POLYMERIC MICELLES COMPRISING GLUCURONIDE-PRODRUGS

Abstract

The invention relates to a polymeric micelle comprising a block copolymer comprising a polyethylene glycol (PEG) hydrophilic block and a hydrophobic block, and a compound according to formula (I) or formula (III) encapsulated within said polymeric micelle and to uses thereof.

##STR00001##

Claims

1. A polymeric micelle comprising a block copolymer comprising a polyethylene glycol (PEG) hydrophilic block and a hydrophobic block, and a compound according to formula (I) or formula (III) encapsulated within said polymeric micelle ##STR00027## wherein X and Y are independently a hydrophilic drug molecule, R.sup.1 is H or —(CH.sub.2).sub.p—CH.sub.3, wherein p is 0, 1, 2 or 3, or R.sup.1 is a drug molecule and L is a spacer molecule comprising 2 to 6 aromatic rings oriented in a linear manner, or 1-5 aromatic rings oriented in a linear manner and one or more aromatic rings as a pendant side group or 1-5 aromatic rings and one or more double carbon-carbon bonds oriented in a linear manner, wherein said rings are each optionally and independently substituted with at least one halogen atom, hydroxyl or alkoxy group, and/or at least one (C1-4)-alkyl.

2. The polymeric micelle according to claim 1 of formula (I) ##STR00028## wherein X is a hydrophilic drug molecule, R.sup.1 is H or —(CH.sub.2).sub.p—CH.sub.3, wherein p is 0, 1, 2 or 3, or R.sup.1 is a drug molecule, and L is a spacer molecule comprising 2 to 6 aromatic rings, wherein said rings are each optionally and independently substituted with at least one halogen atom and/or at least one (C1-4)-alkyl.

3. The polymeric micelle according to claim 1 of formula (III) ##STR00029## wherein X and Y are independently a hydrophilic drug molecule, and R.sup.1 is H or —(CH.sub.2).sub.p—CH.sub.3, wherein p is 0, 1, 2 or 3, or R.sup.1 is a drug molecule.

4. The polymeric micelle according to claim 1 wherein L comprises 2 to 6 aryl rings.

5. The polymeric micelle according to claim 1 wherein the compound is selected from the group consisting of formula's (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii) and (III). ##STR00030## ##STR00031## ##STR00032## ##STR00033## wherein R.sup.1 is H or —(CH.sub.2).sub.p—CH.sub.3, wherein p is 0, 1, 2 or 3, or R.sup.1 is a drug molecule, and wherein n is 2 to 6, m is 1 to 3, and o is 1 or 2.

6. The polymeric micelle according to claim 1, wherein R.sup.1 is H.

7. The polymeric micelle according to claim 1, wherein R.sup.1 is a platinum compound.

8. The polymeric micelle according to claim 1, wherein each hydrophilic drug molecule is selected from the group consisting of an anticancer drug, an antifungal drug, an antibiotic drug and a combination thereof.

9. The polymeric micelle according to claim 1, wherein said hydrophilic drug molecule is selected from the group consisting of anthracyclines, nucleoside or deoxycytidine analogues, topoisomerase I inhibitors, nitrogen mustard alkylating agents, immunomodulators, adjuvants, P-glycoprotein drug efflux pump inhibitors, taxanes, anticancer peptides drug molecules, anticancer nucleic acid compounds and platinum compounds; the hydrophilic drug molecule.

10. The polymeric micelle according to claim 1 wherein at least part of the hydrophobic block contains an aromatic side group.

11. A pharmaceutical composition comprising a polymeric micelle according to claim 1 and at least one pharmaceutically acceptable carrier, diluent or excipient.

12. (canceled)

13. A method for the treatment or prevention of cancer or infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polymeric micelle according to claim 1.

14. (canceled)

15. A method of immunotherapy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polymeric micelle according to claim 1.

16. (canceled)

17. The method according to claim 15 for enhancing efficacy of immunotherapy in a subject suffering from cancer and being treated with said immunotherapy, the method comprising administering to the subject a therapeutically effective amount of a polymeric micelle according to claim 1.

18. (canceled)

19. The polymeric micelle according to claim 4 wherein said aryl rings are independently selected from the group consisting of phenyl rings, naphthyl, biphenyl groups, and combinations thereof.

20. The polymeric micelle according to claim 7, wherein the platinum compound is selected from the group consisting of cisplatin, oxaliplatin and carboplatin.

21. The polymeric micelle according to claim 9, wherein the hydrophilic drug molecule is selected from the group consisting of doxorubicin, daunorubicin, irinotecan and gemcitabine.

22. The polymeric micelle according to claim 10 wherein the aromatic side group is an aryl or heteroaryl.

23. The polymeric micelle according to claim 22 wherein the aromatic side group is selected from the group consisting of benzyl, phenyl and naphthyl.

24. The method according to claim 17, wherein said immunotherapy is antibody-based immunotherapy.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0150] FIG. 1: 1H-NMR spectrum of DOX GA3-DS.

[0151] FIG. 2: HPLC chromatogram of DOX GA3-DS.

[0152] FIG. 3: Conversion of DOX prodrugs to DOX in the presence of beta-GUS.

[0153] FIG. 4: Transmission electron micrographs of micelles loaded with 5 mg/mL doxorubicin double spacer prodrug (left panel) and single spacer prodrug (right panel).

[0154] FIG. 5: Release of DOX and prodrugs from the micelles.

[0155] FIG. 6: In vitro calreticulin (CRT) translocation of 4T1 cancer cells treated with DOX, prodrug and DOX prodrug-loaded micelles. 3A (left panel) shows the higher signal in the flow cytometry plots, which in 3B (right panel) is further quantified.

[0156] FIG. 7: Experimental setup of the in vivo study

[0157] FIG. 8: Quantification of CD3+CD8+ cytotoxic T cells in tumors after treatment assessed by flow cytometry.

[0158] FIG. 9: Quantification of CD3+CD8+ cytotoxic T cells in tumors after treatment assessed by fluorescence microscope.

[0159] FIG. 10: 1H-NMR spectrum of GEM-Phe-mGA3.

[0160] FIG. 11: HPLC chromatogram of GEM-Phe-mGA3.

[0161] FIG. 12: Z-average of GEM, GEM-Phe, and GEM-Phe-mGA encapsulated in micelles as measured by dynamic light scattering (DLS).

[0162] FIG. 13: Loading capacity (LC) and encapsulation efficiency (EE) of GEM, GEM-Phe, and GEM-Phe-mGA encapsulated in micelles.

[0163] FIG. 14: Release of GEM, GEM-Phe, and GEM-Phe-mGA from the micelles over time as assessed by the dialysis method.

EXAMPLES

Example 1. Synthesis and Micelle Loading of a DOX-Glucuronic Acid Double Spacer Prodrug

[0164] Prodrug Synthesis

[0165] The synthesis of the prodrug DOX-GA3 double spacer was divided into 3 parts. Detailed information is shown below.

[0166] Part 1: Synthesis of the Acetyl-Protected Methyl Ester of Glucuronic Acid

[0167] Firstly, methyl 1,2,3,4-tetra-O-acetyl-6-D-glucopyranuronate (3) was prepared by pyridine-catalyzed acetylation from the starting commercially available D-(+)-Glucuronic acid γ-lactone (1) according to Bollenback et al in: The Synthesis of Aryl-D-glucopyranosiduronic Acids, 1954, pp. 3310-3315. And compound (4) with a free anomeric hydroxyl was prepared through catalyzing compound (3) with ammonium carbonate associated with potassium bisulfate according to Bosco et al in: Lewis-Acidic Polyoxometalates as Reusable Catalysts for the Synthesis of Glucuronic Acid Esters under Microwave Irradiation, 2010, pp. 1249-1252, and purified by column chromatography. As show in scheme 1.

##STR00019##

[0168] Part 2: Synthesis of p-t.Butyldimethylsilyloxymethyl Benzoic Acid

[0169] Saponification of the esters from the commercially available compound (5) to carboxylic acids (6), followed by protection using tert-butyldimethylsilyl chloride (TBDMSCl) to yield silyl ethers (8). The specific procedures are shown in scheme 2.

[0170] From (5) to (6). 60 mmol methyl-(p-hydroxymethyl) benzoate (5) and 120 mmol KOH were dissolved in MeOH/water (3/1) mixture. After 2 h of refluxing, MeOH was evaporated and 1M HCl was added. The compound (6) formed crystals and were collected by filtration.

[0171] From (6) to (8). 13.1 mmol of (6), 39.7 mmol of imidazole and 33.2 mmol of t.butyldimethylsilyl chloride were dissolved in dry THF. After overnight reaction under nitrogen, saturated NaHCO.sub.3 was added to the THF solution to saponify the silyl ester. After complete hydrolysis, the water layer was extracted with ethyl acetate and washed with saturated NaCl to get the compound (8).

##STR00020##

[0172] Part 3: Synthesis of the Final DOX-GA3 Double Spacer Prodrug

[0173] Synthesis of Silyl Protection of Methyl-Glucuronide Single Spacer Compound B (Scheme 3-i, See the End of the Protocol)

[0174] 2 g (7.52 mmol) of compound 8, 0.9 g (8.9 mmol) of triethylamine and 2.45 g (8.9 mmol) of diphenylphosphoryl azide (DPPA) were dissolved in 40 ml of dry toluene. The mixture was stirred at 85° C. for 3 h under argon atmosphere. Next, 2 g of compound 4 (6 mmol) in 20 ml of toluene was added to the above mixture and the reaction was kept overnight at room temperature. After evaporation of the solvent, the residue was purified by column chromatography (heptane/ethyl acetate=5/2).

[0175] Synthesis of Deprotected Silyl of Methyl-Glucuronide Single Spacer Moiety C (Scheme 3-ii)

[0176] 7.3 g of compound B is hydrolyzed in a mixture water/THF/acetic acid (1/1/1) for 4 h. Then the mixture is concentrated, the residue was dissolved in dichloromethane and purified by column chromatography (heptane/ethyl acetate=2/3).

[0177] Synthesis of Silyl Protection of Methyl-Glucuronide Double Spacer Compound D (Scheme 3-iii)

[0178] The procedure was similar with the synthesis of compound B. The carboxylic acid group of compound 8 was converted to isocyanate and then 2 g of compound C (4.1 mmol) and 0.42 g of triethylamine (4.1 mmol) in dry toluene and acetonitrile were added. After stirring overnight, the reaction mixture was dissolved in a minimum amount of chloroform and brought on a silica column (heptane/ethyl acetate=2/1).

[0179] Synthesis of Deprotected Silyl of Methyl-Glucuronide Double Spacer Moiety E (Scheme 3-iv)

[0180] Compound D (4 mmol) was dissolved in 45 mL of THF/H.sub.2O/acetic acid (1/1/1) and the solution was stirred for 4 hours. the reaction mixture was concentrated and the residue was dissolved in dichloromethane for further purification by column chromatography (heptane/ethyl acetate=2/3).

[0181] Deprotection of Acetyl of Sugar of Compound E (Scheme 3-v)

[0182] 2.6 g (4.1 mmol) of compound E was dissolved in 50 ml of MeOH, and then 4.6 ml of 1M LiOMe (4.6 mmol Li) in 45 ml MeOH was added dropwise to this solution. After 2 h stirring at 0° C., the reaction mixture was neutralized by adding silica. After concentrating, this reaction mixture was purified continually to get hydroxyl of sugar of compound F by column chromatography (2% MeOH/ethyl acetate followed by 4% MeOH/ethyl acetate).

[0183] Activation of the Terminated-Hydroxyl Group of Compound F (Scheme 3-vi)

[0184] To a solution of 1 g of F in 60 ml of acetonitrile and 8 ml THF was added with 194 mg (2.46 mmol) of pyridine and 454 mg (2.26 mmol) of 4-Nitrophenyl chloroformate. After stirring 2 h at 0° C., the reaction mixture was concentrated and the residue was purified by column chromatography to get compound G (8% MeOH/dichloromethane).

[0185] Synthesis of DOX Prodrug DOX-mGA3 (Scheme 3-vii)

[0186] 265 mg of activated compound G was dissolved in 14 ml of DMF, the above solution was added 226 mg of DOX HCl and 40 mg of triethyl amine After overnight reaction at room temperature, the reaction mixture was purified by column chromatography to get DOX prodrug DOX-mGA3 (4% methanol/ethyl acetate).

[0187] Synthesis of DOX Prodrug DOX-GA3 (Scheme 3-viii)

[0188] Dissolving DOX prodrug DOX-mGA3 in NaHCO.sub.3 solution to hydrolyze the methyl group of the glucuronide, then adding 1M HCl to obtain DOX prodrug DOX-GA3 with —COOH group. The final compound was collected by lyophilization.

##STR00021## ##STR00022##

[0189] The .sup.1H-NMR spectrum of DOX GA3-DS is shown in FIG. 1.

[0190] Mass: [0191] M.sub.fnd=1084.28138 (M+Na) [0192] M.sub.cal=1084.28110 (M+Na)

[0193] .sup.1H NMR (400 MHz, DMSO-d6) δ 14.04 (s, 1H, 6-OH), 13.28 (s, 1H, 11-OH), 10.03 (s, 1H, Ar-NH), 9.74 (s, 1H, Ar-NH), 7.90-7.88 (m, 2H, Ar), 7.64-7.61 (m, 1H, Ar), 7.48 (d, J=8.0 Hz, 2H, Ar), 7.44-7.29 (m, 4H, Ar), 7.22 (d, J=8.3 Hz, 2H, Ar), 6.82 (d, J=8.1 Hz, 1H, 3′—NH), 5.50-5.42 (m, 4H, Glu-2,3,4-OH, and 9-OH), 5.33 (d, J=7.9 Hz, 1H, Glu-1H) 5.21 (m, 1H, 1′-H), 5.05 (s, 2H, Ar-CH.sub.2), 5.00-4.90 (m, 1H, 7-H), 4.87 (s, 2H, Ar-CH.sub.2), 4.71 (d, J=8 Hz, 1H, Glu-5H) 4.57 (s, 2H, 14-CH.sub.2), 4.15 (q, J=6.3 Hz, 1H, 5′-H), 3.99 (s, 3H, 4-OMe), 3.75 (d, J=9.1 Hz, 1H, 3′-H), 3.49-3.22 (m, 2H, Glu 2,3,4-H and 4′H partly hidden under DMSO peak), 3.04-2.92 (m, 2H, 10.sub.eq and 10.sub.ax-H), 2.25-2.06 (m, 2H, 8.sub.ax and 8.sub.eq-H), 1.89-1.75 (m, 1H, 2′.sub.ax-H), 1.52-1.42 (m, 1H, 2′eq-H), 1.12 (q, 3H, J=6.6 Hz, 5′-CH.sub.3).

[0194] FIG. 2 shows the HPLC chromatogram of DOX GA3-DS.

[0195] FIG. 3 shows the conversion of DOX prodrugs to DOX in the presence of beta-GUS.

[0196] Micelle Preparation and Drug Loading with DOX-GA3-DS Prodrug.

[0197] The drug-loaded mPEG-b-p(HPMAmBz) micelles were prepared using a nano-precipitation method. Firstly, different quantities of double spacer prodrug DOX-GA3 (1 mg, 5 mg or 10 mg) with 27 mg mPEG-b-p(HPMAmBz) polymer were dissolved in 1 mL THF. For DOX-GA3(—OH) with single spacer, the compound of 1 mg or 5 mg or 10 mg with 27 mg mPEG-b-p(HPMAmBz) were dissolved in a mixture of 800 μl methanol and 200 μl THF. Then the methanol-THF solution was added dropwise to 1 mL H.sub.2O with stirring at 1000 rpm for 1 minute. The mixture was kept still at room temperature for 24 h to allow for evaporation of THF. Afterward, the DOX-GA3 prodrug-loaded micelles was filtered through 0.45 μm nylon membrane to remove non-encapsulated prodrug.

[0198] For the prodrug-loaded micelles characterization, The Z-average (Zave) size was measured by dynamic light scattering (DLS), the data is shown in the Table 1. To assess the DOX-GA3 prodrug loading content, the prodrug-loaded polymeric micelles were diluted with DMSO for at least 10 times to dissolve the micelles and the prodrugs encapsulated in the micelles, and the concentrations of the prodrugs were subsequently quantified by HPLC analysis using a Waters Acquity system. A gradient method was used, with Eluent A (H.sub.2O with 0.1% TFA) and Eluent B (ACN with 0.1% TFA). A gradient was run with the volume fraction of eluent B increasing from 40 to 95% from 0 to 11 min and subsequently decreasing to 40% from 11 to 14 min. The injection volume was 100 μl and the detection wavelength was 485 nm. Standard solutions of the prodrugs were measured by the HPLC to generate calibration curves. The loading capacity (LC) and encapsulation efficiency (EE) of micelles can be calculated according the formula below. The results are shown in Table 1,

[00001]$\mathrm{LC}=\frac{concentr\mathrm{ation}\mathrm{of}\mathrm{drug}\mathrm{measured}}{concentr\mathrm{ation}\mathrm{of}\left(\mathrm{drug}\mathrm{measured}+\mathrm{polymer}\mathrm{added}\right)}\times 100\%$$\mathrm{EE}=\frac{concentr\mathrm{ation}\mathrm{of}\mathrm{drug}\mathrm{measured}}{concentr\mathrm{ation}\mathrm{of}\mathrm{drug}\mathrm{added}}\times 100\%$

TABLE-US-00001 TABLE 1 Results: final characteristics of drug-loaded micelles feed (mg) size (nm) PDI LC EE DOX 1 average 60 0.08 2.9% 90.9% SD 1 0.02 0.3% 5.2% 5 average 72 0.13 11.6% 78.5% SD 1 0.01 0.9% 4.3% 10 average 85 0.19 16.7% 62.9% SD 1 0.01 0.7% 5.2% DOX single spacer 1 average 54 0.18 2.6% 71.4% SD 4 0.02 0.2% 6.7% 5 average 55 0.13 12.6% 78.1% SD 3 0.04 1.1% 7.9% 10 average 67.57 0.13 24.6% 88.1% SD 1 0.02 0.49% 2.5% DOX double spacer 1 average 47 0.20 3.3% 92.8% SD 3 0.02 0.3% 9.3% 5 average 62 0.23 16.2% 91.7% SD 1 0.02 1.3% 1.2% 10 average 61 0.13 21.5% 75.4% SD 1 0.03 4.2% 18.9%

[0199] As can be seen in Table 1 doxorubicin needs to be loaded as a hydrophobic prodrug to be able to form a uniform micellar particle formulation around 50 nm.

[0200] Transmission electron microscopy images were made of the micelles loaded with the single spacer prodrug of doxorubicin and compared to the micelles loaded with the double spacer doxorubicin prodrug. The results are shown in FIG. 4. As can be seen in the left panel of FIG. 4, the double spacer prodrug of doxorubicin yields a more homogeneous particle size distribution of the micelles than micelles loaded with 5 mg/mL single spacer doxorubicin prodrug (right panel).

[0201] The release of doxorubicin from the micelles was studied with a dialysis method in PBS (pH=7.4) containing 0.2% v/v Tween 80 over 3 days of incubation at 37° C. At pre-set time point, aliquotes were taken from the dialysis medium and the concentration of DOX or prodrugs were analyzed by HPLC. The results are shown in FIG. 5.

[0202] In a further experiment the ability of doxorubicin and doxorubicin double spacer prodrug and doxorubicin double spacer prodrug in polymeric micelles to induce immunogenicity of tumor cells was assessed in vitro. As one of the most studied phenotypes of immunogenic cell death, calreticulin (CRT) translation to outer surface of cancer cells is able to attract antigen presenting cells to endocytose apoptotic cancer cells. CRT translocation was measured by flow cryometry. To assess CRT translocation induced by the formulations, they were added to breast cancer 4T1 cells which were cultured overnight at 37° C. in RPMI 1640 medium containing 10% FBS and 1% penicillin. After incubation, the cells were washed with PBS and then detached. The CRT translocated to the outer surface of the cells was labeled with anti-CRT antibodies. In the flow cytometry plots in FIG. 6A, the cells treated with DOX, DOX prodrug or DOX prodrug-loaded micelles showed higher signal of CRT compared to untreated control or cells treated with DOX but not stained with the antibodies. The quantitative date in FIG. 6B indicates that the prodrug-loaded micelles and prodrug were similarly potent in inducing CRT translocation compared to DOX.

[0203] A further experiment was performed in which doxorubicin, doxorubicin double spacer prodrug and the prodrug in polymeric micelles were evaluated regarding their ability to modulate lymphocytes in the tumors in a model of triple negative breast cancer (4T1). To establish the model, 4T1 cells were inoculated in BALB/c mice. The tumor size reached ˜50-100 mm.sup.3 around 10 days after inoculation. Then the formulations were intravenously injected in the mice at 5 mg/kg in the schedule indicated in FIG. 7 (3 injection in total with 3 days intervals). At day 9 from the first injection, the mice were sacrificed the tumors were collected. The tumors were cut into two pieces, one was used for flow cytometry and the other for immunofluorescence microscopy. For flow cytometry study, the tumors were digested and the cells were suspended in PBS, which were labeled with anti-CD3 and anti-CD8 antibodies. Afterwards, the cell suspensions were washed with PBS and then analyzed by a flow cytometer. The rest of the tumor tissues were cryosectioned and stained with anti-CD4 and anti-CD8 antibodies.

[0204] In FIG. 8, results from T cell quantification of the tumor tissues by flow cytometry were presented. Compared to tumors treated with free DOX or DOX prodrug, the tumors received prodrug-loaded micelles showed significantly higher infiltration of CD3+CD8+ T cells. The higher level of cytotoxic T cells is an important indicator of enhanced cancer immunity.

[0205] In FIG. 9, the main subpopulations of T cells (CD8+/CD4+) were identified and quantified by immunofluorescence microscopy. The amount of cytotoxic T cells (CD8+) in tumors treated with DOX prodrug-loaded micelles was significantly increased than those in DOX and DOX prodrug treated. Moreover, the CD4+ T cells in tumors were also increased by treatments with DOX prodrug or prodrug-loaded micelles compared to free DOX.

Example 2. Synthesis and Micelle Loading of a GEM-Phe-mGA Prodrug

[0206] Prodrug Synthesis

[0207] The synthesis of the prodrug GEM-Phe-mGA was divided into 3 parts. Part 1 and 2 are similar to Example 1. and described in detail above.

[0208] Part 3: Synthesis of the Final GEM-Phe-mGA Prodrug (Scheme 4)

[0209] Synthesis of Gemcitabine-phenylalanine precursor (GEM-Phe) (based on: Bioorganic and Medicinal Chemistry 2018, 26, 5624-5630 and Molecules 2018, 23, 2608-2020)

[0210] First Part: Synthesis of Boc-Protected Phenylalanine GEM (GEM-Phe-Boc) (Scheme 4, Step ix)

[0211] 1-hydroxybenzotriazole (HOBt; 68 mg, 0.50 mmol, 1 eq), N-(tert-butoxycarbonyl)-L-phenylalanine (Boc-Phe-OH; 146 mg, 0.55 mmol, 1.1 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; 125 mg, 0.65 mmol, 1.3 eq) and N,N-diisopropylethylamine (DIPEA; 65 mg, 0.5 mmol, 1 eq) were added to a solution of gemcitabine hydrochloride (GEM-HCl; 150 mg, 0.5 mmol, 1 eq) in a mixture of N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) (3:1, 5 mL) at room temperature under inert atmosphere. The mixture was stirred overnight at 55° C. under inert atmosphere. After the reaction was completed, it was cooled down to room temperature and the solvent removed under reduced pressure. Brine (5 mL) was added and the mixture extracted with ethyl acetate (EtOAc; 3×10 mL). The combined organic layers were washed with a NaHCO.sub.3 solution in water (10 mL) and brine (10 mL). The organic phase was dried over sodium sulfate and evaporated to render a white solid (GEM-Phe-Boc), which was purified by silica column chromatography (EtOAc/Hexane 4:1 with 5% MeOH). Yield: 91% (232.8 mg).

[0212] 1H NMR (400 MHz, MeOD-d4): δ (in ppm) 8.39 (d, J=7.7 Hz, 1H), 7.50 (d, J=7.7 Hz, 1H), 7.35-7.19 (m, 5H), 6.35-6.24 (m, 1H), 4.51 (s, 1H), 4.36-4.32 (m, 1H), 4.04-3.94 (m, 2H), 3.89-3.80 (m, 1H), 3.18 (d, J=9.4 Hz, 1H), 2.98-2.87 (m, 1H), 1.41 (s, 9H). MS (ESI+, MeOH) for [M+Na]+, 533.181 (cal. 533.182). Reversed-phase analytical HPLC (0% MeOH/100% H.sub.2O+0.1% TFA to 100% MeOH/0% H.sub.2O+0.1% TFA in 30 minutes): Rt=21.3 min.

[0213] Boc-Deprotection of GEM-Phe-Boc (GEM-Phe) (Scheme 4, Step x)

[0214] To a solution of the aforementioned GEM-Phe-Boc (112 mg, 0.22 mmol) in EtOAc (6 mL), 1 M HCl (0.3 mL) were added and the solution stirred for 15 min at room temperature. The isolated precipitate was washed with EtOAc (2×10 mL) and the crude purified by column chromatography (MeOH/DCM, 1:9) to afford the desired compound GEM-Phe. Yield: 22% (20 mg).

[0215] 1H NMR (400 MHz, MeOD-d4): δ (in ppm) 7.75 (d, J=7.6 Hz, 1H), 7.27-7.13 (m, 5H), 6.20-6.12 (m, 1H), 5.90 (d, J=7.6 Hz, 1H), 4.95 (dd, J=8.5, 6.1 Hz, 1H), 4.21 (td, J=12.1, 8.3 Hz, 1H), 3.96-3.80 (m, 2H), 3.78-3.64 (m, 1H), 3.18 (dd, J=13.9, 6.1 Hz, 1H), 2.95 (dd, J=13.9, 8.5 Hz, 1H). MS (ESI+, MeOH) for [M+H]+, 411.147 (cal. 411.148); for [M+Na]+, 433.128 (cal. 433.130). Reversed-phase analytical HPLC (0% MeOH/100% H.sub.2O+0.1% TFA to 100% MeOH/0% H.sub.2O+0.1% TFA in 30 minutes): Rt=14.1 min.

##STR00023##

[0216] Second Part: Synthesis of GEMCITABINE Prodrug GEM-Phe-mGA3 (Scheme 5)

[0217] Activation of the Benzyl Alcohol of Compound C: Compound H (Scheme 5, Step xi)

[0218] Compound C (150 mg, 0.42 mmol, 1 eq) was dissolved in a mixture of ACN (15 mL) and THF (2 mL). After cooling the mixture down to 0° C., pyridine (44 μL, 0.55 mmol, 1.3 eq) and 4-nitrophenyl chloroformate (Cl—COOPhNO.sub.2, 102 mg, 0.50 mmol, 1.2 eq) were added. The reaction was stirred for 2 hours and the crude was purified by silica column chromatography (8% MeOH in DCM) to afford compound H. Yield: 37% (80.5 mg).

[0219] 1H NMR (600 MHz, MeOD-d4): δ (in ppm) 8.39-8.23 (m, 2H), 7.58-7.36 (m, 6H), 5.52 (d, J=8.1 Hz, 1H), 5.25 (s, 2H), 4.02 (d, J=9.7 Hz, 1H), 3.76 (s, 3H), 3.58 (t, J=9.3 Hz, 1H), 3.50 (t, J=9.0 Hz, 1H), 3.44 (dd, J=9.1 Hz, 1H). Reversed-phase analytical HPLC (0% MeOH/100% H.sub.2O+0.1% TFA to 100% MeOH/0% H.sub.2O+0.1% TFA in 30 minutes): Rt=20.7 min.

[0220] Synthesis of GEM-Prodrug GEM-Phe-mGA3 (Scheme 5, Step xii)

[0221] Compound H (45 mg, 0.086 mmol, 1.2 eq) was dissolved in anhydrous DMF (2 mL) under nitrogen atmosphere. GEM-Phe (30 mg, 0.072 mmol, 1 eq), TEA (10 μL, 0.072 mmol, 1 eq), HOBt (10 mg, 0.072 mmol, 1 eq) and DIPEA (13 μL, 0.072 mmol, 1 eq) were added to this solution and the mixture kept under stirring overnight at room temperature. The DMF was removed and the crude was purified by silica column chromatography (10% MeOH in DCM) to yield GEM-Phe-mGA3. Yield: 41% (23.6 mg).

[0222] 1H NMR (400 MHz, MeOD-d4): δ (in ppm) 8.36 (d, J=7.6 Hz, 1H), 7.82 (d, J=8.4 Hz, 1H), 7.71 (d, J=8.3 Hz, 1H), 7.30-7.08 (m, 8H), 6.27-6.23 (m, 1H), 5.50 (d, J=8.1 Hz, 1H), 4.96 (m, 2H), 4.56 (dd, J=9.0, 5.7 Hz, 1H), 4.35-4.26 (m, 1H), 3.98 (d, J=9.7 Hz, 2H), 3.81 (dd, J=12.9, 3.2 Hz, 1H), 3.76 (s, 3H), 3.56 (t, J=9.3 Hz, 1H), 3.49 (t, J=9.0 Hz, 1H), 3.46-3.40 (t, J=9.1 Hz, 1H), 3.16-3.08 (m, 1H), 2.96-2.89 (m, 1H). MS (ESI+, MeOH) for [M+Na]+, 816.22 (cal. 816.2152). MS (ESI−, MeOH) for [M+Cl]−, 828.1960 (cal. 828.1943). Reversed-phase analytical HPLC (0% MeOH/100% H.sub.2O+0.1% TFA to 100% MeOH/0% H.sub.2O+0.1% TFA in 30 minutes): Rt=17.9 min.

##STR00024##

[0223] The mass spectrum of GEM-Phe-mGA3 is shown in FIG. 10.

[0224] FIG. 11 shows the HPLC chromatogram of GEM-Phe-mGA3.

[0225] Micelle Preparation and Drug Loading with GEM-Phe-mGA3 Prodrug

[0226] The drug-loaded mPEG-b-p(HPMAmBz) micelles were prepared using a nano-precipitation method. Firstly, single spacer phenylalanine GEM prodrug GEM-Phe-mGA3 (1 mg), GEM (1 mg) and GEM-Phe (1 mg) with 10 mg mPEG-b-p(HPMAmBz) polymer (1:10; drug:polymer ratio) were dissolved in 1 mL THF containing 10% MeOH. Then the THF/MeOH solution was added dropwise to 1 mL H.sub.2O with stirring at 1000 rpm for 1 minute. The mixture was kept still at room temperature for 24 h to allow for evaporation of THF and MeOH. Afterwards, the GEM, GEM-Phe and GEM-Phe-mGA3 prodrug-loaded micelles were filtered through 0.2 μm nylon membrane to remove non-encapsulated prodrug. For the prodrug-loaded micelles characterization, the Z-average (Zave) size was measured by dynamic light scattering (DLS), the data is shown in FIG. 12. To assess the GEM-Phe prodrug and GEM-mGA3 prodrug loading content, the prodrug-loaded polymeric micelles were diluted with MeOH for at least 10 times to dissolve the micelles and the prodrugs encapsulated in the micelles, and the concentration of the prodrugs were subsequently quantified by reversed-phase HPLC analysis using a C18 column. In the case of GEM, between 10-20% of DMSO was added. A gradient method was used, with Eluent A (H.sub.2O with 0.1% TFA) and Eluent B (MeOH with 0.1% TFA). A gradient was run with the volume fraction of eluent B increasing from 0 to 100% from 0 to 30 min. The injection volume was 100 μl and the detection wavelength was 260 nm. The loading capacity (LC) and encapsulation efficiency (EE) of micelles can be calculated according to the formula below. The results are shown in FIG. 13.

LC=(concentration of drug measured)/(concentration of (drug measured+polymer added))×100%

EE=(concentration of drug measured)/(concentration of drug added)×100%

[0227] The release of the compounds from the micelles was studied with a dialysis method in PBS (pH 7.4) containing 30 mg/mL of BSA over 12 h incubation at 37° C. At pre-set time point, aliquots were taken from the dialysis medium and the concentration of GEM or GEM prodrugs were analyzed by HPLC. The results are shown in FIG. 14.

Example 3 Synthesis Bifurcated Glucuronide Structure (for Compounds of Formula (III) of the Invention)

[0228] The bifurcated glucuronide structure can be synthesized as described in Tranoy-Opalinski et al. 2008, for instance as shown in scheme 6 below.

##STR00025## ##STR00026##

REFERENCES

[0229] De Graaf et al. Biochem Pharmacol 2004 Dec. 1; 68(11):2273-81.doi:10.1016/j.bcp.2004.08.004. [0230] De Groot et al. J Org Chem 2001 Dec. 28; 66(26):8815-30. doi: 10.1021/jo0158884. [0231] Houba et al. Br J Cancer. 1998 December; 78(12):1600-6. doi: 10.1038/bjc.1998.729. [0232] Houba et al. Br J Cancer. 2001 February; 84(4):550-7. doi: 10.1054/bjoc.2000.1640. [0233] Leenders et al. Bioorg Med Chem. 1999 August; 7(8):1597-610. doi: 10.1016/s0968-0896(99)00095-4. [0234] D. Neradovic et al. Macromolecules, 2001, 34 (22), pp 7589-7591 [0235] Ruiz-Hernandez et al. Polym Chem. 2014 Mar. 7; (5):1674-1681. doi: 10.1039/C3PY01097J. [0236] Shi et al., Biomacromolecules 2013, 14, 1826-1837 [0237] Talleli et al. Bioconjug Chem 2011 Dec. 21; 22(12):2519-30. doi: 10.1021/bc2003499. [0238] Tranoy-Opalinski et al. Anticancer Agents Med Chem. 2008 August; 8(6):618-37.