Enzymatic Activation of Second-Generation Dendritic Prodrugs

Bioconjugate Chem. 2006, 17, 1432−1440

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Enzymatic Activation of Second-Generation Dendritic Prodrugs: Conjugation of Self-Immolative Dendrimers with Poly(ethylene glycol) via Click Chemistry Anna Gopin,† Sharon Ebner,‡ Bernard Attali,‡ and Doron Shabat†,* School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, and Department of Physiology and Pharmacology, Sackler Faculty of Medical Sciences, Tel-Aviv University, Tel Aviv 69978 Israel. Received June 26, 2006; Revised Manuscript Received September 21, 2006

Single-triggered disassemble dendrimers were recently developed and introduced as a potential platform for a multi-prodrug. These unique structural dendrimers can release all of their tail units through a self-immolative chain fragmentation initiated by a single cleavage at the dendrimer’s core. There are several examples for the bioactivation of first-generation self-immolative dendritic prodrugs. However, enzymatic activation failed for second-generation self-immolative dendrimers. The hydrophobic large molecular structure of the dendritic prodrugs results in aggregation under aqueous conditions and prevented the enzyme from reaching the triggering substrate. Here we show a simple solution for the enzymatic activation of second-generation self-immolative dendrimers. Poly(ethylene glycol) (PEG) was conjugated to the dendritic platform via click chemistry. The poly(ethylene glycol) tails significantly decreased the hydrophobic properties of the dendrimers and thereby prevented aggregate formation. We designed and synthesized a dendritic prodrug with four molecules of the anticancer agent camptothecin and a trigger that can be activated by penicillin-G-amidase. The PEG5000-conjugated, self-immolative dendritic prodrug was effectively activated by penicillin-G-amidase under physiological conditions and free camptothecin was released to the reaction media. Cell-growth inhibition assays demonstrated increased toxicity of the dendritic prodrug upon incubation with the enzyme.

INTRODUCTION Recently, three groups, almost simultaneously, reported the design and synthesis of novel dendritic structures. These dendrimers include a trigger that initiates the fragmentation of the molecule in a self-immolative manner with the consequent release of the tail-high-group units (1-3). All three groups exploited the fact that the dendrimer skeleton can be constructed in such a way that it will disintegrate into known molecular fragments once the disintegration process has been initiated. These unique structural dendrimers can release all of their tail units through a domino-like chain fragmentation, initiated by a single cleavage at the dendrimer’s core (4). Incorporation of drug molecules as the tail units and use of an enzyme substrate as the trigger generates a multi-prodrug unit that is activated with a single enzymatic cleavage. We have shown significant advantage in tumoral cell-growth inhibition by our singletriggered dendritic prodrugs in comparison with classic monomeric prodrugs (5, 6). In another report, we designed and synthesized fully biodegradable dendrimers that disassemble through multienzymatic triggering followed by self-immolative chain fragmentation (7). A practical application for such multitriggered self-immolative dendrons was recently demon* Corresponding author. Tel: +972 (0) 3 640 8340. Fax: +972 (0) 3 640 9293. E-mail: [email protected]. † School of Chemistry. ‡ Department of Physiology and Pharmacology. 1 Abbreviations. ACN, acetonitrile; Boc, tert-butoxycarbonyl; DCM, dichloromethane; DIPEA, diisopropylethyleneamine; DMAP, dimethylaminopyridine; DMF, dimethylformamide; EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; EtOAc, ethyl acetate; He, hexanes; HOBT, 1-hydroxybenzotriazole; NHS, N-hydroxysuccinimide; PEG, poly(ethylene glycol); PNP, p-nitrophenol; tR, retention time; TBSCl, tert-butyldimethylsilyl chloride; TBTA, tris(1-benzyl-1H-[1,2,3]triazol4-ylmethyl)amine; TFA, trifluoroacetic acid; THF, tetrahydrofuran.

strated by the concept of prodrug activation through a molecular OR logic trigger (8). Two options for the triggering/activation of self-immolative dendrimers were proposed. One approach is based on chemical triggering whereas the other is based on enzymatic bioactivation. While chemical activation of first and second generation selfimmolative dendrimers was achieved by all three groups, only ours successfully demonstrated the enzymatic approach with incorporation of substrate triggers for catalytic antibody 38C2 (9-11) and penicillin-G-amidase (12) (PGA). However, when second-generation self-immolative dendrimer were tested for enzymatic activation, no fragmentation was observed at all. We assumed that the hydrophobic structure of the dendritic prodrugs generates aggregation under aqueous conditions, which prevented the enzyme from accessing the triggering substrate (13). Here we report a simple solution to this problem. Enzymatic activation of second-generation self-immolative dendrimers was achieved through conjugation of poly(ethylene glycol) (PEG) to the dendritic platform via click chemistry. The PEG tails significantly decreased the hydrophobicity of the dendrimers and thereby prevented aggregate formation.

EXPERIMENTAL PROCEDURES General. All reactions requiring anhydrous conditions were performed under argon or N2 atmosphere. Chemicals and solvents were either A.R. grade or purified by standard techniques. Thin layer chromatography (TLC): silica gel plates Merck 60 F254; compounds were visualized by irradiation with UV light and/or by treatment with a solution of 25 g phosphomolybdic acid, 10 g Ce(SO4)2·H2O, 60 mL concd H2SO4, and 940 mL H2O, followed by heating. Flash chromatography (FC): silica gel Merck 60 (particle size 0.040-0.063 mm), eluent given in parentheses. 1H NMR: Bruker AMX 200 or 400. The chemical shifts are expressed in δ relative to TMS (δ ) 0 ppm) and the coupling constants J in hertz. The spectra

10.1021/bc060180n CCC: $33.50 © 2006 American Chemical Society Published on Web 10/17/2006

Enzymatic Activation of Dendritic Prodrugs

were recorded in CDCl3 or CD3OD as a solvent at room temp. All reagents, including salts and solvents, were purchased from Sigma-Aldrich. PEG400-azide was received from Polypure (Norway). TBTA was received from the Sharpless laboratory (Scripps, La Jolla). Compound 2. Commercially available 4-hydroxybenzoic acid (2.0 g, 14.5 mmol) was dissolved in DMF. Then EDC (3.3 g, 17.4 mmol), HOBT (1.0 g, 7.3 mmol), and propargyl amine (1.0 mL, 14.5 mmol) were added. The mixture was stirred overnight and monitored by TLC (EtOAc:He ) 2:3). After completion of the reaction, the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc:He ) 2:3) to give compound 2 (1.8 g, 70%) in the form of yellowish oil. 1H NMR (200 MHz, CDCl ) δ ) 7.70 (2H, d, J ) 6.8 Hz); 3 6.81 (2H, d, J ) 6.8); 4.11 (2H, d, J ) 2.5); 2.71 (1H, t, J ) 2.5). 13C (400 MHz, CDCl3) δ ) 167.9, 160.6, 128.8, 124.4, 114.5, 79.5, 70.3, 28.3. MS (FAB): calculated for C10H9NO2 176.0 [M + H +], found 176.0. Compound 3. To a cool 12% NaOH (12 mL) compound 2 (1.8 g, 10.2 mmol) was added while being cooled to 0 °C. Formaldehyde 37% in water (10 mL) was added. The reaction was stirred at 55 °C for 3 days and monitored by TLC (EtOAc: MeOH ) 95:5). After completion the reaction was diluted with EtOAc and washed with ammonium chloride saturated solution. The aqueous layer was washed twice with EtOAc. The combined organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc: MeOH ) 95:5) to give compound 3 (1.9 gr, 80%) in the form of a white solid. 1H NMR (200 MHz, CD OD) δ ) 7.80 (2H, s); 4.91 (4H, 3 s); 4.26 (2H, d, J ) 2.5); 2.70 (1H, t, J ) 2.5). 13C (400 MHz, CD3OD) δ ) 168.1, 156.7, 126.8, 126.0, 124.4, 79.4, 70.2, 60.3, 28.3. MS (FAB): calculated for C12H13NO4 236.0 [M + H +], found 236.0. Compound 4. Compound 3 (713 mg, 3.0 mmol) was dissolved in DMF and cooled to 0 °C. Imidazole (408 mg, 6.0 mmol) and TBSCl (910 mg, 6.0 mmol) were added. The reaction was stirred at room temp for 2 h and monitored by TLC (EtOAc: He ) 2:8). After completion, the reaction was diluted with ether and washed with ammonium chloride saturated solution. The organic layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc:He ) 15:85) to give compound 4 (1.12 g, 80%) in the form of a colorless oil. 1H NMR (400 MHz, CDCl ) δ ) 7.57 (2H, s); 4.87 (4H, s); 3 4.23 (2H, dd, J ) 2.5, J ) 2.6); 2.17 (1H, t, J ) 2.5); 0.95 (18H, s); 0.13 (12H, s). 13C (400 MHz, CDCl3) δ ) 166.7, 156.4, 126.1, 124.5, 79.6, 71.7, 62.7, 29.6, 25.8, 25.6, 18.2, -5.5. MS (FAB): calculated for C24H41NO4Si2 464.2 [M + H +], found 464.2. Compound 5. Compound 4 (1.12 g, 2.4 mmol) was dissolved in dry THF, Et3N (1.0 mL, 7.2 mmol) was added, and the mixture was cooled to 0 °C. Then, p-nitrophenyl chloroformate (581 mg, 2.9 mmol) dissolved in dry THF (10 mL) was added dropwise, and the reaction was stirred for 1 h at room temp and monitored by TLC (EtOAc:He ) 2:8). After completion, the reaction was filtered, the solvent was evaporated, and the crude product was purified by column chromatography on silica gel (EtOAc:He ) 15:85) to give compound 5 (1.35 g, 90%) in the form of a colorless oil. 1H NMR (200 MHz, CDCl ) δ ) 8.43 (2H, d, J ) 8.1); 8.02 3 (2H, s); 7.63 (2H, d, J ) 8.1); 7.01 (1H, m); 4.91 (4H, s); 4.38 (2H, dd, J ) 2.5, J ) 2.6); 2.41 (1H, t, J ) 2.5); 1.08 (18H, s); 0.29 (12H, s). 13C (400 MHz, CDCl3) δ ) 166.4, 155.2, 149.4,

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147.7, 145.5, 133.9, 132.2, 126.3, 125.3, 121.5, 79.2, 71.8, 60.3, 31.5, 25.8, 18.2, -5.5. HRMS (MALDI-TOF): calculated for C31H44N2O8Si2 651.2528 [M + Na+], found 651.2562. Compound 6. Compound 5 (1.5 g, 2.3 mmol) was dissolved in DMF. Mono-Boc-N,N′-dimethylethylenediamine (541 mg, 2.9 mmol) was added. The reaction was stirred at room temperature for 1 h and monitored by TLC (EtOAc:He ) 1:1). After completion, the solvent was removed under reduced pressure and the crude product was purified by column chromatography on silica gel (EtOAc:He ) 2:8) to give compound 6 (1.45 g, 90%) in the form of a colorless oil. 1H NMR (400 MHz, CDCl ) δ ) 7.79 (2H, s); 6.32 (1H, 3 m); 4.68-4.67 (4H, m); 4.27-4.25 (2H, m); 3.61-3.43 (4H, m); 3.24 (2H, s); 3.12 (1H, s); 2.96 (3H, s); 2.32 (1H, bs); 1.511.46 (9H, m); 0.92 (18H, s); 0.08 (12H, s). 13C (400 MHz, CDCl3) δ ) 167.2, 153.1, 153.0, 134.6, 130.8, 125.1, 80.2, 78.8, 72.0, 59.9, 46.4, 46.1, 36.4, 35.9, 35.1, 29.8, 28.3, 25.7, 18.2, -5.5. MS (FAB): calculated for C34H59N3O7Si2 700.4 [M + Na +], found 700.3. Compound 7. Compound 6 (1.5 g, 2.2 mmol) was dissolved in 10 mL of methanol, and amberlist 15 was added. The reaction was stirred at room temperature for 2 h and monitored by TLC (EtOAc:MeOH ) 95:5). After completion, the amberlist was filtered out and the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc:MeOH ) 95:5) to give compound 7 (500 mg, 56%) in the form of a white solid. 1H NMR (200 MHz, CD OD) δ ) 7.78 (2H, s); 4.57 (4H, 3 bs); 4.25-4.23 (2H, m); 3.6-3.4 (4H, m); 3.2 (2H, s); 3.1 (1H, s); 2.96 (3H, s); 2.40 (1H, bs); 1.59-1.54 (9H, m). 13C (400 MHz, CD3OD) δ ) 169.8, 158.9, 157.5, 152.1, 134.1, 130.3, 130.0, 83.2, 83.0, 74.4, 62.5, 50.3, 49.0, 38.7, 37.9, 37.6, 31.3. HRMS (MALDI-TOF): calculated for C22H31N3O7 472.2015 [M + Na+], found 472.2059. Compound 8. Triphosgen (57 mg, 2 mmol) was dissolved in 10 mL of dry THF and was cooled to 0 °C. A mixture of p-nitroaniline (614 mg, 4.5 mmol) and Et3N (622 µL, 4.5 mmol) dissolved in 20 mL dry THF was added dropwise. The reaction was stirred for 20 min, and afterward a mixture of compound 7 (400 mg, 0.9 mmol) and DMAP (274 mg, 2.3 mmol) in dry THF was added dropwise. The reaction was stirred at room temperature overnight and monitored by TLC (EtOAc:He ) 1:1). After completion, the mixture was diluted with EtOAc and was washed with 1 M HCl. The organic layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. Then, the crude product was dissolved in a small amount of DCM and filtered. The filtrate was concentrated, and the product was purified by column chromatography to give compound 8 (478 mg, 68%) in the form of a yellow solid. 1H NMR (400 MHz, CDCl ) δ ) 8.11 ppm (4H, d, J ) 9 3 Hz); 7.9 (2H, s); 7.61 (4H, d, J ) 9); 5.2 (4H, bs); 4.13 (2H, s); 3.66-3.64 (1H, m); 3.4-3.2 (4H, m); 3.2 (2H, s); 3.1 (1H, s); 2.96 (3H, s); 2.46 (1H, bs); 1.37 (9H, s). 13C NMR (400 MHz, CDCl3) δ ) 166.2; 156.4; 155.9; 153.8; 153.5; 144.6; 142.5; 131.2; 130.2; 129.9; 129.8; 124.9; 117.8; 80.6; 79.2; 71.7; 61.6; 46.6; 45.5; 34.3; 34.0; 29.6; 28.3. HRMS (MALDITOF): calculated for C36H39N7O13 [M + Na+] 800.2498; found 800.2454. Compound 10. Previously reported compound 9 (344 mg, 0.9 mmol) was deprotected with 1 mL TFA to remove the Boc group. The excess of the acid was removed under reduced pressure, and the residue was dissolved in 1 mL DMF. Linker 12 (305 mg, 0.75 mmol) was added followed by addition of Et3N (374 µL, 2.7 mmol). The reaction was stirred at room temperature for 1 h and monitored by TLC (EtOAc:He ) 3:1). After completion, the solvent was removed under reduced

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pressure, and the crude product was purified by column chromatography to give compound 10 (380 mg, 69%) in the form of a sticky foam. 1H NMR (200 MHz, CDCl ) δ ) 7.4-7.1 ppm (11H, m); 3 5.11 (2H, bs); 4.5 (4H, s); 3.7 (2H, s); 3.6-3.46 (4H, m); 3.15 (1H, s); 3.01-2.93 (4H, m); 2.3 (3H, s). 13C NMR (200 MHz, CDCl3) δ ) 169.3; 156.1; 137.6; 134.5; 133.4; 132.7; 132.4; 130.8; 130.4; 129.9; 129.6; 128.9; 127.8; 120.0; 67.2; 60.8; 47.6; 46.7; 45.2; 31.6; 21.0. HRMS (MALDI-TOF): calculated for C30H35N3O7 [M + Na+] 572.2367, found 572.2349 Compound 11. Compound 10 (370 mg, 0.67 mmol) was dissolved in 10 mL of dry THF and cooled to 0 °C. Then DIPEA (692.5 mg, 5.36 mmol) was added followed by p-nitrophenyl chloroformate (814 mg, 4.04 mmol) and pyridine (26 mg, 0.34 mmol). The reaction was allowed to warm to room temperature and monitored by TLC (EtOAc:He ) 35:65). After completion, the reaction was diluted with EtOAc and washed with 1 M HCl and with saturated NaHCO3 solutions. The organic layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc:He ) 35:65) to give compound 11 (547 mg, 93%) in a form of a white solid. 1H NMR (200 MHz, CDCl ) δ ) 8.39 ppm (4H, d, J ) 8); 3 7.52-7.39 (15H, m); 5.38 (4H, s); 5.19 (2H, s); 3.7 (2H, s); 3.6-3.46 (5H, m); 3.15 (1H, s); 3.01-2.93 (6H, m); 2.3 (3H, s). HRMS (MALDI-TOF): calculated for C44H41N5O15 [M + Na+] 902.2491; found 902.2475 Compound 1. Compound 8 (200 mg, 0.26 mmol) was deprotected with 1 mL TFA to remove the Boc group. The excess of the acid was removed under reduced pressure, and the residue was dissolved in 1 mL DMF. Compound 11 (75 mg, 0.09 mmol) was added followed by addition of Et3N (50 µL, 0.36 mmol). The reaction was stirred at room temperature for 1 h and was monitored by TLC (EtOAc:He ) 85:15). After completion, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography to give compound 1 (158 mg, 89%) in the form of a light yellow solid. 1H NMR (200 MHz, CDCl3) δ ) 8.23-8.17 ppm (8H, m); 7.8-8.1 (4H, m); 7.7-7.6 (8H,m), 7.5-7.25 (11H, m); 5.25-4.8 (14H, m); 4.3 (4H, bs); 3.8 (2H, bs); 3.75-3.4 (14H, m); 3.25-2.8 (18H, s); 2.4 (3H, bs); 2.3 (2H, s). HRMS (MALDI-TOF): calculated for C94H93N17O31 [M + Na+] 1978.6116; found 1978.6293. Compound 15. Compound 14 (226 mg, 1.28 mmol) was dissolved in DCM. NHS (162 mg, 1.4 mmol) and EDC (269 mg, 1. 4 mmol) were added, and the mixture was stirred at room temp for 15 min and monitored by TLC (EtOAc:He ) 4:6). After completion, the solvent was evaporated and the crude product was purified by column chromatography to give product 15 (310 mg, 88%) in the form of a white solid. 1H NMR (200 MHz, CDCl ) δ ) 8.16 ppm (2H, d, J ) 8.2); 3 7.47 (2H, d, J ) 8.2); 4.4 (2H, s); 2.91 (4H, s). 13C (400 MHz, CDCl3) δ ) 169.1; 161.4; 131.1; 131.0; 128.1; 126.9; 54.0; 25.6. HRMS (MALDI-TOF): calculated for C12H10N4O4 [M + Na+] 297.0572, found 297.0572. Compound 16. PEG5000-amine (500 mg, 0.1 mmol) was dissolved in DMF. Compound 15 (42 mg, 0.15 mmol) was added. The reaction was stirred for 2 h and monitored by TLC (DCM: MeOH ) 9: 1). After completion, the solvent was removed under reduced pressure and the crude product was purified by column chromatography, dissolved in water, filtered and lyophilized to give compound 16 (67%) in the form of a white powder. 1H NMR (200 MHz, CDCl ) δ ) 7.93 ppm (2H, d, J ) 8.2); 3 7.43 (2H, d, J ) 8.2); 4.4 (2H, s); 3.8-3.2 (multiplet signal of PEG).

Gopin et al.

Compound 17. Compound 7 (300 mg, 0.67 mmol) was deprotected with 1 mL TFA to remove the Boc group. The excess of the acid was removed under reduced pressure, and the residue was dissolved in 1 mL DMF. Compound 11 (196 mg, 0.22 mmol) was added followed by addition of Et3N (185 µL, 1.34 mmol). The reaction was stirred at room temperature overnight and monitored by TLC (EtOAc 100% then EtOAc: MeOH ) 8:2). After completion, the solvent was removed under reduced pressure and the crude product was purified by column chromatography (EtOAc:MeOH ) 8:2) to give compound 17 (154 mg, 54%) in the form of sticky foam. 1H NMR (200 MHz, MeOD) δ ) 8.04 ppm (4H, s); 7.65 (2H, m); 7.47-7.20 (9H, m); 5.2-5.0 (6H, m); 4.68-4.5 (8H, m); 4.28 (4H, d, J ) 2.4); 3.78-3.61 (14H, m); 3.27 (3H, s); 3.18-3.03 (15H, m); 2.72 (2H, t, J ) 2.4); 2.31-2.29 (3H, m). HRMS (MALDI-TOF): calculated for C66H77N9O19 [M + Na+] 1322.5228; found 1322.5360. Compound 18. Compound 17 (154 mg, 0.11 mmol) was dissolved in 10 mL of dry THF at 0 °C. Then, DIPEA (254 mg, 1.97 mmol) was added followed by p-nitrophenyl chloroformate (290 mg, 1.44 mmol) and pyridine (10 mg, 0.13 mmol). The reaction was allowed to warm to room temperature and monitored by TLC (EtOAc:He ) 85:15). After completion, the reaction was diluted with EtOAc and washed with 1 M HCl and with saturated NaHCO3 solutions. The organic layer was dried over magnesium sulfate. The solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc:He ) 85:15) to give compound 18 (156 mg, 69%) in the form of a white solid. 1H NMR (400 MHz, CDCl ) δ ) 8.25 ppm (8H, d, J ) 8.3); 3 8.0 (4H, s); 7.37-7.03 (17H, m); 5.3 (8H, bs); 5.18 (2H, s); 5.1 (6H, m); 4.28 (4H, s); 3.8-3.5 (16H, m); 3.3 (4H, s); 3.02.8 (16H, m); 2.3 (2H, s); 2.1-2.0 (3H, m). HRMS (MALDITOF): calculated for C94H89N13O35 [M + Na+] 1982.5476; found 1982.5429. Compound 20. Previously reported CPT-amine-Boc unit 19 (259 mg, 0.5 mmol) was deprotected with 1 mL TFA to remove the Boc group. The excess of the acid was removed under reduced pressure and the residue was dissolved in 1 mL DMF. Compound 18 (150 mg, 0.077 mmol) was added followed by addition of Et3N (140 µL, 1 mmol). The reaction was stirred at room temperature overnight and was monitored by TLC (EtOAc: MeOH ) 8:2). After completion, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography to give compound 20 (188 mg, 75%) in the form of a yellowish solid. 1H NMR (200 MHz, CDCl ) δ ) 8.5 ppm (4H, s); 8.32 (4H, 3 d, J ) 8.4); 8.07-8.03 (6H, m); 7.9 (6H, t, J ) 7); 7.7 (6H, t, J ) 7); 7.6-7.5 (2H, m); 7.4-7.2 (14H, m); 5.9-5.7 (4H, m); 5.5 (4H, m); 5.3-5.1 (19H, m); 4.3-4.2 (4H, m); 3.75-3.5 (25H, m); 3.4-3.25 (14H, m); 3.25-2.99 (25H, m); 2.8-2.75 (3H, m); 2.5-2.25 (18H, m); 1.4 (16H, s); 1.1-0.95 (12H, m). HRMS (MALDI-TOF): calculated for C170H173N25O43 [M + Na+] 3275.2011, found 3275.2221. General Procedure for the “Click Chemistry”. Corresponding PEG-azide (2.2 equiv) was dissolved in DMF. Corresponding dendrimer (1 equiv) was added followed by addition of copper sulfate (0.5 equiv) and TBTA (1equiv). Then, few copper turnings were added, and the reaction was stirred overnight at room temperature. The reaction was monitored by HPLC and after completion the mixture was filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel (DCM: MeOH ) 9:1) to give desired product. Compound 1a. HPLC conditions: C18 reverse phase column, λ ) 348 nm, flow rate 1 mL/min, gradient program: t ) 0 (30% ACN: 70% H2O); t ) 20-25 min (100% ACN).

Enzymatic Activation of Dendritic Prodrugs Scheme 1. Molecular Structure of Second-Generation Self-Immolative Dendron with a Trigger Designed for Activation by PGA (red), Reporter Groups of 4-Nitroaniline (blue), and Acetylene Functional Groups (purple) for Click Conjugation

tR(compound 1) ) 18.5 min, tR(compound 1a) ) 15.5 min. The yield after column chromatography is 52%. HRMS (MALDITOF): calculated for C126H159N23O47 2769.0651 [M + Na+], found 2769.0859. Compound 13. HPLC conditions: C18 reverse phase column, λ ) 360 nm, flow rate 1 mL/min, gradient program: t ) 0 (30% ACN: 70% H2O); t ) 20-25 min (100% ACN). tR(compound 20) ) 14 min, tR(compound 13) ) 11 min (broad peak). After the column chromatography, the product was dissolved in mixture of water:MeOH ) 9:1, filtered, and lyophilized to give pure product (50.4%) in a form of a yellowish powder. Solubility Determination. Compound 13 (2 mg) was suspended in 100 µL of distilled water. The mixture was stirred for 1 h and filtered. The concentration of the filtrate was determined by spectroscopic assay (wavelength 360 nm) with CPT solution as a reference. The observed concentration of solution was 11 mg/mL. Scheme 2. Synthesis of Intermediate Compound 8

Scheme 3. Synthesis of Self-Immolative Dendritic Compound 1


Cell-Growth Inhibition Assays. The human embryonic kidney HEK293 cell line was maintained in DMEM (Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal calf serum, 2 mM L-glutamine, and penicillin (10 U/mL)streptomycin (0.01 mg/mL). The human Molt3 and Jurkat leukemia cell lines were maintained in RPMI 1640 (Biological Industries, Beit Haemek, Israel) supplemented with 20% FCS, 1 mM L-glutamine, and penicillin (10 U/mL)-streptomycin (0.01 mg/mL). Cells were grown in Nunclon Flasks in a humidified atmosphere at 37 °C in 5%CO2. For measurements of growth inhibition by CPT and prodrug 13, the Quick cell Proliferation Assay was used (Biovision). Briefly, Jurkat and Molt3 cells were cultured in 96-well microtiter plate at an initial density of 15 000 cells per well in a final volume of 200 µL medium. Various concentrations of the cytotoxic substance were added to the wells in triplicates, and the extent of cell proliferation was determined 3 days later using the WST-1 reagent. The WST-1 reagent is cleaved to a formazan dye via the mitochondrial dehydrogenase, and the dye produced by viable cells was quantified by measuring absorbance of the dye solution at 440 nm. HEK293 cell line proliferation was evaluated in a similar manner, except that the initial culture density was 2500 cells per well. The cytotoxicity effect of prodrug 13 was evaluated in the presence/absence of 1 µM of the enzyme PGA.

RESULTS AND DISCUSSION Synthesis and Bioactivation of Second-Generation Dendritic Model System. In order to test our approach, we designed and synthesized second-generation dendritic molecule 1 (Scheme 1). The molecule has an enzymatic trigger that is activated by PGA (red), four reporter groups of 4-nitroaniline (blue), and two acetylene functional groups that are ready to be coupled to various PEG-azide units (purple). The synthesis of dendritic molecule 1 was achieved as illustrated in Schemes 2 and 3. 4-Hydroxybenzoic acid was coupled with propargylamine to give amide


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Scheme 4. Conjugation of Dendritic Molecule 1 with PEG400-azide

Scheme 5. Disassembly Pathway of Second-Generation Dendritic Molecule 1a Triggered by Enzymatic Activation of PGA

2, which was further reacted with formaldehyde under aqueous basic conditions to afford compound 3. The latter was selectively protected with two equivalents of tert-butyl-dimethylsilyl chloride to generate phenol 4, which was then activated with p-nitrophenyl chloroformate to give carbonate 5. Reaction of mono-Boc-N,N′-dimethylethylenediamine with carbonate 5 afforded carbamate 6, which was stirred with amberlist 15 in methanol to generate compound 7. The latter was reacted with 4-nitrophenyl isocyanate (generated in situ from 4-nitrophenyl and triphosgene) to give compound 8. Carbamate 9 (1) was then deprotected with trifluoroacetic acid (TFA) and directly reacted with linker 12 to generate compound 10, which was activated with p-nitrophenyl chloroformate to give dicarbonate 11. Compound 8 was deprotected with TFA and immediately reacted with dicarbonate 11 to afford dendritic molecule 1. Dendritic molecule 1 was conjugated with 2 equiv of commercially available PEG400-azide via the copper-catalyzed

click reaction (14) to give conjugate 1a (Scheme 4). The location of the PEG tails in the dendron’s periphery was highly important for aqueous solubility during the dendritic platform fragmentation as presented in Scheme 5. The PEG tails remained attached to the dendron fragments until the reporter groups of 4-nitroaniline were released. Cleavage of the phenylacetamide group by PGA triggered the disassembly of dendron 1a through a known self-immolative reaction sequence (1). Following the enzymatic cleavage, azaquinone methide was rapidly eliminated and decarboxylation occurred, leading to internal cyclization that released a urea derivative and phenol 1b. The latter was disassembled as previously described (1) to generate 2 equiv of phenol 1c, which was further fragmented to release the four reporter groups. In order to prepare aqueous solutions of 1 and 1a, the compounds were initially dissolved in DMSO/Cremophor EL (4/1) and then diluted into water. The final composition of the


Figure 1. Bioactivation of dendron 1a by PGA: (blue) dendron 1a, (green) intermediates, (red) 4-nitroaniline.

solution was 10% organic and 90% aqueous. Dendritic molecules 1 and 1a were then incubated with PGA in phosphatebuffered saline (PBS, pH 7.4) at 37 °C. Control solutions were composed of buffer without the enzyme. The sequential fragmentation illustrated in Scheme 5 was monitored by observing the disappearance of dendrons 1 or 1a and the release of 4-nitroaniline by RP-HPLC. As expected, dendron 1 could not be activated by PGA and remained intact for 72 h (data not shown). However, dendron 1a showed clear activation upon incubation with PGA, and its corresponding peak completely disappeared from the HPLC chromatogram as 4-nitroaniline appeared (Figure 1). No 4-nitroaniline was observed in the control experiment when dendron 1a was incubated in the buffer without PGA. Synthesis and Bioactivation of Second-Generation Dendritic Prodrug System. Motivated by the results obtained with the model system, we evaluated the enzymatic activation of a


second-generation dendritic prodrug with camptothecin (CPT), an anticancer agent, in place of the reporter groups (dendron 13). The antitumor activity of 20(S)-camptothecin, a pentacyclic plant alkaloid, was recognized more than 20 years ago. It was first isolated from the Asian tree Camptotheca acuminata by Wall and co-workers in 1966 (15). CPT exerts its antitumor activity mainly through inhibition of topoisomerase I (16). This enzyme, which is found in all mammals, binds preferentially to double-stranded DNA, cleaving one strand and forming an enzyme-DNA covalent bond between a tyrosine residue and the 3′ phosphate of the cleaved DNA. Drug-induced accumulation of topoisomerase I-DNA complexes was identified as an essential step in the effects of CPT, ultimately leading to cell death by apoptosis (17). There are two problems associated with CPT therapy: the instability of the 20-hydroxy lactone and the insolubility of CPT in aqueous and organic media. Both the lactone ring and the 20-hydroxy group of CPT are critical for its antitumor activity. The 20-hydroxy lactone is readily hydrolyzed at neutral pH, yielding the inactive carboxylate. The 20-hydroxyl generates an intramolecular hydrogen bond with the carbonyl moiety of the lactone, accelerating the hydrolysis of the otherwise stable lactone ring. Masking of the 20-hydroxy group of CPT results in increased stability of the lactone ring, but also in reduced activity of the drug (18). However, a prodrug can be generated from CPT by masking the 20-hydroxyl by a chemical linker that can be selectively removed (19). The masking linker serves two purposes: (1) removal of the intramolecular hydrogen bond, resulting in a stable lactone, and (2) increased solubility of CPT. These circumstances make CPT an ideal drug candidate for the evaluation of our dendritic prodrug system. Second-generation CPT dendritic prodrug 13 was prepared with a trigger that is activated by PGA and PEG5000 tails to allow sufficient aqueous solubility (Scheme 6). The synthesis

Scheme 6. Molecular Structure of Second-Generation, Self-Immolative, Dendritic CPT (blue) Prodrug with a Trigger Designed for Activation by PGA (red)


Gopin et al.

Scheme 7. Synthesis of PEG5000 Azide Derivative

is outlined in Schemes 7 and 8. In order to conjugate PEG5000 with the dendritic platform via a click reaction, we had initially prepared a PEG-azide derivative. Hence, compound 14 was synthesized as previously reported (20) and its N-hydroxysuccinimide ester 15 was prepared (Scheme 7). The latter was reacted with commercially available PEG5000-amine to afford the PEG5000-azide derivative 16. Then, compound 7 (Scheme 8) was deprotected with TFA and directly reacted with linker 12 to generate compound 17, which was activated with p-nitrophenyl chloroformate to give tetracarbonate 18. Four equivalents of CPT derivative 19 (prepared according to published procedure (21)) was reacted with 18 to afford dendritic molecule 20. Click conjugation of azide 16 (2 equiv) with diacetylene 20 afforded conjugate 13. The aqueous solubility of dendritic prodrug 13 was measured as described in the experimental section and found to be over 11 mg/mL. The bioactivation of the CPT dendritic prodrugs 20 and 13 by PGA was then evaluated. The prodrugs were incubated in PBS (pH 7.4) at 37 °C with and without PGA and the release of free CPT was monitored by RP-HPLC. As in the model system described above, the PEG-conjugated prodrug 13 was efficiently activated by PGA, whereas its parent prodrug 20 (without the PEG) remained intact. The release of CPT from dendritic prodrug 13 as a function of time is plotted in Figure 2. No release was observed when prodrug 13 was incubated in the buffer without PGA. Cell-Growth Inhibition Assays. Next we evaluated the ability of dendritic prodrug 13 to inhibit cell proliferation in Scheme 8. Synthesis of Second-Generation Dendritic Prodrug 13

Figure 2. CPT release from dendritic prodrug 13 with PGA (red) and without PGA (blue). Table 1. IC50 (nM) Values from Cell-Growth Inhibition Assays MOLT-3

JURKAT

HEK-293

drug/ prodrug

IC50a

IC50b

IC50a

IC50b

IC50a

IC50b

CPT pro-CPT 13

2.9 4200

2.9 41

3.1 2000

3.1 13

12 4100

12 22

a Cells were incubated in medium with drug/prodrug. b Cells were incubated in medium with drug/prodrug + 1 µM of PGA.

the presence of PGA using three different cell lines: the human T-lineage acute lymphoblastic leukemia cell line MOLT-3, the human leukemia T cell line JURKAT, and the human kidney embryonic HEK-293 cell line. The results are summarized in Table 1, and the full data from the cell assays are presented in Figure 3. PGA effectively activated dendritic prodrug 13, and its toxicity was significantly increased in the cell-growth inhibition assays of three cancerous cell lines. The IC50 of prodrug 13 was between 100- and 1000-fold less than free CPT. When PGA was added, the prodrug was activated and its toxicity approached that of free CPT. The 2-3 log difference between the drug and the prodrug cytotoxicity suggests that relatively high concentration of prodrug may be applied for tumor therapy, decreasing the negative side-effect of chemotherapeutic drugs.



CONCLUSIONS In summary, we have demonstrated the first enzymatic activation of second-generation self-immolative dendrimers. We designed and synthesized a dendritic prodrug with four molecules of the anticancer agent camptothecin and a trigger that allows activation by PGA. Two molecules of PEG5000 were conjugated via click chemistry to the dendritic platform in order to gain aqueous solubility. The second-generation self-immolative dendritic prodrug was effectively activated by PGA under physiological conditions, and free CPT was released to the reaction media. Cell-growth inhibition assays demonstrated toxicity of the dendritic prodrug dependent upon incubation with PGA. Incorporation of a specific enzymatic substrate, cleaved by a protease that is overexpressed in tumor cells, could generate a cancer-cell-specific dendritic prodrug activation system.

ACKNOWLEDGMENT D.S. thanks the Israel Science Foundation, the Israel Ministry of Science “Tashtiot” program, and the Israel Cancer Association for financial support.

LITERATURE CITED

Figure 3. Growth inhibition assay of three human cancerous cell lines, with dendritic prodrug 13 in the presence (9) and absence (0) of PGA; cells were incubated for 72 h.

A dramatic 75% decrease in subcutaneous (s.c.) tumor size has been observed in mice that received a combination of intratumoral injections of activator protein (catalytic antibody 38C2) and systemic treatments with an etoposide prodrug that was 500fold less toxic than its parent drug (10). It has been previously demonstrated that PEG conjugation prevents protein precipitation by two possible mechanisms (22). One possibility is that the PEG group poses a steric hindrance to protein-protein association. A second possibility is that the PEG group shields hydrophobic patches on the protein. Both would significantly reduce protein aggregation and enhance solubility of hydrophobic molecules in water. In the present study even two short PEG400 oligomers were sufficient to permit access of the enzyme to substrate in the dendron’s focal point and allow the bioactivation of the system. However, the PEG400 oligomers were large enough to significantly affect to dendrons’ aqueous solubility. We assumed that the conjugation PEG oligomers with higher mass should achieved sufficient hydrophilicity to solubilize the dendritic prodrug system in water. Indeed, when PEG5000 was conjugated to a secondgeneration dendritic prodrug bearing four molecules of the hydrophobic drug CPT, the conjugate had significant aqueous solubility and PGA efficiently activated it.

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Enzymatic Activation of Second-Generation Dendritic Prodrugs

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