Therapeutic conjugates with sulfated dendrimers for intracellular targeting

Abstract

The present invention relates to novel dendrimer conjugates, methods for their preparation and their use for treatment of diseases. The invention discloses a new method for the delivery of dendrimer conjugates with therapeutically active molecules into the cell by utilizing transmembrane solute carrier proteins enabling uptake of the inventive dendrimer conjugates. Particular subject-matter of the present invention is a conjugate of the formula E-[G-L-D(OSO.sub.3.sup.−M.sup.+).sub.n].sub.m, wherein E is a therapeutic or diagnostic effector molecule, wherein D(OSO.sub.3.sup.−M.sup.+).sub.n is a dendrimer D carrying a number n of sulfate groups OSO.sub.3.sup.−M.sup.+, wherein the number n of sulfate groups is selected from 6 to 96, wherein M is a cationic inorganic or organic counter ion to the anionic sulfate group, wherein L is a linker or spacer between D and E, wherein G is a connecting functional group forming the attachment between L and E, and wherein m is an integer from 1 to 20.

Claims

1. A pharmaceutical composition, comprising a conjugate of the formula
E-[G-L-D(OSO.sub.3.sup.−M.sup.+).sub.n].sub.m, wherein E is a therapeutic or diagnostic effector molecule, wherein D(OSO.sub.3.sup.−M.sup.+).sub.n is a dendrimer D carrying a number n of sulfate groups OSO.sub.3.sup.−M.sup.+, wherein the number n of sulfate groups is selected from 6, 8, 12, 16, 18, 24, 30, 32, 36, 40, 48, 72 or 96, wherein M is a cationic inorganic or organic counter ion to the anionic sulfate group, wherein L is a linker or spacer between D and E, wherein G is a connecting functional group forming the attachment between L and E, and wherein m is an integer from 1 to 20 and wherein each of the dendrimers D of said conjugate has the same molecular weight and wherein the number n of sulfate groups is the same for each dendrimer D, wherein the linker L is covalently bound to a focal point of the dendrimer D at a position, whereby from this focal point, the dendrimer is grown to reach its dendritic structure and wherein the dendrimers D(OSO.sub.3.sup.−M.sup.+).sub.n have the following relations between n and molecular weight, which are calculated for M.sup.+ being a sodium ion: TABLE-US-00012 Molecular weight of number of sulfates (n) D(OSO.sub.3.sup.-M.sup.+).sub.n not exceeding 6  2000 Da 8  2400 Da 12  4000 Da 16  5500 Da 18  6000 Da 24  8000 Da 30 10000 Da 32 11000 Da 36 12000 Da 40 13000 Da 48 16000 Da 72 24000 Da 96  32000 Da.

2. The pharmaceutical composition according to claim 1, wherein the repeating units of monomers to build the dendrimer D are selected from the group consisting of 1,2-substituted glycerol, 1,3-substituted glycerol, pentaerythritol, glucose, mannose, galactose, lysine, tris(hydroxymethyl)aminomethane, tris(propionic acid)aminomethane, 1,1′-bis(hydroxymethyl)-propionic acid, succinic acid, glutaric acid, maleic acid, glycolic acid, diglycolic acid, adipic acid, lactic acid, citric acid, propionic acid (2-aminoethyl)amide, propyleneimine, ethyleneimine, propyleneoxide, and ethyleneoxide.

3. The pharmaceutical composition according to claim 1, wherein the connection of said monomers in the dendrimer D is based on functional groups selected from ether, thioether, carboxylic ester, sulfonylester, sulfonamide, carboxylamide, amine, carbamate, thiocarbamate, urea, thiourea, hydrazone, imine, disulfide, phosphate, phosphonate, triazole, acetal, and ketal.

4. The pharmaceutical composition according to claim 1, wherein D contains terminal groups selected from 1,2-disulfatoalkyl, 1,3-disulfatoalkyl, 1,2,4-trisulfato-3-alkyl, N,N′-di(1-sulfatoalkyl)amine, tris(sulfatomethyl)methyl, and 1,2,3,4,5-pentasulfatoalkyl.

5. The pharmaceutical composition according to claim 1, wherein L is a C.sub.4-100-alkyl group, selected from the group consisting of aliphatic cyclic, branched or linear units in which one or more methylene groups may independently be replaced by a unit selected from the group consisting of O, S, NH, NH—O, C(═O)NH, OC(═O)NH, OC(═O)O, NHC(═O)NH, NHC(═S)NH, C(═NH)NH, C(═O), S(═O).sub.2, S(═O), S(═O.sub.2)O, S—S, CH═N, CH═N—NH, C═N—NHC(═O), OP(═O)(O.sup.−M.sup.+)O, P(═O)(O.sup.−M.sup.+)O, arylene, ethenylene or ethinylene, and triazolylene, in which any hydrogen atom may independently be replaced by methyl, ethyl or hydroxymethyl.

6. The pharmaceutical composition according to claim 1, wherein E is a therapeutic or diagnostic effector molecule.

7. The pharmaceutical composition according to claim 6, wherein the effector molecules are selected from the group consisting of small molecules with a molecular weight of approximately 600 to 2000 Da, peptides, proteins, glycans, and nucleic acids.

8. The pharmaceutical composition according to claim 6, wherein the effector molecule is a therapeutic effector molecule comprising substances which may interfere with intracellular mechanisms of proliferation, apoptosis, synthesis of connective tissue material (e.g. collagen, fibronectin), immune function, senescence, or immune defence.

9. The pharmaceutical composition according to claim 7, wherein the effector molecules are cytostatic agents.

10. The pharmaceutical composition according to claim 7, wherein the proteins are selected from the group consisting of globular proteins, glycoproteins, toxins, enzymes, antibodies, antibody fragments, engineered antibody and protein constructs, wherein said antibodies, antibody fragments, engineered antibody and protein constructs optionally comprise single domain antibodies (sdAb), single chain Fv antibodies (scFv), or single chain-Fv-Fc antibodies (scFv-Fc).

11. The pharmaceutical composition according to claim 6, wherein E is directed against molecules involved in proliferation and apoptosis of tumor cells.

12. The pharmaceutical composition according to claim 1, wherein G is a connecting functional group forming the covalent attachment between E and L, selected from the group consisting of O, S, NH, NH—O, C(═O)NH, OC(═O)NH, OC(═O)O, NHC(═O)NH, NHC(═S)NH, C(═NH)NH, C(═O), S(═O).sub.2, S(═O), S(═O.sub.2)O, S—S, CH═N, CH═N—NH, C═N—NHC(═O), OP(═O)(O.sup.−M.sup.+)O, P(═O)(O.sup.−M.sup.+)O, arylene, ethenylene ethinylene, and triazolylene.

13. The pharmaceutical composition according to claim 1, for use (i) in treating a disease selected from the group comprising cancer, inflammation, autoimmune disease, metabolic disease and fibrosis, or (ii) in anti-proliferative, pro-proliferative, anti-apoptotic, pro-apoptotic, anti-fibrotic, pro-fibrotic, anti-lipogenic, anti-diabetic, immune-stimulatory and anti-aging treatment.

14. The pharmaceutical composition according to claim 1, wherein the conjugate is a defined dendrimer system of a single molecular weight.

15. The pharmaceutical composition according to claim 1, wherein M.sup.+ is a sodium ion.

16. The pharmaceutical composition according to claim 7, wherein the effector molecules are peptide or peptidomimetic structures, wherein said peptide or peptidomimetic structures optionally comprise cyclic or open-chain peptides with natural or non-natural structural modifications.

17. The pharmaceutical composition according to claim 7, wherein the small molecules have a molecular weight of 600 to 2000 Da.

18. The pharmaceutical composition according to claim 1, wherein the effector molecule is a peptide which binds targets selected from the group consisting of p19INK4D, GSK-3, myc, INK4A, p53, KRas, NRas, Hras, p27, KIP1, GSK3 beta, HER4, Src, PTEN, Bcl-2, Bcl-xL, mcl-1 Ataxin-1, catenin, IRAK1, IRAK2, IRAK4, VEGFR1, ZAP70, Aurora A, Aurora B, and Aurora C.

19. The pharmaceutical composition according to claim 18, wherein the effector molecule is a peptide which binds targets selected from the group consisting of myc, Bcl-2, Bcl-xL, mcl-1, catenin, and p53.

20. The pharmaceutical composition according to claim 1, wherein the effector molecule is an alpha-helical peptide derived from apoptosis sensitizing proteins which is selected from the group consisting of BIM, BID, NOXA, and PUMA.

21. The pharmaceutical composition according to claim 1, wherein the effector molecule is a peptide selected from the group consisting of: CGMRPEIWIAQELRRIGDEFNA; CGDMRPEIYI(Aib)QELRRIGD(Aib)Y; CGLSQEQLEHRERSLQTLRDIQRMLF; CGLSKEQLEHRERSLQTLRDIERLL; CGEEQWAREIGAQLRRMADDLNAQYER; CGEDIIRNIARHAAQVGASADRSI; and CPKWILKKATAYILSVQAEEQKL.

22. The pharmaceutical composition according to claim 1, wherein the effector molecule is a toxin polypeptide selected from the group consisting of diphtheria toxin, diphtheria toxin lacking receptor-binding activity, pseudomonas exotoxin A, truncated forms of pseudomonas exotoxin that lacks the receptor-binding domain Ia, ricin toxin, saporin, dianthin, gelonin, thricosanthin, pokeweed antiviral protein (PAP), bouganin, anthrax protective antigen, alpha toxin, abrin, and apoptosis-inducing polypeptides.

23. The pharmaceutical composition according to claim 1, wherein the effector molecule is a toxic polypeptide selected from the group consisting of desoxyribonuciease I (DNase I), desoxyribonuclease II (DNase II), polypeptides targeting alpha-tubulin, polypeptides targeting beta-tubulin, polypeptides targeting dynein, conjugates polypeptides targeting kinesin, polypeptides targeting NEDD.sub.1, polypeptides targeting transforming acidic coiled-coil protein TACC, and polypeptides targeting colonic hepatic tumor overexpresses gene chTOG.

24. The pharmaceutical composition according to claim 1, wherein the effector molecule is bevacizumab or IgG.

25. The pharmaceutical composition according to claim 1, wherein the effector molecule is a protein selected from the group consisting of wild-type p53, wild-type p21, apoptosis-inducing factor 1 (AIF.sub.1), ASK.sub.1, apoptosis-inducing protein (AIP), caspase-2, caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, Bax, serine protease, Smac, cytochrome c, Apaf-1, and apoptin.

Description

FIGURE DESCRIPTION

(1) FIG. 1 depicts exemplary chemical structures of sulfated dendrimers in accordance with the invention.

(2) FIG. 1A: core derivable e.g. from pentaerythritol or TRIS, dendrimer units: glycerol, 1,2-substituted, connected as ether (—O—), 24 sulfate groups,

(3) FIG. 1B: core derivable e.g. from glycerol or 2-amino-propane-1,3-diol, dendrimer units: glycerol, 1,3-substituted, connected as ether (—O—), 32 sulfate groups (sulfate group illustrated as S in bulb, star indicates connection to linker L).

(4) FIG. 2 depicts exemplary chemical structures of sulfated dendrimers in accordance with the invention.

(5) FIG. 2A: core derivable e.g. from pentaerythritol or TRIS, dendrimer units: pentaerythritol and glycerol, 1,2-substituted, connected as ether (—O—), 36 sulfate groups,

(6) FIG. 2B: core 2,2-bis(hydroxymethyl)propionic acid, dendrimer units: 2,2-bis(hydroxymethyl)propionic acid, connected as ester, and glycerol, 1,2-substituted, connected as ether (—O—), 32 sulfate groups.

(7) FIG. 3 depicts exemplary chemical structures of sulfated dendrimers in accordance with the invention.

(8) FIG. 3A: core derivable e.g. from aminomethyl-tris(propionic acid) or hydroxymethyl-tris(propionic acid connected as amide, dendrimer units: glycerol, 1,2-substituted and glycerol 1,3-substituted, connected as ether (—O—), 24 sulfate groups,

(9) FIG. 3B: core derivable e.g. pentaerythritol or TRIS, dendrimer units: propionic acid connected as amide, glycerol 1,3-substituted and glycerol 1,3-substituted, connected as ether (—O—), 24 sulfate groups.

(10) FIG. 4 depicts exemplary chemical structures of sulfated dendrimers in accordance with the invention.

(11) FIG. 4A: core derivable e.g. from tris(propionic acid)methylamine, dendrimer units: pentaerythritol, connection as ester, glycerol, 1,2-substituted connected as ether, 36 sulfate groups,

(12) FIG. 4B: core derivable e.g. from tris(propionic acid)methylamine, dendrimer units: bis(propionic acid)ethylene diamine, connection as amide, triglycerolamine connected as amide, 32 sulfate groups.

(13) FIG. 5 depicts exemplary chemical structures of sulfated dendrimers in accordance with the invention.

(14) FIG. 5A: core derivable e.g. from glycerol or 2-amino-propane-1,3-diol, dendrimer units: glycerol, 1,3-substituted, connected as ether (—O—), succinic acid, connected as ester, 32 sulfate groups,

(15) FIG. 5B: core derivable e.g. from tris(propionic acid)methylamine, dendrimer units: glycerol, 1,3-substituted, succinic acid; 24 sulfate groups.

(16) FIG. 6 depicts exemplary chemical structures of sulfated dendrimers based on sugar moieties in accordance with the invention.

(17) FIG. 6A: core based on monofunctionalized cyclodextrin (here β-cyclodextrin): dendrimers units: glycerol, 1,2-substituted, connected as ether (—O—), 40 sulfate groups,

(18) FIG. 6B: core derivable from e.g. pentaerythritol or TRIS, dendrimer units: propionic acid connected as amide, bis(glucamine) connected as amide, 30 sulfate groups.

(19) FIG. 7 depicts exemplary chemical structure of sulfated dendrimer: core derivable e.g. from pentaerythritol or TRIS; ethylene oxide units, connected via ether bond to pentaerythritol; dendrimer units at pentaerythritol: glycerol, 1,2-substituted, connected via ether; 36 sulfate groups in accordance with the invention

(20) FIG. 8 illustrates cellular uptake of dendrimer conjugates (Example 15) in accordance with the invention.

(21) FIG. 8A depicts a microscopic image of SKBR3 cells showing fluorescence of ICC-d02 as homogeneous distribution allowing visualization of the cell body.

(22) FIG. 8B shows the absorption spectrum of cell lysate of SKBR3 after incubation with ICC-d02 giving 5 Mio molecules/cell recovered from intracellular compartment.

EXAMPLES

(23) The following examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention. It is believed than one skilled in the art can easily ascertain the essential characteristics of this invention and understands the Examples of the invention as exemplary. Thus the below examples are not limiting the subject-matter of the invention.

Example 1

Synthesis of Polyglycerol Dendrimer System with 24 Sulfate Groups and aminodecyl Linker (Compound d01)

Example 1a

Synthesis of 10-azidodecyl-triallylpentaerythritol

(24) ##STR00010##

(25) To a solution of triallylpentaerythritol (5 g, 19.5 mmol) in 50 mL of dry THF, solid NaH (3.9 g, 98 mmol, 60% dispersion in mineral oil). is added portionwise. The mixture is stirred at 40° C. for 3 h, followed by the addition of 0.3 g potassium iodide and a solution of 1,10-dibromodecane (29.3 g, 98 mmol) in 20 mL THF. After 24 h at reflux, the mixture is treated with water, evaporated to dryness, and the residue dissolved in water/dichloromethane. The product is extracted into dichloromethane, dried over Na.sub.2SO.sub.4, and purified by silica chromatography (cyclohexane/ethylacetate) yielding 8.1 g intermediate 10-bromodecyl-triallylpentaerythritol. 8 g (16.8 mmol) of the intermediate is dissolved in DMF (50 mL) and sodium azide (5.6 g, 84 mmol) is added. The mixture is stirred at 80° C. for 24 h, followed by filtration and evaporation of the solvent. Chromatographic purification (cyclohexane, then dichloromethane: methanol 95:5 to 1:1) yielded 4.0 g (55%) product as slightly yellow oil.

Example 1b

Synthesis of 10-azidodecyl-tris(2,3-dihydroxypropyl)pentaerythritol

(26) ##STR00011##

(27) The reaction is conducted according Zieringer et al., ChemPhysChem. 2010, 11, 2617. 10-azidodecyl-triallylpentaerythritol (4.0 g, 9.1 mmol) is dissolved in 300 mL of a mixture of t-butanol/water (1:1). To this mixture trimethylamine-N-oxide dihydrate (9.1 g, 82.4 mmol), citric acid (10.0 g, 47.7 mmol) and potassium osmate dihydrate (0.4 g, 1.1 mmol) is added, followed by stirring at room temp. for 24 h. Ion exchange resin Lewatit K6267 (70 g) is added and the mixture allowed to stir another 1 h. After filtration and washing with t-butanol/water 1:1, the solution is evaporated to dryness and the residue purified by chromatography (RP C-18 Licroprep) using water/methanol in an MPLC system. Yield 3.5 g (71%) of a colorless viscous oil.

Example 1c

Dendrimer Generation from 10-azidodecyl-tris(2,3-dihydroxypropyl)pentaerythritol

(28) The dendrimer is built of a dendron consisting of glycerol monomers (1,2-subst. pattern) employing 24 hydroxy groups by published methods (Zieringer et al., Chem. Phys. Chem. 2010, 11, 2617 and Wyszogrodska et al., Eur. J. Org. Chem. 2008, 53) in 4 reaction steps of alternating allylation with NaH/allybromide and dihydroxylation. The product is 10-azidodecyl-glycerol (OH.sub.24), molecular weight 1873 g/mol, which is sulfated in the next step.

Example 1d

Sulfation of 10-azidodecyl dendron(OH).SUB.24

(29) 100 mg (0.053 mmol) 10-azidodecyl dendron(OH.sub.24) is dissolved in 0.5 mL dry DMF and heated to 60° C. Under stirring, SO.sub.3-pyridine complex (245 mg, 1.54 mmol) is added, followed by 5 h stirring at 60° C. and 18 h at room temp. The reaction mixture is quenched with water, adjusted to pH 9-10 using 1 mM NaOH, filtrated and subjected to ultrafiltration (MWCO 1000) with water. The product 10-azidodecyl dendron(OSO.sub.3.sup.−Na.sup.+).sub.24 (150 mg, 65%) is obtained after lyophilisation. Elementary analysis revealed complete sulfation. N, 0.863, C, 21.70, S, 17.83, H, 3.042, molecular weight 4322 g/mol.

Example 1e

Synthesis of 10-aminodecyl dendron(OSO.SUB.3..SUP.−.Na.SUP.+.).SUB.24 .(Compound d01)

(30) ##STR00012##

(31) To a solution of 10-azidodecyldendron(OSO.sub.3.sup.−Na.sup.+).sub.24 (150 mg, 0.035 mmol) in water/methanol 1:1 (2 mL) TCEP (60 mg, 0.2 mmol) is added and the mixture is stirred at room temp. for 18 h. After evaporation, the residue is dialysed against 20% NaCl and dest. water (reg. cellulose, MWCO 1000), yield 107 mg (72%) of compound d01 after lyophilisation (molecular weight 4296 g/mol).

(32) The example can also be extended to alkane substituents beyond decane, including e.g. the use of 1,6-dibromohexane, 1,8-dibromooctane, 1,11-dibromoundecane, or further dibromo-alkanes employing unsaturated, cyclic or substituted moieties according to the description of the linker L in the general formula E-[G-L-D(OSO.sub.3.sup.−M.sup.+).sub.n].sub.m. Furthermore, the linker can be introduced by a PEG-alkane moiety, such as azido-PEG-alkylbromide/iodide (or mesylate, tosylate), such as azido-PEG.sub.3-10-(CH.sub.2).sub.2-18—Br.

Example 2

Synthesis of Polyglycerol Dendrimer System with 24 Sulfate Groups and Reactive Groups for Bioconjugation from d01 by Reaction with Bifunctional PEG-COOH NHS Esters

Example 2a

(33) Synthesis of Polyglycerol Dendrimer System with 24 Sulfate Groups and Maleimido Reactive Group (Compound d02) from d01 by Reaction with Maleimido-PEG(4)-COOH NHS ester

(34) ##STR00013##

(35) 10 mg (2.33 μmol) of compound d01 is dissolved in 1 mL DMF/water 9:1 and maleimido-PEG(4)-COOH NHS ester (3.0 mg, 5.83 μmol) is added, followed by shaking at 40° C. for 48 h. The product (compound d02) is precipitated by addition of dichloromethane, centrifugation and repeated washing with dichloromethane, followed by lyophilisation from dest. water. The degree of coupling is determined by H-NMR (700 MHz) based on the ration of maleimido signals (2H, s) and aliphatic spacer (multiplets <1.5, 16 H) giving 88% degree of coupling for d02 (MW 4694 g/mol).

Examples 2b-h

(36) The synthesis of polyglycerol dendrimer systems with 24 sulfate groups and further reactive linkers is accomplished by using different bifunctional PEG-COOH NHS esters comprising an azido, propargyl, cyclooctinyl, pyridinyldisulfide, thioacetyl, or maleimido group. The reaction with compound d01 is conducted according to example 2a (yields 70-85%). Table 1 summarizes products d03 to d09.

Example 2i

Synthesis of Polyglycerol Dendrimer System with 24 Sulfate Groups and Isothiocyanate Group for Bioconjugation (Compound d10)

(37) The amino group can alternatively be directly converted into an isothiocyanate group. 10 mg (2.33 μmol) of compound d01 is dissolved in 0.5 mL DMF and di(2-pyridyl)-thionocarbonate (1.1 mg, 4.66 μmol) is added, followed by shaking at 40° C. for 6 h. The product d10 is isolated as described in example 2a. Conversion is monitored by FTIR (2100 cm.sup.−1) (MW 4338 g/mol).

(38) TABLE-US-00002 TABLE 1 Reactive linkers or groups coupled to d01 and characterization of resulting product. (dotted line indicates bond to amino group in d01 forming an amide) Product MW Ratio compound Linker structure (g/mol) linker:dendrimer d03 4870 78 d04 4741 89 d05 4917 68 d06 4657 95 d07 4670 95 d08 4719 90 d09 0 4673 87

(39) The aforementioned dendrimer systems of type d01 should be understood as exemplary and are extendable to the application of other n-numbers as the specific ones here shown, e.g. by doubling the hydroxyl groups via allylation/dihydroxylation giving 48 sulfate groups after sulfation, which give compounds in analogy to d03-d10.

Example 3

Synthesis of Polyglycerol Dendrimer System with 24 Sulfate Groups and NHS Ester Group for Bioconjugation from Example 1d (azido Linker) by Reaction with Bifunctional propargyl-PEG-COOH Linkers

Example 3a

Click Coupling of azido Dendrimer (example 1d) with propargyl-PEG(4)-carboxylic acid-t-butyl ester

(40) To a solution of 100 mg (0.023 mmol) of azido dendrimer (example 1d) in 1 mL of a mixture of water/DMF (1:1), CuSO.sub.4.5H.sub.2O (5.7 mg, 0.023 mmol), ascorbic acid (0.035 mmol), and (0.058 mmol) propargyl-PEG4-COO.sup.tbutylester (Broadphann Ltd., US) is added and the mixture is stirred at 80° C. for 18 h. The residue obtained after lyophilisation is suspended in a mixture of dichloromethane (5 mL), trifluoroacetic acid (3 mL) and water (0.1 mL), stirred for 5 h at 40° C. to obtain the free carboxylic acid group. After evaporation, the residue is suspended in MeOH and precipitated in diethylether, washed with dichloromethane, and isolated by centrifugation. The residue is dialysed against NaCl and dest. water (reg. cellulose, MWCO 1 kDa), yield 80 mg of carboxy dendrimer (MW 4582 g/mol) after lyophilisation.

Example 3b

Synthesis of polyglycerol dendrimer System with 24 Sulfate Groups and NHS ester Group for Bioconjugation (Compound d11)

(41) ##STR00021##

(42) To a solution of 80 mg (17.5 μmol) carboxy dendrimer (example 3a) in 2 mL DMF 6 μL (35 μmol) DIPEA is added, followed by the addition of 13 mg O—(N-succinimidyl)-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HSTU; 35 μmol). After 18 h stirring at room temp., the product is isolated by precipitation with dichloromethane (repeated circles of DMF/CH.sub.2Cl.sub.2), and drying in vacuum, yield 82 mg NHS ester (compound d11; MW 4679 g/mol) as amorphous solid, used without further purification.

(43) The scope of the invention regarding polyglycerol dendrimer systems of type d11 shows that an NHS ester function can be synthesized at polysulfated dendrimers and is not limited to the example and may include other numbers n of sulfate groups, e.g. by doubling the hydroxyl groups via allylation/dihydroxylation giving 48 sulfate groups after sulfation, or other azido-alkylcarboxylic acid derivatives to synthesize NHS esters of sulfated dendrimer systems.

Example 4

Synthesis of Polyglycerol Dendrimer System with 32 Sulfate Groups and Reactive Groups for Bioconjugation (Compound d12-d17)

Example 4a

Modification of Polyglycerol Dendrimer Based on Polyglycerol Dendron [G3.0]-OH (Wyszogrodzka M. et al., Chemistry 2008, 14, 9202) with azidoundecyl Linker

(44) A solution of 0.3 g (0.21 mmol) [G3.0]-OH (16 OH groups, acetal protected) in 2 mL dry THF was reacted with 1,10-dibromodecane and subsequently with NaN.sub.3 as described in example 1a followed by cleavage of acetal groups in MeOH/HCl and purification by RP C-18 chromatography (Licroprep) using water/methanol giving 0.15 g (52%) [G3.0]-O—(CH.sub.2).sub.11—N.sub.3 (MW 1355 g/mol).

Example 4b

Formation of Polyglycerol Dendrimer with Azidodecyl Linker (Example 4a) Through Allylation/Dihydroxylation, Sulfation and Reduction to Give Aminoundecyl-Dendrimer System with 32 Sulfate Groups (Compound d12)

(45) ##STR00022##

(46) The synthesis is accomplished as described in example 1d-e, giving 155 mg of product d12 as colorless amorphous solid (MW 5721 g/mol).

Example 4c-h

(47) The synthesis of polyglycerol dendrimer systems with 24 sulfate groups and further reactive linkers is accomplished by using different bifunctional PEG-COOH NHS esters comprising an azido, propargyl, cyclooctinyl, pyridinyldisulfide, thioacetyl, or maleimido group. The reaction with compound d01 is conducted according to example 2a/4c (yields 65-86%). Table 2 summarizes achievable products d13 to d17.

(48) TABLE-US-00003 TABLE 2 Reactive linkers coupled to d12 and characterization of resulting product. (dotted line indicates bond to amino group in d12 forming amide) Product MW Ratio compound Linker structure (g/mol) linker:dendrimer d13 6119 87 d14 6295 82 d15 6341 75 d16 6082 90 d17 6095 94

(49) The scope of the invention regarding polyglycerol dendrimer systems of type d12 is not limited to the Examples and may include other numbers n of sulfate groups, e.g. by doubling the hydroxyl groups via allylation/dihydroxylation giving 64 sulfate groups after sulfation, which give compounds in analogy to d13-d17.

Example 5

Synthesis of polyglycerol/amido dendrimer System with 24 Sulfate Groups and Reactive Groups for Bioconjugation based on aminomethyl-tris(Propionic Acid) and Triglycerol (Compounds d18-d22)

Example 5a

Synthesis of aminomethyl-tris(Propionic Acid)-tris(Triglycerolamide)

(50) ##STR00028## ##STR00029##

(51) To a solution of nitromethane-trispropionic acid (1.5 g, 5.4 mmol) and DIPEA (3.9 g, 30 mmol) in 15 mL DMF, a solution of HBTU (9.3 g, 24 mmol) in 2 mL DMF is given. After 30 min stirring at room temp. a solution of (7.3 g, 23 mmol) triglycerolamine, acetal-protected (1,3-bis[2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-2-propylamine; according to Wyszogrodzka M. et al., Chemistry 2008, 14, 9202) in 2 mL DMF. is added, followed by stirring at room temp. for 24 h. The reaction mixture is evaporated to dryness and filtrated through a short silica column with cyclohexane/ethylacetate. The product-containing fractions are collected, evaporated, and the residue is dissolved in 20 mL methanol, to which 1.7 mL cone. HCl is added to cleave the acetal protecting groups by stirring another 24 h. After evaporation to dryness, the residue is purified by RP C-18 chromatography (Licroprep) using water/methanol; yield 2.7 g (53%) intermediate product (H-NMR, ESI MS). Reduction of the nitro group to an amino group is accomplished by dissolving 2.7 g intermediate in 120 mL methanol, to which Pd/C (0.3 g) and ammonium formate (1.27 g, 20 mmol) is added. The mixture is hydrogenated at 5 bar/room temp. for 48 h, followed by filtration over celite, evaporation and RP C-18 chromatography as described above, yield 1.9 g (74%) aminomethyl-tris(propionic acid)-tris(triglycerolamide) as viscous oil.

Example 5b

Linker Modification with 11-azidoundecane carboxylic acid

(52) To a solution of 11-azidoundecane carboxylic acid (0.11 g, 0.5 mmol) and DIPEA (0.16 g, 1.2 mmol) in 2 mL DMF, HBTU (0.2 g, 0.56 mmol) is added and the mixture is stirred for 1 h, followed by the addition of aminomethyl-tris(propionic acid)-tris(triglycerolamide) (0.3 g, 0.33 mmol; example 5a). The mixture is stirred at room temp. for 48 h, evaporated to dryness and purified by RP C-18 chromatography (Licroprep) using water/methanol; yield 0.26 g (71%) as viscous oil.

Example 5c

Formation of Polyglycerol/Amido Dendrimer with Azidodecylcarbonyl Linker (Example 5b) Through Allylation/Dihydroxylation, Sulfation and Azide Reduction to Give Aminodecylcarbonyl Dendrimer System with 24 Sulfate Groups (Compound d18)

(53) ##STR00030##

(54) The synthesis is accomplished as described in example 1d-e giving 120 mg of product d18 as colorless amorphous solid (MW 4432 g/mol).

Example 5c-h

(55) The synthesis of polyglycerol/amido dendrimer systems with 24 sulfate groups and further reactive linkers is accomplished by using different bifunctional PEG-COOH NHS esters comprising an azido, propargyl, cyclooctinyl, pyridinyldisulfide, thioacetyl, or maleimido group. The reaction with compound d01 is conducted according to example 2a/4c (yields 65-86%). Table 3 summarizes achievable products d19 to d22. Another option is the derivatization of the amino group with anhydrides, such as diglycolic acid anhydride by known methods to introduce a free carboxylic acid (product d22) or conversation of the amino group into an isothiocyanate group (product d23; see also example 2i).

(56) TABLE-US-00004 TABLE 3 Reactive linkers coupled to d18 and characterization of resulting product. (dotted line indicates bond to amino group in d18 forming an amide) Product MW Ratio compound Linker structure (g/mol) linker:dendrimer d19 4830 90 d20 5006 79 d21 4876 86 d22 4548 98

(57) The scope of the invention regarding polyglycerol dendrimer systems of type d18 is not limited to the examples and may include other numbers n of sulfate groups, e.g. by doubling the hydroxyl groups via allylation/dihydroxylation giving 48 sulfate groups after sulfation, which give compounds in analogy to d19-d22.

Example 6

Synthesis of polyglycerol dendrimer System with 24 Sulfate Groups, NHS ester Reactive Group and Reductively Cleavable Disulfide Unit in the Linker (Compound d24)

(58) ##STR00035##

(59) A solution of compound d09 (example 2h) (25 mg, 5.3 μmol) and 20 mg ion exchange resin Dowex® 50 W in 0.5 mL methanol/DMF 1:1 is shaken for 3 h, filtrated, and evaporated to dryness. The residue is dissolved in PBS buffer (50 mM; pH 7.4, incl. 1 mM EDTA)/methanol 1:1, to which 4-(2-pyridyldithio)butanoic acid (2.4 mg, 10.6 μmol; Widdison et al., J. Med. Chem. 2006, 49, 4392) is added. After 18 h stirring, the residue is evaporated and dialysed against 20% NaCl and dest. water (reg. cellulose, MWCO 1000), yield 21 mg of compound d24 as lyophilisate (MW 4764 g/mol).

Example 7

Synthesis of polyglycerol/amido dendrimer System with 24 Sulfate Groups and Triglycine Motif for Sortase-Mediated Enzymatic Ligation (d25)

(60) ##STR00036##

(61) Boc-GlyGlyGly-OH (Bachem; 25 mg, 0.085 mmol) and DIPEA (11 mg, 0.17 mmol) are dissolved in 3 mL DMF and HATU (38 mg, 0.1 mmol) is added. After 1 h, compound d18 (95 mg, 0.021 mmol) is added. The mixture is stirred at 50° C. for 48 h. The product is precipitated by the addition of diethylether and collected by filtration. The residue is repeatedly washed with dichloromethane, and then suspended in 5 mL of a mixture of dichloromethane/trifluoroacetic acid 1:1, followed by 5 h stirring at room temp.

Example 8

Synthesis of Conjugates of dendrimer Systems with Fluorescent Dye ICC

Example 8a-c

Conjugation of amino dendrimer Compounds d01, d12 and d18 with Cyanine Dye

(62) Cyanine dyes for labeling, such as Cy3 or ICC, employing different functional groups and substitution patterns, are synthetically accessible according to known literature. ICC dye (NHS ester) is a published VIS dye (abs/fluoresc. ˜550 nm/575 nm) (Gröger et al., Bioconjugate Chem 2013, 24, 1507).

(63) ##STR00037##

(64) 10 mg (2.33 μmol) of compound d01 is dissolved in 1 mL DMF/water (9:1) and ICC NHS ester (MiDye550 NHS ester, Iris Biotech., Germany) (8.9 mg, 12 μmol) is added, followed by shaking at 40° C. for 72 h. The product (ICC-d01) is precipitated by addition of dichloromethane, and purified by RP C-18 chromatography (Licroprep) using water/methanol. The degree of coupling is determined by spectrophotometry (ICC dye λ.sub.max at 550 nm, ε 120.000 L.sup.−1 M.sup.−1) giving 54% degree of coupling. The dendrimers d12 and d18 are covalently conjugated with ICC dye NHS ester in analog fashion.

(65) Other fluorescent dye NHS esters can be conjugated as described here, such as cyanine dye ITCC NHS ester, Cy3 to Cy7 NHS esters, rhodamine or fluorescein NHS esters. Also, commonly known isothiocyanate (ITC) derivatives of such dyes, such as FITC, can be used.

(66) Another method for dye conjugation is to use amino dendrimers (e.g. d01, d12, d18) in combination with 2-iminithiolane and maleimido dyes (e.g. ICC maleimide) giving, as example, ICC-IT-d01 or ICC-IT-d18.

Example 9

Synthesis of dendrimer with UV-Detectable Linker (d26)

(67) ##STR00038##

(68) The synthesis of d26 is accomplished in analogy to example 4, using 4,4′-di-(3-bromopropyloxy)benzophenone (Yan et al., Bioorg. Med. Chem. 2013, 21, 508) instead of 1,10-dibromodecane, as linker. Dendrimer d26 can then be further modified as described in examples 4c-h to be used for protein conjugation. 4,4′-di-(3-bromopropyloxy)benzophenone linkers can be used to built up dendrimers of the type of examples 1-3.

Example 10

Synthesis of Dendrimer with VIS-Detectable Cyanine Dye Linker (d28)

Example 10a

Synthesis of Polyglycerol Dendrimer System with 24 Sulfate Groups and Aminohexyl Linker (Compound d27)

(69) Compound d27 is an analog to d01 with aminohexyl group and is synthesized according to example 1a-e using 1,6-dibromohexane as starting material; d27 molecular weight 4240 g/mol.

Example 10b

Conjugation with Indocyanine Moiety as VIS-Detectable Linker and NHS Ester Reactive Group (d28)

(70) ##STR00039##

(71) 5,5′-Dicarboxy-1,1′-dimethylindocarbocyanine, monoacetate is synthesized according to known procedures. To a solution of 0.1 g (0.2 mmol) of this dye and DIPEA (0.11 g, 0.8 mmol) in 3 mL DMF is added a solution of HBTU (0.31 g, 0.8 mmol) in 0.5 mL DMF. After 90 min stirring at room temp., d027 (0.17 g, 0.04 mmol) is added portionwise as a solid and the resulting mixture stirred at 60° C. for 72 h. The product is precipitated by adding 3 mL diethylether and collected by centrifugation. Purification by RP C-18 chromatography (Licroprep) using water/methanol yields 0.12 g intermediate after lyophilisation. Conversion into the NHS ester is conducted by adding HSTU (75 mg, 0.2 mmol) to a solution of intermediate and 35 μL DIPEA in 1 mL DMF and stirring for 18 h at room temp., followed by precipitation in diethylether (repeated circles of DMF/dietyhlether), giving 0.10 g product d28 (mol. weight 4743 g/mol).

Example 11

Synthesis of Conjugates of dendrimer Systems with Proteins and Antibodies

Example 11a

General Procedure for the Conjugation Using Dendrimer Systems with Maleimido Groups (d02, d03, d13, d14, d19, d20)

(72) A solution of 2 mg of protein in 1 mL of 50 mM phosphate buffer pH 7.4/10 mM EDTA is reacted with 10 mol-eq. 2-iminothiolane for 60 min. To this mixture is added 14 mol-eq. of maleimido-functionalized dendrimer (e.g. d02, d03, d13, d14, d19, d20), followed by incubation for 18 h. The mixture is subjected to purification into TRIS-buffered saline (TBS; pH 7.4) using NAP10 column or Slide-A-Lyzer dialysis cassettes (reg. cellulose, MWCO 10 kDa).

Example 11b

General Procedure for the Conjugation Using Dendrimer Systems with Pyridyldisulfide Group (d04, d05, d15, d21)

(73) A solution of 2 mg of protein in 1 mL of 50 mM phosphate buffer pH 7.4/10 mM EDTA is reacted with 10 mol-eq. 2-iminothiolane for 60 min. To this mixture 10 mol-eq. of maleimido-functionalized dendrimer (e.g. d04, d05, d15, d21) is added, followed by incubation for 3 h. The mixture is subjected to purification into TRIS-buffered saline (TBS; pH 7.4) using NAP10 column or Slide-A-Lyzer dialysis cassettes (reg. cellulose, MWCO 10 kDa).

Example 11c

General Procedure for the Conjugation Using Dendrimer Systems with Isothiocyanate or NHS Ester Group (d10, d11, d23)

(74) A solution of 2 mg of protein in 1 mL of PBS pH7.4 is reacted with 10 mol-eq. of isothiocyanate or NHS ester dendrimer (e.g. d10, d11, d23) for 24 h. The mixture is subjected to purification into TRIS-buffered saline (TBS; pH 7.4) using NAP10 column or Slide-A-Lyzer dialysis cassettes (reg. cellulose, MWCO 10 kDa).

Example 12

Synthesis of Conjugates of Dendrimer Systems with Enzymes

Example 12a

Conjugation of Dendrimer Systems with Maleimido Groups (d02, d13, d19) with the Enzyme L-asparaginase

(75) L-asparaginase is a homotetramer of 4 units of 34 kDa molecular weight, which can be modified at a disulfide bridge in each of the monomer units (Balan et al., Bioconjugate Chem 2007, 18, 61). L-asparaginase (10 mg, 0.29 μmol monomer; L-asparaginase 5000E, Medac, Germany) is dissolved in 2 mL of 50 mM phosphate buffer pH8/10 mM EDTA. Dithiotreithol (DTT; 80 mg) is added and the solution shaken for 1 h. Excess DTT is removed via SEC (Sephadex G50; phosphate buffer pH8/10 mM EDTA). To the resulting solution (approx. 8 mg in 4 mL, as measured by UV), a solution of 2.3 μmol (8 mol-eq.) of maleimido dendrimer (e.g. d02, d13, d19) in approx. 0.2 mL of water is added, followed by incubation for 24 h under gentle shaking. Purification is achieved by ultrafiltration (centriprep flasks, reg. cellulose, MWCO 20.000).

Example 12b

(76) Conjugation of dendrimer Systems with Maleimido Group (d02) with the Enzyme DNase

(77) The synthesis is accomplished as described in example 11a yielding DNase-d02 as solution in TBS pH 7.4.

Example 12c

Conjugation of Dendrimer Systems with Isothiocyanate Group (d10) with the Enzyme DNase

(78) To a solution of 2 mg (65 nmol) DNase (desoxyribonuclease I from bovine pancreas, 31 Da) in 1 mL PBS pH7.4 is given 5 mol-eq. isothiocyanate dendrimer d10 (1.4 mg) in 0.2 mL PBS, followed by reaction at room temp. for 18 h. Purification is accomplished in Float-A-Lyzer dialysis cassettes (cellulose ester, MWCO 10 kDa) into TBS pH 7.4.

(79) TABLE-US-00005 TABLE 4 Protein conjugates with dendrimer systems according examples 11 and 12 conjugate product according to protein dendrimer general formula I saporin d02, d13, d19 saporin-d02 saporin-d13 saporin-d19 ovalbumin d02, d03, d20, d28 ova-d02 ova-d03 ova-d20 ova-d28 apoptin d02, d13 apo-d02 apo-d13 diphtheria toxin d02, d13 dph-d02 dph-d13 human serum albumin (HSA) d21 HSA-d21 rCys-Protein G d02, d13, d19 ProtG-d02 ProtG-d13 ProtG-d19 bevacizumab d10, d11, d13 bev-d11 bev-d23 IgG d22 IgG-d22 d28 IgG-d28 L-asparaginase d02, d13, d19 asp-d02 asp-d13 asp-d19 DNase d02, d10 DNase-d02 DNase-d10

(80) Protein conjugates with dendrimer systems according to table 4 can be additionally conjugated with fluorescent dyes in order to detect the conjugates in biological systems. Fluorescence labeling is a known procedure and can be accomplished with a variety of reactive fluorophores. Here, the dye ICC NHS ester (Gröger et al., Bioconjugate Chem 2013, 24, 1507) is used in the examples demonstrating results of cellular uptake by FACS and microscopy.

Example 13

Synthesis of Conjugates of Dendrimer Systems with Small Molecule Peptides and Peptidomimetics

Example 13a

Synthesis of Dendrimer Conjugate with Monomethyl Auristatin E

(81) ##STR00040##

(82) Maleimidocaproyl-monomethyl auristatin E (mc-MMAE) can be synthesized according to the literature (Doronina et al., 2006, 17, 114). Conjugation is achieved by dissolving dendrimer d09 (2 mg, 0.43 μmol) in 0.5 mL of an aqueous 50 mM hydroxylamine solution, followed by adding maleimidocaproyl-monomethyl auristatin E (0.37 mg in 0.5 mL DMF/PBS 1:1). After 18 h incubation at 25° C., the product is precipitated with dichloromethane, followed by HPLC purification (water/methanol) giving MMAE-d09 (1.1 mg).

(83) Other not limiting examples of structures of dendrimers d01 with auristatin effector molecules which are synthesized according to methods described herein may include:

(84) ##STR00041##

Example 13b

Synthesis of dendrimer Conjugate with Stapled Peptide

(85) ##STR00042##

(86) Stapled peptide for activation of p53 pathway (ATSP-3900 with N-terminal β-alanine) can be synthesized according to the literature (Chang et al., PNAS 2013, 110, E3445). Conjugation is achieved by dissolving dendrimer d10 (2 mg, 0.46 μmol) in 0.5 mL 50 mM Phosphate buffer pH8. To this solution, a solution of β-Alanyl-ATSP-3900 (0.65 mg, 0.40 μmol) in 0.2 mL DMF is added, followed by reaction at 40° C. for 24 h. Purification is achieved by HPLC (water/methanol) giving ATSP3900-d10 conjugate (0.8 mg).

Example 13c

Synthesis of dendrimer Conjugates with Alpha-Helical Peptides Derived from Natural Binding Motifs

(87) The peptide sequences P1 to P7 (as C-terminal amides) were synthesized by common solid-phase peptide synthesis. These peptides carry a N-terminal cystein for conjugation of maleimido dendrimers to the thiol group. Conjugation to dendrimers d02, d03, d13, d14, d19 was performed. As example, the procedure employing dendrimer d02 is described:

(88) 1 mg peptide is dissolved in 200 μL, DMF and further diluted with 50 mM phosphate buffer (up to 500 μL). To this solution is added 5 mol-eq. of TCEP (stock solution in water) followed by 0.9 mol-eq. of dendrimer, dissolved in 200-300 μL water. The mixture is shaken gently at 25° C. for 18 h. Purification is achieved by HPLC (water/acetonitrile) or SEC (Sephadex LH-20, water/DMF), or a combination of both, giving conjugates listed in table 5.

(89) TABLE-US-00006 TABLE 5 Peptide conjugates with dendrimers according to example 13 c Synthesized origi- peptide nal sequence for Con- # protein conjugation jugates P1 BIM CGMRPEIWIAQELRRIGDEFNA d02-P1 (SEQ ID NO: 1) P2 BIM CGDMRPEIYI(Aib)QELRRIGD(Aib)Y d02-P2 (SEQ ID NO: 2) P3 Bcl9 CGLSQEQLEHRERSLQTLRDIQRMLF d02-P3, (SEQ ID NO: 3) d03-P3 P4 Bcl9/2 CGLSKEQLEHRERSLQTLRDIERLL d02-P4, (SEQ ID NO: 4) d03-P4 P5 PUMA CGEEQWAREIGAQLRRMADDLNAQYER d02-P5 (SEQ ID NO: 5) P6 BID CGEDIIRNIARHAAQVGASADRSI d02-P6 (SEQ ID NO: 6) P7 c-myc CPKVVILKKATAYILSVQAEEQKL d02-P7 (SEQ ID NO: 7)

Example 14

Synthesis of Conjugates of dendrimer Systems with Small Molecule Inhibitors

Example 14a

Synthesis of dendrimer Conjugate with Staurosporine

(90) ##STR00043##

(91) Staurosporine is a kinase inhibitor which can be conjugated to carrier molecules while maintaining its inhibitory activity. Succinoyl-staurosporine is synthesized as described (Caravatti et al., Bioorg. Med. Chem. Lett. 1994, 4, 399) and conjugated to d01 in DMF using HATU/DIPEA coupling conditions.

Example 14b

Synthesis of dendrimer Conjugate with Maytansine Derivative

(92) ##STR00044##

(93) N.sup.2′-deacetyl-N.sup.2′-(3-mercapto-1-oxopropyl)-maytansine is a published derivative suited for covalent conjugation to biomolecules via disulfide bonds or maleimido groups (Erickson et al. Bioconjugate Chem. 2010, 21, 84). The covalent conjugation to dendrimer systems of types d02, d03, d13, d19, d20 (maleimides) is possible. Exemplary, A solution of N.sup.2′-deacetyl-N.sup.2′-(3-mercapto-1-oxopropyl)-maytansine (5 mg, 6.8 μmol) and dendrimer d02 (24 mg, 5.2 μmol) in 0.5 mL DMF/50 mM phosphate buffer pH7.0+5 mM EDTA (9:1) is reacted for 3 h at room temp., the product precipitated by adding 1 mL dichloromethane. Excess maytansine is removed by repeated circles of precipitation from DMF/dichloromethane, giving 21 mg may-d02 (mol. weight 5432 g/mol).

Example 15

Cellular Uptake of Dendrimer-ICC Dye Conjugates (Example 8) and Dendrimer-Protein Conjugates (Example 11)

(94) The human cancer cell lines A2780 and QGP-1 are cultured in RPMI medium, with 10% fetal calf serum (FCS), 2% glutamine, and penicillin/streptomycin added. All cells are seeded into medium at 1×10.sup.5 cells/ml, cultured at 37° C. with 5% CO.sub.2, and split 1:5 two times a week. The epithelial human cancer cell lines A549, MCF7, HaCaT, and HepG2 are propagated in DMEM medium (PAN Biotech), with 10% fetal FCS, 2% glutamine, and penicillin/streptomycin. Cells are seeded into medium at 1×10.sup.5 cells/ml (37° C. with 5% CO.sub.2, split 1:5 two times a week). HT29 cells are cultured in McCoy's medium (PAN biotech), with 10% FCS), 2% glutamine, and penicillin/streptomycin. Cells are seeded into medium at 1×10.sup.5 cells/ml (37° C. with 5% CO.sub.2, split 1:10 two times a week). For cellular microscopy, cells are seeded at 2×10.sup.5 cells/ml in a 24-well culture plate on glass coverslips (Sigma), cultured for 24 hours at 37° C., then cultured with medium containing 10.sup.−6 M of ICC-d01, ICC-d12, ICC-d18 or respective derivatives with 48 sulfate groups, or dendrimer conjugates with proteins (examples 12 and 13) or 10.sup.−6 M glycerol-ICC (control) for up to 24 hours at 37° C. Afterwards, cells are fixed with cold acetone, rinsed and covered with 4,6-diamidino-2-phenylindole (DAPI, Abcam) for nuclear counterstain. Image acquisition is performed using a Leica DMRB microscope (Leica). Images are taken with a digital camera (Spot 32, Diagnostic Instruments) with the same exposure time for all pictures. FIG. 8A shows an example of intracellular distribution in the cytosolic areas (ICC-d01, example 8). To quantify the cellular uptake mediated by the sulfated dendrimers, ICC-d01 (example 8) is used to incubate 10.sup.7 SKBR3 tumor cells with 10.sup.−6 M ICC-d01 for 24 h. This amount and time is sufficient to measure the supernatant after pooling and cell lysis with DMSO. FIG. 8B shows the resulting absorption spectrum (ICC ext.coeff (559 nm) 150,000 M.sup.−1 cm.sup.−1) giving 10.sup.−17 moles per cell, which corresponds to approx. 5 Mio molecules per cell.

(95) For FACS studies, 2×10.sup.5 cells/ml cells are cultured in 24-well plates with normal culture medium or medium containing different concentrations of test substances for 3 or 24 hours. Thereafter, cells are washed with PBS and detached with 200 μl/well accutase (PAA) and washed two times with PBS. Cells are fixed with 500 μl 3% paraformaldehyde for 10 min at 4° C., stopped with 2 ml PBS and centrifuged with 250×g, for 10 min at 4° C. Supernatants are removed and cells were suspended in 200 μl PBS with 0.5% bovine serum albumin (Roth).

(96) Fixed cells are kept at 4° C. until analysis in a FACS Calibur instrument (Becton-Dickinson). Table 5 shows examples of FACS analysis.

(97) TABLE-US-00007 TABLE 5 Values of uptake measured by FACS, relative to ICC-d01 (range from different cell lines: HT29, HepG2, QGP-1, A431, MCF-7, A2780) after incubation for 24 h. All conjugates were additionally conjugated with ICC NHS ester, and values were corrected by fluorescence due to differing dye-to-protein ratio or dendrimer loading. Compound % uptake ICC-d01 (dendrimer dye only) 100 ICC-IT-d01 (dendrimer dye only)  85-100 ICC-d18 (dendrimer dye only) 110-125 ICC-IT-d18 (dendrimer dye only)  95-110 Sap (without dendrimer) 5-8 Sap-d02 55-70 Diphtheria toxin (without dendrimer)  8-10 Dph-d02 45-68 L-asparaginase (without dendrimer) 4-7 Asp-d02 85-95 DNase (without dendrimer)  4-12 DNase-d02 50-65 IgG (without dendrimer) 3-5 IgG-d02 30-45

Example 16

Activity of Protein-dendrimer Conjugates in Tumor Cells

(98) For cytotoxicty measurements, 2×10.sup.5 cells were incubated with 1 ml culture medium containing increasing concentrations of test substances. After 72 hs treatment, cell number, viability and cell diameter as parameter of apoptotic processes were analyzed in a cell counter and analyzer system (CASY®, Schärfe Systems). In addition, drug cytotoxicity was assessed in vitro using the MTT assay (cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as a test for metabolic activity of the cells. 1×10.sup.4 cells per well were seeded in 96-well plates in 100 μl culture medium containing increasing concentration of the test substance. 10 μl MTT (5 mg/ml in PBS, obtained from Sigma) was added to each well and the plates were incubated for 4 hrs, 24 hrs, 48 hrs or up to 72 h. The resulting formazan product was dissolved with acid isopropanol and the absorbance at a wavelength of 570 nm (Ex570) was read on a Microplate Spectrophotometer (Anthos htII, Microsystems). In a couple of experiments, medium with test substances was removed after 48 hs, cells were counted and identical cell numbers with new medium without test substances were seeded in new plates. MTT assay was performed after further incubation for 72 hours.

(99) As examples, table 6 shows that conjugates of ribosomal-inhibiting toxins exhibit an increased cytotoxicity due to cellular uptake and intracellular activity. Effects are obtainable by using e.g. compounds Sap-d02, Sap-d13 or Sap-d19. Similar effects are obtainable in different tumor cells lines, such as HT29 colon tumor cells, HepG2 liver tumor cells, QGP-1 pancreatic tumor cells, A431 epidermoid tumor cells;

(100) TABLE-US-00008 TABLE 6 IC.sub.50 values of inhibition of cell proliferation measured by MTT test (range from different cell lines: HT29, HepG2, QGP-1, A431, MCF-7, A2780. Compound IC.sub.50 Sap (without dendrimer) >1 μM Sap-d02 2-5 nM Sap-d13 4-8 nM Diphtheria toxin (without dendrimer) >0.5 μM Dph-d02 1-3 nM L-asparaginase (without dendrimer) >1.5 μM Asp-d13 10-20 nM DNase (without dendrimer) >2 μM DNase-d10 4-9 nM

Example 17

Activity of Peptide-Dendrimer Conjugates in Tumor Cells

(101) Cell culture measurements were performed as described in example 16. Table 7 shows results for the different peptide-dendrimer conjugates.

(102) TABLE-US-00009 TABLE 7 IC.sub.50 values of inhibition of cell proliferation measured by MTT test (range from different cell lines: HT29, HepG2, QGP-1, A431, MCF-7, A2780. Compound IC.sub.50 d02-P1 0.9-2.1 μM P1 (peptide without dendrimer) >10 μM d02-P2 0.8-1.7 μM d03-P3 0.6-1.2 μM P3 (peptide without dendrimer) >10 μM d03-P4 0.4-1.1 μM P4 (peptide without dendrimer) >10 μM d02-P5 0.8-2.0 μM P5 (peptide without dendrimer) >10 μM d02-P6 1.2-2.3 μM P6 (peptide without dendrimer) >10 μM d02-P7 1.1-1.8 μM P7 (peptide without dendrimer) >10 μM

Example 18

Selectivity of Intracellular Uptake of Dendrimer Conjugates with Proteins: Effects of Inhibitors on Uptake of L-Asparaginase Conjugate Asp-d02 (Example 12a)

(103) Studies on cellular uptake measured by FACS were conducted as described in example 13. Compound Asp-d13 is incubated at a concentration of 10.sup.−7 M in QGP-1 tumor cells for 3 h and 6 h. Incubation is done in the presence of one of the following two substrates: (1) 0.1 mM genistein, an inhibitor for endocytotic uptake (Rejman et al., Mol. Ther. 2005, 12, 468-474) or (2) 50 mM rifamycin, an inhibitor of uptake via organic anion transporter proteins (OATP; Bi et al., Drug Metab Dispos. 2012, 40, 1085-92). The results demonstrate that FACS signals relative to Asp-d13 without inhibitor (defined as 100%) decrease in the presence of rifamycin to 37-40%, whereas no significant decrease can be measured in the presence of genistein (80-85%), see table 7 for data. For L-asparaginase coupled to a polyglycerolsulfate (mean molecular weight of 12000 Da; used according to WO2011/095311) endocytosis could be identified due to inhibition by genistein.

(104) TABLE-US-00010 TABLE 7 Relative values of inhibition of uptake into QGP-1 cells in presence of rifamycin or genistein. w/o inhibitor +genistein +rifamycin Compound time (%) (%) (%) Asp-d02 3 h 100 81 58 Asp-d02 6 h 100 95 53 Asp-polyglycerolsulfate 3 h 100 50 76 Asp-polyglycerolsulfate 6 h 100 55 72

Example 19

Activity of Protein-Dendrimer Conjugates in Tumor Cells in Comparison to Sulfated Polymer

(105) In this example, saporin conjugate Sap-d02 (example 11) is compared with an analog conjugate using a polyglycerolsulfate of polymeric, nondefined nature with comparable molecular weight (average value at 6000 g/mol; synthesized according to Gröger et al., Bioconjugate Chem 2013, 24, 1507). This polymer is functionalized with the linker maleimido-PEG(4)-COOH NHS ester as described in example 2a, and further purified via RP chromatography. Conjugation to saporin is accomplished as described in example 11. Comparative cytotoxicity measurements are conducted according to example 16 giving IC.sub.50 values from MTT test in 4 different cell lines (see table 8).

(106) TABLE-US-00011 TABLE 8 IC.sub.50 values of inhibition of cell proliferation measured by MTT test (range from different cell lines: HT29, HepG2, QGP-1, MCF-7). Compound IC.sub.50 Sap (without dendrimer) >1 μM Sap-d02 2-5 nM Sap-polyglycerol sulfate 38-45 nM

Example 20

Chemoselective and Site-Specific Conjugation Methods of Dendrimers with Proteins Employing an N-Terminal Cystein (Cystag)

Example 20a

(107) Dendrimers can be conjugated with protein and antibody therapeutics, as well as synthetic peptides, when these polyamino acids are provided with a cystein tag, thereby enabling chemoselective conjugation to cystein with dendrimers carrying maleimido, pyridinyldisulfide or other thiol-selective groups know to the skilled artisan (such as bromoacetyl, vinylsulfone).

(108) General protocol for conjugation of proteins (25-30 kDa) with Cystag: 1 mL of a solution of protein (conc. 2 mg/mL) in PBS containing 1 mM EDTA is incubated with a solution of TCEP in PBS (yielding 1 mM TCEP in the mixture) for 3 h at room temp., followed by the addition of 2 mol-eq. maleimido-containing or pyridinyldisulfide-containing, sulfated dendrimers (e.g. d02-d05, d13-d15) and reaction of further 2 h at room temp. Purification is accomplished in Float-A-Lyzer dialysis cassettes (cellulose ester, MWCO 10 kDa) into TBS pH7.4.

Example 20b

(109) Proteins with N-terminal cystein or cystein surrogates can be fused with other macromolecules employing a thioester moiety by the process of native chemical ligation (NCL) (Wong C T et al, Mol Biosyst. 2013, 9, 826-33). Surprisingly, sulfated dendrimers can be synthesized as thioesters and enable NCL-type conjugation to proteins with N-terminal cystein.

(110) Synthesis of sulfated dendrimers with thioester group: A solution of sulfated dendrimer with NHS ester (d11; 10 mg) in DMF (1 mL) is reacted with 10 mol-eq. of 3-thiopropionic acid methylester (Xiao et al., Bioorg Med Chem Lett 2013, 23, 6046-6051) for 24 h at 40° C. After repeated precipitation in DMF/ethylacetate and lyophilization from dest. water, 11 mg of product d29 were obtained as yellow solid.

(111) General protocol for conjugation of proteins (25-30 kDa) with Cystag: 1 mL of a solution of protein (conc. 2 mg/mL) in NCL-buffer containing MPAA and TCEP in 50 mM phosphate buffer pH7.0 is incubated 2.5 mol-eq. of dendrimer d29 for 24 h at room temp. Purification is accomplished in Slide-A-Lyzer dialysis cassettes (reg. cellulose, MWCO 20 kDa) into PBS pH7.4. The product contains a free cystein according to the mechanism of NCL and can be additionally conjugated to a fluorescence dye (such as ICC maleimide).

Example 21

Chemoselective Conjugation Via Reductive Amination Using a Sulfated Dendrimer with Carbaldehyde Group

Example 21a

Synthesis of Sulfated Dendrimer d31 with Carbaldehyde Group

(112) ##STR00045##

(113) The azido group in dendrimer d06 (example 2e) is reduced to the amine by TCEP. To a solution of d06 (50 mg, 0.011 mmol) in 1 mL of a mixture of water/methanol (1:1) TCEP (20 mg, 0.067 mmol) is added and the mixture is stirred at room temp. for 18 h. After evaporation, the residue is dialysed against 20% NaCl and dest. water (reg. cellulose, MWCO 1000), yield 45 mg (90%) of compound d30 after lyophilisation (molecular weight 4631 g/mol). Modification with a carbaldyehyde is accomplished by reaction with 4-formylbenzoic acid N-hydroxysuccinimidyl ester (Hooker, J M et al., Nano Letters 2007, 7, 2207-2210) in DMF/water, yielding sulfated dendrimer d31 as solid precipitate (molecular weight 4763 g/mol).

Example 21b

Chemoselective Conjugation of Sulfated Dendrimers d31 to Proteins Via Reductive Amination

(114) The process is performed with protein solutions of 2.5 mg/mL in 50 mM phosphate buffer pH7.0. General procedure: A solution of 1 mg protein (0.4 mL) and 3 mol-eq. of dendrimer d31 is treated with a stock solution of sodiumcyanoborohydride (NaBH.sub.3CN) in water yielding a final concentration of 1 mM NaBH.sub.3CN in the mixture, which is gently shaken for 48 h at 20° C. Purification is accomplished in Slide-A-Lyzer dialysis cassettes (reg. cellulose, MWCO 20 kDa) into TBS pH7.4. Dendrimer conjugation is determined by gel electrophoresis, yielding conversion of 50-70% of protein into conjugates (dendrimer-to-protein ratio unknown). Used proteins were ovalbumin, saporin, diphtheria toxin, and an unspecific IgG. Purification is afforded by SEC HPLC. Analysis of dendrimer-to-protein ratio is performed by gel electrophoresis.

Example 22

Conjugation of Sulfated Dendrimer to Proteins Via Acid-Cleavable Bond: Synthesis of Sulfated Dendrimer with Hydrazone Linker and Maleimido Group

(115) ##STR00046##

(116) Dendrimer d31 is fused with EMCH (ε-maleimidocaproic acid hydrazide) according to methods published (Walker G F et al., Molecular Therapy 2005, 11, 418-425).

(117) These types of modification can be extended to other aromatic, aliphatic aldehyde moieties, as well as ketone moieties (such as 4-acetylbenzoic acid) yielding hydrazones, carboxyhydrazones, as well as imines.