Scope of use

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. Thus, 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.

Background of the invention

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.

Given the importance of the complete removal of malignant lesions, it is useful to ensure that the malignant lesions are accurately and completely identified. 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.

Despite the understanding of the importance of tumor removal and the availability of certain identification methods for visualizing the tumor mass, many malignant nodes still elude detection, which leads to relapse of the disease and often death. Thus, there is a need for improved identification of tumors. There are a number of developments aimed at the synthesis of conjugates of a fluorescent dye with a tumor-specific guiding ligand, which is responsible for the accumulation of an attached dye in cancer tumors that overexpress the receptor of this ligand. Examples of 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.

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). As 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). The synthesis of these 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. Depending on the structure of the peptides used, approaches to modify the amino group using isothiocyanate derivatives of fluorescent dyes, or modifying the thiol group using Michael's reaction using maleimide derivatives of fluorescent dyes were used to obtain conjugates with fluorescent labels. 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. Depending on the structure of the peptides used, 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.

However, 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). The synthesis of known compounds is carried out in a similar manner used for the synthesis of conjugates in patent document W02009026177. Depending on the structure of the peptides used, to obtain conjugates with fluorescent labels, methods were used for coupling ligands based on reactions of forming an amide bond with the e-amino group of lysine and/or forming a disulfide derivative obtained by modifying the thiol group of cysteine, as well as forming a thiourea fragment, by reaction isothiocyanate derivative of the dye and the amino group of the linker, also the formation of the target conjugates was achieved by the Michael reaction of the mercapto group of the linker with maleimide derivatives of fluorescent dyes. However, 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.

Closest to the claimed is 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). These compounds are intended for the diagnosis and treatment of diseases associated with cells and/or the vascular system expressing PSMA. Also disclosed are methods and compositions for making and using compounds, methods for administering compounds, as well as kits for administering compounds. The synthesis of these compounds is carried out similarly to the method used for the synthesis of conjugates in patent document W02009026177. For the preparation of conjugates with fluorescent labels, methods were used to junction ligands with a fluorescent label based on the nucleophilic substitution reaction in a substituted polyunsaturated system of a fluorescent dye.

However, despite the diversity of the presented conjugates, all of them are represented by structures containing two or less aromatic fragments promoting hydrophobic interactions between the linker and the structure of the PSMA hydrophobic tunnel. In the experiments, 6 nmol of the fluorescent conjugate was used to visualize 22Rvl tumors.

Summary of the invention

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. Thus, to achieve effective tumor imaging, the dosage of the claimed conjugates was 5 nmol (250 nM/kg). The use of an azido derivative of aminopentanoic acid in the synthesis of the inventive conjugates makes it possible to obtain a PSMA vector with a long hydrophobic linker and protected carboxy groups, which in turn facilitates its modification and reduces the amount of solvents used in the process due to a significant increase in the solubility of the parent compound (PSMA vector with a long hydrophobic linker and protected carboxy groups).

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.

The technical problem is solved by 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):

(HI),

followed by alkylation of the obtained 3-tert-butyl derivative of PSMA-binding ligand to obtain the compound of formula (IV):

followed by the preparation of a compound containing an alkylated 3-tert-butyl derivative of the PSMA ligand and a linker fragment representing an alkyl fragment containing 5 carbon atoms of the formula (V):

which is modified with succinic anhydride, to obtain a derivative acylated with succinic anhydride, then the dipeptides of aromatic amino acid derivatives, which are L- phenylalanyl-L-tyrosine of formula (VI), are obtained to bind with a modified linker fragment,

then receive 3-tert-butyl derivative of the conjugate of the radical PSMA-binding ligand and a modified hydrophobic peptide linker, including fragments of 6-aminohexanoic acid, a fragment of L-phenylalanine, a fragment of L-tyrosine of the formula (VII):

with the subsequent removal of the tert-butyl protective groups of the compound of formula (VII) to obtain a covalently bonded PSMA-binding ligand and a modified hydrophobic peptide linker (II) Next, the copper (I) catalyzed azide-alkyne cycloaddition of compound (II) is reacted with a fluorescent dye containing a terminal triple bond, which results in obtaining the target compound (I) (L. Liang, D. Astruc The copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) “click” reaction and its applications. An overviewCoordination Chemistry Reviews, 255 (2011) 2933-2945).

In this case, 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 reaction of copper (I) catalyzed azide-alkyne cycloaddition of compound (II) with a fluorescent dye containing a terminal triple bond is carried out, which results in the target compound (I) (L. Liang, D. Astruc The copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) “click” reaction and its applications. An overviewCoordination Chemistry Reviews, 255 (2011) 2933-2945).

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. The problem is also solved by 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;

c) removing the unbound conjugate, and

d) illumination/irradiation of a biological sample, where

e) by the presence of fluorescence, it is concluded that there is a detection/presence of a population of pathogenic PSMA expressing cells in a biological sample.

In this case, 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. At the same time, 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. Using the methods of preparation of the sample, 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

(i);

b) illumination/irradiation of the accumulation region of the conjugate with infrared light;

c) by the presence of fluorescence when excited by infrared light, it is concluded that there is a population of pathogenic PSMA expressing cells in the individual.

The problem is also solved by a method of performing a surgical operation in an individual, guided by visualization, including:

a) administering a 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;

b) illuminating the region of accumulation of the compound for its visualization using infrared light;

c) performing surgical resection of areas that fluoresce when excited by infrared light. At the same time, illumination (irradiation) 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.

In other aspects, the invention provides a method for screening anti-cancer agents, comprising the steps of:

1. obtaining test tumor cells (first aliquot) and tissue cells not affected by the tumor (second aliquot);

2. exposure to a candidate agent (anti-cancer agents) over a period of time;

3. measuring the degree of cell proliferation in the first and second aliquot, where a decrease in any cell(s) proliferation in the first aliquot relative to the cell(s) in the second aliquot identifies a strong or active anticancer agent and/or increased or continuous cell(s) proliferation in the first an aliquot relative to the cell(s) in the second aliquot identifies a weak or inactive anticancer agent.

Brief Description of the Drawings

The invention is illustrated by the following drawings, where:

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. 11 shows the accumulation of PSMA-Cy5 in a 22Rvl tumor 24 hours after intravenous administration. Fluorescence signal intensity scale. Color scale: Min = 5.35 ^c 10 ^s ; Max = 1.71 x 10 ^s . Signal intensity: (photon/s/cm ² /sr )/(pW/cm ² ).

FIG. 12 demonstrates the accumulation of PSMA-Cy5 in 22Rvl tumors 24 hours after ex vivo intravenous administration. Fluorescence signal intensity scale. Color scale: Min = 8.18xl0 ⁷ ; Max = 1.48 MO ⁸ . Signal intensity: (photon/s/cm ² /sr)/(pW/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. 14 demonstrates the accumulation of PSMA-Cy7 in 22Rvl tumor 24 hours after intravenous administration. Fluorescence signal intensity scale. Color scale: Min = 1.76 ^c 10 ⁸ , Max = 4.55 x 10 ^s . Signal intensity: (photon/s/cm ² /sr)/(pW/cm ² )

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. Magnification: 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.

The implementation of the invention

The following are definitions of terms used in the description of the present invention. "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.

EDC*HC1 - 1 -Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride

PFPOH - pentafluorophenol

HOBT - hydroxybenzotriazole

HBTU - 3-[Bis(dimethylamino)methyliumyl]-3H-benzotriazole- 1 -oxide hexafluorophosphate

EtOAc/MeOH - ethyl acetate/methanol

DMAP - 4-dimethylaminopyridine

DCM - dichloromethane

DIC - diisopropylcarbodiimide

DMF - dimethylformamide

DIPEA - diisopropylethylamine

TFA - trifluoroacetic acid

DHM - dichloromethane

PBS - sodium phosphate buffer

FBS - Fetal Calf Serum

PPh3 - Triphenylphosphine PyBOP - benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate

THF - tetrahydrofuran

BOC20 - 2-tretbutyl dicarbonate

All reagents used are commercially available; solvent evaporation was performed using a rotary evaporator, under reduced pressure at a bath temperature of about 50°C; monitoring the progress of the reaction was carried out using thin-layer chromatography (TLC), and the reaction time is given for illustration only; the structure and purity of all isolated compounds was confirmed by at least one of the following methods: TLC (plates for TLC with pre-applied silica gel 60 F254 Merck), mass spectrometry or nuclear magnetic resonance (NMR). The product yield is for illustrative purposes only. Flash column chromatography was performed using Merck silica gel 60 (230—400 mesh ASTM). 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.

First 3-tert-butyl derivative of PSMA-binding ligand of formula (III) is obtained:

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. Bioorg Med Chem. 2014, 22(15): 4099—4108.)

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 solvent is then removed from the resulting reaction mixture under reduced pressure, and the target intermediate is isolated using column chromatography (Puriflach SILICA-HP 120G, 50 pm, gradient from 100% petroleum ether to 100% EtOAc for 30 minutes, flow rate = 50 ml/min) 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.

Next, 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).

Preferably, DMF or DMSO is used as the polar aprotic solvent.

Preferably, 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. In this case, the modification by a succinic anhydride of a linker fragment, which represents an alkyl fragment containing 3-5 carbon atoms, 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.

Preferably, dichloromethane or chloroform is used as the non-polar aprotic solvent. Preferably, diisopropylethylamine or triethylamine is used as the non-nucleophilic base.

Production of dipeptides of aromatic amino acid derivatives, which is phenylalanyl- tyrosine, for binding with the modified linker fragment of the compound of formula (V).

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). To a suspension of L-phenylalanine in a solvent mixture of dioxane: water with a water content of not less than 40 vol% at a temperature not exceeding 5 °C was added base in an amount of at least one molar equivalent of 2-tert-butyl dicarbonate BOC20 in an amount no less than one mole equivalent. 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.

Preferably, 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. Upon completion of the reaction, 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.

Preferably, diisopropylethylamine or triethylamine is used as the non-nucleophilic base.

At the third stage, 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. At the end, 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.

Adding 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.

Thus, as a result of the sequence of reactions according to the transformation scheme, the synthesis of 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.

The preparation of 3-tert-butyl derivative of a compound of the PSMA-binding ligand radical and a modified hydrophobic peptide linker comprising 6-aminohexanoic acid fragments, a phenylalanine fragment and a tyrosine fragment of the general formula (VII) was carried out by forming the amide bond of the acylation product of succinic anhydride and dipeptide formula (VI) (FIG. 22).

At least 1 equivalent of dipeptide, HOBT, HBTU and a non-nucleophilic base was added to a solution of the acylation product with succinic anhydride in DMF. The mixture was stirred for at least 24 hours. The solvent was then removed under reduced pressure. The product was isolated using column chromatography. Eluent - EtOAc/MeOH=5:l.

Preferably, 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).

As fluorescent dyes, including alkyne fragments, for azide-alkyne cycloaddition

(regardless of their spectral characteristics), 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.).

The inventive compounds (conjugates) 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.

The term "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. For example, 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. 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 (optical visualization of biological tissue) 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.

As a light source for excitation, you can use any equipment that provides a radiation source with a wavelength corresponding to the fluorescence excitation wavelength for the type of dyes used.

Below is a more detailed description of the claimed invention. The present invention may undergo various changes and modifications understood by a person skilled in the art based on the reading of this description. Such changes do not limit the scope of claims.

Example 1.

Synthesis of l-(6-(-(((l-(3S, 7S, 25S, 28S)-25-benzyl-l,3,7-3-carboxy-12-(4- chlorobenzyl)-28-(4-hydroxybenzyl)-5 , 13 ,20,23 ,26,29-hexaoxo-4,6, 12, 19,24,27,30- heptaazatritriacontan-33 -yl)- 1 H- 1 ,2,3 -triazol-4-yl)methyl) amino)-6-oxohexyl)-3,3-dimethyl- 2-((lE,3E,5E)-5-(l,3,3-trimethyl-5-sulfonate-indoline-2-ylid ene)penta-l,3-diene-l-yl)-3H- indole-l-ium-5-sulfonate (conjugate PSMA-diag MA-207257-Sulfo-Cy5) (FIG. 27)

Pre-synthesized (3S,7S,2S,28S)-3-tert-butyl-33-azido-25-benzyl-12-(3-chlorob enzyl)- 28-(4-hydroxybenzyl)-5, 13,20,23, 26,29-hexaoxo-4,6, 12, 19,24,27,3 O-heptaazatrytriatonate-

1 ,3 ,7-tricoarboxylate. Compound V (160 mg; 0.28 mmol) was dissolved in 10 ml of DCM and the system was purged with argon. Succinic anhydride (37 mg; 0.376) was added, followed by DIPEA (54 pL, 0.315 mmol). Left under stirring for a day. After added 1 ml of methanol and left under stirring for 1.5 hours. The resulting reaction mixture was evaporated. The dry residue was dissolved in 2 ml of DCM, DIPEA (36 mΐ 0.210 mmol) was added. The system was purged with argon. HOBT (24 mg, 0.158 mmol), HBTU (59 mg 0.158 mmol) was added and left to mix for 30 minutes. Next, compound VI dissolved in 500 mΐ DMF (83 mg, 0.158 mmol) was added. The reaction mixture was allowed to stir overnight. The solvent was evaporated under reduced pressure. Purification was performed by the method of column chromatography (ethyl acetate/hexane from 5% to 100% ethyl acetate; methanol: ethyl acetate; from 0% to 100%).

Thus, compound VII was isolated as a yellowish oily substance, the yield was 109 mg.

(32%)

In a round-bottom three-neck flask (29/32, ChemGlass) with a magnetic stirrer, 8 mg

(7.63 pmol) of the obtained compound were placed to dissolve 3 ml of DMF and 3 ml of deionized water. Then a flask was put on an adapter for entering argon (29/32, ChemGlass) and filled into the flask with argon. Then 6 mg of Sulfo-Cy5 alkyne (7.63 pmol), 1.8 mg of sodium ascorbate (9.16 pmol) and 1.9 mg of copper sulfate pentahydrate (7.63 pmol) were added. The resulting mixture was stirred for 24 hours using a magnetic stirrer (Teflon, C). After the reaction, 4.4 mg of ethylenediaminetetraacetic acid (EDTA) (15.26 mthoΐ) was added to the reaction mixture, for complexation of copper ions, after which the solvent was evaporated under reduced pressure (on a rotary evaporator). Then the product was purified by the method of reversed phase chromatography: Puriflash 15C18HP-F0012, deionized water/acetonitrile system, from 5% acetonitrile to 100% acetonitrile for 25 minutes, then for 5 minutes washing with methanol, flow rate - 20 ml/min. The resulting product was analyzed by NMR spectroscopy, HPLC-MS, and mass spectrometry. An NMR spectrum of 'H MA-207257-Sulfo-Cy5 (400 MHz, DMSO-d6, d, ppm) is shown in FIG. 1.

12.44 (br. s, 2H, COOH), 9.16 (s, 1H, NH), 8.21-8.49 (m, 3H, CH (Ar)), 8.05-8.21 (m, 1H, CH (Ar)), 7.85-8.05 (m, 3H, NH+CH (Ar)), 7.55-7.72 (m, 2H, NH+CH (Ar)), 6.89- 7.48 (m, 12H, NH+CH (Ar)), 6.45-6.89 (m, 3H, NH+CH (Ar)), 6.08-6.45 (m, 3H, NH+CH (Ar)), 3.71-4.67 (m, 1H, CH+CH2), 3.48-3.71 (m, 2H, CH+CH2), 3.24-3.49 (m, 4H, CH+CH2), 3.10-3.24 (m, 2H, CH+CH2), 2.82-3.09 (m, 7H, CH+CH2), 2.52-2.82 (m, 5H, CH+CH2), 2.12-2.41 (m, 7H, CH+CH2), 1.78-1.98 (m, 3H, CH+CH2), 1.58-1.78 (m, 1H, CH+CH2), 0.75-1.58 (m, 17H, CH+CH2+CH3).

HPLC chromatogram of MA-207257-Sulfo-Cy5 (FIG. 2)

ESI-HRMS Compound MA-207257-Sulfo-Cy5 (FIG. 3)

m/z calculated for [M-2H] ² - 862.8320; found: 862.8331

Example 2.

Synthesis of l-(6-(-(((l-(3S,7S,25S,28S)-25-benzyl-l,3,7-3-carboxy-12-(4- chlorobenzyl)-28-(4-hydroxybenzyl)-5,13,20,23,26,29-hexaoxo- 4,6,12,19,24,27,30- heptaazatritriacontan-33 -yl)- 1 H- 1 ,2,3 -triazol-4-yl)methyl)amino)-6-oxohexyl)-3 ,3 -dimethyl- 2-((E)-2-((E)-3-((E)-2-(l,3,3-trimethyl-5- sulfonate indolin-2-ylidene) ethyldien)cyclohex-l- ene-l-yl) 3H-indol-l-ium-5-potassium sulfonate (conjugate PSMA-diag MA-207257-Sulfo- Cy7) (FIG. 28)

In a round-bottom three-neck flask (29/32, ChemGlass) with a magnetic stirrer were placed 8 mg (3S,7S,2S,28S)-tri-tert-butyl-33-azido-25-benzyl-12-(3-chlor obenzyl)-28-(4- hydroxybenzyl)-5, 13,20,23,26,29-hexa-oxo-4,6, 12, 19,24,27,30-heptaazatry triaconate- 1 ,3,7- tricoarboxylate (7.63 pmol), 3 ml of DMF and 3 ml of water were added to dissolve. Adapter for entering argon (29/32, ChemGlass). 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. Next, the solvent was evaporated and purified by reverse phase chromatography: Puriflash 15C18HP-F0012, deionized water/acetonitrile system (chd, Reachim), from 5% acetonitrile to 100% acetonitrile for 25 minutes, then 5 minutes rinsing with methanol, the flow rate is 20 ml/min. The resulting product was analyzed by NMR spectroscopy, HPLC-MS, and mass spectrometry.

An NMR spectrum of Ή MA-207257-Sulfo-Cy7 (400 MHz, DMSO-d6, d, ppm) is shown in FIG. 4. 8.21-8.26 (m, 1H, NH), 7.88 (s, 1H, NH), 7.74 (s, 2H, NH+CH (Ph)), 7.67-7.70 (m, 2H, CH (Ph)) 7.61-7.63 (m, 2H, CH (Ph)), 7.15-7.29 (m, 7H, CH (Ph)), 6.99 (m, 1H, CH (Ph)), 6.61 (m, 1H, NH), 6.12-6.15 (m, 1H, NH), 4.24-4.34 (m, 4H, CH), 4.01-^1.09 (m, 2H, CH (BZ)), 3.79 (m, 1H, CH ₂ ), 3.65 (m, 1H, CH ₂ ), 3.60 (m, 3H, CH ₂ ), 2.88-3.06 (m, 6H, CH ₂ ), 2.66 (m, 2H, CH ₂ ), 2.32 (m, 1H, CH ₂ ), 2.17 (m, 2H, CH ₂ ), 1.81-1.91 (m, 4H, CH ₂ ), 1.64 (m, 15H, CH ₂ ), 1.54 (m, 5H, CH ₂ ), 1.33 (m, 5H, CH ₂ ), 1.22 (m, 2H, CH ₂ ), 1.14-1.18 (m, 1H, CH ₂ ), 1.02-1.10 (m, 1H, CH ₂ ).

HPLC chromatogram of MA-207257-Sulfo-Cy7 (FIG. 5)

ESI HRMS Compound MA-207257-Sulfo-Cy7 (FIG. 6)

m/z calculated for [M-2H] ² - 895.8555; found: 895.8561

Example 3.

(3R,7S,25S,28R)-25-benzyl-12-(3-chlorobenzyl)-33-(4-((3', 6'-dihydroxy-3-oxo-3H- spiro [isobenzofuran- 1 ,9'-xanthene] -4-ylcarboxyamido methyl)- 1 H- 1 ,2,3 -triazol- 1 -yl)-28-(4- hydroxybenzyl)-5, 13,20,23,26,29-hexaoxo-4, 6,12,19,24,27,30-heptaazatritriacontane- 1 -

1,3,7-3-carboxylic acid (PSMA-DIAG MA-207257-FAM conjugate) (FIG. 29)

In a round-bottom three-neck flask (29/32, ChemGlass) with a magnetic stirrer, 270 mg (244 _ji mol) of the obtained compound were placed to dissolve 18 ml of DMF and 6 ml of deionized water. Then a flask was put on an adapter for entering argon (29/32, ChemGlass) and filled into the flask with argon. Then 117 mg of FAM-5 alkyne (269 mhioΐ), 21 mg of sodium ascorbate (108 pmol) and 27 mg of copper sulfate pentahydrate (108 pmol) were added. The resulting mixture was stirred for 24 hours using a magnetic stirrer (Teflon, C). After completion of the reaction, 63 mg of ethylenediaminetetraacetic acid (EDTA) (216 mthoΐ) was added to the reaction mixture, for complexation of copper ions, after which the solvent was evaporated under reduced pressure (on a rotary evaporator). Then the product was purified by the method of reversed phase chromatography: Puriflash 15C18HP-F0012, deionized water/acetonitrile system, from 5% acetonitrile to 100% acetonitrile for 25 minutes, then for 5 minutes washing with methanol, flow rate - 20 ml/min. The resulting product was analyzed by NMR spectroscopy, HPLC-MS, and mass spectrometry.

An NMR spectrum of 'H MA-207257-FAM (400 MHz, DMSO-d6, d, ppm) is shown in FIG. 7.

12.44 (br. s, 3H, COOH), 10.17 (br. s, 1H, CON), 9.38 (c, 1H, C(O)NH), 8.48 (s, 1H, C(O)NH), 8.30-8.24 (m, 2H, C(O)NH) 8.10-8.02 (m, 3H, C(O)NH), 7.77-7.65 (m, 2H, C(0)NH+ArH), 7.65-7.64 (m, 2H, C(0)NH+ArH), 7.35-7.30 (m, 5H, C(0)NH+ArH), 7.18- 7.15 (m, 3H, C(0)NH+ArH), 4.67^1.54 (m, 5H, C(0)NH+ArH), 4.31^1.29 (m, 2H, NH (urea)), 4.55-4.45 (m, 4H, PhCH ₂ NC(0)), 4.28 (s, 3H, PhCH ₂ NC(0)), 4.07^.05 (m, 3H, CH, CH), 3.17-3.16 (m, 3H, CH ₂ ), 3.00-2.98 (m, 6H, CH ₂ ), 2.70-2.65 (m, 3H, CH ₂ ), 2.23- 2.19 (m, 8H, CH ₂ ), 1.91-1.88 (m, 3H, CH ₂ ), 1.49-1.10 (m, 11H, CH ₂ ).

HPLC chromatogram of MA-207257-FAM (FIG. 8).

ESI-HRMS for C74H80CIN1 1O19 of MA-207257-FAM compound (FIG. 9).

m/z calculated for [M-2H] ² 729.7576; found: 729.7586

The resulting conjugates were investigated to evaluate the effectiveness of imaging prostate tumors using fluorescent conjugates PSMA-Diag-Cy5 and PSMA-Diag-Cy7.

We used the PC-3 and 22Rvl cell lines. 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.

The study of the efficiency of PC3 and 22Rvl tumors imaging.

Experiments were performed on animals. Concentrate of the conjugate in dimethyl sulfoxide was preliminarily prepared. For this purpose, the obtained conjugate PSMA-diag MA-207257-Sulfo-Cy5, MA-207257-FAM or PSMA-diag MA-207257-Sulfo-Cy7 was dissolved to a concentration of 10 mM/ml in dimethyl sulfide. The concentrate can be stored at a temperature of -20 °C to +4 °C for no more than 14 days. Before the experiment, a solution was prepared by diluting the concentrate with saline to a conjugate content of 50 nM/ml. The animals were injected in a dosage of 250 nM/kg (0.448 mg/kg) in a volume of 100 mΐ into the tail vein, after which they conducted a study on an IVIS Spectrum-CT optical imaging system with excitation/emission filters:

1) For Cy5 dye: 605/660; 605/680; 605/700; 640/680; 640/700; 640/720; 640/740; 675/720; 675/740; 675/760; 675,780.

2) For Cy7 dye: 675/720; 675/740; 675/760; 675/780; 675/800; 710/760; 710/780; 710/800; 710/820; 710/840; 745/800; 745/820; 745/840;

The study was carried out in the following time intervals after drug administration: 30 minutes, and 1 , 8, 24 hours. After 24 hours, the animals were taken out of the experiment by an overdose of anesthesia, excision of the peritoneum was performed, and the IVIS Spectrum CT system was tested with excitation/emission filters:

1) For Cy5 dye: 605/660; 605/680; 605/700; 640/680; 640/700; 640/720; 640/740; 675/720; 675/740; 675/760; 675,780.

2) For Cy7 dye: 675/720; 675/740; 675/760; 675/780; 675/800; 710/760; 710/780; 710/800; 710/820; 710/840; 745/800; 745/820; 745/840;

The obtained images were processed in the Living Image 4.5 program for the purpose of spectral separation of specific channels and autofluorescence.

Experimental results obtained in 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).

Dynamics of PSMA accumulation.

Since tissues actively expressing the PSMA receptor are the main target of the conjugate, the 22RV1 tumor model was chosen as such a tumor. However, it is known that 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. In order to answer this question, it was decided to conduct experiments on animals that were implanted with both PSMA positive 22RV 1 tumor and PSMA negative PC3 tumor. 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).

Analysis of the data obtained shows that in the first hour there are no significant differences in the accumulation of the drug between PSMA-positive and PSMA-negative tumors. At the same time, the most intense signal is observed 30 minutes after the injection, and its level gradually decreases. The most significant differences are observed after 24 hours. This picture is probably explained by the fact that after the introduction, most of the conjugate is in the bloodstream, including the capillaries of the skin and internal organs, which ensures a high overall signal level. Over time, the concentration of the conjugate in the blood decreases due to its removal from the body, presumably by the kidneys in the urine. After 24 hours, almost all of the non-specifically bound conjugate is removed from the body, and accumulation is observed only in the organs where receptors are expressed that ensure its capture, in particular, in the 22RV 1 tumor. The data obtained on live animals show that in the 22RV1 tumor, a twofold increase in the signal intensity is observed (FIG. 11). Separately, it is worth noting that the skin and tissues actively absorb light, which leads to a weakening of the signal, so it was decided to take pictures of each animal separately, having performed an autopsy and freeing the tumor from the surrounding skin and tissues. These conditions are more close to the conditions of intraoperative diagnosis during abdominal or laparoscopic surgery, since there are no additional layers of tissue between the light source/receiver. An analysis of the accumulation of conjugate in tumors in this way showed even more significant differences between PSMA-negative and PSMA-positive tumors (FIG. 12).

An analysis of accumulation in ex vivo tumors showed that a tumor of type 22RV1 accumulates on average 14±2 times more conjugate than in a tumor of type PC3.

Dynamics of accumulation of PSMA-Su7 in tumors.

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).

Despite the fact that in vivo, the signal values between PC3 and 22RV 1 tumors also differed by 2 times, in general, the signal level from tumors was more distinguishable against the background of other tissues (FIG. 14). This is due to a lower level of intrinsic fluorescence of the tissues in the near-infrared range of the spectrum, where fluorescence of the dye Cy7 is observed.

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. In general, for both conjugates, 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.

Dynamics of accumulation of free dyes Cy5 and Cy7 in tumors

Since the difference in accumulation of the conjugate shown earlier can only be explained by the chemical nature of the Cu5 and Cy7 dyes themselves, the distribution of these dyes without conjugation with PSMA was also investigated. For both dyes, a significantly faster signal decrease in tumors with time was shown, which can be explained by differences in pharmacokinetics and faster elimination. Also, after 24 hours, neither in vivo nor ex vivo found any significant difference in accumulation between PSMA-positive 22RV1 and PSMA-negative PC3 (FIG. 15, FIG. 16). A comparative analysis of the level of accumulation of the claimed compounds (conjugates) and free dyes in PSMA-positive tumors also showed that the conjugates accumulate much better compared to free dyes (FIG. 17, FIG. 18). At the same time, the ratio of the intensities of the conjugate/ffee dye for PSMA-Cy5 and Cy5 was 8:1, and for PSMA- Cy7 and Cy7 - 150:1.

Thus, a specific accumulation of the PSMA-Cy5 and PSMA-Cy7 fluorescent conjugates was shown on the PC3 and 22RV1 tumor models, while the intensity ratio in the 22RV1 and PC3 tumors 24 h after intravenous administration of the conjugates was (14±2): 1 and (5±1):1 for PSMA-Cy5 and PSMA-Cy7, respectively, and the ratio of signal intensity conjugate/ffee dye was 8:1 and 150:1 for PSMA-Cy5 and PSMA-Cy7, respectively. This indicates the selectivity of the accumulation of synthesized conjugates in tissues and tumors with the expression of PSMA. And also about the specificity of accumulation, which is caused precisely by the presence of the PSMA ligand in the structure of the fluorescent conjugate.

The ability of the claimed compounds (conjugates) to visualize in histological sections of the cell depending on the PSMA representation on their surface was evaluated. Visualization was performed using the optical method (fluorescence microscopy). 1) Histological sections of subcutaneous PC3 and 22Rvl xenograft tumors (xenograft mouse model of prostate cancer). 22Rvl and PC3 cell lines have different levels of PSMA expression on the cell surface. According to literature data, 22Rvl is a PSMA-positive cell line, the PC3 cell line does not carry PSMA, and is PSMA negative. 2) histological sections of acinar adenocarcinoma of the human prostate gland (Gleason 3-5), histological sections were obtained on the basis of Moscow City Oncological Hospital No. 62.

Logistics: High-speed histological processor Leica Peloris II Tissue Tek Prisma highspeed histological processor, Sakura Nikon Ti-U inverted microscope General laboratory equipment (water and air thermostats, pipettors, aspirators, vortex, etc.). The following reagents were used: buffered formalin 10%, Xylene, Dapi (Dako), Ethanol 95%.

To obtain histological sections, biological material (PC3 and 22Rvl xenograft tumors) were fixed in 10% buffered formalin. Tissue wiring was performed using a Leica Peloris II high speed histological processor as follows:

- embedded in paraffin (melting point 56-58 °C);

- carried out microtomy and drying of sections in a vertical position in a thermostat for 30 minutes; - for each xenograft tumor, one of the prepared sections was stained with hematoxylin- eosin, followed by embedding under the film.

Histological sections of subcutaneous PC3 and 22Rvl xenograft tumors and histological sections of human prostate gland adenocarcinoma were treated with the claimed compound (conjugate). The conjugate was applied at 100 mΐ per section (at a concentration of 30 mM in a dilution buffer solution) and incubated for 1 hour in a humid chamber at room temperature. As a positive control for detecting PSMA expression, commercial antibodies on PSMA Monoclonal Antibody (1H8H5) were used in accordance with the manufacturer's recommendations. The primary antibodies were imaged on PSMA using the commercial Bond Polymer Refine Detection kit system (Leica Biosystems). In addition to the PSMA immunohistochemical study, 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. We took out the Coplin vessel, cooled it at room temperature for 15 minutes, processed it with wash buffer (Wash Buffer) for 2x3 minutes, removed the buffer from the glass and carefully wiped off the remains of the buffer around the cut with filter paper. Then 3-5 drops of cold Pepsin (DAKO) were applied to the cut for 15 minutes, treated with wash buffer (Wash Buffer) for 2x3 minutes, then the claimed compound was applied (at a concentration of 30 mM in dilution buffer) 100 mΐ per section, incubated for 1 h in a humid chamber at room temperature, were treated with wash buffer (Wash Buffer) - 2x3 min at room temperature in a dark place, and then with 95% alcohol 3x2 min. Sections were dried in air (in a dark place) for 20 minutes. DAPI (Dako) was applied with 5-7 mΐ on the cut and quickly covered with a cover glass 24x24 mm. The samples were incubated in a dark place for at least 15 minutes before the start of visualization. Visualization was performed using a microscope.

During the visualization of histological sections PC3 and 22Rvl xenograft tumors after treatment with the claimed compounds bearing FAM fluorescent label shows that the compound accumulated in the cytoplasm and membrane xenograft tumor cells, the accumulation present in PC3 xenograft tumors, and in the case 22Rvl xenograft tumors (FIG. 24).

When conducting research trials with histological sections of human prostate adenocarcinoma (Gleason 3-5), expression of PSMA by human adenocarcinoma cells was confirmed, while increasing the Gleason score resulted in an increase in the representation of PSMA in adenocarcinoma cells, which is consistent with literature data. In imrnunohistochemical analysis of human prostate adenocarcinoma, Gleason 3, luminal/apical membrane staining was observed in the tubular structures of the prostate adenocarcinoma. Attention is drawn to the range of intensity - from the absence of staining to moderate intensity.

An imrnunohistochemical analysis showed luminal/apical membrane staining in tubular structures of prostate adenocarcinoma. It was shown that in structures with Gleason 4 and 5, the intensity of staining is significantly higher than in structures with Gleason 3.

Thus, in accordance with the conducted research tests of the inventive compounds, it was shown that with their use it is possible to visualize prostate cancer cells when conducting analysis of fixed cells/tissue in a histological section.

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.

To assess the contribution of PSMA mediated ligand capture by cells to the total fluorescence detected by a flow cytometer, partial blocking of PSMA was performed. 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.

Three cell lines with different levels of PSMA expression on the surface of LNCaP, 22Rvl, and PC3 cells were selected. According to literature data, the highest level of PSMA expression is characteristic of LNCaP, low level of PSMA expression is characteristic of 22Rvl, and the PC3 cell line does not carry PSMA, is PSMA negative.

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 mΐ of RPMI medium, incubated under standard culture conditions overnight. Next, the cells were washed with cold RPMI medium containing 1% fetal bovine serum.

For each conjugate, the analysis included 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 mΐ 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 mΐ of RPMI medium (1% FBS) were added, these wells with cells were also incubated under standard culture conditions for 1 hour. After the time of incubation, 80 mΐ of RPMI medium (1% FBS) containing the conjugate to be analyzed at a concentration of 40 mM was added to each well to achieve a final concentration of 4 mM in the well. The samples were incubated under standard culture conditions for 30 minutes. Cells that were incubated with RPMI medium (1% FBS) with an equivalent DMSO content for 30 minutes were included as control samples.

Cells were removed from the plastic surface by trypsinization, trypsin inactivation was performed using PBS containing 10% FBS. The cells were centrifuged and washed twice in PBS (10% FBS), the resulting cells were resuspended in 500 mΐ of PBS and then used for flow cytometry (Becton Dickinson FACSAria III).

The accumulation of the fluorescence signal associated with the arrival of the labeled conjugate PSMA-207257-Sulfo-Cy5 was analyzed in three cell lines. The results of analysis using flow cytofluorometry are shown in FIG. 25.

As can be seen from FIG. 25 and FIG. 26 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.

In the experiment with partial blocking of PSMA by preincubation with the ligand PSMA-207257, it was shown that in the cell line LNCaP, having the maximum representation of PSMA on the cell surface, there was a decrease in the accumulation of the conjugate PSMA-207257-Sulfo-Cy5 by 4.1 times. A pronounced decrease in the penetration of the conjugate PSMA-207257-Sulfo-Cy5 was also characteristic of 22RV1 cells. For PC3 cells, a slight decrease in the accumulation of fluorescent ligand was observed when there was an excess of PSMA-207257 ligand in the incubation medium (FIG. 26). Thus, in the experiment with partial blocking of PSMA, the selective accumulation of PSMA-207257-Sulfo-Cy5 conjugate by cells was confirmed, depending on the expression of PSMA on its surface. Thus, the penetration of the PSMA-207257-Sulfo-Cy5 conjugate into cells depends on the expression of PSMA on the cell surface. The data obtained indicates the possibility of differential determination of PSMA expressing cells with the claimed compounds, MA-207257-FAM and MA-207257-Sulfo-Cy5 with subsequent histological analysis.

Study of the pharmacokinetics of the drug PSMA-Deag in plasma of rabbits and rats after a single injection.

The study was conducted on healthy conscious Soviet Chinchilla male rabbits, 2.5 to 3 months old and weighing 2.0 to 2.5 kg and healthy conscious white outbred adult male rats aged about 2 months, weighing 190-210 g and was aimed at determining the concentration of a specific substrate conjugate for PSMA by the diagnostic agent Cy7 in the blood plasma of rabbits and rats after a single injection of PSMA-Diag.

The study was conducted in one group of each animal species, including 6 rabbits and

7 rats.

To study the pharmacokinetic study of the drug, a study was conducted on the dynamics of changes in its concentration in the blood; therefore, blood samples were taken from the test animals (followed by release of blood plasma) at certain intervals after drug administration.

The composition of the investigated drug PSMA-Diag (PSMA-207257-Sulfo-Cy7):

Conjugate specific substrate for prostate specific membrane antigen (PSMA) with diagnostic agent Cy7.

Solubilizer Pluronic FI 27: in the amount of five times the amount of the conjugate by weight.

Solvent: Hemodez infusion solution.

Dimethyl sulfoxide: in the amount of 5% of the prepared volume.

Immediately prior to administration, 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. To prepare the solution, 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. Then, 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.

Based on this and taking into account the interspecific dose conversion factor, 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.

Table 1 Scheme of the pharmacokinetic study with intravenous route of administration

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

(13 samples from each animal) during the study.

Immediately after taking a blood sample, 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.

All plasma samples were frozen in an upright position and stored at a temperature not higher than -20 °C prior to analysis.

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.

Each method was previously validated for the following parameters: • sensitivity,

• selectivity,

• matrix effect,

• calibration range,

• accuracy,

• precision,

• assessment of recovery rate the analyte,

• breeding test,

• stability.

In calculating the pharmacokinetic parameters, the actual time of blood sampling was used.

Concentration values “below the limit of determination” (BLQ) calculations of pharmacokinetic parameters and descriptive statistics are considered as missing values.

Calculation of pharmacokinetic parameters and the construction of pharmacokinetic curves, as well as statistical analysis of pharmacokinetic data were performed using validated software Phoenix Winnonlin (Version 8.0).

For the concentration of the studied drug at each time point and all pharmacokinetic parameters, the following parameters of descriptive statistics were calculated: arithmetic mean, geometric mean, standard deviation of the average result, coefficient of variation, median, minimum and maximum values, range (see Tables 2 and 3).

During the study, sufficient data were obtained on the change in the concentration of the PSMA-DIAG preparation in the blood plasma of animals to determine the required pharmacokinetic parameters. The data are presented in Tables 4 and 5 for rabbits and rats, respectively.

Table 2. The concentration of PSMA-Diag in the blood plasma of rabbits and relevant descriptive statistics able 2. The concentration of PSMA-Diag in the blood plasma of rabbits and relevant descriptive statistics

Table 3. Concentrations of PSMA-Diag in rat plasma and relevant descriptive statistics

Table 4: The obtained pharmacokinetic parameters in plasma of rabbits

Table 5. The obtained pharmacokinetic parameters in the blood plasma of rats

Acute toxicity study

The study of the acute toxicity of the drug PSMA-Diag - a conjugate of a specific substrate for prostate specific membrane antigen (PSMA) with the diagnostic agent Cy7 was performed using outbred male mice with at least two toxic doses after a single administration of the test drugs.

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.

In the study of acute toxicity, the solutions of the studied drug in the infusion solution Hemodez with the addition of the Pluronic FI 27 and DMSO solubilizer were used. To prepare the solution, 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. Then, the required volume of the Hemodez solution was measured into the vial and mixed. Before the administration, the required volume of a 1% starch solution was added to the sample of the starch and mixed until a homogeneous suspension was obtained.

In the experiment on the determination of acute toxicity, 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.

When assessing acute toxicity, the animals' behavior, appearance, physical activity, reaction to external stimuli were monitored, physiological functions were evaluated, and body weight dynamics were determined.

During the experiment, the observation of animals was carried out continuously for the first 30 minutes after administration, then hourly, for 4 hours, then after 24 hours and then daily for 14 days.

Table 6. Acute toxicity study design by intravenous route of administration

Before the start of the experiment to determine acute toxicity, the animals 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. For the control group, Pluronic Solution in Hemodez was used. 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).

The results of the experiment are summarized in Table 7.

Table 7. The results of the observations with a single intravenous administration of PSMA-DIAG to male rats

The dependence of mortality on the dose of the drug and the magnitude of the average lethal dose for male mice has not been established due to the absence of death in the experimental groups.

As a result of the acute toxicity study, the following conclusions were made:

1. The maximum tolerated dose for mice was 89.7 mg/kg

2. No toxic effect of the drug is shown.

3. Analysis of the internal organs did not reveal morphological changes.

As a result of the study of acute toxicity, it was established that the PSMA conjugate with Cy7 does not have a toxic effect in a dosage exceeding the effective 2000 times.

Thus, the experiments showed that the claimed conjugates have a high affinity and selectivity of action against PSMA expressing cells, with low toxicity. These compounds allow you to expand the arsenal of diagnostic tools for imaging cells with high level of PSMA expression.

The finished dosage form for the practical use of the invention The finished dosage form 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. Examples of monosaccharides that may be mentioned are glucose, mannose, galactose, fructose and sorbose, examples of disaccharides that may be mentioned are sucrose, lactose, maltose and trehalose, and raffinose is an example of a trisaccharide that may be mentioned. Also, 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%.

If 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.

If the reconstituted solution is not already isotonic due to the osmotic properties of the conjugate, then 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.

In addition, 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. 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:

Example 1

Weigh 8.5 g of sodium chloride, 1.00 g of potassium hydrogen phosphate (K2HPO4), and 2.00 g of potassium dihydrogen phosphate (K2HPO4) add 1.14 kg of water for injection. Add 100 g of mannitol. Stirred on a magnetic stirrer, at 400 rpm until complete dissolution of 10 minutes, add 20 mg of propyl 4-hydroxybenzoate of sodium, 180 mg of methyl 4- hydroxybenzoate of sodium. Stirred on a magnetic stirrer, at 400 rpm until completely dissolved for 10 minutes. 0.25 g of PSMA-DIAG MA-207257-SULFO-CY7 substance is added. Stirred on a magnetic stirrer at 400 rpm until the substance is completely dissolved for 1 hour.

Example 2

Weigh 8.5 g of sodium chloride, 1.00 g of potassium hydrogen phosphate (K2HPO4), and 2.00 g of potassium dihydrogen phosphate (K2HPO4) add 1.14 kg of water for injection. Add 100 g of mannitol. Stirred on a magnetic stirrer, at 400 rpm until completely dissolved for 10 minutes. 0.25 g of PSMA-DIAG MA-207257-SULFO-CY7 substance is added. Stirred on a magnetic stirrer at 400 rpm until the substance is completely dissolved for 1 hour.

Example 3

Weigh 8.5 g of sodium chloride, 1.00 g of potassium hydrogen phosphate (K2HPO4), and 2.00 g of potassium dihydrogen phosphate (K2HPO4) add 1.14 kg of water for injection. Add 100 g of mannitol. Stirred on a magnetic stirrer, at 400 rpm until completely dissolved for 10 minutes. 20 mg of propyl 4-hydroxybenzoate of sodium, 180 mg of methyl 4- hydroxybenzoate of sodium, 10 mg of disodium edetate are added. Stirred on a magnetic stirrer, at 400 rpm until completely dissolved, 10 minutes. 0.25 g of PSMA-DIAG MA- 207257-SULFO-CY7 substance is added. Stirred on a magnetic stirrer, at 400 rpm until the substance is completely dissolved for 1 hour.

Next, the solutions prepared according to the examples are filtered through a 0.45 p prefilter filter into a suitable, pre-sterilized glass vessel. Next, the resulting solutions are filtered through a sterilizing filter with a pore size of 0.2 pm in a sterile receiving vessel. Next, 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. At the end of the freezing mode, 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. At the end of the main drying, 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.

Table 8 The study of the stability of the finished dosage form containing PSMA-

DIAG MA-207257-SULFO-CY7