CZ2021423A3 - Fluorescently labelled polymer for visualizing tumours, preparing and using it - Google Patents

Abstract

The presented solution relates to a fluorescent polymer to which a fluorescent label is bound in an amount from 0.1 to 10 mol. %, and whose fluorescence is activated in tumor tissue to provide significant visualization of tumor tissue for diagnostic procedures or for image-guided, navigated surgery. Advantageously, the fluorescent polymer is targeted by specific molecules to tumor cells and tumor neovasculature. The presented solution further relates to a method of producing a fluorescent polymer and its use as a fluorescent probe in medical diagnostics as a contrast agent for tumor visualization, whole-body imaging and/or for image-guided surgery.

Description

Fluorescently labeled polymer for visualization of tumors, method of its preparation and its use

Field of technology

The invention relates to a directed fluorescent polymer with activatable fluorescence composed of a polymer carrier with side chains with fluorophores intended for visualization of tumor tissue. The use of the polymer is aimed at highlighting the tumor tissue for subsequent surgical removal or non-invasive monitoring of the progress of the cancer.

Current state of the art

Precise and complete resection of the entire tumor without unnecessary removal of adjacent healthy tissue is a prerequisite for a successful outcome of oncological surgery. Unfortunately, the visual distinction between malignant and healthy tissue with the naked eye is often very difficult, if not impossible, for the surgeon. However, radical resection with adequate negative margins is one of the most important factors influencing the prognosis of patients with, for example, Head and Neck Squamous Cell Carcinoma (HNSCC). Usually, the safe margin is defined as greater than 5.0 mm. In the context of oncological endoscopic surgery, the precise setting of tumor boundaries remains a key issue. At the moment, it is possible to partially use non-invasive imaging, such as autofluorescence (AF) of tumor tissues, for imaging tumor margins. However, the method using AF is not accurate and is only used for certain types of cancer. There is currently no method that works reliably to guide the surgeon during surgery. Fluorescence imaging (Fluorescence Imaging, FI) is based on the illumination of tissue with light that excites near-infrared (NIR) or short-wave infrared (Shortwave infrared, SWIR) fluorophores or contrast agents. Due to the permeability of light in the NIR and SWIR region through tissues, up to several cm, it is possible to use these fluorophores for visualization as part of marking tumor foci.

In the past few decades, various polymeric materials have been studied as biocompatible, non-immunogenic and non-toxic biomaterials for medical applications. Biomaterials designed as vectors for the transport of biologically active molecules, materials for tissue engineering and diagnostics have been and are being studied. Systems for the targeted transport and controlled release of drugs, so-called drug delivery systems (DDS), are intensively studied, in which polymeric forms of drugs are studied as new formulations of "classic" drugs with the aim of ensuring an increased therapeutic function for antineoplastic, anti-inflammatory and antimicrobial treatment while simultaneously elimination of side effects of treatment. Many described DDS are based on water-soluble or amphiphilic polymer carriers carrying low molecular weight active molecules, e.g. covalently bound by biodegradable linkers, intended for controlled release and activation of the carried active molecules in desired tissues or cells. Much attention has been focused on biocompatible, non-toxic and non-immunogenic copolymers based on N-(2hydroxypropyl)methacrylamide (pHPMA), poly(ethylene glycol) (PEG), poly (caprolactone) (PCL), poly (lactid acid) / poly (acid lactide-co-glycolide) (PLA / PLGA) and their copolymers as carriers of biologically active molecules and directing groups. In general, polymeric carriers of bioactive molecules are designed to optimize the pharmacokinetics of active molecules, prolong their blood circulation time, improve localization in tumors, inflammation and/or target cells, reduce secondary toxicity and immunogenicity, and solubilize water-insoluble active molecules. Active carrier molecules can be released/activated by enzymatic hydrolysis, reduction, or pH-induced hydrolysis by lowering the pH from 7.4 (blood) to 5 - 6.5 (endosomes / lysosomes; tumor microenvironment; inflammatory environment). To increase accumulation in the target tissue, e.g., tumor tissue, while minimizing side effects, targeted systems containing targeting groups, e.g., antibodies or oligopeptides, or systems with a structure and size enabling "passive" targeting based on

- 1 CZ 2021 - 423 A3 of the EPR effect (increased permeability and retention) using carriers with high molecular weight (HMW), e.g. star polymers, nanoparticles or micelles.

Recently, pHPMA-based polymeric therapeutics and diagnostics have been developed that have increased accumulation in solid tumors. These systems, therapeutics carrying antitumor drugs, e.g., doxorubicin, paclitaxel, have been found to have anticancer activity in various tumor models in mice. Polymeric systems containing both the drug and a rigidly bound fluorescent label were not only therapeutically active, but were able to monitor tumor development using fluorescence imaging (FI). Recently, linear pHPMA constructs containing NIR dye Dyomics-782 and EGF (epidermal growth factor) targeting oligopeptides GE-7 and GE-11 have been described. The in vivo biodistribution study showed very promising results in tumor visualization. The polymer probe was mainly localized at the tumor border, showing the potential for image-guided surgery.

Recently, high molecular weight delivery systems for fluorescence imaging and tumor surgery have been described, for example WO 2020/245447 A1 (fluorescent polypeptides). These high-molecular systems are used for imaging tumor tissue and although they meet the condition of biocompatibility and excludability, none of them bring a selective increase in fluorescence locally in the tumor tissue.

- 2 CZ 2021 - 423 A3

The essence of the invention

The present invention relates to a directed fluorescent polymer, which forms a polymer probe with the possibility of activating a fluorescent signal at the site of a tumor. The fluorescent polymer according to the present invention enables targeted accumulation in tumor tissue and subsequent activation of a fluorescent signal for visualization of tumors. The polymer system itself contains a polymer carrier, a fluorescent label linked by a bond, which is hydrolytically, enzymatically or reductively degradable in the tumor environment, possibly further directing the group to tumor or endothelial cells, and possibly a fluorescence quencher. The resulting fluorescent polymer enables signal amplification and the achievement of a significant tumor/healthy tissue contrast in the vicinity of the tumor by activating the fluorescence of the transported label in the tumor tissue. While the fluorescence yield of the fluorescent label drops significantly after binding the fluorophore to the polymer carrier, due to the self-quenching of fluorescent molecules localized in the vicinity due to non-radiative energy transfer between the fluorophores, after the release of the label from the polymer carrier in the tumor tissue, the fluorescence signal is restored again, leading to to a significant increase in the signal versus noise ratio in the tumor environment. This surprising effect of increasing the fluorescence signal contrast between healthy and tumor tissue has not been described before. Copolymers based on A-(2-hydroxypropyl)methacrylamide (HPMA) are suitable precursors for the preparation of the described fluorescent polymers with activatable fluorescence. Functional groups are introduced along the chain of this copolymer, to which suitable fluorophore derivatives can be attached via biodegradable links and directing groups. The entire system according to the present invention thus enables increased visualization of tumor foci for subsequent surgical resection guided by a fluorescent signal. Another use is the application of fluorescent polymers to monitor the progression and regression of cancer during treatment with another therapy. It is advantageous to use fluorescent polymers for tumors that are not localized in the depth of the tissue or that require endoscopic techniques, e.g. head and neck tumors, breast tumors, colorectal tumors and melanomas.

The subject of the present invention is a fluorescent polymer useful for tumor visualization, which contains a semitelechelic random linear copolymer, wherein the semitelechelic random linear copolymer is selected from the group including polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(A-(2hydroxypropyljmethacrylamide), in which from 0, 1 to 10 mol % of monomer units, preferably 0.4 to 6 mol % of monomer units, more preferably 0.5 to 4 mol % of monomer units, based on the total number of monomer units, statistically replaced by monomer units of the general formula (I)

ΟΗ _3η

Γ°

HN

And /

B fluoro for (I), where

A is selected from the group consisting of a straight or branched carbon alkylenyl chain having from 1 to 7 carbons ((C1 to C7)alkylenyl); -(CH 2 ) _P -(C(O)-NH-(CH 2 ) _r ) _P -;

-3CZ 2021 - 423 A3

-(CH2)p-(C(O)-NH-(CH2) _r )pC(O)-; -(CH 2 ) _P -(C(O)-NH-(CH 2 ) _r )pC(O)-NH-C ₆ H 4 -CH 2 -OC(O)-; a -(CH ₂ ) _P C(O)-NH-(CH2) _P -L-(CH2)p-(C(O)-NH-(CH2)r)pC(O)-NH-C ₆ H4- CH2-OC(O)-, where L is a coupling containing a triazole bridge, formed for example by the reaction of a propargyl or DBCO group with an azide group; Thus, L can have, for example, the following structure:

or

where p is an integer ranging from 1 to 5, and r is selected from 1, 2 and 3;

wherein one or more hydrogen atoms in the CH 2 groups of the substituent A may be further substituted with the same or different side chains of a natural amino acid; these chains are preferably methyl, isopropyl, isobutyl, -CH(CH ₃ )(CH ₂ CH ₃ ), -CH ₂ OH, -CH(OH)(CH ₃ ), -CH 2 -(C ₆ H ₄ )OH, -(CH ₂ ) ₂ -S-CH ₃ , CH ₂ SH, -(CH ₂ )4-NH ₂ , -CH 2 COOH, -CH ₂ C(O)NH ₂ , -(CH ₂ ) ₂ COOH, -(CH ₂ )2C(O)NH ₂ , -(CH ₂ ) ₃ NHC(=NH)(NH ₂ ), -(CH ₂ ) ₃ NH-C(=O)(NH ₂ ); benzyl;

B is selected from the group consisting of bond,

fluorophore (fluorophore or its amino derivative, NCS ester or N-hydroxysuccinimidyl derivative) has a molecular weight in the range of 350 to 1500 g/mol, an excitation wavelength in the range of 300 to 850 ^-C nm and an emission wavelength in the range of 350 to 1200 nm, and is covalently bound to =CH-, or

-S-S- group of the substituent B of the monomer unit of the general formula I of the semitelechelic statistical linear copolymer through its primary amino group, or NCS group, Nhydroxysuccinimidyl group, keto group, aldehyde group or disulfide group, while the groups formed after the binding of fluorophore are subsequently part of group B;

wherein when B is a bond, then A is -(CH 2 ) _P -(C(O)-NH-(CH 2 ) _r ) _P -C(O)-; or

-(CH ₂ ) _P -(C(O)-NH-(CH 2 ) _r ) _P -C(O)-NH-C ₆ H 4 -CH 2 -OC(O)-; or

-(CH ₂ ) _P -C(O)-NH-(CH2) _P -L-(CH2) _P -(C(O)-NH-(CH2) _r ) _P -C(O)-NH-C6H4- CH ₂ -OC(O)-;

and wherein the molecular weight M _n of the fluorescent polymer ranges from 6000 to 100,000 g/mol, preferably from 10,000 to 60,000 g/mol, more preferably from 15,000 to 40,000 g/mol (corresponding to 100 to 280 monomer units) , more preferably from 20,000 to 30,000 g/mol (corresponding to 134 to 210 monomer units).

The fluorophore is thus attached to the side chain of the semitelechelic random linear copolymer by a labile hydrazone or disulfide bond or a peptide bond (Val-Cit (NH-CH(C(CH3) ₂ )-C(=O)-NH-CH((CH ₂ )3 -NH-C(=O)-NH ₂ )-C(=O)-), Val-Cit-Aba coupling (-NH-4CZ 2021 - 423 A3

CH(C(CH3)2)-C(=O)-NH-CH((CH2)3-NH-C(=O)-NH2)-C(=O)-NH-C6H4-CH2-O-C(= O)-), which is hydrolytically, enzymatically and/or reductively degradable in the tumor environment.

Natural amino acids are 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 are chains attached to the alpha-carbon of an amino acid.

The term "alkylenyl" means a divalent linear or branched carbon chain, for example -CH 2 , -(CH 2 ) 2 -, -(CH 2 ) 5 - or -(CH 2 ) 2 -(CH(CH 3 ))-CH 2 -.

By fluorophore is meant an organic molecule with at least one aromatic nucleus capable of fluorescence, attached to the substituent B by a covalent bond. A fluorophore is a fluorescent label or a derivative thereof, containing an amino group, a keto group or an S-S bond, added to the fluorophore structure by reacting the fluorescent label with a selected oxoacid or reagent containing an activated disulfide, for example by reacting with 3-(2-pyridyldithio)propionate,

5-cyclohexyl-5-oxopentanoic acid, 4-(2-oxopropyl)benzenecarboxylic acid and 4-oxo-4-(2-pyridyl)butanoic acid. After binding the fluorophore to the substituent B of the general formula (I), the groups formed by this reaction are part of the group B defined above.

In a preferred embodiment, the fluorophore is selected from the group comprising (2E)-2-[(E)-3-(7azaniumylidene-2-tert-butylchromen-4-yl)prop-2-enylidene]-1-[6-(2, 5-dioxopyrrolidin-1-yl)oxy6-oxohexyl]-3,3-dimethylindole-5-sulfonate (DY-615), 2-[3-[2-tert-butyl-7-[ethyl(3sulfonatopropyl)azaniumylidene]chromene Sodium -4-yl]prop-2-enylidene]-1-(5-carboxypentyl)-3,3-dimethylindole-5-sulfonate (DY-633), 3-(3-carboxypropyl)-2-[3-(9- Sodium ethyl 6,8,8-trimethyl-2-phenylpyrano[3,2-g]quinolin-4-ylidene)prop-1-enyl]-3-methyl-1-(3-sulfonatopropyl)indol-1-ium-5-sulfonate (DY-676), 3-(3-carboxypropyl)-2-[3-[8,8-dimethyl-2-phenyl-6(sulfonatomethyl)-9-(3-sulfonatopropyl)pyrano[3,2-g] Sodium quinolin-4-ylidene]prop-1-enyl]-3-methyl 1-(3-sulfonatopropyl)indol-1-ium-5-sulfonate (DY-678), 2-[5-(2-tert-butyl- 7-diethylazaniumylidenechromen-4-yl)penta-2,4-dienylidene]-1-(5-carboxypentyl)-3,3dimethylindole-5-sulfonate (DY-730), 2-[5-(2-tert-butyl-9- ethyl 6,8,8-trimethylpyrano[3,2g]quinolin-4-ylidene)penta-1,3-dienyl]-1-(5-carboxypentyl)-3,3-dimethylindol-1-ium-5-sulfonate sodium (DY-750), 3-(3-carboxypropyl)-2-[5-(9-ethyl-6,8,8-trimethyl-2-phenylpyrano[3.2g]quinolin-4-ylidene)penta-1 Sodium ,3-dienyl]-3-methyl-1-(3-sulfonatopropyl)indol-1-ium-5-sulfonate (DY-776), 2-[5-[4-tert-butyl-7-[ethyl( Sodium 3-sulfonatopropyl)azaniumylidene]chromen-2yl]penta-2,4-dienylidene]-3-(3-carboxypropyl)-3-methyl-1-(3-sulfonatopropyl)indole-5-sulfonate (DY-782), 1-[2-[dimethoxy(phenyl)methyl]phenoxy]-3,4-diphenylisoquinoline (CY-7), 1Hbenz[e]indolium, 2-[2-[3-[2-(1,3-dihydro- 1,1,3-trimethyl-2H-benz[e]indol-2-ylidene)ethylidene]1-cyclohexen-1-yl]ethenyl]-3-[6-[(2,5-dioxo-1-pyrrolidinyl) oxy]-6-oxohexyl]-1,1dimethylisoquinoline (CY-7.5), 4-[(2E)-2-[(2E,4E,6E)-7- [1,1-dimethyl-3-(4sulfonatobutyl)benzo Sodium [e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3yl]butane-1-sulfonate (ICG), 2,3, 5-trichloro-4-[2-[[6-(2,5-dioxopyrrolidin-1-yl)oxy-6oxohexyl]amino]-2-oxoethyl]sulfanyl-6-[6,7,7,19,19, 20-Hexamethyl-9,17-bis(sulfonatomethyl)2-oxa-20-aza-6-azoniapentacyclo[12.8.0.03,12.05,10.016,21]docosa-1(14),3,5,8,10,12 . Optionally, fluorophores are modified with oxoacids using the amide reaction of the amino group of the fluorophore with the activated carboxyl of the oxoacid, the resulting product is an oxoderivative of the fluorophore. Optionally, the fluorophore is modified with cystamine using an amide bond between the cystamine and the fluorophore. Optionally, the fluorophore is modified with N-(5-Azidopentanoyl-valyl-citrulyl)-4-aminobenzyl (4-nitrophenyl) carbonate or with N-(5-Azidopentanoyl-valyl-citrulyl) by reaction with its amino group.

- 5 CZ 2021 - 423 A3

An example of a fluorophore that can be successfully attached to group B of general formula (I) are commercially available: DY-782, DY-782-NHS ester, DY-728-carboxylic acid, DY-728-maleimide, DY728-amine, DY- 728-azide, DY-676, DY-676-NHS ester, DY-676-carboxylic acid, DY-676maleimide, DY-676-amine, Cy7, Cy7-NHS ester, Cy7-amine, Cy7.5, Cy7.5 -NHS ester, Cy7,5-amine, Cy7,5-azide. The fluorophore can be linked to group B via an oligopeptide link of 2 to 5 amino acids that can be enzymatically degradable by lysosomal enzymes, a pH-sensitive hydrolytically degradable hydrazone bond or a reductively biodegradable disulfide bond, which enables the controlled release of the fluorophore in the tumor environment. An oligopeptide linker of 2 to 5 amino acids can be, for example, Val-Cit or Val-CitAba.

The end groups of the resulting fluorescent polymer contain portions of the polymerization initiator and transfer agent molecules. The initiator can be, for example, 2,2'-azobis[N-(2carboxyethyl)-2-methylpropionamidine] (V-70)) and the transfer agent can be, for example, N-(3azidopropyl)-4-ethylsulfanylcarbothioylsulfanyl-4-methyl-pentanamide (CTA -N3)), or their derivatives formed by reaction with, for example, 2,2'-azobisisobutyronitrile (AIBN). The end groups of the copolymer therefore contain an azide group, which can subsequently be further modified by reaction with dibenzocyclooctyne-maleimide (DBCO-MI) to another reactive maleimide functional group, by reaction with dibenzocyclooctyne-carboxyl (DBCO-Cbx), which is subsequently modified with thiazolidine-2- thione to the thiazolidine-2-thione reactive functional group, by reaction with dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO-NHS) to the Nhydroxysuccinimidyl ester reactive functional group.

During biodistribution, the fluorescent polymer defined above is directed to the tissues of solid tumors due to its high molecular weight and the longer circulation time during which the fluorescent polymer accumulates in solid tumors (EPR effect - Enhanced Permeability and Retention Effect).

In one embodiment, the fluorescent polymer further contains directing groups to enhance targeting of the fluorescent polymer to tumor tissues. These directing groups can be attached to the terminal groups of the linear chain of the fluorescent polymer and/or they can be attached to the side chains.

The directing group for directing the fluorescent polymer to the tumor cells of head and neck tumors, breast tumors, melanomas and colorectal tumors or to tumor endothelium cells is selected from the group comprising oligopeptides with a number of amino acids from 3 to 20, and proteins, preferably antibodies, more preferably monoclonal antibodies targeting tumor cells of head and neck tumors, breast tumors, melanomas and colorectal tumors or tumor endothelial cells.

In a preferred embodiment, oligopeptides with a number of amino acids from 3 to 20 are selected from the group including YESIKVAVS, SIGYPLP, RGD and cRGD (tumor endothelium), CPLHQRPMC (prostate tumors), NPVVGYIGERPQYRDL called GE7 and YHWYGYTPQNVI called GE11 (to the EGF receptor), WHYPWFQNWAMA ( head and blood tumor cells), DMPGTVLP (breast tumors), CNGRC, cyclic RGDfK, HEWSYLAPYPWF and SYSMEHFRWGKPV. Oligopeptides and proteins that target tumor tissues in the body are known to those skilled in the art.

In a preferred embodiment, the monoclonal antibodies are selected from the group including herceptin, erbitux, daratuzumab, trastuzumab.

In embodiments where the directing groups are attached to the terminal groups of the linear chain of the fluorescent polymer, these directing peptides and/or proteins are attached by conjugation of the amino group or introduced propargyl, DBCO, or SH groups of the directing peptide/protein with an azide, maleimide, thiazolidine-2-thione, or by the N-hydroxysuccinimidyl ester end group of the linear chain of the fluorescent polymer.

- 6 CZ 2021 - 423 A3

In an embodiment where the directing groups are attached to the side chains of the fluorescent polymer, the fluorescent polymer defined above also contains monomeric units of the general formula (Π) (II) where A is defined above,

X is bond,

-S-S-, L, where L is as defined above; and Z is a targeting group for directing the fluorescent polymer to tumor cells of head and neck tumors, breast tumors, melanomas, and colorectal tumors, or to tumor endothelial cells, also defined above, and attached to the X group via an amide, maleimide, azide, or propargyl bond, preferably the directing group is attached to the X group via azide or propargyl. These directing groups are thus covalently bound to =CH-, , -CH=CH-, CH=CH- in the DBCO structure, N3 or -S-S- group of the substituent X of the monomer unit of the general formula (II) of the semitelechelic random linear copolymer through its primary amino group , or an introduced azide, propargyl, DBCO or SH group, whereby the groups formed after the binding of the directing group are subsequently part of the X group.

In this embodiment, the amount of monomer units of general formula (II) is from at least one monomer unit to 8 mol. %, based on the total number of monomer units.

The total number of monomeric units of general formulas (I) and (II) in the fluorescent polymer is no more than 10 mol. %, based on the total number of monomer units.

The directing group may optionally be modified prior to conjugation to the fluorescent polymer to contain at least one group selected from -NH2, -SH, sDBCO (sulfodibenzocyclooctyne group), DBCO (dibenzocyclooctyne group), azide, propargyl to which the fluorescent polymer is covalently attached by means of a functional group, preferably selected from X-hydroxysuccinimidyl ester, thiozolidine-2-thione amide, malenimide, azide, DBCO, sDBCO, or propargyl. Modifications of oligopeptides and proteins to introduce -NH 2 , -SH, sDBCO, DBCO or azide into the structure are known to those skilled in the art.

In a preferred embodiment, the semitelechelic random linear copolymer is poly(V-(2-hydroxypropyl-methacrylamide).

The fluorescent polymer can further contain from 0 to 9.9 mol. % of monomer units of the general formula (III), based on the total number of monomer units,

CZ 2021 - 423 A3 ch ₃

And /

HN (III) wherein A is as defined above and C is selected from the group consisting of -SS-(CH 2 ) 2 -OH, -C(=O)-NH(CH ₂ ) _and -CH ₂ (OH); -C(=O)-NH-(CH ₂ ) _b -CH(OH)-CH ₃ ; -C(=O)-NH-(CH ₂ )b-CH(OH)-(CH ₂ )c-CH ₃ ; and -NH-C(=O)-CH ₃ , wherein a is an integer from 0 to 4, A is an integer from 0 to 3, and c is an integer from 1 to 4; and while the total number of monomer units of the general formula (I), (II) and (III) in the fluorescent polymer is no more than 10 mol. %, based on the total number of monomer units.

Preferably, the substituent C is selected from the group consisting of -C(=O)-NH-CH2(OH); -C(=O)-NHCH(OH)-CH ₃ ; -C(=O)-NH-(CH ₂ ) 2 -CH ₂ (OH); -NH-C(=O)-CH ₃ .

In a preferred embodiment, A is selected from the group consisting of ethane-1,2-diyl (-CH2-CH2-); propane 1,3-diyl (-CH 2 -CH 2 -CH 2 -); butane-1,4,-diyl (-CPEh-); pentane-1,5-diyl (-CH 2 ) 5 -); hexane-1,6-diyl (CH ₂ ) 6 -); heptane-1,7-diyl (-CH ₂ ) ₇ ; -CH ₂ -C(=O)-NH-CH ₂ - (derived from the dipeptide Gly-Gly); -CH ₂ C(=O)-NH-CH(CH ₂ -CH(CH ₃ ) ₂ )-C(=O)-NH-CH ₂ - (derived from the tripeptide Gly-Leu-Gly); -CH ₂ C(=O)-NH-CH(CH ₂ Ph)-C(=O)-NH-CH ₂ - (derived from the tripeptide Gly-Phe-Gly); -CH ₂ -C(=O)NH-CH(CH2-CH(CH ₃ ) ₂ )-C(=O)-NH-CH(CH ₂ Ph)-C(=O)-NH-CH2- (derived from the tetrapeptide Gly-Leu-Phe-Gly); -CH2-C(=O)-NH-CH(CH ₂ Ph)-C(=O)-NH-CH(CH2-CH(CH ₃ ) ₂ )-C(=O)-NHCH(CH ₂ Ph) -C(=O)-NH-CH ₂ - (derived from the pentapeptide Gly-Phe-Leu-Phe-Gly); -(CH ₂ ) ₄ -C(=O)NH-CH(C(CH ₃ ) ₂ )-C(=O)-NH-CH((CH 2 ) ₃ -NH-C(=O)-NH 2 )- C(=O)-NH-C ₆ H4-CH2-O- C(=O)(derived from N-(5-azidopentanoyl-valyl-citrulyl)-4-aminobenzyl carbonyl), -(CH2) ₄ -C( =O)NH-CH(C(CH ₃ ) ₂ )-C(=O)-NH-CH((CH ₂ ) ₃ -NH-C(=O)-NH ₂ )-C(=O)- ( derived from N-(5-azidopentanoyl-valyl-citrulyl).

In one embodiment, the fluorescent polymer is a linear random copolymer of HPMA and monomer units of general formula (I), or (II) and (III). This linear random copolymer can be represented by the general formula (IV), wherein the molecular weight M _n of the random linear copolymer is in the range of 6,000 to 100,000 g/mol, preferably in the range of 15,000 to 45,000 g/mol (corresponding to 100 up to 300 monomer units), more preferably the molecular weight is in the range from 20,000 to 40,000 g/mol (corresponding to 134 to 280 monomer units);

(IV),

-8CZ 2021 - 423 A3 where A, B, C and Z are defined above, where the total number of monomer units in the fluorescent polymer is in the range from 100 to 300, the representation of units of the general formula (I) in this statistical linear copolymer is from 0, 1 to 10 mol %, based on the total number of monomer units, which corresponds to the number of 1 to 30 monomer units of general formula (I), and the representation of units of general formula (II) in this statistical linear copolymer is from 0 to 8 mol %, based on the number of monomer units, which corresponds to the number of 0 to 24 monomer units of the general formula (II), and the representation of the units of the general formula (III) in this statistical linear copolymer is from 0 to 9.9 mol%, based on the number of monomer units, which corresponds to the number of 0 up to 29 monomer units of the general formula (III), while the total number of monomer units of the general formula (I) and (II) and (III) is no more than 10 mol. %, based on the total number of monomer units, which corresponds to a maximum total number of 30 units of formula (I) + formula (II) + formula (III). The remaining monomer units, numbering approximately from 270 to 298, are HPMA monomer units. The end groups of the resulting linear copolymer of general formula (IV) contain parts of the polymerization initiator and transfer agent molecules as mentioned above.

The fluorescent polymer is a semitelechelic random linear copolymer that can be prepared by radical polymerization. The end groups of the resulting linear copolymer are blocked with, for example, 2,2'-azobisisobutyronitrile (AIBN).

The subject of the present invention is therefore also a method of preparing a fluorescent polymer (fluorescent polymer with activatable fluorescence and possibly with an attached directing group), which includes the following steps:

a) provision of fluorescent polymer monomers,

b) polymerization of these monomers,

c) linking the fluorophore to the statistical linear copolymer

d) optionally linking the directing structure to a fluorescent polymer, wherein the directing structure is selected from the group including oligopeptides with the number of amino acids from 3 to 20 and proteins, especially monoclonal antibodies; whereby the directing structure can be attached to the end of the linear chain and/or to the side chains of the fluorescent polymer (as part of the monomer unit of general formula (II)).

and a) Providing linear copolymer monomers includes providing monomers selected from the group consisting of acrylamide, methacrylamide, acrylate, methacrylate, N-(2-hydroxypropylmethacrylamide (HPMA), and monomers of general formula (V)

And /

D (V), which is a precursor for the monomeric units of general formula (I), (II) and (III), where A is defined above;

and D is -carbonyl-thiazoline-2-thione group (TT), (4-nitrophenyl)oxy group, (2,3,4,5,6pentafluorophenyloxy group, (succinimidyl)oxy group, carboxyl, hydrazide, azide,

-9CZ 2021 - 423 A3 activated disulfide group or NH2-group. The amino group and the hydrazide group may optionally be protected by a protecting group, for example a tert-butoxycarbonyl (Boc) protecting group. Carbonyl means -C(=O)- group.

N-(2-Hydroxypropyl)methacrylamide (HPMA) and other monomers of the acrylamide, methacrylamide, acrylate, and methacrylate types are commercially available. The provision of these monomers therefore means their commercial acquisition.

Substances of the general formula (V) were either obtained from commercially available sources, e.g. methacryloyl6-aminopropylamine protected on the amine groups by tert-butyloxycarbonyl groups (MA-Pr-NH-Boc), or were prepared according to the procedures indicated in the literature (Etrych T., et al., N(2-Hydroxypropyl)methacrylamide-Based Polymer Conjugates with pH-Controlled Activation of Doxorubicin. I. New Synthesis, Physicochemical Characterization and Preliminary Biological Evaluation, J.Appl.Pol.Sci. 109, 3050-3061 (2008) , V. Šubr, et al., Synthesis and properties of new N-(2-hydroxypropyl)-methacrylamide copolymers containing thiazolidine-2-thione reactive groups, React. Funct. Polym. 66 (12) (2006) 1525-1538. ). The provision of substances of general formula (V) therefore means either their commercial acquisition or synthesis.

ad b) The polymerization of the monomers of the statistical linear copolymer is carried out by controlled radical RAFT polymerization (reversible addition-fragmentation chain transfer) of monomers from step a) with a content of 0.1 to 10 mol. % of monomers of the general formula (V), and at least 90 mol. % (90 to 99.9 mol. %) of monomer units, selected from the group including N-(2-hydroxypropyl)methacrylamide (HPMA), acrylamide, methacrylamide, acrylate, methacrylate, preferably selected from the group including HPMA, acrylamide, methacrylamide, most preferably HPMA. The reaction typically takes place at a temperature in the range of 30 to 100°C, preferably 40 to 80°C, and a solvent preferably selected from the group consisting of dimethylsulfoxide, dimethylacetamide, dimethylformamide, methanol, ethanol, dioxane, tetrahydrofuran, propanol, tert-butanol or their mixture.

The reaction is initiated by an initiator, preferably selected from the group including in particular the azoinitiators 2,2'-azobis(2-methylpropionitrile) (AIBN), 4,4'-azobis(4-cyanopentanoic acid) (ACVA), 2,2'-azobis (4-methoxy-2,4-dimethylpentanenitrile) (V70), optionally in the presence of a transfer agent, preferably selected from the group containing 2-cyano-2-propylbenzodithioate, 4-cyano-4-(thiobenzoylthio)pentanoic acid, 2-cyano -2-propyldodecyltrithiocarbonate, 2-cyano-2-propylethyltrithiocarbonate, 4-cyano-4[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, [4-(3-azidopropylamino)-1-cyano1-methyl-4-oxo-butyl]benzenecarbodithioate, N- (3-Azidopropyl)-4-cyano-4-ethylsulfanylcarbothioylsulfanylpentanamide, [1-cyano-1-methyl-4-oxo-4-(2-thioxothiazolidin-3yl)butyl]benzenecarbodithioate and 2-ethylsulfanylcarbothioylsulfanyl-2- methyl 5-oxo-5-(2-thioxothiazolidin-3-yl)pentanenitrile. The Mn molar mass of linear random copolymers thus prepared is in the range from 4000 to 100,000 g/mol, preferably 25,000 to 50,000 g/mol. The resulting semitelechelic linear copolymer contains terminal reactive groups (for example, azide and TT).

The prepared linear copolymer from step b) can optionally be further subjected to the removal of protecting groups protecting the hydrazide groups of the side chains (for example, Boc groups). Deprotection can be accomplished by established procedures known to those skilled in the art, such as removal of the Boc group with trifluoroacetic acid or by heating the copolymer in water. The resulting copolymer/product can be stored without the risk of its decomposition.

The product of this step is thus a semitelechelic random linear copolymer, which contains a linear polymer selected from the group consisting of polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(N-(2-hydroxypropyl)methacrylamide) in which from 0.1 to 10 mol. % monomer units statistically replaced by a monomer unit of the general formula (VI)

- 10 CZ 2021 - 423 A3 * A

D (knows) where A and D are defined above.

and c) binding of the fluorophore to group D of the statistical linear copolymer from step b) is carried out by conjugation of carbonyl-thiazoline-2-thione groups, (4-nitrophenyl)oxy groups, (2,3,4,5,6pentafluorophenyloxy groups, (succinimidyl) oxy groups, carboxyl, hydrazide, azide, activated disulfide groups and NH2-groups (while the amino group and the hydrazide group, in case it was protected by a protecting group in the previous step, must be protected from this conjugation) of monomeric units of the general formula (VI) of semitelechelic of the statistical linear copolymer from step b), with a low molecular weight fluoro form. A fluoro form is a fluorescent label or a derivative thereof that contains suitable reactive groups (eg amine, carboxyl, activated carboxyl, activated disulphide or keto group) and which can be used in free form or as a salt with an acid, eg HCl; wherein a low molecular weight fluorophore is a fluorophore having a molecular weight in the range of 350 to 1,500 g/mol. The fluorophore is selected according to excitation and emission wavelengths, with excitation wavelengths ranging from 300 to 850 nm and emission wavelengths ranging from 350 to 1200 nm; any molecule of a low-molecular-weight fluorophore linked to a linear copolymer by an amide, disulfide or hydrazone bond.

The product of this step is thus a semitelechelic random linear copolymer containing monomer units of general formula (I), (VI), and further containing monomer units selected from the group including acrylamide, methacrylamide, acrylate, methacrylate and N-(2-hydroxypropyl-methacrylamide).

ad dl) linking the directing structure to the monomer units of the general formula (VI) of the semitelechelic random linear copolymer from step c) - step of conjugation of the directing structure with introduced amino groups, SH, azide, propargyl sulfodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups to group D defined above. The targeting structure is selected from the group comprising oligopeptides with a number of amino acids from 3 to 20, and proteins, preferably antibodies, more preferably monoclonal antibodies, targeting tumor cells of head and neck tumors, breast tumors, melanomas and colorectal tumors or tumor endothelium cells, wherein oligopeptides are attached using a click reaction of the terminal azide with DBZO groups on the polymer; antibodies are linked by a click reaction of the terminal maleimide group of the semitelechelic fluorescent polymer with the thiol group of the antibody molecule introduced by mild reduction.

In a preferred embodiment, oligopeptides with a number of amino acids from 3 to 20 are selected from the group including YESIKVAVS, SIGYPLP, RGD and cRGD (tumor endothelium), CPLHQRPMC (prostate tumors), NPWGYIGERPQYRDL called GE7 and YHWYGYTPQNVI called GE11 (to the EGF receptor), WHYPWFQNWAMA ( head and blood tumor cells), DMPGTVLP (breast tumors), CNGRC, cyclic RGDfK, HEWSYLAPYPWF and SYSMEHFRWGKPV. Oligopeptides and proteins that target tumor tissues in the body are known to those skilled in the art.

In a preferred embodiment, the monoclonal antibodies are selected from the group including herceptin, erbitux, daratuzumab, trastuzumab. Peptide and protein structures with introduced aminos

- 11 CZ 2021 - 423 A3 groups, azide, sulfodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups; groups of the fluorescent polymer react with -NH2, propargyl, sDBCO, or DBCO groups present on the directing structure in minutes and in high yield, and the resulting conjugate retains its biological activity unaffected.

and d2) linking the directing structure to the ends of the semitelechelic statistical linear fluorescent polymer from step c)

- a step of conjugation of the directing structure with amino groups introduced by SH, azide, propargyl, sulfodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups to the terminal group of the linear polymer, which can be a meleinimidyl group, a TT group, azide, propargyl, DBCO, or sDBCO. The routing structure is defined above.

Any unreacted D groups (thiazolidine-2-thione groups, (2,3,4,5,6pentafluorophenyl)oxy groups, (succinimidyl)oxy groups) can be removed by reaction with an amino alcohol selected from the group consisting of NH2-(CH2)a- CH 2 (OH); NH2-(CH2)b-CH(OH)CH3; NH2-(CH2)b-CH(OH)-(CH2)c-CH3; where a is an integer from 0 to 4, b is an integer from 0 to 3 and c is from 1 to 4, preferably with 1-aminopropan-2-ol, and/or any unreacted NH2 groups can be removed by reaction with acetythiazolidin- 2-thione, and/or any unreacted disulfide groups are removed by reaction with 2-Hydroxyethanethiol;

to form a group C selected from the group consisting of carboxyl; hydrazide; azide; -S-S-(CH2)2-OH, C(=O)-NH-(CH2)a-CH2(OH); -C(=O)-NH-(CH 2 ) b -CH(OH)-CH 3 ; -C(=O)-NH-(CH2)b-CH(OH)(CH2)c-CH3; and -NH-C(=O)-CH3, wherein a is an integer from 0 to 4, b is an integer from 0 to 3, and c is an integer from 1 to 4, preferably forming the group -C(=O) -NH-CH2(OH), -C(=O)-NH-CH(OH)CH3, -C(=O)-NH-(CH2)2-CH2(OH) or -NH-C(=O) -CH3. Carboxyls, hydrazides and azides do not need to be removed.

The product of this step is a fluorescent polymer according to the present invention, i.e. a semitelechelic random linear copolymer, containing monomeric units of the general formula (I), optionally (II), and optionally (III), defined above, and further containing monomeric units selected from the group comprising acrylamide , methacrylamide, acrylate, methacrylate and N-(2-hydroxypropyl)methacrylamide.

The subject of the present invention is also a star-shaped fluorescent polymer that contains a multivalent carrier to which at least one fluorescent polymer according to the present invention is bound, wherein the multivalent carrier is selected from the group including poly(amidoamine) dendrimer of the second or third generation, 2,2-bis( second to fourth generation hydroxymethyl)propion dendrimer or dendron; polyols with the number of hydroxyl groups from 2 to 8, glycerol, penta-erythritol, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol.

In one embodiment, the star fluorescent polymer is a copolymer of general formula (VII) that contains a multivalent carrier and at least one fluorescent polymer of general formula (IV), defined above.

- 12 CZ 2021 - 423 A3

where the multivalent carrier is a poly(amidoamine) (PAMAM) dendrimer (for example with an ethylenediamine, 1,4-diaminobutane, 1,6-diaminohexane, 1,12-diaminododecane, cystamine) dendrimer of the second or third generation or 2,2-bis(hydroxymethyl )propion dendrimer or dendron (for example, with a trimethylol propane core) of the second to fourth generation; polyols with the number of hydroxyl groups from 2 to 8, for example ethylene glycol, polyethylene glycol, trimethylolpropane (1,1,1-tris(hydroymethyl)propane), glycerol, penta-erythritol, bis(2hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol;

wherein A, B, C and Z are as defined above;

Y is selected from the group consisting of a primary amino group; primary hydroxy groups; alkyne groups with a number of carbons from 3 to 6, e.g. propargyl; and a cyclooctyne group, which may optionally be further independently substituted by one or more groups selected from an alkyl group having 1 to 6 carbons and an aryl having 6 carbons, e.g. DBCO;

W is an amide bond between the primary amino group Y of the multivalent carrier and the carboxyl terminal group of the polymer of general formula (IV); or an ester bond between the primary hydroxy group Y of the multivalent support and the carboxyl end group of the polymer of the general formula (IV) or a triazole coupling, formed by the reaction of the azide end group of the polymer of the general formula (IV) and the alkyne or cycloalkyne group Y of the dendrimer or dendron (multivalent support).

A star-shaped fluorescent copolymer therefore consists of a central carrier molecule (for example PAMAM dendrimer, 2,2-bis(hydroxymethyl)propion dendrimer or dendron or polyol) to the end groups of which at least one chain of fluorescent polymer is bound, preferably a linear statistical copolymer of the general formula (IV ), defined above. End groups of the multivalent carrier are amino groups, hydroxy groups or alkyne groups with a number of carbons from 3 to 6, e.g. propargyl, or a cyclooctyne group, which can optionally be further independently substituted by one or more groups selected from an alkyl group with a number of carbons from 1 to 6 and an aryl with a carbon number of 6, e.g. DBCO, and a fluorescent polymer (for example, a linear copolymer of formula (IV) is attached to them via an amide, ester or triazole linkage.

Preferably, the star-shaped fluorescent copolymer contains from 2 to 48 linked fluorescent polymer chains, more preferably 3 to 32 linked fluorescent polymer chains, most preferably 4 to 24 linked fluorescent polymer chains. In the most preferred embodiment, the fluorescent polymer is a linear statistical copolymer of general formula (IV).

The molar mass of the Mn star fluorescent copolymer is preferably in the range of 60,000 to 1,000,000 g/mol, preferably 70,000 to 400,000 g/mol. The molar mass of each chain of the fluorescent polymer bound to the multivalent carrier is from 6000 to 100,000 g/mol, preferably 40,000 to 70,000 g/mol, while the molar mass of the multivalent carrier itself,

- 13 CZ 2021 - 423 A3, which is part of the star-shaped fluorescent copolymer, does not exceed 50,000 g/mol. This ensures that after the disintegration of the star-shaped fluorescent copolymer into a multivalent carrier and a fluorescent polymer in the tumor tissue and after the release of the fluorophore, all the resulting fragments have a molecular weight below the renal filtration limit and are therefore easily removed from the body by renal filtration.

The method of preparing the star polymer of general formula (VII) is shown in Scheme 1.

Scheme 1. Schematic of the preparation of a star copolymer, where n is an integer ranging from 1 to 48, where Y is a primary amino group, a hydroxy group, a C3-C6 alkyne group, or a cyclooctyne group of poly(amidoamine) or 2,2-bis(hydroxymethyl)propion of a dendrimer or dendron or polyol;

V is an azide or TT terminal reactive group introduced by a transfer agent to the end of the polymer chain of the fluorescent polymer of general formula (IV) during RAFT polymerization;

W is an amide bond, an ester bond or a triazole bond formed by the reaction of the azide of the polymer of general formula (IV) and the alkyne group or cycloalkyne group of the multivalent carrier.

Examples of schematic structures of multivalent carriers for the preparation of star-shaped fluorescent polymers are shown in Scheme 2 below:

- 14 CZ 2021 - 423 A3

(ϋ)

- 15 CZ 2021 - 423 A3

- 16CZ 2021 - 423 A3 mh ₂

(in)

Scheme 2: Examples of structures of multivalent carriers for the synthesis of star-shaped fluorescent copolymer: i) schematic representation of bis-MPA dendron with DBCO groups, ii) PAMAM dendrimer with amino groups, iii) bis-MPA dendrimer with propargyl groups, iv) polyols with hydroxyl number from 4 to 8; and v) an example of the structure of a star-shaped fluorescent copolymer with a PAMAM dendrimer support, in which the chains containing the linear fluorescent polymer are marked schematically with a wavy line. The maximum number of linear fluorescent polymer chains that can be attached to a multivalent carrier (for example, a dendrimer) is equal to the number of terminal groups of the multivalent carrier (in this case, 16 terminal amino groups of the dendrimer).

The subject of the present invention is also a method of preparing the star-shaped fluorescent polymer defined above, comprising the following steps:

i) providing a multivalent carrier selected from the group consisting of second or third generation poly(amidoamine) dendrimer, 2,2-bis(hydroxymethyl)propion dendrimer or second to fourth generation dendron; polyols with the number of hydroxyl groups from 2 to 8, glycerol, pentaerythritol, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol, porphyrin derivatives; terminated by groups Y selected from primary amino groups, keto groups, azide groups, hydroxy groups, alkyne groups with a number of carbons from 3 to 6, e.g. propargyl; and cyclooctyne groups, which may optionally be further independently substituted by one or more groups selected from an alkyl group having 1 to 6 carbon atoms and an aryl having 6 carbon atoms, e.g. DBCO.

Said multivalent carriers are commercially available, optionally terminal DBCO groups can be prepared by reaction of terminal primary amino groups with DBCO-NHS. The provision therefore means the commercial acquisition of a multivalent carrier, possibly followed by the modification of its end groups.

ii) preparation of a random linear copolymer which contains a linear polymer selected from the group consisting of polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(V-(2hydroxypropyljmethacrylamide), in which from 0.1 to 10 mol.% of the monomer units are statistically replaced by a monomer unit of general formula (VI) which contains terminal reactive groups (for example, azide, DBCO, hydrazide, amino groups and TT), this preparation is described above under the preparation of a linear fluorescent polymer;

- 17CZ 2021 - 423 A3 iii) step of grafting terminal reactive functional groups (for example, azide or TT) of the statistical linear copolymer prepared in step ii) to the Y groups of the multivalent carrier from step i), to form a star-shaped polymer, which contains in its arms a statistical linear copolymer, selected from the group including polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(N-(2-hydroxypropyl)methacrylamide), in which from 0.1 to 10 mol. % monomer units statistically replaced by a monomer unit of the general formula (VI). The grafting reaction of polymers onto multivalent carriers takes place in a solvent preferably selected from the group including dimethylsulfoxide, dimethylacetamide, dimethylformamide, methanol and ethanol. The molar mass Mn of the star-shaped polymers prepared in this way is in the range from 60,000 to 1,000,000 g/mol, preferably from 100,000 to 400,000 g/mol.

iv) linking the fluorophore to group D of the statistical linear copolymer from step iii) by conjugation of carbonyl-thiazoline-2-thione groups, (4-nitrophenyl)oxy groups, (2,3,4,5,6-pentafluorophenyl)oxy groups, (succinimidyl )oxy groups, carboxyl, hydrazide, azide, activated disulfide groups or NH2-groups of monomer units of the general formula (VI) with an amine, carboxyl, activated carboxyl, activated disulfide or keto group of the fluorophore; wherein both the fluorophore and its attachment to group D are described above; to form a fluorescent star-shaped copolymer containing monomeric units of general formula (I) and (VI) and further containing monomeric units selected from the group including acrylamide, methacrylamide, acrylate, methacrylate and N-(2-hydroxypropyl)methacrylamide;

The binding of the fluorophore to the D group is carried out by conjugation of free hydrazide groups, amino groups, activated sidulfide groups or thiazolidine-2-thione (TT) groups of the monomeric units of the general formula (VI) of the random linear copolymer described above with a low-molecular fluorescent label or its derivative ( fluorophore) which contains suitable reactive groups (for example an amine, carboxyl, activated carboxyl or keto group) and which can be used in free form or in the form of a salt with an acid, for example HCl; wherein a low molecular weight fluorophore is a fluorophore having a molecular weight in the range of 350 to 1,500 g/mol. The fluorophore is selected according to excitation and emission wavelengths, with excitation wavelengths ranging from 300 to 850 nm and emission wavelengths ranging from 350 to 1200 nm; any low molecular weight fluorophore molecule is bound to the linear copolymer by an amide, disulfide or hydrazone bond.

v) optionally binding the directing structure defined above (oligopeptides with the number of amino acids from 3 to 20 or protein, especially monoclonal antibodies), to groups D of the monomer unit of the general formula (VI) of the fluorescent star-shaped copolymer from step iv).

Binding of the directing structure (peptide structures with introduced amino, azide, sulfodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups) is carried out by reacting the D group of the monomer unit of general formula (VI) with -NH2, propargyl, sDBCO or DBCO groups present on the directing structure . The reaction takes place in the order of minutes and with a high yield, and the resulting conjugate retains its biological effect unaffected.

vi) optionally attaching a directing group to the linear chain ends of the fluorescent star polymer from step iv) or v);

Binding of the directing structure (peptide structures with amino groups or introduced azide, maleinimidyl, propargyl, sulfodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups) is carried out by reaction of the terminal group, TT group, azide, propargyl, DBCO or sBCO group or SH group, with groups present on the directing structure. The reaction takes place in the order of minutes and with a high yield, and the resulting conjugate retains its biological effect unaffected.

- 18 CZ 2021 - 423 A3 vii) Any unreacted thiazolidine-2-thione groups, (4-nitrophenyl)oxy groups, (2,3,4,5,6pentafluorophenyl)oxy groups, (succinimidyl)oxy groups can be removed by reaction with an amino alcohol selected from the group consisting of NH 2 -(CH 2 ) _and -CH 2 (OH); NH2-(CH2)b-CH(OH)CH3; NH 2 -(CH 2 ) b -CH(OH)-(CH 2 ) _c -CH 3 ; where a is an integer from 0 to 4, Z? is an integer from 0 to 3 and is from 1 to 4;, preferably with 1-aminopropan-2-ol, and/or the unreacted NH 2 groups can be removed by reaction with acetylthiazolidin-2-thione. Activated disulfide groups are removed by reaction with mercaptoethanol. Carboxyls, hydrazides and azides do not need to be removed.

The present invention further relates to the use of fluorescent probes for visualization of tumors for diagnostic purposes and for image-guided surgery.

Fluorescent probe means a fluorescent polymer according to the present invention defined above, a star-shaped fluorescent polymer according to the present invention defined above, and also a statistical linear fluorescent copolymer selected from the group including polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(A-(2-hydroxypropyl) )methacrylamide) in which from 0.1 to 10 mol. % of monomer units, based on the total number of monomer units, statistically replaced by monomer units of the general formula (la) fluorophore (la), where

A is selected from the group consisting of a straight or branched carbon alkylenyl chain having from 1 to 7 carbons ((C1 to C7)alkylenyl); -(CH2) _P- (C(O)-NH-(CH2) _r )p-;

-(CH ₂ )p-(C(O)-NH-(CH ₂ )r)pC(O)-; -(CH ₂ )p-(C(O)-NH-(CH 2 ) _r )pC(O)-NH-C 6 H 4 -CH ₂ -OC(O)-; and (CH2)pC(O)-NH-(CH2)pL-(CH2)p-(C(O)-NH-(CH2)r)pC(O)-NH-C ₆ H4-CH2-OC(O )-, where L is a coupling containing a triazole bridge, formed for example by the reaction of a propargyl or DBCO group with an azide group; Thus, L can have, for example, the following structure:

N=N or

- 19 CZ 2021 - 423 A3

where p is an integer ranging from 1 to 5, and r is selected from 1, 2 and 3;

wherein one or more hydrogen atoms in the CH 2 groups of the substituent A may be further substituted with the same or different side chains of a natural amino acid; these chains are preferably methyl, isopropyl, isobutyl, -CH(CH ₃ )(CH ₂ CH ₃ ), -CH ₂ OH, -CH(OH)(CH ₃ ), -CH ₂ (C ₆ H ₄ )OH, -(CH ₂ ) ₂ -S-CH ₃ , -CH ₂ SH, -(CH ₂ ) ₄ -NH ₂ , -CH ₂ COOH, -CH ₂ C(O)NH ₂ , -(CH ₂ ) ₂ COOH, (CH ₂ ) ₂ C(O)NH ₂ , -(CH ₂ ) ₃ NH-C(=NH)(NH ₂ ), -(CH ₂ ) ₃ NH-C(=O)(NH ₂ ); benzyl;

B is selected from the group consisting of the bond, -C(=O)-NH-, -NH-C(=O)-,

and wherein the fluorophore has a molecular weight in the range of 350 to 1500 g/mol, an excitation wavelength in the range of 300 to 850 nm, and an emission wavelength in the range of 350 to 1200 nm, and is ^-ch covalently bonded to -ΝΗ -, -C(=O)-, =CH-, or -S-S-group of the substituent B of the monomer unit of the general formula (la) of the semitelechelic random linear copolymer via its primary amino group, or NCS group, N-hydroxysuccinimidyl group, keto group or disulfide group, whereby the groups formed after the fluoro forum are subsequently formed are part of group B;

and wherein the molecular weight M _n of the statistical linear fluorescent copolymer is in the range of 6000 to 100,000 g/mol.

In one embodiment, the fluorescent probe may carry a combination of a fluorophore whose fluorescence is activated in the tumor and a targeting group to increase the specificity of tumor tissue visualization. In this embodiment, the statistical linear fluorescent copolymer further contains at least one directing group for directing the fluorescent probe to tumor tissues, which is selected from the group including oligopeptides with the number of amino acids from 3 to 20 and proteins for directing the fluorescent polymer to tumor cells of head and neck tumors, breast tumors, melanomas and colorectal tumors or to tumor endothelium cells. The directing group can be attached to the terminal group of the linear chain of the fluorescent probe or it can be part of the monomeric unit (Ila)

-20CZ 2021 - 423 A3

CH3

UO

HN

AND

B' of (Ila), where A and B are as defined above;

Z is a directing group for directing the fluorescent polymer to tumor tissues, covalently bonded to the -NH-, -C(=O)-, =CH-, or -S-S- group of the substituent B of the monomer unit of the general formula (Ila) semitelechelic statistical of a linear copolymer through its primary amino group, or a keto group or a disulfide group, whereby the groups formed after the linking of the directing group are subsequently part of group B;

whereby the amount of monomer units of the general formula (Ila) in the fluorescent polymer is from at least one monomer unit to 8 mol. %, based on the total number of monomeric units of the fluorescent probe;

while the total number of monomer units of general formula (Ia) and (Ila) in the fluorescent probe is no more than 10 mol. %, based on the total number of monomer units.

In one embodiment, the statistical linear fluorescent copolymer may further comprise at least one monomeric unit of the general formula (IIIa)

CH _3n

(lilac) where

A is defined above;

C is selected from the group consisting of -C(=O)-NH-(CH ₂ ) _and -CH ₂ (OH); -C(=O)-NH-(CH ₂ ) _b -CH(OH)CH ₃ ; -C(=O)-NH-(CH ₂ ) _b -CH(OH)-(CH ₂ ) _c -CH ₃ ; and -NH-C(=O)-CH ₃ , where a is an integer from 0 to 4, Z? is an integer from 0 to 3 and is an integer from 1 to 4, -SS-(CH ₂ ) ₂ -OH ;

wherein the amount of monomer units of the general formula (IIIa) in the semitelechelic linear fluorescent copolymer is from at least one monomer unit to 9.9 mol. %, based on the total number of monomeric units of the fluorescent polymer;

-21 CZ 2021 - 423 A3 and while the total number of monomer units of the general formula (Ia), (Ila) and (Ila) in the statistical linear fluorescent copolymer is no more than 10 mol. %, based on the total number of monomer units.

The fluorescent probe can also be a star-shaped fluorescent polymer that contains a multivalent carrier to which at least one statistical linear fluorescent copolymer containing monomer units of the general formula (Ia), or (Ila) or (Ila) defined above is bound.

The multivalent carrier is selected from the group consisting of a second or third generation poly(amidoamine) dendrimer, a 2,2-bis(hydroxymethyl)propion dendrimer, or a second to fourth generation dendron; polyols with the number of hydroxyl groups from 2 to 8, glycerol, penta-erythritol, bis(2hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol, porphyrin derivatives.

The method of preparing fluorescent probes according to the present invention is analogous to the methods of preparing fluorescent polymer and star-shaped fluorescent polymer described above. The input monomers are selected from the group including acrylamide, methacrylamide, acrylate, methacrylate, X-(2-hydroxypropyl)methacrylamide and monomer of general formula (Va)

HN

AND

D (Va);

where A is as defined above and D is selected from the group consisting of carbonyl-thiazoline-2-thione group, (4-nitrophenyl)oxy group, (2,3,4,5,6-pentafluorophenyl)oxy group, (succinimidyl)oxy group , a carboxyl group, a hydrazide group, an azide group, a disulfide group and an NH2-group, whereby the amino group and the hydrazide group can optionally be protected by a protecting group.

The subject of the present invention is therefore the use of the fluorescent probe according to the present invention in medical diagnostics, whole-body imaging and/or fluorescence-guided surgery, preferably in the diagnosis and monitoring of the success of treatment in cancer diseases, diseases of the hematopoietic system (leukemia, lymphomas, hematopoietic failure) and the immune system . Fluorescent probes according to the present invention can be used, for example, in whole-body imaging techniques based on fluorescence detection for the detection of tumor tissue; in fluorescence-guided surgery for marking and imaging fluorescence in target structures of organs and body tissues.

Thus, the fluorescent probes are activatable linear and star-shaped fluorescent polymers according to the present invention, in which the fluorophore is bound to the polymer carrier by a biologically degradable amide, hydrazone or disulfide bond, and they allow to significantly extend the circulation time of the bound fluorophore in the organism, which enables on a high-molecular polymer carrier deliver the fluorophore to the tumor and release it here in its original form. Another advantage of these activatable fluorescent probes according to the invention is the activation of fluorescence, which is given by the release of the fluorophore from the polymer carrier.

-22 CZ 2021 - 423 A3

The polymer activatable fluorescent probes according to the invention are further characterized by the fact that the binding of the fluorophore to the polymer chain is relatively stable, it releases up to 10% of the fluorophore in 24 h during transport in the bloodstream and body fluids, and is hydrolytically, enzymatically or reductively cleavable in the tumor environment and inside target tumor cells in lysosomes. This means that the fluorophore is transported through the bloodstream in an inactive, low-fluorescence-active form, and its release and activation of fluorescence mainly occur after entering the tumor tissue or after penetration into the target tumor cells. Activation of the fluorophore only in the target cells leads to a significant increase in fluorescence in the tumor, to an increase in the tumor/healthy tissue contrast and thus to a clear visualization of the tumor tissue. By binding the fluorophore to the polymer chain, there will be a significant increase in the molecular weight of the contrast agent and thus an increase in its circulation time in the bloodstream and thus an increase in its bioavailability. Responsible for the targeted transport to the tumor or tumor cells is a polymer carrier prepared preferably based on HPMA copolymers, whose molecular weight and thus the efficiency of accumulation in the tumor tissue can be controlled by changes in the structure of the polymer carrier (linear polymer, high molecular weight biodegradable star-shaped polymer). Due to the increased molecular weight of the polymer carrier, the entire conjugate is accumulated in solid tumors due to the EPR effect. Polymeric probes can advantageously also be directed actively by means of linked directing structures, oligopeptides and proteins, which further increases the selectivity of accumulation in tumor tissue and supports an increase in tumor/healthy tissue contrast.

Summary

The subject of the invention is a directional and activatable fluorescent polymer and a star-shaped fluorescent polymer for enhancing the visualization of solid tumors. Due to their hydrodynamic size in solution and targeting to receptors on tumor cells, they are significantly accumulated in solid tumors, resulting in a significant enrichment of the tumor tissue with the present fluorophore. In addition, the carried fluorophore will be released in the tumor tissue environment, which results in an increase in the fluorescence signal due to a decrease in fluorescence quenching, which is associated with the binding of the fluorophore to the polymer system. As a result, there is a very significant increase in the fluorescence contrast between tumor and non-tumor tissue, which significantly contributes to the visualization of tumor tissue during guided tumor surgery. Contrast enhancement significantly advances the boundaries of guided surgery compared to the current state.

Clarification of drawings

Giant. 1: Fluorescence intensity of the fluorophore before and after hydrolysis of the pol-PYR-Cy7 conjugate in Example 10.

Giant. 2: Rate of release of fluorophore from conjugates with DY-676 attached to the polymer by pH-sensitive linkers with different structures in Example 11.

Giant. 3: Confocal microscopy images - Example 13.

Giant. 4: In vivo fluorescence imaging of polymeric systems with fluorescent label Dyomic 676; A - polymer system with a label firmly bound by an amide bond to a non-degraded linker; B- polymer system with a label bound via a hydrazone coupling formed by OPB-Dy676 - Example 14

Examples of embodiments of the invention

Examples of the synthesis of intermediates and conjugates according to the invention

Example 1: Synthesis of polymer precursors

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The copolymer poly(HPMA-co-Ma-AP-TT) was prepared by controlled solution radical copolymerization of HPMA (93 mol%, 100 mg) and 3-(3-Methacrylamidopropanoyl)thiazolidine-2thione (Ma-AP-TT) (7 mol% , 14.0 mg) performed in the presence of the initiator 2,2'-azobis(4methoxy-2,4-dimethylvaleronitrile) (V70) and the transfer agent 4-cyano-4thiobenzoylsulfanylpentanoic acid (CTA). The polymerization mixture was dissolved in tert-butyl alcohol (751 μL), CTA dissolved in 10% v/v DMA (83 μL) and all transferred to a glass ampoule where the mixture was bubbled with Ar and the ampoule sealed. After 24 h at 40 °C, the polymer was isolated by precipitation into acetone/diethyl ether, the precipitate was then washed with diethyl ether and dried in vacuo. The terminal dithiobenzoate groups were removed from the copolymer by reaction with AIBN (10-fold molar excess) in DMSO (15% polymer solution) under an argon atmosphere for 3 h at 70 °C in a sealed ampoule. The polymer conjugate was isolated by precipitation into acetone. The precipitate was washed with diethyl ether and dried in vacuo to dryness. The prepared poly(HPMA-co-Ma-APTT) conjugate had M„ = 28,000 g/mol, D = 1.05, TT content 5.9 mol%.

The precursor for the click reaction was prepared by reacting poly(HPMA-co-Ma-AP-TT) (100 mg, 39.3 μmol) with DBCO-NH2 (5.5 mg, 19.9 μmol) in DMA (1 mL) in the presence of DIPEA base (3.4 μL , 19.9 μmol). The progress of the reaction was monitored using HPLC. In the case of some polymers prepared for binding of labels by amide bond, the polymer poly(HPMA-co-Ma-AP-TT-co-MA-AP-DBCO) was separated, and in the case of polymers for binding of the label via click chemistry, the remaining TT groups on the polymer were removed by adding 1-aminopropan-2-ol (3.0 μl, 39.3 μmol). The polymer precursor poly(HPMA-co-Ma-AP-DBCO) was precipitated into acetone/diethyl ether and reprecipitated from MeOH, washed with diethyl ether and dried. The same polymer precursors were also used for directional conjugates with either an enzymatically cleavable fluorophore or control conjugates with a rigidly bound fluorophore.

Polymer precursors poly(HPMA-co-Ma-X-TT), where X = GG, GLG, GFG, GLFG, GFLFG, and poly(HPMA-co-Ma-XX-NH-NH-Boc), where XX= - CH 2 -CH 2 -(C=O)-(ethyl); -CH 2 -CH 2 -CH 2 (C=O)-(propyl); -CH 2 ) 4 -(C=O)-(butyl); -CH 2 ) 5 -(C=O)-(pentyl); -CH 2 ) 6 -(C=O)-(hexyl); -CH 2 ) 7 (C=O)-(heptyl); GG, GLG, GFG, GLFG, GFLFG, were prepared analogously according to this procedure.

Poly(HPMA-co-Ma-XX-NH-NH-Boc) precursors were used for the binding of conjugates with fluorescent labels bound via pH-sensitive hydrazone couplings, where the Boc protecting groups were removed in TFA and the poly(HPMA) polymer was precipitated after 10 min -co-Ma-XX-NH-NH2) into diethyl ether, precipitated from MeOH and dried. For the synthesis of directed conjugates with a pH-sensitive hydrazone bond, part of the hydrazide groups was unreacted with DBCO-NHS, and for the subsequent click reaction with the peptide, poly(HPMA-co-MaXX-NH-NH2-co-MaXX-NH-NH-DBCO) was used .

The disulfide polymer was prepared by reacting poly(HPMA-co-MaXX-NH-NH2) with succinimidyl 3-(2pyridyldithio)propionate) (SPDS) to form the polymer precursor poly(HPMA-co-Ma-XXNH-NH-PDS).

The characteristics of the prepared polymer precursors are shown in Table 1.

Table 1.

Conjugate structure M _w (g/mol) IP Content of reactive groups (mol%) poly(HPMA-co-Ma-AP-TT) 28,000 1.05 5.9 poly(HPMA-co-Ma-GG-TT) 27,500 1.10 6.1 poly(HPMA-co-Ma-GFG-TT) 26,800 1.12 5.8 poly(HPMA-co-Ma-GLFG-TT) 28,400 1.12 5.8 poly(HPMA-co-Ma-GFLFG-TT) 27,800 1.13 5.6 poly(HPMA-co-Ma-Ethyl-NH-NHi) 35,300 1.08 8.2

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poly(HPMA-co-Ma-Propyl-NH-NH ₂ ) 35,000 1.10 8.1 poly(HPMA-co-Ma-Pentyl-NH-NH ₂ ) 35,500 1.07 8.2 poly(HPMA-co-Ma-Heptyl-NH-NH ₂ ) 35,300 1.08 8.0 poly(HPMA-co-Ma-Hexyl-NH-NH ₂ ) 35,000 1.08 8.2 poly(HPMA-co-Ma-Hexyl-NH-NH-PDS) 36,200 1.10 7,8

Example 2: Synthesis of a star-shaped polymer with a triazole spcjka

The synthesis of the star-shaped copolymer took place in two steps. First, a reactive copolymer p(HPMA-co-Ma-Pentyl-NHNH-Boc)-N3 was prepared using the transfer agent azide-CTA, N(3-azidopropyl)-7-ethylsulfanylcarbothioylsulfanyl-7-methyl-pentanamide, containing an azide group, in a similar manner to the reactive copolymer in Example 1. Further, the Boc groups were removed by boiling in water and the polymer was subsequently reacted with a bisMPA dendrimer containing terminal DBCO or propargyl groups sp(HPMA-co-Ma-Pentyl-NHNH ₂ )-N3 in methanol for 2 h. The resulting star-shaped polymer conjugate was precipitated into acetone and dried to constant weight. Characterization of the resulting star-shaped polymer conjugate: A'/„=250,000 g/mol, £>=1.20, content of hydrazide groups=8% mol. By changing the polymer/dendrimer core ratio and changing the dendrimer generation, it is possible to control polymer systems in a wide range. Similarly, for the preparation of star-shaped conjugates, it is also possible to use a derivatized bis-MPA dendron or a PAMAM dendrimer, on which they are introduced

DBCO or propargyl groups. Star-shaped copolymers with polymers containing other linkers were prepared in a similar manner.

Scheme 3. Synthesis and structure of star copolymers

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Example 3: Synthesis of a star-shaped conjugate with an ester spcjka

The synthesis of the star-shaped copolymer took place in three steps. In the first step, pentaerythritol was modified using dibenzocyclooctyne-N-hydroxysuccinimidyl ester. Furthermore, a reactive copolymer p(HPMA-co-Ma-Pentyl-NHNH-Boc)-N3 was prepared using the transfer agent azide-CTA, X-(3-azidopropyl)-7-ethylsulfanylcarbothioylsulfanyl-7-methyl-pentanamide, containing an azide group , in a manner similar to the reactive copolymer in Example 1. Further, the Boc groups were removed by boiling in water and subsequently the polymer was reacted with pentaerythritol containing the terminal DBCO groups sp(HPMA-co-Ma-Pentyl-NHNH2)-N3 in methanol for 2 h. The resulting star-shaped polymer conjugate was precipitated into acetone and dried to constant weight. Characterization of the resulting star-shaped polymer conjugate: A/ _w = 1,80,000 g/mol, £>=1.25, content of hydrazide groups = 8% mol. Star-shaped copolymers with polymers containing other polyols were prepared in a similar manner.

Scheme 4: An example of the structure of a star-shaped copolymer based on pentaerythritol and a linear copolymer.

Example 4: Synthesis of a star-shaped conjugate with an amide spcjka

The synthesis of this star-shaped conjugate took place in two steps. First, the reactive copolymer p(HPMA-co-Ma-Pentyl-NHNH-Boc)-TT was prepared using the transfer agent TTCTA, [l-cyano-l-methyl-4-oxo-4-(2-thioxothiazolidin-3-yl )butyl]benzenecarbodithioate, containing a TT group, in a similar way to the copolymer in Example 1. In the second step, the bis-MPA dendrimer containing amino groups was reacted with sp(HPMA-co-Ma-Pentyl-NH-NH)-TT in methanol for 2 h. The resulting star-shaped polymer conjugate was precipitated into acetone and dried to constant weight. Characterization of the resulting star-shaped polymer conjugate: A£ _w =220,000 g/mol, £>=1.18, content of hydrazide groups = 8% mol. By changing the ratio

-26CZ 2021 - 423 A3 polymers/dendrimer core and by changing the dendrimer generation, it is possible to control a wide range of polymer systems. Similarly, it is possible to use bis-MPA dendron or PAMAM dendrimer with amino groups for the preparation of star-shaped conjugates.

Scheme 5: Example of a 2nd generation bis-MPA dendron structure with a linear copolymer linked via an amide bond. The maximum number of linear copolymer chains that can be attached to a dendron is equal to the number of terminal groups of the dendron (in this case 4 terminal amino groups).

Example 5: Preparation of fluorophore derivatives

Preparation of the derivative Az-Val-Cit-Aba-DY-67 6 and Az-Val-Cit-DY-676

5-Azidopentanoyl-valyl-citrulline (Az-Val-Cit-OH)

The dipeptide derivative was prepared by solid-phase peptide synthesis on 2-chlorotrityl chloride resin (0.5 g, substitution 1 mmol/g) by reacting 0.2 M solutions of amino acids (Fmoc-Cit-OH, FmocVal-OH, 5-azidopentanoic acid) . The product was cleaved from the resin with a 30% solution of HFIP in DCM, yielding 121 mg (0.23 mmol, 46%) of the azido-peptide derivative. ESI MS (calcd 399.5, found 422.5 M+Na). According to HPLC, 1 peak is visible at _r = 1.39 min.

N-(5-Azidepentanoyl-valyl-citrullyl)-DY-676 (Az-Val-Cit-DY-676)

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Az-Val-Cit-OH (0.5 mg, 1.2 pmol) and the amino derivative DY-676 (1 mg, 1.2 pmol) were dissolved in DMA (0.5 mb) with DIC (0.3 pL, 1.8 pmol) and HOBt (0.3 mg, 1.8 pmol). The progress of the reaction was monitored by 5 HPLC. After 24 h, the dye and the coupler reacted and, according to HPLC, there was one peak corresponding to the product Az-Val-Cit-DY-676.

N-(5-Azidopentanoyl-valyl<atrolyte)-4-aminobenzyl alcohol (Az-Val-Cit-Aba)

Az-Val-Cit-OH (80 mg, 0.2 mmol), 4-aminobenzyl alcohol (26 mg, 0.21 mmol), 1-hydroxybenzotriazole (35 mg, 0.23 mmol), and DIC (36 μL, 0.23 mmol) were dissolved in a 2 mL mixture DMF: DCM (3:2), the reaction mixture was stirred for 1 h at 0 °C and then allowed to react for 16 h at 25 °C.

The solvent was concentrated under reduced pressure and the product was isolated by precipitation into diethyl ether, filtered and purified on preparative HPLC (Chromolith C18, linear gradient water-acetonitrile 0-100%). The reaction yield was 53 mg (0.11 mmol, 55%) of a white powder. ESI MS (calcd 504.6, measured M+H=505.6). One peak at vt _r =1 .74 min is visible on HPLC.

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N-(5-Azidecpentanoyl-valyl-citruly)-4-aminobenzyl (4-nitrophenyi) carbonate (Az-Val-Cit-AbaNpc)

Az-Val-Cit-Aba (50 mg, 0.1 mmol) was dissolved in DMA (0.15 mL), 4-nitrophenyl chloroformate (60 mg, 0.3 mmol) was dissolved in DCM (0.5 mL), the solutions were mixed and pyridine (0.15 mL) was added. The progress of the reaction was monitored by HPLC, the product was isolated on preparative HPLC (Chromolith C18, linear gradient water-acetonitrile 0-100%), the yield of the reaction was 50 mg (75 mmol, 10 75%) of a white powder. ESI MS (calcd 669.7, measured M+H=670.7). On HPLC, there is one peak at t _r =2.55 min.

N-[N'-(5-Azidopentanoyl-valyl-citrullate)-4-aminobenzyloxycarbonate] DY-676

Az-Val-Cit-Aba-Npc (0.8 mg, 1.2 pmol) and the amino derivative DY-676 (1 mg, 1.2 pmol) were dissolved in DMA (0.5 mL) with DIPEA (0.25 pL, 1.4 pmol). The progress of the reaction was monitored by HPLC. After 24 h

-29CZ 2021 - 423 A3 the dye and the coupler reacted and, according to HPLC, one peak corresponding to the product Az-Val-Cit-AbaDY-676 was formed.

Derivatives with other fluorescent labels (Dyomics782, Dyomics-633, Dyomics-781, Dyomics-776, Dyomics-777, Dyomics-778, Dyomics 780, Cyanine-7, Cyanine-7.5, Cysanine-5, Cyanine- 5.5, indocyanine green).

Example 6: Derivatization of ketoacids

Carboxylic keto acids such as 5-cyclohexyl-5-oxopentanoic (COP), 4-(2-oxopropyl)benzenecarboxylic (OPB) and 4-oxo-4-(2-pyridyl)butanoic (PYR) were activated by reaction with thiazolidine-2 -thione, this reaction is shown in Scheme 6.

O

DOC, DMAP

-18 °C (4 °C), THF, 20 h

Scheme 6

100 mg (0.56 mmol) of OPB acid and 132 mg (0.67 mmol, 1.2x mol. equiv. to the keto acid) of ΛξΑ'-dicyclohexylcarbodiimide DCC were weighed, these weights were dissolved in 0.78 ml of tetrahydrofuran (THF) (2.45 dm ³ -mol ^-1 ). Next, 39.4 mg (0.59 mmol, 1.05 x mol. equiv. to the keto acid) of thiazolidine-2-thione TT and 2 mg (catalytic amount) of 4-(dimethylamino)pyridine (DMAP) were weighed, these weights were dissolved in 0.30 mL THF (1.12 dnT-moF ¹ ).

Subsequently, the solutions were cooled to -18 °C and mixed after twenty minutes. With constant stirring, this temperature was maintained for one hour, and the temperature was 4°C for the next twenty hours. Subsequently, the reaction mixture was freed from A, A'-dicyclohexylurea by filtration, the filtrate was evaporated under reduced pressure and dissolved in ethyl acetate. This procedure was repeated once more and the product was purified by double crystallization in a mixture of ethyl acetate:dichloromethane (vol. 1/1). The yields of these reactions range between 30 and 50%.

The same procedure was performed with COP and PYR.

The activated acids were then derivatized with an amino derivative of a fluorescent molecule, e.g. Cyanine7 (Cy7) or Dyomics 676 (DY-676). 1.39 mg (4.65 pmol) of COP acid and 3.35 mg (4.65 pmol, 1.0 mol eq. to the keto acid) of Cy7 were weighed. Both weights were dissolved in 1.193 ml (256.5 dm ³ -mol ^-1 ) of solvent (dimethylformamide - methanol, vol. 3/1) and then mixed. A 2.6M solution of sodium hydroxide (1.10 mol. equiv. to the keto acid) was added to the solution. The reaction was carried out at room temperature for three hours with constant stirring. The progress of the reaction was monitored using HPLC and TLC (ethyl acetate). The reaction was terminated by evaporation of the solvent. The yields of these reactions range between 94 and 99%.

The same procedure was performed with OPB and PYR.

Example 7: Preparation of peptide azido derivatives

Oligopeptides GE-7, GE-11, CNGRC, cyclic RGDfK, HEWSYLAPYPWF and SYSMEHFRWGKPV were prepared by solid-phase synthesis using a microwave peptide synthesizer using the standard Fmoc method from the C-terminus of the peptide using an N-Fmoc-protected amino acid (2.5 eq.), DIC (2.5 eq.) as activator and Oxymy (2.5 eq.) as base in

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DMF. After binding of the last N-Fmoc-amino acid and removal of the Fmoc group, 5-azidopentanoic acid (2.5 eq.), PyBOP (2.5 eq.) as an activator, and DIPEA (5 eq.) as a base were attached.

Example 8: Preparation of polymer conjugates containing a fluorescent label by polymer analogue reaction

The polymer conjugate poly(HPMA-co-Ma-GFLG-DY-676) with an aminolytically bound dye via a GFLG linker was prepared by reacting the amino derivative of the dye (2 mg) with the polymer precursor poly(N-(2-hydroxypropyl)methacrylamide-co- N-methacryloylglycyl-leucylphenylalanyl-glycine thiazolidine-2-thione) (poly(HPMA-co-Ma-GLFG-TT) (98 mg) in 1 mL DMA in the presence of DIPEA base (eq. to the dye). The reaction was monitored by HPLC and after binding all the free dye, the remaining TT groups on the polymer were removed by the addition of 1-aminopropan-2-ol (eq. to TT at the beginning of the reaction). The reaction mixture was purified by gel filtration (Sephadex LH20) and the polymer fraction was concentrated and precipitated into an acetone mixture /diethyl ether and washed with diethyl ether, the precipitate dried to constant weight.The dye content was determined and the molar mass was measured by SEC of the prepared polymer conjugate.

A control polymer conjugate poly(HPMA-co-Ma-AP-DY-676) with a tightly bound dye without a degradable sequence was also prepared from the polymer precursor poly(N-(2hydroxypropyl)methacrylamide-co-methacrylamidopropanoyl)thiazolidine-2-thione) (poly(HPMA-co-Ma-AP-TT). The contents of fluorescent labels in the polymer conjugates are shown in Table 2.

The polymer conjugate poly(HPMA-co-Ma-AP-DBCO-Az-ValCit-Aba-DY-676) was prepared by a two-step synthesis. In the first step, (poly(HPMA-co-Ma-AP-TT) was converted to poly(HPMA-co-Ma-AP-DBCO) by reaction with DBCO-NH2 in the presence of DIPEA base (eq. to DBCONH2). In the second step, to the precursor poly(HPMA-co-Ma-AP-DBCO) (48.6 mg) dissolved in 300 μl of DMA, a solution of the modified dye Az-Val-Cit-Aba-DY-676 was added to form a triazole bridge. After the disappearance of the peak of the free dye according to HPLC, the reaction mixture was precipitated into acetone/diethyl ether 2:1, the precipitate washed with diethyl ether and dried. The precipitate was dissolved in MeOH and purified by gel filtration (Sephadex LH20). The polymer fraction was concentrated and precipitated into diethyl ether. The dye content was determined and molar mass measured by SEC of prepared polymer conjugate.

Example 9: Preparation of conjugates with a pH-sensitive bond

The polymer conjugate with a pH-sensitive bond poly(HPMA-co-Ma-Heptyl-NH-NH=COP-DY-676) was prepared by the reaction of the keto acid-modified dye COP-Dy-676 (2 mg) and the polymer precursor poly(HPMA-co -Ma-Pentyl-NH-NH2) (98 mg) in 1 mL of MeOH with 40 μL of acetic acid and stirred at room temperature for 48 h. The progress of the reaction was monitored by HPLC and TLC (CHClb/MeOH/acetic acid - 8/2/1). Then the reaction products were purified by column gel chromatography using Sephadex LH-20 gel, methanol was used as the mobile phase. The product fractions were concentrated by evaporation of methanol under reduced pressure and precipitated into ethyl acetate, centrifuged and dried to constant weight. The yields of these reactions range between 50 and 70%. Subsequently, the fluorophore content was determined spectrophotometrically and the molar mass was measured using SEC.

Example 10: Preparation of directed conjugates

Peptide-directed conjugates were prepared by click reaction of an azido-derivative of the GE-7 peptide with a polymeric precursor with DBCO groups, which was prepared from the precursor poly(HPMA-coMa-GFLG-TT) converted to poly(HPMA-co-Ma-GFLG-TT- co-Ma-GFLG-DBCO) by reaction with 0.5-4 mol% DBCO-NH2 in the presence of DIPEA base (eq. to DBCO-NH2). After binding the fluorophore, see Example 8, the azido-derivative of the peptide was bound by an uncatalyzed click reaction in DMA. In the same way, other directed conjugates of azido-derivative peptides GE-11, CNGRC, were prepared by cyclic

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RGDfK, HEWSYLAPYPWF and SYSMEHFRWGKPV. A control directed conjugate with tightly bound dye was prepared from poly(HPMA-co-Ma-AP-TT) as described above.

Peptide-directed conjugates with a pH-sensitive bond were prepared as in Example 9, and further DBCO-NHS was bound to the remaining hydrazide groups in the presence of DIPEA base (eq. to DBCO-NHS) in methanol. The azido-derivative of the peptide was then attached to the polymer conjugate by a click reaction via DBCO.

Antibody-directed conjugates were prepared from the semitelechelic precursor poly(HPMAco-Ma-Pentyl-NH-NH-Boc)-TT, terminated with a reactive TT group prepared using the transfer reagent TT-CTA as in Example 4. The terminal TT group was reacted with N- (2-aminoethyl)maleimide was converted to maleimide, which was used, after deprotection of the Boc protecting group and attachment of the fluorophore, for reaction with the reduced antibody rituximab.

The antibody was reduced by gentle reduction with dithiothreitol in PBS buffer and after reduction was purified from free dithiothreitol on a PD10 column. It was subsequently mixed with a polymer conjugate bearing a hydrazone-linked fluorescent label and a terminal maleimide group.

The prepared targeted conjugates, see Table 2, were characterized, the dye content was determined and the peptide/antibody content was determined by amino acid analysis. The molar mass could not be measured due to the interaction of the laser used in the GPC detectors with the fluorophore, which made it impossible to determine these characteristics.

Example 11: Fluorescence quenching on a polymer

Fluorescence quenching was experimentally performed by measuring the fluorescence intensity of the fluorescent polymer poly(HPMA-co-Ma-Acap-NH-NH=COP-DY-676) in a phosphate buffer (0.3M, pH 7.4) with a fluorescent polymer concentration of 0.1 g^dm ³ and fluorescence intensity of this solution after total hydrolysis. This was achieved by adding concentrated vinegar (¼ of the original volume) to the original solution. This dilution was compensated for by multiplying this intensity by 1.25x. It was found that after binding there is a significant decrease in the fluorescence of the bound fluorophores, and the fluorescence is restored again after release from the polymer, see Fig. 1. An experiment was similarly carried out with polymer conjugates with hydrazone-bound OPB and PYR derivatives of the fluorescent label DY-676 and also with identical derivatives of other fluorescent labels, e.g. DY-782, Cy-7, DY-767, and then with the fluorescent label DY- 676 or CY-7 bound via enzymatically cleavable linkers GLG, GFG, GLFG, GFLFG, GFLG, Val-Cit and Val-Cit-Aba, where significant quenching of the fluorescence signal upon binding to the polymer and subsequent activation of fluorescence upon incubation with the model was also observed by the lysosomal enzyme cathepsin B.

The activation of the fluorescent signal can be used to increase the fluorescence within the tumor tissue in tumor cells, where after passive or active accumulation, the fluorophore is released either due to the reduced pH of the tumor tissue or due to the activity of lysosomal enzymes after the conjugates enter the tumor cells. This activation of the signal subsequently leads to a significant increase in the contrast of the fluorescent signal in the tumor environment and beyond. The increase in contrast can be used within the framework of navigated tumor surgery, when the improved tumor/non-tumor tissue contrast will allow the surgeon to clearly highlight the tumor mass and therefore to perform a precise resection of this tissue.

Example 12: Release of fluorophores from polymer systems

Fluorophore release was measured by HPLC. Fluorescent polymers with a hydrazone-linked fluorophore were incubated in phosphate buffer (0.3M, pH 7.4) at a concentration of 0.1 g^dm ³ at 37°C. At 0, ½, 1, 2, 4, 6, 8 and 10 hours, the amount of released fluorophore was measured. From Fig. 2 it can be seen that the release rate of the fluorophore is dependent on the pH and the type of coupling. Cleavage at physiological pH 7.4 is slower than cleavage at acidic pH, simulating tumor tissue.

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A significant increase in the fluorescence intensity caused by the release of the fluorophore from the polymer will therefore only occur in the tumor tissue, where there will be an increase in the contrast between the tumor and healthy tissue. Fluorescent polymers with a fluorescent label bound via enzymatically degradable linkers were incubated in phosphate buffer (KH2PO4, NaOH, pH=6.0, 0.001 M EDTA and 0.01 M 5 glutathione)) at a concentration of 0.1 g^dm ³ at 37° C in the presence of cathepsin B (2*10 ⁷ mol l ^-1 ).

At times 0, 1, 2, 8 and 24 hours, the amount of released fluorophore was measured. After cleavage with cathepsin B, the release of the fluorophore and a significant increase in fluorescence were detected.

Example 13: Physical characterization of conjugates

The fluorophore content was determined spectrophotometrically and the fluorophore content in the conjugates was determined: pol-COP-Cy7 (ε = 91,000 dm ³ ^mol ^- ^cm ^-1 ) 0.4 wt. %, pol-OPB-Cy7 (ε = 83,000 dm ³ ^mol ^- ^cm ^-1 ) 1.8 wt. % and pol-PYR-Cy7 (ε = 60,000 dm ³ ^mol ^-b cm ^-1 ) 0.4 wt. %.

Table 2

Conjugate structure Fluorophore content (wt%) Peptide/antibody content (wt%) poly(HPMA-co-Ma-Heptyl-NH-NH=COP-Cy7) 0.5 - poly(HPMA-co-Ma-Heptyl-NH-NH=COP-DY-676) 1.4 - poly(HPMA-co-Ma-Heptyl-NH-NH=OPB-Cy7) 1.8 - poly(HPMA-co-Ma-Heptyl-NH-NH=OPB-DY676) 1 - poly(HPMA-co-Ma-Heptyl-NH-NH=PYR-Cy7) 1 - poly(HPMA-co-Ma-Heptyl-NH-NH=PYR-DY676) 0.9 - poly(HPMA-co-Ma-AP-DBCO-Az-ValCit-Aba-DY676) 1.8 - poly(HPMA-co-Ma-GFLG-DY-676) 1.8 - poly(HPMA-co-Ma-AP-DY-676) 1.9 - poly(HPMA-co-Ma-Heptyl-NH-NH=OPB-Cy7-co-MaHeptyl-NH-NH-DBCO-cRGDfK) 1.5 10.2 poly(HPMA-co-Ma-GFLG-DY-676-co-Ma-GFLGDBCO-cRGDfK) 1.7 11.2 poly(HPMA-co-Ma-Heptyl-NH-NH=OPB-Cy7-co-MaAcap-NH-NH-DBCO-GE-7) 1.6 12.3 poly(HPMA-co-Ma-AP-DBCO-Az-Val-Cit-Aba-Cy7co-Ma-AP-DBCO-GE-7) 1.5 10.3 poly(HPMA-co-Ma-GFLG-DY-676-co-Ma-GFLG- DBCO-GE-11) 1.7 12.1 poly(HPMA-co-Ma-GFLG-DY-676-co-Ma-GFLGDBCO-HEWSYLAPYPWF) 1.7 12.1

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PAMAM-P-OPB-Cy7 1.2 - Bis-MPA-P-OPB-Cy7 1.3 - Rituximab-(poly(poly(HPMA-co-Ma-Heptyl-NHNH=OPB-Cy7))6 1.2 44.3 Erbitux-(poly(poly(HPMA-co-Ma-Heptyl-NH- NH=PYR-DY-676))6 1.3 45.1

Example 14: In Vitro Testing

Cells with the fluorescent polymer poly(HPMA-co-Ma-Acap-NH-NH=OPB-Cy7) at a concentration of 5 mg^dm ³ were incubated at 37 °C for 6 and 24 h. Then fluorescence was measured using a confocal microscope . From Fig. 3. it is noticeable that in 24 h there will be a significant increase in fluorescence due to the cleavage of the fluorophore, as the polymer conjugate had enough time to penetrate the cells. On the contrary, at the time of 6 h, it is clear that no hydrolysis or enzymatic cleavage of the couplings has yet occurred, and therefore no release.

Example 15: In vivo testing

An in vivo experiment was performed on athymic nude mice (Hs1Cpb:NMRI-Foxn1 ^nu ) bearing DLD-1 colorectal tumor. Mice were injected with the fluorescent polymers poly(HPMA-co-MaAP-DY-676) or poly(HPMA-co-Ma-Acap-NH-NH=OPB-DY676), which had a fluorescent label attached either by an amide bond to a non-degradable linker in the lateral chains of the polymer carrier, or by a pH-sensitive link (hydrazone bond). After 24 h, the mice were examined using a non-invasive imaging system, Fig. 4. It was found that while in mice with a fixed label the fluorescence is spread very homogeneously within the body of the mouse with a slightly increased intensity at the tumor site (Fig. 4A), so in mice with administered pH-sensitive fluorescent polymer (Fig. 4B) it was found , that the fluorescence is mainly localized in the tumor tissue and that the contrast with respect to the healthy tissue is significantly higher than in the stable system.

Claims (14)

1. A fluorescent polymer for tumor visualization that contains a semitelechelic random linear copolymer selected from the group consisting of polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(A-(2-hydroxypropyl)methacrylamide) in which from 0.1 to 10 mol. % of monomer units, based on the total number of monomer units, statistically replaced by monomer units of the general formula (I) ϊ AND B fluorophores (I) where A is selected from the group consisting of a linear or branched carbon alkylenyl chain having from 1 to 7 carbons; -(CH2)p-(C(O)-NH-(CH2) _r )p-; -(CH ₂ )p-(C(O)-NH-(CH ₂ ) _r )pC(O)-; -(CH ₂ )p-(C(O)-NH-(CH 2 ) _r )pC(O)-NH-C 6 H 4 -CH ₂ -OC(=O)-; α-(CH ₂ )pC(O)-NH-(CH ₂ )pL(CH ₂ )p-(C(O)-NH-(CH2) _r )pC(O)-NH-C6H4-CH ₂ -OC (O)-, where L is a linkage containing a triazole bridge; where p is an integer ranging from 1 to 5, and r is selected from 1, 2 and 3; wherein one or more hydrogen atoms in the CH 2 groups of the substituent A may be further substituted with the same or different side chains of a natural amino acid; and wherein the fluorophore has a molecular weight in the range of 350 to 1500 g/mol, an excitation wavelength in the range of 300 to 850 nm, and an emission wavelength in the range of 350 to 1200 nm, and is covalently bonded to =CH-, -- or the -S-S- group of the substituent B of the monomer unit of the general formula I of the semitelechelic random linear copolymer through its primary amino group, or the NCS group, the N-hydroxysuccinimidyl group, the keto group or the disulfide group, whereby the groups formed after the binding of fluorophorum are subsequently part of the B group ; and wherein the molecular weight M _n of the fluorescent polymer ranges from 6000 to 100,000 g/mol; and wherein when B is a bond, then A is -(CH 2 ) _P -(C(O)-NH-(CH 2 ) _r )pC(O)-; or -(CH ₂ )p-(C(O)-NH-(CH 2 ) _r )pC(O)-NH-C 6 H 4 -CH ₂ -OC(O)-; or -(CH2)pC(O)-NH-(CH2)pL-(CH2)p-(C(O)-NH-( _CH2 )r)pC(O)-NFI-C6H4-CH2-OC(O) -. 2. Fluorescent polymer according to claim 1, which further contains at least one directing group for directing the fluorescent polymer to tumor tissues, which is selected from the group including oligopeptides with a number of amino acids from 3 to 20 and proteins for directing the fluorescent polymer to tumor cells of head and neck tumors , breast tumors, melanomas and colorectal tumors or to tumor endothelium cells. -35CZ 2021 - 423 A3 3. The fluorescent polymer according to claim 2, wherein the directing group is attached to the terminal group of the linear chain of the fluorescent polymer. 4. Fluorescent polymer according to claim 2 or 3, which further contains monomer units of general formula (II) c ² r° HN % A X ^x z (Π) where A is as defined in claim 1; O O JI , V nh d . A ,n _x A 1 X expression, 1 , Av ,-S-S-, L, where L is defined in the claim 1; Z is a directing group for targeting the fluorescent polymer to tumor tissues, covalently bound to =CH-,_______I, -CH=CH-, -CH=CH- in the structure DBCO, N3 or -S-S group of the substituent X of the monomeric unit of general formula (II ) of a semitelechelic random linear copolymer via an amide bond, a maleimide, an azide, or a propargyl, whereby the groups formed after the linking of the directing group are subsequently part of the X group; whereby the amount of monomer units of general formula (II) in the fluorescent polymer ranges from at least one monomer unit to 8 mol. %, based on the total number of monomeric units of the fluorescent polymer; while the total number of monomer units of the general formula (I) and (II) in the fluorescent polymer is no more than 10 mol. %, based on the total number of monomer units. 5. A fluorescent polymer according to any one of the preceding claims, wherein the semitelechelic random linear copolymer is poly(A-(2-hydroxypropyl)methacrylamide). 6. A fluorescent polymer according to any one of the preceding claims, wherein A is selected from the group consisting of ethane-1,2-diyl; propane-1,3-diyl; butane-1,4,-diyl; pentane-1,5-diyl; hexane-1,6-diyl; heptane-1,7-diyl; -CH ₂ -C(=O)-NH-CH ₂ -; -CH ₂ -C(=O)-NH-CH(CH ₂ -CH(CH ₃ ) ₂ )-C(=O)-NH-CH ₂ -; CH ₂ -C(=O)-NH-CH(CH ₂ Ph)-C(=O)-NH-CH ₂ -; -CH ₂ -C(=O)-NH-CH(CH ₂ -CH(CH ₃ ) ₂ )-C(=O)-NHCH(CH ₂ Ph)-C(=O)-NH-CH ₂ -; -CH ₂ -C(=O)-NH-CH(CH ₂ Ph)-C(=O)-NH-CH(CH ₂ -CH(CH ₃ ) ₂ )C(=O)-NH-CH(CH ₂ Ph)-C(=O)-NH-CH ₂ -; -(CH ₂ ) ₄ -C(=O)-NH-CH(C(CH ₃ ) ₂ )-C(=O)-NHCH((CH ₂ )3-NH-C(=O)-NH ₂ ) -C(=O)-NH-C ₆ H ₄ -CH ₂ -OC(=O); -(CH ₂ ) ₄ -C(=O)-NH-CH(C(CH ₃ ) ₂ )-C(=O)-NH-CH((CH ₂ )3-NH-C(=O)-NH ₂ )-C(=O)-. 7. Fluorescent polymer according to any one of the preceding claims, which further contains at least one monomer unit of general formula (III) -36CZ 2021 - 423 A3 CH. HN A C where (III) A is defined in claim 1; C is selected from the group consisting of -C(=O)-NH-(CH ₂ ) _and -CH ₂ (OH); -C(=O)-NH-(CH ₂ ) _b -CH(OH)CH ₃ ; -C(=O)-NH-(CH ₂ ) _b -CH(OH)-(CH ₂ ) _c -CH ₃ ; and -NH-C(=O)-CH ₃ , where a is an integer from 0 to 4, Z? is an integer from 0 to 3 and is an integer from 1 to 4, or -SS-(CH ₂ ) ₂ -OH; whereby the amount of monomer units of general formula (III) in the fluorescent polymer is from at least one monomer unit to 9.9 mol. %, based on the total number of monomeric units of the fluorescent polymer; and while the total number of monomer units of the general formula (I), (II) and (III) in the fluorescent polymer is no more than 10 mol. %, based on the total number of monomer units. 8. Fluorescent polymer according to any one of the preceding claims, which is a linear random copolymer of general formula (IV) (IV) where A, B and C are as defined above; wherein the total number of monomer units in the fluorescent polymer ranges from 100 to 300, and wherein the number of units of general formula (I) ranges from 1 to 30; the number of units of general formula (II) is in the range from 0 to 24 and the number of units of general formula (III) is in the range from 0 to 29, whereby the total number of monomer units (I), (II) and (III) is 30 at most. 9. A method for preparing a fluorescent polymer according to any one of claims 1 to 8, characterized in that it contains the following steps: i) controlled radical polymerization with reversible-fragmentation chain transfer - RAFT - 90 to 99.9 mol. % of monomers selected from the group including acrylamide, methacrylamide, acrylate, methacrylate, V-(2-hydroxypropyl jmethacrylamide; and 0.1 to 10 mol. % of the monomer of the general formula (V) -37CZ 2021 - 423 A3 > (V) wherein A is as defined in claim 1; D is selected from the group consisting of carbonyl-thiazoline-2-thione group, (4-nitrophenyl)oxy group, (2,3,4,5,6-pentafluorophenyl)oxy group, (succinimidyl)oxy group, carboxyl group, hydrazide group , an azide group, a disulfide group and an NH2-group, wherein the amino group and the hydrazide group may optionally be protected by a protecting group; to form a random linear copolymer that contains a linear polymer selected from the group consisting of polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(V-(2hydroxypropyljmethacrylamide), in which from 0.1 to 10 mol.% of the monomer units are statistically replaced by a monomer unit of the general formulas (VI) (know ) ii) optionally removing protecting groups from group D of monomeric units of general formula (VI); iii) linking the fluorophore to group D of the random linear copolymer by conjugation of carbonylthiazoline-2-thione groups, (4-nitrophenyl)oxy groups, (2,3,4,5,6-pentafluorophenyl)oxy groups, (succinimidyl)oxy groups, carboxylic, hydrazide, azide, activated disulfide groups or NH2-groups of monomeric units of general formula (VI) with an amine, carboxyl, activated carboxyl, activated disulfide or keto group of the fluorophore; wherein fluoro is as defined in claim 1; iv) optionally attaching a directing group to the D group of the monomer of general formula (VI), wherein the directing group is defined in claim 2; v) optionally attaching a directing group to the linear chain ends of the fluorescent polymer, wherein the directing group is as defined in claim 2; vi) removal of any unreacted thiazolidine-2-thione groups, (4-nitrophenyl)oxy groups, (2,3,4,5,6-pentafluorophenyl)oxy groups, (succinimidyl)oxy groups by reaction with an amino alcohol selected from the group consisting of NH2 -(CH 2 ) _and -CH 2 (OH); NH2-(CH2)b-CH(OH)CH3; NH 2 -(CH 2 ) b -CH(OH)-(CH 2 ) _c -CH 3 ; where a is an integer from 0 to 4, Z? is an integer from 0 to 3 and is from 1 to 4, preferably with 1-aminopropan-2-ol, and/or removal of unreacted NH 2 groups by reaction with acetylthiazolidin-2-thione; optionally removal of activating disulfide groups by reaction with mercaptoethanol; to form a fluorescent polymer according to any one of claims 1 to 8. -38CZ 2021 - 423 A3 10. A star-shaped fluorescent polymer that contains a multivalent carrier to which at least one fluorescent polymer according to any of the preceding claims 1 to 8 is bound, wherein the multivalent carrier is selected from the group comprising a second or third generation poly(amidoamine) dendrimer, 2,2- second to fourth generation bis(hydroxymethyl)propion dendrimer or dendron; polyols with the number of hydroxyl groups from 2 to 8, glycerol, penta-erythritol, bis(2hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol, porphyrin derivatives. 11. Star fluorescent polymer according to claim 10, which has the general formula (VII), (VII) where A, B, C, Z and the fluorophore are as defined above; Y is selected from the group consisting of a primary amino group; primary hydroxy groups; alkyne groups having from 3 to 6 carbons; a cyclooctyne group, which may optionally be further independently substituted by one or more groups selected from alkyl having 1 to 6 carbons and aryl having 6 carbons; DBCO Group; W is an amide bond between the primary amino group Y of the multivalent carrier and the carboxyl terminal group of the polymer of general formula (IV); or an ester bond between the primary hydroxy group Y of the multivalent support and the carboxyl end group of the polymer of the general formula (IV) or a triazole coupling, formed by the reaction of the azide end group of the polymer of the general formula (IV) and the alkyne or cycloalkyne group of the Y of the multivalent support. 12. Star fluorescent polymer according to claim 10 or 11, which has a molar mass of Mn in the range of 60,000 to 1,000,000 g/mol, the molar mass of the multivalent carrier itself being at most 50,000 g/mol. 13. A method for preparing a star fluorescent polymer according to any one of claims 10 to 12, characterized in that it contains the following steps: i) preparation according to claim 9 of a statistical linear copolymer, which contains a linear polymer selected from the group consisting of polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(V-(2-hydroxypropyl)methacrylamide), in which from 0.1 to 10 mol. % monomer units statistically replaced by a monomer unit of general formula (VI); and which contains terminal reactive groups, preferably azide, DBCO, hydrazide, amino groups and TT; ii) the step of grafting the terminal reactive functional groups of the random linear copolymer prepared in step i) to the Y groups of a multivalent support, selected from the group comprising a second or third generation poly(amidoamine) dendrimer, a 2,2-bis(hydroxymethyl)propion dendrimer or a second to fourth generation; polyols with the number of hydroxyl groups from 2 to 8, glycerol, penta-erythritol, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol; terminated by groups Y, selected from primary amino groups, keto groups, azide groups, hydroxy groups, alkyne groups with a number of carbons from 3 to 6; and cyclooctyne groups, which may optionally be further independently substituted by one or more groups selected from an alkyl group having 1 to 6 carbon atoms and an aryl having 6 carbon atoms; to form a star-shaped polymer, which in its arms contains a statistical linear polymer, selected from the group including polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(V-(2-hydroxypropyl)methacrylamide), in which from 0.1 to 10 mol. % -39CZ 2021 - 423 A3 monomer units statistically replaced by a monomer unit of the general formula (VI). iii) optional removal of protecting groups from group D monomer units of general formula (VI); iv) linking the fluorophore to group D of the star-shaped polymer from step ii) by conjugation of carbonylthiazoline-2-thione groups, (4-nitrophenyl)oxy groups, (2,3,4,5,6-pentafluorophenyl)oxy groups, (succinimidyl)oxy groups , carboxyl, hydrazide, azide, activated disulfide groups or NH2-groups of monomeric units of the general formula (VI) with an amine, carboxyl, activated carboxyl, activated disulfide or keto group of the fluorophore; wherein the fluorophore is as defined in claim 1; to form a fluorescent star-shaped copolymer containing monomeric units of the general formula (I) and (VI) and further containing monomeric units selected from the group including acrylamide, methacrylamide, acrylate, methacrylate and A-(2-hydroxypropyl)methacrylamide; v) optionally attaching a directing group to the D group of the monomer of general formula (VI), wherein the directing group is defined in claim 2; vi) optionally attaching a directing group to the linear chain ends of the fluorescent polymer, wherein the directing group is as defined in claim 2; vii) removal of any unreacted thiazolidine-2-thione groups, (4-nitrophenyl)oxy groups, (2,3,4,5,6-pentafluorophenyl)oxy groups, (succinimidyl)oxy groups by reaction with an amino alcohol selected from the group consisting of NH2 -(CH 2 ) _and -CH 2 (OH); NH2-(CH2)b-CH(OH)CH3; NH 2 -(CH 2 ) b -CH(OH)-(CH 2 ) _c -CH 3 ; where a is an integer from 0 to 4, ů is an integer from 0 to 3, and c is from 1 to 4, preferably with 1-aminopropan-2-ol, and/or removal of unreacted NH2 groups by reaction with acetythiazolidin-2-thione ; removal of activated disulfide groups by reaction with mercaptoethanol; to form a star-shaped fluorescent polymer according to claim 10, 11 or 12. 14. A fluorescent probe for use in medical diagnostics as a contrast agent for tumor visualization, whole-body imaging and/or for image-guided surgery, particularly in fluorescence imaging techniques, preferably selected from the group including non-invasive imaging, image-guided surgery, microscopy, fluorescence flow cytometry , and in the diagnosis of tumor, inflammatory and bacterial diseases, whereby the fluorescent probe is selected from the group including: - fluorescent polymer according to any one of claims 1 to 8; - star fluorescent polymer according to claim 10 to 12; - statistical linear fluorescent copolymer, selected from the group including polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate and poly(A-(2-hydroxypropyl)methacrylamide), in which from 0.1 to 10 mol. % monomer units, based on the total number of monomer units, statistically replaced by monomer units of the general formula (la) ch ₃ _. fluorophore (la) where A is defined in claim 1; B is selected from the group consisting of the bond, -C(=O)-NH-, -NH-C(=O)-, -40CZ 2021 - 423 A3 ------------------------, -S-S-; and wherein the fluorophore has a molecular weight in the range of 350 to 1500 g/mol, an excitation wavelength in the range of 300 to 850 nm, and an emission wavelength in the range of 350 to 1200 nm, and is covalently bonded to -NH-, -C(=O)-, =CH-, or -S-S- group of the substituent B of the monomer unit of general formula (la) of the semitelechelic random linear copolymer through its primary amino group, or NCS group, N-hydroxysuccinimidyl group, keto group or disulfide group, while the groups formed after the fluorophores have been established are subsequently part of group B; and wherein the molecular weight M _n of the fluorescent random linear copolymer is in the range of 6000 to 100,000 g/mol; - a star fluorescent polymer that contains a multivalent carrier to which at least one statistical linear fluorescent copolymer is bound, containing monomeric units of the general formula (1a), wherein the multivalent carrier is selected from the group comprising a poly(amidoamine) dendrimer of the second or third generation, 2, Second to fourth generation 2-bis(hydroxymethyl)propion dendrimer or dendron; polyols with the number of hydroxyl groups from 2 to 8, glycerol, penta-erythritol, bis(2hydroxyethyl)aminotris(hydroxymethyl)methane, dipentaerythritol, porphyrin derivatives.