RU2696096C2 - Low-molecular conjugates of antitumour agents and highly selective ligands of asialoglycoprotein receptor for therapy of oncological liver pathologies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine and concerns a ligand characterized by asialoglycoprotein receptor affinity and intended for delivering an antitumour compound used in therapy of hepatocellular carcinoma, which is a N-acetyl galactosamine derivative. Group of inventions also relates to a compound for therapy of hepatocellular carcinoma, which is a conjugate of said ligand with an antitumour compound; method of obtaining said compound by reacting [3+2] azide-alkyne cycloadaptation.

EFFECT: group of inventions provides treating hepatocellular carcinoma.

27 cl, 15 ex, 3 dwg, 1 tbl

Description

Technical field

The present invention relates to the field of organic and medical chemistry, as well as molecular biology, and relates to a method for producing a new class of biologically active substances - low molecular weight covalently coupled conjugates of antitumor compounds with highly selective ligands of the asialoglycoprotein receptor (ASGP-R), which can be used as medicines for treatment of hepatocellular carcinoma (HCC).

State of the art

Hepatocellular carcinoma ranks 5th in prevalence and 3rd in mortality among human cancers. More than 600,000 new cases of the disease are diagnosed annually in the world [Kumar Y. et al. Transarterial therapies for hepatocellular carcinoma: a comprehensive review with current updates and future directions // Asian Ras J Cancer Prev. - 2016. - T. 17. - C. 473-478]. Due to the wide range of side effects and the high general toxicity of anticancer drugs, an urgent task is to find systems for their targeted delivery to the affected tissue. Targeted delivery is a promising method for modifying drug agents, which allows improving the pharmacological profile, localizing therapeutic agents in the target tissue, organ or cell, increasing the therapeutic index and the effectiveness of drugs by increasing its effective concentration and lowering the total body content [Maklakova S. Yu. et al. A new approach to the synthesis of ligands of asialoglycoprotein receptor for targeted delivery of oligonucleotides to hepatocytes // Russian Chemical Bulletin. - 2015. - T. 64. - No. 7. - C. 1655-1662]. Asialoglycoprotein receptor (ASGP-R) is one of the most convenient targets for targeted delivery of drug agents to liver cells. This is due to factors such as: 1) high selectivity of ASGP-R with respect to galactose derivatives, 2) location of ASGP-R only on the surface of hepatocytes and 3) high concentration (more than 500,000 receptors on each cell) [Spiess M. The asialoglycoprotein receptor : a model for endocytic transport receptors // Biochemistry. - 1990. - T. 29. - No. 43. - C. 10009-10018; Stockert R.J. The asialoglycoprotein receptor: relationships between structure, function, and expression // Physiological reviews. - 1995. - T. 75. - No. 3. - C. 591-609]. An important advantage of ASGP-R is the mechanism of receptor-mediated endocytosis, due to which the drug enters the hepatocyte and is released from the delivery molecule, and the receptor returns to the membrane surface within an hour [Bareford L.M., Swaan P.W. Endocytic mechanisms for targeted drug delivery // Advanced drug delivery reviews. - 2007. - T. 59. - No. 8. - C. 748-758]. A characteristic feature of ASGP-R is its increased expression in the cell in the presence of biotin [Collins J.C. et al. Biotin-dependent expression of the asialoglycoprotein receptor in HepG2 // J. Biol. Chem. 1988. V. 263. No. 23. P. 11280-11283]. In this regard, the targeted delivery of therapeutic molecules to the liver is possible with the help of address systems that include a tissue-specific ligand based on N-acetylgalactosamine and its derivatives [Maklakova S. Yu. et al. A new approach to the synthesis of ligands of asialoglycoprotein receptor for targeted delivery of oligonucleotides to hepatocytes // Russian Chemical Bulletin. - 2015. - T. 64. - No. 7. - C. 1655-1662].

R. et al. Assessment of the integrity of poly (caprolactone)-b-poly (ethylene oxide) micelles under biological conditions: a fluorogenic-based approach // Langmuir. - 2006. - T. 22. - №. 8. - C. 3570-3578;

R., Eisenberg A., Maysinger D. Block copolymer micelles as delivery vehicles of hydrophobic drugs: micelle-cell interactions // Journal of drug targeting. - 2006. - T. 14. - №. 6. - C. 343-355; Burt H.M. et al. Development of copolymers of poly (D,L-lactide) and methoxypolyethylene glycol as micellar carriers of paclitaxel // Colloids and Surfaces B: Biointerfaces. - 1999. - T. 16. - №. 1. - C. 161-171]; 3) сравнительно короткое время полувыведения [Hong М. et al. Efficient tumor targeting of hydroxycamptothecin loaded PEGylated niosomes modified with transferrin // Journal of Controlled Release. - 2009. - T. 133. - №. 2. - C. 96-102].There are a number of examples of systems for the targeted transport of antitumor drugs to fcc cells using ASGP-R ligands [Ivanenkov YA et al. Modern approaches for treatment of liver diseases via targeted drug delivery strategies // Russ Chem Rev. - 2017. - T. 86]. The vast majority are copolymers, nanoparticles, liposomes or micelles modified with galactose or N-acetylgalactosamine residues. For example, a PK2 polymeric carrier developed by Pfizer and tested in phase I / II in patients with primary or metastatic liver cancer [Seymour LW et al. Hepatic drug targeting: phase I evaluation of polymer-bound doxorubicin // Journal of clinical oncology. - 2002. - T. 20. - No. 6. - C. 1668-1676]. Doxorubicin (Dox) was coupled via a cleavable tetrapeptide sequence to an N- (2-hydroxypropyl) methacrylamide copolymer containing fragments of N-acetylgalactosamine. It was demonstrated that in this way it is possible to provide selective transport of doxorubicin into cells, and the drug was recommended for further research (phase II), which nevertheless was suspended as a result. The main disadvantages of high molecular weight directed transport systems (micelles, nanoparticles, liposomes, etc.) are: 1) low permeability through the cell membrane and blood vessel walls [Ivanenkov YA et al. Modern approaches for treatment of liver diseases via targeted drug delivery strategies // Russ Chem Rev. - 2017 .-- T. 86; Goodman T.T., Olive PL, Pun SH Increased nanoparticle penetration in collagenase-treated multicellular spheroids // International journal of nanomedicine. - 2007. - T. 2. - No. 2. - C. 265]; 2) low stability in blood plasma [

R. et al. Assessment of the integrity of poly (caprolactone) -b-poly (ethylene oxide) micelles under biological conditions: a fluorogenic-based approach // Langmuir. - 2006. - T. 22. - No. 8. - C. 3570-3578;

R., Eisenberg A., Maysinger D. Block copolymer micelles as delivery vehicles of hydrophobic drugs: micelle-cell interactions // Journal of drug targeting. - 2006. - T. 14. - No. 6. - C. 343-355; Burt, HM et al. Development of copolymers of poly (D, L-lactide) and methoxypolyethylene glycol as micellar carriers of paclitaxel // Colloids and Surfaces B: Biointerfaces. - 1999. - T. 16. - No. 1. - C. 161-171]; 3) a relatively short half-life [Hong M. et al. Efficient tumor targeting of hydroxycamptothecin loaded PEGylated niosomes modified with transferrin // Journal of Controlled Release. - 2009. - T. 133. - No. 2. - C. 96-102].

In contrast to high molecular weight delivery systems, low molecular weight conjugates of ASGP-R ligands with antitumor drugs in which the vector fragment and the active substance are connected using a relatively small biodegradable fragment (linker) are practically not described in the literature. Although the addition of a galactose fragment to the structure of low molecular weight conjugates is quite common [Ma Y. et al. Galactose as Broad Ligand for Multiple Tumor Imaging and Therapy // Journal of Cancer. - 2015. - T. 6. - No. 7. - C. 658-670; Melisi D. et al. D-Galactose as a vector for prodrug design // Current topics in medicinal chemistry. - 2011. - T. 11. - No. 18. - C. 2288-2298], these works do not consider it as an ASGP-R ligand, but in particular suggest an increase in solubility in aqueous solutions and an affinity for another receptor. The invention is the first case describing conjugates of antitumor drug compounds linked to an ASGP-R-specific ligand via a relatively short biodegradable linker. The synthesis of new asialoglycoprotein receptor ligands based on N-acetylgalactosamine derivatives was performed, as well as the synthesis of their covalently bound low molecular weight conjugates with anticancer drugs and biological tests of the obtained conjugates on HepG2, Hek293 cell lines.

Disclosure of invention

An object of the present invention is to provide new low molecular weight covalently coupled conjugates of antitumor drug compounds with asialoglycoprotein receptor ligands based on N-acetylgalactosamine derivatives for the treatment of liver cancer.

The technical result is to obtain new compounds with a pronounced affinity for the asialoglycoprotein receptor and toxicity to cell lines of hepatocellular carcinoma, low toxicity to other tissues and improved bioavailability of the active antitumor compound.

The problem is solved by low molecular weight conjugates of antitumor compounds with ligands of the asialoglycoprotein receptor based on derivatives of N-acetylgalactosamine, the general structural formula:

where the ligand is a derivative of N-acetylgalactosamine of structural formulas 1, 2, 3:

a "Antineoplastic agent" (antitumor compound) is a compound containing a free hydroxyl or amino group, forming a covalent bond with a ligand and having an antitumor effect for the treatment of liver cancer. In this case, it is preferable to use paclitaxel or gemcitabine, or cytarabine, or azacitidine as an antitumor compound containing a free hydroxyl compound, and doxorubicin (Dox * HCl) or daunorubicin is preferably used as an antitumor compound containing a free amino group. The ligands of formulas 1, 2 and 3 are characterized by affinity for the asialoglycoprotein receptor and are intended for the delivery of antitumor compounds used in the treatment of hepatocellular carcinoma.

The method of obtaining low molecular weight conjugates consists in carrying out two sequential reactions, first the carbodiimide synthesis reaction by mixing the antitumor compound and the alkynyl acid with the terminal triple bond, taken from the calculation that at least 1.0 equivalent of the alkynyl acid with the terminal triple bond is taken per 1 molar equivalent of the antitumor compound , in the presence of an activator of a carboxyl group, taken on the basis of 1 molar equivalent of alkynyl acid, take 1 equivalent of an activator a carboxyl group in a medium that provides a carbodiimide synthesis reaction and isolation of the intermediate by column chromatography — an antitumor compound modified with alkynyl acid with a teminal triple bond, and then the reaction of [3 + 2] azide-alkynyl cycloaddition by mixing the intermediate and the component, containing an azido group, in the presence of a catalyst - a compound of monovalent copper, in an environment that provides the reaction of cycloaddition, while 1 the molar equivalent of the intermediate product uses at least 1.0 equivalent of the component containing the azido group, and at least 0.2 equivalent of the catalyst, after completion of the reaction, the medium is removed and the final product is isolated by column chromatography. It is preferable to use paclitaxel, or gemcitabine, or cytarabine, or azacytidine, or doxorubicin (Dox * HCl), or daunorubicin as an antitumor compound, while prop-2-inic acid, but-3-, is used as an alkynyl acid with a terminal triple bond inoic acid, pent-4-inoic acid, hex-5-inoic acid, hept-6-inoic acid, and as a medium providing the reaction of carbodiimide synthesis, use DMF (dimethylformamide), CH ₂ Cl ₂ (dichloromethane), CHCl ₃ (chloroform), tetrohydrofuran, ClCH ₂ CH ₂ Cl ( 1,2-dichloroethane). As an activator of the carboxyl group, it is preferable to use carbodiimide-soluble in a medium providing the reaction of carbodiimide synthesis, carbodiimides, namely 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), N, N'-diisopropylcarbodiimide (DIC), N, N '-dicyclohexylcarbodiimide (DCC). Isolation of the intermediate product is preferably carried out using direct column chromatography in a system with a concentration gradient of CH ₂ Cl ₂ : MeOH (10: 1) → MeOH. For the [3 + 2] azide-alkynyl cycloaddition reaction, it is preferable to use the N-acetylgalactosamine derivative selected from the group consisting of 1-Azido-1-deoxy-2-acetamido-2-deoxy-β- as the component containing the azido group D-galactopyranose, 1-O- (2'-azidoethyl) -2-acetamido-2-deoxy-β-D-galactopyranose, 1-O-allyl-2-acetamido-2-deoxy-6-azido-6-deoxy β-D-galactopyranose. In this case, as a compound of monovalent monovalent copper, it is preferable to use monovalent salts of copper soluble in a medium providing a cycloaddition reaction, namely, copper iodide (CuI), copper bromide (CuBr), copper chloride (CuCl), and as a medium providing a flow [3 + 2] azide-alkynyl cycloaddition reactions, it is preferable to use DMF, a mixture of water-tetrohydrofuran in a volume ratio of 1: 1, dimethyl sulfoxide (DMSO). To isolate the final product, a low molecular weight conjugate, it is preferable to use reverse phase column chromatography in an H ₂ O-MeCN system.

To obtain a low molecular weight conjugate with an antitumor compound comprising an amine available for modification, preferably doxorubicin or daunorubicin, a ligand of the formulas 1, 2 and 3 is first obtained by reaction of the [3 + 2] azide-alkine cycloaddition by mixing the compound containing the terminal an alkyl fragment, and a compound containing an azido group, taken in a molar ratio of 1: 1, in the presence of a catalyst — a salt of monovalent copper, taken at the rate of 1 mol the equivalent component containing the azido group, at least 0.25 equivalents of the catalyst in the medium providing the cycloaddition reaction, after the reaction is complete, the medium is removed and the ligand is isolated by column chromatography, followed by the carbodiimide synthesis reaction between the obtained ligand and the antitumor compound available for modifications with an amino group taken in a 1: 1 molar ratio in the presence of an activator of a carboxyl group, taken at the rate of 1 molar equivalent gand according to claim 1, at least 1 equivalent of an activator, in DMF, after the reaction, DMF is removed and the conjugate is isolated by column chromatography. Moreover, DMF, a mixture of water-tetrohydrofuran in a volume ratio of 1: 1, DMSO are used as a medium providing the course of the reaction of [3 + 2] azide-alkine addition. As a compound containing a terminal alkynyl moiety, it is preferable to use alkynyl acids with a terminal triple bond, namely prop-2-inic acid, but-3-inic acid, pent-4-inic acid, hex-5-inic acid, hept- 6-acid, and as a compound containing an azido group, it is preferable to use derivatives of N-acetylgalactosamine, namely 1-Azido-1-deoxy-2-acetamido-2-deoxy-β-D-galactopyranose, 1-O- ( 2'-azidoethyl) -2-acetamido-2-deoxy-β-D-galactopyranose, 1-O-allyl-2-acetamido-2-deoxy-6-azido-6-d zoksi-β-D-galactopyranose. It is preferable to use monovalent copper salts soluble in a medium providing the cycloaddition reaction, copper salts, namely CuI, CuBr, CuCl. Moreover, to isolate the obtained ligand, it is preferable to use direct column chromatography in a system with a concentration gradient of CH ₂ Cl ₂ : MeOH (10: 1) → MeOH. When carrying out the carbodiimide synthesis reaction, it is preferable to use DMF-soluble carbodiimides as an activator of the carboxyl group, namely 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), N, N'-diisopropylcarbodiimide (DIC), N, N'- dicyclohexylcarbodiimide (DCC). To isolate the resulting conjugate, it is preferable to use two sequential column chromatography direct column chromatography system with a concentration gradient of CH ₂ Cl ₂ : MeOH (20: 1) → MeOH and reverse phase column chromatography in a system of H ₂ O-MeCN.

Brief Description of the Drawings

In FIG. 1 shows the pictures taken with a microscope, conjugates (4) - A, (5) - B, (6) - C. The incubation time is 24 hours.

In FIG. 2 shows images of untreated Hepg2 cells that were fixed, permeabilized and immunized with anti-α-tubulin antibody

In FIG. Figure 3 presents images of the Hepg2 cell line treated with conjugates (8-10). Cells were fixed, permeabilized and immunized with anti-α-tubulin antibody. On the pictures: A - cells after treatment with conjugate 8 (6 hours), B - cells after treatment with conjugate 8 (39 hours), C - cells after treatment with conjugate 9 (3 hours), D - cells after treatment with conjugate 9 (17 hours) , E - cells after treatment with conjugate 10 (24 hours), F - cells after treatment with conjugate 9 (17 hours).

The implementation of the invention

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

“Asialoglycoprotein Receptor (ASGP-R)” is a transmembrane protein predominantly expressed by hepatocytes, selectively recognizing derivatives of D-galactose and D-galactosamine and participating in their transport into the cell.

A “conjugate” is a complex formed by covalent bonds between an antitumor compound and its carrier ligand.

“Ligand” is a chemical compound that forms a non-covalent complex with one or another biomolecule (most often a protein, for example, a cellular receptor, but sometimes, for example, with DNA) and produces, due to this binding, certain biochemical, physiological or pharmacological effects.

"Antitumor drug compound (agent)" - a substance used as an active ingredient in drugs for the treatment of cancer. Examples of used antitumor agents and their covalent bond with the claimed ligand are shown in scheme (I)

where "Ligand" (ligand) is the above-described derivatives of N-acetylgalactosamine with structural formulas 1, 2, 3.

All reagents used are commercially available; the solvent was evaporated using a rotary evaporator under reduced pressure at a bath temperature of about 50 ° C; the progress of the reaction was monitored by thin layer chromatography (TLC), and the reaction time is indicated for illustration only; the structure and purity of all isolated compounds was confirmed by at least one of the following methods: TLC (TLC plates with 60 F ₂₅₄ Merck pre-coated silica gel), mass spectrometry, or nuclear magnetic resonance (NMR). The product yield is for illustration only. Flash column chromatography was performed using Merck silica gel 60 (230-400 mesh ASTM). High resolution mass spectra (HRMS) were recorded on a Bruker microTOF II spectrometer. NMR spectra were recorded on Bruker Avance-400 instruments (operating frequency 400.1 and 100.6 MHz for ¹ N and ¹³ C, respectively) and Agilent 400-MR (operating frequency 400.0 and 100.6 MHz for ¹ N and ¹³ C, respectively) using deuterated chloroform (99.8% D) or methanol (99.9%) or DMSO (99.9% D) as a solvent, unless otherwise indicated, relative to tetramethylsilane (TMS) as an internal standard, ppm (ppm); The usual abbreviations used are as follows: s - singlet, d - doublet, t - triplet, q - quartet, m - multiplet, width - wide and so on.

A method for producing conjugates of antitumor drug compounds with ligands of an asialoglycoprotein receptor based on derivatives of N-acetylgalactosamine for the treatment of liver cancer is carried out by a carbodiimide synthesis reaction followed by a [3 + 2] azide-alkynyl cycloaddition reaction. For antitumor compounds with an amino group available for modification, a synthesis route is possible with the initial production of the ligand by the reaction of [3 + 2] azide-alkine cycloaddition and the subsequent carbodiimide synthesis reaction between the ligand and the antitumor compound.

The formation of an asialoglycoprotein receptor ligand conjugate based on derivatives of N-acetylgalactosamine with antitumor compounds containing an amino and hydroxyl group, accessible for acylation with alkynyl acid with a terminal triple bond, consists of two sequential reactions. First, the antitumor compound is modified with an alkyno acid with a terminal triple bond using a carbodiimide synthesis reaction, which is carried out by mixing the antitumor compound and an alkynic acid with a terminal triple bond in the presence of an activator of a carboxyl group in a medium providing a carbodiimide synthesis reaction, with 1 molar equivalent anti-tumor compounds take at least 1 equivalent of acid and at least 1.0 equivalent of activator carboxy yl group. Then, an anticancer compound modified with an alkynic acid with a terminal triple bond is isolated by direct column chromatography in a system with a concentration gradient of CH ₂ Cl ₂ : MeOH (10: 1) → MeOH. Then, the [3 + 2] azide-alkine cycloaddition reaction is carried out by mixing the modified antitumor compound containing the terminal alkyl fragment and the compound containing the azido group in a molar ratio, based on the fact that per 1 molar equivalent of the compound containing the azido group is used at at least 1.0 equivalent of a compound containing a terminal alkyl moiety. The reaction is carried out in the presence of a catalyst - a compound of monovalent copper, taken on the basis of 1 molar equivalent of a compound containing an azido group, at least 0.2 equivalent of a catalyst is taken, while the reaction is carried out in an environment that dissolves the compounds. After the reaction, the medium is removed, followed by isolation of the product by column chromatography.

As an antitumor compound, paclitaxel, or gemcitabine, or cytarabine, or azacitidine, or doxorubicin, or daunorubicin is used.

As the alkynyl acid with a terminal triple bond, prop-2-inic acid, but-3-in-acid, pent-4-in-acid, hex-5-in-acid, hept-6-in-acid are used.

As the medium providing the reaction of carbodiimide synthesis, dimethylformamide, dichloromethane, chloroform, tetrohydrofuran, 1,2-dichloroethane are used.

As an activator of the carboxyl group, carbodiimides: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), N, N'-diisopropylcarbodiimide (DIC), N, N'-dicyclohexylcarbodiimide are used as soluble in the carboxyl group; (DCC).

To isolate the intermediate, direct column chromatography was used in a system with a concentration gradient of CH ₂ Cl ₂ : MeOH (10: 1) → MeOH.

As the medium providing the course of the [3 + 2] azide-alkine cycloaddition reaction, DMF, a mixture of water-tetrohydrofuran in a volume ratio of 1: 1, DMSO or any other solvent that provides dissolution and is inert to the components of the mixture are used.

As a component containing an azido group, derivatives of N-acetylgalactosamine: 1-Azido-1-deoxy-2-acetamido-2-deoxy-β-D-galactopyranose, 1-O- (2'-azidoethyl) -2-acetamido -2-deoxy-β-D-galactopyranose, 1-O-allyl-2-acetamido-2-deoxy-6-azido-6-deoxy-β-D-galactopyranose.

As a catalyst — compounds of monovalent copper, any salt of monovalent copper, which is soluble in the medium providing the reaction of cycloaddition, is used. For example, CuI, CuBr, CuCl, and the like.

To isolate the obtained product, reverse phase column chromatography in a H ₂ O-MeCN system is used.

The upper limit of the content of the reagents used is not limited, because an excess of any reagent does not reduce the yields of the reactions; however, with a large excess, additional purification of the reaction products may be necessary.

For antitumor compounds with a modifiable amino group, ligand addition is carried out as follows.

At the first stage, the inventive ligands are obtained by carrying out the [3 + 2] azide-alkynyl cycloaddition reaction by mixing a compound containing a terminal alkyl fragment and a compound containing an azido group, using 1.0 molar equivalent of a compound containing an azido group at least 1.0 equivalent of a compound containing a terminal alkyl moiety. The reaction is carried out in the presence of a catalyst - a compound of monovalent copper, taken in an amount of at least 0.25 equivalent of catalyst per 1.0 molar equivalent of a compound containing an azido group. The reaction is carried out in an environment that provides dissolution of the components. Moreover, DMF, a water-tetrohydrofuran mixture in a volume ratio of 1: 1, DMSO or any other solvent that provides dissolution and is inert to the components of the mixture are used as a medium providing dissolution of the compounds. After the reaction, the medium is removed, followed by isolation of the product by column chromatography.

As compounds containing a terminal alkynyl moiety, alkynyl acids with a terminal triple bond are used. The length of the carbon fragment of the acid does not matter, the alkynyl acid with the terminal triple bond may be prop-2-inic acid, but-3-inic acid, pent-4-inic acid, hex-5-inic acid, hept- 6-in, etc.

As a component containing an azido group, derivatives of N-acetylgalactosamine are used: 1-Azido-1-deoxy-2-acetamido-2-deoxy-β-D-galactopyranose, 1-O- (2'-azidoethyl) -2- acetamido-2-deoxy-β-D-galactopyranose, 1-O-allyl-2-acetamido-2-deoxy-6-azido-6-deoxy-β-D-galactopyranose.

As a catalyst — compounds of monovalent copper, any salt of monovalent copper, which is soluble in the medium providing the reaction of cycloaddition, is used. For example, CuI, CuBr, CuCl, etc.

To isolate the obtained product, direct column chromatography was used with a concentration gradient system of CH ₂ Cl ₂ : MeOH (10: 1) → MeOH.

The formation of the conjugate of the obtained ligand of the asialoglycoprotein receptor based on derivatives of N-acetylgalactosamine with antitumor compounds with an amine available for modification consists in carrying out a carbodiimide synthesis reaction by mixing a compound containing a carboxyl group - a ligand of the formula 1, or 2, or 3, respectively, and the antitumor compound, including an amino group, for example, doxorubicin or daunorubicin, in a molar ratio of 1: 1, in the presence of an activator of a carboxyl group oops. The amount of activator is taken at the rate of 1.0 molar equivalent of an antitumor agent at least 1.1 equivalent of an activator. The carbodiimide synthesis reaction is carried out in an environment that provides dissolution of the compounds and is inert to the components of the mixture. It is preferable to use DMF. After the reaction, the medium is removed, followed by isolation of the product by column chromatography.

As activators of the carboxyl group, any activator of the carboxyl group of the carbodiimide series is used, for example: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), N, N'-diisopropylcarbodiimide (DIC), N, N'-dicyclohexylcarbodiimide (DCC) and so on

To isolate the resulting product, two sequential column chromatography was used, direct column chromatography on a system with a concentration gradient of CH ₂ Cl ₂ : MeOH (20: 1) → MeOH, and reverse phase column chromatography in an H ₂ O-MeCN system.

The following examples illustrate but do not limit the present invention.

Example 1. Obtaining 4- (1- (3-acetamido-4,5-dihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) -1H-1,2,3-triazol-4-yl) butanoic acid (1).

N. et al. Iron (III) chloride as an efficient catalyst for stereoselective synthesis of glycosyl azides and a cocatalyst with Cu (0) for the subsequent click chemistry // Chemical Communications. - 2011. - T. 47. - №. 37. - C. 10440-10442], в 15 мл сухого ДМФА добавили 60 мг (0,528 ммоль, 1,2 экв.) гекс-5-иновой кислоты и 21 мг (0,11 ммоль, 0,25 экв.) CuI. Полученную смесь перемешивали в течение 48 часов, затем ДМФА удалили на роторном испарителе. Полученное вещество очищали методом колоночной хроматографии в системе с градиентом концентраций CH₂Cl₂:МеОН (10:1) → МеОН. В результате получили соединение 1 в виде светло-желтого аморфного вещества 125 мг (79%).To a solution of 108 mg (0.44 mmol) of 1-Azido-1-deoxy-2-acetamido-2-deoxy-β-D-galactopyranose, obtained according to the procedure described in [

N. et al. Iron (III) chloride as an efficient catalyst for stereoselective synthesis of glycosyl azides and a cocatalyst with Cu (0) for the subsequent click chemistry // Chemical Communications. - 2011. - T. 47. - No. 37. - C. 10440-10442], 60 mg (0.528 mmol, 1.2 eq.) Of hex-5-in acid and 21 mg (0.11 mmol, 0.25 eq.) Of CuI were added to 15 ml of dry DMF . The resulting mixture was stirred for 48 hours, then DMF was removed on a rotary evaporator. The resulting material was purified by column chromatography in a system with a concentration gradient of CH ₂ Cl ₂ : MeOH (10: 1) → MeOH. As a result, compound 1 was obtained as a pale yellow amorphous substance 125 mg (79%).

¹ H NMR (400 MHz, CD ₃ OD, δ, ppm): 8.03 (s, 1H, H-16); 5.75 (d, 1H, J = 9.8 Hz, H-5); 4.51-4.63 (m, 1H, H-1); 4.06 (d, 1H, J = 3.1 Hz, H-2); 4.21-4.30 (m, 1H, H-3); 3.73-3.97 (m, 4H, H-3,4,7); 2.68-2.80 (m, 2H, H-17); 2.20-2.32 (m, 2H, H-19); 1.88-2.01 (m, 2H, H-18); 1.79 (s, 1H, -NHAc).

HRMS (m / z) for C ₁₄ H ₂₂ N ₄ O ₇ : [M + H] ⁺ 359.1561 (found), 359.1562 (calc.); [M + Na] ⁺ 381.1381 (found), 381.1381 (calc.).

Example 2. Obtaining 4- (1- (2 - ((3-acetamido-4,5-dihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) ethyl) -1H-1,2, 3-triazol-4-yl) butanoic acid (2).

To a solution of 30 mg (0.103 mmol) of 1-O- (2'-azidoethyl) -2-acetamido-2-deoxy-β-D-galactopyranose, obtained according to the procedure described in [Hasegawa A. et al. Studies on the thioglycosides of N-acetylneuraminic acid 10: Synthesis of S- (α-sialosyl) - (2 → 6) -O-2-acetamido-2-deoxy-β-d-hexopyranosyl ceramide and its related compounds // Journal of carbohydrate chemistry. - 1992. - T. 11. - No. 3. - C. 319-331], 13.9 mg (0.124 mmol, 1.2 equiv.) Of hex-5-in acid and 4.9 mg (0.026 mmol, 0.25 equiv.) Were added to 15 ml of dry DMF. ) CuI. The resulting mixture was stirred for 48 hours, then DMF was removed on a rotary evaporator. The resulting material was purified by column chromatography in a system with a concentration gradient of CH ₂ Cl ₂ : MeOH (10: 1) → MeOH. The result was a compound (2) in the form of a light yellow amorphous substance 31 mg (75%).

¹ H NMR (400 MHz, CD ₃ OD, δ, ppm): 8.21 (s, 1H, H-19); 4.67-4.77 (m, 2H, H-1.2); 4.40 (d, 1H, J = 8.4 Hz, H-5); 4.21-4.30 (m, 1H, H-3); 3.90-4.03 (m, 2H, H-4, 13); 3.80-3.90 (m, 1H, H-13); 3.48-3.80 (m, 4H, H-7.14); 2.86 (t, 2H, J = 7.4 Hz, H-20); 2.45 (t, 2H, J = 7.4 Hz, H-22); 1.92-2.08 (m, 5H, H-21, -NHAc).

HRMS (m / z) for C ₁₆ H ₂₆ N ₄ O ₈ : [M + H] ⁺ 403.1819 (found), 403.1823 (calc.).

Example 3. Obtaining 4- (1 - ((5-acetamido-6- (allyloxy) -3,4-dihydroxytetrahydro-2H-pyran-2-yl) methyl) -1H-1,2,3-triazol-4- il) butanoic acid (3).

To a solution of 30 mg (0.105 mmol) of 1-O-Allyl-2-acetamido-2-deoxy-6-azido-6-deoxy-β-D-galactopyranose, obtained according to the previously described method [Wong C.N. et al. A library approach to the discovery of small molecules that recognize RNA: use of a 1, 3-hydroxyamine motif as core // Journal of the American Chemical Society. - 1998. - T. 120. - No. 33. - C. 8319-8327] in 15 ml of dry DMF, 14.1 mg (0.125 mmol, 1.2 equiv.) Of hex-5-in acid and 5 mg (0.026 mmol, 0.25 equiv.) Of CuI were added. The resulting mixture was stirred for 48 hours, then DMF was removed on a rotary evaporator. The resulting material was purified by column chromatography in a system with a concentration gradient of CH ₂ Cl ₂ : MeOH (10: 1) → MeOH. As a result, compound (3) was obtained as a pale yellow amorphous substance (28 mg, 67%).

¹ H NMR (400 MHz, CD ₃ OD, δ, ppm): 7.99 (s, 1H, H-19); 5.65-5.78 (m, 1H, H-14); 5.04-5.19 (m, 2H, H-15); 4.81 (d, 1H, J = 3.6 Hz, H-2); 4.63-4.71 (m, 2H, H-1.5); 4.30 (dd, 1H, J = 3.5 and 11.0 Hz, H-3); 4.19-4.24 (m, 1H, H-4), 3.95 (d, 1H, J = 2.6 Hz, H-7), 3.84 (dd, 1H, J = 3.1 and 11.0 Hz, H-7), 3.63-3.75 (m, 2Н, Н-13), 2.74-2.86 (m, 2Н, Н-20), 2.32-2.42 (m, 2Н, Н-22), 1.91-2.05 (m, 5Н, Н-21, -NHAc )

HRMS (m / z) for C ₁₇ H ₂₆ N ₄ O ₇ : [M + H] ⁺ 399.1876 (found), 399.1874 (calculated); [M + Na] ⁺ 421.1692 (found), 421.1694 (calc.).

Example 4. Obtaining compound (4).

To a solution of 6 mg (0.01 mmol) of doxorubicin hydrochloride in 6 ml of dry DMF was added 3.95 mg (0.01 mmol, 1 equiv.) Of compound (1) in the presence of 1.15 mg (0.011 mmol) of Et ₃ N, 1.31 mg (0.011 mmol, 1.1 eq.) Of NHS; 2.11 mg (0.011 mmol, 1.1 eq.) Of EDC in an inert atmosphere. The resulting mixture was stirred for 24 hours, then DMF was removed on a rotary evaporator. The obtained substance was purified by two successive chromatographies: 1) column chromatography on silica gel (gradient elution with CH ₂ Cl ₂ : MeOH (20: 1) → MeOH), 2) HPLC in a reversed-phase system (H ₂ O - AcCN). The result was conjugate (4) in the form of a dark red powder (7.3 mg, 80%).

¹ H NMR (400 MHz, CD ₃ OD, δ, ppm): 7.72-8.04 (m, 3H, H-1.6, 61); 7.56 (d, 1H, J = 7.6 Hz, H-2); 5.61 (d, 2H, J = 9.2 Hz, H-51); 5.09-5.22 (m, 1H, H-32); 4.65 (s, 2H, H-20); 4.46 (t, 1H, J = 10.0 Hz, H-53); 4.09-4.33 (m, 3H, H-54.55.56); 4.04 (s, 5H, H-29.57); 3.69-3.87 (m, 5H, H-31.34.35.36, OH); 3.62 (br s, 1H, OH); 2.91-3.18 (m, 2H, H-45); 2.27-2.44 (m, 2H, H-16); 2.06-2.22 (m, 2H, H-42); 1.84-2.03 (m, 4H, H-44.33); 1.77 (s, 3H, -NHAc); 1.20-1.34 (m, 5H, H-18.38).

HRMS (m / z) for C ₄₁ H ₄₉ N ₅ O ₁₇ : [M + H] ⁺ 884.3196 (found), 884.3196 (calc.); [M + Na] ⁺ 906.3025 (found), 906.3016 (calc.).

Example 5. Obtaining compound (5).

To a solution of 6 mg (0.01 mmol) of Dox * HCl in 6 ml of dry DMF was added 4.16 mg (0.01 mmol, 1 equiv.) Of compound (2) in the presence of 1.15 mg (0.011 mmol, 1.1 equiv.) Et ₃ N, 1.31 mg (0.011 mmol, 1.1 equiv.) NHS, 2.11 mg (0.011 mmol, 1.1 equiv.) EDC in an inert atmosphere. The resulting mixture was stirred for 24 hours, then DMF was removed on a rotary evaporator. The obtained substance was purified by two successive chromatographies: 1) column chromatography on silica gel (gradient elution with CH ₂ Cl ₂ : MeOH (20: 1) → MeOH), 2) HPLC in a reversed-phase system (H ₂ O - AcCN). The result was conjugate (5) in the form of a dark red powder (6.7 mg, 70%).

¹ H NMR (400 MHz, CD ₃ OD, δ, ppm): 8.00 (d, 1H, J = 7.5 Hz, H-6); 7.85 (t, J = 7.5 Hz, H-1); 7.74 (s, 1H, H-61); 7.58 (d, 1H, J = 7.5 Hz, H-2); 5.43 (m, 1H, OH); 5.19 (m, 1H, H-32); 4.74 (m, 2H, H-55); 4.61 (s, 2H, H-20); 4.51 (t, 2H, J = 6.2 Hz, H-56); 4.33 (d, 1H, J = 8.4 Hz, H-47); 4.28 (d, 1H, J = 6.6 Hz, H-43); 4.08-4.22 (m, 2H, H-44.45); 4.04 (s, 3H, H-29); 3.66-3.96 (m, 6H, H-31.34.35.36.46, OH); 3.62 (br s, 1H, OH); 3.51-3.57 (m, 1H, H-49); 3.44-3.50 (m, 1H, H-49); 3.00-3.18 (m, 2H, H-62); 2.37 (m, 2H, H-16); 2.23 (t, 2H, J = 7.8 Hz, H-64); 1.85-2.05 (m, 7H, -NHAc, H-33.63); 1.24-1.34 (m, 5H, H-18.38).

HRMS (m / z) for C ₄₃ H ₅₃ N ₅ O ₁₈ : [M + H] ⁺ 928.3458 (found), 928.3456 (calculated); [M + Na] ⁺ 950.3277 (found), 950.3270 (calc.).

Example 6. Obtaining compound (6).

To a solution of 10 mg (0.017 mmol) of Dox * HCl in 10 ml of dry DMF was added 6.87 mg (0.017 mmol, 1 equiv.) Of compound (3) in the presence of 1.92 mg (0.019 mmol, 1.1 equiv.) Et ₃ N, 2.18 mg (0.019 mmol, 1.1 equiv.) NHS, 3.64 mg (0.019 mmol, 1.1 equiv.) EDC in an inert atmosphere. The resulting mixture was stirred for 24 hours, then DMF was removed on a rotary evaporator. The obtained substance was purified by two successive chromatographies: 1) column chromatography on silica gel (gradient elution with CH ₂ Cl ₂ : MeOH (20: 1) → MeOH), 2) HPLC in a reversed-phase system (H ₂ O - AcCN). As a result, the conjugate (6) was obtained as a dark red powder (6 mg, 38%). ¹ H NMR (400 MHz, CD ₃ OD, δ, ppm): 7.94 (d, 1H, J = 7.5 Hz, H-6); 7.83 (t, J = 7.5 Hz, H-1); 7.73 (s, 1H, H-59); 7.56 (d, 1H, J = 7.5 Hz, H-2); 5.56-5.72 (m, 1H, H-54); 5.42 (m, 1H, OH); 4.96-5.23 (m, 3H, H-32.55); 4.75 (m, 2H, H-53); 4.48-4.56 (m, 2H, H-20); 4.20-4.33 (m, 2H, H-41.45); 4.08-4.19 (m, 1H, H-42.43); 4.02 (s, 3H, H-29); 3.88 (s, 1H, OH); 3.74-3.84 (m, 1H, H-44); 3.46-3.70 (m, 5H, H-31.34.35.36.47); 2.95-3.18 (m, 2H, H-64); 2.11-2.42 (m, 4H, H-16, 61); 1.81-2.07 (m, 5H, -NHAc, H-63); 1.54-1.76 (m, 2H, H-33).

HRMS (m / z) for C ₄₄ H ₅₃ N ₅ O ₁₇ : [M + H] ⁺ 924.3509 (found), 924.3543 (calc.); [M + Na] ⁺ 946.3340 (found), 946.3329 (calc.).

Example 7. Synthesis of paclitaxel monoacylated with hex-5-inic acid (7). [[Pilkington-Miksa M. et al. Design, synthesis, and biological evaluation of novel cRGD-paclitaxel conjugates for integrin-assisted drug delivery // Bioconjugate chemistry. - 2012. - T. 23. - No. 8. - C. 1610-1622.]

To a solution of 40.9 mg (47.87 μmol) of paclitaxel in 20 ml of dry CH ₂ Cl _{2 was} added 5.9 mg (52.66 μmol, 1.1 eq.) Of hex-5-inic acid and 8.2 mg ( 52.66 μmol, 1.1 eq.) EDC, as well as a catalytic amount of DMAP in an inert atmosphere. The resulting mixture was stirred for 24 hours at room temperature, then CH ₂ Cl _{2 was} removed on a rotary evaporator. The obtained substance was purified by reverse phase column chromatography on an INTERCHIM PURIFLASH 430 instrument on a PURIFLASH C18-HP 15UM F0012 reverse phase column in a system with a concentration gradient of H ₂ O: MeCN (1: 0) → H ₂ O: MeCN (0: 1 ) As a result, compound (7) was obtained as a white powder (41.3 mg, 91%).

¹ H NMR (400 MHz, CDCl ₃ , δ, ppm): 8.15 (d, 2H, J = 7.2 Hz, H-ortho- (Ar1)); 7.75 (d, 2H, J = 7.3 Hz, H-ortho- (Ar3)); 7.62 (t, 1H, J = 7.4 Hz, H-para- (Ar1)); 7.57-7.48 (m, 3H, Aromatics); 7.47-7.32 (m, 7H, Aromatics); 6.90 (d, 1H, J = 9.2 Hz, NH); 6.23-6.32 (m, 2H, H-10, 13); 5.97 (dd, 1H, J = 2.9 and 9.2 Hz, H-3 '); 5.69 (d, 1H, J = 7.1 Hz, H-2); 5.51 (d, 1H, J = 3.1 Hz, H-2 '); 4.98 (dd, 1H, J = 1.8 and 9.5 Hz, H-5); 4.45 (dd, 1H, J = 6.7 and 10.8 Hz, H-7); 4.33 (d, 1H, J = 8.4 Hz, H-20); 4.21 (d, 1H, J = 8.6 Hz, H-20); 3.82 (d, 1H, J = 7.1 Hz, H-3); 2.48-2.70 (m, 3H, H-6, 4``,); 2.47 (s, 3H, 4-OAc); 2.13-2.44 (m, 8H, H-14, 6``, 10-OAc, H-2 ''); 2.00-1.78 (m, 6H, H-6.18, 3``); 1.69 (s, 3H, H-19); 1.24 (s, 3H, H-17); 1.14 (s, 3H, H-16).

Example 8. Synthesis of conjugate paclitaxel (8).

N. et al. Iron (III) chloride as an efficient catalyst for stereoselective synthesis of glycosyl azides and a cocatalyst with Cu (0) for the subsequent click chemistry // Chemical Communications. - 2011. - T. 47. - №. 37. - С. 10440-10442] в присутствии 0,4 мг (2,11 мкмоль, 0,2 экв.) CuI и 0,2 мг (2,11 мкмоль, 0,2 экв.) Et₃N в инертной атмосфере. Полученную смесь перемешивали в течение 24 часов, затем ДМФА удалили на роторном испарителе. Полученное вещество очищали методом ВЭЖХ в обращено-фазовой системе (Н₂О - AcCN). В результате получили конъюгат (8) в виде белого порошка (10 мг, 79%).To a solution of 10 mg (10.54 μmol) of compound (7) in 7 ml of dry DMF was added 2.6 mg (10.54 μmol, 1 equiv.) Of 1-Azido-2-acetamido-2-deoxy-3,4, 6-tri-O-acetyl-β-D-galactopyranose obtained according to the procedure described in [

N. et al. Iron (III) chloride as an efficient catalyst for stereoselective synthesis of glycosyl azides and a cocatalyst with Cu (0) for the subsequent click chemistry // Chemical Communications. - 2011. - T. 47. - No. 37. - S. 10440-10442] in the presence of 0.4 mg (2.11 μmol, 0.2 equiv.) CuI and 0.2 mg (2.11 μmol, 0.2 equiv.) Et ₃ N in inert atmosphere. The resulting mixture was stirred for 24 hours, then DMF was removed on a rotary evaporator. The resulting substance was purified by HPLC in a reversed-phase system (H ₂ O — AcCN). The result was conjugate (8) in the form of a white powder (10 mg, 79%).

¹ H NMR (400 MHz, CDCl ₃ , δ, ppm): 8.04 (d, 2H, J = 7.8 Hz, H-ortho- (Ar1)); 7.99 (m, 1H, -NH-); 7.82 (s, 1H, H-6``); 7.69 (d, 2H, J = 6.5 Hz, H-ortho- (Ar3)); 7.29-7.57 (m, 11H, Aromatics); 6.26 (s, 1H, H-10); 6.08 (t, 1H, J = 8.7 Hz, H-13); 5.84-5.91 (m, 1H, H-3 '); 5.60-5.68 (m, 2H, H-2, H-10``); 5.34-5.42 (m, 1H, H-2 '); 4.91 (d, 1H, J = 9.8 Hz, H-5); 4.12-4.42 (m, 4H, H-7.20, 12``); 3.93-4.00 (m, 1H, H-13``); 2.53-2.73 (m, 3H, H-6.4``); 2.30-2.50 (m, 5H, 4-Ac, 2``); 2.10-2.27 (m, 5H, H-14, 10-Ac); 1.75-2.06 (m, 6H, H-6.3``, -NHAc); 1.65 (s, 3H, H-18); 1.60 (s, 3H, H-19); 1.11 (s, 3H, H-17); 1.07 (s, 3H, H-16).

HRMS (m / z) for C ₆₁ H ₇₁ N ₅ O ₂₀ : [M + H] ⁺ 1194.4771 (found), 1194.4765 (calc.); [M + Na] ⁺ 1216.4585 (found), 1216.4585 (calc.).

Example 9. Synthesis of paclitaxel conjugate (9).

To a solution of 10 mg (10.54 μmol) of compound (7) in 7 ml of dry DMF was added 3.1 mg (10.54 μmol, 1 equiv.) 1-O- (2'-azidoethyl) -2-acetamido-2 -deoxy-β-D-galactopyranose obtained according to the method described in [Hasegawa A. et al. Studies on the thioglycosides of N-acetylneuraminic acid 10: Synthesis of S- (α-sialosyl) - (2 → 6) -O-2-acetamido-2-deoxy-β-d-hexopyranosyl ceramide and its related compounds // Journal of carbohydrate chemistry. - 1992. - T. 11. - No. 3. - C. 319-331], in the presence of 0.4 mg (2.11 μmol, 0.2 equiv.) CuI and 0.2 mg (2.11 μmol, 0.2 equiv.) Et ₃ N in inert atmosphere. The resulting mixture was stirred for 24 hours, then DMF was removed on a rotary evaporator. The resulting substance was purified by HPLC in a reversed-phase system (H ₂ O — AcCN). As a result, the conjugate (9) was obtained in the form of a white powder (7 mg, 54%).

¹ H NMR (400 MHz, CDCl ₃ , δ, ppm): 8.07 (d, 2H, J = 7.5 Hz, H-ortho- (Ar1)); 8.00 (m, 2H, -NH-, H-6``); 7.71 (d, 2H, J = 7.0 Hz, H-ortho- (Ar3)); 7.30-7.60 (m, 12H, Aromatics, -NH-); 6.27 (s, 1H, H-10); 6.09 (t, 1H, J = 8.7 Hz, H-13); 5.87 (d, 1H, J = 4.4 Hz, H-3 '); 5.63 (d, 1H, J = 7.0 Hz, H-2); 5.33-5.42 (m, 1H, H-2 '); 4.93 (d, 1H, J = 9.3 Hz, H-5); 4.08-4.56 (m, 9H, H-7.20, 11``, 13 '', 15``, 16 '', + OH); 3.67-3.98 (m, 7H, H-3, 10``, 17 '', 18``, 19 ''); 2.62-2.77 (m, 3H, H-6.4``); 2.31-2.52 (m, 5H, 4-Ac, 2``); 2.09-2.26 (m, 5H, H-14, 10-Ac); 1.76-1.99 (m, 9H, H-6.18.3``, NHAc); 1.62 (s, 1H, H-19); 1.19 (s, 3H, H-17); 1.14 (s, 3H, H-16).

HRMS (m / z) for C ₆₃ H ₇₅ N ₅ O ₂₁ : [M + H] ⁺ 1238.5035 (found), 1238.5027 (calculated); [M + Na] ⁺ 1260.4848 (found), 1260.4847 (calc.).

Example 10. Synthesis of paclitaxel conjugate (10).

To a solution of 6.5 mg (6.85 μmol) of compound (7) in 7 ml of dry DMF was added 2.2 mg (6.85 μmol, 1 equiv.) 1-O-Allyl-2-acetamido-2-deoxy- 6-azido-6-deoxy-β-D-galactopyranose obtained according to the previously described method [Wong S.N. et al. A library approach to the discovery of small molecules that recognize RNA: use of a 1, 3-hydroxyamine motif as core // Journal of the American Chemical Society. - 1998. - T. 120. - No. 33. - C. 8319-8327] in the presence of 0.3 mg (1.58 μmol, 0.2 eq.) CuI and 0.2 mg (1.58 μmol, 0.2 eq.) Et ₃ N in inert atmosphere. The resulting mixture was stirred for 24 hours, then DMF was removed on a rotary evaporator. The resulting substance was purified by HPLC in a reversed-phase system (H ₂ O — AcCN). The result was conjugate (31) in the form of a white powder (6.5 mg, 77%).

1 H NMR (400 MHz, CD3OD, δ, ppm): 8.11 (d, 2H, J = 7.2 Hz, H-ortho- (Ar1)); 7.80 (d, 2H, J = 7.2 Hz, H-ortho- (Ar3)); 7.77 (s, 1H, H-6``); 7.62-7.70 (m, 2H, -NH-, H-para- (Ar1)); 7.38-7.60 (m, 9H, Aromatics); 7.27 (t, 1H, J = 7.1 Hz, H-para- (Ar2)); 6.43 (s, 1H, H-10); 6.08 (t, 1H, J = 9.1 Hz, H-13); 5.84 (d, 1H, J = 6.1 Hz, H-3 '); 5.70-5.81 (m, 1H, H-19``); 5.65 (d, 1H, J = 7.2 Hz, H-2); 5.46 (d, 1H, J = 6.0 Hz, H-2 '); 4.95-5.15 (m, 3H, H-5.20``); 4.59-4.66 (m, 1H, H-11``); 4.17-4.39 (m, 4H, H-7, 13``, 20); 4.09 (dd, 1H, J = 4.7, 13.3 Hz, H-14``); 3.76-4.00 (m, 5H, N-3, 15``, 16 '', 18``); 3.60 (dd, 1H, J = 3.0, 10.7 Hz, H-10``); 3.42-3.50 (m, 1H, H-10``); 2.68-2.79 (m, 2H, H-4``); 2.44-2.54 (m, 3H, H-2``, 6); 2.40 (s, 3H, 4-OAc); 2.11-2.23 (m, 5H, H-14, 10-OAc); 1.76-2.06 (m, 7H, H-6.3``, 18, -NHAc); 1.66 (s, 3H, H-19); 1.14 (s, 6H, H-16, 17).

HRMS (m / z) for C ₆₄ H ₇₅ N ₅ O ₂₀ : [M + H] ⁺ 1234.5084 (found), 1234.5078 (calc.); [M + Na] ⁺ 1256.4896 (found), 1256.4898 (calc.).

Example 11. Measurement of the dissociation constant of the ligand-ASGP-R complex

The experiment was carried out on a Biacore X100 instrument (Biacore AB, Uppsala, Sweden) using a CM5 carrier chip consisting of a gold plate coated with a layer of carboxymethylated dextran. The surface of the chip includes two flow cells: the analyzed protein is immobilized on one, the second cell is the comparison cell.

A buffer mixture containing 150 mM NaCl, 50 mM CaCl ₂ , 50 mM Tris, pH 7.4 was used. Both cells were activated with a mixture containing 0.05 M N-hydroxysuccinimide (NHS) and 0.2 M N-ethyl-N'-dimethylaminopropyl carbodiimide (EDC) for 7 minutes, the flow rate of the mixture was 10 μl / min. The surface of cell 1 was not modified. On the surface of cell 2, the rabbit liver ASGRP enzyme was immobilized, dissolved in buffer: 10 mM Tris-HCl, pH 7.0. The enzyme solution was supplied for 15 minutes at a rate of 10 μl / min. Both cells were treated with 0.1 M ethanolamine solution (pH 8.5) for deactivation. The amount of ASGPR immobilized protein is 2000 RU.

Sampling and preparation

The ligands of formulas 1, 2, 3, tested for affinity for ASGP-R protein, were dissolved in a working buffer mixture (150 mM NaCl, 50 mM CaCl ₂ , 50 mM Tris, pH 7.4). Dimethyl sulfoxide (DMSO) was added to the samples with low solubility, up to a volume equal to half the volume of the used buffer mixture. Higher concentrations of DMSO were not used due to intolerance to the protein structure of extreme conditions and the danger of irreversible denaturation.

Each ligand was presented in a wide range of concentrations, from 10 ^-2 M to 5 * 10 ^-11 M. The flow rate was 20 μl / min, the ligand was supplied for 180 s (binding time), and complex dissociation was studied for 60 s. For recovery, the carrier chip was treated with 20 μl of a 20 mM EDTA solution. The experiments were carried out at 25 ° C. All buffer solutions were degassed and filtered.

The results are shown in table 1.

The data obtained indicate a greater affinity for the obtained ligands for ASGP-R with respect to the native ligand, N-acetylgalactosamine.

Example 12. The study of the toxicity of conjugates in relation to cell lines of hepatocellular carcinoma.

The results of the study are presented in table 1. Experimental data on the cytoxicity of paclitaxel itself on the HepG2 cell line are presented by Luo et al. [Luo D. et al. Effects of Bcl-2 and Bcl-XL protein levels on chemoresistance of hepatoblastoma HepG2 cell line // Biochemistry and Cell Biology. - 2000. - T. 78. - No. 2. - S. 119-126.], And also obtained in this work.

For the obtained conjugates, the measured cytotoxicity values (CC ₅₀ ) on the cell line of hepatocellular carcinoma are comparable with the cytotoxicity value for the antitumor compound.

Example 13. The study of the stability of conjugates 8-10 in models of biological media at pH 5.0 and 7.4.

Buffer solutions (HCOOH, NH ₄ OH) were prepared with pH 5.0 and 7.4. After dissolving 200 μg of the conjugate in 2 ml of buffer, 200 μl of the resulting solution was taken at the specified time points (0; 0.5; 1; 2; 4; 8; 12; 24 hours). Then the samples were subjected to freezing at -20 ° C. After that, the samples were studied by HPLC-MS on a Shimadzu Prominence LC-20 instrument with a Shimadzu LCMS-2020 single quadrupole mass spectrometer with a double ionization source DUIS-ESI-APCI. The analytical and preparative column was Phenomenex Luna 3u C18 100A.

For all the conjugates studied, the peak area of the substance over time remained the same within the error range, which implies that the conjugates obtained do not undergo degradation and are stable in these biological media.

Example 14. Studies of the accumulation of doxorubicin and conjugates 4-6 in cell culture HepG2 by fluorescence microscopy.

Fluorescence microscopy was carried out using the EVOS FL Cell Imaging System. With a 40-fold increase. In FIG. 1 shows the pictures taken with a microscope, conjugates (4) - A, (5) - B, (6) - C. The incubation time is 24 hours. As shown by the presented images, conjugates accumulate in the cytoplasm and, therefore, are delivered by ligands into the cell.

Example 15. Analysis of the morphology of the nucleus and cytoskeleton of a cell culture line of hepatocellular carcinoma (HepG2) when exposed to conjugates 8-10 using immuno-fluorescence microscopy.

HepG2 cells were grown on poly-L-lysine coated glass coverslips for 24 hours, followed by exposure to paclitaxel and conjugates at a concentration of 1 μmol. After 24 hours of treatment, the cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) in PBS for 10 minutes, washed three times in PBS, and then incubated for 1 hour with blocking solution (1% bovine serum albumin (BSA), 1% Triton X-100 in PBS). Cells were then incubated overnight with Alexa Fluor 488 Mouse Monoclonal Antibody alpha-tubulin (Invitrogen # 322588) at 2 μg / ml. Cells were washed twice with PBS, and the nuclei were stained with 4 ', 6-diamidino-2-phenylindole (DAPI) (Invitrogen). Cells were observed using a Zeiss Axiovert 200 M microscope.

Untreated cells showed typical nuclear and cytoskeletal structures with the formation of normal mitotic spindles. Microtubules organized as diffuse in the cytoplasm (Fig. 2).

After processing the cells with paclitaxel, morphological changes are characteristic: mitoses with more than two poles; many abnormal nuclei (micronuclei or multicore), altered structure of microtubules (thicker and denser bundles of microtubules). Similar cellular morphological changes were observed for paclitaxel-treated conjugates (FIG. 3).

Claims (28)

1. The ligand, characterized by affinity for the asialoglycoprotein receptor and intended for the delivery of the antitumor compound used in the treatment of hepatocellular carcinoma, which is a derivative of N-acetylgalactosamine of the formulas 1, 2 and 3

2. A compound for the treatment of hepatocellular carcinoma, characterized in that it is a ligand conjugate according to claim 1 with an antitumor compound containing a free hydroxyl or amino group forming a covalent bond with the ligand. 3. The compound according to claim 2, characterized in that paclitaxel, or gemcitabine, or cytarabine, or azacytidine is used as an antitumor compound containing a free hydroxyl group. 4. The compound according to claim 2, characterized in that doxorubicin or daunorubicin is used as an antitumor compound containing a free amino group. 5. A method for producing a compound according to claim 2, characterized in that two sequential reactions are carried out, first the carbodiimide synthesis reaction by mixing the antitumor compound and alkynyl acid with a terminal triple bond, taken from the calculation that at least 1.0 molar equivalent of the antitumor compound is taken at least 1.0 molar equivalent of alkynyl acid, in the presence of an activator of a carboxyl group, taken from the calculation - 1.0 equivalent of activator is taken per 1.0 molar equivalent of alkynyl acid , in a medium providing a carbodiimide synthesis reaction, followed by isolation of the intermediate by column chromatography — an anticancer compound modified with an alkynyl acid with a terminal triple bond, and then the reaction of a [3 + 2] azide-alkynyl cycloaddition by mixing the intermediate and the component containing azido group, which is used as a derivative of N-acetylgalactosamine selected from the group comprising 1-azido-1-deoxy-2-acetamido-2-deoxy-β-D-galak support, 1-O- (2'-azidoethyl) -2-acetamido-2-deoxy-β-D-galactopyranose, 1-O-allyl-2-acetamido-2-deoxy-6-azido-6-deoxy-β -D-galactopyranose, in the presence of a monovalent copper compound as a catalyst, in a medium providing a cycloaddition reaction, with at least 1.0 equivalent of the component containing the azido group and at least 0.2 equivalent of the catalyst per 1.0 molar equivalent of the intermediate, after the passage of the reaction removes the medium, followed by isolation of the final Ukta by column chromatography. 6. The method according to claim 5, characterized in that paclitaxel, or gemcitabine, or cytarabine, or azacitidine, or doxorubicin, or daunorubicin is used as an antitumor compound. 7. The method according to claim 5, characterized in that propyn-2-inic acid, but-3-inic acid, pent-4-inic acid, hex-5-inic acid, hept are used as alkynyl acids with a terminal triple bond -6-inic acid. 8. The method according to p. 5, characterized in that as the medium providing the reaction of carbodiimide synthesis, dimethylformamide, dichloromethane, chloroform, tetrohydrofuran, 1,2-dichloroethane are used. 9. The method according to p. 5, characterized in that as an activator of the carboxyl group, soluble in a medium providing the reaction of carbodiimide synthesis, carbodiimides are used. 10. The method according to p. 9, characterized in that as carbodiimides use 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N, N'-diisopropylcarbodiimide, N, N'-dicyclohexylcarbodiimide. 11. The method according to p. 5, characterized in that for the isolation of the intermediate product using direct column chromatography in a system with a concentration gradient of CH ₂ Cl ₂ : MeOH (10: 1) → MeOH. 12. The method according to p. 5, characterized in that as a compound of monovalent copper using monovalent salts of copper, soluble in a medium that provides the passage of the cycloaddition reaction. 13. The method according to p. 12, characterized in that as monovalent salts of copper using copper iodide, or copper bromide, or copper chloride. 14. The method according to p. 5, characterized in that as the medium providing the reaction [3 + 2] azide-alkyne cycloaddition, use DMF, a mixture of water-tetrohydrofuran in a volume ratio of 1: 1, DMSO. 15. The method according to p. 5, characterized in that for the selection of the final product using reverse phase column chromatography in a system of H ₂ About-MeCN. 16. The method of producing the conjugate according to claim 2, characterized in that the ligand according to claim 1 is obtained by carrying out the reaction [3 + 2] of an azide-alkyne cycloaddition by mixing a compound containing a terminal alkyl moiety and a compound containing an azido group taken in molar 1: 1 ratio, in the presence of a catalyst - a monovalent copper salt, calculated on the basis of 1.0 molar equivalent of a component containing an azido group, at least 0.25 equivalent of a catalyst in a reaction medium after passing through the reaction removes the medium and the ligand is isolated by column chromatography, followed by the carbodiimide synthesis reaction between the obtained ligand and the antitumor compound with an amine group available for modification, taken in a 1: 1 molar ratio, in the presence of a carboxyl group activator, calculated at a 1.0 molar basis the ligand equivalent is taken at least 1.0 molar equivalent of the activator of the carboxyl group in DMF, after completion of the reaction, DMF is removed and the resulting conjugate is isolated by column th chromatography. 17. The method according to p. 16, characterized in that as the medium providing the reaction [3 + 2] azide-alkyno addition, use DMF, a mixture of water-tetrohydrofuran in a volume ratio of 1: 1, DMSO. 18. The method according to p. 16, characterized in that as an antitumor compound with an amino group available for modification, doxorubicin or daunorubicin is used. 19. The method according to p. 16, characterized in that as the compound containing the terminal alkyne fragment, alkynyl acids with a terminal triple bond are used. 20. The method according to p. 19, characterized in that as alkynyl acid with a terminal triple bond use prop-2-inic acid, but-3-inic acid, pent-4-inic acid, hex-5-inic acid, hept -6-inic acid. 21. The method according to p. 16, characterized in that as the compound containing the azido group using derivatives of N-acetylgalactosamine: 1-azido-1-deoxy-2-acetamido-2-deoxy-β-D-galactopyranose, 1-O- (2'-azidoethyl) -2-acetamido-2-deoxy-β-D-galactopyranose, 1-O-allyl-2-acetamido-2-deoxy-6-azido-6-deoxy-β-D-galactopyranose. 22. The method according to p. 16, characterized in that as salts of monovalent copper, copper salts are used that are soluble in a medium providing the cycloaddition reaction. 23. The method according to p. 22, characterized in that as the salts of copper using copper iodide, or copper bromide, or copper chloride. 24. The method according to p. 16, characterized in that for the isolation of the ligand using direct column chromatography in a system with a concentration gradient of CH ₂ Cl ₂ : MeOH (10: 1) → MeOH. 25. The method according to p. 16, characterized in that as an activator of the carboxyl group, DMB-soluble carbodiimides are used. 26. The method according to p. 25, characterized in that as carbodiimides use 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N, N'-diisopropylcarbodiimide, N, N'-dicyclohexylcarbodiimide. 27. The method according to p. 16, characterized in that two sequential column chromatography is used to isolate the conjugate: direct column chromatography in a system with a concentration gradient of CH ₂ Cl ₂ : MeOH (20: 1) → MeOH and reverse phase column chromatography in the system H ₂ O-MeCN.

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