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WO2021002771A1 - Conjugué de colorant fluorescent pour la visualisation de cellules exprimant le psma - Google Patents

Conjugué de colorant fluorescent pour la visualisation de cellules exprimant le psma Download PDF

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Publication number
WO2021002771A1
WO2021002771A1 PCT/RU2020/000096 RU2020000096W WO2021002771A1 WO 2021002771 A1 WO2021002771 A1 WO 2021002771A1 RU 2020000096 W RU2020000096 W RU 2020000096W WO 2021002771 A1 WO2021002771 A1 WO 2021002771A1
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Prior art keywords
psma
conjugate
cells
formula
compound
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Inventor
Aleksei Ehduardovich MACHULKIN
Anastasiia Alekseevna USPENSKAIA
Anton Petrovich BER
Stanislav Aleksandrovich PETROV
Ehmil Iulaevich IAMANSAROV
Aleksandr Valerevich FINKO
Olga Olegovna KRASNOVSKAIA
Ekaterina Alekseevna NIMENKO
Nikolai Iurevich ZYK
Ian Andreevich IVANENKOV
Dmitrii Aleksandrovich SKVORTSOV
Aleksandr Sergeevich EROFEEV
Petr Vladimirovich GORELKIN
Elena Kimovna Beloglazkina
Elena Sergeevna KHAZANOVA
Aleksandr Georgievich Mazhuga
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Obshchestvo S Ogranichennoi Otvetstvennostiu Izvarino Farma
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Obshchestvo S Ogranichennoi Otvetstvennostiu Izvarino Farma
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes

Definitions

  • the invention relates to the field of organic and medical chemistry, oncology and relates to a new class of compounds for imaging cells and tissues expressing PSMA, including, for example, prostate cancer cells.
  • the invention relates to markers of prognostic or clinical significance in the diagnosis and treatment of cancer and the use of drugs for the treatment of cancer.
  • Prostate cancer is the most common malignant disease in men and is the second leading cause of death in the western world. As of 2018, the total number of new cases of prostate cancer detection in the world was more than 1,250 thousand people, and more than 350 thousand deaths caused by this disease were recorded. The increase in the number of patients compared to 2015 was 8.0%.
  • Surgical removal of a malignant neoplasm is one of the most common and effective treatments for primary cancer therapy. Resection of all detectable malignant lesions leads to the absence of a detectable recurrence of the disease in approximately 50% of all cancer patients and may prolong life expectancy or reduce the prevalence of the disease in patients with identified cancer recurrence. Not surprisingly, surgical methods for achieving cell removal more quantitatively are currently attracting more and more attention.
  • One of the methods for identifying malignant tissue during surgery is the use of fluorescent dyes, which passively pass from the primary tumor to the draining lymph nodes.
  • tumor-targeted ligands used for this latter purpose include folic acid, which is specific for folic acid receptor (FR) positive tumors of the ovary, kidney, lung, endometrium, breast and colon, and DUPA, which can deliver the attached fluorescent dyes selectively for cells expressing a prostate-specific membrane antigen (PSMA), i.e. to prostate cancer cells and to cells neovascularizing other solid tumors.
  • PSMA prostate-specific membrane antigen
  • Conjugates for the specific binding of prostate specific membrane antigen (PSMA) for the delivery of therapeutic, diagnostic and imaging agents including a ligand for PSMA (B), a linker (L) and a drug (D), where the linker is covalently bound to the drug, are known in the art and a ligand, and where the linker contains a chain of at least seven atoms (W02009026177, 2009-02-26).
  • a ligand in particular, DUPA can act, and the drug is in one of the embodiments of the invention, an imaging agent selected from the group of fluorescent dyes (Oregon Greens, AlexaFluor, fluorescein, BODIPY fluorescent agents, rhodamines, DyLight fluorescent agents).
  • conjugates which consists in obtaining a 3 -tret-butyl-protected derivative of ligand DUPA, is also disclosed.
  • a peptide linker is then synthesized using solid phase synthesis, where at the final stage a 3-tert-butyl- protected derivative of the ligand DUPA, obtained earlier, is introduced into the structure of the polypeptide, the subsequent isolation of the polypeptide in the presence of trifluoroacetic acid can also remove the tert-butyl, and/or tert-butyloxycarbonyl and/or trityl protecting groups.
  • the linker can be long hydrophobic polypeptide derivatives based on aromatic amino acids: L-Phe-L-Phe-Glu-1, 2-diaminopropyl L-Phe.
  • the disadvantage is the absence of any substituents in the aromatic fragment closest to the active site, the presence of which can significantly affect the binding of these conjugates with PSMA.
  • the prior art contains a conjugate containing a ligand PSMA (B), a linker (L), and a nucleotide (N), where the linker is covalently linked to the nucleotide and ligand, and the linker contains a chain of at least seven atoms (WO2010045598, 2010-04-22).
  • the nucleotide is selected from the group consisting of siRNA, microRNA and methylated RNA, while the nucleotide additionally contains an imaging agent selected from the group consisting of Oregon Greens, AlexaFluor, fluoresceins, BODIPY fluorescent agents, rhodamines, DyLight fluorescent agents.
  • This conjugate is used to target prostate cancer cells.
  • the synthesis is carried out similarly to the method used for the synthesis of conjugates in patent document W02009026177.
  • methods for coupling ligands with siRNA, miRNA and methylated RNA with a fluorescent label based on reactions of forming 3-thioxuccinamide-lyl alkyl derivatives were used to obtain conjugates with fluorescent labels.
  • the disadvantage is the absence of any substituents in the aromatic fragment closest to the active site, whose presence can significantly affect the binding of these conjugates with PSMA, as well as its absence in a number of conjugates.
  • the prior art contains a conjugate containing a PSMA ligand (B), a linker (L), and a preparation (D) in which the ligand includes one or more carbon-sulfur double bonds, phosphorus-sulfur, a single bond of a phosphorus-sulfur, a thioester linkage or combinations thereof, and where the linker is covalently bound to the drug, the ligand, and where the linker contains a chain of at least seven methylene units, in particular the linker contains one or more phenylalanine residues, each of which can be independently replaced (WO2011106639, 2011- 09-01).
  • the drug can be, in particular, an imaging agent selected from the group of fluorescent dyes (Oregon Greens, AlexaFluor, fluorescein, BODIPY fluorescent agents, rhodamines, DyLight fluorescent agents).
  • an imaging agent selected from the group of fluorescent dyes (Oregon Greens, AlexaFluor, fluorescein, BODIPY fluorescent agents, rhodamines, DyLight fluorescent agents).
  • the disadvantage is the absence of any substituents in the aromatic fragment closest to the active site for conjugates containing three aromatic fragments in the linker structure, the presence of which can significantly affect the binding of these conjugates with PSMA, as well as its absence in a number of conjugates.
  • a compound of the general formula B-X-Y-Z where the compound (B) capable of binding to a prostate specific membrane antigen (PSMA), a hydrocarbon chain or a hydrocarbon chain with heteroatoms (X), at least one amino acid, or its derivative (Y) and infrared dye (Z) (near-IR range of spectrum) (WO2017044584, 2017- 03-16).
  • PSMA prostate specific membrane antigen
  • X hydrocarbon chain or a hydrocarbon chain with heteroatoms
  • Z infrared dye
  • a technical problem that the invention is directed to is the development of new diagnostic compounds for the visualization of pathogenic cells or tissues expressing PSMA, including the PSMA ligand with a linker and a fluorescent dye, its preparation and use.
  • the technical result of the claimed group of inventions is the high affinity and selectivity of the action of the claimed conjugates in relation to PSMA expressing cells.
  • These conjugates allow you to expand the arsenal of diagnostic tools for imaging cells with high PSMA expression, allowing for selective binding to cancer cells and achieving a high signal- to-noise ratio.
  • the dosage of the claimed conjugates was 5 nmol (250 nM/kg).
  • the key feature of the claimed conjugate is the presence of a long hydrophobic linker in the structure, as well as additional aromatic fragments, the presence of which contributes to better binding of the claimed conjugate to the protein target, due to the involvement of additional interactions between the conjugate and the hydrophobic pockets in the structure of the hydrophobic tunnel of the protein target.
  • a compound for visualizing pathogenic cells or tissues expressing PSMA which is a covalently linked PSMA-binding ligand based on a urea derivative of the DCL structure and a modified hydrophobic peptide linker, including a fragment of 6-aminohexanoic acid, associated with fluorescent dyes of various nature (cyanine dyes, derivatives of fluorescein and others) of formula (I): where R is a fluorescent dye.
  • Fluorescent dyes containing alkynyl groups are selected from fluorescent dyes (regardless of their spectral characteristics), which can be used to obtain conjugates with compound II, by the reaction of azide-alcine cycloaddition. Fluorescent dyes are selected from structures containing an alkyne structural fragment that is reactive for their modification, for example, fluorescein and related analogs, boron-dipyrromethene fluorescent agents, rhodamine fluorescent agents, cyanic dyes (including trade names Oregon Green, AlexaFluor, fluorescent means BODIPY, DyLightLight.
  • the problem is also solved by a method for producing a compound for visualizing populations of pathogenic PSMA expressing cells of formula (I), including the synthesis of a tritretbutille derivative of PSMA-binding ligand of formula (III):
  • the alkylated 3-tert-butyl derivative of the PSMA ligand is obtained by reductive animation with m-chlorobenzaldehyde;
  • the compound of formula (IV) is obtained by acylation of the 6-azidohexanoic acid derivatives to obtain the azide derivative of the alkylated derivative of the PSMA ligand, followed by reduction of the azide to an amino group.
  • the reduction reaction of the azide to the amino group is carried out in the presence of triphenylphosphine and water in THF solution, or in a solution of methanol using hydrogen in the presence of palladium on carbon as a catalyst.
  • Modification of a linker fragment representing an alkyl fragment containing 5 carbon atoms with an succinic anhydride is carried out by acylation of the amino group with succinic anhydride in the presence of non- nucleophilic bases using diisopropylethylamine or triethylamine.
  • the preparation of 3-tert- butyl derivative of the conjugate of formula (V) is carried out by acylation of a derivative of compound (III), acylated with succinic anhydride, of a dipeptide of formula (IV), and the removal of 3-tert-butyl protective groups is carried out in the presence of 9-11% TFA for 15- 17 hours in dichloromethane.
  • the problem is also solved by a composition for visualizing populations of pathogenic PSMA expressing cells, comprising a conjugate of formula (I) and a pharmaceutically acceptable carrier, vehicle or diluent.
  • a method of detecting/determining the presence of a population of pathogenic PSMA expressing cells in a biological tissue sample including: a) sample processing with a composition comprising a conjugate of formula (I); b) incubating the sample for the time required binding of the conjugate with at least one pathogenic cell;
  • the tissue is a tumor or lymph node.
  • Samples obtained from patients are preferably biopsy samples.
  • a biopsy is a diagnostic test that involves removing cells or tissues for examination.
  • the tissue is usually examined by a pathologist under an optical microscope.
  • the illumination/irradiation is performed using a laser of an optical microscope at a wavelength of light in the visible and/or infrared region, ranging from approximately 450 to 900 nm.
  • staining and sensing well known in the art, it is possible to show the structure of the cells and to detect specific proteins associated with them, and their localization within the detected sample.
  • the problem is also solved by a method of intraoperative detection/determination of the presence of a population of pathogenic PSMA expressing cells in an individual, including: a) intravenous administration of a composition comprising a conjugate of formula
  • the problem is also solved by a method of performing a surgical operation in an individual, guided by visualization, including:
  • composition comprising a compound of formula (I) under conditions and for a period of time sufficient to allow the compound to interact with target cells;
  • illumination is performed using a tomographic system, a manual optical imaging system, surgical glasses or an intraoperative microscope, at a wavelength of light in the visible and infrared regions of approximately 450 to 900 nm.
  • the information can be used to predict whether a patient’s solid tumor is sensitive to anticancer therapy.
  • This aspect of the invention preferably allows stratification of patients with a malignant neoplasm. This makes it possible to identify the optimal anticancer therapy or treatment regimen for a given individual patient.
  • the invention provides a method for screening anti-cancer agents, comprising the steps of:
  • test tumor cells first aliquot
  • tissue cells not affected by the tumor second aliquot
  • FIG. 1 shows the NMR spectrum *H MA-207257-Sulfo-Cy5 (abscissa axis - chemical shift (ppm), the ordinate axis - normalized intensity).
  • FIG. 2 shows the HPLC chromatogram of MA-207257-Sulfo-Cy5.
  • FIG. 3 shows the ESI HRMS mass spectrum of MA-207257-Sulfo-Cy5 (abscissa axis is a mass/charge ratio (m/z), ordinate axis is the intensity).
  • FIG. Figure 4 shows the *H MA-207257-Sulfo-Cy7 Cy5 NMR spectrum (abscissa axis - chemical shift (ppm), ordinate axis - normalized intensity).
  • FIG. 5 shows the HPLC chromatogram of MA-207257-Sulfo-Cy7.
  • FIG. 6 shows the ESI HRMS mass spectrum of MA-207257-Sulfo-Cy7 (the abscissa axis is the mass/charge ratio (m/z), the ordinate axis is the intensity).
  • FIG. Figure 7 shows the 'H MA-207257-FAM NMR spectrum (abscissa axis - chemical shift (ppm), ordinate axis— normalized intensity).
  • FIG. 8 shows the HPLC chromatogram of MA-207257-FAM.
  • FIG. 9 shows the ESI HRMS mass spectrum of MA-207257-FAM (abscissa axis is mass/charge ratio (m/z), ordinate axis is intensity).
  • FIG. 10 shows the accumulation of the PSMA-Cy5 conjugate in 22RV1 and PC3 tumors, where BBH are the results of accumulation in the PC3 tumor, Hi and in the 22RV1 tumor.
  • 24h_0 is the level of accumulation obtained by analyzing animals ex vivo (abscissa axis: time (h), ordinate axis: total intensity, (f/s)/(pV/cm 2 )).
  • FIG. 13 shows the accumulation of the PSMA-Cy7 conjugate in 22Rvl and PC3 tumors, where HH are the results of accumulation in the PC3 tumor, BH and in the 22RV 1 tumor. 24h_0 - the level of accumulation obtained in the analysis in animals ex vivo.
  • FIG. 15 shows the accumulation of free dye Cy5 in tumors 22Rvl and PC3, where HH are the results of accumulation in a PC3 tumor, and IHi - in a 22RV 1 tumor. 24h_0 - the level of accumulation obtained in the analysis in animals ex vivo.
  • FIG. 16 shows the accumulation of free dye Cy7 in 22Rvl and PC3 tumors, where HH are the results of accumulation in a PC3 tumor, and ⁇ - in a 22RV 1 tumor. 24h_0 - the level of accumulation obtained in the analysis in animals ex vivo.
  • FIG. Figure 17 shows the accumulation of the PSMA-Cy5 conjugate and the free Cy5 dye in the 22RV 1 tumor, where HH are the results of the accumulation of the PSMA-Cy5 conjugate Hi - the free Cy5 dye. 24h_0 - the level of accumulation obtained in the analysis in animals ex vivo.
  • FIG. 18 shows the accumulation of the PSMA-Cy7 conjugate and the free Cy7 dye in a 22RV 1 tumor, where HH are the results of the accumulation of the PSMA-Cy5 conjugate 1HS - the free Cy5 dye. 24h O - the level of accumulation obtained in the analysis in animals ex vivo.
  • FIG. 19-23 shows the general synthesis scheme of the inventive conjugate.
  • FIG. 24 shows micrographs of histological sections of PC3 (A)and 22Rvl (B)xenograft tumors, staining with the claimed compound with the FAM dye.
  • FIG. 25 shows a histogram of Cy5 signal expression by cell populations in lines 22RV1, LNCaP, PC3 when exposed to the PSMA-207257-Sulfo-Cy5 conjugate, where the population of cells carrying the Cy5 signal and not carrying the Cy5 signal are indicated.
  • FIG. 26 shows a graphical representation of the percentage of cells carrying the Cy5 fluorescent signal, where EH are the results in PC3 tumor B - 22RV1, Q - LNCaP (left column). Blocking - the results of the experiment with pre-incubation with an excess of ligand PSMA-207257 for 60 min, in the corresponding cell lines (right column).
  • FIG. 27 shows the synthesis scheme for MA-207257-Sulfo-Cy5 conjugate
  • FIG. 28 shows the synthesis scheme for MA-207257-Sulfo-Cy7 conjugate
  • FIG. 29 shows the synthesis scheme for the MA-207257-FAM conjugate.
  • PSMA is a transmembrane glycoprotein type II with a mass of -100 kDa, consisting of 750 amino acids. This protein consists of a short intracellular region (1-18 amino acids), a transmembrane domain (19— 43 amino acid residues) and a large extracellular domain (44-750 amino acid residues). This protein has a high expression in the tissues of the prostate gland, therefore it is a promising target for targeted delivery.
  • High- resolution mass spectra (HRMS) of positive ions were recorded on a Jeol GCMate II spectrometer with an ionization energy of 70 eV.
  • NMR spectra were recorded on Bruker Avance-400 instruments (operating frequency 400.1 and 100.6 MHz for 'H and 13 C, respectively) and Agilent 400-MR (operating frequency 400.0 and 100.6 MHz for *H and 13 C, respectively) using deuterated chloroform (99.8% D) or DMSO (99.9% D) as a solvent, unless otherwise indicated, relative to tetramethylsilane (TMS) as an internal standard, parts per million (ppm); the usual abbreviations used are: s - singlet, d - doublet, t - triplet, q - quartet, m - multiplet, w - wide and so on.
  • the compound of formula (III) can be obtained by a method known in the art (Ryan P. Murelli, Andrew X. Zhang, Julien Michel, William L. Jorgensen, David A. Spiege. Chemical Control Over Immune Recognition: A Class of Antibody-Recruiting Molecules (ARMs) that Target Prostate Cancer. J. AM. CHEM. SOC. 2009, 131, 17090-17092) (FIG. 19). Then, the resulting 3-tert-butyl derivative of the PSMA binding ligand is alkylated to obtain the compound of formula (IV).
  • the alkylation reaction is carried out by reductive amination with m-chlorobenzaldehyde (Jan Tykvart, Jiri Schimer, Jitka Bafinkova, Petr Pachl, Lenka Postova-Slavetinska, Pavel Majer, Jan Konvalinka, Pavel Sacha. Rational design of urea- based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery.
  • m-chlorobenzaldehyde Jan Tykvart, Jiri Schimer, Jitka Bafinkova, Petr Pachl, Lenka Postova-Slavetinska, Pavel Majer, Jan Konvalinka, Pavel Sacha.
  • the preparation of the compound of formula (V) is carried out by acylation of 5- azidohexanoic acid derivatives to obtain an azide derivative of an alkylated derivative of PSMA-ligand (V) followed by reduction of the azide to the amino group, while the reduction of the azide to the amino group is carried out in the presence of triphenylphosphine and water in a THF solution or in methanol solution using hydrogen in the presence of palladium on carbon as a catalyst.
  • the acylation reaction is carried out in a polar aprotic solvent medium dissolving the starting amine (IV), azido acid and non-nucleophilic base, taken from the calculation that at least 1 molar equivalent of amine is taken at least 1 molar equivalent of azido acid and base, as well as at least 100 molar equivalent polar aprotic solvent; at least 1 molar equivalent of PyBOP is added to the mixture while stirring; the mixture is stirred at room temperature until the starting amine (IV) disappears.
  • the upper limit of the reagents used is not limited, since an excess of any reagent does not reduce the reaction yields, but with a large excess, additional purification of the reaction products may be necessary.
  • the reduction reaction of the azido group to the amino group in THF/water medium with a water content of at least 10 vol. % in which the resulting azido-derivative and triphenylphosphine are taken taken from the calculation that at least 1 molar equivalent of the azido-derivative takes at least 1.5 molar equivalents of triphenylphosphine, as well as at least 50 molar equivalents of solvent mixture (THF/water) on the water.
  • the reaction mixture was heated at a temperature of at least 45 °C until the starting azido derivative disappeared.
  • the solvent was removed under reduced pressure. Purification was performed by the column chromatography method (triethylamine: methylene chloride: methanol; from 1%: 98%: 1% to 1%: 89%: 10%) (FIG. 20).
  • DMF or DMSO is used as the polar aprotic solvent.
  • diisopropylethylamine or triethylamine is used as the non-nucleophilic
  • Modification with an succinic anhydride of a linker fragment representing an alkyl fragment containing 5 carbon atoms to obtain a derivative of compound (V) acylated with succinic anhydride is carried out by an acylation reaction with succinic anhydride of the amino group in the presence of non- nucleophilic bases.
  • Diisopropylethylamine or triethylamine is used as non-nucleophilic bases.
  • the reaction of acylation with succinic anhydride is carried out in a non-polar aprotic solvent medium by dissolving the starting amine (III), succinic anhydride and non- nucleophilic base, calculated on the basis that 1 mole equivalent of amine anhydride requires at least 1 molar equivalent of succinic anhydride and non-nucleophilic base, as well as not less than 100 molar equivalents of non-polar aprotic solvent, the resulting mixture is stirred at room temperature until the starting amine (V) disappears.
  • dichloromethane or chloroform is used as the non-polar aprotic solvent.
  • diisopropylethylamine or triethylamine is used as the non-nucleophilic base.
  • the synthesis of the dipeptide (VI) is illustrated in the diagram shown in FIG. 21.
  • Synthesis of a PSMA vector fragment based on the L-phenylalanyl-L-tyrosine dipeptide derivative (L-Phe-L-Tyr, VI) was carried out according to scheme (c) (FIG. 21).
  • the resulting mixture was stirred at room temperature for at least 4 days.
  • the reaction mixture was concentrated under vacuum of a rotary evaporator until the organic solvent was removed. Then a hydrochloric acid solution with a concentration of at least 1 mol/L was added to the aqueous residue to a pH of no more than 4 and extracted with ethyl acetate. The combined organic phase was washed with a saturated solution of NaHC03 and NaCl, dried with Na2SC>4 and concentrated in vacuo. Then repeatedly boiled off with dichloromethane. The reaction product was obtained as a colorless amorphous substance.
  • sodium bicarbonate, sodium carbonate, sodium hydroxide or potassium hydroxide is used as the base.
  • EDOHC1 (at least 1 eq.), PFPOH (at least 1 eq.) was added to a solution of the compound Boc-L-Phe in dichloromethane, and stirred for at least 12 hours at room temperature. Further purification was performed using column chromatography on a silica gel column (eluent - dichloromethane). The reaction product (yellow oily substance) was dissolved in a mixture of THF: water (at least 30% volume of water) and L-tyrosine (at least 1 eq.) was added with stirring. A solution of a non-nucleophilic base was added dropwise to the obtained solution (at least 1 eq.) and stirred for at least 12 hours at room temperature.
  • the reaction mixture was concentrated under vacuum of a rotary evaporator until complete removal of the organic solvent.
  • the residue in the flask was acidified with a solution of HC1 with a concentration of at least 1 mol/L to a pH of at least 4 and extracted with ethyl acetate.
  • the combined organic phase was washed with a saturated solution of NaHCCb and NaCl, dried with Na2SC>4 and concentrated in vacuo.
  • the obtained colorless amorphous residue was dissolved in a minimum amount of dichloromethane and hexane was added dropwise with stirring until the precipitation ceased.
  • the precipitate was filtered and resuspended in hexane in an ultrasound bath, then re-filtered.
  • diisopropylethylamine or triethylamine is used as the non-nucleophilic base.
  • the process of activation of the carboxyl group of the compound Boc-L-Phe-L-Tyr was repeated, followed by interaction with azidopropylamine (at least 1 eq.) for at least 24 hours at room temperature in dichloromethane.
  • the crude reaction mass was chromatographed on a silica gel column, and an intermediate dipeptide amide was obtained, which was involved in the removal reaction of tert-butoxycarbonyl protection with a 10% solution of trifluoroacetic acid in anhydrous dichloromethane.
  • TFA was carried out with cooling in a water bath with ice at a temperature of not more than 10 °C, followed by gradual heating of the reaction mixture to room temperature. Deprotection should be carried out at room temperature for at least 3 hours.
  • linker VI was carried out, which was subsequently used to obtain highly specific PSMA vectors.
  • the developed synthesis methods are distinguished by environmental compatibility, good yields of target products, high selectivity of processes and do not require the use of special equipment or expensive reagents.
  • diisopropylethylamine or triethylamine is used as the non-nucleophilic base.
  • the preparation of a compound of the PSMA-binding ligand radical and a modified hydrophobic peptide linker of general formula (II) was carried out by removing the 3-tert- butyl protecting groups of the compound of formula (VII). Removal of 3-tert-butyl protecting groups was carried out in the presence of 9-11% by volume TFA for 15-17 hours in dichloromethane (FIG. 22).
  • the preparation of the conjugate of formula (I) is carried out by the reaction of an azide-alkyne cycloaddition catalyzed by copper ions (I) obtained in situ.
  • the reaction is carried out using 0.1-1 molar equivalents of copper sulfate pentahydrate (relative to ligand II) and 0.3-3 molar equivalents of sodium ascorbate in a DMF/H2O mixture (4:1 concentration) with a water content of 10-50 vol. %, as well as 0.9- 1.1 molar equivalents of a derivative of a fluorescent agent containing a terminal alkyne group (FIG. 23).
  • fluorescent dyes including alkyne fragments, for azide-alkyne cycloaddition
  • dyes selected from the following groups can be used (without limitation): Oregon Green (for example, Oregon Green 488, Oregon Green 514, etc.), AlexaFluor (for example, AlexaFluor 488, AlexaFluor 647, etc.), fluorescein and related analogues, BODIPY fluorescent agents (for example, BODIPY FI, BODIPY 505, etc.), rhodamine fluorescent agents (tetramethylrodamine, etc.), DyLight fluorescent agents (for example, DyLight 680, DyLight 800, etc.); cyan dyes (for example Heptamethine IR-780, Heptamethine IR-808, IRDye® 800CW, DY-675, DY-676, DY-677, DY-678, Cy5, Cy7, etc.).
  • Oregon Green for example, Oregon Green 488, Oregon Green 514, etc.
  • AlexaFluor for example, AlexaFluor 488, AlexaFluor
  • inventive compounds can be used separately or in combination with other compounds suitable for the diagnosis, visualization and/or treatment of diseases caused by PSMA expressing cells.
  • the compounds of the present invention may be used for visualization of nearly all solid tumors expressing PSMA, including lung tumor, renal cell, glioblastoma, pancreas, bladder, sarcoma, melanoma, breast, colon, germ cell, pheochromocytoma, esophageal and stomach tumors. Also, in accordance with the present invention, it is possible to visualize some benign lesions and tissues, including endometria, and the chronic peptic ulcer of the esophagus (Barret syndrome).
  • PSMA is often expressed in capillary vascular endothelial cells in the precancerous and intratumoral regions of various malignant tumors, thus the compounds of the present invention and imaging methods using such compounds are suitable for visualizing such malignant tumors.
  • pharmaceutically acceptable refers to a non-toxic material that does not interact with the action of the active ingredient of the pharmaceutical composition.
  • “Pharmaceutically acceptable carrier” refers to a biocompatible solution, which was sufficiently has characteristics such as sterility, p[Eta], isotonicity, stability and the like, and may include anysolvents and diluents and including sterile saline, sodium chloride solution for injection, Ringer's solution for injection, dextrose solution for Injection, dextrose solution and sodium chloride for injection, Ringer's lactate solution for injection and other aqueous buffer solutions dispersive media, coatings, antibacterial and antifungal agents, isotonic agents and the like.
  • a pharmaceutically acceptable carrier may also contain stabilizers, preservatives, antioxidants, or other additives that are well known to those skilled in the art, or other excipient known in the art.
  • Pharmaceutical acceptable salts refer to derivativesof disclosed compounds wherein the parent compound is modified so as to obtain non-toxic acid or base salts of this compound.
  • examples of pharmaceutically acceptable salts include, but are not limited to, salts of mineral or organic acids, obtained from basic residues, such as amines, alkaline or organic salts of acidic residues, such as carboxylic acids, and the like.
  • Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, formed, for example, from non-toxic inorganic or organic acids.
  • conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymalonic, phenylacetic, glutamic, benzoic, salicylic, methanesulfonic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, HOOC-(CH2)n-COOH, wherein n has a value of 0-4, and the like.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, ni
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains a basic or acidic group, by conventional chemical methods. Typically, such salts can be obtained by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base (such as hydroxide, carbonate, Na, Ca, Mg bicarbonate or the like) or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the above two solvents. Non- aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally used where practicable.
  • a list of additional suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985).
  • Detection of pathogenic PSMA expressing cells is carried out by intravenous administration to an individual of a composition comprising a compound of formula (I) that was previously required for distribution to the tissue of the claimed conjugate and its interaction with PSMA the subsequent in vivo irradiation of a part of an individual’s body containing the affected tissue with light having at least one excitation light wavelength in the range of from about 450 to about 900 nm to observe fluorescence emanating from a conjugate that is specifically bound and/or captured by the affected tissue, and the region in which the bound compound is detected by the presence of fluorescence indicates the presence of a pathogenic cell population in area under study.
  • Populations of pathogenic PSMA expressing cells are prostate tumor cells, metastatic prostate tumor cells or lymph node cells in which tumor cells (metastases) are present.
  • the time elapsed since the introduction of the conjugate of the invention to the subject before the start of the evaluation using the fluorescence imaging method of the present invention varies depending on the type of fluorescent contrast agent for the near infrared region of the spectrum.
  • the administration to an individual a composition comprising of a conjugate (compound) of formula (I) is preferably carried out from 4 to 72 hours prior to surgery.
  • the time range is set based on how long it will be enough for the fluorescent agent to accumulate in the tumor so that it can be visualized during the operation.
  • the inventive diagnostic method or imaging method allows the surgeon or practitioner to simultaneously see, observe, visualize the affected or abnormal tissues to facilitate the procedure of biopsy or surgical excision.
  • Determining (detecting) the presence of pathogenic PSMA expressing cells can also be carried out in remote tissues, organs by placing a tissue sample in a composition containing a compound of formula (I), incubating the sample for the time required for distribution to the tissue of the claimed conjugate and its interaction with PSMA, followed by washing the sample with a solution to remove unbound conjugate and irradiating the sample with light, exciting the fluorescent dye used.
  • the region in which fluorescence is observed is an area containing pathogenic PSMA expressing cells.
  • Incubation of the sample to determine the presence of pathogenic cell populations is carried out for 1-12 hours, depending on the size of the sample of the tissue/organ being examined, such as a fluorescent dye.
  • the optimal incubation time can be determined by comparing with standard immunohistochemical staining using antibodies containing a fluorescent label of the appropriate fluorescence range. Incubation with the use of fluorescent conjugates occurs until a fluorescence signal is obtained comparable to that of antibodies.
  • Flask was filled with argon. Then 6 mg of Sulfo-Cy5 alkyne (7.63 pmol; Lumiprobe), 1.8 mg of sodium ascorbate (9.16 pmol; SigmaAldrich) and 1.9 mg pentahydrate of copper sulfate (7.63 pmol; SigmaAldrich) were added. The resulting mixture was stirred for 24 hours.
  • the resulting conjugates were investigated to evaluate the effectiveness of imaging prostate tumors using fluorescent conjugates PSMA-Diag-Cy5 and PSMA-Diag-Cy7.
  • PC-3 is the most common model of androgen- independent prostate cancer. Since this line is highly malignant and insensitive to androgens, PC-3 is an excellent experimental model for exploring new therapies for human prostate cancer.
  • a new prostate cancer cell line, 22Rvl was obtained from the CWR22R xenograft line. 22Rvl expresses PSA and is sensitive to dihydrotestosterone, respectively, this cell line is androgen-dependent, unlike PC-3.
  • Cy5 dye 605/660; 605/680; 605/700; 640/680; 640/700; 640/720; 640/740; 675/720; 675/740; 675/760; 675,780.
  • Cy5 dye 605/660; 605/680; 605/700; 640/680; 640/700; 640/720; 640/740; 675/720; 675/740; 675/760; 675,780.
  • the obtained images were processed in the Living Image 4.5 program for the purpose of spectral separation of specific channels and autofluorescence.
  • mice can be extrapolated to the further use of the claimed human conjugates (Guidelines for preclinical studies of drugs. Rev. A.N. Mironov. Part 1. - M., 2012).
  • the 22RV1 tumor model was chosen as such a tumor.
  • tumor tissues are capable of nonspecific accumulation of xenobiotics, therefore a study on animals carrying only 22RV1 tumor could not give an unambiguous answer about the specificity and level of accumulation.
  • the results of a quantitative analysis of the accumulation of the PSMA-Cy5 conjugate in the 22RV1 and PC3 tumors are presented below (Ha fkG. 10).
  • the results obtained for the PSMA-Cy7 conjugate in general, repeat the results obtained for PSMA-Cy5. As in the previous case, in the interval of 0-8 h after intravenous administration, most of the conjugate is in the bloodstream, and a significant difference in accumulation can be detected only after 24 h (FIG. 13).
  • the ex vivo accumulation study also showed an increase in accumulation specificity, and the signal intensity in the 22RV 1 tumor was 5 times greater than in the PC3 tumor.
  • the signal level 24 hours after injection was comparable and amounted to 1.5*10 9 and 1.9*10 9 ( ⁇ J>/C)/(MKB/CM 2 ) for PSMA-Cy5 and PSMA-Cy7, respectively.
  • a standard hemotoxylin-eosin staining was performed for each sample using the Tissue-Tek Prisma multistainer. Dewaxing and staining sections were performed as follows. Initially, xylene treatment was carried out at 3x10 minutes, then with 95% alcohol, 3x5 minutes, rinsed in dH20, then treatment with wash buffer (Wash Buffer) - 2x3 minutes, and then Pretreatment solution (DAKO) at 98 °C for 15 min in a water bath.
  • Wash Buffer wash buffer
  • DAKO Pretreatment solution
  • Sections were dried in air (in a dark place) for 20 minutes.
  • DAPI Dermatin-Naphthalate
  • the samples were incubated in a dark place for at least 15 minutes before the start of visualization. Visualization was performed using a microscope.
  • the differential ability of the analyzed conjugates to penetrate into the cells was also assessed depending on the representation of PSMA on the surface. Internalization of the fluorescent ligand in the cells was detected using flow cytofluorometry.
  • Partial blocking of PSMA was achieved by incubating the analyzed cells with an excess of ligand, identical in structure, but free from the fluorescent label.
  • Wells of a 12-well plate for further culture of LNCaP cells were coated with L- polylysine (Sigma) for 1 hour, and then the wells were twice washed with PBS.
  • Cells (LNCaP, 22Rvl, and PC3 cell cultures) were seeded into a 12-well plate, 2x105 cells were placed into a well in 800 m ⁇ of RPMI medium, incubated under standard culture conditions overnight. Next, the cells were washed with cold RPMI medium containing 1% fetal bovine serum.
  • LNCaP cells three replications
  • 22Rvl cells three replications
  • PC3 cells three replications
  • the experiment was carried out in triplicate with partial blocking of PSMA (with an excess of the analyzed ligand) and in the experiment without additional loading with an excess of ligand.
  • Excess ligand was achieved by adding to the cells 720 m ⁇ of RPMI medium (1% FBS) containing 400 mM (100-fold concentration excess) of a fluorescent unlabeled ligand identical in structure to the analyzed one. Incubation was performed under standard culture conditions for 1 hour. We used wells with cells as samples of comparison, in which 720 m ⁇ of RPMI medium (1% FBS) were added, these wells with cells were also incubated under standard culture conditions for 1 hour.
  • Cy5 fluorescent signal accumulation with the introduction of the PSMA-207257-Sulfo-Cy5 conjugate was mediated by the representation of PSMA on the cell surface. So, the highest Cy5 fluorescent signal accumulation was characteristic of the LNCaP cell line (98.4% of cells), 57.5% of the cells of the 22Rvl cell line, and 41.1% for PC3 cell line.
  • composition of the investigated drug PSMA-Diag PSMA-207257-Sulfo-Cy7:
  • Solubilizer Pluronic FI 27 in the amount of five times the amount of the conjugate by weight.
  • Dimethyl sulfoxide in the amount of 5% of the prepared volume.
  • a solution of the investigational drug was prepared for intravenous use in an infusion solution Gemodez-N with the addition of the Pluronic F127 solubilizer and DMSO.
  • sterile DMSO was added to the sterile sample of the preparation and the solubilizer in the amount of 5% of the required total volume of solvent, stirred and left on for ultrasound treatment at 40 °C until dissolved.
  • the required volume of the Hemodez-N solution was measured into the vial and gently mixed, drawing the solution into the syringe and discharging it back.
  • the concentration of the solution was 250 nmol/kg. The solution was used only freshly prepared.
  • the studied drug was administered to rabbits at a dose of 0.0122 mg/kg, and to rats - 0.0224 mg/kg.
  • the scheme of the study of the pharmacokinetics of the drug is presented in Table 1.
  • Rat blood samples were taken from the tail vein in a volume of 0.2 ml into polyethylene micro test tubes with a capillary volume of up to 0.5 ml, by truncating the tip of the tail. K2EDTA is used as an anticoagulant. In total, during the study, 91 blood samples were taken from 7 animals (13 samples from each animal).
  • Rabbit blood samples were taken from the marginal ear vein of rabbits in a volume of 1.5-1.7 ml in 3.0 ml polyethylene tubes using a butterfly needle, individual for each animal. K2EDTA is used as an anticoagulant. In total, 78 blood samples were taken from 6 animals
  • the tube was gently inverted several times to mix the contents (anticoagulant). Then the tubes were placed vertically in a stand rod. The plasma was separated by centrifugation at 1500 g for 10 minutes at a temperature of +4 °C. Centrifugation was performed no later than 15 minutes after the collection of each blood sample. The resulting plasma from each tube was transferred to Eppendorf-type plastic tubes, labeled with the animal number and the time point number of the sample.
  • Determination of the studied drugs in plasma samples of rats and rabbits was carried out by high performance liquid chromatography with tandem mass spectrometric detection with preliminary isolation of the analyte from the biomaterial.
  • the experiment used white outbred male mice (130 individuals) about 2 months old with a mass of 19-21 g.
  • the main task of the experiment is to determine the LD50 value and describe potential target organs and systems, to determine the maximum tolerated dose.
  • the solutions of the studied drug in the infusion solution Hemodez with the addition of the Pluronic FI 27 and DMSO solubilizer were used.
  • sterile DMSO was added to a sterile sample of the preparation with a five-fold amount of Pluronic in the amount of 5% of the required total volume of solvent, stirred and left on for ultrasound treatment at 40 °C until dissolved.
  • the required volume of the Hemodez solution was measured into the vial and mixed.
  • the required volume of a 1% starch solution was added to the sample of the starch and mixed until a homogeneous suspension was obtained.
  • each animal was administered one dose of the test drug intravenously in a volume not exceeding the maximum allowable amount for a single injection, and if necessary, fractionally.
  • the design of the acute toxicity study is presented in Table 6.
  • mice were deprived of food and water for 2 hours, and then weighed. Then each animal was injected intravenously with the study drug according to the study design, in a volume not exceeding the allowable one-time injection volume: 0.5 ml for mice, 2 ml for rats, and fractionally if necessary.
  • Pluronic Solution in Hemodez was used for the control group. The duration of observation of laboratory animals was 14 days. During this period, visible signs of intoxication were assessed. The concentration of the study drug is 250 nM/kg (0.448 mg/kg).
  • mice 1.
  • the maximum tolerated dose for mice was 89.7 mg/kg
  • the finished dosage form for the practical use of the invention is intended for practical use of the invention and is a lyophilized pharmaceutical preparation that includes the PSMA-diag conjugate in accordance with the invention and may also include excipients, buffers and preservatives.
  • the drug allows to obtain the drug in the form of a solution for injection of the conjugate in accordance with the invention, which corresponds to its clinical needs according to its concentration.
  • the excipient used in the preparation can be a mono-, di-, or trisaccharide.
  • monosaccharides that may be mentioned are glucose, mannose, galactose, fructose and sorbose
  • disaccharides examples of disaccharides that may be mentioned are sucrose, lactose, maltose and trehalose
  • raffinose is an example of a trisaccharide that may be mentioned.
  • the specified filler may be a sorbitol, as well as any other substance with a suitable glass transition temperature.
  • the filler is present in the preparation in a concentration of about 50-99%.
  • the drug includes buffer solutions, then they should, in general, be physiologically tolerated substances that are acceptable to establish the desired pH value.
  • the amount of buffer substances is chosen so that after restoring the lyophilized preparation, for example, using water for injection, the resulting aqueous solution has a buffer concentration of from 5 mmol/1 to 20 mmol/1, preferably about 10 mmol/1.
  • Preferred buffer solutions are citrate buffer solution or phosphate buffer solution.
  • Acceptable phosphate buffer solutions are solutions of phosphoric acid salts of mono- and/or disodium and potassium, such as disodium hydrogen phosphate or potassium dihydrogen phosphate, as well as mixtures of sodium and potassium salts, such as, for example, disodium hydrogen phosphate and potassium dihydrogen phosphate mixtures.
  • the excipients used for stabilization are an isotonic agent, preferably a physiologically tolerable salt, such as, for example, sodium chloride or potassium chloride, or a physiologically tolerable polyol, such as, for example glucose or glycerin may also be present at the concentration required to establish isotonicity.
  • a physiologically tolerable salt such as, for example, sodium chloride or potassium chloride
  • a physiologically tolerable polyol such as, for example glucose or glycerin may also be present at the concentration required to establish isotonicity.
  • the drug may also include physiologically tolerable excipients, such as, for example, antioxidants, such as ascorbic acid or glutathione, preservatives, such as phenol, cresol, methyl or propyl paraben, chlorbutanol, thiomersal or benzalkonium chloride, polyethylene glycols (PEG), such as PEG 3000, 3350, 4000 or 6000, or cyclodextrins, such as hydroxy propyl- b-cyclodextrin, sulfobutylethyl- -cyclodextrin or g-cyclodextrin, chilators, such as disodium edetate.
  • antioxidants such as ascorbic acid or glutathione
  • preservatives such as phenol, cresol, methyl or propyl paraben, chlorbutanol, thiomersal or benzalkonium chloride
  • PEG polyethylene glycols
  • the preparation according to the invention can be obtained by preparing an aqueous preparation comprising a conjugate as an active ingredient, as well as an excipient and, if desired, pharmaceutical auxiliaries such as buffer salts and preservatives, followed by lyophilization of the solution.
  • the finished dosage form is composed, but not limited to the following examples:
  • the solutions prepared according to the examples are filtered through a 0.45 p prefilter filter into a suitable, pre-sterilized glass vessel.
  • the resulting solutions are filtered through a sterilizing filter with a pore size of 0.2 pm in a sterile receiving vessel.
  • sterile vials are filled with a sterile solution of the drug, pre-sealed with a rubber stopper, so that there is a possibility of air exchange with the contents of the vial during the drying process.
  • the vials prepared in this way are placed in freeze drying. The freezing mode is started, up to the temperature of -70 °C for 3 hours.
  • the dryer automatically switches to the main drying mode, at which a pressure of 0.2-0.6 mbar is created in the chamber, and the temperature of the shelf rises to -30 °C, in this mode the product was dried for 24-72 hours.
  • the final drying is turned on, the pressure gradually reaches 0.012 mbar.
  • the duration of the final drying mode is 3—4 hours.
  • the stability of the finished dosage form was studied in natural storage at -20 °C for 1 year.
  • the results of the analyzes showed that the finished dosage form is stable in all the quality indicators studied during the studied shelf life. Data on the stability shown in Table 8.

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Abstract

La présente invention se rapporte au domaine de la chimie organique et médicinale, ainsi que de la biologie moléculaire, et concerne une nouvelle classe de composés pour l'imagerie de cellules et de tissus exprimant l'antigène membranaire spécifique de la prostate (PSMA), tels que les cellules du cancer de la prostate. L'invention porte sur de nouveaux conjugués de diagnostic pour la visualisation de cellules ou de tissus pathogènes exprimant le PSMA, comprenant le ligand du PSMA avec un lieur et un colorant fluorescent, son procédé de préparation et son utilisation. Le résultat technique de l'invention est l'affinité et la sélectivité élevées de l'action des conjugués par rapport aux cellules exprimant le PSMA. Ces composés permettent d'élargir la panoplie d'outils de diagnostic pour des cellules d'imagerie avec un niveau élevé d'expression du PSMA. L'utilisation d'un dérivé azido d'acide aminopentanoïque permet d'obtenir un vecteur de PSMA avec un lieur hydrophobe long et des groupes carboxy protégés, ce qui à son tour facilite sa modification, augmente le rendement et réduit la quantité de solvants utilisés dans le procédé en raison d'une augmentation significative de la solubilité du composé de départ (vecteur de PSMA avec lieur long hydrophobe et groupes carboxy protégés). La principale caractéristique du conjugué de l'invention est la présence d'un lieur hydrophobe long dans la structure, ainsi que de fragments aromatiques supplémentaires, dont la présence contribue à une meilleure liaison du conjugué de l'invention à la cible protéique, du fait de l'implication d'interactions supplémentaires entre le composé et les poches hydrophobes dans la structure du tunnel hydrophobe de la cible protéique.
PCT/RU2020/000096 2019-07-02 2020-02-26 Conjugué de colorant fluorescent pour la visualisation de cellules exprimant le psma Ceased WO2021002771A1 (fr)

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