Soluble Camptothecin Derivatives Prepared by Click Cycloaddition

Standing on the shoulders of Hermann Staudinger: Post-polymerization modification from past to present. Kemal Arda Günay , Patrick Theato , Harm-Anto...
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Bioconjugate Chem. 2007, 18, 263−267

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TECHNICAL NOTES Soluble Camptothecin Derivatives Prepared by Click Cycloaddition Chemistry on Functional Aliphatic Polyesters Bryan Parrish and Todd Emrick* Polymer Science & Engineering Department, University of Massachusetts, Conte Center for Polymer Research, Amherst, Massachusetts 01003. Received July 6, 2006; Revised Manuscript Received October 19, 2006

Aliphatic polyesters are of interest as biomaterials and drug-delivery vehicles, as their ability to degrade under physiological conditions provides a mechanism for both drug release and clearance of the polymer from the body. Presented here is the synthesis of a polyester-drug graft copolymer conjugate, enabled by click cycloaddition of azide-functionalized camptothecin derivatives with alkyne-functionalized aliphatic polyesters. Further grafting of residual alkyne groups with azide-terminated poly(ethylene oxide) gave a water-soluble polyester-camptothecin conjugate. Control over PEGylation and drug loading, inherent to the graft copolymer design, opens versatile routes to new materials with potential utility in polymer therapeutics.

INTRODUCTION Central issues associated with chemotherapy include the methods and mechanisms by which drugs are introduced to the body and delivered to cancer tissue (1-3). As potent anticancer drugs such as doxorubicin, camptothecin, and paclitaxel have poor aqueous solubility, and in some cases short-term aqueous stability, introduction of these drugs into the bloodstream is enhanced by modification of their chemical structures or by their encapsulation in liposomal or polymeric formulations (4). In order to improve delivery efficiency and avoid high levels of dosing to achieve therapeutic drug levels, new conjugation methods to attach drugs to polymer materials are needed (56). A number of polymer-drug conjugates have been reported to enhance drug solubility and stability in aqueous media, in turn providing longer circulation time of the drug in the bloodstream relative to the nonconjugated drugs (7-10). In addition, the high molecular weight of the polymer-drug conjugates enables tumor targeting by the enhanced permeability and retention effect (11). Polymer-drug conjugates that utilize poly(ethylene glycol) (PEG), N-(2-hydroxypropyl)methacrylamide (HPMA), and poly(glutamic acid) (PGA) as the polymeric component have been investigated, as have a variety of polymer architectures including linear, graft, and dendritic systems (1217). The conjugates are too large to permeate the vasculature of healthy tissue, and thus preferably enter the leaky vasculature of cancerous tissue, where they accumulate due to poor lymphatic drainage in this environment. Following localization in cancer tissue, the polymer-drug conjugates can be taken up by cancer cells through endocytosis, then may be delivered to the lysosomal compartment. The relatively acidic environment of the lysosome makes attractive the use of acid-cleavable linking groups between the polymer and the drug (18). Here, we present the synthesis of a water-soluble form of camptothecin by its conjugation to a PEGylated aliphatic polyester graft copolymer. Aliphatic polyesters are particularly * Corresponding author. E-mail: [email protected].

attractive as biomaterials and drug delivery vehicles, as their in vivo degradation products are relatively benign (19). However, conventional aliphatic polyesters are insoluble in water, and thus not inherently well-suited for aqueous-based injectable formulations. Moreover, water-soluble PEG-aliphatic polyester diblock copolymers have drug carrying capabilities limited to physical encapsulation and/or substitution at the PEG chain end(s) with one (or two) drug molecules.

EXPERIMENTAL PROCEDURES Materials. 6-Bromohexanoic acid (98%), bromotris(triphenylphosphine) copper(I) (98%), 20(S)-camptothecin (95%), copper(II) sulfate pentahydrate (>98.0%), N,N′-dicyclohexylcarbodiimide (DCC) (99%), N,N-diisopropylethylamine (g99%), (4(dimethylamino)pyridine (DMAP) (99%), and sodium azide (99.5%) were obtained from Aldrich. Sodium ascorbate (crystalline) was purchased from Source Naturals. Dialysis tubing (Spectra/Por Membrane MWCO 6-8000) was obtained from VWR. CH2Cl2 was washed according to standard procedures and distilled over CaH2. All other materials were used without further purification. Instrumentation. NMR spectra were recorded on CDCl3 solutions using a Bruker DPX300 or Bruker Avance400 spectrometer (ω13C ) 0.25*ω1H) with residual solvent signal as calibration. Molecular weights and polydispersity indices were measured by gel permeation chromatography in THF relative to polystyrene standards (Scientific Polymer Products Mp ) 503, 700, 1306, 2300, 4760, 12 400, 196 700, and 556 000 g/mol) on a system equipped with a three-column set (Polymer Laboratories 300 × 7.5 mm; 5 µm; 10-5, 10-4, and 10-3 Å pore sizes) and a refractive-index detector (HP 1047A) at room temperature with a flow rate of 1 mL/min. High-resolution mass spectral (HRMS) data were obtained on a JEOL JMS700 MStation. IR absorbance data was obtained on a Perkin-Elmer Spectrum One FT-IR spectrometer equipped with a universal ATR sampling accessory. Synthesis of 6-Azidohexanoic Acid (2). 6-Bromohexanoic acid (5.0 g, 25.6 mmol) and sodium azide (8.37 g, 128 mmol)

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were dissolved in DMSO and stirred at room temperature for 8 h. The reaction mixture was then dissolved in CH2Cl2, washed with water, brine, and NaHCO4(aq), dried over MgSO4, and concentrated by rotary evaporation. Residual DMSO was removed by Kugelrohr distillation at 120 °C. Distillation at 160 °C gave 6-azidohexanoic acid 2 as a colorless liquid (2.98 g, 74%). MS-ESI (m/z): [M + Na]+ calculated for C6H11N3O2 180.1, found 180.1. 1H NMR (CDCl3, 300 MHz): δ (CHCl3 ) 7.26 ppm) 11.43 (br, 1H, COOH), 3.28 (t, 2H, CH2N3), 2.38 (t, 2H, CH2COOH), 1.65 (m, 4H, CH2CH2CH2CH2CH2), 1.43 (m, 2H, CH2CH2CH2CH2CH2) ppm. 13C NMR (CDCl3, 75 MHz): δ (CHCl3 ) 77.0 ppm) 180.1 (CdO), 51.2 (CH2N3), 33.9 (CH2COOH), 28.5 (CH2CH2N3), 26.2 (CH2CH2CH2N3), 24.2 (CH2CH2COOH) ppm. IR(ATR): NdNdN 2090 cm-1. Synthesis of Camptothecin Azide (3) Camptothecin (100 mg, 0.27 mmol), 6-azidohexanoic acid 2 (85 mg, 0.54 mmol), and DMAP (4 mg) were dissolved in CH2Cl2 (5 mL). DCC (110 mg, 5.4 × 10-1 mmol) was added, and the solution was stirred at room temperature for 8 h. The reaction mixture was then precipitated into diethyl ether (50 mL) and isolated by filtration. Recrystallization from MeOH/CH2Cl2 (95:5) gave camptothecin azide 3 as a slightly yellow, crystalline solid (114 mg, 87%). HRMS-EI (m/z): [M]+ calculated for C26H25N5O5 487.186, found 487.186. 1H NMR (CDCl3, 300 MHz): δ (CHCl3 ) 7.26 ppm) 8.40 (s, 1H, C-16H), 8.23 (d, J ) 8.4 Hz, 1H, C-18H), 7.95 (d, J ) 8.4 Hz, 1H, C-21H), 7.84 (t, J ) 6.8 Hz, 1H, C-19H), 7.67 (t, J ) 7.6 Hz, 1H, C-20H), 7.21 (s, 1H, C-25H), 5.70 (d, J ) 17.2 Hz, 1H, C-11H2), 5.43 (d, J ) 17.2 Hz, 1H, C-11H2), 5.29 (s, 2H, C-14H), 3.22 (t, J ) 6.8 Hz, 2H, C-1H2), 2.51 (m, 2H, C-5H2), 2.28 (m, 1H, C-8H2) 2.16 (m, 1H, C-8H2), 1.67 (m, 2H, C-2H2), 1.62 (m, 2H, C-4H2), 1.43 (m, 2H, C-3H2), 0.98 (t, J ) 7.2 Hz, 2H, C-9H3) ppm. 13C NMR (CDCl3, 75 MHz): δ (CHCl3 ) 77.0 ppm) 172.5 (CdOlactone C-10), 167.7 (CdOester C-6), 157.5, 152.5, 149.0, 146.4, 146.0, 131.4, 130.9, 129.7, 128.6, 128.4, 128.3, 128.2, 120.4, 96.0, 75.9, 67.2, 51.3 (CH2N3 C-1), 50.1, 33.7, 32.0, 28.6, 26.2, 24.3, 7.7 (CH3 C-9) ppm. IR (ATR): NdNdN 2096 cm-1. Synthesis of Polyester-Camptothecin Conjugate (4). Aliphatic polyester 1 (52 mol % acetylene, Mn ) 9.2 × 103 g/mol, PDI ) 1.17, 500 mg, 2.1 mmol acetylene) was dissolved in CH2Cl2 (2 mL) in a small reaction vessel. Camptothecin azide 3 (205 mg, 0.42 mmol, N,N-diisopropylethylamine (31 µL, 0.16 mmol), and bromotris(triphenylphosphine) copper(I) (77 mg, 8.4 × 10-2 mmol), to target 20 mol % camptothecin grafting, were then added sequentially, and the reaction mixture was stirred at room temperature for 48 h. The crude reaction mixture was precipitated into ether (50 mL) and isolated by filtration. The resulting solid was then dissolved in CH2Cl2 (2 mL) and dialyzed against pure CH2Cl2 (400 mL) for 48 h. Removal of the solvent and washing with cold hexanes (5 mL) afforded polyester-camptothecin conjugate 4 as a slightly yellow, viscous liquid (580 mg, 82%). GPC (THF): Mn ) 14.3 × 103 g/mol, PDI ) 1.19, 18 mol % camptothecin grafting. 1H NMR (CDCl3, 300 MHz): δ (CHCl3 ) 7.26 ppm) 8.41 (s, 1H, camptothecin 3 C-16H), 8.20 (d, J ) 8.3 Hz, 1H, camptothecin 3 C-18H), 7.94 (d, J ) 8.3 Hz, 1H, camptothecin 3 C-21H), 7.86 (t, J ) 6.9 Hz, 1H, camptothecin 3 C-19H), 7.67 (m, 1H, camptothecin 3 C-20H), 7.37 (m, 1H, R2CdCH), 7.19 (s, 1H, camptothecin 3 C-25H), 5.68 (d, J ) 15.2 Hz, 1H, camptothecin 3 C-11H2), 5.40 (d, J ) 15.3 Hz, 1H, camptothecin 3 C-11H2), 5.29 (s, 2H, camptothecin 3 C-14H), 4.25 (br, 2H, CH2NR2 camptothecin 3), 4.04 (br, 4H, CH2OCdO polyester), 3.22 (t, J ) 6.6 Hz, 2H, camptothecin 3 C-1H2), 3.01 (br, 2H, R2CHCH2 polyester), 2.51 (m, 2H, camptothecin 3 C-5H2), 2.35 (br, 4H, CdOCH polyester + CdOCH2 polyester + camptothecin 3 C-8H2), 2.15 (m, 1H, camptothecin 3 C-8H2), 2.03 (m, 1H, CtCH polyester), 1.65 (br m, 12H, CH2CH2CH2O polyester

Parrish and Emrick

+ CH2CH2CH2CH2O polyester + camptothecin 3 C-2H2 + camptothecin 3 C-4H2), 1.36 (br m, 4H, CH2CH2CH2O polyester + camptothecin 3 C-3H2), 0.95 (t, J ) 7.1 Hz, 2H, camptothecin 3 C-9H3) ppm. 13C NMR (CDCl3, 75 MHz): δ (CHCl3 ) 77.0 ppm) 174.2 (CdO polyester), 173.4 (CdO polyester), 172.5 (CdO camptothecin 3 C-10), 167.6 (CdO camptothecin 3 C-6), 157.5, 152.5, 149.0, 146.4, 146.0, 144.5, 131.4, 130.9, 129.7, 128.6, 128.3, 128.3, 128.2, 122.5, 120.4, 96.0, 75.9, 67.2, 64.6, 64.1, 53.3, 50.1, 49.9, 44.7, 34.1, 33.7, 32.1, 28.6, 28.4, 27.6, 26.2, 25.9, 25.4, 24.5, 24.3, 7.7 (CH3 camptothecin 3 C-9) ppm. Synthesis of PEG-Grafted Polyester-Camptothecin Conjugate (5). Polyester-camptothecin conjugate 4 (580 mg, 1.4 mmol acetylene) was dissolved in a minimal amount of acetone and added by syringe to a rapidly stirred solution of azidefunctionalized PEG-1100 (1.8 g, 1.5 mmol) in water (2 mL). Sodium ascorbate (120 mg, 6.0 × 10-1 mmol) and copper(II) sulfate (150 mg, 6.0 × 10-1 mmol) were added, and the resulting dispersion was heated to 80 °C overnight. The crude reaction mixture was then diluted with water (50 mL) and extracted five times with CH2Cl2 (50 mL). The combined organics were dried over MgSO4 and concentrated by rotary evaporation. The resulting solid was then dissolved in PBS (2 mL) and dialyzed against pure PBS for 48 h to remove residual PEG-azide. After removal of the solvent, the product was dissolved in acetone (2 mL) and precipitated into cold hexanes (50 mL). The product was isolated by filtration and dried in a vacuum oven overnight to give PEG-grafted polyester-camptothecin conjugate 5 as an off-white powder (1.80 g, 76%). GPC (THF): Mn ) 23.5 × 103 g/mol, PDI ) 1.20. 1H NMR (CDCl3, 300 MHz): δ (CHCl3 ) 7.26 ppm) 8.42 (s, 1H, camptothecin 3 C-16H), 8.17 (d, J ) 8.3 Hz, 1H, camptothecin 3 C-18H), 7.94 (d, J ) 8.3 Hz, 1H, camptothecin 3 C-21H), 7.84 (t, J ) 6.7 Hz, 1H, camptothecin 3 C-19H), 7.66 (m, 1H, camptothecin 3 C-20H), 7.51 (br, 1H, R2CdCH PEG), 7.46 (br, 1H, R2CdCH camptothecin), 7.18 (s, 1H, camptothecin 3 C-25H), 5.66 (d, J ) 14.9 Hz, 1H, camptothecin 3 C-11H2), 5.40 (d, J ) 14.9 Hz, 1H, camptothecin 3 C-11H2), 5.28 (s, 2H, camptothecin 3 C-14H), 4.48 (m, 2H, R2NCH2), 4.26 (br, 2H, CH2NR2 camptothecin 3), 4.02 (br, 2H, CH2OCdO polyester), 3.84 (t, J ) 5.15 Hz, 2H, R2NCH2CH2), 3.61 (br m, 96H, CH2CH2O PEG), 3.43 (t. J ) 5.44 Hz, 2H, CH2OCH3 PEG), 3.37 (s, 3H, CH3 PEG), 3.20 (t, J ) 6.6 Hz, 2H, camptothecin 3 C-1H2), 3.00 (br, 2H, R2CHCH2 polyester), 2.48 (m, 2H, camptothecin 3 C-5H2), 2.27 (br, 4H, CdOCH polyester + CdOCH2 polyester + camptothecin 3 C-8H2), 2.15 (m, 1H, camptothecin 3 C-8H2), 1.66 (br m, 12H, CH2CH2CH2O polyester + CH2CH2CH2CH2O polyester + camptothecin 3 C-2H2 + camptothecin 3 C-4H2), 1.35 (br m, 4H, CH2CH2CH2O polyester + camptothecin 3 C-3H2), 0.91 (t, J ) 7.1 Hz, 2H, camptothecin 3 C-9H3) ppm. 13C NMR (CDCl3, 75 MHz): δ (CHCl3 ) 77.0 ppm) 174.1 (CdO polyester), 173.3 (CdO polyester), 172.5 (CdO camptothecin 3 C-10), 167.7 (CdO camptothecin 3 C-6), 157.5, 152.3, 149.1, 146.4, 146.0, 144.5, 131.3, 130.9, 129.7, 128.6, 128.2, 128.3, 128.2, 122.5, 120.4, 96.0, 75.9, 71.7 (CH2OCH3 PEG), 70.4 (CH2CH2O PEG), 69.2, 67.2, 64.7, 64.1, 58.6 (CH3 PEG), 53.3, 50.1, 49.8, 44.7, 34.0, 33.7, 32.0, 28.6, 28.4, 27.5, 26.2, 25.9, 25.4, 24.5, 24.3, 7.8 (CH3 camptothecin 3 C-9) ppm.

RESULTS AND DISCUSSION We recently reported the preparation of novel, water-soluble PEGylated aliphatic polyester graft copolymers by the controlled ring-opening polymerization of alkyne-functionalized lactones, followed by PEGylation of the polyester by click chemistry with azide-terminated PEG (20). Tailored graft densities of PEG were achieved by copolymerization of unfunctionalized lactones (i.e., -caprolactone) with the acetylene-functionalized derivatives. These aliphatic polyesters proved soluble in water when ∼25

Technical Notes

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Figure 1. Synthesis of azide-functionalized camptothecin derivative 3 by carbodiimide coupling of camptothecin with 6-azidohexanoic acid 2.

mol % of the monomer units in the backbone were grafted with PEG 1100 g/mol (∼70 wt % PEG), using click chemistry. This provides the opportunity to intentionally leave unreacted acetylene in the backbone for additional click cycloaddition reactions. Click chemistry is useful for functionalization of aliphatic polyesters, as unlike the wide variety of acid- or baseinduced chemistries, the mild nature of click chemistry allows it to occur in the absence of polyester degradation. The particular click chemistry employed here is the Cu(I)-catalyzed cycloaddition of azides and alkynes to give triazoles, which has been demonstrated, for example, by Sharpless and Fokin (21) to be efficient, selective, and tolerant of many functional groups, and thus advantageous not only for maintaining the integrity of the polyester backbone following conjugation but also potentially for conjugation of a variety of therapeutic drugs to the polymer backbone, as such drugs often contain numerous types of functionality that could interfere with conventional organic coupling reactions. In this study, we demonstrate the synthesis of water-soluble PEG- and camptothecin-functionalized aliphatic polyesters, where both PEGylation and camptothecin grafting is accomplished using click chemistry. For the camptothecin coupling, it was necessary to prepare the requisite azide derivative of the drug. Sn(II)-mediated ring-opening copolymerization of R-propargyl-δ-valerolactone and -caprolactone was conducted to give aliphatic polyester 1 with a nearly 1:1 ratio of the two monomers, as confirmed by integration of the 1H NMR spectrum of the polymer. Gel permeation chromatography of polyester 1, performed in THF against polystyrene molecular weight standards, gave an estimated number-average molecular weight (Mn) of 9200 g/mol and a polydispersity index (PDI, Mw/Mn) of 1.17. Click cycloaddition of camptothecin to the alkynefunctionalized polyester required the synthesis of an azidefunctionalized camptothecin derivative. For this, esterification of camptothecin at the 20-OH position was carried out, as upon cleavage of the ester bond in aqueous-based delivery applications, the drug would be released in its active, unmodified form. Moreover, Greenwald and co-workers recently reported that PEG-esterification of camptothecin at the 20-OH position led to stabilization of the drug in its lactone form under physiological conditions, as characterized by NMR and UV-vis spectroscopy (22). Buffer hydrolysis experiments at different pH values further demonstrated that camptothecin remained in its lactone form until exocylic ester cleavage, at which point the active drug was released. Thus, on the basis of Greenwald’s important study, we chose to integrate azides onto camptothecin using an ester linkage at the 20-OH position, for both enhanced aqueous stability of the conjugate and the ultimate release of free, unmodified camptothecin, leaving behind the degradable aliphatic polyester. Azide-functionalized camptothecin 3 was prepared as shown in Figure 1. 6-Azidohexanoic acid 2 was obtained by nucleophilic substitution of 6-bromohexanoic acid with sodium azide in 74% yield after purification by Kugelrohr distillation. Compound 2 was characterized by 1H NMR spectroscopy (methylene triplet -CH2N3 at 3.28 ppm, shifted slightly relative to the -CH2Br methylene group of the starting material at 3.42

ppm), 13C NMR spectroscopy (-CH2N3 at 51.2 ppm), infrared spectrometry (azide signal at 2108 cm-1), and mass spectrometry (MS ESI (m/z) [M + Na]+ calcd 180.1, found 180.1). While azide 2 was reported previously as a synthetic intermediate (23), its detailed synthesis and characterization were not. R,ω-Azidecarboxylic acid 2 was then used to esterify camptothecin at the 20-OH position, using dicyclohexylcarbodiimide coupling conditions. This esterification occurred readily at room temperature in dichloromethane to give azide-functionalized camptothecin 3 in 87% yield after purification by crystallization from 95:5 MeOH/CH2Cl2. The structure of 3 was confirmed by 1H and 13C NMR spectroscopy, FTIR spectrometry (azide signal at 2096 cm-1), and high-resolution mass spectrometry (HRMS-EI (m/ z): [M]+ calculated for C26H25N5O5 487.186, found 487.186. Coupling of acetylene-functionalized polyester 1 with camptothecin azide 3 was then performed by targeting 20 mol % camptothecin incorporation (or one camptothecin per five monomer repeat units). Initial attempts to use aqueous click chemistry for this coupling were unsuccessful, due to the poor aqueous solubility of both components. Thus, organic soluble copper(I) catalysts for click chemistry were used as alternatives (24-25); bromotris(triphenylphosphine) copper(I) with N,Ndiisopropylethylamine in dichloromethane solution was found to be effective for the cycloaddition of camptothecin azide 3 and acetylene-functionalized polyester 1, as shown in Figure 2a. The reaction was complete in 48 h, at which point the crude polymer-drug conjugate was precipitated into ether. The resulting solid was dissolved in CH2Cl2 and dialyzed against pure CH2Cl2 using a Spectra/Por Membrane MWCO 6-8000. Removal of the solvent and washing with cold hexanes gave polyester-camptothecin conjugate 4 as a slightly yellow, viscous liquid in 82% yield. 1H NMR spectroscopy of 4 revealed signals characteristic of both camptothecin and the aliphatic polyester backbone, with new signals at 7.37 and 4.25 ppm, corresponding to the triazole proton R2CdCHR and the methylene group adjacent to the triazole CH2NR, respectively. The relative integrations of the signals at 4.04 ppm (CH2O polyester backbone) and 4.25 ppm (CH2-triazole) indicated a nearly quantitative incorporation of camptothecin azide 3 into the polyester backbone. GPC analysis in THF indicated a substantial increase in molecular weight, from Mn ≈ 9200 g/mol before conjugation to ∼14 300 after conjugation of the drug. The narrow polydispersity (PDI ) 1.19) of the polyester-camptothecin product demonstrated that these click cycloaddition conjugation conditions caused no substantial degradation of the polyester, a key feature of click chemistry that makes it especially attractive for polyester modification. While these hydrophobic polymer-drug conjugates can in principle be used to prepare hydrophobic aliphatic polyester microparticles with covalently bound camptothecin, further grafting, for example, with PEG, is necessary to impart water solubility to the conjugate. In the 1H NMR spectrum of 4, the signal at 2.03 ppm (-CtCsH) indicated the presence of substantial residual alkyne (∼30-35 mol %) available for further coupling, as expected from the use of camptothecin azide 3 as the limiting reagent. Click cycloaddition of these residual acetylene groups with azide-terminated PEG (19) (i.e., CH3O-

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Figure 2. (a) Preparation of hydrophobic polyester-camptothecin conjugate 4 by click cycloaddition of azide-functionalized 3 to acetylenefunctionalized copolymer 1 and (b) preparation of hydrophilic PEG-grafted polyester-camptothecin conjugate 5 by further grafting of conjugate 3 with PEG-azide.

Figure 3. Gel permeation chromatography traces (THF relative to polystyrene standards) of acetylene-functionalized polyester 1, polyestercamptothecin conjugate 4, and PEG-grafted polyester-camptothecin conjugate 5.

PEG-N3; Mn ) 1100), as shown in Figure 2b, was performed successfully in water. The acetylene-containing polyester was dissolved in a minimal amount of acetone, then added to a rapidly stirred, aqueous solution of azide-terminated PEG to ensure good dispersion. Copper sulfate and sodium ascorbate were then added, and the resulting reaction mixture heated to 80 °C and stirred overnight. Purification of the PEGylated camptothecin-polyester conjugate 5 was performed by extraction into CH2Cl2, dialysis in phosphate buffered saline, and finally precipitation into cold hexanes to give a slightly yellow, semicrystalline solid in 74% yield. Successful grafting of both the PEG and camptothecin components onto the aliphatic polyester backbone was confirmed by 1H NMR spectroscopy of 5, by the triazole proton resonance from the camptothecin (7.36 ppm, R2CdCHR) and PEG (7.51 ppm, R2CdCHR) grafts, as well as methylene signals at 4.26 and 4.48 ppm, corresponding to the R2NCH2 groups from the camptothecin and PEG grafts, respectively. The relative integrations of the NMR signals at 4.46 (2H-PEG), 4.23 (2H-camptothecin), and 4.04 (2Hpolyester backbone) ppm indicated a grafting density, in terms of percent monomers containing the grafts, of ∼18% camptothecin and ∼33% PEG. GPC analysis of 5, shown in Figure 3, gave an estimated Mn of ∼23 500 g/mol, a significant increase over the polyester-camptothecin conjugate 4. Moreover, the narrow polydispersity of 4 was maintained in 5 (PDI ≈ 1.20),

again confirming the absence of degradation reactions during the coupling procedure. Importantly, PEG grafting had a tremendous influence on the solubility properties of the polyester-camptothecin conjugate 5, which proved highly soluble in water, as well as many organic solvents (e.g., acetone, DMSO, and CH2Cl2), due to the amphiphilicity of PEG. The water solubility of this conjugate, coupled with the biodegradable features of the polyester backbone, holds potential for enhancing the ability of camptothecin to be used in polymer therapeutics. Future studies will center on active targeting of the conjugate by the incorporation of oligopeptide sequences (26), as well as pH-dependent release of camptothecin from the backbone and in vitro cytotoxicity toward cancer cell lines. In addition, the convenient synthesis of camptothecin azide 3 reported here may prove useful for click cycloaddition to other polymer scaffolds, oligopeptide sequences, and/or modified proteins.

ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the Army Research Laboratory through a MURI award, the National Science Foundation (Collaborative Research in Chemistry award, CHE-0404575), and a gift from PMUSA.

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Technical Notes Supporting Information Available: NMR spectra of 6-azidohexanoic acid 2, azide-functionalized camptothecin derivative 3, hydrophobic polyester-camptothecin conjugate 4, and PEGgrafted polyester-camptothecin conjugate 5. This material is available free of charge via the Internet at http://pubs.acs.org.

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