The subject of this invention is a new compound, the method for its production, a pharmaceutical solution which contains the new compound, a method for determining the presence of cancer, a cancer test kit and the use of hydrolysis of the new compound for cancer detection.

Solid-phase synthesis of peptides was first introduced in the 1980s by Bruce Merfield, who was awarded a Nobel Prize for his achievement.

Proteasome is a multi-catalytic complex of endopeptidase (EC 3.4.25.1), which is responsible for non-lysosomal removing of proteins which are damaged or incorrectly formed due to a mutation or external factors which cause oxidative stress. The compounds which have been examined until now and which enabled to denote the activity of proteasome 20S or 26S are short peptides which contain 4-5 amino acid residues. These amino acid residues contain a fluorescent group on the C-terminus. These compounds allow to determine the activity of proteasome, including the presence of proteasome or another proteolytic enzyme. When the C-terminus fluorescent group is released, it tends to be highly fluorescent. Examples of sequences for such fluorogenic substrates are described in the work by Ma. This kind of compounds was used to indicate the activity of proteasome in plasma. Leu-Leu-Val-Tyr-AMC, AMC is aminocoumarin.

The overview of literature indicates that the main subject of studies monitoring the level of protseasome activity are circulatory system cancers. The great majority of peptide derivatives which are proteasome substrates was described in laboratories like Promega or Molecular Probes. Alternatively, the measurement of proteasome concentration is performed using immunochemical techniques (ELISA tests), which are based on determining a particular proteasome subunit by a particular type of antibodies. However, this procedure is time-consuming and it leads to detecting the whole proteasome population, not its actively enzymatic fraction.

The examined and obtained compound, the marked peptide, is characterised by a high value of specificity constant (of 4,3xl0 ⁶ IVfW) and limits of determination and detection, proper for this kind of markings, which are 10 pM and 0.4 pM, respectively. These values are the evidence that the compound is hydrolysed by proteasome with high efficiency, moreover, even in the presence of 10 pM of this enzyme. The whole procedure of proteasome activity detection takes little time (30 minutes), is not a complex one (2 solutions need to be mixed), and little amount of an easily accessible biological material such as human urine is needed (the minimal volume is 50 μΐ). The basic advantage of the obtained compound and the method related to it is the possibility of diagnosing bladder cancer by using the procedure described below. A positive result (fluorescence growth in time) means that the urine contains active proteasome, and this strongly correlates with the presence of cancer.

The P. 408905 invention revealed the new compound, whose hydrolysis in the position no. 5 by proteasome 20s is used to detect cancer, particularly bladder cancer. However, the compound claimed in this invention is more selective than the previous one, which means it is more cancer-sensitive (3.2 more times sensitive in the determination limit and 12.5 times more sensitive in detection). Moreover, it can detect smaller amounts of proteasome 20s. The new compound is a peptide, which means it is not degradable.

The aim of this invention is to provide a new compound and methods which could be used to diagnose cancer, especially bladder cancer.

The subject of this application is the compound of the following formula: ABZ- Dap(02(Cbz))-Dap(01)-Dap(02)-Arg-ANB-NH2, the method for its synthesis, and its use. In the synthesis of this compound, new building blocks, which are derivatives of diamino acids (Dap), were used. They were modified by the functionalised residues of mono-ethylene or diethylene glycol (PEG) (their proposed general name is DAPEG (DAP+PEG)). An example of their synthesis is shown in diagram 1.

The subject of this invention is a new compound of the following formula:

ABZ'-Dap(02(Cbz)) ² -Dap(01) ³ -Dap(02) ⁴ -Arg ⁵ -ANB ⁶ -NH ₂ where:

ABZ stands for 2-aminobenzoic acid

DAP stands for diammopropanoic acid (Dap) derivatives, modified by the functionalised residues of mono-ethylene or diethylene glycol (PEG)

ANB stands for 5-amino-2-nitrobenzoic acid

Furthermore, the subject of this invention is the method for the production of the

aforementioned compound, which has the following stages: a. removing the Fmoc group from the amino group of TENTA GEL S RAM resin by shaking out with 20% solution of piperidine in DMF;

b. adding the ANB molecule to the carrier prepared using the above-mentioned

method;

c. in the next stage, attaching Fmoc-Arg(Pbf) to ANB-resin;

d. then removing the Fmoc group from the amino group Arg(Pbf)-ANB-resin using 20% solution of piperidine in DMF;

e. attaching Fmoc-Dap(Mtt) to the above-mentioned resin, using the standard procedure with HBTU and DIPEA (1 :2);

f. in the next step, removing the Mtt group from Fmoc-Dap(Mtt)-Arg(Pbf)- ANB-resin using 2% TFA solution in methylene chloride (DCM) with 1% of triisopropylsilane, blending time: 15 minutes;

g. after this period of time, adding 3 drops of trifluoro acetic acid (TFA) to the resin sample; observing the appearance of a yellow colour— the colour appeared due to the presence of free Mtt groups; h. repeating the procedure until the solution was colourless;

i, at the next stage, rinsing the resin three times with diisopropylethylamine solution in DMF (30%) and thus obtaining Fmoc-Dap-Arg(Pbf)-ANB-resin, to which PEG/DIPCI HOBt solution in DMF/NMP (1 :1, v/v) blend was then added;

j. performing the reaction of 02(Boc) residue acylation until the result of the Kaiser test was negative, which indicated that there were no free amino groups on the resin;

k. then removing the Fmoc group and attaching another Fmoc-Dap(Mtt) residue; the whole procedure was performed again while using different PEG residues— in the position no. 3 Ol(Boc) was used, while in the position no. 4 02(Cbz) was used;

1. completing synthesis of peptidomimetic by attaching the ABZ group;

m. removing the obtained peptidomimetic from the resin with simultaneous removing of the side casings from the resin, using TFA/phenol/triisopropylsilane/H ₂ 0 88:5:2:5, v/v), obtaining ABZ- Dap(02(Cbz))-Dap(01 )-Dap(02) Arg-ANB-NH ₂ .

The metod where at stage b), before attaching the ANB to the solid carrier, resin with a free amino group was prepared by rinsing it three times in a 5% solution of N- methylmorpholine (NMM) in DMF, then in DMF (also three times), next, in a round- bottom flask, ANB was dissolved in 40 ml of DMF and HBTU, DMAP, and DIPEA, were added in sequence in the following molar ratio:

ANB : HBTU : DMAP : DIPEA (3 : 3 : 2 : 6). This solution was mixed with resin, and the reaction was performed for 3 hours; afterwards, the resin was drained under reduced pressure and rinsed. The procedure of attaching ANB to the polymere was repeated twice.

Method where at stage c) the process was commenced by dissolving the ninefold excess of the particular derivative in pyridine (22.2 ml) (for l of peptidyl resin there are 10 ml of pyridine), then the mixture of aminoacid in the pyridine was mixed with a portion of resin with sedimented 5-amino-2-nitrobenzoic acid; then it was cooled in an ice bath (prepared by mixing 1 part by weight of ice, 1 part by weight of NH ₄ C1 and 1 part by weight of NaN0 ₃ ) to the temperature of -15° C. Then the ninefold excess of phosphoryl chloride (POCI3) was added, and the whole mixture was then stirred on a magnetic stirrer for 20 minutes. Then, the temperature of the reaction was brought to the room temperature for the next 30 minutes. Then, the flask with the mixture was placed in an oil bath and was heated for 6 hours in the temperature of 40 °C. After the completion of the reaction, the resin with the first amino acid residue attached was drained on a Schott funnel under reduced pressure. Then it was rinsed with methanol (MeOH) and chlorom ethane.

The subject of the invention is also the pharmaceutical solution for detecting cancer which contains an active substance and a buffer solution, where the new above- mentioned compound is the active substance and the buffer solution has the pH of 7-9. The solution where TRIS HC1 is the buffer solution.

The solution which detects cancer; in particular bladder cancer.

The subject of this invention is also the method of determining the presence of cancer, and it includes in vitro analysis of a human urine sample, to which a new compound, determined in claim 1, is added, and/or it incubates with the inhibitor of trypsic subunit PR671A.

The method, where in 1 minute, 60 minutes the measurement of the fluorescence intensity produced due to release of a fragment which contains the fluorescent tag ABZ- Dap(02(Cbz))-Dap(01)-Dap(02)-Arg, is performed.

The method which detects cancer; particularly bladder cancer.

Furthermore, the subject of the invention is the cancer test kit, which detects bladder cancer positively, and contains the new compound, described above, and the inhibitor of trypsic subunit PR671A.

The use of hydrolysis of the new compound described above in position 5 by proteasom 20s for cancer detection, in particular bladder cancer.

The tenns used above and in the description and patent claims have the following meaning:

ABZ - 2-aminobenzoic acid

ANB - 5-amino~2-nitrobenzoic acid

Dap - derivatives of diaminopropionic acids

DAPEG - the compound which contains Dap and PEG residues PEG - functionalised mono-ethylene or di ethylene glycol residues DIPCI - diisopropylcarbodiimide

PEG = (02(Cbz) - 8-(benzyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (02(Cbz). 01(Boc) - (5-(t-butyloxycarbonyI-amino)-3-oxapentanoic acid

Description of figures:

Fig.l - it presents the synthesis of a sample DAPEG residue

Fig.2 - it presents building blocks of the library which contain DAPEG residues.

Fig.3 - it presents an example of a synthesis of a compound which contains DAPEG residues

Fig.4 - it presents the chemical formula for the compound— a substrate selectively hydrolysed by trypsin-like subunit of proteasome 20S.

Fig.4 A - it presents the mass spectrum of the ABZ-Dap(02(Cbz))-Dap(01)- Dap(02)Arg-ANB-NH ₂ compound (molecular mass: 1283). 1282.6 ions (pseudo- molecular) and the fragment ion M- 15 are present.

Fig.5 - it presents the influence of the inhibitors on the level of hydrolysis of the compound, where PR523A - carfilzomib, inhibitor of chymotryp sin-like proteasome 20S subunit, NCOOl inhibitor of caspase-like subunit of proteasome 20S, PR671A inhibitor of trypsin-like subunit of proteasome 20S, epoxomicin inliibitor of all the three activities of proteasome 20S.

Fig.6 - it presents the titration curve of the compound by reducing the concentration of proteasome 20S. The obtained limit of detection was 10 ^{~! 1} M, which corresponds to 5xl0 ^"16 g.

Fig.7 - it presents the hydrolysis of the ABZ-Dap(02(Cbz))-Dap(01)-Dap(02)-Arg- ANB-NH2 compound in the presence of proteasome 20S. Time A) 1 minute B) 60 minutes. Peak with retention time of 16.15 is the ABZ-Dap(Cbz)-Dap(0)l-Dap(Cbz)- Arg-ANB-NH ₂ compound, peak with retention time of 15.23 is a fragment of ABZ- Dap(Cbz)-Dap(01)-Dap(Cbz)-Arg-OH. Fig.8 - it presents the hydrolysis of the ABZ-Dap(02(Cbz))-Dap(01)-Dap(02)-Arg- ANB-NH2 compound in human urine (A) healthy (B) diagnosed with bladder cancer. Peak with retention time of 16.15 is an ABZ-Dap(02(Cbz))-Dap(01)-Dap(02)-Arg- ANB-NH2 compound, peak with a retention time of 15.23 is a fragment of ABZ- Dap(02Cbz)-Dap(01)-Dap(02)-Arg-OH.

Fig.9 - it presents the influence of the inhibitors on the hydrolysis of the compound by the urine sample with cancer diagnosis.

Fig.10 - it presents the preliminary diagnosis of bladder cancer. Samples 1-21 are taken from patients diagnosed with bladder cancer. 22-28 are control samples taken from healthy patients.

Fig.ll - it presents the SDS PAGE gel, coloured with silver (A) Western blot analysis (B) of samples taken from healthy patients (22-24) and patients with cancer (2,5,8,10,14,16,20,21). Murine monoclonal antibody anti-20S IgGl was used as the primary antibody, and IgG-HRP rabbit anti-mouse antibody as the secondary one. The signal was obtained by using the chemiluminescent substrate.

The invention is illustrated by the following example of production, which does not limit it.

Example

Synthesis of peptidodimetic ABZ-Dapf02fCbz))-Papf01)-Dapf02VArg-ANB-

The synthesis of peptidodimetic ABZ-Dap(02(Cbz))-Dap(01)-Dap(02)-Arg-ANB- N¾, is divided into stages:

1 ) The first one is to remove the Fmoc group from the amino group of TENTA GEL S RAM resin through shaking out with 20% solution of piperidine in DMF. In the next step, the ANB (5-amino 2-nitrobenzoic acid) molecule is attached to the carrier prepared this way. Prior to attaching ANB to the solid carrier, the resin with the free amino group was prepared by rinsing it three times with 5% solution of N-methylmorpholine (NMM) in DMF, then in DMF (also three times), next, in a round-bottom flask, ANB was dissolved in 40 ml of DMF and HBTU, DMAP, and DIPEA were added in sequence in the following molar ratio:

ANB : HBTU : DMAP : DIPEA (3 : 3 : 2 : 6). This solution was mixed with resin, and the reaction was performed for 3 hours; afterwards, the resin was drained under reduced pressure and rinsed. The procedure of attaching ANB to the polymere was repeated twice.

) In the next step, Fmoc-Arg(Pbf) was added to the ANB-resin. The process was commenced by dissolving the ninefold excess of the particular derivative in pyridine (22.2 ml) (for Ig of peptidyl resin there are 10 ml of pyridine), then the mixture of the amino acid in pyridine was mixed with a portion of resin with sedimented 5-amino-2-nitrobenzoic acid; then it was cooled in an ice bath (prepared by mixing 1 part by weight of ice, 1 part b weight of NH ₄ C1 and 1 part by weight of NaN0 ₃ ) to the temperature of -15° C. Then ninefold excess of phosphoryl chloride (POCl ₃ ) was added, and the whole mixture was then stirred on a magnetic stirrer for 20 minutes. Then, the reaction was brought to the room temperature for the next 30 minutes. Then the flask with the mixture was placed in an oil bath and heated for 6 hours in the temperature of 40 °C. After the completion of the reaction, the resin with the first attached amino acid residue was drained on a Schott funnel under reduced pressure. Then it was rinsed with methanol (MeOH) and chloromethane.

Then the Fmoc group was removed from the amino group Arg(Pbf)-ANB-resin using 20% solution of piperidine in the DMF. ) Fmoc-Dap(Mtt) was then attached to the above-mentioned resin, using the standard procedure with HBTU and DIPEA (1 :2). In the next step, the Mtt group was removed from Fmoc-Dap(Mtt)-Arg(Pbf)-ANB-resin using the 2% TFA solution in methylene chloride (DCM) with 1% of triisopropylsilane, blending time: 15 minutes, after this period of time, 3 drops of trifiuoro acetic acid (TFA) were added to the resin sample; the appearance of a yellow colour was observed — the colour appeared due to the presence of free Mtt groups. The procedure was repeated until the solution was colourless.

) At the next stage, the resin was rinsed three times with the diisopropylethylamine solution in DMF (30%) and thus Fmoc-Dap-Arg(Pbf)- ANB-resin was obtained, to which the PEG/DIPCI/HOBt solution in the DMF/NMP (1 :1, v/v) mixture was then added, where DIPCI stands for diisopropylcarbodiimide and PEG is 8-(tertbutyloksyl-amino)-3,6~dioxaoctanoic acid (02(Boc). The reaction of residue acylation was performed until the result of the Kaiser test was negative, which indicated that there were no free amino groups on the resin. Then, the Fmoc group was removed and another Fmoc- Dap(Mtt) residue was attached; the whole procedure was performed again while using different PEG residues— in the position no. 3 Ol(Boc) was used, while in the position no. 4 02(Cbz) was used.

5) the synthesis of the peptidomimetic was completed by attaching the ABZ group.

The obtained peptidomimetic was then removed from the resin with simultaneous removing of the side casings from the resin, using TFA/phenol/triisopropylsilane/H20 88:5:2:5, v/v), obtaining ABZ-Dap(02(Cbz))-Dap(01)-Dap(02)Arg-ANB-NH ₂ .

New compound identity/characteristics

The obtained compound undergoes a selective hydrolysis by the human trypsin subunit of proteasome 20S. The results of the experiment in which the obtained compound was incubated with proteasome 20S with selectively blocked enzyme subunits as: PR523A, carfilzomib selective chymotrypsin subunits inhibitors, NCOOl selective caspare subunit inhibitor, and PR671 A trypsin subunit inhibitor— all clearly indicate that there is no hydrolysis of the compound (no fluorescence growth) for the system only when the PR671A inhibitor is used (Fig. 5). Moreover, lack of activity of proteasome incubated in the presence of epoxomicin (inliibitor of all the three units) confirms that the elaborated compound is hydrolysed by proteasome 20S.

Tirating the obtained compound with decreasing amounts of proteasome 20S (Fig. 6) indicates that a noticeable growth of fluorescence (signal to noise ratio is 3:1) is observed at the concentration of 10 pM, which is 5x10 ^"16 g of proteasome in the probe.

Fig. 7 illustrates (A) a HPLC RP analysis of the obtained compound, with the signals at about 10 ^th minute coming from the buffer ingredient (buffer is 50 mM TRIS-HCl pH 8.2 with 0.02% of SDS (sodium dodecyl sulphate), where (B) HPLC RP analysis of the incubated compound in the presence of commercially available human proteasome 20S for 60 minutes was used. Fig. 8. illustrates a similar analysis, but healthy human urine samples (A) and urine samples of a patient diagnosed with bladder cancer (B) replaced proteasome 20S. The analysis of both Figures clearly indicates that the obtained compound, when exposed to human proteasome 20S, decomposes into two f agments ANB-NH2 and fluorescent ABZ-Dap(02(Cbz)-Dap(01)-Dap(02)-Arg-OH.

This is confirmed by figure 9, where the probe from fig. 8B (bladder cancer) and 8A (cancer- free) was analysed, and 8B incubated with trypsin subunit inhibitor P 671A. The results obtained clearly indicate that there is no decomposition of the analysed compound in a healthy patient's urine, whereas in the urine of a patient diagnosed with cancer the compound decomposes. Furthermore, this decomposition is hindered by selective trypsin subunit inhibitor.

During the next stage, 21 samples of urine from patients diagnosed with bladder cancer, and 8 cancer-free samples were analysed. The results of such an analysis indicate that the fluorescence grew in 20 out of 21 samples with urine of patients diagnosed with bladder cancer.

Selected samples were analysed using immunochemical techniques (Western blot), where proteasome 20S was subject to detection. Figure 11 indicates that the positive response (the presence of proteasome) was obtained for all samples taken from patients diagnosed with cancer, whereas in healthy patients, this enzyme was not found.

Literature:

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2. Groll M, Bochtler M., Brandstetter H., Clausen T., Huber R., Molecular machines for protein degradation, ChemBioChem., 6, 222-256, 2005.

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4. Ostrowska H, Hempel D, Holub M, Sokolowski J, Kloczko J. Assessment of circulating proteasome chymotrypsin-like activity in plasma of patients with acute and chronic leukemias. Clin Biochem. 2008 Nov;41(16-17):1377-83

5. www.promega.com Ma W, Kantarjian H, O'Brien S, Jilani I, Zhang X, Estrov Z, et al. Enzymatic activity of circulating proteasomes correlates with clinical behavior in patients with chronic lymphocytic leukemia. Cancer 2008;112: 1306-12.