METHODS FOR THE CONJUGATION OF ANTHRACYCLINES TO CARBOHYDRATE POLYMERIC CARRIERS

1 . A method for conjugating anthracycline to a carbohydrate polymer comprising:

providing (1) a carbohydrate polymer comprising at least one alkyne moiety bound directly or indirectly thereto, and (2) an anthracycline derivatized to a hydrazone azide; or

providing (1) a carbohydrate polymer comprising at least one hydrazone azide moiety bound directly or indirectly thereto, and (2) an anthracycline comprising at least one alkyne moiety bound directly or indirectly thereto; and

reacting the carbohydrate polymer with the anthracycline in the presence of a Cu 1 catalyst and an amine ligand to form a 1,3-triazole linkage between the anthracycline and the carbohydrate polymer,

wherein the 1,3-triazole linkage results from a 1,3-cycloaddition between the azide and the alkyne.

2 . The method of claim 1 , wherein the carbohydrate polymer is selected from the group consisting of cellulose, dextran, and mannan.

3 . The method of claim 1 , wherein the anthracycline comprises doxorubicin.

4 . A method of synthesizing an anthracycline carbohydrate polymer construct comprising:

providing an alkyne carbohydrate polymer;

providing an anthracycline hydrazone azide; and

reacting the alkyne carbohydrate polymer with the anthracycline hydrazone azide in the presence of a Cu 1 catalyst and a ligand to form a 1,3-triazole linkage between the anthracycline and carbohydrate polymer,

wherein the 1,3-triazole linkage results from a 1,3-cycloaddition between the azide of the anthracycline hydrazone azide and the alkyne of the alkyne carbohydrate polymer.

5 . The method of claim 4 , wherein the anthracycline hydrazone azide is formed by a step comprising derivatizing the anthracycline by reacting the anthracycline with a hydrazide via condensation to form the anthracycline hydrazone azide.

6 . The method of claim 4 , wherein the hydrazide is 4-azidobenzohydrazide.

7 . The method of claim 4 , wherein the anthracycline comprises doxorubicin.

8 . The method of claim 4 , wherein the alkyne carbohydrate polymer construct is synthesized by a step comprising reacting an amine carbohydrate polymer comprising cellulose, dextran, or mannan, with an alkyne under dehydrative conditions to form the alkyne carbohydrate polymer.

9 . The method of claim 8 , wherein the alkyne is hexynoic acid.

10 . The method of claim 4 , wherein the ligand is an amine ligand.

11 . The method of claim 10 , wherein the amine ligand has the following structure:

12 . The method of claim 11 , wherein the ligand is synthesized through the reaction of ((2-benzimidazolyl)methyl)amine and 2-(chloromethyl)pyridine in the presence of Et 3 N.

13 . The method of claim 4 , wherein the ligand and Cu 1 catalyst are present at a ratio of between about 1:5 to about 5:1.

14 . The method of claim 4 , wherein the step of reacting the alkyne carbohydrate polymer with the anthracycline hydrazone azide is carried out in the presence of sodium ascorbate.

15 . The method of claim 14 , wherein the Cu 1 catalyst and ascorbate are present at a ratio of between about 1:5 to about 9:10.

16 . The method of claim 4 , wherein the alkyne and the Cu 1 catalyst are present at a ratio of between about 1:1 to about 5:1.

17 . The method of claim 4 , wherein the reacting step is performed in the presence of DMSO and water, and wherein the DMSO and water are present at a ratio of between about 4:1 to about 20:1.

18 . The method of claim 4 , wherein loading of anthracycline to the anthracycline carbohydrate polymer construct can be controlled by varying the ratio of alkyne to the anthracycline hydrazone azide.

19 . The method of claim 18 , wherein as the ratio of the alkyne to anthracycline hydrazone azide increases, the loading of anthracycline to the anthracycline carbohydrate polymer construct decreases.

20 . The method of claim 4 , wherein the alkyne carbohydrate polymer is a mannosylated alkyne carbohydrate polymer having a molecular weight (Mw) of from about 1 kDa to about 50 kDa.

21 . The method of claim 4 , wherein the alkyne carbohydrate polymer has the structure of formula (I):

wherein

each X is independently H, L2-R, or L3-Y;

each L2 and L3 are independently amino terminated leashes comprising the formula (CH 2 ) p S(CH 2 ) q —NH—, wherein p and q are integers from 0 to 5;

each R independently comprises a mannose-binding C-type lectin receptor targeting moiety, or H;

each Y independently comprises a terminal alkyne moiety, or H; and

n is an integer greater than zero,

wherein at least one R comprises a mannose-binding C-type lectin receptor targeting moiety selected from the group consisting of mannose, fucose, and n-acetylglucosamine;

wherein at least one Y comprises the terminal alkyne moiety; and

wherein each unit of n may be the same or different.

22 . The method of claim 21 , wherein each X is independently H,

and wherein each bond 1A is connected to any —OH group in formula (I).

23 . The method of claim 4 , wherein the reacting step is carried out at a pH of from about 6.5 to about 10.

24 . The method of claim 4 , wherein the anthracycline is released from the anthracycline carbohydrate polymer construct at a pH of about 5.5 or below.

25 . The method of claim 4 , wherein the anthracycline carbohydrate polymer has the structure of formula (II):

wherein

each X is independently H, L1-A, L2-R, or L3-Y;

each L1, L2 and L3 are independently amino terminated leashes comprising the formula —(CH 2 ) p S(CH 2 ) q —NH—, wherein p and q are integers from 0 to 5;

each A independently comprises an anthracycline hydrazone azide moiety, or H;

each R independently comprises a mannose-binding C-type lectin receptor targeting moiety, or H;

each Y independently comprises a terminal alkyne moiety, or H; and

n is an integer greater than zero,

wherein at least one A comprises the anthracycline hydrazone azide moiety;

wherein at least one R comprises a mannose-binding C-type lectin receptor targeting moiety selected from the group consisting of mannose, fucose, and n-acetylglucosamine;

wherein at least one Y comprises the terminal alkyne moiety; and

wherein each unit of n may be the same or different.

26 . The method of claim 25 , wherein each X is independently H,

and wherein each bond 1A is connected to any —OH group in formula (II).

27 . A pharmaceutical composition comprising:

a compound having the structure of formula (II):

wherein

each X is independently H, L1-A, L2-R, or L3-Y;

each L1, L2 and L3 are independently amino terminated leashes comprising the formula —(CH 2 ) p S(CH 2 ) q —NH—, wherein p and q are integers from 0 to 5;

each A independently comprises an anthracycline hydrazone azide moiety, or H;

each R independently comprises a mannose-binding C-type lectin receptor targeting moiety, or H;

each Y independently comprises a terminal alkyne moiety, or H; and

n is an integer greater than zero,

wherein at least one A comprises the anthracycline hydrazone azide moiety;

wherein at least one R comprises a mannose-binding C-type lectin receptor targeting moiety selected from the group consisting of mannose, fucose, and n-acetylglucosamine;

wherein at least one Y comprises the terminal alkyne moiety;

wherein each unit of n may be the same or different; and

a pharmaceutically acceptable carrier thereof.

28 . The pharmaceutical composition of claim 27 , wherein each X is independently is H,

and wherein each bond 1A is connected to any —OH group in formula (II).

29 . The pharmaceutical composition of claim 27 , wherein the compound of formula (II) has a molecular weight (Mw) of greater than about 5 kDa.

30 . The pharmaceutical composition of claim 27 , wherein the anthracycline comprises doxorubicin.

31 . The pharmaceutical composition of claim 27 , wherein the anthracycline is released from the composition at a pH of about 5.5 or below.