Fluorescent Cypate Conjugate of Hyaluronic Acid or Salt Thereof, Hydrophobized Conjugate, Methods of Preparation and Use Thereof

Abstract

The present invention relates to the fluorescent conjugate of hyaluronic acid containing cypate or salts thereof, the hydrophobized conjugate, methods of the preparation and use thereof in medicinal applications for in vivo imaging and the treatment of neoplasms.

Claims

1. A fluorescent conjugate of hyaluronic acid or a salt thereof of the general formula I, ##STR00009## wherein R.sup.+ is H.sup.+ or physiologically acceptable salts selected from the group containing Na.sup.+, K.sup.+, Mg.sup.2+ or Ca.sup.2+, R.sup.1 is H or a cypate residue of the formula II, where is in the place of covalent bond of cypate residue of the formula II ##STR00010## one R.sup.1 being cypate residue of formula II in at least one repeated unit providing that if there is R.sup.1 cypate residue of formula II in the unit, then the other R.sup.1 in the unit are H, and wherein n is an integer in the range 2 to 625.

2. The fluorescent conjugate of claim 1, where the residue of cypate of the formula II is in the position 6 of the glucosamine part of the fluorescent conjugate of the hyaluronic acid or the salt thereof of the general formula I.

3. The fluorescent conjugate of claim 1 or claim 2, where the degree of substitution of the residue of cypate of the formula II in the conjugate of hyaluronic acid or the salt thereof of the general formula I is from 0.1 to 2%, preferably 1.0%.

4. The fluorescent conjugate of any one of claims 1 to 3 that it absorbs the light in the area of 570 nm to 790 nm and it emits the light in the area of 680 nm to 850 nm, preferably at 850 nm.

5. The fluorescent conjugate of hyaluronic acid or the salt thereof of any one of claims 1 to 3, wherein R.sup.+, R.sup.1 and n are, as defined in claim 1, applying at the same time that in at least one repeated unit at least one R.sup.1 is C(O)R.sup.2, where R.sup.2 is C.sub.xH.sub.y substituent, where x is an integer in the range of 5 to 17 and y is an integer 11 to 35, whereas it is linear or branched, saturated or unsaturated C.sub.6-C.sub.18 aliphatic chain.

6. The fluorescent conjugate of claim 5, wherein the degree of substitution of C(O)R.sup.2 in the conjugate of hyaluronic acid or the salt thereof of the general formula I is from 3 to 70%, preferably 5 to 12%.

7. The fluorescent conjugate of claim 5 or claim 6, that it absorbs the light in the range of wavelengths 570 nm to 790 nm and it emits the light at 680 to 850 nm, preferably 850 nm.

8. A method of preparation of the conjugate of any one of claims 1 to 4, characterized in, that cypate I of the formula III ##STR00011## is activated with N,N-carbonyl diimidazole (CDI) in the aprotic polar solvent selected from the group containing dimethyl sulfoxide, dimethyl formamide, formamide or acetonitrile, preferably dimethyl sulfoxide; resulting in a reactive intermediate mono-imidazolide of the formula IV ##STR00012## that reacts with hyaluronic acid or the salt thereof in the presence of the organic base, that is either generated in situ in the form of imidazole, or it is added to the reaction mixture, the added organic base being selected from the group containing 1,4-diazabicyclo[2.2.2]octan, N,N,N,N-tetramethyl-1,6-hexandiamine, N-methyl morfolin, imidazole, triethylamine, or N,N-diisopropyl ethylamine, preferably imidazole generated in situ; and polar aprotic solvent, as is defined above.

9. The method of claim 8, characterized in, that the activation of cypate is performed at the temperature in the range 20 C. to 60 C., preferably at 22 C. to 25 C.; for 10 minutes to 20 hours, preferably 0.5 to 2 hours

10. The method of claim 8, characterized in, that the formation of the conjugate of hyaluronic acid or the salt thereof is performed at temperature 40 C. to 80 C., preferably 40 C. to 60 C., more preferably 60 C.; for 12 to 48 hours, preferably 24 hours.

11. The method of any one of claims 8 to 10, characterized in, that the molar ratio of cypate I:hyaluronic acid or the salt thereof:N,N-carbonyl diimidazole:the organic base is 0.5:1:0.5:0.5 to 3.5 in the reaction mixture, preferably the weight ratio is 0.5:1:0.5:1.

12. A method of a preparation of the fluorescent conjugate of any one of claims 5 to 7, characterized in, that the activation of the fat acid of the general formula V is performed
R.sup.2COOH(V), wherein R.sup.2 is C.sub.xH.sub.y, whereas x is an integer in the range 5 to 17 and y is an integer 11 to 35 and C.sub.xH.sub.y is linear or the branched, saturated or unsaturated chain; using the substituted or unsubstituted benzoyl chloride of the general formula VI ##STR00013## wherein R.sup.3 is one or more substituents selected from the group containing H, NO.sub.2, COOH, halogenides, C.sub.1-C.sub.6 alkyl alkoxy, preferably H; in the presence of the organic base selected from the group containing 1,4-diazabicyclo[2.2.2]octan, N,N,N,N-tetramethyl-1,6-hexandiamine, N-methyl morfolin, triethylamin or N,N-diisopropyl ethylamine, preferably triethylamine; and the polar solvent selected from the group containing isopropyl alcohol, tetrahydrofuran, preferably isopropyl alcohol to form the reactive anhydride of the general formula VII ##STR00014## wherein R.sup.2 and R.sup.3 are, as is defined above, that it esterifies the fluorescent conjugate of hyaluronic acid or the salt thereof of the general formula (I), as defined in any one of claims 1 to 3, in the presence of the organic base, preferably amine selected from the group containing (1,4-diazabi-cyclo[2.2.2]octan), N,N,N,N-tetramethyl-1,6-hexandiamine, N-methyl morfolin, imidazole, triethylamin or N,N-diisopropyl ethylamine, more preferably triethylamine; the mixture of water and the polar solvent miscible with water selected from the group containing isopropyl alcohol, dimethyl sulfoxide or tetrahydrofuran, preferably isopropyl alcohol.

13. The method of claim 12, characterized in, that the activation of the fat acid of the general formula V is performed for 0.5 to 24 hours, at the temperature in the range 0 C. to 60 C., preferably 0.5 hours at the temperature from 0 C. to 25 C., and the esterification of the fluorescent conjugate of hyaluronic acid or the salt thereof is performed for 0.5 to 2 hours, preferably 2 hours, at the temperature in the range of 22 C. to 25 C.

14. The method of claim 12 or claim 13 characterized in, that the amount of the organic base corresponds to 2 to 6 molar equivalents, preferably 4 molar equivalents per the dimer of hyaluronic acid or the salt thereof; the amount of the substituted or unsubstituted benzoyl chloride corresponds to 0.2 to 2.0 molar equivalents, preferably 0.6 molar equivalents per the dimer of hyaluronic acid or the salt thereof; the amount of the fat acid corresponds to 0.2 to 2.0 molar equivalents, preferably 0.6 molar equivalents of hyaluronic acid or the salt thereof.

15. The method of any one of claims 12 to 14 characterized in, that the amount of water in the mixture water and the polar solvent miscible with water is 50 to 80% v/v, preferably 50% v/v.

16. The fluorescent conjugate of any one of claims 1 to 7 for use in medicinal applications for in vivo imaging of the conjugate distribution, preferably for in vivo imaging of organs or neoplasms.

17. The fluorescent conjugate of claim 16 for use in the intravenous, intraperitoneal or subcutaneous application.

18. The fluorescent conjugate of claim 16 for use in administration for in vivo imaging of non-palpable and/or palpable tumors.

19. The fluorescent conjugate of claim 18 for use in the intravenous, intraperitoneal administration.

20. A composition on the basis of aggregated fluorescent conjugate of any one of claims 5 to 7 characterized in that it contains an aggregate of fluorescent conjugates and at least one or more nonpolar agents, preferably drugs and/or nanoparticles.

21. The composition of claim 20 characterized in that the drug is a cytostatic, preferably doxorubicin or paclitaxel.

22. The composition of claim 20 characterized in that the fluorescent conjugate is the conjugate of any one of claims 5 to 7, where R.sup.1 C(O)C.sub.17H.sub.33 and nanoparticles are superparamagnetic nanoparticles.

23. The composition of any one of claims 20 to 22 characterized in that it contains 2 to 15 wt. %, preferably contains 2 to 6 wt. % of nonpolar compounds in respect to weight content of the fluorescent conjugate of hyaluronic acid or the salt thereof.

24. The composition of any one of claims 20 to 23 for use in medicinal applications for in vivo imaging of neoplasms.

25. The composition of any one of claims 20 to 23 for use in the treatment of neoplasms.

Description

DESCRIPTION OF FIGURES IN DRAWINGS

[0053] FIG. 1: .sup.1H NMR (D.sub.2O) conjugate HA-Cypate.

[0054] FIG. 2: DOSY NMR spectrum (D.sub.2O) conjugate HA-Cypate.

[0055] FIG. 3: Chromatogram record of SEC-MALLS (HA-Cypate 14,000 g/mol) conjugate HA-Cypate (Example 3).

[0056] FIG. 4: Chromatogram record of SEC-MALLS (HA-Cypate 14,000 g/mol) conjugate HA-Cypate (Example 7).

[0057] FIG. 5: Chromatogram record of SEC-MALLS (HA-Cypate 58,000 g/mol) conjugate HA-Cypate (Example 8).

[0058] FIG. 6: Chromatogram record of SEC-MALLS (HA-Cypate 72,000 g/mol) conjugate HA-Cypate (Example 9).

[0059] FIG. 7: The emission spectrum of the fluorescence of conjugate HA-Cypate in the aqueous solution at the excitation 650, 660, 665 and 670 nm.

[0060] FIG. 8: The emission fluorescence of the conjugate in the aqueous solution at the excitation with laser k=632.8 nm without (left panel) and in a combination with the filter transmitting wavelengths over =635 nm (right panel).

[0061] FIG. 9: In vivo fluorescent imaging: HA-Cypate applied subcutaneously. The place of application is indicated with letter S. Figures show the detection of emission using the different excitation and emission filters.

[0062] FIG. 10: In vivo fluorescent imaging in time after the intraperitoneal application of HA-Cypate.

[0063] FIG. 11: In vivo fluorescent imaging in time after the intravenous application of HA-Cypate-C18:1.

[0064] FIG. 12: In vivo fluorescent imaging in time after the intravenous application of HA-Cypate-C18:1 (mouse with tumor, tumor cells indicated with chemiluminescent luciferase).

[0065] FIG. 13: The evaluation of in vivo luminescence of tumor after the administration of (i) HA-Cypate-C18:1 (=HA cyp), (ii) HA-Cypate-C18:1+doxorubicin (=HA cyp dox), (iii) HA-Cypate-C18:1+doxorubicin+spion (=HA cyp dox+spions).

[0066] FIG. 14: The comparison of spleen and liver weight after the administration of (i) HA-Cypate-C18:1 (=HA cyp), (ii) HA-Cypate-C18:l+doxorubicin (=HA cyp dox), (iii) HA-Cypate-C18:1+doxorubicin+spion (=HA cyp dox+spions).

[0067] FIG. 15: In vivo fluorescent imaging in time after the intraperitoneal application of HA-Cypate-C18:1 (mouse with tumor, tumor cells indicated with luminescent luciferase).

[0068] FIG. 16: The mass spectrum of dimer HA-Cypate obtained from the enzymatic degradation of the conjugate HA-Cypate with the hyaluronan lyase.

EXAMPLES OF THE EMBODIMENTS OF THE PRESENT INVENTION

Description of Instrumentation

[0069] NMR spectra were recorded on BRUKER AVANCE 500 at frequency 500.13 MHz (.sup.1H).

[0070] For the processing of experimental data was used the software by Bruker TOPSPIN 1.2 or software SpinWorks 3.1. For the interpretation of the spectra from NMR analyses were used abbreviations: s (singlet), d (doublet), t (triplet), m (multiplet). For UV/Vis spectra measurement in the wavelength range 190-800 nm was used UV/Vis spectrophotometer Varian Cary 100. Fluorescent spectra were recorded on the apparatus PTI Quantarnaster 400. ESI-MS analyses of Cypate were performed on the mass spectrophotometer with ionic trap amaZon X (BrukerDaltonics) equipped with the electrospray ionizing source and the quadrupole mass analyzer. Measurements were performed in the positive and negative mode. The structure of conjugate of HA-cypate was confirmed using LC-MS analysis of the conjugate after its enzymatic cleaving with hyaluronan lyase. The mixture of oligosaccharides was separated on column Kinetex 1.7 um F5 100A (Phenomenex) using gradient 0.1% HCOOH in H.sub.2O and acetonitrile. The detection was performed on Synapt G2-Si in negative resolution mode with ionization with electrospray. Analysis of samples and molecule mass of initial hyaluronan was determined using the method SEC-MALLS (HPLC Alliance) with UV/VIS 2489 and the refractometry detector RID 2414 and the detector of light scattering mini DAWN TREOS. Data were processed using the software Astra Version 5.3.4.20 (Wyatt Technology EuropeGmbH). All in vivo imaging analyses were carried out on the apparatus IVIS Luminia XR Series III) on a laboratory mice of the strain Balb/c.

Example 1. Synthesis of 3-(2-carboxyethyl)-1,2,2-trimethyl-1H-benzo[e]indolium-bromide

[0071] 2.0 g (9.6 mmol) of 1,1,2-trimethyl-1H-benzindol and 2.2 g (14.3 mmol) 3-bromopropanoic acid were dissolved in 10 ml 1,2-dichlorobenzen and under the constant stirring warmed at 115 C. for 16 hours. The crude reaction mixture was cooled down to the room temperature and the resulting precipitate was washed with 1,2-dichloromethane (1050 ml). The final product was separated using the filtration, dried under vacuum on the rotary evaporator (RE) and it was obtained in the form of light gray crystal powder (yield 2.2 g (64%)).

[0072] .sup.1H NMR (DMSO-d.sub.6, 500 MHz): 8.38 (d, J=8.35, 1H.sub.arom), 8.29 (d, J=9.00, 1H.sub.arom), 8.23 (d, J=8.35, 1H.sub.arom), 8.18 (d, J=9.00, 1H.sub.arom), 7.80-7.71 (m, 2H.sub.arom), 4.79 (t, J=6.95, 2H, CH.sub.2), 3.05 (t, J=6.95, 2H, CH.sub.2), 2.98 (s, 3H, CH.sub.3), 1.76 (s, 6H, CH.sub.3) ppm

[0073] ESI-MS: .sup.+MS [M].sup.+=282

Example 2. Preparation of Cypate

[0074] 0.8 g (2.8 mmol) glutaconic dialdehyde dianiline hydrochloride was dissolved in 8 ml 1,2-dichloromethane and tempered to 0-5 C. 521 l (5.5 mmol) of the anhydride of acetic acid, 481 l (2.8 mmol) and DIPEA in the small volume of 1,2-dichlormethan (0.5 ml) was added dropwise into the solution and the reaction mixture was left while cooling and stirring to react for 3 hours. In the meantime 2 g of (5.5 mmol) 3-(2-carboxyethyl)-1,2,2-trimethyl-1H-benzo[e]indolium-bromide prepared in Example 1 and 0.9 g (11.0 mmol) of sodium acetate in the mixture of solvents acetonitrile/water 95/5 of volume 15 ml were brought to reflux and the first reaction mixture was added dropwise. The reaction proceeded for 18 hours under stirring at reflux in dark. The crude reaction mixture was cooled down to the room temperature, washed with 400 ml ethyl-acetate and 400 ml 1M HCl, the product was filtrated and dried under the low pressure. The resulting product was obtained in the form of dark green crystal powder yielding 1.62 g (88%).

[0075] .sup.1H NMR (DMSO-d.sub.6, 500 MHz): 8.25 (d, J=8.75, 2H), 8.07-7.97 (m, 6H), 7.82 (t, J=12.65, 1H), 7.73 (d, J=8.75, 2H), 7.69-7.62 (m, 2H), 7.54-7.48 (m, 2H), 6.60 (t, J=12.65, 2H), 6.47 (d, J=13.75, 2H), 4.43 (bt, 4H CH.sub.2), 2.77 (t, J=6.9, 4H CH.sub.2), 1.92 (s, 12H CH.sub.3) ppm

[0076] ESI-MS: .sup.+MS [M].sup.+=625; .sup. MS [M-2H].sup.=623 m/z

[0077] UV/Vis: .sub.abs.max=782 nm (MeOH)

Example 3: Preparation of Conjugate HA-Cypate

[0078] 87 mg (0.13 mmol, 0.5 eqv.) Cypate from Example 2 was dissolved in 2 ml DMSO, 22 mg (0.13 mmol, 0.5 eqv.) N,N-carbonyl diimidazole was added and under the constant stirring was let activate 2 hours at the room temperature. In the meantime 100 mg (0.27 mmol, 1 eqv.) acid form of hyaluronic acid M.sub.w 14,000 g/mol was disolved in DMSO at 60 C. Then 15 l (0.13 mmol, 0.5 eqv.) N,N-methylmorfoline and the first reaction mixture without the previous isolation was added into the solution. The reaction proceeded under the constant stirring in dark at 60 C. 24 hours. The reaction was stopped by adding ten-fold of 100% isopropyl alcohol (AIPA) in respect to the initial volume of the reaction mixture and the saturated solution of NaCl, when precipitation of the desired product occurred. The crude product was purified with 5100 ml AIPA, dissolved in 50 ml demineralized water and transferred into dialysis tube. The protonized form of HA was neutralized 1.sup.st day in 0.5% solution of NaCl and 0.5% NaHCO.sub.3, further dialyzed 2.sup.nd and 3.sup.rd day in the demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as green lyofilizate of mass 89 mg (87%).

[0079] .sup.1H NMR (D.sub.2O) (FIG. 1): cypate: 8.80 (s, 2H), 8.50-8.47 (m, 2H), 8.46-8.43 (m, 2H), 8.29-8.19 (m, 2H), 8.16-8.10 (m, 2H), 8.08-7.89 (m, 2H), 7.88-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.46 (m, 2H), 5.16, 5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.02 (12H, CH.sub.3, overlaid with signals of HA), HA: 4.62-4.39 (m, 2H anomer), 4.01-3.28 (m, 10H skeletal), 2.02 (s, 3H, CH.sub.3) ppm

[0080] DOSY (D.sub.2O): (FIG. 2)

[0081] SS=1.5% (determined from .sup.1H NMR)

[0082] SEC-MALLS-LS-UV/Vis-RI: (FIG. 3)

[0083] Fluorimeter: .sub.em.max=695 nm (at .sub.exc.=665 nm; H.sub.2O); (FIG. 7)

[0084] Fluorescence of the conjugate excited with the laser wavelength =632.8 nm (FIG. 8)

[0085] ESI-MS: [M-H].sup.=984 m/z (FIG. 16, dimer detected after the enzymatic degradation of the conjugate with lyase)

Example 4: Preparation of Conjugate HA-Cypate

[0086] 87 mg (0.13 mmol, 0.5 eqv.) Cypate from Example 2 was dissolved in 2 ml DMSO, 22 mg (0.13 mmol, 0.5 eqv.) N,N-carbonyl diimidazole was added and under the constant stirring let to activate 30 min. at the room temperature. 100 mg (0.27 mmol, 1 eqv.) of the acid form of hyaluronic acid of M.sub.w 14,000 g/mol in DMSO at 60 C. was added. The reaction proceeded under the constant stirring in dark at 60 C. for 24 hours. The reaction was stopped by addition of ten-fold volume of 100% isopropyl alcohol (AIPA) in respect to the initial volume of the reaction mixture and the saturated solution of NaCl, then the precipitation of the desired product occurred. The crude product was purified with 5100 ml AIPA, dissolved in 50 ml demineralized water and transferred into the dialysis tube. The protonized form of HA was neutralized 1.sup.st day in 0.5% solution NaCl and 0.5% NaHCO.sub.3, further dialyzed 2.sup.nd and 3.sup.rd day in demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as green lyofilizate of mass 89 mg (87%).

[0087] .sup.1H NMR (D.sub.2O) (FIG. 1): cypate: 8.80 (s, 2H), 8.50-8.47 (m, 2H), 8.46-8.43 (m, 2H), 8.29-8.19 (m, 2H), 8.16-8.10 (m, 2H), 8.08-7.89 (m, 2H), 7.88-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.46 (m, 2H), 5.16, 5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.02 (12H, CH.sub.3, overlaid with signals of HA), HA: 4.62-4.39 (m, 2H anomer), 4.01-3.28 (m, 10H skeletal), 2.02 (s, 3H, CH.sub.3) ppm

[0088] SS=1% (determined from .sup.1H NMR)

[0089] Fluorimeter: .sub.em.max=695 nm (at .sub.exc.=665 nm; H.sub.2O)

Example 5: Preparation of Conjugate HA-Cypate

[0090] 17 mg (0.03 mmol, 0.1 eqv.) Cypate from Example 2 was dissolved in 1 ml DMSO, 6 mg (0.04 mmol, 0.15 eqv.) N,N-carbonyl diimidazole was added and under the constant stirring was let to activate 30 min. at the room temperature. 100 mg (0.27 mmol, 1 eqv.) of the acid form of hyaluronic acid of M.sub.w 14,000 g/mol in DMSO was dissolved at 60 C. The reaction proceeded under the constant stirring in dark at 60 C. 24 hours. The reaction was stopped with the addition of ten-fold volume of 100% isopropyl alcohol (AIPA) in respect to the initial reaction mixture volume and the saturated solution of NaCl, then the precipitation of the desired product occurred. The crude product was purified with 5100 ml AIPA, dissolved in 50 ml demineralized water and transferred into the dialysis tube. The protonized form of HA was neutralized 1.sup.st day in 0.5% solution of NaCl and 0.5% NaHCO.sub.3, further dialyzed 2.sup.nd and 3.sup.rd day in the demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as green lyofilizate of mass 92 mg (90%).

[0091] .sup.1H NMR (D.sub.2O): cypate: 8.80 (s, 2H), 8.50-8.47 (m, 2H), 8.46-8.43 (m, 2H), 8.29-8.19 (m, 2H), 8.16-8.10 (m, 2H), 8.08-7.89 (m, 2H), 7.88-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.46 (m, 2H), 5.16, 5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.02 (12H, CH.sub.3, overlaid with signals of HA), HA: 4.62-4.39 (m, 2H anomer), 4.01-3.28 (m, 10H skeletal), 2.02 (s, 3H, CH.sub.3) ppm

[0092] SS=0.7% (determined from .sup.1H NMR)

[0093] Fluorimeter: .sub.em,max=695 nm (at .sub.exc.=665 nm; H.sub.2O)

Example 6: Preparation of Conjugate HA-Cypate

[0094] 122 mg (0.19 mmol, 0.7 eqv.) Cypate from Example 2 was dissolved in 1 ml DMSO, 34 mg (0.21 mmol, 0.8 eqv.) N,N-carbonyl diimidazole was added and under the constant stirring was let to activate 30 min. at the room temperature. 100 mg (0.27 mmol, 1 eqv.) of the acid form of hyaluronic acid of M.sub.w 14,000 g/mol in DMSO was dissolved at 60 C. The reaction proceeded under the constant stirring in dark at 60 C. 24 hours. The reaction was stopped with the addition of ten-fold volume of 100% isopropyl alcohol (AIPA) in respect to the initial reaction mixture volume and the saturated solution of NaCl, then the precipitation of the desired product occurred. The crude product was purified with 5100 ml AIPA, dissolved in 50 ml demineralized water and transferred into the dialysis tube. The protonized form of HA was neutralized 1st day in 0.5% solution of NaCl and 0.5% NaHCO.sub.3, further dialyzed 2.sup.nd and 3.sup.rd day in the demineralized water. The tube content was frozen and then lyophilized. The resulting product was obtain in the form of hyaluronan as green lyofilizate of mass 85 mg (88%).

[0095] .sup.1H NMR (D.sub.2O): cypate: 8.80 (s, 2H), 8.50-8.47 (m, 2H), 8.46-8.43 (m, 2H), 8.29-8.19 (m, 2H), 8.16-8.10 (m, 2H), 8.08-7.89 (m, 2H), 7.88-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.46 (m, 2H), 5.16, 5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.02 (12H, CH.sub.3, overlaid with signals of HA), HA: 4.62-4.39 (m, 2H anomer), 4.01-3.28 (m, 10H skeletal), 2.02 (s, 3H, CH.sub.3) ppm

[0096] SS=1.3% (determined from .sup.1H NMR)

[0097] Fluorimeter: .sub.em.max=695 nm (at .sub.exc.=665 nm; H.sub.2O)

Example 7: Preparation of Conjugate HA-Cypate

[0098] 87 mg (0.13 mmol, 0.5 eqv.) Cypate from Example 2 was dissolved in 2 ml DMSO, 22 mg (0.13 mmol, 0.5 eqv.) CDI was added and under the constant stirring was let to activate 2 h at the room temperature. 100 mg (0.27 mmol, 1 eqv.) the acid form of hyaluronic acid of M.sub.w (14,000 g/mol) was dissolved in DMSO at 60 C. Then was 138 l (0.79 mmol, 3 eqv.) DIPEA and the first reaction mixture added to this solution. The reaction proceeded under the constant stirring in dark at 60 C. 24 hours.

[0099] The reaction was stopped by the addition of ten-fold volume of AIPA in respect to the initial volume of the reaction mixture and the saturated solution of NaCl, then occurred the precipitation of the desired product. The product was purified with 5100 ml AIPA, dissolved in 50 ml demineralized water and transferred in the dialysis tube. The protonized form of HA was neutralized 1.sup.st day in 0.5% solution of NaCl and 0.5% NaHCO.sub.3, further dialyzed 2.sup.nd and 3.sup.rd day in demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as lyofilizate of mass 86 mg (84%).

[0100] .sup.1H NMR (D.sub.2O): cypate: 8.81 (s, 2H), 8.50-8.47 (m, 2H), 8.46-8.43 (m, 2H), 8.29-8.19 (m, 2H), 8.16-8.10 (m, 2H), 8.08-7.89 (m, 2H), 7.88-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.46 (m, 2H), 5.16, 5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.03 (12H, CH3, overlaid with signals of HA), HA: 4.62-4.38 (m, 2H anomer), 4.01-3.26 (m, 10H skeletal), 2.03 (s, 3H, CH.sub.3) ppm

[0101] SS=1.5% (determined from .sup.1H NMR)

[0102] SEC-MALLS-LS-UV/Vis-RI: (FIG. 4)

[0103] Fluorimeter: .sub.em.max=695 nm (at .sub.exc.max=665 nm; H.sub.2O)

Example 8: Preparation of Conjugate HA-Cypate

[0104] 87 mg (0.13 mmol, 0.5 eqv.) Cypate from Example 2 was dissolved in 2 ml DMSO, 22 mg (0.13 mmol, 0.5 eqv.) CDI was added and under the constant stirring let activate for 2 h at the room temperature. 100 mg (0.27 mmol, 1 eqv.) of the acid form of hyaluronic acid of Mw (5.810.sup.4 g/mol) in DMSO was dissolved at 60 C. Into this solution was then added 15 l (0.13 mmol, 0.5 eqv.)N-methyl morfolin and the first reaction mixture without the previous purification. The reaction proceeded under the constant stirring in dark at 60 C. 24 hours.

[0105] The reaction was quenched by the addition of ten-fold of AIPA in respect to the initial volume of the reaction mixture and saturated solution of NaCl, then the precipitation of the desired product occurred. The crude product was purified with 5100 ml AIPA, dissolved in 50 ml demineralized water and transferred into the dialysis tube. The protonized form of HA was neutralized 1.sup.st day in 0.5% solution NaCl and 0.5% NaHCO.sub.3, further dialyzed 2.sup.nd and 3.sup.rd day in demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as green lyofilizate of mass 95 mg (93%).

[0106] .sup.1H NMR (D.sub.2O): cypate: 9.2 (m, 2H), 8.82-8.77 (m, 2H), 8.46-8.43 (m, 2H), 8.29-8.19 (m, 2H), 8.16-8.10 (m, 2H), 8.08-7.89 (m, 2H), 7.89-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.46 (m, 2H), 5.16, 5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.03 (12H, CH.sub.3, overlaid with signals of HA), HA: 4.64-4.38 (m, 2H anomer), 4.04-3.21 (m, 10H skeletal), 2.03 (s, 3H, CH.sub.3) ppm

[0107] [SS=1.0% (determined from .sup.1H NMR)

[0108] SEC-MALLS-LS-UV/Vis-RI: (FIG. 5)

[0109] Fluorimeter: .sub.em.max=695 nm (at .sub.exc.=665 nm; H.sub.2O))

Example 9: Preparation of Conjugate HA-Cypate

[0110] 87 mg (0.13 mmol, 0.5 eqv.) Cypate from Example 2 was dissolved in 2 ml DMSO, 22 mg (0.13 mmol, 0.5 eqv.) N,N-carbonyl diimidazole was added and with the constant stirring let activate for 2 h at the room temperature. 100 mg (0.27 mmol, 1 eqv.) acid form of hyaluronic acid of Mw (7.210.sup.4 g/mol) in DMSO was dissolved at 60 C. 15 l (0.13 mmol, 0.5 eqv.)N-methyl morfoline and the first reaction mixture was then added into this solution. The reaction proceeded with the constant stirring in dark at 60 C. 24 hours.

[0111] The reaction was stopped by addition of ten-fold volume of AIPA in respect to the initial volume of the reaction mixture and the saturated solution of NaCl, then occurred the precipitation of the desired product. The crude product was purified with 5100 ml 100% AIPA, dissolved in 50 ml demineralized water and transferred into the dialysis tube. The protonized form of HA was neutralized 1.sup.st day in 0.5% solution NaCl and 0.5% NaHCO.sub.3, further dialyzed 2.sup.nd and 3.sup.rd day in demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as lyofilizate of mass 96 mg (94%).

[0112] .sup.1H NMR (D.sub.2O): cypate: 9.2 (m, 2H), 8.83-8.5 (m, 2H), 8.45-8.43 (m, 2H), 8.30-8.18 (m, 2H), 8.16-7.99 (m, 2H), 7.99-7.89 (m, 2H), 7.89-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.46 (m, 2H), 5.16, 5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.03 (12H, CH3, overlaid with signals of HA), HA: 4.64-4.38 (m, 2H anomer), 4.04-3.21 (m, 10H skeletal), 2.03 (s, 3H, CH.sub.3) ppm

[0113] SS=1.0% (determined from .sup.1H NMR)

[0114] SEC-MALLS-LS-UV/Vis-RI: (FIG. 6)

[0115] Fluorimeter: .sub.em.max=695 nm (at .sub.exc.=665 nm; H.sub.2O))

Example 10: Preparation of Conjugate HA-Cypate

[0116] 87 mg (0.3 mmol, 0.5 eqv.) Cypate from Example 2 was dissolved in 2 ml DMSO, 22 mg (0.13 mmol, 0.5 eqv.) N,N-carbonyl diimidazole was added and under the constant stirring let activate 2 h at the room temperature. 100 mg (0.27 mmol, 1 eqv.) of the acid form of hyaluronic acid of Mw (2.510.sup.5 g/mol) was dissolved in DMSO at 40 C. N-methyl morfolin and first reaction mixture was then added 15 l (0.13 mmol, 0.5 eqv.) into this solution. The reaction proceeded under the constant stirring in dark at 40 C. 24 hours

[0117] The reaction was stopped by the addition of ten-fold volume of AIPA in respect to the initial volume of the reaction mixture and the saturated solution of NaCl, then the precipitation of desired product occurred. The crude product was purified with 5100 ml AIPA, dissolved in 50 ml demineralized water and transferred into the dialysis tube. The protonized form of HA was neutralized 1.sup.st day in 0.5% solution NaCl and 0.5% NaHCO.sub.3, further dialyzed 2.sup.nd and 3.sup.rd day in demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as lyofilizate of mass 93 mg (92%).

[0118] .sup.1H NMR (D.sub.2O): cypate: 9.2 (m, 2H), 8.83-8.5 (m, 2H), 8.45-8.43 (m, 2H), 8.30-8.17 (m, 2H), 8.16-7.99 (m, 2H), 7.99-7.89 (m, 2H), 7.89-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.42 (m, 2H), 5.16, 5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.03 (12H, CH.sub.3, overlaid with signals of HA), HA: S 4.62-4.38 (m, 2H anomer), 4.01-3.26 (m, 10H skeletal), 2.03 (s, 3H, CH.sub.3) ppm

[0119] SS=0.5% (determined from .sup.1H NMR)

[0120] Fluorimeter: .sub.em.max=695 nm (at .sub.exc.=665 nm; H.sub.2O)

Example 11: Esterification of HA-Cypate with Hexanoic Acid

[0121] 300 mg (0.73 mmol, 1 eqv.) of the conjugate HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 13 ml AIPA, 382 l (2.20 mmol, 3 eqv.) DIPEA and 4.5 mg (0.04 mmol, 0.05 eqv) DMAP were then added. 165 l (1.32 mmol, 1.8 eqv.) hexanoic acid was dissolved in 2 ml AIPA, 255 l (1.46 mmol, 2 eqv.) DIPEA 153 l (1.32 mmol, 1.8 eqv.) benzoyl chloride was added and let react under the constant stirring for 30 minutes at 0 C. After the period of time was everything quantitatively transferred to the first reaction mixture, then the esterification proceeded for 2 hours at the room temperature.

[0122] The reaction was quenched by addition of the high excess of AIPA and the saturated solution of NaCl, whereas the precipitation of product occurred. The crude product was washed with 4200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO.sub.3, 0.5% NaCl and further 8 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 230 mg (73%).

[0123] .sup.1H NMR additional signals compared with example 3 (D.sub.2O, 500 MHz): 2.4 (m, 2H, .sub.CH.sub.2), 1.6 (m, 2H, .sub.CH.sub.2), 1.31 (m, 4H, .sub.CH.sub.2), 0.8 (m, 3H, CH.sub.2CH.sub.3) ppm

[0124] SS (acylation)=70% (determined from .sup.1H NMR)

[0125] Fluorimeter: .sub.em.max=700 nm (at .sub.exc.=665 nm; H.sub.2O)

Example 12: Esterification of HA-Cypate with Palmitic Acid

[0126] 300 mg (0.73 mmol, 1 eqv.) of the conjugate of HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water, and then was added 306 l (2.20 mmol, 3 eqv.) TEA and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP were added. In the meantime 94 mg (0.37 mmol, 0.5 eqv.) of hexadecanoic acid dissolved in 3 ml THF, 153 l (1.10 mmol, 1.5 eqv.) TEA, 43 l (0.37 mmol, 0.5 eqv.) benzoyl chloride was added and let react under the constant stirring for 30 minutes at the room temperature. After the period of time was everything quantitatively transferred to the first reaction mixture, the esterification then proceeded for 2 hours at the room temperature.

[0127] The reaction was quenched by addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO.sub.3 and 0.5% NaCl and further for 48 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 189 mg (61%).

[0128] .sup.1H NMR additional signals to example 3 (D.sub.2O, 500 MHz): 2.76-2.66 (m, 2H, .sub.CH.sub.2), 1.64-1.48 (m, 2H, .sub.CH.sub.2), 1.31-1.08 (m, 24H, CH.sub.2), 0.98-0.78 (m, 3H, CH.sub.2CH.sub.3) ppm

[0129] SS acylation=7% (determined from .sup.1H NMR)

[0130] Fluorimeter: .sub.em.max=710 nm (at .sub.exc.=685 am; H.sub.2O)

Example 13: Esterification of HA-Cypate with Palmitic Acid

[0131] 200 mg (0.49 mmol, 1 eqv.) of the conjugate of HA-Cypate prepared according to Example 3 was dissolved in 10 ml demi water, and then 136 l (0.98 mmol, 2 eqv.) TEA and 3 mg (0.02 mmol, 0.04 eqv.) DMAP were added. 38 mg (0.16 mmol, 0.3 eqv.) of palmitic acid dissolved in 3 ml THF, 68 l (0.48 mmol, 1 eqv.) TEA, 17 l (0.16 mmol, 0.3 ekv.) benzoyl chloride was added and let react with the constant stirring for 30 minutes at the room temperature. After the period of time was everything quantitatively transferred to the first reaction mixture, the esterification then proceeded for 2 hours at the room temperature.

[0132] The reaction was quenched by the addition of high excess of AIPA and saturated solution of NaCl, whereas the precipitation of product occurred. The crude product was washed with 4200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO.sub.3 and 0.5% NaCl and further for 48 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 76 mg (37%).

[0133] .sup.1H NMR (D.sub.2O, 500 MHz) additional signals to Example 3: 2.76-2.66 (m, 2H, .sub.CH.sub.2), 1.64-1.48 (m, 2H, .sub.CH.sub.2), 1.31-1.08 (m, 24H, CH.sub.2), 0.98-0.78 (m, 3H, CH.sub.2CH.sub.3) ppm

[0134] SS (acylation)=5% (determined from .sup.1H NMR)

[0135] Fluorimeter: .sub.em.max=710 nm (at .sub.exc.=685 nm; H.sub.2O)

Example 14: Esterification of HA-Cypate with Palmitic Acid

[0136] 200 mg (0.49 mmol, 1 eqv.) of the conjugate HA-Cypate prepared according to Example 3 was dissolved in 10 ml demi water, and then 255 l (1.47 mmol, 3 eqv.) DIPEA and 3 mg (0.02 mmol, 0.04 eqv.) DMAP were added. 38 mg (0.15 mmol, 0.3 eqv.) palmitic acid was dissolved in 3 ml THF, 85 l (0.4-9 mmol, 1 eqv.) DIPEA, 17 l (0.15 mmol, 0.3 eqv.) benzoyl chloride was added and let react under the constant stirring for 30 minutes at the room temperature. After the period of time was everything quantitatively transferred to the first reaction mixture, the esterification then proceeded for 2 hours at the room temperature.

[0137] The reaction was quenched by the addition of high excess of AIPA and the saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 h against 0.5% NaHCO.sub.3 and 0.5% NaCl and further for 48 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 93 mg (45%).

[0138] .sup.1H NMR (D.sub.2O, 500 MHz) additional signals to Example 3: 2.76-2.66 (m, 2H, .sub.CH.sub.2), 1.64-1.48 (m, 2H, .sub.CH.sub.2), 1.31-1.08 (m, 24H, CH.sub.2), 0.98-0.78 (m, 3H, CH.sub.2CH.sub.3) ppm

[0139] SS (acylation)=5% (determined from .sup.1H NMR)

[0140] Fluorimeter: .sub.em.max=710 nm (at .sub.exc.=685 nm; H.sub.2O)

Example 15: Esterification of HA-Cypate with Oleic Acid

[0141] 300 mg HA-Cypate (0.73 mmol, 1 eqv.) from Example 3 was dissolved in 15 ml demi water and 13 ml AIPA, 306 l (2.20 mmol, 3 eqv.) TEA and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP were then added. 186 l (0.59 mmol, 0.8 eqv.) cis-octadec-9-enoid acid dissolved in 2 ml AIPA, 306 l (2.20 mmol, 3 eqv.) TEA, 68 l (0.59 mmol, 0.8 eqv.) benzoyl chloride was added and everything was let react under the constant stirring for 30 minutes at the room temperature. After the period of time was everything quantitatively transferred to the first reaction mixture, esterification then proceeded for 2 hours at the room temperature.

[0142] The reaction was quenched by the addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of product occurred. The crude product was washed 4200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO.sub.3 and 0.5% NaCl and further 48 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 210 mg (66%).

[0143] .sup.1H NMR (D.sub.2O, 500 MHz) additional signals to Example 3: 5.41-5.34 (m, 2H, CHCH), 2.48-2.40 (m, 2H, CH.sub.2CO), 1.68-1.54 (m, 2H, CH.sub.2CH.sub.2, CO), 1.40-1.23 (m, 24H, (CH.sub.2).sub.12), 0.91-0.68 (m, 3H, CH.sub.2CH.sub.3) ppm

[0144] SS (acylation)=10% (determined from .sup.1H NMR)

[0145] Fluorimeter: .sub.em.max=710 nm (at .sub.exc.=685 nm; H.sub.2O)

Example 16: Esterification of HA-Cypate with Oleic Acid

[0146] 300 mg (0.73 mmol, 1 eqv.) of the conjugate of HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 13 ml AIPA, then 306 l (2.20 mmol, 3 eqv.) TEA and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP were added. 116 l (0.36 mmol, 0.5 eqv.) of cis-octadec-9-enoic acid dissolved in 2 ml AIPA, 153 l (1.08 mmol, 1.5 eqv.) TEA, 43 l (0.36 mmol, 0.5 eqv.) benzoyl chloride was added and let react under the constant stirring for 30 minutes at the room temperature. After the period of time everything was quantitatively transferred to the first reaction mixture, the esterification then proceeded for 2 hours at the room temperature.

The reaction was quenched with the addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO.sub.3 and 0.5% NaCl and further for 48 hours in demineralized water. The product was obtained in the form of green lyofilisate yielding 205 mg (73%).

[0147] .sup.1H NMR (D.sub.2O, 500 MHz) additional signals to Example 3: 5.41-5.34 (m, 2H, CHCH), 2.48-2.40 (m, 2H, CH.sub.2CO), 1.68-1.54 (m, 2H, CH.sub.2CH.sub.2, CO), 1.40-1.23 (m, 24H, (CH.sub.2).sub.12), 0.91-0.68 (m, 3H, CH.sub.2CH.sub.3) ppm

[0148] SS (acylation)=8% (determined from .sup.1H NMR)

[0149] Fluorimeter: .sub.em.max=710 nm (at .sub.exc.=685 nm; H.sub.2O)

Example 17: Esterification of HA-Cypate with Oleic Acid

[0150] 300 mg (0.73 mmol, 1 eqv.) of the conjugate of HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 13 ml AIPA, 382 l (2.20 mmol, 3 eqv.) DIPEA and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP were added. 139 l (0.45 mmol, 0.6 eqv.) of cis-octadec-9-enoic acid dissolved in 2 ml AIPA, 229 l (1.32 mmol, 1.8 eqv.) DIPEA, 51 l (0.44 mmol, 0.6 eqv.) benzoyl chloride was added and let react under the constant stirring 30 minutes at 0 C. After the given period was everything quantitatively transferred to the first reaction mixture, and the esterification proceeded for 2 hours at the room temperature.
The reaction was quenched by the addition of high excess of AIPA and saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO.sub.3 and 0.5% NaCl and further for 48 hrs in demineralized water. The product was obtained in the form of green lyofilizate yielding 204 mg (65%).

[0151] .sup.1H NMR (D.sub.2O, 500 MHz) additional signals to Example 3: 5.41-5.34 (m, 2H, CHCH), 2.48-2.40 (m, 2H, CH.sub.2CO), 1.68-1.54 (m, 2H, CH.sub.2CH.sub.2, CO), 1.40-1.23 (m, 24H, (CH.sub.2).sub.12), 0.91-0.68 (m, 3H, CH.sub.2-CH.sub.3) ppm

[0152] SS (acylation)=12% (determined from .sup.1H NMR)

[0153] Fluorimeter: .sub.em.max.=710 nm (at .sub.exc.=685 nm; H.sub.2O)

Example 18: Esterification of HA-Cypate with Oleic Acid

[0154] 300 mg (0.73 mmol, 1 eqv.) of the conjugate HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 11 ml AIPA, then was 306 l (2.20 mmol, 3 eqv.) TEA and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP added. 231 l (0.73 mmol, 1 eqv.) of cis-octadec-9-enoic acid dissolved in 2 ml AIPA, 204 l (1.46 mmol, 2 eqv.) TEA, 85 l (0.73 mmol, 1 eqv.) benzoyl chloride were added and let react with the constant stirring for 30 minutes at the room temperature. After the given period of time everything was quantitatively transferred into the first reaction mixture, and the esterification proceeded for 2 hours at the room temperature.

The reaction was quenched by the addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4200 ml AIPA, further dissolved in the demineralized water and transferred into dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO.sub.3 and 0.5% NaCl and further for 48 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 226 mg (70%).

[0155] .sup.1H NMR (D.sub.2O, 500 MHz) additional signals to Example 3: 5.41-5.34 (m, 2H, CHCH), 2.48-2.40 (m, 2H, CH.sub.2CO), 1.68-1.54 (m, 2H, CH.sub.2CH.sub.2, CO), 1.40-1.23 (m, 24H, (CH.sub.2).sub.12), 0.91-0.68 (m, 3H, CH.sub.2-CH.sub.3) ppm

[0156] SS (acylation)=12% (determined from 11H NMR)

[0157] Fluorimeter: .sub.em.max.=710 nm (at .sub.exc.=685 nm; H.sub.2O)

Example 19: Esterification of HA-Cypate with Oleic Acid

[0158] 300 mg (0.73 mmol, 1 eqv.) of the conjugate HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 11 ml AIPA, then 375 l (1.76 mmol, 2.4 eqv.) N,N,N,N-tetramethyl-1,6-hexanediamine and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP was added. 139 l (0.44 mmol, 0.6 eqv.) of cis-octadec-9-enoic acid dissolved in 2 ml AIPA, 375 l (1.76 mmol, 2.4 eqv.) N,N,N,N-tetramethyl-1,6-hexanediamine, 51 l (0.44 mmol, 0.6 eqv.) benzoyl chloride were added and let react under the constant stirring for 30 minutes at the room temperature. After the given period everything was quantitatively transferred into the first reaction mixture, and the esterification proceeded for 2 hours at the room temperature.

The reaction was quenched by the addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO.sub.3 and 0.5% NaCl and further for 48 hrs in demineralized water. The product was obtained in the form of green lyofilizate yielding 217 mg (70%).

[0159] .sup.1H NMR (D.sub.2O, 500 MHz) additional signals to Example 3: 5.41-5.34 (m, 2H, CHCH), 2.48-2.40 (m, 2H, CH.sub.2CO), 1.68-1.54 (m, 2H, CH.sub.2CH.sub.2, CO), 1.40-1.23 (m, 24H, (CH.sub.2).sub.12), 0.91-0.68 (m, 3H, CH.sub.2CH.sub.3) ppm

[0160] SS (acylation)=6% (determined from .sup.1H NMR)

[0161] Fluorimeter: .sub.em.max.=710 nm (at .sub.exc.=685 nm; H.sub.2O)

Example 20: Esterfication of HA-Cypate with Oleic Acid

[0162] 300 mg (0.73 mmol, 1 eqv.) of the conjugate HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 11 ml AIPA, and then 241 l (2.20 mmol, 3 eqv.)N-methyl morfoline and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP were added. 139 l (0.44 mmol, 0.6 eqv.) cis-octadec-9-enoic acid dissolved in 2 ml AIPA, 193 l (1.76 mmol, 2.4 eqv.)N-methyl morfoline, 51 l (0.44 mmol, 0.6 eqv.) benzoyl chloride were added and let react under the constant stirring for 30 minutes at the room temperature. After the given period was everything quantitatively transferred into the first reaction mixture, and the esterification proceeded for 2 hours at the room temperature.

The reaction was quenched by the addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO.sub.3 and 0.5% NaCl and further for 48 hrs in demineralized water. Product was obtained in the form of green lyofilizate yielding 264 mg (82%).

[0163] .sup.1H NMR (D.sub.2O, 500 MHz) additional signals to Example 3: 5.41-5.34 (m, 2H, CHCH), 2.48-2.40 (m, 2H, CH.sub.2CO), 1.68-1.54 (m, 2H, CH.sub.2CH.sub.2, CO), 1.40-1.23 (m, 24H, (CH.sub.2).sub.12), 0.91-0.68 (m, 3H, CH.sub.2-CH.sub.3) ppm

[0164] SS (acylation)=12% (determined from .sup.1H NMR)

[0165] Fluorimeter: .sub.em.max=710 nm (at .sub.exc.=685 nm; H.sub.2O)

Example 21: Esterification of HA-Cypate with Oleic Acid

[0166] 300 mg (0.73 mmol, 1 eqv.) of the conjugate HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 11 ml AIPA, and then 246 mg (2.20 mmol, 3 eqv.) DABCO and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP were added. 139 l (0.44 mmol, 0.6 eqv.) cis-octadec-9-enoic acid dissolved in 2 ml AIPA, 148 mg (1.32 mmol, 1.8 eqv.) DABCO, 51 l (0.44 mmol, 0.6 eqv.) benzoyl chloride were added and let react with the constant stirring for 30 minutes at the room temperature. After the given period everything was quantitatively transferred into the first reaction mixture, and the esterification proceeded for 2 hours at the room temperature.

The reaction was quenched by the addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of product occurred. The crude product was washed with 4200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO.sub.3 and 0.5% NaCl and further 48 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 183 mg (60%).

[0167] .sup.1H NMR (D.sub.2O, 500 MHz) additional signals to Example 3: 5.41-5.34 (m, 2H, CHCH), 2.48-2.40 (m, 2H, CH.sub.2CO), 1.68-1.54 (m, 2H, CH.sub.2CH.sub.2, CO), 1.40-1.23 (m, 24H, (CH.sub.2).sub.12), 0.91-0.68 (m, 3H, CH.sub.2-CH.sub.3) ppm

[0168] SS (acylation)=3% (determined from .sup.1H NMR)

[0169] Fluorimeter: .sub.em.max=710 nm (at .sub.exc.=685 nm; H.sub.2O)

Example 22: Loading of Hydrophobized HA-Cypate with Nonpolar Compound

[0170] 150 mg of the acylated conjugate of HA-cypate prepared according to Example 16 was 2 hours dissolved in 15 ml demi water under the constant stirring on the magnetic stirrer. Then was gradually added 10 mg paclitaxel in 2 ml chloroform and the resulting mixture was evaporated (RE) to dry and then hydrated with demi water (15 ml). Unbound paclitaxel was removed with the filtration through 1.0 m glass filter and the resulting product was lyofilized.
The amount of unbound paclitaxel (HPLC determination): 4.2% (wt.)

Example 23: Loading of Hydrophobized HA-Cypate with Nonpolar Compound

[0171] 150 mg of the acylated conjugate HA-cypate prepared according to Example 18 was 2 hours dissolved in 15 ml demi water with the constant stirring on the magnetic stirrer. Then 15 mg doxorubicin was gradually added in 2 ml chloroform and the resulting mixture was first sonicated (pulse sonication cca 15 min, 200 W, amplitude 65%, cycle 0.5 s) until reaching of the homogenous mixture and then evaporated (RE) to dry and then hydrated with demi water (15 ml). Unbound doxorubicin was removed with filtration through 1.0 m glass filter and the resulting product was lyofilized.
The amount of unbound doxorubicin (HPLC determination): 7.5% (wt.)

Example 24: Loading of Hydrophobized HA-Cypate with Nonpolar Compound

[0172] 150 mg of the acylated conjugate HA-cypate prepared according to Example 18 was 2 hours dissolved in 15 ml demineralized water with the constant stirring on the magnetic stirrer. Then 2 mg of spions on the basis of iron (Fe.sub.2O.sub.3, Fe.sub.3O.sub.4) (5 mm) and 20 mg doxorubicin was gradually added in 5 ml chloroform and the resulting mixture was first sonicated (pulse sonication, cca 15 min, 200 W, amplitude 65%, cycle 0.5 s) until reaching homogenous mixture and then evaporated (RE) to dry and then hydrated with demi water (15 ml). Unbound doxorubicin was removed with filtration through 1.0 m glass filter and the resulting product was lyofilized.
The amount of unbound doxorubicin (HPLC determination): 6.5% (wt.), unbound spions: 2% (wt.)

Example 25: In Vivo Experiment Fluorescence of Conjugate HA-Cypate

[0173] For in vivo experiments was HA-Cypate, prepared according to Example 3, dissolved in the saline solution (c=3.8 mg derivative in 100 l solution) and then sterilized by filtration (0.22 m). 50 l (FIG. 9) and/or intraperitoneally 150 l (FIG. 10) sterile solution of the given derivative was subcutaneously applied to the laboratory mouse of the type Balb/c in narcosis. Fluorescence was detected using the combination of different excitation wavelengths and emission filters.

[0174] FIG. 9 a 10 show the sufficient intensity of fluorescence of the derivative after the subcutaneous and intraperitoneal application for in vivo imaging. The imaging can be performed using the various excitation wavelengths (530-745 nm), and filters for the emission (ICG, Cy5.5). The filter DsRed cannot be used. From FIG. 10 the stability of the derivative as fluorophore after intraperitoneal administration is further apparent.

Example 26: In Vivo Experiment Fluorescence of Conjugate HA-Cypate-C18:1

[0175] HA-Cypate-C18:1 (SS=10%) prepared according to Example 15 was dissolved in phosphate buffer (c=3.6 mg of the conjugate in 100 l solution and sterilized by filtration (0.22 m). 100 l of such prepared solution was applied intravenously to two model mice Balb/c (in narcosis) and observed its fluorescence in vivo for the period of 7 days (FIG. 11).

[0176] FIG. 11 show that HA-Cypate-C18:1 is distributed after i.v, administration in the healthy mouse especially into liver. Fluorescence of the conjugate is sufficient for in vivo imaging, fluorescence of the conjugate is further very stable, after one administration can be imaging performed for 2 weeks.

Example 27: In Vivo Experiment Fluorescence of Conjugate HA-Cypate-C18:1

[0177] HA-Cypate-C18:1 (SS=10%) prepared according to Example 15 was dissolved in the saline solution (c=1.8 mg of the conjugate in 100 l solution) and sterilized by the filtration (0.22 m). 100 l of such prepared solution was applied intravenously to 3 model mice Balb/c (in narcosis) with non-palpable breast tumor (orthotopic administration 4T1 luc cells, displayable through chemiluminescence-detection of luciferase activity after i.p. injection of luciferin) and observable its fluorescence in vivo for period 72 hours (FIG. 12).

[0178] FIG. 12 shows that HA-Cypate-C18:1 is distributed after i.v. administration into liver and further after 24 hours into very small (non-palpable) tumor. In tumor, there is the accumulation of HA-Cypate-C18:1 growing with time and the presence of the conjugate in tumor is very significant even after 15 days after the administration of the conjugate. Fluorescence of the conjugate in vivo is thus very stable and the conjugate can be used to imaging of very small tumors and further to tumor observation in time. In left panel of the figure is the control image of tumor with the luminescent imaging (the detection of luciferase activity).

Example 28: In Vivo ExperimentFluorescence of Conjugate HA-Cypate-C18:1

[0179] To 9 model mice Balb/c (in narcosis) were orthotopically administered 4T1 luc cells, (cells displayable with chemiluminescence-detection of luciferase activity after i.p. application of luciferin) and breast tumor was let grow for 14 days. 14.sup.th day were mice divided into 3 groups (1 group=3 animals). To every group was 14.sup.th, 21.sup.th and 28.sup.th day intravenously applied 1 mg of selected conjugate in 100 l phosphate buffer and then: to first group was applied HA-Cypate-C18:1 prepared in Example 15, to second group HA-Cypate-C18:l loaded with doxorubicin, prepared in Example 23 and to third group HA-Cypate-C18:1 loaded with doxorubicin and spions, prepared in Example 24. The radiation was evaluated for 35 days in vivo (FIG. 13), then mice were sacrificed and the tumor and spleen weight in individual groups was compared (FIG. 14).

[0180] FIG. 13 shows that the tumor grew most in the group, to which only HA-Cypate was administered. Slower grow was detected in contrast in groups, to which HA-Cypate-C18:1+doxorubicin, and/or HA-Cypate-C18:1+doxorubicin+spion was applied.

[0181] FIG. 14 shows, that the first (not treated) group had the highest spleen and tumor weight. In given model, increased spleen indicates progression of disease (duPre et al., Experimental and Molecular Pathology 2007, 82, 12-24). The second (treated) and the third (treated) group had spleen significantly smaller and tumor smaller compared to the first group. The smallest tumor size was found in the third group. The carrier system can thus not only image but also cure the tumor and fills the function of theranostatic.

Example 29: In Vivo ExperimentFluorescence of Conjugate HA-Cypate-C18:1

[0182] HA-Cypate-C18:l (SS=10%) prepared according to Example 15 was dissolved in the saline solution (c=0; 0.625; 1.25; 2.5 mg of the conjugate in 100 l solution) and sterilized by the filtration (0.22 m). 100 l of such prepared solutions was applied intraperitoneally to 4 model mice Balb/C (in narcosis) with the breast tumor (35.sup.th day after orthotopic administration of 4T1 luc cells, displayable with chemiluminescence detection of luciferase activity after i.p. injection of luciferin) and observed its fluorescence in vivo for 7 days (FIG. 15).

[0183] FIG. 15 shows, that HA-Cypate-C18:1 is distributed after the i.p. administration into tumor and liver. The tumor is without any problem displayable in vivo at minimum for 7 days after the administration of the conjugate in all concentrations used. In left panel of the figure is the control image of the tumor with the luminescent imaging (detection of luciferase). The right panel of the figure shows 4 mice, to which the solution of the derivative was administeredfrom left to right grows the concentration: (0; 0.625; 1.25; 2.5 mg of the conjugate administered in 100 l solution).