WO2020017984A1 - Composés chimiques destinés à être utilisés en tant que marqueurs de diagnostic d'une inflammation et de néoplasmes - Google Patents

Composés chimiques destinés à être utilisés en tant que marqueurs de diagnostic d'une inflammation et de néoplasmes Download PDF

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WO2020017984A1
WO2020017984A1 PCT/PL2019/050004 PL2019050004W WO2020017984A1 WO 2020017984 A1 WO2020017984 A1 WO 2020017984A1 PL 2019050004 W PL2019050004 W PL 2019050004W WO 2020017984 A1 WO2020017984 A1 WO 2020017984A1
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abz
pna
val
denotes
resin
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Adam LESNER
Natalia Gruba
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Uniwersytet Gdanski
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the field of the invention is diagnosis in vitro of inflammatory processes, the mechanism initiates in vivo as a result of immune responses of immune system blood cells and diagnostics of neoplasms in vitro, especially epithelial neoplasms in particular tumors.
  • the invention relates to markers for use in in vitro diagnosis of inflammatory processes and diagnostic markers for use in in vitro diagnosis of epithelial neoplasms at a very early stage.
  • the invention relates to compounds for diagnostic use - for use in diagnosis of inflammatory conditions and in the early diagnosis of epithelial neoplasms, wherein these compounds are used as a diagnostic agent - diagnostic marker - to detect the pathophysiological process in the biological material of mammals, especially human blood and urine in vitro.
  • the invention relates to a diagnostic kit containing at least two new compounds - a diagnostic reagent for use in detecting immune changes, including predicting the development of epithelial cell neoplasms at a very early stage of the disease, even before the process of neoplasm processes - in diagnosis of inflammatory process, or for use in diagnosis of initiated neoplasms process with simultaneous inflammatory reaction, and a marker for use in the detection of additional inflammatory processes regardless of presence of epithelial neoplasms, e.g. bacterial or viral infection that is additionally present.
  • the invention also relates to a method for the preparation of new compounds based on a chain of amino acids while the compounds are used for diagnosis of inflammatory processes and / or neoplasms especially epithelial neoplasms.
  • the object of the invention is to provide a sensitive and specific diagnostic markers for use in diagnosis of a neoplasm in particular tumor, epithelial cancer and including bladder cancer, urinary tract cancer, urethral cancer or other cancers accompanied by an increase in proteolytic enzymes, and which can be observed in the disease process in vitro in the biological material, especially the urine of the patient, at a very early stage of pathogenesis, i.e. even at the inflammatory reaction stage that are direct cause of neoplasm formation.
  • the new amino acid-containing compounds developed during the invention study are useful as a diagnostic markers that only in the presence of proteolytic enzymes that are the result of an inflammatory reaction and / or neoplasms e.g.
  • epithelial neoplasms, tumor, and which enzymes are released and present in the biological material exhibit chromogenic properties and/or fluorogenic measured in the 300-500 nm waveband, especially 380-430.
  • the color properties are observed during the enzymatic hydrolysis of these compounds in the biological material in vitro, especially the urine and blood of the patient in whom the epithelial neoplasms is present or is during formation, including the inflammatory process or whether the inflammatory response itself or an inflammatory response additionally accompanied by neoplastic disease has developed. In the case of neoplasm, this process is associated with the effects of neoplasms - cancer, i.e.
  • Part of the compounds according to the invention makes it possible to detect even the already inflammatory process, also by hydrolysing process, including the release of enzymes from the inflammatory focus during the only immunological process, which involves triggering a color reaction detected by measuring the absorbance in the 300-500 nm wavelength range, especially 380-430.
  • the study also developed a compound of the formula ABZ-Met-Lys-Val-Trp-pNA, where ABZ is 2-aminobenzoic acid and pNA is para-nitroaniline, while the compound is for use as marker for detecting neoplasms, in particular epithelial neoplasms, cancer.
  • the inflammatory process detected by the compounds mentioned above as first may also be correlated at the time of the appearance of the neoplasm around which the inflammation occurs. Detection of inflammation may also be useful in the case of infection with bacteria or viruses in the presence or absence of neoplasms in particular epithelial neoplasms.
  • Both processes diagnosed by the proposed compounds are accompanied by / results of enzymatic hydrolysis of the markers - as a result of the release of enzymes during immunological processes - inflammatory process, taking place in vivo and/or proteolytic enzymes released as a result of the development of neoplasms in particular epithelial neoplasms including cancer.
  • This process is observed by monitoring the released ANB- NH 2 molecule (5-amino-2-benzoic acid amide) or pNA (p-nitroaniline) at a wavelength of 300-400, preferably 380-430 nm, respectively, that is, hydrolysis occurs within the 5th position of the compound - after the 5th amino acid in the sequence.
  • the present invention provides a compound defined by the general formula: ABZ 1 -Leu 2 -Glu 3 -Pro 4 -Val 5 -X 6 , wherein: ABZ denotes 2-aminobenzoic acid, X is ANB- H 2 or pNA, where ANB-NH 2 denotes 5-amino-2-benzoic acid amide, ANB - 5-amino-2-benzoic acid and pNA denotes 4-nitroaniline.
  • the present invention also provides a compound of the formula: ABZ 1 -Met 2 -Lys 3 -Val 4 -Trp 5 -pNA 6 , wherein: ABZ is 2-aminobenzoic acid, and pNA means 4-nitroaniline.
  • the present invention also provides a process for the preparation of ABZ 1 -Leu 2 -Glu 3 -Pro 4 -Val 5 -A]S[B-]SlH2 6 , where: ABZ is 2-aminobenzoic acid, ANB-NH2 is 5-amino-2-benzoic acid amide, and this compound is obtained in the synthesis on a solid support and the method is carried out in the following steps: a) preparation of a resin enabling the conversion of an acid into an amide, b) deprotection - removal of the protective group,
  • the invention also relates to a process for the preparation of the compound ABZ 1 -Leu 2 -Glu 3 -Pro 4 -Val 5 -pNA 6 , where: ABZ is 2-aminobenzoic acid, pNA is 4-nitroaniline, and the compound is obtained in the synthesis partly on solid support and partly in buffer or only on a solid support, and the method is carried out in the following steps: a) preparation of 2-chloro-chlorotrityl resin,
  • the invention also relates to a process for the preparation of the compound ABZ 1 -Met 2 -Lys J -Val 4 -Trp 5 -pNA 6 , where: ABZ is 2-aminobenzoic acid and pNA is 4-nitroaniline, and the compound is obtained in solid phase synthesis and partly in buffer or only on a solid support and the method is carried out in the following steps: a) preparation of 2-chloro-chlorotrityl resin,
  • the subject of the invention is a diagnostic marker of the general formula ABZ 1 -Leu 2 -Glu 3 -Pro 4 -Val 5 -X 6 , where: ABZ is 2-aminobenzoic acid, X is ANB-NH 2 or pNA, where ANB-NH 2 is 5-amino-2-benzoic acid amide, pNA is 4-nitroaniline, for use in the in vitro diagnosis of inflammation and/or neoplasms in particular cancer, especially epithelial neoplasms.
  • the subject of the invention is a diagnostic marker of the general formula ABZ 1 -Met 2 -Lys 3 -Val 4 -Trp 5 -pNA 6 , where: ABZ is 2-aminobenzoic acid and pNA is
  • 4-nitroaniline for use in the in vitro diagnosis of neoplasms in particular cancer, especially epithelial neoplasms.
  • the subject of the invention is a diagnostic kit for use in the in vitro early diagnosis of neoplasm, especially epithelial neoplasms and/or inflammatory disease, comprising at least one compound of general formula ABZ 1 -Leu 2 -Glu 3 -Pro 4 -Val 5 -X 6 and at least one compound of formula ABZ’-Mer-Lys’-Va ⁇ -Trp ⁇ -pNA 6 , where: ABZ is 2-aminobenzoic acid, X is ANB-NH 2 or pNA, where ANB-NH 2 is
  • 5-amino-2-benzoic acid amide, pNA is 4-nitroaniline.
  • the subject of the invention is a method for in viti'o diagnosing epithelial cancer and/or an inflammatory process in a mammalian biological material, characterized in that as a diagnostic marker, at least one compound of the general formula ABZ 1 -Leu 2 -Glu 3 -Pro 4 -Val 5 -X 6 is for use a marker of the immune response and additionally for diagnosis of neoplasms, where: ABZ is 2-aminobenzoic acid, X is ANB-NH 2 or pNA, where ANB-NH 2 is 5-amino-2-benzoic acid amide, pNA is 4-nitroaniline.
  • This compound is added and incubated in the biological material of the mammal, especially urine or human blood, then absorbance is measured at a wavelength in the range of 300 to 500 nm. Demonstration of color and increase in absorbance indicates a positive result.
  • the color is the result of the enzymatic hydrolysis of the compound especially in the position 5 - after the fifth amino acid in turn, under the influence of proteolytic enzymes present in the biological material in vitro as a result of an immune response and/or neoplasms in vivo.
  • the absorbance reading time is determined depending on the amount of compound added. The reading can take place after about 60 minutes.
  • the absorbance is measured at 380-430 nm.
  • the subject of the invention is a method for in vitro diagnosing of epithelial neoplasm in biological material of a mammal, characterized according to the invention in that at least one compound of the general formula ABZ 1 -Met 2 -Lys 3 -Val 4 -Trp 5 -pNA 6 is for use as a marker of neoplasm - the diagnostic marker; where: ABZ is 2-aminobenzoic acid, pNA is 4-nitroaniline.
  • the compound is added to the biological material, then absorbance is measured at a wavelength in the range of 300 to 500 nm. Demonstration of color and increase in absorbance indicates a positive result.
  • the color is the result of the enzymatic hydrolysis of the compound, especially at the position 5 - after the fifth amino acid in turn, under the influence of proteolytic enzymes present in the biological material in vitro as a result of the in vivo tumor-related process.
  • the absorbance reading time is determined depending on the amount of compound added. The reading can take place after about 60 minutes.
  • the absorbance is measured at 380-430 nm.
  • the method for in vitro diagnosing and differentiating epithelial neoplasms and the inflammatory process in a mammalian biological material is characterized in that at least one compound of the general formula ABZ 1 -Leu 2 -Glu 3 -Pro 4 -Val 5 -X 6 is for use as a diagnostic marker - the marker for use in diagnosing of the immune response and in addition a neoplasm, and at least one compound of the general formula ABZ 1 -Met 2 -Lys 3 -Val 4 -Trp 5 -pNA 6 for use as a marker for neoplasm diagnosing; wherein ABZ is 2-aminobenzoic acid, pNA is 4-nitroaniline.
  • the compounds are added to the biological material, each into a separate sample, then absorbance is measured at a wavelength in the range of 300 to 500 nm.
  • the increase in absorbance indicates a positive result.
  • the color is the result of the enzymatic hydrolysis of the compound especially in the position 5 - after the fifth amino acid in turn, under the influence of proteolytic enzymes present in the biological material in vitro as a result of an immune response and/or tumor in vivo.
  • the absorbance reading time is determined depending on the amount of compound added. The reading can take place after about 60 minutes.
  • the absorbance is measured at 380-430 nm.
  • a method for the preparation of ABZ 1 -Leu 2 -Glu 3 -Pro 4 -Val 5 -ANB-]S!H2 6 wherein: ABZ is 2-aminobenzoic acid, ANB-NFL is 5-amino-2-benzoic acid amide, according to the embodiment, the method is carried out in the following stages: swelling of the amide resin and:
  • a method for the preparation of the compound ABZ 1 -Leu 2 -Glu 3 -Pro 4 -Val 3 -pNA 6 wherein: ABZ is 2-aminobenzoic acid, pNA means para-nitroaniline, according to embodiment is carried out in the following steps: a) swelling process of 2-chloro-chlorotritric resin
  • a compound of the formula ABZ 1 -Met 2 -Lys 3 -Val 4 -Trp 5 -pNA 6 is obtained, wherein: ABZ is 2-aminobenzoic acid and pNA is 4-nitroaniline using other amino acids.
  • the compound is obtained in the synthesis on a solid support and the method is carried out in the following steps: a) preparation of 2-chloro-chlorotrityl resin,
  • the invention enables the detection of epithelial neoplasm at an early stage of development - in biological material in vitro - especially urine or blood.
  • the invention additionally allows for the diagnosis of an immune response independently or in addition to neoplams.
  • Epithelial neoplasms in particular tumor - means the one whose cells originate from the epithelium of the external and internal secretion glands and from the glands of these glands, e.g. epithelial tumor of the urinary tract.
  • Chromogenic properties - means the formation of a colored product
  • Fluorogenic properties - means the formation of a fluorescent product
  • Fig. 1 - shows the rate of hydrolysis of the ABZ-Leu-Glu-Pro-Val-ANB-NH 2 substrate in urine samples diagnosed with neoplasm. Arabic numerals are the number of the randomly selected urine sample.
  • Fig. 2 - shows the rate of hydrolysis of the ABZ-Leu-Glu-Pro-Val-pNA substrate in urine samples diagnosed with neoplasm. Arabic numerals are the number of the randomly selected urine sample.
  • Fig. 3 - shows the hydrolysis rates of the ABZ-Leu-Glu-Pro-Val-ANB-NFp substrate depending on the presence of an inhibitor of human neutrophilic elstasis, which enzyme is a cross-fertilization marker in a human urine sample with confirmed inflammation and/or neoplasm of the epithelium of the urinary tract.
  • Fig. 4 - shows the dependence of the degree of hydrolysis of the ABZ-Leu-Glu-Pro-Val-ANB NH 2 substrate from the pH environment
  • Fig. 5 - shows the hydrolysis efficiency of the ABZ-Leu-Glu-Pro-Val-ANB-NH 2 substrate depending on the degree of compaction of human urine with the confirmed urinary epithelial carcinoma.
  • the first stage of the synthesis was to obtain a chromogenic peptide that was obtained by solid phase synthesis - on a solid support - using Fmoc/tBu chemistry, i.e. using covers.
  • an amide resin e.g. TentaGel S RAM from RAPP Polymere (Germany) e.g. with a deposition of 0.23 mmol/g.
  • the compound was synthesized in a manual manner using a laboratory shaker.
  • the reactors were a syringe ended with sinter for synthesis in a solid phase with a capacity of 25 ml.
  • the peptide synthesis was performed on a Rapp Polymere resin TentaGel S RAM with a deposition of 0.23 mmol/g.
  • a resin was prepared, including the loosening of the resin through a series of washes.
  • Subsequent removal of the amine Fmoc protecting group from the carrier was carried out with a 20% piperidine solution in NMP.
  • a cycle of solvent washes was carried out.
  • a chloranil test was carried out.
  • the chloranil test consisted of transferring, by means of a spatula, several resin beads from the reactor - a syringe into a glass ampoule, to which was then added IOOmI of a saturated solution of p-chloranil in toluene and 50m1 of fresh acetaldehyde. After 10 minutes, the color control of the grains was carried out.
  • the first step in the synthesis of the peptide library - a mixture of peptides was the deposition of ANB per lg of resin. Prior to attaching the chromophore, the resin used for the reaction was washed with the following solvents: DMF, DCM and again DMF, after which the Fmoc-shield was removed from the carrier functional group.
  • One Fmoc shield removal cycle included the following steps:
  • the corresponding amino acid derivative (9-fold molar excess relative to the resin deposition) was dissolved in pyridine and transferred to a flask containing ANB embedded resin. The whole was cooled to -l5°C (ice bath: 1 part NH 4 CI weight, 1 part NaN0 3 weight, 1 part ice weight). After reaching the desired temperature, POCI 3 (in a 1 : 1 ratio to the amount of amino acid derivative used) was added and the mixture 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 completion of the reaction, the resin was filtered under reduced pressure, washed with DMF and MeOH and allowed to dry.
  • the resin together with the attached ANB residue in the reactor was washed with DMF and then the Fmoc shield from the amine group was deprotected to attach the protected amino acid derivative of glycine.
  • the ABZ-Leu-Glu-Pro-Val-ANB-NH 2 peptide amide was removed from the support with the simultaneous removal of the side shields by means of a mixture of: TFA : phenol : water : TIPS (88 : 5 : 5 : 2, v / v / v / v) in a round bottom flask on a magnetic stirrer.
  • TFA in water B 80% acetonitrile in A), flow 1 ml/min, UV detection at 226 nm.
  • the chloranil test consisted of transferring, by means of a spatula, several resin beads from the reactor - a syringe into a glass ampoule, to which was then added 100 pl of a saturated solution of p-chloranil in toluene and 50 m ⁇ of fresh acetaldehyde. After 10 minutes, the color control of the grains was carried out.
  • the first step in the synthesis of the peptide library was the deposition of Fmoc-Pro on lg of resin. Prior to attachment of the amino acid derivative, the resin used to react with the following solvents was washed: DMF (dimethylformamide), DCM (methylene chloride) and again DMF, after which the Fmoc shield was removed from the carrier functional group.
  • DMF dimethylformamide
  • DCM methylene chloride
  • the resin together with the attached Fmoc-Pro residue in the reactor was washed with DMF and then the Fmoc shield from the amine group was deprotected to attach the protected amino acid derivative of glutamic acid.
  • the protected ABZ-Leu-Glu (OtBu) -Pro-OH peptide was removed from the carrier together with retaining the side shields with a mixture of acetic acid: TFE (trifluoroethanol):DCM (2:2:6, v/v/v) ) in a round bottom flask on a magnetic stirrer.
  • the method of mixed anhydrides was used to synthesize Fmoc-Val-pNA.
  • 2 mmol Fmoc-Val was dissolved in anhydrous tetrahydrofuran (THF) in the presence of 2 mmol N-methylmorpholine (NMM).
  • NMM N-methylmorpholine
  • the carboxyl group of the amino acid derivative was activated with 2 mmol of isobutyl chloride.
  • 3 mmoles of p- nitroaniline was added. Reactions were carried out for 2 hours at -15°C, followed by a day at room temperature. After the completion of the reaction, the solvent was evaporated and the dry residue was dissolved in ethyl acetate.
  • the resulting solution was washed successively with saturated aqueous NaCl, 10% citric acid, 5% sodium bicarbonate.
  • the solution obtained was dried over anhydrous sodium sulfate, the ethyl acetate was distilled off under reduced pressure, and the dried residue was dried in a vacuum desiccator over P 2 0 5 and KOH.
  • the protected ABZ'-Leu ⁇ Glu (OtBu) 3 -Pro 4 -OH peptide was dissolved in a small amount of DCM, subsequently activated with TFFH (tetramethylfluoroformamide) for 30 minutes at 0 ° C. Then a catalytic amount of DMAP and Fmoc-Val-pNA was added.
  • TFFH tetramethylfluoroformamide
  • HPLC conditions RP Bio Wide Pore Supelco C8 column 250 mm 4 mm, phase system A
  • the incubation time is 60 minutes
  • the diagnostic marker obtained according to example 1 or 2 was added to the urine with a diagnosed inflammation, confirmed by the detection of a marker of inflammation such as neutrophil elastase.
  • a marker of inflammation such as neutrophil elastase.
  • this marker can be used as a marker of inflammation, as well as a marker of inflammation and epithelial neoplasms, cancer, in the presence of neoplasms/cancer and confirmed inflammation.
  • the color was observed - absorbance reading at a wavelength around 410 nm - in the 380-430 nm range.
  • the proteolytic activity of the enzymes present in the examined biological material is proportional to the intensity of the observed absorbance signal released by the hydrolysis of the ANB peptide bond.
  • substrate hydrolysis of ABZ 1 -Leu 2 -Glu 3 -Pro 4 -Val 5 -A B-NH2 6 takes place at position 5, where we obtain ABZ 1 -Leu 2 -Glu 3 -Pro 4 -Val 5 and ANB-NH 2 6 .
  • proteolytic enzymes Proteolytic activity is dependent on the presence of specific compounds called inhibitors (proteolytic enzymes). Their incubation with a given biological material allows us to pre- determine the class of enzymes present in the tested samples. For this purpose, we have added effective inhibitory concentrations of certain classes of proteolytic enzymes to the combined urine samples of the ill persons - patients (urothelial epithelial neoplasms, in particular tumor). The result of this experiment shows figure 3.
  • Carfizomib also inhibits proteolysis to some extent, hence the conclusion that the enzymatic activity in the urine of people diagnosed with bladder neoplasms, in particular cancer is caused by the enzyme (s) belonging to a different type from that listed in the table above (fig. 3).
  • the obtained biological material human urine with a confirmed urinary tract epithelium
  • Amicon filter columns were used.
  • a four times folded material with activity about 2 times higher than the starting material fig. 5
  • the fact of a linear response to the increasing concentration of the enzyme confirms the selectivity of the compound obtained by us.
  • example 1 and 2 are for use in diagnosis - their use in the diagnosis of inflammation and in the diagnosis of epithelial neoplasms, in particular cancer.
  • Preliminary results indicate that these compounds may be used to identify inflammation - an immune response that accompanies epithelial neoplasm, cancer as well as to diagnose an additional inflammatory condition e.g. caused by an infection that accompanies neoplasm or in the absence of neoplasms, e.g. cancer, i.e. the recognition of the immune response itself, which, e.g., can lead to a disease process including neoplasm, in particular cancer.
  • an additional inflammatory condition e.g. caused by an infection that accompanies neoplasm or in the absence of neoplasms, e.g. cancer, i.e. the recognition of the immune response itself, which, e.g., can lead to a disease process including neoplasm, in particular cancer.
  • the mechanism of action of the new compounds - diagnostic markers - according to the invention is based on enzymatic hydrolysis at a peptide site, thereby liberating the A B-TS(H 2 -5-amino-2-nitrobenzoic acid amide or pNA, which has an absorbance at a wavelength of 300-500 nm, especially 380-430 nm.
  • Example 4
  • ABZ means 2-aminobenzoic acid
  • pNA means 4-nitroaniline.
  • the first step in the synthesis was to obtain a protected peptide that was obtained by solid phase synthesis using Fmoc/tBu chemistry.
  • the synthesis of the compound was carried out manually using a laboratory shaker.
  • the peptide synthesis was performed on a 2-chloro-chlorotinyl resin from Iris BIOTECH GMBH (Germany) with a deposition of 1.6 mmol Cl /g.
  • the resin was loosened through a series of washes.
  • Subsequent removal of the amine Fmoc protecting group from the carrier was carried out with a 20% piperidine solution in NMP.
  • a cycle of solvent washes was carried out.
  • a chloranil test was carried out.
  • the chloranil test consisted of transferring, by means of a spatula, several resin beads from the reactor - a syringe into a glass ampoule, to which was then added 100 pl of a saturated solution of p-chloranil in toluene and 50 m ⁇ of fresh acetaldehyde. After 10 minutes, the color control of the grains was carried out.
  • the first step in the synthesis of the peptide library was the deposition of Fmoc-Val on lg of resin. Prior to attachment of the amino acid derivative, the resin used to react with the following solvents was washed: DMF (dimethylformamide), DCM (methylene chloride) and again DMF, after which the Fmoc shield was removed from the carrier functional group.
  • DMF dimethylformamide
  • DCM methylene chloride
  • the resin together with the attached Fmoc-Val residue in the reactor was washed with DMF and then the Fmoc shield from the amine group was deprotected to attach the protected amino acid derivative of glutamic acid.
  • the protected ABZ-Met-Lys (Boc)-Val-OH peptide was removed from the carrier together with retaining the side shields with a mixture of: acetic acid: TFE (trifluoroethanol):DCM (2:2:6, v/v/v) in a round bottom flask on a magnetic stirrer.
  • the method of mixed anhydrides was used to synthesize Fmoc-Trp-pNA.
  • 2 mmol Fmoc-Trp was dissolved in anhydrous tetrahydrofuran (THF) in the presence of 2 mmol N-methylmorpholine (NMM).
  • NMM N-methylmorpholine
  • the carboxyl group of the amino acid derivative was activated with 2 mmol of isobutyl chloride. After 10 minutes of activation, 3 mmoles of p- nitroaniline was added. Reactions were carried out for 2 hours at -l5°C, followed by a day at room temperature. After the completion of the reaction, the solvent was evaporated and the dry residue was dissolved in ethyl acetate.
  • HPLC conditions RP Bio Wide Pore Supelco C8 column 250 mm 4 mm, phase system A
  • the ABZ-Met-Lys-Val-Trp-pNA compound was analyzed for use in the diagnosis of epithelial neoplasm similar to that described in example 3.
  • the analysis was carried out by assessing the protolytic activity of human urine in samples of ill persons: with confirmed epithelial neoplasms, in particular cancer and in the control group: without inflammation and cancer.
  • the diagnostic kit contains at least two compounds: markers which are described in the above examples, in that the kit contains at least one compound A - a marker of inflammation and epithelial neoplasm with the general formula ABZ 1 -Leu 2 -Glu 3 -Pro 4 -Val 5 -X 6 and compound B - epithelial neoplasm marker of the formula ABZ'-Me ⁇ -Lys’-Vaf ⁇ -Tipri-pNA, where: ABZ is 2-aminobenzoic acid, pNA means p-nitroaniline.
  • ABZ 2-aminobenzoic acid
  • pNA means p-nitroaniline.
  • the measurement of the increase in absorbance was monitored in the 380-430 nm range, especially 410 nm, for 60 minutes.

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Abstract

La présente invention concerne un composé de formule générale : ABZ1-Leu2-Glu3-Pro4-Val5-X6, dans laquelle : ABZ représente l'acide 2-aminobenzoïque, X représente ANB-NH2 ou pNA, ANB-NH2 étant un amide d'acide 5-amino-2-benzoïque, pNA étant 4-nitroaniline. L'invention concerne un composé de formule générale : ABZ1-Met2-Lys3-Vai4-Trp5-pNA6, dans laquelle : ABZ représente l'acide 2-aminobenzoïque, pNA représente 4-Nitroaniline. L'invention concerne l'utilisation diagnostique des composés pour le diagnostic in vitro de néoplasmes et/ou d'inflammation, un procédé de préparation de ces composés, et un kit de diagnostic contenant des marqueurs destinés à être utilisés dans le diagnostic in vitro d'un néoplasme et d'une inflammation.
PCT/PL2019/050004 2018-07-14 2019-01-21 Composés chimiques destinés à être utilisés en tant que marqueurs de diagnostic d'une inflammation et de néoplasmes WO2020017984A1 (fr)

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PLP.426332 2018-07-14
PL426332A PL236125B1 (pl) 2018-07-14 2018-07-14 Związki chemiczne mające zastosowanie jako markery diagnostyczne stanu zapalnego i nowotworów, sposób ich otrzymywania, marker diagnostyczny i metoda diagnozowania procesów zapalnych i nowotworów nabłonkowych

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WO2003064620A2 (fr) * 2002-01-31 2003-08-07 Irm, Llc Substrats de l'hepsine et promedicaments
WO2009154510A1 (fr) * 2008-06-19 2009-12-23 Автономная Некоммерческая Организация "Иhctиtуt Молекулярной Диагностики" Système de test permettant de diagnostiquer le cancer de la prostate et méthode de diagnostic du cancer de la prostate
PL225341B1 (pl) * 2014-07-17 2017-03-31 Univ Gdański Nowy związek, sposób jego otrzymywania, roztwór farmaceutyczny zawierający nowy związek, sposób określania obecności choroby nowotworowej, zestaw do wykrywania nowotworów oraz zastosowanie hydrolizy nowego związku do wykrywania nowotworów

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WO2003064620A2 (fr) * 2002-01-31 2003-08-07 Irm, Llc Substrats de l'hepsine et promedicaments
WO2009154510A1 (fr) * 2008-06-19 2009-12-23 Автономная Некоммерческая Организация "Иhctиtуt Молекулярной Диагностики" Système de test permettant de diagnostiquer le cancer de la prostate et méthode de diagnostic du cancer de la prostate
PL225341B1 (pl) * 2014-07-17 2017-03-31 Univ Gdański Nowy związek, sposób jego otrzymywania, roztwór farmaceutyczny zawierający nowy związek, sposób określania obecności choroby nowotworowej, zestaw do wykrywania nowotworów oraz zastosowanie hydrolizy nowego związku do wykrywania nowotworów

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