Technical field

The invention relates to multivalent water-soluble or micellar polymers, i.e. homopolymers or copolymers, with carbohydrate structures containing a terminal monosaccharide in galacto- configuration, which are effective as inhibitors of galectins. These glycopolymers can be used as medicaments, in therapy and prevention of cancer diseases involving overproduction of galectins, especially of human galectin-3 (Gal-3).

Background Art

In recent years, the development of anticancer drugs has been shifting from classical low-molecular- weight drugs to the search for new drugs or drug forms, in which the active compound preferentially acts at the desired site of action. Nowadays, monoclonal antibodies enter clinical applications due to their high efficiency in the treatment of various malignancies. Nevertheless, their use is often connected with toxic effects for patients. Besides antibodies, the targeted forms of drugs can be utilized for compounds, of which side effects can harm the healthy parts of the body. The use of polymer carriers, particularly water-soluble or micelle-forming polymers, for targeted drug delivery is an important possible solution to this situation. High molecular weight of polymers bearing active compounds prevents fast elimination from the organism by glomerular filtration. The prolonged blood circulation time and even total concentration level in the organism improve the biological impact of the system. Also, the high-molecular-weight of polymer system increases accumulation in solid tumors by the EPR (enhanced permeability and retention) effect.

The EPR effect can be utilized for the targeted tumor accumulation of the macromolecular carrier with a bound drug. In the past, various drug delivery systems utilizing the EPR effect were developed, e.g. polymer micelles, liposomes, nanoparticles, nanocapsules, or water-soluble polymer conjugates. Unlike water-soluble polymer systems, polymer micelles are usually prepared by self-assembly of amphiphilic diblock copolymers to high-molecular-weight micelles forming colloid solutions. One of the most important water-soluble polymer carrier systems is based on polymers containing N-(2- hydroxypropyl)methacrylamide units (HPMA polymers). The only one and main disadvantage complicating the use of HPMA polymers in human medicine is their non-biodegradable carbon chain, which limits their molecular weight. Only polymers with a molecular weight below the limit of renal threshold (50,000 - 60,000 g/mol) can be used as polymer carriers. Polymers above this threshold are not effectively and sufficiently eliminated from the body, and thus their use as drug carriers would lead to undesired accumulation in the body. The increase of molecular weight of HPMA polymers, and thus prolongation of their circulation in the organism, is enabled by the introduction of biodegradable linkers between HPMA polymer chains. Suitable linkers comprise mainly enzymatically, hydrolytically or reductively degradable linkers. Structures formed by linking such polymer blocks are called multiblock, grafted or star polymer structures. Optionally, the amphiphilic HPMA polymers self-assembling into supramolecular micellar structures can also be used. They are disassembled after the concentration decrease below the critical micellar concentration and eliminated from the body as short polymers via renal filtration.

Recently, it was shown that HPMA polymer can be used as a carrier of several scFv chains of anti- CD20 and induced apoptosis of CD20 positive cells due to its multivalency (Kopecek J. Adv. Drug Delivery Rev. 2013, 65, 49-59). This concept described as drug-free therapeutics has been tested in the USA. The efficacy of this system consists of the multivalency of the polymer structure.

Galectin-3 (Gal-3) is a lectin belonging to the family of galectins - glycan-binding proteins with a terminal b-galactoside. Currently, considerable attention is paid to this galectin due to its significant effect on processes affecting tumor growth, such as metastasis, overcoming the body immune response, mRNA splicing, gene expression, apoptosis and inflammation. Gal-3 is expressed in many tumor tissues and cells, both intracellularly and extracellularly, both when it is 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 worse 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 through blood circulation into the whole body. The increased presence of Gal-3 is directly correlated to a worse prognosis for cancer, e.g., in thyroid tumors, colon, head and neck tumors or brain tumors. One reason may be the effect of free Gal-3 on the apoptosis of anti-tumor T cells. Due to its increased expression in tumor tissue, Gal- 3 can serve as a therapeutic marker and at the same time as a therapeutic target. Also, its presence in the bloodstream or in the 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, Guha P. PNAS 2013, 110 (13), 5052- 5057).

In recent years, various modifications of the basic carbohydrate ligands of galectins was studied, especially based on galactose, lactose (Ga1b4G1c) or N -acetyllactosamine (Ga1b4G1cNAc), as well as their effect of these modifications on the affinity of prepared glycomimetics to individual galectins, especially to biomedically most studied galectin- 1 and -3. 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 to galectins. Structural analogs of N-acetyllactosamine and thiodigalactosides (TDG; Ga1b1- 1bGa1) have developed into a large group of potent glycomimetic galectin inhibitors resistant against 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. In the past, these complex oligosaccharide structures and other, simpler carbohydrates based on lactose, galactose or N-acetyllactosamine without the mentioned substitutions have also been presented in multivalent mode on a number of carriers, such as peptides, polymers, oligonucleotides, fullerenes and calixarenes, as well as dendrimers and nanoparticles. Multivalent presentation often significantly increased the low affinity of the monovalent carbohydrate. Although high affinities to 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) in view of the possibility of active targeting to selected cancer cell lines, some of which expressed Gal-3. In the cases described, the effect of the conjugates on Gal-3 inhibition was not considered and the correlation of the obtained results with Gal-3 expression on the cells was not conclusive. In the inventors' publication (Bojarova P. et al. J. Nanobiotechnol. 2018, 16, 73), a series of HPMA conjugates with the single disaccharide LacdiNAc were synthesized and their inhibitory effect (IC ₅₀ in the order of mM) on Gal-3 was demonstrated in an ELISA assay. It is the aim 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 their practical use.

Disclosure of the invention

Object of the present invention is the structure, synthesis, and use of novel polymer drug with well- controllable content of substituted carbohydrates. The content of the substituted carbohydrates is controlled by the amount of carbohydrate component added to polymer carrier, because it is directly proportional. The novel medicament shows prolonged pharmacokinetics and enhanced tumor accumulation due to the presence of the polymer component, together with strong inhibition of galectins.

Glycopolymers bearing substituted carbohydrates according to this invention showed not only a higher affinity to Gal-3 in binding assays such as ELISA, compared to the glycopolymers known from the state of the art, but mainly exhibited a significantly stronger effect in biological experiments as proved in the comparative example 40. When compared with the closest state of the art, the glycopolymers according to the invention are considerably more efficient in inhibition of Gal-3, which has direct impact on the anticancer immune response or migration of cancer cells.

Contrary to all previously prepared systems, the glycopolymers according to the invention are the only substance with inhibition effect to Gal-3 that can be used for antitumor therapy in vivo due to their suitable pharmacokinetics ensured in particular by the polymer carrier, biocompatibility, in vivo stability, and well-reproducible production process. Within the framework of this invention, a high activity of the prepared glycopolymers was demonstrated not only in inhibition of apoptosis induced in the cells of the immune system by Gal-3 but also in suppression of tumor cell migration, which has not been demonstrated yet in systems of this type. Both activitites are directly involved in tumor treatment and in suppression of the metastatic process.

One of the important characteristics of the system according to the invention is a polymer chain formed by inert, neutral, water-soluble HPMA-based polymer, which does not interact with the organism. The polymer is used as a multivalent carrier for attachment of substituted carbohydrates to achieve enhanced interaction with lectins and also a more suitable pharmacokinetics of substituted carbohydrates. These effects are attained by the combination with specific substituted carbohydrates. The structure of the glycopolymer according to the invention is based on polymer carriers based on HPMA polymers bearing covalently bound substituted carbohydrates. Their interaction with galectins has a multivalent character owing to the polymer carrier, and avidity to galectins when binding occurs. The described strong interaction with galectins leads to the biological effect which includes several processes: the protective effect on immune cells by suppressing apoptosis induced by Gal-3, and the inhibition of migration of tumor cells connected with the inhibition of the metastatic spread.

The term „drug“ or „medicament“ means a compound with intrinsic therapeutic effect, or adjuvant or immunomodulator.

The term „polymer“ means copolymer and homopolymer. Namely, the term ,,HRMA polymer" includes HPMA copolymer and HPMA homopolymer.

Therefore, the object of the present invention is a glycopolymer containing a polymer carrier based on HPMA polymer containing from 0.5 to 25 mol. % of:

- structural units of formula I:

wherein

Y ¹ is selected from a group comprising C1-C8 alkylene; phenylene; -(CH ₂ ) _q -(C(O)-NH- (CH ₂ ) _r ) _p -, wherein p=l to 5, and q and r are independently selected from 1, 2, and 3; wherein Y ¹ is optionally substituted by one or more side chains of naturally occurring amino acid(s) wherein the side chains can be the same or different; Y ¹ is preferably selected from the group comprising -CH ₂ -CH ₂ - and -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -;

Y ² is selected from a group comprising a bond, carbamoyl, carbamoyl-( C1-C8 -alkylene), phenylene; wherein carbamoyl means the group -C(=O)-NH- or -NH-C(=O)-;

Linker is selected from the group comprising 1,2,3-triazolylene, (C1-C6 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 bound to the substituent Y ² via the group -C(=O)- or via a covalent bond, said cyclooctynyl or azacyclooctynyl being substituted with at least one halogen atom and/or cyclopropane or conjugated with at least one benzene ring, wherein said cyclooctynyl or azacyclooctynyl is further conjugated with triazolyl (for example composed of 3,4,5,13-tetrazatetracyclo[13.4.0.02,6.07,12]nonadeca-1(15), 2(6),3,7(12),8,10,16,18-octaene- 13-carbaldehyde motif);

Substituted carbohydrate is a substituent derived from the substituted carbohydrate of formula IIIp described herein below (in the description of step d) of the manufacturing process) by the reaction of terminal amine, azide, alkynyl, aminoethylureidyl or aminoethylthioureidyl group, wherein the groups formed by the reaction of these terminal groups form a part of the Linker; and wherein the Substituted carbohydrate is not lactose (Ga1b4G1c), LacNAc (Ga1b4G1cNAc) nor LacdiNAc (Ga1NAcb4G1cNAc); and/or

- terminal groups of HPMA polymer chain, said terminal groups being of formula -S- succinimide-(CH ₂ ) _r -Linker-Substituted carbohydrate or -C(CN)(CH ₃ )-(C1-C4 alkylene)-Linker- Substituted carbohydrate; wherein r is selected from the group comprising 1, 2 and 3, and Linker and Substituted carbohydrate are defined above. „Naturally occurring amino acids“ mean herein 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. „Side chains" mean chains bound to the alpha carbon atom of the amino acid.

Substituted carbohydrate derived from the substituted carbohydrate of formula IIIp described herein below (within the framework of step d) of the manufacturing process) by the reaction of terminal amino, azido, alkynyl, aminoethylureidyl or aminoethylthioureidyl group, wherein the groups formed by the reaction of these terminal groups form a part of the Linker, is preferably a substituted carbohydrate of formula III: wherein U, V and W and R4 are selected from the following combinations for Ilia, Illb, IIIc, Illd, Ille:

Ilia: wherein V and W is a bond, and wherein U is 1 -thio -b-D-galactopyranosyl substituted at atom C- 2 with R ⁴ group, at atom C-3 with -Z-Y-X-R ¹ group combination and at atom C-6 with -OR ⁶ group;

Illb: wherein V and W is a bond and wherein U is 4-O-D-glucopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with R ⁴ group;

IIIc: wherein V and W is a bond and wherein U is 3-O-D-galactopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with R ⁴ group; Illd: wherein V and W is a bond and wherein U is 3-O-D-glucopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with R ⁴ group; Ille: wherein U is 4-O-D-glucopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with substituent V, wherein V is 3-O-D-galactopyranosyl substituted at atom C-2 with R ^3v group and at atom C-1 with substituent W, wherein W is 4-O-D-glucopyranosyl substituted at atom C-2 with R ^3w group and at atom C-1 with R ⁴ group; and wherein

R ¹ is selected from a group comprising hydrogen, phenyl, phenyl substituted with at least one halogen atom, phenyl substituted with at least one nitro group, phenyl substituted with at least one carboxy group, phenyl substituted with at least one C1-C5 alkoxy 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 with at least one amino group, phenyl substituted with at least one C1-C5 aminoalkyl group, phenyl substituted with at least one hydroxy group, naphthyl, naphthyl substituted with at least one halogen, biphenyl, carbohydrate, C2-C6 heteroaryl containing at least one heteroatom selected from O, S, N, sulfo group, coumaryl, benzoyl, C2-C5 alkynyl, C1-C5 alkyl with a bond to Linker, a bond to Linker, phenyl with a bond to Linker, phenyl-(C1-C5)alkyl with a bond to Linker;

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

Y is CH ₂ or a bond,

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

R ² , R ^3u , R ^3v , R ^3w are independently selected from a group comprising hydroxy group, acetamido group, C1-C5 acylamido group, 1-O-a-L-fucosyl,

R ⁴ is selected from a group comprising hydroxy group, acetamido group, C1-C5 acylamido group, 1-O-a-L-fucosyl, -NH-bond to Linker, C1-C5 alkoxy with a bond to Linker, a bond to Linker, -O-bond to Linker, phenyloxy group with a bond Linker located on phenyl,

R ⁵ and R ⁶ are independently selected from a group comprising hydrogen, sulfo group, 2-O-a -sialyl, C1-C5 alkyl with a bond to Linker, a bond to Linker; wherein the substituted carbohydrate of formula III contains exactly one bond to Linker, preferably as a part of a substituent selected from R ¹ , R ⁵ or R ⁶ when U is 1 -thio -b-D-galactopyranosyl, and preferably as a part of substituent R ⁴ in all other combinations.

Halogen herein means an atom selected from fluorine, chlorine, bromine, iodine. In addition to poly(HPMA) chains, the glycopolymer can also contain branching units, for example, amidoamine units suitable for preparation of dendri meric (star-like) polymer cores of poly(amidoamine) (PAMAM), or 2,2-bis(hydroxymethyl)propionic units suitable for the formation of dendrimer core. An example of a star-like glycopolymer can contain a PAMAM core and arms of poly(HPMA) chains containing terminal groups and/or monomer units of formula I as described herein above.

In some embodiments, the glycopolymer is a glycopolymer based on HPMA polymers containing from 0.5 to 5 mol.% of:

- structural units of formula I: wherein

Y ¹ is selected from agroup comprising C1-C8 alkylene; phenylene; -(CH ₂ ) _q -(C(O)-NH-(CH ₂ ) _r ) _p - , wherein p=1 to 5, and q and r are independently selected from 1, 2, and 3; wherein Y ¹ is optionally substituted with one or more side chains of naturally occurring amino acid wherein the side chains can be the same or different;

Y ² is selected from a group comprising a bond, carbamoyl, carbamoyl-(C1-C8-alkylene), and phenylene; wherein carbamoyl means the group -C(=O)-NH- or the group -NH-C(=O)-;

Linker is selected from the group comprising 1,2,3-triazolylene, (C1-C6 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

3,4,5,13-tetrazatetracyclo[13.4.0.02,6.07,12]nonadeca-l(1 5),2(6),3,7(12),8,10,16,18-octaene- 13-carbaldehyde bound via the group -C(=O)- or directly covalently bound to the substituent Y ² ;

Substituted carbohydrate is substituted carbohydrate of formula III; and/or

- terminal groups of structural formula -S-succinimide-(CH ₂ ) _r -Linker-Substituted carbohydrate or -C(CN)(CH ₃ )-(C1-C4 alkylene)-Linker-Substituted carbohydrate; wherein r is selected from a group comprising 1, 2 and 3, and Linker and Substituted carbohydrate are defined above; wherein Substituted carbohydrate of formula III is wherein the combination U, V and W and R4 corresponds to formula Ilia wherein U is 1 -thio -b-D-galactopyranosyl substituted at atom C-2 with R ⁴ group, at atom C-3 with the moiety -Z-Y-X-R ¹ and at atom C-6 with -OR ⁶ ; V and W are a bond; and wherein

R ¹ is selected from a group comprising hydrogen, phenyl, phenyl substituted with at least one halogen atom, phenyl substituted with at least one nitro group, phenyl substituted with at least one carboxy group, phenyl substituted with at least one C1-C5 alkoxy 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 with at least one hydroxy group, naphthyl, naphthyl substituted with at least one halogen, biphenyl, carbohydrate, C2-C6 heteroaryl containing at least one heteroatom selected from O, S, N, sulfo group, coumaryl, benzoyl, C1-C5 alkyl with a bond to Linker, a bond to Linker, phenyl with a bond to Linker, phenyl-(C1-C5)alkyl with a bond to Linker;

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

Y is CH ₂ or a bond,

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

R ² is selected from a group comprising hydroxy group, acetamido group, C1-C5 acylamido group, 1- O-a-L-fucosyl,

R ⁴ is selected from a group comprising hydroxygroup, acetamidogroup, C1-C5 acylamidogroup, 1-O- a-L-fucosyl,

R ⁵ and R ⁶ are independently selected from a group comprising hydrogen, sulfo group, 2-O-a-sialyl; while Substituted carbohydrate of formula III contains exactly one bond to Linker as a part of substituent R ¹ .

In some embodiments, the glycopolymer is a glycopolymer based on HPMA polymer containing from 0.5 to 5 mol.% of - structural units of formula I: wherein

Y ¹ is selected from a group comprising C1-C8 alkylene; phenylene; -(CH ₂ ) _q -(C(O)-NH- (CH ₂ ) _r ) _p -, wherein p=1 to 5, and q and r are independently selected from 1, 2, and 3; wherein Y ¹ is optionally substituted with one or more side chains of naturally occurring amino acid wherein the side chains can be identical or different;

Y ² is selected from a group comprising a bond, carbamoyl, carbamoyl-(C1-C8-alkylene), and phenylene; wherein carbamoyl means group -C(=O)-NH- or group -NH-C(=O)-; Linker is selected from a group comprising 1,2,3-triazolylene, (C1-C6 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 3,4,5,13- tetrazatetracyclo[13.4.0.02,6.07,12]nonadeca-1(15),2(6),3,7( 12),8,10,16,18-octaene-13- carbaldehyde bound to the substituent Y ² via the group -C(=O)- or via a covalent bond; Substituted carbohydrate is a substituted carbohydrate of formula III; and/or

- terminal groups of formula -S-succinimide-(CH ₂ ) _r -Linker-Substituted carbohydrate or - C(CN)(CH ₃ )-(C1-C4 alkylene)-Linker-Substituted carbohydrate; wherein r is selected from a group comprising 1, 2 and 3, and Linker and Substituted carbohydrate are defined above; wherein Substituted carbohydrate of formula III is wherein the combination of U, V and W and R4 corresponds to formula Illb or IIIc or Illd wherein in formula Illb: U is 4-O-D-glucopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with R ⁴ group; V and W is a bond; wherein in formula IIIc: U is 3-O-D-galactopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with R ⁴ group; V and W is a bond; wherein in formula Illd: U is 3-O-D-glucopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with R ⁴ group; V and W is a bond; and wherein

R ¹ is selected from a group comprising hydrogen, phenyl, phenyl substituted with at least one halogen atom, 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 with at least one hydroxy group, naphthyl, naphthyl substituted with at least one halogen, biphenyl, carbohydrate, C2-C6 heteroaryl containing at least one heteroatom selected from O, S, N, coumaryl and benzoyl;

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

Y is CH ₂ or a bond,

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

R ² , R ^3u are independently selected from a group comprising hydroxy group, acetamido group, C1-C5 acylamido group, 1-O-a-L-fucosyl,

R ⁴ is selected from a group comprising -NH-bond to Linker, C1-C5 alkoxy with a bond to Linker, a bond to Linker, -O-bond to Linker, phenyloxy group with a bond to Linker on the phenyl moiety;

R ⁵ is selected from a group comprising hydrogen, sulfo group, 2-O-a-sialyl.

In some embodiments, the glycopolymer is a glycopolymer based on HPMA polymer containing from 0.5 to 5 mol.% of - structural units of formula I:

wherein

Y ¹ is selected from a group comprising C1-C8 alkylene; phenylene; -(CH ₂ ) _q -(C(O)-NH- (CH ₂ ) _r ) _p -, wherein p=l to 5, and q and r are independently selected from 1, 2, and 3; whereas Y ¹ is optionally substituted with one or more side chains of naturally occurring amino acid wherein the side chains can be the same or different;

Y ² is selected from a group comprising a bond, carbamoyl, carbamoyl-(C1-C8-alkylene), and phenylene; wherein carbamoyl means the group -C(=O)-NH- or the group -NH-C(=O)-;

Linker is selected from a group comprising 1,2,3-triazolylene, (C1-C6 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 3,4,5,13- tetrazatetracyclo[13.4.0.02,6.07,12]nonadeca-l(15),2(6),3,7( 12),8,10,16,18-octaene-13- carbaldehyde bound to the substituent Y ² via the group -C(=O)- or via a covalent bond; Substituted carbohydrate is a substituted carbohydrate of formula III; and/or - terminal groups of formula -S-succinimide-(CH ₂ ) _r -Linker-Substituted carbohydrate or -

C(CN)(CH ₃ )-(C1-C4 alkylene)-Linker-Substituted carbohydrate; wherein r is selected from the group comprising 1, 2 and 3, and Linker and Substituted carbohydrate are defined above; wherein the Substituted carbohydrate of formula III is wherein the combination of U, V and W and R4 corresponds to formula Ille wherein in formula Ille: U is 4-O-D-glucopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with substituent V, wherein V is 3-O-D-galactopyranosyl substituted at atom C-2 with R ^3v group and at atom C-1 with substituent W, wherein W is 4-O-D-glucopyranosyl substituted at atom C- 2 with R ^3w group and at atom C-1 with R ⁴ group; and wherein

R ¹ is selected from a group comprising hydrogen, phenyl, phenyl substituted with at least one halogen atom, 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 with at least one hydroxy group, naphthyl, naphthyl substituted with at least one halogen, biphenyl, carbohydrate, C2-C6 heteroaryl containing at least one heteroatom selected from O, S, N, coumaryl and benzoyl;

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

Y is CH ₂ or a bond,

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

R ² , R ^3u , R ^3v , R ^3w are independently selected from the group comprising hydroxy group, acetamido group, C1-C5 acylamido group, 1-O-a-L-fucosyl,

R ⁴ is selected from the group comprising C1-C5 alkoxy with a bond to Linker, a bond to Linker, -O- bond to Linker, phenyloxy group with a bond to Linker on the phenyl moiety,

R ⁵ is selected from the group comprising hydrogen, sulfo group, 2-O-a-sialyl.

The process of preparation of gly copolymers according to the invention comprises the following steps: a) provision of monomers for polymer carrier, and optionally of branching units, b) polymerization of monomers (and optionally the branching units) of the polymer carrier, c) optional step of polymer-derived reactions, d) attachment of Substituted carbohydrate.

The step of the provision of monomers includes the provision ofN-(2-hydroxypropyl)methacrylamide (HPMA) and the provision of methacryloyl(aminoacyl) esters, and optionally of branching units. HPMA is commercially available and its synthesis was published (for example Chytil P. et al. Eur. J. Pharm. Sci. 2010, 41 (3-4), 472-482). Commercially available monomers are for example N- aminoethylmethacrylamide, N-aminopropylmethacrylamide, or their tBoc protected analogs.

The synthesis of other functionalized monomers derived from HPMA can be described as follows. The functionalized monomers, methacroylated compounds, have formula II wherein

Y ¹ is selected from a group comprising C1-C8 alkylene; phenylene; -(CH ₂ ) _q -(C(O)-NH- (CH ₂ ) _r ) _p -, wherein p=1 to 5, and q and r are independently selected from 1, 2, and 3; wherein Y ¹ is optionally substituted with one or more side chains of naturally occurring amino acid wherein the side chains can be the same or different;

Y ² is selected from a group comprising a bond, carbamoyl, carbamoyl-(C1-C8-alkylene), and phenylene; wherein carbamoyl means the group -C(=O)-NH- or the group -NH-C(=O)-;

Y ³ is selected from a group comprising primary amine (Nth), tBoc substituted amine, azide, terminal C2-C8 alkynyl, phenyl substituted with at least one azide or C2-C4 alkynyl; and cyclooctynyl or azacyclooctynyl substituted with at least one halogen atom, cyclopropane or conjugated with at least one benzene ring, for example, 11,12-didehydrodibenzo[b,f]azocin- 5(6H)-yl or (1R ,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl, wherein the substituted cyclooctynyl or azacyclooctynyl is bound via a carbonyl group; -carbonyl-thiazoline-2-thione group (TT); - carbonyl-4-nitrophenoxy group; -carbonyl-2, 3, 4, 5, 6-pentafluorophenoxy group; -carbonyl- succinimidyl group; or COOH group; wherein carbonyl is -C(=O)- group.

The branching units are commercially available, in some cases also branching or star-like cores for dendrimer polymer are commercially available.

The next step of the process of the invention is step b), i.e. the step of synthesis of polymer carriers by polymerization of monomers, optionally with branching units. Polymer carrier usually contains a statistical polymer containing 0.5 to 25 mol.% of monomer units of formula II and/or terminal groups of the main polymer chain described above, and at least 75 mol% (75 to 99.5 mol%) of different units, which includes monomer units derived from HPMA and optionally branching units. Typically, polymerization is carried out at the temperature range from 30 to 100 °C, preferably from 40 to 80 °C, in a solvent preferably selected from the group containing water, aqueous buffers, dimethyl sulfoxide, dimethylacetamide, dimethylformamide, methanol, ethanol, dioxane, tert-butyl alcohol and their mixtures. The polymerization is typically initiated by an initiator, preferably selected from the group comprising azoinitiators 2,2'-azobis(2-methylpropionitrile) (AIBN), 4,4'-azobis(4-cyanopentanoic acid) (ACVA), 2,2'-azobis(4-methoxy-2,4-dimethylpentanenitril) (V70), in the presence of a chain transfer agent, preferably selected from the group comprising 2-cyano-2-propylbenzodithioate, 4- cyano-4-(thiobenzoylthio)pentanoic acid, 2-cyano-2-propyldodecyltrithiocarbonate, 2-cyano-2- propylethyltrithiokarbonate and 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulphanyl]pentanoic acid. Molecular weight M _n of these polymers is typically in the range from 4,000 to 100,000 g/mol, preferably from 20,000 to 50,000 g/mol.

Optionally, a step of removal of polymer terminal groups bearing sulfur from the polymers polymerized in the presence of chain transfer agents can be involved. Polymers reacted with the excess of azoinitiators from the group of polymerization initiators described above. Then, the polymer carrier is terminated by the radical residue formed by the initiator disintegration. Typically, the reaction is carried out at temperature between 50 to 100 °C, preferably from 60 to 80 °C, in a solvent preferably selected from the group comprising dimethyl sulfoxide, dimethylacetamide, and dimethylformamide.

Optional step c) includes the introduction of azide or alkynyl group (i) by the reaction of Y ³ group of the polymer when this group is a leaving group (i.e. it contains a thiazoline-2-thione group (TT), 4- nitrophenoxy group, 2,3,4,5,6-pentafluorophenoxy group, succinimidyl group, or OH group) with an amino-group containing compound (e.g. amino(C1-C8 alkane)) terminated by a group selected from azide, ethynyl, phenyl substituted by at least by one azide, C2-C5 terminal alkynyl, or substituted cyclooctyne, preferably 3-amino-1-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)propan -1-one, or N- [(1R,8S,9)-bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8 -diamino-3,6-dioxaacetate; or (ii) by the reaction of Y ³ group of the polymer when this group is a primary amino group with carboxy(C1- C8 alkane) terminated with a group selected from azide, ethynyl, phenyl substituted by at least by one azide, C2-C5 terminal alkynyl, or substituted cyclooctyne, preferably 6-(11,12- didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoic acid, or its N-hydroxysuccinimidyl ester, or(1R,8S,9)-bicyclo[6.1 0]non-4-yn-9-yl methyl N-succinimidyl carbonate or functional derivatives of this carboxyalkane containing readily leaving group formed preferably by TT, 4-nitrophenoxy, 2,3,4,5,6-pentafluorophenoxy, or succinimidyl group. These polymer-derived reactions are typically carried out at laboratory temperature, in a solvent preferably selected from a group comprising dimethyl sulfoxide, dimethylacetamide, dimethylformamide, methanol, and ethanol.

In a preferred embodiment, the polymer carrier further contains 0.5 to 12 mol% of further structural units derived from formula II, wherein Y ¹ and Y ² are defined above, and Y ³ is selected from a group comprising carbonyl-hydrazono-(C12-C18 alkanone), carbonyl-hydrazono-5a-cholestanone, carbonyl- hydrazono-cholest-4-en-3-one, or a substituent derived from keto-derivatives of cholesterol; or Y ³ is selected from a group comprising carbonyl-(C9-C15 alkoxy), carbonyl-cholesteryl, or cholesterol- derived compounds such as 7-dehydrocholesterol, and vitamin D; or Y ³ is selected from a group comprising carboxamido-(C10-08 alkyl), carbohydrazido-(C10-08 alkyl), carboxamido-(C10-08 alkenyl), carbohydrazido-(C10-08 alkenyl), containing at least one double bond (C=C), preferably derived from oleic acid, linoleic acid or linolenic acid.

Polymer carriers can have a linear, branched, or cross-linked structure in general.

In case of the linear structure, the polymer carrier usually contains 0.5 to 25 mol% of monomer units of formula II and/or terminal groups described above, and at least 75 mol% of HPMA monomer units. In case of the branched or cross-linked structure, the polymer carrier usually contains 0.5 to 25 mol% of monomer units of formula II and/or terminal groups described above, and at least 75 mol% of HPMA monomer units and branching units.

In some embodiments of polymer carriers having a branched structure, only a part of functional groups suitable for the attachment of substituted carbohydrates is located along the polymer chain, or monomers of formula II may not be present at all. The functional groups suitable for the attachment of substituted carbohydrates, or at least part thereof, can be formed by the terminal groups located at one end of HPMA arms, whereas the second polymer end is bound to a multivalent molecule, e.g. dendrimer, e.g. PAMAM dendrimer core, or core based on 2,2-bis(hydroxymethyl)propionic dendrimer. The first step of preparation in these embodiments is the polymerization of HPMA, which is typically carried out at temperatures in the range from 30 to 100°C, preferably from 40 to 80 °C, in a solvent preferably selected from the group comprising water, aqueous buffers, dimethyl sulfoxide, dimethylacetamide, dimethylformamide, methanol, ethanol, dioxane, tert-butyl alcohol and their mixtures, initiated by an initiator, preferably selected from the group of azoinitiators AIBN, ACVA, V70, in the presence of a chain transfer agent, preferably containing carboxylic group or its functional derivative and containing readily leaving group formed preferably by TT, 4-nitrophenoxy, 2, 3, 4,5,6- pentafluorophenoxy, or succinimidyl group; wherein the chain transfer agent is preferably selected from a group comprising 4-cyano-4-(thiobenzoylthio)pentanoic acid, 4-cyano-4- [(dodecylsulfanylthiocarbonyl)sulphanyl]pentanoic acid, 1-cyano-1-methyl-4-oxo-4-(2- thioxothiazolidin-3-yl) butyl dithiobenzoate, or 2-cyano-5-oxo-5-(2-thioxo-1,3-thiazolidin-3-yl)pentan- 2-yl ethylcarbontrithioate. In the second step, the polymers are bound via an amide bond to a multivalent compound bearing terminal primary amino groups, preferably selected from poly(amidoamine) (PAMAM), and 2,2-bis(hydroxymethyl)propionic acid-based dendrimer, wherein the reaction is usually carried out in a solvent preferably selected from a group comprising dimethylsulfoxide, dimethylacetamide, dimethylformamide, methanol, and ethanol. The dendrimer can form up to 3 mol% of the resulting polymer conjugate. Subsequently, the remaining amino groups are blocked in situ, using a low-molecular amino-reactive compound, preferably using acetic anhydride. In the third step, the sulphur-containing groups located at the opposite polymer end are either reduced by sodium borohydride followed by the addition of SH groups to N-derived maleimide compound, preferably propynylmaleimide, or azido-PEG3 -maleimide, wherein the reaction is carried out in a solvent preferably selected from a group comprising dimethyl sulfoxide, dimethylacetamide, dimethylformamide, methanol, and ethanol. Alternatively, the sulphur-containing groups are reacted with the excess of azoinitiators bearing carboxylic acid, or its functional derivatives containing readily leaving group comprising preferably TT, 4-nitrophenoxy, 2,3,4,5,6-pentafluorophenoxy, or succinimidyl group, at temperatures in the range from 50 to 100°C, preferably from 60 to 80 °C, in a solvent preferably selected from a group comprising dimethyl sulfoxide, dimethylacetamide, and dimethylformamide. Molecular weight M _n of these polymers is typically in the range from 60,000 to 1,000,000 g/mol, preferably from 100,000 to 400,000 g/mol.

The final step d) of the preparation is attachment of Substituted carbohydrate selected from group comprising substituted carbohydrates of formula IIIp: wherein the combination of U, V and W and R4 is selected from following options Ilia, Illb, IIIc, Illd, Ille:

IlIa: wherein V and W is a bond, and wherein U is 1 -thio -b-D-galactopyranosyl substituted at atom C- 2 by R ⁴ group, at atom C-3 with a moiety -Z-Y-X-R ¹ and at atom C-6 with -OR ⁶ group;

Illb: wherein V and W is a bond and wherein U is 4-O-D-glucopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with R ⁴ group;

IIIc: wherein V and W is a bond and wherein U is 3-O-D-galactopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with R ⁴ group; Illd: wherein V and W is a bond and wherein U is 3-O-D-glucopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with R ⁴ group; Ille: wherein U is 4-O-D-glucopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with substituent V, wherein V is 3-O-D-galactopyranosyl substituted at atom C-2 with R ^3v group and at atom C-1 with substituent W, wheinre W is 4-O-D-glucopyranosyl substituted at atom C-2 with R ^3w group and at atom C-1 with R ⁴ group; and wherein

R ¹ is selected from a group comprising hydrogen, phenyl, phenyl substituted with at least one halogen atom, phenyl substituted with at least one nitro group, phenyl substituted with at least one carboxy group, phenyl substituted with at least one C1-C5 alkoxy group, phenyl substituted with at least one C2-C5 alkynyl, phenyl substituted with at least one C1-C5 azidoalkyl, phenyl substituted with at least one azidogroup, 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 with at least one amino group, phenyl substituted with at least one C1-C5 aminoalkyl group, phenyl substituted with at least one hydroxy group, naphthyl, naphthyl substituted with at least one halogen, biphenyl, carbohydrate, C2-C6 heteroaryl containing at least one heteroatom selected from O, S, N, sulfo group, coumaryl, benzoyl, C2-C5 alkynyl;

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

Y is CH ₂ or a bond,

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

R ² , R ^3u , R ^3v , R ^3w are independently selected from a group comprising hydroxy group, acetamido group, C1-C5 acylamido group, 1-O-a-L-fucosyl,

R ⁴ is selected from a group comprising hydroxy group, amino group, C2-C5 alkynyloxy group, azide, C1-C5 azidoalkoxy group, aminoethylureidyl, aminoethylthioureidyl, aminophenyloxy group and azidophenyloxy group,

R ⁵ and R ⁶ are independently selected from a group comprising hydrogen, sulfo group, 2-O-a-sialyl, C1-C5 azidoalkyl, C2-C5 alkynyl and C1-C5 aminoalkyl; wherein the substituted carbohydrate of formula IIIp contains exactly one terminal amino group, azido group or C2-C5 alkynyl, preferably as a part of substituent selected from R ¹ , R ⁵ or R ⁶ if U is 1 -thio -b- D-galactopyranosyl, and preferably as a part of substituent R ⁴ in all other combinations.

In formulas (III) and (IIIp):

The carbohydrate in substituent R ¹ is preferably a monosaccharide, more preferably 1-O-a-D- galactopyranosyl or 2-O-a-sialyl.

When V and W is a bond and U is 1 -thio -b-D-galactopyranosyl substituted at atom C-2 with R ⁴ group, at atom C-3 with the moiety -Z-Y-X-R ¹ and at atom C-6 with -OR ⁶ group (option Ilia), preferably R ² is hydroxy group and R ⁴ is also hydroxy group.

When V and W is a bond and U is 4-O-D-gIucopyranosyl substituted at atom C-2 with R ^3u group and at atom C-1 with R ⁴ group (option Illb), preferably R ² is selected from the group comprising hydroxy group and acetamido group and R ^3u is acetamido group.

When V and W is a bond and U is 3-O-D-gaIactopyranosyI substituted at atom C-2 with R ^3u group and at atom C-1 with R ⁴ group (option IIIc), preferably R ² is hydroxy group and R ^3u is acetamido group. When V and W is a bond and U is 3-O-D-gIucopyranosyI substituted at atom C-2 with R ^3u group and at atom C-1 with R ⁴ group (option Illd), preferably R ² is hydroxy group and R ^3u is acetamido group. When U is 4-O-D-gIucopyranosyI substituted at atom C-2 with R ^3u group and at atom C-1 with substituent V, wherein V is 3-O-D-gaIactopyranosyI substituted at atom C-2 with R ^3v group and at atom C-1 with substituent W, wherein W is 4-O-D-gIucopyranosyI substituted at atom C-2 with R ^3w group and at atom C-1 with R ⁴ group (option Ille), preferably R ² , R ^3v and R ^3w are independently selected from the group comprising hydroxy group and acet amido group and R ^3u is acetamido group.

Substituted carbohydrates are bound either by amide bond or via azide-alkyne cycloaddition, the so called „click“ reaction (forming Linker from the group Y ³ using substituent of the substituted carbohydrate comprising terminal amine, azide, alkyne, aminoethylureidyl or aminoethylthioureidyl group, typically substituent R ¹ , or R ⁴ , or R ⁵ , or R ⁶ ) to polymer carriers. Aminolytic reaction is usually carried out in an aprotic solvent, preferably selected from a group comprising dimethyl sulfoxide, dimethylacetamide and dimethylformamide. The cycloadition reaction is preferably catalyzed, preferably using copper(I) ionts, or can be catalyst-free. This reaction is usually carried out in water, or in solvent mixtures comprising water, aqueous buffer, alcohol and aprotic solvent, preferably selected from a group comprising dimethyl sulfoxide, dimethylacetamide and dimethylformamide or their mixtures. In case of Cu-catalyzed cycloaddition reaction, the reaction mixture contains Cu(I) or Cu(II) cations, preferably due to the presence of CuSCL or CuBr, and a reduction agent, preferably ascorbic acid or sodium ascorbate. In all cases, preferable purification procedure consists in gel filtration using a column (e.g. filled with Sephadex G-25) in water, eventually followed by purification from copper cations, preferably using complexation with 8-quinoIinoI, and then by another gel filtration using a column (e.g. filled with Sephadex LH-20) in methanol as a mobile phase. An important benefit of the invention is an appropriate presentation of the substituted carbohydrate on the polymer carrier wherein the content the the substituted carbohydrate in the glycopolymer ranges from 0.5 to 25 mol%, preferably from 3 to 18 mol%. The multivalent presentation of substituted carbohydrates on the polymers enables multivalent interaction with Gal-3, and thus significantly increases binding activity to Gal-3 and to all galectins in general.

Object of the invention is further an immunomodulatory effect of the polymer conjugates with substituted carbohydrates due to the binding to Gal-3 by the multivalent interaction described above. The application of polymer carriers with carbohydrates results in un-binding of extracellular Gal-3 from tumor tissue, thereby inhibiting Gal-3-induced T lymphocyte apoptosis in the tumor, which in turn enables the enhancement of the immune response against tumor cells directly inside the tumor. The binding of the polymer carriers with substituted carbohydrates to Gal-3 directly produced by tumor cells further leads to a decrease of the tumor cell migration. In a number of types of tumor cells, Gal-3 has the ability to increase the probability of metastasis formation through inhibition of cell-to-substrate and cell-to-cell binding. Thus, the application of the polymeric conjugates with substituted carbohydrates leads to a decrease in cell motility and cell migration in the metastatic sites, via binding to Gal-3.

A further object of the invention is a pharmaceutical composition, which is characterized by the composition comprising at least one glycopolymer according to the invention, said glycopolymer containing a substituted carbohydrate and at least one pharmaceutically acceptable excipient selected from the group comprising fillers, antiadhesives, binders, coating agents, coloring agents, disintegrants, flavorings, lubricants, preservatives, sweeteners, absorbents.

A yet further of the invention is a glycopolymer according to the invention and/or its pharmaceutical composition for use as a medicament for the treatment of solid tumors and/or lymphomas and/or leukemias, in particular colorectal carcinoma, prostate carcinoma, breast carcinoma, melanoma, lymphoma, leukemia.

An object of the invention is also a glycopolymer according to the invention and/or its pharmaceutical composition for the use as an adjuvant for antitumor therapy.

The invention further provides a method of treatment of solid tumors and/or lymphomas and/or leukemias, in particular colorectal carcinoma, prostate carcinoma, breast carcinoma, melanoma, lymphoma, leukemia, said method comprising administering at least one glycopolymer according to the invention to a subject in need of such treatment.

Brief Description of Drawings

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

Fig. 2: Inhibition of galectin-3 -induced apoptosis of Jurkat cells by conjugate P11d (the content of substituted carbohydrate 20 was 9.7 mol%).

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

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

Fig. 5: Inhibition of galectin-3-induced apoptosis of Jurkat cells by conjugate P14 (the content of substituted carbohydrate 23 was 9.5 mol%).

Fig. 6: Inhibition of galectin-3-induced apoptosis of Jurkat cells by conjugate P15 (the content of substituted carbohydrate 26 was 7.3 mol%).

Fig. 7: Inhibition of galectin-3-induced apoptosis of Jurkat cells by conjugate P16 (the content of substituted carbohydrate 27 was 4.7 mol%).

Fig. 8: Inhibition of galectin-3 -induced apoptosis of Jurkat cells by conjugate HPMA with simple disaccharide LacdiNAc (content of LacdiNAc was 12.3 mol%).

Fig. 9: Inhibition of migration and proliferation of murine (4T1, B16-F10) and human (DLD-1) tumor cells by conjugates Plla and P11c.

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)prop-2- enamide; 3-(3-methacrylamidopropanoyl)thiazolidine-2-thione; MA group, N-methacryloyl group; tBoc group, tert-butoxycarbonyl group; TT group, thiazoline-2-thione group; TBAB, tetra-n- butylamoniumbromide; TEA, triethylamine; THPTA, tris-hydroxypropyltriazolylmethylamine ligand; V70, 2,2'-azobis(4-methoxy-2,4-dimethylpentanenitrile); LacdiNAc (Ga1NAcb4G1cNAc); LacNAc (Ga1b4G1cNAc); lactose (Ga1b4G1c), 4T1 (murine mammary carcinoma cell line); B16-F10 (murine melanoma cell line); CT26 (murine colorectal carcinoma 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-lymphoma cell line); LNCaP (human prostate adenocarcinoma cell line); OVCAR-3 (human ovarian adenocarcinoma cell line); PC3 (human prostate adenocarcinoma cell line); Raji (human Burkitt's lymphoma cell line); SU-DHL-5 (human B- lymphoma cell line); SU-DHL-6 (human B-lymphoma cell line);

Examples of carrying out the invention Example 1 : Synthesis of monomers N-(2-hydroxypropyl)methacrylamide (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): d 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). N-Methacryloylpropynyl amine (MA-propynyl) was prepared according to the previously described procedure (Lynn G.M. et al., Biomacromolecules, 2019, 20 (2), 854—870). The product was chromatographically pure. ¹ H-NMR (300 MHz, (CD ₃ ) ₂ SO, 296 K): d 1.85 (s, 3H, CH ₃ ), 3.05 (s, 1H, ºCH), 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): d 1.20-1.27 (m, 2H, CH ₂ -g), 1.40-1.54 (m, 4H, CH ₂ -b, CH ₂ -d), 1.82 (s, 3H, CH ₃ ), 2.28 (t, 2H, CH ₂ - a), 3.04-3.34 (m, 2H, CH ₂ -e), 3.57 (s, 3H, OCH ₃ ), 5.28 and 5.60 (d, 2H, CH ₂ =), 7.88 (br, 1H, NH).

Cholest-5en-3b-yl 6-methacrylamido hexanoate (MA-AH-chol) was prepared according to the previously described procedure (Chytil P. et al. J. Controlled Release 2008, 127/2, 121-130). The product was chromatographically pure. Melting point 98 - 100 °C, Elemental analysis: Calculated: C 78.25 %, H 10.83 %, N 2.47 %; Found C 78.73 %, H 10.85 %, N 2.34 %, ¹ H-NMR (CDC1 ₃ ): d 5.81 br, 1H (NH); d 5.65 and 5.29 d, 2H (CH ₂ =C(CH ₃ )CO); d 4.58 m, 1H (CO-O-CH-(CH ₂ ) ₂ ); d 3.30 m, 1H (CH ₂ -NH); selected intensities from the cholesterol part of molecule: d 5.35 t, 1H (C=CH-CH ₂ ); d 0.66 s, 3H (C(18)H ₃ ).

Example 2: Synthesis of statistical copolymer poly(HPMA-co-MA-AP-TT) by radical polymerization

(P1)

833 mg of HPMA (5.82 mmol), 167 mg of MA-AP-TT (0.646 mmol) and AIBN (160 mg; 0.974 mmol) were dissolved in 6.2 mL dimethyl sulfoxide. The reaction mixture was poured into a glass ampoule, bubbled with argon and sealed. After 6 h in a thermostat-controlled water bath at 60°C, the ampoule was cooled, and the reaction mixture was poured into an excess of acetone (150 mL). The polymer was filtered off and purified by re-precipitation from methanol (6 mL) into an excess of acetone (150 mL). The polymer was isolated by filtration and dried under vacuum. Yield 850 mg, 85 %; molecular weights M _w = 23,900 g/mol, M _n = 12,100 g/mol, D = 1.98.

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

800 mg of HPMA (5.59 mmol) was dissolved in terbt-utanol (5.52 mL) and mixed with a solution of 160 mg of MA-AP-TT (0.621 mmol), 4.97 mg of AIBN (17.7 mmol), and 7.84 mg of 2-cyanopropan-

2-yl dithibenzoate (35.5 mmol) in 1.38 mL of dimethyl sulfoxide. The reaction mixture was poured into a glass ampoule, bubbled with argon and sealed. After 16 h in a thermostat-controlled water bath at 70 °C, the ampoule was cooled, and the reaction mixture was poured into an excess of acetone (150 mL). The polymer was filtered off and purified by reprecipitation from methanol (6 mL) into an excess of a mixture of acetone and diethyl ether (3:1; 120 mL). The polymer was isolated by filtration and dried under vacuum. Yield 730 mg, 76 %; molecular weights M _w = 22,900 g/mol, M _n = 20,600 g/mol, D = 1.11.

Example 4: Removal of polymer terminal groups originated from transfer agents 700 mg of polymer P2 and 70 mg of AIBN were dissolved in 5 mL dimethyl sulfoxide, poured into a glass ampoule, bubbled with argon and sealed. After 2 h in a thermostat-controlled water bath at 80 °C, the ampoule was cooled and opened. The polymer was isolated by precipitation into a mixture of 150 mL acetone and purified by reprecipitation from methanol (6 mL) into an excess of a mixture of acetone and diethyl ether (3:1; 120 mL). The polymer was filtered off and dried under vacuum. The yield of polymer was 621 mg.

Example 5: Synthesis of statistical copolymer poly(HPMA-co-MA-propynyl) by controlled RAFT radical polymerization (P3) 264 mg of HPMA (1.84 mmol) and 12 mg of N-methacryloylpropynylamine (MA-propynyl) (97.0 mmol) were dissolved in 1.3 mL of distilled water and mixed with a solution of 1.3 mg ACVA (4.7 mmol) and 2.6 mg of 4-cyano-4-(thiobenzoylthio)pentanoic acid (9.5 mmol) dissolved in 0.65 mL of dioxane. The reaction mixture was poured into a glass ampoule, bubbled with argon and sealed. After 7 h in a thermostat-controlled water bath at 70 °C, the ampoule was cooled, and the reaction mixture was poured into an excess of acetone (150 mL) and centrifuged. The purification was carried out by gel filtration using Sephadex LH-20 in methanol. The polymer was filtered off and purified by reprecipitation from methanol (6 mL) into an excess of a mixture of acetone and diethyl ether (3:1; 120 mL). The polymer was isolated by precipitation into diethyl ether, filtered off and dried under vacuum. Yield 127 mg, 46 %; molecular weights M _w = 22,900 g/mol, M _n = 20,600 g/mol, D = 1.11.

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

600 mg of the polymer precursor (containing 0.54 mmol TT groups) was dissolved in 6 mL of dimethylformamide and 40 mL of propynylamine (0.65 mmol) and 108 mL of N-ethyldiisopropyl amine (0.65 mmol) were added under stirring at room temperature. The reaction was carried out for 16 h at room temperature. The polymer was purified from low-molecular-weight compounds by gel filtration using Sephadex LH-20 in methanol. Polymer was isolated by precipitation into an excess of acetone (120 mL). Yield 560 mg; molecular weight: M _w = 21,800 g/mol, M _n = 20,200 g/mol, D = 1.08.

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

500 mg of HPMA (3.49 mmol) was dissolved in 3.4 mL of terbt-utanol and mixed with a solution of 2.8 mg of 2-[1-cyano-1-methyl-4-oxo-4-(2-thioxo-thiazolidin-3-yl)-buty lazo]-2-methyl-5-oxo-5-(2- thioxothiazolidin-3-yl)-pentanenitrile (5.4 mmol) and 4.4 mg of 1-cyano-1-methyl-4-oxo-4-(2- thioxothiazolidin-3-yl) butyl ester of dithiobenzoic acid (10.7 mmol) in 0.85 mL of dimethyl sulfoxide. The reaction mixture was poured into a glass ampoule, bubbled with argon and sealed. After 6 h in a thermostat-controlled water bath at 70°C, the ampoule was cooled, and the reaction mixture was poured into an excess of acetone (120 mL). The polymer was filtered off and purified by reprecipitation from methanol (6 mL) into an excess of a mixture of acetone and diethyl ether (3:1; 120 mL). The polymer was isolated by filtration and dried under vacuum. Yield 345 mg, 69 %; molecular weights M _w = 29,800 g/mol, M _n = 27,100 g/mol, D = 1.10.

340 mg of polymer precursor (containing 11.2 mmol terminal TT groups) was dissolved in 2.2 mL of dimethyl sulfoxide and added to 20 wt% methanol solution of 1.5 mg of PAMAM dendrimer (1.4 mmol dendrimer G2 with diaminobutane core). The reaction was carried out at room temperature under stirring and quenched after 1.5 h by the addition of 50 mL of acetic anhydride. The polymer was purified from low-molecular-weight compounds by gel filtration using Sephadex LH-20 in methanol. The polymer was isolated by precipitation into an excess of acetone (120 mL), filtered off and dried under vacuum. The yield of the conjugation reaction was 85 % of star copolymer.

100 mg of the star polymer precursor was dissolved in 1 mL of methanol. 10 mg of solid sodium borohydride was added under stirring. After 1 h at room temperature, 10 mg of propynylmaleimide dissolved in 0.2 mL of methanol was added in situ. After another 1 h of reaction, the polymer was purified from low-molecular-weight compounds by gel filtration using Sephadex LH-20 in methanol. Polymer was isolated by precipitation into an excess of acetone (25 mL), filtered off and dried under vacuum. Molecular weight: M _w = 210,000 g/mol, M _n = 210,000 g/mol, D = 1.19. In a similar way, samples with the 2,2-bis(hydroxymethyl)propionic acid-based dedrimer were prepared.

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

500 mg of HPMA (3.49 mmol) was dissolved in 3.4 mL of tert-utanol and mixed with a solution of 2.8 mg of 2-[1-cyano-1-methyl-4-oxo-4-(2-thioxo-thiazolidin-3-yl)-buty lazo]-2-methyl-5-oxo-5-(2- thioxothiazolidin-3-yl)-pentanenitrile (5.4 mmol) and 4.4 mg of 1-cyano-1methyl-4-oxo-4-(2- thioxothiazolidin-3-yl) butyl ester of dithiobenzoic acid (10.7 mmol) in 0.85 mL of dimethyl sulfoxide.

The reaction mixture was poured into a glass ampoule, bubbled with argon and sealed. After 6 h in a thermostat-controlled water bath at 70 °C, the ampoule was cooled, and the reaction mixture was poured into an excess of acetone (120 mL). The polymer was filtered off and purified by reprecipitation from methanol (6 mL) into an excess of a mixture of acetone and diethyl ether (3:1; 120 mL). The polymer was isolated by filtration and dried under vacuum. Yield 345 mg, 69 %; molecular weights M _w = 29,800 g/mol, M _n = 27,100 g/mol, D = 1.10.

340 mg of polymer precursor (containing 11.2 mmol terminal TT groups) was dissolved in 2.2 mL of dimethyl sulfoxide and added to 20 wt% of methanol solution of 1.5 mg of PAMAM dendrimer (1.4 mmol dendrimer G2 with diaminohutane core). The reaction was carried out at room temperature under stirring and quenched after 1.5 h by the addition of 50 mL of acetic anhydride. The polymer was purified from low-molecular-weight compounds by gel filtration using Sephadex LH-20 in methanol. The polymer was isolated by precipitation into an excess of acetone (120 mL), filtered off and dried under vacuum. The yield of the conjugation reaction was 85 % of star copolymer. 100 mg of the star polymer precursor and 10 mg of 2-[1-cyano-1-methyl-4-oxo-4-(2-thioxo- thiazolidin-3-yl)-butylazo]-2-methyl-5-oxo-5-(2-thioxothiazo lidin-3-yl)-pentanenitrile were dissolved in 0.7 mL of dimethyl sulfoxide, poured into a glass ampoule, bubbled with argon and sealed. After 3 h in a thermostat-controlled water bath at 70 °C, the ampoule was cooled and opened. The polymer was isolated by precipitation into a mixture of 25 mL of acetone and purified by reprecipitation from methanol (1 mL) into an excess of a mixture of acetone and diethyl ether (3:1; 25 mL). The polymer was filtered off and dried under vacuum. Molecular weight: M _w = 205,000 g/mol, D = 1.20. Similarly, samples with the 2,2-bis(hydroxymethyl)propionic acid-based dendrimer were prepared. Example 9: Synthesis of statistical copolymer poly(HPMA-co-MA-AP-azadibenzocyclooctyne) by conjugation with azadibenzocyclooctynamine with poly(HPMA-co-MA-AP-TT) (P7)

200 mg of the polymer precursor (containing 0.11 mmol TT groups) was dissolved in 2 mL of dimethylformamide. 32 mg of azadibenzocyclooctynamine (0.12 mmol) dissolved in 0.1 mL of dimethylformamide and 22 mL of N-ethyldiisopropylamine (0.13 mmol) were added under stirring at room temperature. The reaction was carried out for 16 h at room temperature. The polymer was purified from low-molecular-weight compounds by gel filtration using Sephadex LH-20 in methanol. Polymer was isolated by precipitation into an excess of acetone (50 mL), filtered off and dried under vacuum. Yield 184 mg; molecular weight: M _w = 28,000 g/mol, M _n = 25,200 g/mol, D = 1.11.

Example 10: Synthesis of statistical copolymer poly(HPMA-co-MA-AP-propylazide) by conjugation with 3 -azido-1 -propylamine with poly(HPMA-co-MA-AP-TT) (P8)

300 mg of the polymer precursor P2 was dissolved in 3.5 mL of methanol. 40 mL of 3-azido-1- propylamine (0.40 mmol) was added under stirring at room temperature. Then, 92 mL of N- ethyldiisopropylamine (0.53 mmol) was slowly dropped within 30 min. After 20 h at room temperature, 40 mL of 1-aminopropan-2-ol (0.29 mmol) was added and stirred for another 30 min. The polymer was purified from low-molecular-weight compounds by gel filtration using Sephadex LH-20 in methanol. Polymer was isolated by precipitation into an excess of acetone (50 mL), filtered off and dried under vacuum. Yield 184 mg; molecular weight: M _w = 23,700 g/mol, M _n = 21,100 g/mol, D = 1.1. The content of azide groups was 16.7 mol%.

Example 11: Synthesis of 3'-O-[4-(azidomethyl)benzyl]--D-galactopyranosyl-(1® 1-3-O-(4-{[4-

(bromomethyl)-1H-1,2,3-triazol-1-yl]methyl}benzyl)-1-thio -b-D-galactopyranoside (3)

Starting compound b-D-galactopyranosyl-(1® 1)-1-thio -D-galactopyranoside (1) reacted in presence of dibutyltinoxide (Bu2SnO) with an excess of bromide to form selectively disubstituted C-3, C-3’ compound 2. Reaction took place in the presence of phase-transfer catalyst tetra-n-butylammonium bromide (TBAB) and N,N-diisopropylethylamine (DIPEA) in dry dioxane at increased temperature (82 - 84 °C). Following was the cycloaddition reaction between terminal azido group and propargyl bromide (0.5 eq.) catalyzed by Cul in the presence of tris-hydroxypropyltriazolylmethylamine ligand (THPTA) that yielded compound 3.

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

Disacharide 6 was prepared by chemoenzymatic synthesis from acceptor 2-azidoethyl 2-acetamido-2-deoxy-b-D-glucopyranoside (5) and donor p-nitrophenyl 2-acetamido-2-deoxy-b-D-galactopyranoside (4) catalyzed by mutant Tyr470His b-N-acetylhexosaminidase from Talaromyces flavus (Tyr470His TfHex) (Bojarova P. et al. J Nanobiotechnol. 2018, 16, 73). Benzyl group was selectively installed at CP-3 position by reaction of 6 with benzylbromide in the presence of dibutyltin oxide (Bu ₂ SnO) to form substituted carbohydrate 7.

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

Starting compound b-D-galactopyranosy-(1® 1-)-1-thio-b-D-alactopyranoside (1) reacted in the presence of tin complex (Bu ₂ SnO) with an excess of propargyl bromide to form selectively disubstituted C-3, C-3’ compound 8. Reaction took place in the presence of phase-transfer catalyst (TBAB) in dry dioxane at elevated temperature (82 - 94 °C). Second reaction step based on Cu(I)- catalyzed azide-alkyne cycloaddition with 4-bromophenyl azide and THPTA as an additive was performed in a mixture of tert-butyl alcohol and water to give compound 9.

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

Starting disaccharide 12 was prepared by chemoenzymatic synthesis from acceptor ( tert - butoxycarbonylamino)ethylthioureidyl 2-acetamido-2-deoxy-b-D-glucopyranoside (11) (Bojarova P. et al. Molecules 2019, 24, 599) and donor p-nitrophenyl b-D-galactopyranoside (10) under catalysis of recombinant b3-galactosidase from Bacillus circulans. Disacharide 12 reacted in the presence of dibutyltinoxidu (Bu ₂ SnO) with an excess of propargylbromide to form selectively C-3' substituted compound 13. Reaction took place in the presence of phase-transfer catalyst (TBAB) in dry dioxane at elevated temperature (82 - 94 °C). Following reaction step based on Cu(I)-catalyzed azide-alkyne cycloaddition between terminal alkyne 13 and 4-bromophenyl azide and THPTA as an additive was performed in a mixture of tert- butyl alcohol and water to give compound 14. Following amino group deprotection using 1M HC1 at 4 °C for 48 h gave compound 14.

Example 15: Synthesis of b-D-alNA-(1®4)-b-D- lcNA-(1®3)-b-D-a-(1®4)-b-D-lcNAc-1- O-(2-aminoethylthioureidyl) (LacdiNAc-LacNAc-linker-NH ₂ ; 20) Starting disaccharide 16 was prepared by chemoenzymatic synthesis from acceptor ( tert - butoxycarbonylamino)ethylthioureidyl 2-acetamido-2-deoxy-b-D-glucopyranoside (11) (Bojarova P. et al. Molecules 2019, 24, 599) and donor p-nitrophenyl b-D-galactopyranoside (10) under catalysis of recombinant b4-galactosidase from Bacillus circulans . Disacharide 16 was used as an accceptor for the glycosylation with b-D-GlcNAc unit under the catalysis of recombinant b-N-acctylhcxosaminidasc BbhI from Bifidobacterium bifidum, selective for b(1 3) bond formation. The prepared trisaccharide 18 was further used as an akceptor for glycosylation b-D-GalNAc under the catalysis by selective mutant b-N-acetylhexosaminidase from Talaromyces flavus to afforf tetrasacharide 19. Susequent deprotection of the amino group of tetrasaccharide 19 under the formation of compound 20 was performed in 1M HC1 at 4 °C for 48 h. Alternatively, i tis possible to prepare this compound using recombinant glykosylu transferases according to the procedures described in the literature (Laaf D. et al. Bioconjug. Chem. 2017, 28, 2832-2840).

Example 16: Synthesis of 3-O-benzyl-2-deoxy-b-D-galactopyranosyl-(1 4)-2-acetamido-2-deoxy-b- D-glucopyranosyl azide (23) Disacharide 22 was prepared by chemoenzymatic synthesis from 2-acetamido-2-deoxy-b-D- glucopyranosyla zide (21) as an acceptor and p-nitrophenyl 2-deoxy-b-D-galactopyranoside (10) as a donor under the catalysis by b4-galactosidase from Bacillus circulans (Tavares M. R. et al. Biomacromolecules 2020, 21, 2, 641-652). Benzyl group was selectively installed onto C'-3 position by reacting 22 with benzylbromide in the presence of dibutyltin oxide (Bu ₂ SnO) to form substituted carbohydrate 23. Example 17: Synthesis of 3-O-(coumarylmethyl)-b-D-galactopyranosyl-(1 4)-b-D-glucopyranosyl azide (26)

Lactose (24) was peracetylated in mixture of acetic annhydride and dry pyridine. Peracetate formed was then brominated with HBr/ AcOH at 0 °C in dry dichloromethane. Azido group was installed onto C-1 using NaN while phase-transfer catalyzed by tetrabutylammonium hydrogensulfate. Silica column chromatography followed by Zemplen deprotection gave rise to b-D-lactosyl azide 25.

Azide 25 reacted in presence of dibutyltin oxide (Bu SnO) with excess of 3- (bromomethyl)coumarine to form selectively substituted C-3’ disacharide 26. Reaction took place in the presence of phase-transfer catalyst tetra-n-butylammonium bromide (TBAB) and N,N- diisopropylethylamine (DIPEA) in dry dioxane at elevated temperature (86 °C).

Example 18: Synthesis of 3-O-propynyl-b-D-galactopyranosyl-(1® 1)3-O-[(4-bromophenyl)-1H- (1,2,3 -triazol-4-yl) methyl] - b -D-galactopyranoside (27)

Starting compound b-D-galactopyranosyl(1® 1)-1-thio-b-D-galactopyranoside (1) reacted in the presence of tin complex (Bu ₂ SnO) with an excess of propargyl bromide to form selectively disubstituted C-3, C-3’ compound 8. Reaction took place in the presence of phase-transfer catalyst (TBAB) in dry dioxane at elevated temperature (82 - 94 °C). Second reaction step based on Cu(I)- catalyzed azide-alkyne cycloaddition with t-butylphenyl azide and triethylamine (TEA) as an additive was performed in a mixture of tert-butyl alcohol and water to give compound 27. Example 19: Synthesis of conjugate of substituted carbohydrate 15 with poly(HPMA-co-MA-AP-TT)

(P9)

25 mg of the polymer precursor P2 (containing 14.6 mmol TT groups) and 9.2 mg of compound 15 (13.1 mmol) were dissolved in 0.8 mL dimethylacetamide and bubbled by argon. After the addition of

2.3 mL N-ethyldi isopropyl amine (13.1 mmol), the reaction mixture was stirred at room temperature for 20 h. Then, 1.1 mL of 1-aminopropan-2-ol (14.6 mmol) was added and stirred for 1 h. The polymer conjugate was purified from low-molecular-weight compounds by gel filtration using Sephadex LH- 20 in methanol. Methanol was removed from the polymer fraction by vacuum distillation, the polymer was dissolved in water and isolated by freeze -drying. Yield 25.4 mg; molecular weight: M _w = 25,400 g/mol, M _n = 23,600 g/mol, D = 1.08. The content of the carbohydrate in the conjugate was 5.0 mol%.

Example 20: Synthesis of conjugate of substituted carbohydrate 3 with poly(HPMA-co-MA-AP- propynyl) (P10) 2 mg CuSO ₄ ·5H ₂ O (16 mmol) dissolved in 25 mL of distilled water was added to the solution of 25 mg poly(HPMA-co-MA-AP- propynyl) , P4, (containing 17.4 m mol propynyl groups), 1.6 mg sodium ascorbate (16.2 mmol) and 12.9 mg compound 3 (16.2 mmol) in 225 mL distilled water. The reaction mixture was bubbled with argon before and after the addition of copper sulphate and stirred for 1 h at room temperature. Then, the solution was diluted by 1 mL of 5% solution of disodium salt of ethylenediaminetetraacetic acid, the polymer conjugate was purified by gel filtration using Sephadex G-25 in water and freeze-dried. The conjugate was dissolved in 2 mL of methanol and 8-quinolinol was added in excess. After 20 min, the sample solution was purified from copper by gel filtration using Sephadex LH-20 column in methanol. Methanol was removed from the polymer fraction by vacuum distillation, the polymer was dissolved in water and isolated by freeze -drying. Yield: 29.4 mg; 84.0%; Molecular weights: M _w = 27,500 g/mol, M _n = 25,200 g/mol, D = 1.09. The content of the carbohydrate in the conjugate was 9.8 mol%.

Example 21: Synthesis of conjugate of substituted carbohydrate 20 with poly(HPMA-co-MA-AP-TT)

(Pll)

Polymer conjugate P11 was prepared with different content of the carbohydrate. 20 mg of the polymer precursor P2 (containing 16.4 mmol TT groups) and 2.7 mg of compound 20 (3.0 mmol) for the preparation of P11a; 5.3 mg of compound 20 (5.9 mmol) for the preparation of P11a; 9.1 mg of compound 20 (10.2 mmol) for the preparation of P11c or 10.3 mg of compound 20 (11.2 mmol) for the preparation of P11d were dissolved in 1.28 mL of the mixture of dimethylacetamide and dry methanol (3:1) and bubbled by argon. After the addition of 0.8 mL N-ethyldiisopropylamine (4.8 mmol), the reaction mixture was stirred at room temperature for 20 h. Then, 40 mL of l-aminopropan-2-ol (20 mmol) was added and stirred for 30 min. The polymer conjugate was separated from low-molecular- weight compounds by gel filtration using Sephadex LH-20 in methanol. Methanol was removed from the polymer fraction by vacuum distillation, the polymer was dissolved in water and isolated by freeze-drying. Characteristics are shown in Table 1. Table 1. Characteristics of prepared gly copolymers containing substituted carbohydrate 20

†Molecular weight and dispersity were determined using GPC, as it is described in Example 35.

Example 22: Synthesis of conjugate of substituted carbohydrate 7 with poly(HPMA-co-MA-AP- azadibenzocyklooctyne) (P12)

25 mg of poly(HPMA-co-MA-AP-azadibenzocyclooctyne), P7, (containing 8.9 mmol azadibenzocyclooctyne groups) was dissolved in 300 mL of methanol, mixed with the solution of 6.3 mg of compound 7 (11.0 mmol) dissolved in 200 mL of methanol and bubbled by argon. After 20 h of stirring at room temperature, the polymer conjugate was purified from low molecular weight compounds by gel filtration using Sephadex LH-20 in methanol. Polymer fraction was concetrated to 1 mL by vacuum distillation and the polymer was isolated by precipitation into an excess of ethyl acetate (40 mL). The polymer was filtered off and dried under vacuum. Yield 24.7 mg, 80.1 %; molecular weight: M _w = 30,800 g/mol, M _n = 28,000 g/mol, D = 1.10. The content of the carbohydrate in the conjugate was 5.1 mol%.

Example 23: Synthesis of conjugate of substituted carbohydrate 9 with poly(HPMA-co-M A- AP- propylazide) (P13)

Polymer conjugate P13 was prepared with different contents of the carbohydrate portion. 15 mg of polymer precursor P8 was dissolved in 150 mL of dimethylformamide and mixed with 3.54 mg (5.6 mmol) or with 6.3 mg (9.9 mmol) of substituted carbohydrate 9 dissolved in 300 mL of dimethylformamide for the preparation of P13a or P13b, respectively. Then, 0.88 mg (6.2 mmol) or 2.42 mg (12.9 mmol) of CuBr was added for the preparation of P13a, or P13b, respectively, and stirred at room temperature. After 20 h, 8-quinolinol was added in excess, stirred for 30 min and diluted by 2 mL of methanol. Polymer conjugate was purified by gel filtration using Sephadex LH-20 column in methanol. Methanol was removed from the polymer fraction by vacuum distillation, the polymer was dissolved in water and freeze-dried. P13a: Yield: 15.3 mg; molecular weights: M _w = 28,600 g/mol, M _n = 25,900 g/mol, D = 1.1. The content of the carbohydrate portion in the conjugate was 4.5 mol%. P13b: Yield: 17.4 mg; molecular weights: M _w = 30.600 g/mol, M _n = 28,100 g/mol, D = 1.1. The content of the carbohydrate in the conjugate was 8.9 mol%.

Example 24: Synthesis of conjugate of substituted carbohydrate 23 with poly(HPMA-co-MA-AP- propylazide) (P14)

13 mg of polymer precursor P8 was dissolved in 300 mL of dry methanol and mixed with 4.3 mg (8.7 mmol) of substituted carbohydrate 23 and 1.5 mg (9.5 mmol) CuBr dissolved in a mixture of dimethylformamide and methanol (4:1) under stirring at room temperature. After 20 h, 8-quinolinol was added in excess, stirred for 30 min and diluted by 2 mL of methanol. Polymer conjugate was purified by gel filtration using Sephadex LH-20 column in methanol. Methanol was removed from the polymer fraction by vacuum distillation, the polymer was dissolved in water and isolated by freeze- drying. Yield: 15 mg; molecular weights: M _w = 34,600 g/mol, M _n = 25,900 g/mol, D = 1.3. The content of the carbohydrate in the conjugate was 9.5 mol%.

Example 25: Synthesis of conjugate of substituted carbohydrate 26 with poly(HPMA-co-MA-AP- propylazide) (PI 5)

30 mg of polymer precursor P8 was dissolved in 150 mL of dry methanol and mixed with 5.8 mg (11.3 mmol) of substituted carbohydrate 26 and 2.0 mg (12.4 mmol) of CuBr dissolved in 150 mL of dimethylformamide under stirring at room temperature. After 20 h, 8-quinolinol was added in excess, stirred for 30 min and diluted by 2 mL methanol. Polymer conjugate was purified by gel filtration using Sephadex LH-20 column in methanol. Methanol was removed from the polymer fraction by vacuum distillation, the polymer was dissolved in water and isolated by freeze -drying. Yield: 26 mg; molecular weights: M _w = 38,400 g/mol, M _n = 29,600 g/mol, D = 1.3. The content of the carbohydrate in the conjugate was 7.3 mol%. Example 26: Synthesis of conjugates of substituted carbohydrate 27 with poly(HPMA-co-MA-AP- propylazide) (P16)

First, 50 mg of polymer precursor P8 was dissolved in 600 mL of dry methanol. Half of this solution was used for each conjugate. The polymer solution was added into the carbohydrate 27 solution (6.2 mg, 10.2 mmol) in 200 mL of fresh DMF. After homogenization in argon atmosphere for 30 min, CuBr (1.6 mg, 11.3 mmol) was added as a powder and the reaction mixture was kept under argon atmosphere for more 1 h. The reaction was carried out for 20 h at 24 °C, then, 8-quinolinol was added in excess. After shaking during 30 min, 1 mL methanol was added and quinolinol was filtered out. The sample solutions were purified using a Sephadex LH-20 column with methanol elution and UV detection. The collected fractions were concentrated under vacuum, diluted in water and purified in PD-10 (water elution). After concentrating the sample under strong vacuum at 35 °C for 2 h, the glycoconjugates were diluted in water and freeze-dried. The yield was 22.7 mg, 73%; M _w = 68,200 g/mol, M _n = 55,700 g/mol, D = 1.2. The content of the carbohydrate in the conjugate was 4.7 mol%.

Example 27: Synthesis of conjugate of substituted carbohydrate 3 with star copolymer containing PAM AM dendrimer core and poly(HPMA)-propynyl arms (P17)

1 mg of CuSO ₄ ·5H ₂ O (8 mmol) dissolved in 25 mL of distilled water was added to the solution of 25 mg of star copolymer containing PAMAM dendrimer core and poly(HPMA)- propynyl arms, P5, (containing 1.3 m mol propynyl groups), 1 mg of sodium ascorbate (10.1 mmol) and 2 mg of compound 3 (2.5 mmol) dissolved in 225 mL of distilled 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 by 1 mL of 5% solution of disodium salt of ethylenediaminetetraacetic acid, the polymer conjugate was purified by gel filtration using Sephadex G-25 in water and freeze-dried. The conjugate was dissolved in 2 mL of methanol and 8-quinolinol was added in excess. After 20 min, the sample solution was purified from copper by gel filtration using Sephadex LH-20 column in methanol. Methanol was removed from the polymer fraction by vacuum distillation, the polymer was dissolved in water and isolated by freeze-drying. Yield: 23.1 mg; 85%; molecular weights: M _w = 220,000 g/mol, D = 1.12. The content of the carbohydrate in the conjugate was 0.7 mol%.

Example 28: Synthesis of conjugate of substituted carbohydrate 23 with star copolymer containing PAMAM dendrimer core and poly(HPMA)-propynyl arms (P18)

1 mg of CuSO ₄ ·5H ₂ O (8 mmol) dissolved in 25 mL of distilled water was added to the solution of 25 mg star copolymer containing PAMAM dendrimer core and poly(HPMA)- propynyl arms, P5, (containing 1.3 mmol propynyl groups), 1 mg of sodium ascorbate (10.1 mmol) and 1 mg of compound 23 (2 mmol) dissolved in 225 mL of distilled 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 by 1 mL of 5% solution of disodium salt of ethylenediaminetetraacetic acid, the polymer conjugate was purified by gel filtration using Sephadex G-25 in water and freeze-dried. The conjugate was dissolved in 2 mL of methanol and 8-quinolinol was added in excess. After 20 min, the sample solution was purified from copper by gel filtration using Sephadex LH-20 column in methanol. Methanol was removed from the polymer fraction by vacuum distillation, the polymer was dissolved in water and isolated by freeze-drying. Yield: 22.4 mg; 83%; molecular weights: M _w = 225,000 g/mol, D = 1.13. The content of the carbohydrate in the conjugate was 0.7 mol%.

Example 29: Synthesis of conjugate of substituted carbohydrate 20 with star copolymer containing PAMAM dendrimer core poly(HPMA)-TT arms (P19)

25 mg of the polymer precursor P6 (containing 1.4 mmol TT groups) and 1.6 mg of substituted carbohydrate 20 (1.7 mmol) were dissolved in 1.2 mL of a mixxture dimethylacetamide and dried methanol (3:1) and bubbled with argon. After the addition of 0.6 mL of N-ethyldiisopropylamine (3.4 mmol), the reaction mixture was stirred at room temperature for 20 h. Then, 1 mL of 1-aminopropan-2- ol (13 mmol) was added and stirred for 0.5 h. The polymer conjugate was purified from low molecular weight compounds by gel filtration using Sephadex LH-20 in methanol. Methanol was removed from the polymer fraction by vacuum distillation, the polymer was dissolved in water and isolated by freeze-drying. Yield 21.8 mg, 82 %; molecular weight: M _w = 215,000 g/mol, D = 1.15. The content of the carbohydrate in the conjugate was 0.7 mol%.

Example 30: Synthesis of statistical copolymer poly(HPMA-co-MA-AP-TT-co-MA-AH-chol) by controlled RAFT radical polymerization ( P20 )

979 mg of HPMA (6.84 mmol) and 91.4 mg of MA-AH-chol (0.16 mmol) were dissolved in 9 mL of tert-butanol (5.52 mL) and mixed with a solution of 180.8 mg of MA-AP-TT (0.70 mmol), 6.2 mg of V-70 (20 mmol), and 8.8 mg of 2-cyanopropan-2-yl dithibenzoate (40 mmol) in 1 mL of dimethylacetamide. The reaction mixture was poured into a glass ampoule, bubbled with argon and sealed. After 16 h in a thermostat-controlled water bath at 40°C, the ampoule was cooled, and the reaction mixture was poured into an excess of acetone (150 mL). The polymer was filtered off and purified by reprecipitation from methanol (6 mL) into an excess of a mixture of acetone and diethyl ether (3:1; 120 mL). The polymer was isolated by filtration and dried under vacuum. Yield 937 mg, 75 %; molecular weights M _w = 23,000 g/mol, M _n = 20,800 g/mol, D = 1.10.

Example 31: Synthesis of statistical copolymer poly(HPMA-co-MA-AP-propynyl-co-MA-AH-chol) by conjugation of propynyl amine withpoly(HPMA-co-MA-AP-TT-co-MA-AH-chol) (P21 )

300 mg of the polymer precursor P20 (containing 0.27 mmol TT groups) was dissolved in 3 mL of dimethylformamide and 20 mL of propynylamine (0.325 mmol) and 54 mL of N-ethyldi isopropyl amine (0.32 mmol) were added under stirring at room temperature. The reaction was carried out for 16 h at room temperature. The polymer was purified from low molecular weight compounds by gel filtration using Sephadex LH-20 in methanol. Polymer was isolated by precipitation into an excess of acetone (60 mL). Yield 270 mg; molecular weight: M _w = 22,300 g/mol, M _n = 19,500 g/mol, D = 1.14.

Example 32: Synthesis of conjugate of substituted carbohydrate 3 with poly(HPMA-co-MA-AP- propynyl-co-MA-AH-chol) (P22)

2 mg of CuSO ₄ ·5H ₂ O (16 m mol ) dissolved in 25 mL of distilled water was added to the solution of 25 mg of poly(HPMA-co-MA-AP-propynyl-co-MA-AH-chol), P21, (containing 17.4 mmol propynyl groups), 1.6 mg of sodium ascorbate (16.2 mmol) and 12.9 mg of compound 3 (16.2 mmol) in 225 mL of distilled water. The reaction mixture was bubbled by argon before and after the addition of copper sulfate and stirred for 1 h at room temperature. Then, the solution was diluted by 1 mL of 5 % solution of disodium salt of ethylenediaminetetraacetic acid, the polymer conjugate was purified by gel filtration using Sephadex G-25 in water and freeze-dried. The conjugate was dissolved in 2 mL of methanol and 8-quinolinol was added in excess. After 20 min, the sample solution was purified from copper by gel filtration using Sephadex LH-20 column in methanol. Methanol was removed from the polymer fraction by vacuum distillation, the polymer was dissolved in water and isolated by freeze drying. Yield: 28.2 mg; 81 %; Molecular weights: M _w = 28,100 g/mol, M _n = 25,000 g/mol, D = 1.12. The content of the carbohydrate in the conjugate was 9.3 mol%. Example 33: Synthesis of conjugate of substituted carbohydrate 23 with poly(HPMA-co-MA-AP- propynyl-co-MA-AH-chol) (P23)

2 mg of CuSO ₄ ·5H ₂ O (16 mmol) dissolved in 25 mL distilled water was added to the solution of 25 mg poly(HPMA-co-MA-AP-propynyl-co-MA-AH-chol), P21, (containing 17.4 mmol propynyl groups), 1.6 mg of sodium ascorbate (16.2 mmol) and 8.1 mg of compound 23 (16.2 mmol) in 225 mL of distilled water. The reaction mixture was bubbled by argon before and after the addition of copper sulfate and stirred for 1 h at room temperature. Then, the solution was diluted by 1 mL of 5 % solution of disodium salt of ethylenediaminetetraacetic acid, the polymer conjugate was purified by gel filtration using Sephadex G-25 in water and freeze-dried. The conjugate was dissolved in 2 mL of methanol and 8-quinolinol was added in excess. After 20 min, the sample solution was purified from copper by gel filtration using Sephadex LH-20 column in methanol. Methanol was removed from the polymer fraction by vacuum distillation, the polymer was dissolved in water and isolated by freeze- drying. Yield: 28.2 mg; 81 %; Molecular weights: M _w = 27,300 g/mol, M _n = 24,800 g/mol, D = 1.10. The content of the carbohydrate in the conjugate was 9.0 mol%.

Example 34: Synthesis of conjugate of substituted carbohydrate 20 with poly(HPMA-co-MA-AP-TT- co-MA-AH-chol) (P24) 20 mg of the polymer precursor P20 (containing 15.6 mmol TT groups) and 5.3 mg of substituted carbohydrate 20 (5.9 mmol) were 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 mL of N-ethyldiisopropylamine (4.8 mmol), the reaction mixture was stirred at room temperature for 20 h. Then, 1.5 mL of 1-aminopropan- 2-ol (20 mmol) was added and stirred for 0.5 h. The polymer conjugate was purified from low molecular weight compounds by gel filtration using Sephadex LH-20 in methanol. Methanol was removed from the polymer fraction by vacuum distillation, the polymer was dissolved in water and isolated by freeze -drying. Yield 22.5 mg; molecular weight: M _w = 24,900 g/mol, M _n = 22,600 g/mol, D = 1.10. The content of the carbohydrate in the conjugate was 4.8 mol%.

Example 35: Example of characterization of polymer precursors and conjugates Prepared copolymers, polymer precursors and their conjugates with substituted carbohydrates were characterized by determination of weight-average and number-average molecular weight (Mw, Mn) and dispersity index (D) using gel permeation chromatography (GPC) equipped with a UV detector (Shimadzu, Japan), refractive index (RI) detector (Optilab REX, Wyatt Technology Corp., USA) and multi-angle light scattering detector (DAWN Heleos-II, Wyatt Technology Corp., USA) using TSK 3000 Super SW column and a mixture of methanol (80 %) and 0.3 M acetic buffer of pH 6.5 (20 %) as a mobile phase. The concentration of samples was 3 mg/mL.

The content of TT groups was determined spectrophotometrically using UV-VIS spectrophotometer Specord 205 (Analytik Jena, Germany) in methanol (e ₃₀ = 10 800 L.mol-, cm ^-1 according to the literature (Subr V. et al. Biomacromolecules 2006, 7 (1), 122-130).

The content of triple bonds and conjugated substituted carbohydrates was determined with nuclear magnetic resonance (NMR) using spectrometer Bruker Avance III 600 MHz in water.

Example 36: ELISA

Afinity of Gal-3 to substituted carbohydrates and their conjugates with polymers was determined by compatitive ELISA assay (Bojarova P. et al. J Nanobiotechnol. 2018, 16, 73; Bumba L. et al. Int. J. Mol. Sci. 2018, 19, 372). This assay gives preliminary information on the strength of binding of Gal-3 to glycocopolymers; however, is should be considered only as rough information and consider primarily the biological effect of glycopolymers (see Examples 38-41-). Asialofetuin (Sigma Aldrich, Steinheim, Nemecko; 0,1 mM in PBS buffer, 50 mL, 5 mmol per well) was immobilized in the wells od F16 Maxisorp NUNC-Immuno Modules (Thermo Scientific, Roskilde, Danmark). Then, wells were blocked with BSA (2 % w/v) in PBS (1 h, lab.t.). Then, a mixture of the tested compound in various concentrations and Gal-3 (total volume 50 mL; 4.5 mM final concentration of Gal-3) was added to the wells and incubated for 2 h. The detection of bound Gal-3 was performes using monoclonal anti-His ₆ - IgG1 mouse antibody conjugated to horseradish peroxidase (Roche Diagnostics, Mannheim, Germany) dissolved in PBS (1:1000, 50 mL, 1 h, lab.t.)· The substrate solution TMB One (Kem-En- Tec, Taastrup, Danmark) was used to start colorimetric reaction of the conjugated peroxidase. This reaction was stopped by adding 3M HC1 (50 mL). The binding signal of bound Gal-3 was determined spectrophotometrically at 450 nm (Spectra Max Plus, Molecular Devices, Sunnyvale, CA, USA). The acquired results were analyzed using software Prism 7.0 (GraphPad, USA) and evaluated as ICso-

Table 2. Inhibition potencial of selected compounds and conjugates (ICso) determined with ELISA

*Content of substituted carbohydrates was determined by ¹ H-NMR. 1 ¹ Adopted from the literature (Bdcker S. et al. Biomolecules 2015, 5, 1671-1696)

Example 37: Quantification of Gal- 3 production by tumor cell lines

Example 38: Inhibition of binding of external galectin-3 on the surface of galectin- 3 -expressing cells by glycopolymers The ability of glycopolymers to inhibit binding of external Gal-3 on the cell surface was demonstrated in inhibitory binding assay using flow cytometry. In the assay we used protein construct GaI-3-AVI. This construct carries amino acid sequence Avi-tag at its N-terminus that enables selective binding of biotin molecule and its subsequent detection using fluorescently labeled conjugate streptavidin- phycoerythrin (L. Bumba et al. Int. J. Mol. Sci. 2018, 19, 372). As found by immunochemical assay using ELISA, the binding properties of native Gal-3 a GaI-3-AVI construct are identical. The binding of glycopolymers to Gal-3 inhibited the binding of Gal-3 to the surface of HEK293 cells (immortalized cell line of human embryonal kidney cells), which exhibited a strong expression of Gal- 3 (see Example 37) and also a strong capability to bind free Gal-3 from the solution.

A likvots of GaI-3-AVI construct (final concentration 10 mg/mL) were mixed with increasing concentration of gly copolymer P11b (3 mM - 100 mM) or lactose (3 nM - 100 mM) as a positive control and incubated for 30 min on ice in PBS buffer containing 1% bovine serum albumin (BSA). This mixture was added to the suspension of HEK293 cells (10 ⁶ /mL) and slowly mixed on ice for 30 min. Then cells were washed with PBS buffer and labeled with streptavidin-phycoerytrin conjugate (Biolegend, USA). The strength of binding of GaI-3-AVI construct on the surface of HEK293 bunek was analyzed by flow cytometry and quantified as relative intensity fo fluorescence at 575 nm. Table 3. Inhibition potential of glycopolymer P11b (IC ₅₀ ) determined in binding inhibition assay with cell line HEK293 using flow cytometry

Example 39: Inhibition of galectin-3 induced apoptosis of human T cells by glycopolymers The ability of glycopolymers to efficiently inhibit Gal-3 induced apoptosis was demonstrated by flow cytometry using Annexin V/ propidium iodide apoptosis test. Experiments were performed with immortalized human T lymphocytes Jurkat. It was demonstrated that human Gal-3 produced by tumor cells into the environment induced apoptosis of this cell line.

In in vitro experiments, Jurkat cells were preincubated with various concentrations of glycopolymers P11 and P13 (0.1, 1, 5, 10, and 50 mM) for 5 min and then 10 mM Gal-3 was added to the cells. Glycopolymers bound free Gal-3 from the solution (cell environment), and thus the Gal-3 could not induce apoptosis of Jurkat cells. The efficiency of inhibition of Gal-3 by glycopolymers was determined as a level of cell apoptosis. Pure HPMA polymer without substituted carbohydrates was used as a control.

Glycopolymers inhibited Gal-3 induced apoptosis. The maximal inhibitory effect was achieved in concentration 5 mM or 1 mM in the case of tested conjugates P11b or P11d (see Fig. 1 and 2). Pure HPMA polymer without substituted carbohydrates showed also a subtle protective effect at concentration 50 mM.

Glycopolymer P13 also showed a high inhibitory effect (see Fig. 3 and 4). Even glycopolymers P14 (see Fig. 5), P15 (see Fig. 6) and P16 (see Fig. 7) showed inhibitory effect.

Example 40 (comparative): Inhibition of galectin-3 induced apoptosis of human T cells ( Jurkat ) by glycopolymer carrying simple disaccharide LacdiNAc

Table 4. Characteristics of the comparative glycopolymer carrying simple disaccharide FacdiNAc

†Molecular weight and dispersity were determined using GPC as described in Example 35. Fig. 8 represents the inhibition of galectin-3 induced apoptosis of Jurkat cells by conjugate of HPMA polymer with simple disaccharide LacdiNAc (content of LacdiNAc was 12.3 mol%, see Table 4). The same results was achieved using analogous conjugate with the content of LacdiNAc 8.4 mol%. It was the identical disaccharide as in publication (Bojarova P. et al. J. Nanobiotechnol. 2018, 16, 73). It is evident, that this conjugate demonstrated a considerably lower ability of inhibition of Jurkat cell apoptosis in vitro than conjugates with substituted disaccharides, which are objects of the invention.

Example 41: Inhibition of migration of human and murine tumor cells

The migration of selected tumor cells, which expressed Gal-3, was studied using a so-called scratch test. This test is based on seeding of tested cell culture onto culture dish. When the culture achieves 70-80 % growth, the cells are scratched from the surface by the end of the 1 mL -plastic tip in the length of 1 cm and width of ca. 0.5-1 mm. Subsequently, the culture medium, in which the cells grew, is changed for fresh medium including tested compounds. The migration of cells is then determined by speed and extent of growth of previously created gap.

The inhibitory effect of polymers P11a containing 2.6 mol% and P11c containing 7.2 mol% of tetrasaccharide 20 was determined after its addition to culture media of murine breast cancer (4T1) cells, murine melanoblastoma cell line (B16F10) and human colorectal cancer (DLD1) cells. Polymer conjugates were added in final concentration 10 mM or 20 mM. After 24 and 48 h, the area, which stayed uncovered by cells, was measured, and then the difference in comparison with the control group without the addition of polymer conjugate was evaluated. Fig. 9 represents the difference in the width of the overgrown zone of the control sample without polymer conjugate to that in which HPMA polymer (pHPMA) itself was added or samples with polymer conjugates. In the case of 4T1 and B16F10, both conjugates induced inhibition of migration towards control also in the case of sample incubated with polymer carrier. In the DLD1 cells, only the conjugate with higher molar content of carbohydrate induced inhibition of migration in contrast to the control and polymer carrier.