CZ2020133A3 - Glycopolymer, preparing it and using it as a medicine - Google Patents

Abstract

The invention relates to multivalent glycopolymers based on HPMA polymers (copolymers and homopolymers) with saccharide structures containing a terminal monosaccharide in the galacto-configuration, which are effective as galectin inhibitors. These glycopolymers can be used as drugs for the treatment and prevention of cancers associated with overproduction of galectins, especially human galectin-3 (Gal-3).

Description

APPLICATION OF THE INVENTION

Published according to §31 of the Act. 527/1990 Sb.

(22) Registered: 09.09.2019 (40) Publication Date of Published Application: 05.05.2021 (Bulletin No. 18/2021) (21) Document Number:

2020-133 (13) Document's type: A3 (51) Int. Cl .:

(19)

CZECH REPUBLIC

OFFICE OF INDUSTRIAL PROPERTY

C08F 20/58

C08F 20/60

C07H 3/04

A61K 31/74

A61P 35/00 (2006.01) (2006.01) (2006.01) (2006.01) (2006.01) (71) Applicant:

Institute of macromolecules! chemistry AS CR, v. v. i.,

Prague 6, Břevnov, CZ

Institute of Microbiology AS CR, v. V. I., Prague 4, Krč, CZ (72)

RNDr. Pavla Bojarová, Ph.D., Prague 6, Střešovice, CZ

Mgr. Tomas Vasicek, 94911 Nitra, SK

Mgr. Petr Chytil, Ph.D., Kladno, Kročehlavy, CZ Mgr. Marcela Filipova, Trebechovice pod Orebem, CZ

Mgr. Olga Janoušková, Ph.D., Brandýs nad LabemStará Boleslav, Brandýs nad Labem, CZ

RNDr. Tomáš Etrych, Ph.D., DSc., Klínec, CZ M.R. Tavares, Prague 10, Vršovice, CZ (74) Representative:

HARBER IP s.r.o., Dukelských hrdinů 567/52, 170 00 Prague 7, Holešovice

CZ 2020 -133 A3 (54) Title of the application for the invention:

Glycopolymer, method of its preparation and its use as a medicine (57)

The invention relates to multivalent glycopolymers based on HPMA polymers (copolymers and homopolymers) with saccharide structures containing a terminal monosaccharide in the galactoconfiguration, which are effective as galectin inhibitors. These glycopolymers can be used as drugs for the therapy and prevention of cancers associated with overproduction of galectins, especially human galectin-3 (Gal-3).

CZ 2020 - 133 A3

Glycopolymer, method of its preparation and its use as a medicine

Field of technology

The invention relates to multivalent water-soluble or micellar polymers, i.e. homopolymers and copolymers, with saccharide structures containing a terminal monosaccharide in the galactoconfiguration, which are effective as galectin inhibitors. These glycopolymers can be used as drugs for the therapy and prevention of cancers associated with overproduction of galectins, especially human galectin-3 (Gal-3).

Prior art

Recently, the development of drugs in anticancer therapy has shifted from conventional low-molecular-weight drugs to the search for new drugs or dosage forms enabling targeted action of the active substance, especially in the place where a therapeutic effect is needed. Monoclonal antibodies, which are relatively effective in various types of malignancies, are currently entering clinical use as drugs, but their use is often associated with relatively strong toxic effects for the patient. In addition to antibodies, targeted forms of the drug can be used mainly for such substances, the side effects of which can lead to damage to healthy parts of the body. The use of polymeric carriers, especially water-soluble or micellar polymers, for the targeted delivery of drugs is one of the important options for solving this problem. The high molecular weight of the polymer with the active ingredient prevents rapid excretion from the body by glomerular filtration. This significantly extends the circulation time in the blood and the overall residence time in the body, leading to increased bioavailability of the system. In addition, the high molecular weight of the polymer system leads to increased accumulation in solid tumors due to the enhanced permeability and retention (EPR) effect.

This effect can be used in the case of binding of the drug to the macromolecular carrier for its targeted accumulation in the tumor. In the past, a number of systems have been developed that are based on the use of the EPR effect to target drugs, such as polymeric micelles, liposomes, nanoparticles, nanocapsules or water-soluble polymeric conjugates. Unlike soluble polymer systems, polymeric micelles are usually prepared by self-assembling amphiphilic diblock copolymers into high molecular weight micellar formations forming colloidal solutions. Among the water-soluble systems, one of the most important groups is polymer systems prepared on the basis of polymers containing N- (2-hydroxypropyl) methacrylamide units (HPMA polymers). The only and major drawback limiting the use of HPMA polymers as drug carriers in human medicine is their non-cleavable carbon chain and the associated limitation of the molecular weights useful for preparing a polymeric carrier to a molecular weight of less than 50,000 to 60,000 g / mol (below the excretion limit). Unfortunately, polymers above this limit are not efficiently and sufficiently efficiently excreted from the body, and therefore their use as drugs would lead to an undesirable accumulation of polymer in the body. Increasing the molar mass of HPMA polymers, and thus prolonging their circulation in the body, is possible by interposing biodegradable bonds between the individual blocks of HPMA copolymers. Enzymatically, hydrolytically or reductively cleavable linkers are particularly suitable. Structures created by joining blocks together are called multiblock, grafted, or star-shaped. A variant can also be the preparation of an amphiphilic HPMA polymer, which self-assembles into a supramolecular micellar structure and disintegrates itself into short copolymers which can be eliminated from the organism after the concentration falls below the critical micellar concentration.

It has recently been reported that HPMA polymer can serve as a carrier for several antiCD20 scFv chains and, due to its multivalence, can cause apoptosis of CD20 positive cells (Kopeček J. Adv. Drug Delivery Rev. 2013, 65,49-59). This concept has been described as a "drug-free therapeutics" and is currently being tested in the United States. The basis for the efficiency of this system is the multivalence of the polymer given the very structure of the polymer used.

-1 CZ 2020 - 133 A3

Galectin-3 (Gal-3) is a lectin belonging to the family of galectins - glycan-binding proteins with a terminal β-galactoside. Currently, this galectin is receiving considerable attention due to its significant effect on processes affecting tumor growth, such as metastasis, overcoming the body's immune response, mRNA splicing, gene expression, apoptosis and inflammation. Gal-3 is expressed by a number of tumor tissues and cells, both intracellularly and extracellularly, where it is both associated with glycostructures on the cell surface and is released into the intercellular environment. Increased expression of Gal-3 in tumor tissues (Thijssen V. L. et al. Biochim. Biophys. Acta 2015, 1855, 235-247) leads to a worsened prognosis of the disease and an increased risk of metastasis. Furthermore, the key role of extracellular Gal-3 produced in the tumor microenvironment in suppressing the T cell immune response against tumor cells and reducing NK cell function has been demonstrated, which in turn leads to the spread of tumor growth and metastasis. Inhibition of extracellular Gal-3 has been shown to protect immune system T cells against Gal-3-induced apoptosis.

Gal-3 produced by tumor cells can be localized primarily in the nucleus, cytoplasm, and membrane. Extracellular Gal-3 is produced in the tumor microenvironment and also by blood throughout the body. The increased presence of Gal-3 is directly correlated with a worse prognosis for cancer, eg in thyroid tumors, some colon, head and neck tumors, or brain tumors. One reason may be the effect of free Gal-3 on anti-tumor T cell apoptosis. Due to its increased expression in tumor tissue, Gal-3 can serve as a therapeutic marker and at the same time a therapeutic target. Also, its presence in the bloodstream or micro-tumor environment can be monitored with respect to the development of cancer; influencing the level of free Gal-3 may affect the effectiveness of the anti-tumor immune response (Sano H. et al. J. Immunol. 2000, 165 (4), 2156-2164, GuhaP. PNAS 2013, 110 (13), 5052-5057).

In recent years, various modifications of the basic carbohydrate ligands of galectins - galactose, lactose (Gaip4Glc) or N-acetyllactosamine (Gaip4GlcNAc) - and the effect of these modifications on the affinity of prepared glycomimetics for individual galectins, especially biomedically most studied . The introduction of an aromatic group at the C-3 parent galactose residue has been demonstrated to be the most advantageous for increasing the affinity for galectins. Structural analogs of N-acetyllactosamine and thiodigalactosides (TDG; Gaipi-ipGal) have developed into a large group of potent glycomimetic inhibitors of galectins resistant to enzymatic degradation in vivo, which is an important factor for future clinical application. Multivalent presentation of these glycomimetics on bovine serum albumin as a carrier has also been described.

In addition to the compounds already mentioned, a number of other carbohydrates have been described in the literature as galectin ligands. These are in particular structures based on poly-N-acetyllactosamine. These complex oligosaccharide structures and other, simpler saccharides based on lactose, galactose or N-acetyllactosamine without the mentioned substitutions have also been exhibited in the past in multivalent presentation on a number of carriers, such as peptides, polymers, oligonucleotides, fillers and calixarenes, as well as dendrimers and nanoparticles. . Multivalent presentation often significantly increased the low affinity of the monovalent saccharide. Although high affinities for galectins have been demonstrated in some cases by ELISA, this has always been a model system that cannot be used for in vivo applications. None of the multivalent carriers prepared so far offers a unique combination of the properties of the glycopolymers according to the present invention (in particular good definability and reproducibility of the preparation, biocompatibility, in vivo stability and advantageous pharmacokinetics).

HPMA polymers carrying the anticancer drug doxorubicin and covalently linked simple mono- and disaccharides (lactose, galactose, galactosamine) have been studied in the past (David A. Pharmaceut. Res. 2002, 19, 1114-1122; David A. et al. Eur. J Cancer 2004, 40, 148-157) for the possibility of active targeting to cells of selected cancer lines, some of which expressed Gal3. In the cases described, the effect of the conjugates on the inhibition of Gal-3 was not considered and the correlation of the obtained results with the expression of Gal-3 on the cells was not conclusive. In the publication of the authors of this

Of the invention (Bojarová P. et al. J. Nanobiotechnol. 2018, 16, 73) a series of HPMA conjugates with a single disaccharide LacdiNAc was synthesized and their inhibitory effect (IC50 in the order of μΜ) on Gal-3 was demonstrated in ELISA determination. It is an object of the present invention to provide glycopolymers with an improved inhibitory effect on Gal-3, while having suitable properties for preparation and application, which are necessary conditions for the possibility of practical use.

The essence of the invention

The present invention relates to the structure, synthesis and use of a novel polymeric drug with a well-controlled content of bound substituted carbohydrates. The content of bound substituted saccharides is regulated by the amount of saccharide component added to the polymeric carrier, as it is proportional to it. The new drug appears to have prolonged pharmacokinetics and increased accumulation in the tumor due to the content of the polymeric component, as well as strong inhibition of galectins.

Glycopolymers bearing the substituted carbohydrates of the present invention not only have a higher affinity for Gal-3 in ELISA-type binding assays than glycopolymers known in the art, but above all have a demonstrably stronger effect in biological assays, as demonstrated in Comparative Example 26. The prior art is thus clearly more effective in inhibiting Gal-3, which has a direct impact on the anti-tumor immune response and tumor cell migration.

Unlike all previously prepared systems, the glycopolymers of the invention are the only Gal-3 inhibitory agent that can be used in vivo for the treatment of tumor growth, due to the adequate pharmacokinetics provided by the polymeric component, biocompatibility, in vivo stability, and good reproducibility of the preparation. . In the context of the present invention, a strong effect of the prepared glycopolymers has been demonstrated not only on the suppression of the induction of apoptosis in cells of the immune system, but also a hitherto undiagnosed effect on the suppression of tumor cell migration. Both of these effects have a direct impact on the treatment of tumors and the suppression of the metastatic process.

One important feature of the system of the present invention is the polymer chains formed by an inert, uncharged, non-interacting water-soluble HPMA-based polymer, which is used as a multivalent carrier to bind substituted carbohydrates to achieve increased lectin interaction, as well as improved pharmacokinetics of supported substituted carbohydrates. . In combination with specific substituted carbohydrates, the effects described herein are then achieved.

The structure of the glycopolymers according to the invention is based on polymeric carriers based on HPMA polymers, to which substituted saccharides are covalently attached, the interaction of which with galectins is multivalent due to the polymeric carrier and avidity binds to said galectins. The described strong interaction with galectins leads to the biological effect itself, which consists in several points, namely in the protective effect on cells of the immune system against apoptosis induced by Gal-3, in the inhibition of tumor cell migration and the associated inhibition of metastatic spread.

The term "drug" as used herein means a substance directly having therapeutic effects, or an adjuvant, or an immunomodulator.

The term "polymer" includes both copolymers and homopolymers. In particular, the term "HPMA polymer" includes both HPMA copolymer and HPMA homopolymer.

The present invention therefore relates to a glycopolymer comprising a polymeric carrier based on HPMA polymers with a content of from 0.5 to 25 mol. %

- structural units of formula I:

-3GB 2020 - 133 A3

Linker

Substituted saccharide (I), wherein

Y ¹ is selected from the group consisting of alkylene having 1 to 8 carbon atoms; phenylene; - (CH 2) _q (C (O) -NH- (CH ₂ ) _r ) p -, wherein p = 1 to 5, aqar are independently selected from 1, 2 and 3; wherein Y ¹ may be optionally substituted with one or more natural amino acid side chains, wherein the side chains may be the same or different; Y ¹ is preferably selected from the group consisting of -CH 2 -CH 2 - and -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -;

Y ² is selected from the group consisting of a bond, carbamoyl, carbamoyl- (C 1 -C 8 -alkylene), and phenylene; wherein carbamoyl means the group -C (= O) -NH- and the group -NH-C (= O) -;

The linker is selected from the group consisting of 1,2,3-triazolylene, (C 1 -C 6 alkyl) -1,2,3-triazolylene; NH-C (= O) -NH- (CH ₂ ) 2 -NH-C (= O) -; -NH-C (= S) -NH- (CH ₂ ) 2 -NH-C (= O) -; or ^{cyclooctynyl or azacyclooctynyl substituted by at least one halogen, cyclopropane or conjugated to at least one benzene ring via the group Y 2} via the group -C (= O) - or only covalently bonded, said cyclooctynyl or azacyclooctynyl being further conjugated to triazolyl, for example formed 3,4,5,13-tetrazatetracyclo [13.4.0.02,6.07,12] nonadeca-1 (15), 2 (6), 3,7 (12), 8,10,16,18-octane-13carbaldehyde motif);

A substituted saccharide is a substituent derived from a substituted saccharide of formula III described herein below by reacting a terminal amine, azide, alkynyl, aminoethylureidyl or aminoethylthioureidyl group, the groups formed by the reaction of said terminal groups being part of a Linker;

or

- end groups of an HPMA polymer chain having the formula -S-succinimide- (CH 2) _r -Linker-Subst. saccharide or -C (CN) (CH3) - (C1-C4 alkylene) -Linker-Subst. carbohydrates; wherein r is selected from the group consisting of 1, 2 and 3, and Linker and Subst. carbohydrate are defined above.

By natural amino acids are meant naturally occurring acids: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, cysteine, glutamine, glycine, proline, tyrosine, alanine, aspartic acid, asparagine, glutamic acid, serine, selenocysteine. The side chains are chains attached to the alpha-carbon of the amino acid.

In addition to poly (HPMA) chains, the glycopolymer may also contain branching units, for example amidoamine units suitable for the preparation of dendrime (star) polymer cores.

-4EN 2020 - 133 A3 poly (amidoamine) (PAMAM), or 2,2-bis (hydroxymethyl) propionic units suitable for the formation of dendrimeric nuclei. For example, a glycopolymer of a star structure may comprise a core of PAMAM and side chains of poly (HPMA) containing the above-mentioned end groups and / or monomeric units of formula I.

The process for preparing the glycopolymers of the present invention comprises the following steps:

a) polymerization of monomers of polymeric carrier and possibly branching units,

b) an optional step of polymer-analogous reactions, c) coupling of a substituted saccharide.

Monomers include N- (2-hydroxypropyl) methacrylamide (HPMA) and methacryloyl (aminoacyl) esters, and optionally branching units. HPMA is commercially available and its synthesis is published (e.g. Chytil P. et al. Eur. J. Pharm. Sci. 2010, 41 (3-4), 472-482). Commercially available monomers are furthermore, for example, β'-aminoethylmethacrylamide. N-aminopropylmethacrylamide, or their Boc-protected analogs.

The synthesis of other functionalized HPMA-derived monomers can be described as follows. Functionalized monomers, methacrylated compounds, can be described by formula II

(Π), where

Y ¹ is selected from the group consisting of alkylene having 1 to 8 carbon atoms; phenylene; - (CH 2) _q (C (O) -NH- (CH ₂ ) _r ) p -, wherein p = 1 to 5, aqar are independently selected from 1, 2 and 3; wherein Y ¹ may be optionally substituted with one or more natural amino acid side chains, wherein the side chains may be the same or different;

Y ² is selected from the group consisting of a bond, carbamoyl, carbamoyl- (C 1 -C 8 -alkylene), and phenylene; wherein carbamoyl means the group -C (= O) -NH- and the group -NH-C (= O) -;

Y ³ is selected from the group consisting of primary amine (NH 2), / Boc substituted amine, azide, terminal alkynyl having 2 to 8 carbon atoms, phenyl substituted with at least one azide or C 2 -C 4 alkynyl; and cyclooctynyl or azacyclooctynyl substituted with at least one halogen, cyclopropane or conjugated to at least one benzene ring, such as (11,12-didehydrodibenzo [b, f] azocin-5 (6H) -yl) or (1R, 85.9%). bicyclo [6.1.0] non-4-yn-9-yl; wherein said substituted cyclooctynyl or azacyclooctynyl is attached via a carbonyl group; -carbonyl-thiazoline-2-thione group (TT), carbonyl-4-nitrophenoxy group, -carbonyl-2,3,4,5,6-pentafluorophenoxy group, -carbonylsuccinimidyl group, and COOH group; wherein the carbonyl is a -C (= O) - group.

Branch units are usually commercially available, in some cases branched or star cores for dendrime polymers are also commercially available.

The first step of the process according to the invention is step a), i.e. the step of synthesizing polymeric supports by polymerizing monomers, optionally with branching units. The polymeric carrier usually contains a statistical

-5GB 2020 - 133 A3 polymer containing from 0.5 to 25 mol% of monomer units of general formula II and / or terminal units of the main polymer chain described above, and at least 75 mol% (75 to 99.5 mol%) of other units, which includes monomeric units derived from HPMA and optionally branching units. The polymerization typically takes place at a temperature in the range of 30 to 100 ° C, preferably 40 to 80 ° C, in a solvent preferably selected from the group consisting of water, aqueous buffers, dimethyl sulfoxide, dimethylacetamide, dimethylformamide, methanol, ethanol, dioxane, tert-butyl alcohol or mixtures, initiated by an initiator, preferably selected from the group consisting in particular of the azo initiators 2,2'-azobis (2-methylpropionitrile) (AIBN), 4,4'-azobis (4-cyanopentanoic acid) (ACVA), 2,2'- azobis (4-methoxy-2,4-dimethylpentanenitrile) (V70), in the presence of a transfer agent, preferably selected from the group consisting of 2-cyano-2-propylbenzodithioate, 4-cyano-4- (thiobenzoylthio) pentanoic acid, 2-cyano -2-propyldodecyltrithiocarbonate, 2-cyano-2-propylethyltrithiocarbonate and 4-cyano-4 - [(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid. The molecular weight M _{n of the} polymers thus prepared is in the range from 4000 to 100,000 g / mol, preferably 20,000 to 50,000 g / mol.

Optionally, a step may be included to remove the sulfur-containing end groups from the polymers in the preparation of which a transfer agent has been used, in which the polymers are reacted with an excess of azo initiator from the group of polymerization initiators described above. The polymeric support is then terminated by a residue from the decomposition radical used by the initiate. The reaction typically proceeds at a temperature in the range of 50 to 100 ° C, preferably 60 to 80 ° C, and in a solvent preferably selected from the group consisting of dimethyl sulfoxide, dimethylacetamide and dimethylformamide.

Optional step b) is the introduction of an azide or alkyl group (i) by reacting the Y ³ group with a polymer if it melts a leaving group (i.e. it is selected from a thiazoline-2-thione group (TT), a 4-nitrophenoxy group, 2,3,4, 5,6-pentafluorophenoxy groups, succinimidyl groups, and OH groups), with an amino compound (e.g., an amino (C 1 -C 8 alkane)) terminated by a group selected from azide, ethynyl, phenyl substituted with at least one azide, C 2 -C 5 terminal alkynyl, or substituted cyclooctyne, preferably 3-amino-1- (11,12-didehydrodibenzo [b, f] azocin-5 (6H) yl) propan-1-one, or N- | (1 A. 8.S'.9.s) -bicyclo 16.1.0] non-4-yn-9-ylethyloxycarbonyl] -1,8-diamino-3,6-dioxaoctane; or (ii) reacting the Y ³ group on the polymer, if it is a primary amino group, with a carboxy (C 1 -C 8 alkane) terminated by a group selected from azide, ethynyl, phenyl substituted with at least one azide, C 2 -C 5 alkynyl, or substituted cyclooctyl, preferably 6- (11,12-didehydrodibenzo [b, f] azocin-5 (6H) -yl) -6-oxohexanoic acid or its N-hydroxysuccinimidyl ester and (1R, 8S, 95) -bicyclo [6.1.0] non-4-yn-9-ylmethyl A-succinimidyl carbonate, or functional derivatives of this carboxyalkane containing a leaving group consisting preferably of TT, 4-nitrophenoxy, 2,3,4,5,6-pentafluorophenoxy, or a succinimidyl group. These polymer-analogous reactions typically take place at room temperature in a solvent preferably selected from the group consisting of dimethyl sulfoxide, dimethylacetamide, dimethylformamide, methanol and ethanol.

One preferred embodiment of the polymeric carrier is such that it may optionally further comprise 0.5 to 12 mol% of other structural units derived from formula II, wherein Y ¹ and Y ² are as defined above, but Y ³ is selected from the group consisting of carbonyl -hydrazono- (C12-C18 alkanone), carbonyl-hydrazono-5a-cholestanone, carbonyl-hydrazono-cholest-4-en-3-one, or another substituent derived from cholesterol ketoderivatives; or is selected from the group consisting of carbonyl- (C 9 -C 15 alkoxy), carbonyl-cholesteryl, or other cholesterol-derived compounds, e.g., 7-dehydrocholesterol, or vitamin D; or is selected from the group consisting of carboxamido- (C10-C18 alkyl) and carbohydrazido- (C10-C18 alkyl), or carboxamido- (C10-C18 alkenyl) acarbohydrazido- (C10-C18 alkenyl) containing at least one double bond (C = C18 alkenyl) ), preferably derived from oleic, linoleic, linoleic acid.

The polymeric support may generally have a linear, branched, or cross-linked structure.

-6GB 2020 - 133 A3

The linear structure is usually a polymeric support containing 0.5 to 25 mol% of the monomeric units of formula II and / or the terminal units described above, and at least 75 mol% of the HPMA monomeric units.

In the case of a branched or crosslinked structure, at least 75 mol% of HPMA monomer units and branch units are present.

In some embodiments of the polymeric branched support, only a portion of the functional groups suitable for linking the substituted saccharides are distributed along the polymer chain, or even the monomers of formula II are not present at all. Functional groups suitable for linking substituted saccharides, or at least a portion thereof, may be in the form of end groups located at one end of the polymer chain of the HPMA polymer, the other end of the polymer chain being attached to a multivalent molecule such as a dendrimer such as a PAMAM dendrimer core or core based on 2,2-bis (hydroxymethyl) propion dendrimer. The first preparation step in such embodiments is HPMA polymerization, which typically takes place at a temperature in the range of 30 to 100 ° C, preferably 40 to 80 ° C, in a solvent preferably selected from the group consisting of water, aqueous buffers, dimethylsulfoxide, dimethylacetamide, dimethylformamide , methanol, ethanol, dioxane, tert-butyl alcohol or mixtures thereof, initiated by an initiator, preferably selected from the group consisting in particular of azo initiators AIBN, ACVA, V70, in the presence of a carboxyl group-containing transfer agent or a functional derivative thereof TT, 4-nitrophenoxy, 2,3,4,5,6-pentafluorophenoxy, or succinimidyl; the transfer agent is preferably selected from the group consisting of 4-cyano-4- (thiobenzoylthio) pentanoic acid, 4-cyano-4 - [(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid, 1-cyano-1-methyl-4-oxo-4- (2-thioxothiazolidine- 3-yl] butyl dithiobenzoate, 2-cyano-5-oxo-5- (2-thioxo-1,3-thiazolidin-3-yl) pentan-2-yl ethyl carbonotrithioate In the second step, the polymers are amide-linked to a multivalent compound bearing terminal primary amino groups, preferably poly (amidoamine) (PAMAM), or 2,2-bis (hydroxymethyl) propion dendrimer, the reaction typically proceeding in a solvent preferably selected from the group consisting of dimethylsulfoxide, dimethylacetamide, dimethylformamide, methanol and ethanol. Up to 3 mol% of the content of the resulting polymeric conjugate Subsequently, the remaining primary amino groups are blocked in situ by a low molecular weight aminoreactive compound, preferably acetic anhydride.In the third step, the sulfur-containing groups at the other end of the polymer are either red sodium borohydride and the SH groups are added in situ to an A-derived maleimide, preferably propynylmaleimide or azido-PEG3-maleimide, the reaction taking place in a solvent preferably selected from the group consisting of water, dimethylacetamide, dimethylformamide, methanol and ethanol. Optionally, the sulfur-containing groups at the other end of the polymer are reacted with an excess of an azo initiator bearing a carboxyl group, or a functional derivative thereof containing a leaving group consisting preferably of TT, 4-nitrophenoxy, 2,3,4,5,6-pentafluorophenoxy, or succinimidyl, at a temperature in the range from 50 to 100 ° C, preferably 60 to 80 ° C and a solvent preferably selected from the group consisting of dimethyl sulfoxide, dimethylacetamide and dimethylformamide. The molecular weight M of the polymers thus prepared is in the range from 60,000 to 1,000,000 g / mol, preferably 100,000 to 400,000 g / mol.

The final step c) of the preparation is the coupling of a substituted saccharide selected from the group consisting of substituted saccharides of general formula III:

HO ^ OR ⁵

R ²

U — VWR ⁴ (III),

-7 CZ 2020 - 133 A3 where the combination of U, V and W is selected from the following options lila, Illb, lile, Illd, lile:

Z OH /

XR ¹ (IIIa) IIIa: wherein V and W are only a bond, and wherein U is 1-β-β-D-galactopyranosyl substituted on C-2 by R ⁴ , on C-3 by -ZYXR ¹ and on the C-6 atom by the group -OR ⁶ ;

. A-q

R ^3u (Illb)

IIIb: wherein V and W is a bond, and wherein U is 4-OD-glucopyranosyl substituted at C-2 group, R ^3u and Cl with R ^4;

(lile) lile: wherein V and W are only a bond and wherein U is 3-OD-galactopyranosyl substituted on C-2 by R ^3u and on Cl by R ⁴ ;

Illd: wherein V and W are only a bond and wherein U is 3-OD-glucopyranosyl substituted on C-2 by R ^3u and on Cl by R ⁴ ;

(Me) lile: wherein U is 4-OD-glucopyranosyl substituted on C-2 by R ^3u and on Cl by V, wherein V is 3-OD-galactopyranosyl substituted on C-2 by R ^3v and

-8EN 2020 - 133 A3 of the Cl atom by the substituent W, wherein W is 4-OD-glucopyranosyl substituted on the C-2 atom by the group R ^3w and on the Cl atom by the group R ⁴ ;

and where

R ¹ is selected from the group consisting of hydrogen, phenyl, phenyl substituted with at least one halogen, phenyl substituted with at least one nitro group, phenyl substituted with at least one carboxy group, phenyl substituted with at least one C 1 -C 5 alkoxy group, phenyl substituted with at least one C 2 -C 5 alkynyl, phenyl substituted with at least one C 1 -C 5 azidoalkyl, phenyl substituted with at least one azido group, phenyl substituted with at least one halo (C 1 -C 5) alkyltriazolyl, phenyl substituted with at least one sulfo group, phenyl substituted with at least one cyano group, phenyl substituted with at least one amino group, phenyl substituted with at least one C 1 -C 5 aminoalkyl, phenyl substituted with at least one hydroxy, naphthyl, naphthyl substituted with at least one halogen, biphenyl, saccharide, C 2 -C 6 heteroaryl containing at least one heteroatom selected from O, S, N, sulfo, coumaryl, benzoyl and C 2 -C 5 alkynyl;

X is [1,2,3] -triazole or a bond

Y is CH 2 or a bond,

Z is O or [1,2,3] -triazole.

R ² , R ^3u , R ^3v , R ^3w are independently selected from the group consisting of hydroxy, acetamido, C 1 -C 5 acylamido, 1-OaL-fucosyl,

R ⁴ is selected from the group consisting of hydroxy, amino, C 2 -C 5 alkynyloxy, azide, C 1 -C 5 azidoalkoxy, aminoethylthioureidyl, aminophenyloxy and azidophenyloxy,

R ⁵ and R ⁶ are independently selected from the group consisting of hydrogen, sulfo, 2-O a-sialyl, C 1 -C 5 azidoalkyl, C 2 -C 5 alkynyl and C 1 -C 5 aminoalkyl;

wherein the substituted saccharide of formula III always contains exactly one terminal amino group, azido group or C 2 -C 5 alkynyl, preferably as part of a substituent selected from R ¹ , R ⁵ or R ⁶ , when U is 1-ηο-β-D -galactopyranosyl, and preferably as part of the substituent R ⁴ in all other combinations;

and wherein the substituted saccharide of formula III is not lactose (Ga ^ 4GlcNAc), LacNAc (Ga ^ 4GlcNAc) or LacdiNAc (GalNAcE4GlcNAc).

The saccharide in the substituent R ¹ is preferably a monosaccharide, more preferably 1-OaD-galactopyranosyl or 2-Oa-sialyl.

When V and W is a bond and U is 1- [d] -D-galactopyranosyl substituted on C-2 by R ⁴ , on C-3 by -ZYXR ¹ and on C-6 by -OR ⁶ (variant IIIa), preferably R ² is hydroxy and R ⁴ is also hydroxy.

When V and W is a bond and U is 4-OD-glucopyranosyl substituted on C-2 by R ^3u and on Cl by R ⁴ (variant IIIb), preferably R ² is selected from the group consisting of hydroxy and acetamido and R ^3u is an acetamido group.

When V and W is a bond and U is 3-OD-galactopyranosyl substituted on C-2 by R ^3u and on Cl by R ⁴ (variant IIIc), preferably R ² is hydroxy and R ^3u is acetamido.

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When V and W are a bond and U is 3-OD-glucopyranosyl substituted on C-2 by R ^3u and on Cl by R ⁴ (variant IIId), preferably R ² is hydroxy and R ^3u is acetamido.

When U is 4-OD-glucopyranosyl substituted on C-2 by R ^3u and on Cl by V, where V is 3-OD-galactopyranosyl substituted on C-2 by R ^3v and on Cl by W, where W is 4-OD-glucopyranosyl substituted on C-2 by R ^3w and on Cl by R ⁴ (variant IIIe), preferably R ² , R ^3v and R ^3w are independently selected from the group consisting of hydroxy and acetamido and R ^3u is acetamido group.

Substituted saccharides are attached to the polymeric support either by an amide bond or by an azidalkyne cycloaddition, a click reaction formed by a linker from group Y ³ and a substituent on a substituted saccharide containing terminal amine, azide, alkynyl, or aminoethylthioureidyl groups, typically R ¹ or R ⁴ or R ⁵ or R ⁶ ). The aminolytic reaction typically takes place in an aprotic solvent, preferably selected from the group consisting of dimethyl sulfoxide, dimethylacetamide and dimethylformamide. The cycloaddition reaction is preferably catalyzed, preferably by copper ions, or may be uncatalyzed. The reaction typically takes place in water, optionally in mixed solvents consisting of water, aqueous buffers, alcohols, an aprotic solvent, preferably selected from the group consisting of dimethyl sulfoxide, dimethylacetamide and dimethylformamide, or mixtures thereof. In the case of a copper-catalyzed cycloaddition reaction, the reaction mixture contains copper or copper cations, preferably due to the presence of CuSO 4 or CuBr, and a reducing agent, preferably ascorbic acid or sodium ascorbate. In all cases, it is advantageous to use purification by gel filtration on a column (eg packed with Sephadex G-25) in water and optionally further purification from copper cations, preferably by complexation with 8-quinolinol, and subsequent purification on the column (eg Sephadex LH-20) with methanol as mobile phase.

An important benefit of the invention is the appropriate presentation of the substituted saccharide on a polymeric support, the content of which in the glycopolymer being 0.5 to 25 mol%, preferably 3 to 18 mol%. The multivalent arrangement of substituted carbohydrates on polymers allows a multivalent interaction with Gal-3, and thus significantly increases the binding activity to Gal-3 and galectins in general.

The invention further relates to the immunomodulatory effect of polymeric conjugates with substituted carbohydrates due to binding to Gal-3 by the multivalent interaction described above. The application of polymeric carriers with carbohydrates binds extracellular Gal-3 in the tumor tissue, thereby inhibiting Gal-3-induced T cell apoptosis in the tumor, which allows for an enhanced immune response against tumor cells directly in the tumor. Binding of polymeric carriers with bound substituted carbohydrates to Gal-3 directly produced by tumor cells further leads to a reduction in tumor cell migration. Because Gal-3 increases the likelihood of metastasis in some tumor cell types by inhibiting the binding of cells to the substrate and cells to each other, the application of polymeric carbohydrate conjugates via Gal-3 binding reduces the possibility of cell migration to metastatic foci and reducing their motility.

The present invention provides a pharmaceutical composition comprising a glycopolymer of the invention comprising an active substituted saccharide and at least one pharmaceutically acceptable excipient selected from the group consisting of fillers, antiadhesives, binders, coatings, colorants, swelling agents, flavoring agents, lubricants, preservatives, sweeteners, sorbents.

The invention relates to a glycopolymer according to the invention and / or a pharmaceutical composition thereof for use as a medicament for the treatment of solid tumors and / or lymphoma and / or leukemia, in particular colorectal cancer, prostate cancer, breast cancer, melanoma, lymphoma, leukemia.

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The invention also relates to a glycopolymer according to the invention and / or a pharmaceutical composition thereof for use as an adjuvant in antitumor therapy.

Explanation of drawings

Giant. 1: Inhibition of galectin-3-induced apoptosis of Jurkat cells by P11b conjugate (substituted saccharide 20 content was 4.9 mol%).

Giant. 2: Inhibition of galectin-3-induced Jurkat cell apoptosis by Plld conjugate (substituted saccharide 20 content was 9.7 mol%).

Giant. 3: Inhibition of galectin-3-induced apoptosis of Jurkat cells by P13a conjugate (substituted saccharide 9 content was 5.1 mol%).

Giant. 4: Inhibition of galectin-3-induced apoptosis of Jurkat cells by P13b conjugate (substituted saccharide 9 content was 8.9 mol%).

Giant. 5: Inhibition of galectin-3-induced apoptosis of Jurkat cells by conjugate HPMA with single disaccharide LacdiNAc (LacdiNAc content was 12.3 mol%).

Giant. 6: Inhibition of migration and proliferation of mouse (4T1, B16-F10) and human (DLD-1) tumor cells by Pila and Lung conjugates.

List of abbreviations

ACVA, 4,4'-azobis (4-cyanopentanoic acid); AIBN, 2'-azobis (2-methylpropionitrile); Gal-3, galectin-3; HPMA, N- (2-hydroxypropyl) methacrylamide; MA-AP-TT, 3- (3-methacrylamidopropanoyl) thiazolidine-2-thione; MA-propynyl, 2-methyl-N- (prop-2-yn-1-yl) prop2-enamide; 3- (3-methacrylamidopropanoyl) thiazolidine-2-thione; MA group, N-methacryloyl group; tert-butoxycarbonyl group, TT group, thiazoline-2-thione group, TBAB, tetra-n-butylammonium bromide, THPTA, trishydroxypropyltriazolylmethylamine ligand, V70, 2,2'-azobis (4-methoxy-2,4-dimethylpentanenitrile), LacdiNAc (GalNAcP4 LacNAc (Gaip4GlcNAc), lactose (Gaip4Glc), 4T1 (mouse breast tumor cell line), B16-F10 (mouse skin melanoma cell line), CT26 (mouse colorectal cancer cell line), DLD-1 (human colorectal adenocarcinoma cell line) HEK293 (human embryonic kidney cell line), HT-29 (human colorectal adenocarcinoma cell line), Jurkat (immortalized T cell line), LNCaP (human prostate adenocarcinoma cell line), OVCAR3 (human ovarian adenocarcinoma cell line), PC3 (human prostate adenocarcinoma cell line); Burkitt's lymphoma line); SU-DHL-5 (human B-lymphoma cell line); SU-DHL-6 (human B-lymphoma cell line);

Examples of embodiments of the invention

Example 1: Synthesis of N- (2-hydroxypropyl) methacrylamide (HPMA) monomers

HPMA was prepared according to the previously described procedure (Chytil P. et al. Eur. J. Pharm. Sci 2010, 41 (3-4), 473-482). The product was chromatographically pure. 1 H-NMR (300 MHz, (CD ₃ ) ₂ SO, 296 K): δ 1.00-1.02 (d, 3H, CHOH-CH ₃ ), 1.85 (s, 3H, CH ₃ ), 3 , 00-3.12 (m, 2H, CH ₂ ), 3.64-3.73 (m, 1H, CH), 4.68-4.70 (d, 1H, OH), 5.30 and 5 , 66 (d, 2H, CH ₂ =), 7.59 (br, 1H, NH).

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Β'-Methacryloylpropynylamine (MA-propynylamine) was prepared according to the previously described procedure. (Lynn GM et al., Biomacromolecules, 2019, 20 (2), 854-870) The product was chromatographically pure. 1 H-NMR (300 MHz, (CD ₃ ) ₂ SO, 296 K): δ 1.85 (s, 3H, CH ₃ ), 3.05 (s, 1H, = CH), 3.88 (d, 2H , -CH ₂ -), 5.37 and 5.68 (d, 2H, = CH ₂ ), 8.37 (s, 1H, NH).

3- (3-Methacrylamidopropanoyl) thiazolidine-2-thione (MA-AP-TT)

MA-AP-TT was prepared according to the previously described procedure (Subr V. et al. Biomacromolecules 2006, 7 (1), 122-130). The product was chromatographically pure. 1 H-NMR (300 MHz, (CD ₃ ) ₂ SO, 295 K): δ 1.20-1.27 (m, 2H, CH ₂ -y), 1.40-1.54 (m, 4H, CH _2- p, CH ₂ -b), 1.82 (s, 3H, CH ₃ ), 2.28 (t, 2H, CH ₂ a), 3.04-3.34 (m, 2H, CH ₂ - s), 3.57 (s, 3H, OCH ₃ ), 5.28 and 5.60 (d, 2H, CH ₂ =), 7.88 (br, 1H, NH).

Example 2: Synthesis of random copolymers by poly (HPMA-co-MA-AP-TT) radical polymerization (P1)

833 mg HPMA (5.82 mmol), 167 mg MA-AP-TT (0.646 mmol) and AIBN (160 mg; 0.974 mmol) were dissolved in 6.2 mL dimethyl sulfoxide. The polymerization mixture was bubbled with argon and sealed in a glass ampoule. After 6 h in a water bath thermostated at 60 ° C, the ampoule was cooled and opened. The polymer was isolated by precipitation into excess acetone (150 mL) and reprecipitated from methanol (6 mL) into acetone-diethyl ether (3: 1; 120 mL). The polymer was obtained by filtration and drying under vacuum. Yield 850 mg, 85%; molar mass M _w = 23,900 g / mol, M _a = 12,100 g / mol, D = 1.98.

Example 3: Synthesis of random copolymers of poly (HPMA-co-MA-AP-TT) controlled by RAFT radical polymerization (P2)

800 mg HPMA (5.59 mmol) was dissolved in 5.52 mL tert-butyl alcohol and mixed with a solution of 160 mg MA-AP-TT (0.621 mmol), 4.97 mg AIBN (17.7 pmol) and 7.84 mg 2-cyanopropan-2-yl dithioate (35.5 pmol) in 1.38 mL of dimethyl sulfoxide. The polymerization mixture was bubbled with argon and sealed in a glass ampoule. After 16 h in a water bath thermostated at 70 ° C, the ampoule was cooled and opened. The polymer was isolated by precipitation into excess acetone (150 mL) and reprecipitated from methanol (6 mL) into acetone-diethyl ether (3: 1; 120 mL). The polymer was obtained by filtration and

-12 CZ 2020 - 133 A3 drying under vacuum. Yield 730 mg, 76%; molar mass M _w = 22,900 g / mol, M _n = 20,600 g / mol, D = 1.11.

Example 4: Removal of end groups on a polymer derived from a transfer agent

700 mg of P2 polymer bearing terminal dithiobenzoate groups and 70 mg of AIBN initiate were dissolved in 5 mL of dimethyl sulfoxide, purged with argon and sealed in a glass ampoule. After 2 h in a water bath thermostated at 80 ° C, the ampoule was cooled and opened. The polymer was isolated by precipitation into excess acetone (150 mL) and reprecipitated from methanol (6 mL) into acetone diethyl ether (3: 1; 120 mL). The polymer was obtained by filtration and drying under vacuum. The yield of polymer was 621 mg.

Example 5: Synthesis of a random copolymer of poly (HPMA-co-MA-propynyl) controlled by RAFT radical polymerization (P3)

264 mg of HPMA (1.84 mmol) and 12 mg of N-methacryloylpropynylamine (MA-propynyl) (97.0 pmol) were dissolved in 1.3 mL of distilled water and mixed with a solution of 1.3 mg of ACVA (4.7 pmol). ) and 2.6 mg of 4-cyano-4- (thiobenzoylthio) pentanoic acid (9.5 pmol) in 0.65 mL of dioxane. The polymerization mixture was bubbled with argon and sealed in a glass ampoule. After 7 h in a water bath thermostated at 70 ° C, the ampoule was cooled and opened. The polymer was isolated by precipitation into excess acetone (150 mL) and centrifuged. Purification was performed by gel filtration using a column packed with Sephadex LH-20 in methanol. The polymer was obtained by precipitation into diethyl ether, filtration and drying under vacuum. Yield 127 mg, 46%; molar mass = 22,900 g / mol. M, = 20,600 g / mol, D = 1.11.

Example 6: Synthesis of a poly (HPMA-co-MA-AP-propynyl) random copolymer by conjugation of propynylamine with poly (HPMA-co-MA-AP-TT) (P4)

600 mg of the polymer precursor (containing 0.54 mmol of TT groups) was dissolved in 6 mL of dimethylformamide, and 40 μL of propynylamine (0.65 mmol) and 108 μL of N-ethyldiisopropylamine (0.65 mmol) were added to the solution with stirring at room temperature. ). The reaction was run for 16 h at room temperature. The polymer was purified from low molecular weight impurities by gel filtration on a Sephadex LH-20 column in methanol. The polymer was isolated by precipitation into excess acetone (120 mL), filtration and drying. Yield: 560 mg; molar mass: = 21,800 g / mol, M _n = 20,200 g / mol, D = 1.08.

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Example 7: Synthesis of star copolymers containing PAMAM dendrimer core and poly (HPMA) -propynyl (P5) arms

500 mg of HPMA (3.49 mmol) was dissolved in 3.4 mL of tert-butyl alcohol and mixed with a solution of 2.8 mg of 2- [1-cyano-1-methyl-4-oxo-4- (2-thioxo-thiazolidine) -3-yl) -butylazo] -2-methyl-5-oxo-5- (2-thioxothiazolidin-3-yl) -pentanenitrile (5.4 pmol) and 4.4 mg of 1-cyano-1-methyl-4-oxo Dithiobenzoic acid -4- (2-thioxothiazolidin-3-yl) butyl ester (10.7 pmol) in 0.85 mL dimethyl sulfoxide. The polymerization mixture was bubbled with argon and sealed in a glass ampoule. After 6 h in a water bath thermostated at 70 ° C, the ampoule was cooled and opened. The polymer was isolated by precipitation into excess acetone (120 mL) and precipitated from methanol (6 mL) into acetone-diethyl ether (3: 1; 120 mL). The polymer was obtained by filtration and drying under vacuum. Yield: 345 mg, 69%; molar mass: M "= 29,800 g / mol, M _n = 27,100 g / mol, D = 1.10.

340 mg of the polymer precursor (containing 11.2 pmol of terminal TT groups) was dissolved in 2.2 mL of dimethyl sulfoxide and added to a 20 wt% methanolic solution of 1.5 mg of PAMAM dendrimer (1.4 pmol of dendrimer G2 with a diaminobutane core). After stirring at room temperature for 1.5 h, the reaction was quenched by the addition of 50 μL of acetic anhydride. The polymer was purified from low molecular weight impurities by gel filtration on a Sephadex LH-20 column in methanol. The polymer was isolated by precipitation into excess acetone (120 mL), followed by filtration and drying. The yield of the conjugation reaction was 85% star copolymers.

100 mg of the star polymer polymer precursor was dissolved in 1 mL of methanol. 10 mg of powdered sodium borohydride was added to the stirred polymer solution. After stirring for 1 h at room temperature, 10 mg of propynyl maleimide dissolved in 0.2 mL of methanol was added in situ. After 1 h of reaction, the polymer was purified from low molecular weight impurities by gel filtration on a Sephadex LH-20 column in methanol. The polymer was isolated by precipitation into excess acetone (25 mL), followed by filtration and drying. Molar mass: A / _w = 210,000 g / mol, D = 1.19. In a similar manner, samples were prepared using a 2,2-bis (hydroxymethyl) propionic dendrimer as a core.

Example 8: Synthesis of star copolymers containing PAMAM dendrimer core and poly (HPMA) -TT (P6) arms

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500 mg of HPMA (3.49 mmol) was dissolved in 3.4 mL of tert-butyl alcohol and mixed with a solution of 2.8 mg of 2- [1-cyano-1-methyl-4-oxo-4- (2-thioxo-thiazolidine) -3-yl) -butylazo] -2-methyl-5-oxo-5- (2-thioxothiazolidin-3-yl) -pentanenitrile (5.4 pmol) and 4.4 mg of 1-cyano-1-methyl-4-oxo Dithiobenzoic acid -4- (2-thioxothiazolidin-3-yl) butyl ester (10.7 pmol) in 0.85 mL dimethyl sulfoxide. The polymerization mixture was bubbled with argon and sealed in a glass ampoule. After 6 h in a water bath thermostated at 70 ° C, the ampoule was cooled and opened. The polymer was isolated by precipitation into excess acetone (120 mL) and precipitated from methanol (6 mL) into acetone-diethyl ether (3: 1; 120 mL). The polymer was obtained by filtration and drying under vacuum. Yield: 345 mg, 69%; Molar masses: M _w = 29,800 g / mol, Μ _Ά = 27,100 g / mol, D = 1.10.

340 mg of the polymer precursor (containing 11.2 pmol of terminal TT groups) was dissolved in 2.2 mL of dimethyl sulfoxide and added to a 20 wt% methanolic solution of 1.5 mg of PAMAM dendrimer (1.4 pmol dendrimer G2 with diaminobutane core). After stirring at room temperature for 1.5 h, the reaction was quenched by the addition of 50 μL of acetic anhydride. The polymer was purified from low molecular weight impurities by gel filtration on a Sephadex LH-20 column in methanol. The polymer was isolated by precipitation into excess acetone (120 mL), followed by filtration and drying. The yield of the conjugation reaction was 85% of the star copolymer.

100 mg of a star polymeric precursor and 10 mg of 2- [1-cyano-1-methyl-4-oxo-4- (2-thioxothiazolidin-3-yl) -butylazo] -2-methyl-5-oxo-5- (2 -thioxothiazolidin-3-yl) -pentanenitrile was dissolved in 0.7 mL of dimethyl sulfoxide, purged with argon and sealed in a glass vial. After 3 h in a water bath thermostated at 70 ° C, the ampoule was cooled and opened. The polymer was isolated by precipitation into excess acetone (25 mL) and precipitated from methanol (1 mL) into acetone-diethyl ether (3: 1; 25 mL). The polymer was obtained by filtration and drying under vacuum. Molar mass: A / _w = 205,000 g / mol, D = 1.20. In a similar manner, samples were prepared using a 2,2-bis (hydroxymethyl) propionic dendrimer as a core.

Example 9: Synthesis of a random copolymer of poly (HPMA-co-MA-AP-azadihenzocyclooctyne) by conjugation of azadibenzocyclooctynamine with poly (HPMA-co-MA-AP-TT) (P7)

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200 mg of a polymeric precursor (containing 0.11 mmol of TT groups) was dissolved in 2 mL of dimethylformamide, and 32 mg of azadibenzocyclooctyamine (0.12 mmol) dissolved in 0.1 mL of dimethylformamide and 22 μL of Nethyldiisopropylamine ( 0.13 mmol). The reaction was run for 16 h at room temperature. The polymer was purified from low molecular weight impurities by gel filtration on a Sephadex LH-20 column in methanol. The polymer was isolated by precipitation into excess acetone (50 mL), followed by filtration and drying. Yield: 184 mg; Molar masses: M _w = 28,000 g / mol, M _a = 25,200 g / mol, D = 1.11.

Example 10: Synthesis of a random copolymer of poly (HPMA-co-MA-AP-propyl azide) by conjugation of 3azido-1-propylamine with poly (HPMA-co-MA-AP-TT) (P8)

HN

N,

300 mg of polymeric precursor P2 was dissolved in 3.5 mL of methanol and stirred at rt. 40 μL of 3-azido-1-propylamine (0.40 mmol) were added. After 30 min, 92 μL of N-ethyldiisopropylamine (0.53 mmol) was slowly added dropwise to the reaction mixture. After 20 h, the reaction at lab.t. 40 μL of 1-aminopropan-2-ol (0.29 mmol) was added and stirred for another 30 min. The polymer was purified from low molecular weight impurities by gel filtration on a Sephadex LH-20 column in methanol. The polymer was isolated by precipitation into excess acetone (50 mL), followed by filtration and drying. Yield: 234 mg; Molar masses: M _w = 23,700 g / mol, M _n = 21,100 g / mol, D = 1.1; azide group content 16.7 mol%.

Example 11: Synthesis of 3'-O- [4- (azidomethyl) benzyl] -β-D-galactopyranosyl- (1H-1) -3-O- (4 - {[4- (bromomethyl) -1H-1, 2,3-triazol-1-ylmethyl [benzyl] -1-thio-β-D-galactopyranoside (3)

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The starting material PD-galactopyranosyl) - (1R) -1-thio-PD-galactopyranoside (1) was reacted in the presence of dibutyltin oxide (Bu2SnO) with an excess of bromide to give selectively C-3, C-3 '5 disubstituted compound 2. Reaction was performed in the presence of a tetra-n-butylammonium bromide (TBAB) phase transfer catalyst in dry dioxane at elevated temperature (82-84 ° C). Subsequently, compound 3 was prepared by cycloaddition click reaction of the terminal azide function and the alkyne group of propynyl bromide (0.5 eq.) Catalyzed by Cu (I) in the presence of trishydroxypropyltriazolylmethylamine ligand (THPTA).

Example 12: Synthesis of 2-azidoethyl-3-O-benzyl-2-acetamido-2-deoxy-β-D-galactopyranosyl (1-> 4) -2-acetamido-2-deoxy-β-D-glucopyranoside (7)

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NHAc

Tyr470His High pH 5..0.5 h

θ I T8AB

I BujSnO

I DiPEA benzyl bromide dry dioxane

I 24h, src i

Disaccharide 6 was prepared by chemoenzyme synthesis from the acceptor 2-azidoethyl-2-acetamido-2-deoxy-PD-glucopyranoside (5) and the donor p-nitrophenyl-2-acetamido-2-deoxy-β-D5 galactopyranoside (4) catalyzed by the mutant Tyr470HisP- A-acetylhexosaminidase from Talaromyces flavus (Tyr470His 7 / Hcx) (Bojarová P. et al. J Nanobiotechnol. 2018, 16, 73). Benzyl was selectively introduced at C'-3 by reaction of 6 with benzyl bromide in the presence of dibutyltin oxide (Bu 2 SnO) to give the substituted saccharide 7.

Example 13: Synthesis of 3-O-propynyl-βD-galactopyranosyl- (1R) -3-O - [(4-bromophenyl) -1H- (1,2,3-triazol-4-yl) methyl] -PD- galactopyranoside (9)

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; TBAB

BujSrO

DiPEA propargytteransid ϊ sbcIS ^ dfoxan ♦ 2 <O ^ TC

; CuSOj

L - ^^ fear sftW

TWTA i ^ - ^ íiphesrsy & rĚmš ί ^ (siy ^ kCiMH _s a ξύΐ) * aec ..: Mh ma

The starting material PD-galactopyranosyl) - (1R) -1-indo-β-D-galactopyranoside (1) was reacted in the presence of a tin complex (Bu2SnO) with an excess of propynyl bromide to give selectively C5 3, C-3 'disubstituted compound 8. The reaction was performed in the presence of a phase transfer catalyst (TBAB) in dry dioxane at elevated temperature (82-94 ° C). The second reaction step based on Cu (I) -catalyzed azide-alkyne cycloaddition of 4-bromophenyl azide catalyzed by THPTA was performed in a mixture of tert-butyl alcohol and water to give compound 9.

Example 14: Synthesis of 2-aminoethylthioureidyl-3-O - [(4-bromophenyl) -1H- (1,2,3-triazol-4-yl) methyl] PD-galactopyranosyl- (1H-3) -2- acetamido-2-deoxy-PD-glucopyranoside (15)

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TBAS

BugSnO

SPEA ^ ίιγ ^ φ ^ η ·: T: 32 -34'0

<7 «S0i :. : ΐ <χ! $ κ · 5: ^ ss ^ iý

WTÁ tf í * iWW ^ lM ₂ Q € 1řt5

W HCA 4 $ 6. ίί · ⁵ C

The starting disaccharide 12 was prepared by chemoenzyme synthesis from the acceptor (tert-butoxycarbonylamino) ethylthioureidyl-2-acetamido-2-deoxy-PD-glucopyranoside (11) (Bojarová P. et al. Molecules 2019, 24, 599) and donorup-nitrophenyl-PD-galactopyranoside (10) catalyzed by recombinant β3-galactosidase from Bacillus circulans. Disaccharide 12 was reacted in the presence of dibutyltin oxide (Bu 2 SnO) with an excess of propynyl bromide to give selectively C-3 'substituted compound 13. The reaction was performed in the presence of a phase transfer catalyst (TBAB) in dry dioxane at elevated temperature (82-94 ° C). A subsequent reaction step based on a Cu (I) -catalyzed cycloaddition between terminal alkyne 13 and p-azidophenyl bromide in the presence of THPTA was performed in a mixture of tert-butyl alcohol and water to give compound 14.

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Subsequent deprotection of the amino group to give compound 15 was performed in 1M HCl at 4 ° C for 48 h.

Example 15: Synthesis of pD-GalNAc- (1-> 4) -4-D-GlcNAc- (1-> 3) -4-D-Gal- (1-> 4) -4-D-GlcNAc-10 (2-aminoethylthioureidyl) (LacdiNAc-LacNAc-linker-NH2; 20)

The starting disaccharide 16 was prepared by chemoenzyme synthesis from the acceptor (tert-butoxycarbonylamino) ethylthioureidyl-2-acetamido-2-deoxy-β-D-glucopyranoside (11) (Bojarová P. et al. Molecules 2019, 24, 599) and donorup-nitrophenyl-β -D-galactopyranoside (10) catalyzed by recombinant β4-galactosidase from Bacillus circulans. Disaccharide 16 was used as an acceptor for glycosylation by a β-D-GlcNAc unit catalyzed by recombinant β-Α-acetylhexosaminidase Bbhl from Bifidobacterium bifidum, selective for β (1 → 3) bond formation. The resulting trisaccharide 18 was further used as an acceptor for glycosylation of β-D-GalNAc catalyzed by selective mutant β-A-acetylhexosaminidase from Talaromyces flavus to give tetrasaccharide 19. Subsequent deprotection of the amino group in tetrasaccharide 19 to give compound 20 was performed in 1M HCl at 4 ° C. for 48 h. Alternatively, this compound can be prepared using recombinant glycosyltransferases according to literature procedures (Laaf D. et al. Bioconjug. Chem. 2017, 28, 2832-2840).

Example 16: Synthesis of Substituted Carbohydrate Conjugate 15 Together (HPMA-co-MA-AP-TT) (P9)

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mg of polymer P2 (containing 14.6 pmol of TT groups) and 9.2 mg of compound 15 (13.1 pmol) were dissolved in 0.8 mL of dimethylacetamide and purged with argon. After the addition of 2.3 μL of Nethyldiisopropylamine (13.1 pmol), the reaction mixture was stirred for 20 h at room temperature. Then 1.1 μl of 1-aminopropan-2-ol (14.6 pmol) was added and allowed to react for 1 h. The polymer conjugate was purified from low molecular weight impurities by gel filtration on a Sephadex LH-20 column in methanol. The polymer fraction was freed from methanol by vacuum distillation and the polymer conjugate was isolated by lyophilization. Yield: 25.4 mg; 83.4%; Molar masses: M _w = 25400 g / mol, M _a = 23600 g / mol, D = 1.08; The content of the sugar component in the conjugate was 5.0 mol%.

Example 17: Synthesis of conjugate of substituted saccharide 3 with poly (HPMA-co-MA-APpropynyl) (P10)

To 2 mg of CuSO 4 5H 2 O (16 pmol) dissolved in 25 μL of water was added to a solution of 25 mg of poZy (HPMA-co-MA-AP-propynyl), P4, (17.4 pmol of propynyl groups), 1.6 mg of sodium ascorbate (16.2 pmol) and 12.9 mg of compound 3 (16.2 pmol) dissolved in 225 μL of water. The reaction mixture was bubbled with argon before and after the addition of copper sulfate and stirred for 1 h at room temperature. Then the solution was diluted with 1 mL of a 5% solution of ethylenediaminetetraacetic acid disodium salt, the polymer conjugate was purified by gel filtration on a Sephadex G-25 column in water and lyophilized. The conjugate was dissolved in 2 mL of methanol and excess 8-quinolinol was added. After 20 min, the sample was

- 22 CZ 2020 - 133 A3 purified from residual copper by gel filtration on a Sephadex LH-20 column in methanol. After evaporation of the solvent, the polymer was dissolved in water and lyophilized. Yield: 29.4 mg; 84.0%; Molar masses: M _w = 2Ί 500 g / mol, Mn = 25,200 g / mol, D = 1.09; The content of the sugar component in the conjugate was 9.8 mol%.

Example 18: Synthesis of 20 saccharide substituted saccharide conjugate (HPMA-co-MA-AP-TT) (Pil)

The Pil polymer conjugate was prepared with different sugar content. 20 mg of polymer P2 (containing 16.4 pmol of TT groups) and 2.7 mg of substituted saccharide 20 (3.0 pmol) in the case of the preparation of Pila, 5.3 mg of substituted saccharide 20 (5.9 pmol) in the case of the preparation of P11b, 9.1 mg of substituted saccharide 20 (10.2 pmol) in the case of the preparation of Lung, or 10.3 mg of substituted saccharide 20 (11.2 pmol) in the case of the preparation of P1d was dissolved in 1.2 mL of a mixture of dimethylacetamide and dry methanol (3 : 1) and bubbled with argon. After the addition of 0.8 μL of Nethyldiisopropylamine (4.8 pmol), the reaction mixture was stirred for 20 h at room temperature. Then 1.5 μl of 1-aminopropan-2-ol (20 pmol) was added and allowed to react for 0.5 h. The polymer conjugate was purified from low molecular weight impurities by gel filtration on a Sephadex LH-20 column in methanol. The polymer fraction was freed from methanol by vacuum distillation and the polymer conjugate was isolated by lyophilization. The characteristics are listed in Table 1.

Table 1. Characteristics of prepared glycopolymers bearing a substituted saccharide 20

The name of the conjugate Reaction yield (%) Sugar content (mol%) * M _w (g / mol) y (g / mol) y £> t Saw 88 2.6 22500 21700 1.04 Pllb 90 4.9 24500 22400 1.09 Lung 91 7.2 28300 27300 1.03 Plld 90 9.7 28200 26300 1.07

* The content of substituted carbohydrates was determined by 1 H-NMR.

The molar masses and dispersions were determined by GPC as described in Example 21.

Example 19: Synthesis of conjugate of substituted saccharide 7 with poly (HPMA-co-MA-APazadibenzocyclooctyne) (P12)

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mg of poZy (HPMA-co-MA-AP-azadibenzocyclooctyne), P7, (8.9 pmol of azadibenzocyclooctyne groups) was dissolved in 300 μL of methanol, mixed with a solution of 6.3 mg of substituted saccharide 7 (11.0 pmol) in 200 pL of methanol and bubbled with argon. After stirring at room temperature, the polymeric conjugate was purified by gel filtration on a Sephadex LH-20 column in methanol. The polymer fraction was concentrated in vacuo to 1 mL and the polymer was isolated by precipitation into excess ethyl acetate (40 mL) and filtration followed by drying to constant weight. Yield: 24.7 mg; 80.1%; Molar masses: M _w = 30,800 g / mol, A / n = ίο 28,000 g / mol, D = 1.10; The content of the sugar component in the conjugate was 5.1 mol%.

Example 20: Synthesis of a conjugate of substituted saccharide 9 with poly (HPMA-co-MA-APpropyl azide) (P13)

Br

The polymeric conjugate P13 was prepared with different contents of the sugar component. 15 mg of polymer precursor P8 was dissolved in 150 μL of dimethylformamide and mixed with 3.54 mg (5.6 pmol),

-24EN 2020 - 133 A3 or 6.3 mg (9.9 pmol) of substituted saccharide 9 in 300 dimethylformamide for the preparation of P13a or P13b, respectively. Then 0.88 mg (6.2 pmol) or 2.42 mg (12.9 pmol) of CuBr in the case of P13a or P13b, respectively, were added and stirred at rt. After 20 h, excess 8-quinolinol was added, the solution was stirred for another 30 min and diluted with 2 mL of methanol. The polymer conjugate was purified by gel filtration on a Sephadex LH-20 column in methanol. The polymer-containing fraction was evaporated, the product dissolved in water and lyophilized. P13a: Yield: 15.3 mg; _{molar mass: M n} = 28,600 g / mol, M n = 25,900 g / mol, D = 1.1; the sugar content in the conjugate was 4.5 mol%. P13b: Yield: 17.4 mg; molar mass: M _w = 30,600 g / mol, M _n = 28,100 g / mol, D = 1.1; the content of the sugar component in the conjugate was 8.9 mol%.

Example 21: Example of characterization of polymeric precursors and conjugate

The prepared copolymers, polymer precursors and their conjugates with substituted carbohydrates were characterized by determining the weight and number average molar masses (M _w , Mj) and the corresponding dispersion index (£>) by gel permeation chromatography (GPC) on a system equipped with a UV detector (Shimadzu, Japan ), An RI detector (Optilab REX, Wyatt Technology Corp., USA) and a multi-angle light scattering detector (DAWN Heleos-II, Wyatt Technology Corp., USA). For characterization, a TSK 3000 Super SW column was used for SEC and a mixture of MeOH (80%) and 0.3 M acetate buffer pH 6.5 (20%) as the mobile phase. The sample concentration was 3 mg / ml in all cases.

The content of TT groups was determined spectrophotometrically on a UV-VIS spectrophotometer Specord 205 (Analytik Jena, Germany) in methanol (ssos = 10,800 Lmol fcm ¹ ) according to the literature (Subr V. et al. Biomacromolecules 2006, 7 (1), 122-130 ).

The content of triple bonds and conjugated substituted saccharides was determined by nuclear magnetic resonance (NMR) on a Bmker Avance III 600 MHz spectrometer in water.

Example 22: ELISA

The affinity of Gal-3 for substituted saccharides and their conjugates with polymers was determined using a competitive ELISA assay (Bojarová P. et al. J Nanobiotechnol. 2018, 16, 73; Bumba L. et al. Int. J. Mol. Sci. 2018 , 19, 372). This assay provides preliminary information on the strength of Gal-3 binding to glycopolymers, but should be considered as a guide only and should take into account in particular the biological effect of glycopolymers (see Examples 25-27). Asialofetuin (Sigma Aldrich, Steinheim, Germany; 0.1 μΜ in PBS buffer, 50 μL, 5 pmol per well) was immobilized overnight in the wells of F16 Maxisorp NUNC-Immuno Modules (Thermo Scientific, Roskilde, Denmark). The wells were then blocked with BSA (2% w / v) dissolved in PBS (1 h, lab.t.). Subsequently, a mixture of test compound at various concentrations and Gal-3 (total volume 50 μL; 4.5 pM final concentration of Gal-3) was added to the wells and incubated for 2 hours. Detection of bound Gal-3 was performed using a horseradish peroxidase-conjugated mouse anti-His6-IgG1 monoclonal antibody (Roche Diagnostics, Mannheim, Germany) dissolved in PBS buffer (1: 1000, 50 μL, 1 h, lab.t.). TMB One substrate solution (Kem-En-Tec, Taastrup, Denmark) was used to initiate the colorimetric reaction of the conjugated peroxidase. The reaction was quenched by the addition of 3 M HCl (50 μL). The binding signal of bound Gal-3 was determined spectrophotometrically at 450 nm (Spectra Max Plus, Molecular Devices, Sunnyvale, CA, USA). The obtained results were analyzed using Prism 7.0 software (GraphPad, USA) and evaluated as IC50.

Table 2. Inhibitory potential of selected compounds and conjugates (IC50) determined by ELISA

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The name of the conjugate Sugar content [mol%] * IC ₅₀ per glycan [μΜ | IC _{50 of} glycopolymer | Lactose - 132 - Substituted saccharide 20 - 7.21 - Glycopolymer Saw 2.6 0.22 0.066 Glycopolymer Pllb 4.9 0.14 0.023 Glycopolymer Lung 7.2 4.8 0.51 Glycopolymer Plld 9.7 4.4 0.40 Substituted carbohydrate 9 - 16 - Glycopolymer P13a 4.5 28 4.2 Glycopolymer P13b 8.9 6.9 0.62

* The content of substituted carbohydrates was determined by 1 H-NMR.

¹ Taken from the literature (Bócker S. et al. Biomolecules 2015, 5, 1671-1696)

Example 23: Quantification of Gal-3 production in selected tumor lines.

Cell line Detection of Gal-3 using flow cytometry Gal-3 expression detected by Western blot Membrane localization Membrane localization Intracellular localization 4T1 Not tested + ++ B16-F10 Not tested + ++ CT26 Not tested - ++ DLD-1 ++ + ++ HEK293 ++ + ++ HT-29 + Not tested Not tested Jurkat Not tested - - LNCaP - - - OVCAR-3 ++ + ++ PC3 + + ++ Paradise - Not tested Not tested SU-DHL-5 - - - SU-DHL-6 + Not tested Not tested

Explanations: Gal-3 expression rate - = undetectable, "+" = significant, "++" = strongly pronounced.

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Example 24: Inhibition of external galectin-3 binding to the surface of galectin-3-expressing cells by glycopolymers

The ability of glycopolymers to inhibit the binding of external Gal-3 to the cell surface was demonstrated by an inhibition binding assay by flow cytometry. The assay used a Gal-3-AVI protein construct bearing the Avi-tag amino acid sequence at its N-terminus, which allows targeted binding of the biotin molecule and its subsequent detection using a fluorescently labeled streptavidin-phycoerythrin conjugate (L. Bumba et al. Int. J. Mol. Sci. 2018, 19, 372). As determined by ELISA, the binding properties of the native Gal3 and Gal-3-AVI constructs are identical. Binding of glycopolymers to Gal-3 inhibited the binding of Gal-3 to the surface of HEK293 cells (immortalized human germline kidney cell line), which showed strong Gal-3 expression (see Example 23) and a large capacity to balance free Gal-3 from solution. .

Aliquots of the Gal-3-AVI construct (final concentration 10 pg / mL) were mixed with increasing concentrations of Plib glycopolymers (3 pM - 100 pM) or lactose (3 nM - 100 mM) as positive controls and incubated for 30 minutes on ice. in PBS buffer supplemented with 1% bovine serum albumin (BSA). This mixture was added to a suspension of HEK293 cells (10 ⁶ / mL) and stirred slowly on ice for 30 min. Subsequently, the cells were washed with PBS buffer and labeled with streptavidin-phycoerythrin conjugate (Biolegend, USA). The binding strength of the Gal-3-AVI construct to the surface of HEK293 cells was analyzed by flow cytometry and quantified as the relative fluorescence intensity at 575 nm.

Table 3. Inhibitory potential of Plib glycopolymers (IC50) determined in an inhibition binding assay with the HEK293 cell line by flow cytometry

The name of the conjugate Sugar content [mol%] * IC50 of glycopolymers [μΜ] Lactose - 225 Glycopolymer Pllb 4.9 0.057

* The content of substituted carbohydrates was determined by 1 H-NMR.

Example 25: Inhibition of Galectin-3-Induced Human T Cell Apoptosis by Glycopolymers The ability of glycopolymers to effectively inhibit Gal-3-induced apoptosis was demonstrated by the annexin NI propidium iodide apoptotic flow cytometry assay. The experiments were performed on the immortalized human T cell line Jurkat. Human Gal-3 produced by tumor cells into the environment has been shown to induce apoptosis in this line.

In in vitro experiments, Jurkat cells were preincubated with various concentrations of Pil and P13 glycopolymers (0.1; 1; 5; 10 and 50 μΜ) for 5 minutes and then 10 μΜ Gal-3 was added. Glycopolymers balanced free Gal-3 from solution (around the cells) and thus could not induce apoptosis of Jurkat cells. The inhibition efficiency of Gal-3 glycopolymers was determined as a measure of cell apoptosis. Pure HPMA polymer without substituted carbohydrates was used for the control experiment.

Glycopolymers inhibited Gal-3-induced apoptosis. The maximum inhibitory effect was achieved at a concentration of 5 μΜ or 1 μΜ for the tested conjugate P11b or Plld (see Figures 1 and 2). Pure HPMA polymer without substituted carbohydrates also showed a slight protective effect at a concentration of 50 μΜ.

Glycopolymer P13 also showed a high inhibitory effect (see Figures 3 and 4).

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Example 26 (comparative): Inhibition of galectin-3-induced apoptosis of human T cells (Jurkat) by a glycopolymer carrying a single disaccharide LacdiNAc

Table 4. Characteristics of a comparative glycopolymer bearing a single disaccharide LacdiNAc

The name of the conjugate Sugar content (mol%) * M _w (g / mol) t (g / mol) t IC ₅₀ per glycan Fum] IC 50 per glycopolymer Fum] LacdiNAcHPMA 12.3 30100 28000 1.07 38.5 2.3

* Carbohydrate content was determined by 1 H-NMR.

Molecular weights and dispersions were determined by GPC as described in Example 21.

Giant. 5 shows the inhibition of galectin-3-induced apoptosis of Jurkat cells by HPMA polymer conjugate with the single disaccharide LacdiNAc (LacdiNAc content was 12.3 mol%, see Table 4). The same results were obtained with the analogous conjugate containing 8.4 mol% LacdiNAc. It is the same disaccharide as in the publication (Bojarová P. et al. J. Nanobiotechnol. 2018, 16, 73). It is evident that in cellular assays, this conjugate showed a significantly lower ability to inhibit Jurkat cell apoptosis than the conjugates with substituted disaccharides of the invention.

Example 27: Inhibition of human and mouse tumor cell migration

The migration of selected tumor cells that express Gal-3 was studied using the so-called scratch test. This test is based on placing the examined cell culture on a culture dish, at its 70-80% growth, the cells are scraped from the surface with a tip of 1 mL of plastic tip from a surface about 1 cm long and about 0.5-1 mm wide. Subsequently, the medium in which the cells are cultured is changed and replaced with fresh medium containing the test substances. Cell migration is then monitored by the rate and degree of overgrowth of the gap formed.

The inhibitory effect of Pila polymers containing 2.6 mol% and Plic containing 7.2 mol% of tetrasaccharide was monitored after addition to the medium of mouse breast tumor cells (4T1), mouse melanoblastoma cell line (B16F10) and human colorectal cancer cells (DLD1). Polymeric conjugates were added at a concentration of 10 μΜ or 20 pM. After 24 and 48 h, the area that remained ungrown with cells was measured and the difference compared to the control group without added polymeric conjugate was monitored. Giant. 6 shows the differences in overgrown zone width of a control sample to which no polymeric conjugate (control) was added, a sample to which HPMA polymer alone (pHPMA) was added, and samples with conjugates. In the case of 4T1 and B16F10 cells, both conjugates caused inhibition of migration compared to the control even in the sample incubated with the polymeric carrier. In DLD1 cells, only the conjugate with a higher molar proportion of the sugar component caused an inhibition of migration different from the control and the polymeric carrier.

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Claims (11)

1. Glycopolymer based on HPMA polymers with a content of from 0.5 to 25 mol% - structural units of formula I: Linker substituted carbohydrate (s), where Y ¹ is selected from the group consisting of alkylene having 1 to 8 carbon atoms; phenylene; - (CH ₂ ) _q (C (O) -NH- (CH ₂ ) _r ) p -, wherein p = 1 to 5, aqar are independently selected from 1, 2 and 3; wherein Y ¹ may be optionally substituted with one or more natural amino acid side chains, wherein the side chains may be the same or different; Y ² is selected from the group consisting of a bond, carbamoyl, carbamoyl- (C 1 -C 8 -alkylene), and phenylene; wherein carbamoyl means the group -C (= O) -NH- and the group -NH-C (= O) -; The linker is selected from the group consisting of 1,2,3-triazolylene, (C 1 -C 6 alkyl) -1,2,3-triazolylene; NH-C (= O) -NH- (CH ₂ ) 2 -NH-C (= O) -; -NH-C (= S) -NH- (CH ₂ ) 2 -NH-C (= O) -; or to the substituent Y ² via the group -C (= O) - or only covalently bonded 3,4,5,13-tetrazatetracyclo [13.4.0.02,6.07,12] nonadeca-1 (15), 2 (6), 3 , 7 (12), 8,10,16,18-octane-13-carbaldehyde; The substituted saccharide is a substituted saccharide of formula III; or - end groups of the formula -S-succinimide- (CH 2) _r -Linker-Substituted saccharide or C (CN) (CH 3) - (C 1 -C 4 alkylene-Linker-Substituted saccharide, wherein r is selected from the group consisting of 1, 2 and 3, and the Linker and Substituted Carbohydrate are as defined above; wherein the substituted saccharide of formula III is - 29 CZ 2020 - 133 A3 (ΠΙ), wherein the combination of U, V and W and R 4 corresponds to the formula IIIb or Ilic or Illd (IIIb) wherein in formula IIIb, U is 4-OD-glucopyranosyl substituted on C-2 by R ^3u and on Cl by R ⁴ ; V and W is a bond; (IIIc) wherein in formula IIIc U is 3-OD-galactopyranosyl substituted on C-2 by R ^3u and on Cl by R ⁴ ; V and W is a bond; (Illd); wherein in formula IIId U, 3-OD-glucopyranosyl is substituted on C-2 by R ^3u and on Cl by R ⁴ ; V and W is a bond; and where R ¹ is selected from the group consisting of phenyl, phenyl substituted with at least one halogen, phenyl substituted with at least one nitro group, phenyl substituted with at least one carboxy group, phenyl substituted with at least one halogen (C1-C5) alkyltriazolyl, phenyl substituted with at least one sulfo group, phenyl substituted with at least one cyano group phenyl substituted by at least one hydroxy group, naphthyl, naphthyl substituted by at least one halogen, biphenyl, saccharide, C2-C6 heteroaryl containing at least one heteroatom selected from O, S, N, coumaryl, benzoyl; X is [1,2,3] -triazole or a bond Y is CH ₂ or a bond, -30EN 2020 - 133 A3 Z is O or [1,2,3] -triazole. R ² , R ^3u are independently selected from the group consisting of hydroxy, acetamido, C 1 -C 5 acylamido, 1-OaL-fucosyl, R ⁴ is selected from the group consisting of C 1 -C 5 alkoxy linked to the Linker, linked to the Linker, -O-linked to the Linker, phenyloxy linked to the Linker on the phenyl, R ⁵ is selected from the group consisting of hydrogen, sulfo, 2-Oa-sialyl. Glycopolymer according to claim 1, which comprises polymer chains formed by HPMA polymer and branching units, the branching units being preferably amidoamine units or 2,2-bis (hydroxymethyl) propionic units. The glycopolymer according to claim 1 or 2, which further comprises 0.5 to 12 mol% of weight units of the general formula corresponding to the general formula I, wherein Y ¹ and Y ² are as defined in claim 1, but the structure -Linker-Subst the saccharide is replaced by a substituent selected from the group consisting of carbonyl-hydrazone- (C12-C18 alkanone), carbonyl-hydrazono-5α-cholestanone, carbonyl-hydrazono-cholest-4-en-3-one; carbonyl- (C9-C15 alkoxy), carbonyl-cholesteryl, carbonyl-7-dehydrocholesteryl; carboxamido- (C10-C18 alkyl), carbohydrazido- (C10-C18 alkyl), carboxamido- (C10-C18 alkenyl) and carbohydrazido- (C10-C18 alkenyl), containing at least one double bond (C = C). Process for the preparation of a glycopolymer according to any one of claims 1 to 3, characterized in that it comprises the following steps: a) polymerization of functionalized monomers of general formula II (Π), where Y ¹ is selected from the group consisting of alkylene having 1 to 8 carbon atoms; phenylene; - (CH 2) _q (C (O) -NH- (CH ₂ ) _r ) p -, wherein p = 1 to 5, aqar are independently selected from 1, 2 and 3; wherein Y ¹ may be optionally substituted with one or more natural amino acid side chains, wherein the side chains may be the same or different; Y ² is selected from the group consisting of a bond, carbamoyl, carbamoyl- (C 1 -C 8 -alkylene), and phenylene; wherein carbamoyl means the group -C (= O) -NH- and the group -NH-C (= O) -; Y ³ is selected from the group consisting of primary amine (NH 2), tBoc substituted amine, azide, terminal alkynyl having 2 to 8 carbon atoms, phenyl substituted with at least one azide or C 2 -C 4 alkynyl; (11,12-didehydrodibenzo [b, f] azocin-5 (6H) -yl) or (1R, 8S, 95) bicyclo [6.1.0] non-4-yn-9-yl, which is bonded via a carbonyl a group; -carbonyl-thiazoline-2-thione group (TT), -carbonyl-4-nitrophenoxy group, -carbonyl-2,3,4,5,6-pentafluorophenoxy group, -carbonyl-succinimidyl group, and COOH group; wherein carbonyl is -C (= O) -, And A- (2-hydroxypropyl) methacrylamide (HPMA), and optionally methacryloyl (aminoacyl) esters and / or branching units; at a temperature in the range of 30 to 100 ° C, preferably 40 to 80 ° C, in a solvent selected from the group consisting of water, aqueous buffers, dimethylsulfoxide, dimethylacetamide, dimethylformamide, methanol, ethanol, dioxane, tert-butyl alcohol or mixtures thereof, initiation with an initiator, preferably selected from the group comprising in particular the azo initiators 2,2'-azobis (2-methylpropionitrile), 4,4'-azobis (4-cyanopentanoic acid), 2,2'-azobis (4-methoxy-2,4-dimethylpentanenitrile) ) in the presence of a transfer agent; b) an optional step of polymer-analogous reactions, c) coupling of a substituted saccharide selected from the group consisting of substituted saccharides of formula III: y__R ⁴ (III), wherein the combination of U, V and W and R 4 corresponds to the formula IIIb or IIIe or IIId (Illb) (lile) and where R ¹ is selected from the group consisting of phenyl, phenyl substituted by at least one halogen, phenyl substituted by at least one nitro group, phenyl substituted by at least one carboxy group, phenyl substituted by at least one C 1 -C 5 alkoxy group, phenyl substituted by at least one halogen (C 1 -C 5) alkyltriazolyl, phenyl substituted at least one sulfo group, phenyl substituted by at least one cyano group, phenyl substituted by at least one hydroxy group, Naphthyl, naphthyl substituted with at least one halogen, biphenyl, saccharide, C2-C6 heteroaryl containing at least one heteroatom selected from O, S, N, sulfo, coumaryl, and benzoyl; X is [1,2,3] -triazole or a bond Y is CH ₂ or a bond, Z is O or [1,2,3] -triazole. R ² , R ^3u are independently selected from the group consisting of hydroxy, acetamido, C 1 -C 5 acylamido, 1-Oa-fucosyl, R ⁴ is selected from the group consisting of amino, C 2 -C 5 alkynyloxy, azide, C 1 -C 5 azidoalkoxy, aminoethylthioureidyl, aminophenyloxy and azidophenyloxy, R ⁵ is selected from the group consisting of hydrogen, sulfo, 2-Oa-sialyl. Process according to Claim 4, characterized in that step a) of the synthesis of polymeric supports by polymerization of monomers is carried out in the presence of a transfer agent selected from the group consisting of 2-cyano-2-propylbenzodithioate, 4-cyano-4- (thiobenzoylthio) pentanoic acid, 2-cyano 2-propyldodecyltrithiocarbonate, 2-cyano-2-propylethyltrithiocarbonate and 4-cyano-4 - [(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid. Process according to Claim 4, characterized in that step a) of the synthesis of polymeric supports by polymerization of monomers and branching units is carried out in the presence of a carboxyl group-containing transfer agent or a functional derivative thereof containing a leaving group consisting preferably of thiobenzoylthio, 4-nitrophenoxy, 2, 3,4,5,6-pentafluorophenoxy, or a succinimidyl group; wherein the transfer agent is preferably selected from the group consisting of 4-cyano-4- (thiobenzoylthio) pentanoic acid, 4-cyano-4 - [(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid, 1-cyano-1-methyl-4-oxo-4- (2-thioxothiazolidin-3-yl) butyl dithiobenzoate, 2-cyano-5-oxo-5- (2-thioxo-1,3-thiazolidin-3-yl) pentan-2-yl ethylcarbonitrithioate, and in a second step, the polymers obtained in the first step are reacted. step with a poly (amidoamine) or 2,2-bis (hydroxymethyl) propionic dendrimer to form an amide bond between the HPMA and the dendrimer, the reaction being carried out in a solvent selected from the group consisting of dimethylsulfoxide, dimethylacetamide, dimethylformamide, methanol and ethanol, followed by in the remaining primary amino groups are blocked by a low molecular weight aminoreactive compound, preferably acetic anhydride, in the third step the sulfur-containing groups at the other end of the polymer are either reduced with sodium borohydride and SH groups are added in situ to the N-derived maleimide, preferably propynylmaleimide. azido-PEG3-maleimide, wherein the reaction is performed in a solvent selected from the group consisting of water, dimethylacetamide, dimethylformamide, methanol and ethanol; or the sulfur-containing groups at the other end of the polymer are reacted with an excess of azo-initiator bearing a carboxyl group, or a functional derivative thereof containing a leaving group consisting preferably of thibenzoylthio, 4-nitrophenoxy, 2,3,4,5,6-pentafluorophenoxy, or succinimidyl group at a temperature in the range of 50 to 100 ° C, preferably 60 to 80 ° C, and a solvent preferably selected from the group consisting of dimethyl sulfoxide, dimethylacetamide and dimethylformamide. The method of claim 4, further comprising, in step a), the step of removing sulfur-containing end groups from the polymers in the preparation of which a transfer agent is used in which the polymers are reacted with an excess of azo initiator from the group consisting of 2, 2'-azobis (2-methylpropionitrile), 4,4'-azobis (4-cyanopentanoic acid), 2,2'-azobis (4 -33EN 2020 - 133 A3 methoxy-2,4-dimethylpentanenitrile), to form a polymeric support terminated by a radical residue formed by the decomposition of the initiator used. Process according to any one of claims 4 to 7, characterized in that step b) of further introducing an azide or alkyl group is carried out by a process selected from: (i) reacting the Y ³ group on the polymer, if it is a leaving group, with an amino compound terminated by a group selected from azide, ethynyl, phenyl substituted with at least one azide, C 2 -C 5 terminal alkynyl, or substituted cyclooctyl, preferably 3-amino-1- (1,12,12-dihydro-dibenzo [b, f] azocin-5 (6H) -yl) -propan-1-one, or (1 ', 8', 8 ', 5'), bicyclo | 6.1.0 non-4-yn9-ylmethyloxycarbonyl] -1,8-diamino-3,6-dioxaoctane; (ii) reacting the Y ³ group on the polymer, if it is a primary amino group, with a carboxy (C 1 -C 8 alkane) terminated by a group selected from azide, ethynyl, phenyl substituted with at least one azide, C 2 -C 5 alkynyl, or substituted cyclooctyl, preferably 6 - (11,12-dehydrodibenzo [b, f] azocin-5 (6H) -yl) -6-oxohexanoic acid, or its N-hydroxysuccinimidyl ester and (17R, 8S, 95) -bicyclo [6.1.0] non-4-yne -9-ylmethyl A-succinimidyl carbonate, or functional derivatives of this carboxyalkane containing a well-leaving group preferably consisting of TT, 4-nitrophenoxy, 2,3,4,5,6-pentafluorophenoxy, or a succinimidyl group. A glycopolymer according to any one of claims 1 to 3 for use as a medicament, adjuvant, or immunomodulator. A glycopolymer according to any one of claims 1 to 3 for use in the treatment of solid tumors, lymphoma or leukemia; or to inhibit tumor cell migration and suppress metastatic spread of cancer; or to inhibit the induction of apoptosis in immune system cells in a tumor. Pharmaceutical composition, characterized in that it comprises a glycopolymer according to any one of claims 1 to 3 and at least one pharmaceutically acceptable excipient selected from the group consisting of fillers, anti-adhesives, binders, coatings, dyes, swelling agents, flavoring agents, lubricants, preservatives, sweeteners, sorbents.