derived from prostate cancer cells. Furthermore, the invention provides a kit comprising the compound of the invention and a measurement buffer. In addition, the invention provides the use of the compound, the in vitro method or the kit of the invention for the detection of prostate cancer, or for monitoring a subject that is suspected of having prostate cancer, has an increased risk of developing prostate cancer, or has had prostate cancer. The invention also provides the use of the compound of the invention in a method of treating prostate cancer, the method comprising carrying out the in vitro method for detecting protease activity in a subject's body fluid, and treating prostate cancer in a subject for which protease activity, has been detected.
Claims
1. A compound characterized by formula 1:
X1-Trp-Glu-Gly-Asn-X2 (formula 1), wherein cleavage of the compound into a fragment 1 comprising X1 and a fragment 2 comprising X2 generates a detectable signal; wherein X1 comprises or consists of a component C1 and X2 comprises or consists of a component C2; wherein C1 and C2 are a pair of a fluorescence donor and a fluorescence acceptor; wherein the detectable signal is optical signal, and wherein the detectable signal is generated upon spatial separation of C1 and C2 by hydrolytic cleaving the peptide Trp-Glu-Gly-Asn.
2. The compound according to claim 1, wherein the sequence Trp-Glu-Gly-Asn is accessible for a hydrolytic enzyme, in particular a hydrolytic enzyme cleaving the compound into X1-Trp-Glu-Gly-Asn--OH (fragment 1) and NH2-X2 (fragment 2).
3. The compound according to claim 1, wherein one of C1 and C2, in particular C2, is a chromophore having an absorption maximum 1 (AM1) at a wavelength 1, and the compound has an absorption maximum 2 (AM2) at a wavelength 2 that is different from wavelength 1.
4. The compound according to claim 1, wherein the pair of C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ)/pNA, ABZ/ANB-NH2, ABZ/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/DANSYL, ABZ/Tyr(3-NO2), in particular the pair of C1 and C2 is selected from ABZ/pNA and ABZ/ANB-NH2.
5-12. (canceled)
13. A kit comprising a compound according to claim 1 and a measurement buffer.
14. (canceled)
15. A method of treating prostate cancer, the method comprising the steps of: a. carrying out the method according to claim 16, and b. treating prostate cancer in a subject for which protease activity, in particular increased protease activity has been detected in step a.
16. An in vitro method for detecting protease activity in a subject's body fluid, comprising contacting the body fluid with the compound according to claim 1 and detecting a signal, wherein the body fluid may comprise a hydrolytic enzyme, in particular a protease, derived from prostate cancer cells.
17. The in vitro method according to claim 16 for detecting the presence or absence of prostate cancer in a subject, wherein the presence of protease activity in the body fluid indicates the presence of prostate cancer and the absence of protease activity in the body fluid indicates the absence of prostate cancer.
18. The in vitro method according to claim 16 for the diagnosis of prostate cancer.
19. The in vitro method according to any of claim 16, wherein the body fluid is urine.
20. The method according to any of claim 16, wherein the compound is provided at a concentration of 0.1-10 mg/ml, particularly 0.25-7.5 mg/ml, more particularly 0.5-5 mg/ml, more particularly 0.75-2 mg/ml, even more particularly about 1 mg/ml, in a measurement buffer having neutral or alkaline pH, preferably physiological pH, and the body fluid sample is added to the compound at a ratio of 1:2 to 1:10, particularly 1:3 to 1:8, more particularly 1:4 to 1:6, even more particularly about 1:5.
21. The method according to any of claim 16 wherein detecting the signal comprises measuring absorbance or fluorescence, particularly measuring absorbance intensity at 300-500 nm, more particularly 380-430 nm, preferably for 40-60 min at 25-40° C., in particular at 36-38° C.
22. The compound according to claim 2, wherein one of C1 and C2, in particular C2, is a chromophore having an absorption maximum 1 (AM1) at a wavelength 1, and the compound has an absorption maximum 2 (AM2) at a wavelength 2 that is different from wavelength 1.
23. The compound according to claim 2, wherein the pair of C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ)/pNA, ABZ/ANB-NH2, ABZ/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/DANSYL, ABZ/Tyr(3-NO2), in particular the pair of C1 and C2 is selected from ABZ/pNA and ABZ/ANB-NH2.
24. The compound according to claim 3, wherein the pair of C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ)/pNA, ABZ/ANB-NH2, ABZ/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/DANSYL, ABZ/Tyr(3-NO2), in particular the pair of C1 and C2 is selected from ABZ/pNA and ABZ/ANB-NH2.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0102]
[0103]
[0104]
[0105]
[0106]
EXAMPLES
[0107] The invention is illustrated by the following, non-limiting examples of implementation.
Example 1: Synthesis of Compound ABZ.SUP.1.-Trp.SUP.2.-Glu.SUP.3.-Gly.SUP.4.-Asn.SUP.5.-ANB-NH.SUB.2..SUP.6
1. Preparation of the Chromogenic Peptide
[0108] a) The first stage of the synthesis was to obtain a chromogenic peptide by solid phase synthesis—on a solid carrier, using Fmoc/tBu chemistry, i.e. using the protection.
[0109] A compound with the sequence ABZ.sup.1-Trp.sup.2-Glu.sup.3-Gly.sup.4-Asn.sup.5-ANB-NH.sub.2.sup.6, where ABZ is 2-aminobenzoic acid, ANB-NH.sub.2 is amide of 5-amino-2-benzoic acid, and ANB is 5-amino-2-benzoic acid, obtained in the process of chemical synthesis in the solid phase using the following amino acid derivatives.
Boc-ABZ, Fmoc-Trp(Trt), Fmoc-Glu(OtBu), Fmoc Gly Fmoc-Asn(Trt), ANB
[0110] The synthesis of the compound, i.e. a diagnostic marker for the detection of prostate cancer, whose diagnosis is associated with the hydrolysis of this compound under the influence of proteolytic enzymes, was carried out on a solid carrier enabling the conversion of 5-amino benzoic acid into ANB-NH.sub.2 amide: [0111] e.g. amide resin, for instance TentaGel S RAM from RAPP Polymere (Germany), e.g. with a deposit of 0.23 mmol/g.
[0112] It is possible to use other commercially available amide resins, including Rink Amide (Germany).
[0113] The compound was synthesized manually using a laboratory shaker. For most of the steps, a 25 mL sintered syringe for solid phase synthesis was used as a reactor.
[0114] All the obtained final compounds contained ABZ 2-aminobenzoic acid in position 1 of their sequence (i.e. at the N-terminus), and ANB 5-amino-2-nitrobenzoic acid molecule in position 6 (at the C-terminus). ABZ acts as a fluorescence donor, while ANB (5-amino-2-benzoic acid) acts as a fluorescence quencher and chromophore. The peptides contained at least (and preferably) one reactive site in their sequence, located between the amino acid residues Arg-ANB-NH.sub.2: at the 5 position of the compound. The synthesis, involving the attachment of amino acid derivatives, is carried out from the residue 6 to 1, i.e. from the C- to N-terminus. [0115] b) Deposition of ANB on TentaGel S RAM resin:
[0116] Peptide synthesis was performed on TentaGel S RAM resin (Rapp Polymere) with a deposition of 0.23 mmol/g. In the first stage, the resin was prepared, including its loosening by the wash cycle. Subsequently, the Fmoc amino group protection was removed from the carrier with a 20% solution of piperidine in NMP, and a solvent wash cycle was carried out. To confirm the presence of free amino groups, a chloranil test was performed.
Solvent Wash Cycle:
[0117] DMF 1×10 minutes
IsOH 1×10 minutes
DCM 1×10 minutes
Removal of Fmoc Protection:
[0118] DMF 1×5 minutes
20% piperidine in NMP 1×3 minutes
20% piperidine in NMP 1×8 minutes
Solvent Wash Cycle:
[0119] DMF 3×2 minutes
IsOH 3×2 minutes
DCM 3×2 minutes [0120] c) Chloranil test:
[0121] The chloranil test consisted of transferring (using a spatula) several grains of resin from the reactor—a syringe, into a glass ampoule, to which 100 μL saturated solution of p-chloranil in toluene and 50 μL fresh acetaldehyde were added. After 10 minutes, the control of grains colour was carried out.
[0122] At this stage, after performing the test, a green grain colour was obtained, which indicated the presence of free amino groups. After confirming the removal of 9-fluorenylmethoxycarbonyl protection from the resin, it was possible to proceed to the next stage, the attachment of the ANB derivative (5-amino-2-nitrobenzoic acid) [0123] d) Deposition of 5-amino-2-nitrobenzoic acid on a solid carrier
[0124] The first step in the synthesis of the peptide library—a mixture of peptides, was ANB deposition on 1 g of resin. Before attaching the chromophore, the resin used for the reaction was washed with the following solvents: DMF, DCM and again DMF, after which the Fmoc protection was removed from the carrier functional group. One cycle of removing Fmoc protection included the following steps:
Removal of Fmoc Protection:
[0125] 20% piperidine in NMP 1×3 minutes
20% piperidine in NMP 1×8 minutes [0126] e) Washing
DMF 3×2 minutes
IsOH 3×2 minutes
DCM 3×2 minutes [0127] f) Chloranil test for the presence of free amino groups.
[0128] The resin with a free amino group was washed with a 5% solution of N-methylmorpholine (NMM) in DMF, followed by DMF. The procedure of removing Fmoc protection and the wash cycle were performed in a Merrifield vessel. In a separate flask, ANB was dissolved in DMF, and TBTU, DMAP, and finally diisopropylethylamine (DIPEA) were subsequently added in the following excess in relation to polymer deposition: ANB/TBTU/DMA/DIPEA, 3:3:2:6. The mixture thus prepared was added to the resin and stirred for 3 hours. The resin was filtered off under reduced pressure, washed with DMF, DCM and isopropanol, and the entire acylation procedure was repeated twice. Hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium (HATU), and then hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N′N′-tetramethyluronium (HBTU) were used to carry out subsequent reactions of ANB attachment to the resin. In the last step, the resin was washed successively with DMF, DCM and isopropanol, and air dried. [0129] g) Attachment of the C-terminal amino acid residue to ANB
[0130] The corresponding amino acid derivative (9-fold molar excess relative to resin deposition) was dissolved in pyridine and transferred to the flask containing the resin with ANB deposited. The whole was cooled to −15° C. (ice bath: 1 part by weight NH.sub.4Cl, 1 part by weight NaNO.sub.3, 1 part by weight ice). After reaching the desired temperature, POCl.sub.3 was added (in a 1:1 ratio to the amount of amino acid derivative used) and the whole was stirred on a magnetic stirrer: 20 minutes at −15° C., 30 minutes at room temperature, and 6 hours at 40° C. (oil bath). After completing the reaction, the resin was filtered off under reduced pressure, washed with DMF and MeOH, and left to dry.
[0131] In the next step, the residue (alanine) was attached in the P2 position.
[0132] Every attachment of amino acid residues was preceded by washing the resin with DMF for 5 minutes. Diisopropylcarbodiimide was used as a coupling agent in subsequent attachments. The procedure was repeated twice.
[0133] After each acylation, a resin wash cycle started, followed by the chloranil test to monitor the attachment of the amino acid derivative to the free amino groups of the resin.
Solvent Wash Cycle:
[0134] DMF 3×2 minutes
IsOH 3×2 minutes
DCM 3×2 minutes
Chloranil Test:
[0135] As a result of the tests, after the first two couplings, the colour of the grains was first green and then grey, so it was necessary to carry out another acylation, as a result of which the grains of the resin tested with the chloranil test were colourless. This indicated the attachment of ANB to TentaGel S RAM resin, which enabled moving to the next stage of peptide synthesis. [0136] h) Attachment of further protected amino acid residues:
[0137] The resin together with the attached ANB residue in the reactor was washed with DMF, followed by deprotection of the Fmoc from the amino group to attach the protected amino acid alanine derivative.
Removal of Fmoc Protection:
[0138] DMF 1×5 minutes
20% piperidine in NMP 1×3 minutes
20% piperidine in NMP 1×8 minutes
Solvent Wash Cycle:
[0139] DMF 3×2 minutes
IsOH 3×2 minutes
DCM 3×2 minutes
Chloranil Test:
[0140] The chloranil test produced a positive result, as evidenced by the green colour of the resin grains. This enabled moving to the next stage—attachment of the Fmoc-Thr (tBu)-OH amino acid residue.
Attachment of the Amino Acid Derivative:
[0141] The process of coupling was preceded by washing the resin in DMF. The composition of the coupling mixture remained unchanged when attaching the protected glutamic acid residue.
[0142] At the end of each acylation, a solvent wash cycle was performed according to the given procedure, which was followed by a chloranil test for the presence of free amino groups in the solution.
Solvent Wash Cycle:
[0143] DMF 3×2 minutes
IsOH 3×2 minutes
DCM 3×2 minutes
Chloranil Test:
[0144] The resin grains during the test carried out after the second acylation were colourless, which enabled moving to the next stage of the synthesis, i.e. the introduction of another protected amino acid derivative—threonine and 2-aminobenzoic acid molecule. The coupling processes followed the procedure discussed earlier.
[0145] Tests carried out after attaching the above-mentioned residues showed positive results: the resin grains were colourless.
2. Removal of the Peptide from the Carrier
[0146] After synthesis, the amide of ABZ-Thr-Thr-Ala-Arg-ANB-NH.sub.2 peptide was removed from the carrier, along with the simultaneous removal of the side protection with the mixture: TFA:phenol:water:TIPS (88:5:5:2, v/v/v/v) in a round-bottomed flask on a magnetic stirrer.
[0147] After 3 hours, the contents of the flask were filtered off under reduced pressure on a sintered (Schott) funnel and washed with diethyl ether. The resulting sediment was centrifuged on a SIGMA 2K30 centrifuge (Laboratory Centrifuges) for 20 minutes. The precipitate obtained after centrifugation is dissolved in water by means of ultrasound and lyophilised.
Identity/Characteristics of a New Compound—HPLC Analysis, MS
[0148] HPLC conditions: RP Bio Wide Pore Supelco C8 250 mm 4 mm column, A phase system 0.1% TFA in water, B: 80% acetonitrile in A), flow rate 1 ml/min, UV detection at 226 nm.
[0149] Obtaining the compound was confirmed.
Example 2: Preparation of the Compound with the Formula: ABZ1-Thr2-Thr3-Ala4-Arg5-pNA6
[0150] The process is carried out in a similar manner to that described in Example 1, except that the corresponding amino acid derivatives and additional substituents are used, and the process is carried out partly in solution and partly on a solid carrier.
Preparation of p-Nitroanilide Ala [0151] a) The first stage of the synthesis was to obtain a protected peptide by solid phase synthesis using Fmoc/tBu chemistry.
[0152] ABZ.sup.1-Thr(tBu).sup.2-Thr(tBu).sup.3-Ala.sup.4-OH compound, where ABZ is 2-aminobenzoic acid, was obtained by solid phase chemical synthesis using the following amino acid derivatives:
Boc-ABZ, Fmoc-Thr(tBu), Fmoc-Ala.
[0153] The compound was synthesized on a solid carrier: [0154] 2-chloro-chlorotriyl resin, e.g. from Iris BIOTECH GMBH (Germany), with deposition of 1.6 mmol Cl/g groups.
[0155] The compound was synthesized manually using a laboratory shaker. Throughout all stages, a 25 ml sintered syringe for solid phase synthesis was used as the reactor.
[0156] Peptide synthesis was carried out on a carrier: 2-chloro-chlorotrityl resin, e.g. from Iris BIOTECH GMBH (Germany), with deposition of 1.6 mmol Cl/g groups. In the first stage, the resin was loosened in a wash cycle. Subsequently, the Fmoc amino group protection was removed from the carrier with a 20% solution of piperidine in NMP. Then a solvent wash cycle was carried out. To confirm the presence of free amino groups, a chloranil test was performed.
Solvent Wash Cycle:
[0157] DMF 1×10 minutes
IsOH 1×10 minutes
DCM 1×10 minutes
Removal of Fmoc Protection:
[0158] DMF 1×5 minutes
20% piperidine in NMP 1×3 minutes
20% piperidine in NMP 1×8 minutes
Solvent Wash Cycle:
[0159] DMF 3×2 minutes
IsOH 3×2 minutes
DCM 3×2 minutes [0160] b) Chloranil test:
[0161] The chloranil test consisted of transferring (with a spatula) several grains of resin from the reactor—a syringe, into a glass ampoule, and adding 100 μL saturated solution of p-chloranil in toluene and 50 μL fresh acetaldehyde. After 10 minutes, the control of grains colour was carried out.
[0162] At this stage, after the test, a green colour of grains was obtained, which indicated the presence of free amino groups. After confirming the removal of the 9-fluorenylmethoxycarbonyl protection from the resin, the attachment of Fmoc-Ala derivative was initiated. [0163] c) Embedding Fmoc-Ala on a solid carrier
[0164] The first step in the synthesis of the peptide library was the deposition of Fmoc-Ala on 1 g of resin. Before attachment of the amino acid derivative, the resin used for the reaction was washed with the following solvents: DMF (dimethylformamide), DCM (methylene chloride) and again with DMF, after which the Fmoc-protection was removed from the carrier functional group. One cycle of Fmoc-protection removal included the following steps:
Removal of Fmoc Protection:
[0165] 20% piperidine in NMP 1×3 minutes
20% piperidine in NMP 1×8 minutes
Washing
[0166] DMF 3×2 minutes
IsOH 3×2 minutes
DCM 3×2 minutes
Chloranil Test for the Presence of Free Amino Groups.
[0167] The resin with a free amino group was washed with DMF. In a separate flask, Fmoc-Ala was dissolved in DMF. Next, TBTU, DMAP, and finally diisopropylethylamine (DIPEA) were added in excess to the polymer deposition: Fmoc-Pro/TBTU/DMAP/DIPEA, 3:3:2:6. The obtained mixture was added to the resin and stirred for 3 hours. The resin was filtered off under reduced pressure, washed with DMF, DCM and isopropanol, and the entire acylation procedure was repeated twice. Hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium (HATU), followed by hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium (HBTU) were used to conduct subsequent reactions of Fmoc-Pro attachment to the resin. In the last step, the resin was washed successively with DMF, DCM and isopropanol, and air dried. [0168] c. Attachment of further protected amino acid residues:
[0169] The resin together with the attached Fmoc-Ala residue in the reactor was washed with DMF, followed by deprotection of Fmoc from the amino group to attach the protected threonine derivative.
Removal of Fmoc Protection:
[0170] DMF 1×5 minutes
20% piperidine in NMP 1×3 minutes
20% piperidine in NMP 1×8 minutes
Solvent Wash Cycle:
[0171] DMF 3×2 minutes
IsOH 3×2 minutes
DCM 3×2 minutes
Chloranil Test:
[0172] The chloranil test produced a positive result, as evidenced by the green colour of the resin grains. This enabled moving to the next stage—attachment of the Fmoc-Thr (tBu)-OH amino acid residue.
Attachment of the Amino Acid Derivative:
[0173] The process of coupling was preceded by washing the resin in DMF. The composition of the coupling mixture remained unchanged when attaching the protected glutamic acid residue.
[0174] At the end of each acylation, a solvent wash cycle was performed according to the given procedure, which was followed by a chloranil test for the presence of free amino groups in solution.
Solvent Wash Cycle:
[0175] DMF 3×2 minutes
IsOH 3×2 minutes
DCM 3×2 minutes
Chloranil Test:
[0176] The resin grains during the test carried out after the second acylation were colourless, which enabled moving to the next stage of the synthesis, i.e. the introduction of another protected amino acid derivative—threonine and 2-aminobenzoic acid molecule. The coupling processes followed the procedure discussed earlier.
[0177] Tests carried out after attaching the above-mentioned residues showed positive results: the resin grains were colourless. [0178] d) Removal of peptide from the carrier while maintaining side group protection
[0179] After completing the synthesis, the protected ABZ-Thr(tBu)-Th (tBu)-Ala-OH peptide was removed from the carrier, retaining the side protection with the mixture: acetic acid:TFE (trifluoroethanol):DCM (2:2:6, v/v/v) in a round-bottomed flask on a magnetic stirrer.
[0180] After 2 hours, the contents of the flask were filtered off under reduced pressure on sintered (Schott) funnels, washing with the astringent mixture. The solution was washed with hexane (1:10 v/v), evaporated under reduced pressure and then lyophilised. [0181] e) Chemical synthesis of paranitroanilide Arg derivatives
[0182] The mixed anhydrides method was used to synthesize Fmoc-Arg(Pbf)-pNA. In the first step, 2 mmol of Fmoc-Arg (Pbf) were dissolved in anhydrous tetrahydrofuran (THF) in the presence of 2 mmol of N-methylmorpholine (NMM). The carboxyl group of the amino acid derivative was activated with 2 mmol of isobutyl chloride. After 10 minutes of activation, 3 mmol of p-nitroaniline were added. Reactions were carried out for 2 hours at −15° C., and then for 24 hours at room temperature. After completing the reaction, the solvent evaporated and the dry residue was dissolved in ethyl acetate. The resulting solution was washed successively with saturated aqueous NaCl solution, 10% citric acid, and 5% sodium bicarbonate. The resulting solution was dried over anhydrous sodium sulphate, ethyl acetate was distilled off under reduced pressure, and the dry residue was dried in a vacuum desiccator over P.sub.2O.sub.5 and KOH. [0183] f) Coupling of the protected peptide with paranitroanilide Arg (Pbf)
[0184] The protected ABZ′-Thr(tBu).sup.2-Thr (tBu).sup.3-Ala.sup.4-OH peptide was dissolved in a small amount of DCM, and subsequently activated with TFFH (tetramethylfluoroformamide) for 30 minutes at 0° C. Then a catalytic amount of DMAP and Arg (Pbf)-pNA was added. The reaction was carried out for 24 hours at room temperature, after which the solvent evaporated. The resulting solution was poured with the mixture removing the side protection: TFA:phenol:water:TIPS (88:5:5:2, v/v/v/v) and mixed in a round bottom flask on a magnetic stirrer for 3 hours.
[0185] After this time, cold diethyl ether was added to the flask, and the resulting precipitate was centrifuged in a high speed centrifuge at 5,000 rpm for 20 minutes. The precipitate obtained after centrifugation was dissolved in water by means of ultrasound and then lyophilised.
Identity/Characteristics of a New Compound—HPLC Analysis, MS
[0186] HPLC conditions: RP Bio Wide Pore Supelco C8 250 mm 4 mm column, A phase system 0.1% TFA in water, B: 80% acetonitrile in A), flow rate 1 mL/min, UV detection at 226 nm. Obtaining of the compound was confirmed.
Assay Condition
[0187] The study on the application of new compounds was performed on a group of 25 patients diagnosed with prostate cancer. For this purpose, the compound with formula: ABZ-Trp-Glu-Gly-Asn-ANB-NH2 or ABZ-Trp-Glu-Gly-Asn-pNA was dissolved in dimethyl sulfoxide (at a concentration of 0.5 mg/mL); 50 μL of this solution was mixed with 120 μl buffer (200 mM Tris-HCl, pH 8.0) and 80 μL of urine of a person with prostate cancer. The measurement was made in a 96-well plate designed for measuring absorbance, and each sample was analysed in triplicate at 37° C. The duration of the measurement was 60 minutes. During the measurement, the wavelength characteristic of the released chromophore (ANB-NH2 or pNA) was monitored at 405 nm (range 380-430 nm).
[0188] As a result of the measurement, the colour of the solution increased over time in all urine samples from patients diagnosed with prostate cancer. The observed absorbance increase over time was different for each of the tested samples. A different effect was obtained for 15 samples of healthy people, as none of the 15 urine samples tested had an increase in absorbance in the diagnostic range.
[0189] The analysis confirmed the use of the compounds, according to the examples, in the diagnosis of prostate cancer. The mechanism of action of the new compound is based on its enzymatic hydrolysis in such a place, which leads to the release of free chromophore molecules, respectively, ANB-NH2—amide of 5-amino-2-nitrobenzoic acid