Synthesis and Biological Studies of 5-Aminolevulinic Acid-Containing

Nov 3, 2001 - Photodynamic therapy (PDT) is a nonthermal technique for inducing tissue damage with ..... The mixture was stirred under argon for 30 mi...
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Bioconjugate Chem. 2001, 12, 980−988

Synthesis and Biological Studies of 5-Aminolevulinic Acid-Containing Dendrimers for Photodynamic Therapy Sinan H. Battah,† Cheng-Ean Chee,‡ Hiroaki Nakanishi,‡ Sandra Gerscher,‡ Alexander J. MacRobert,‡ and Christine Edwards*,† Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex, CO4 3SQ, UK, and National Medical Laser Centre, Royal Free and University College Medical School, University College London, UK. Received March 29, 2001; Revised Manuscript Received September 25, 2001

Using a convergent growth approach, a series of novel 5-aminolevulinic acid (ALA)-containing dendrimers have been synthesized. In these molecules, ALA residues are attached to the periphery by ester linkages, with amide bonds connecting the dendrons. Three first-generation dendrimers, bearing either 6 or 9 ALA residues, were synthesized by attachment of a tris(Boc-protected ALA)containing wedge (1) to a di- or tripodent aromatic, or tripodent aliphatic core. Two second generation 18-ALA-containing dendrimers were also synthesized using a 3,3′-iminodipropionic acid spacer unit between wedge 1 and the aromatic core. These compounds differed only in the distance between the core and the linker unit. The Boc-protected dendrimers were deprotected using trifluoroacetic acid and isolated as their TFA salts. The potential of these ALA ester dendrimers as macromolecular prodrugs for photodynamic therapy has been demonstrated in the tumorigenic keratinocyte PAM 212 cell line.

INTRODUCTION

Photodynamic therapy (PDT) is a nonthermal technique for inducing tissue damage with light following administration of a light-activated photosensitizing drug which can be selectively retained in malignant or diseased lesions relative to normal adjacent tissue (1). Activation of the photosensitizer results in the generation of reactive oxygen species (ROS), mainly singlet oxygen via interaction of the sensitizer triplet state with molecular oxygen. The ROS oxidize intracellular substrates such as lipids and amino acid residues leading to cell death. The most widely studied application of PDT to date is in cancer therapy. However, using conventional exogenous sensitizers, skin photosensitivity can be prolonged, and damage to normal tissue is rarely avoidable due to poor tumor selectivity. The use of 5-aminolevulinic acid (ALA) is a relatively new approach in PDT. ALA is a natural precursor of protoporphyrin IX (PpIX), an effective photosensitizer, and cellular concentrations of PpIX can be increased by the administration of 5-aminolevulinic acid (ALA) (2). A certain amount of tumor selectivity can be achieved since porphobilinogen deaminase (a rate-limiting enzyme in heme biosynthesis) activity is increased (3) and ferrochelatase (the enzyme which catalyzes the conversion of photoactive PpIX into inactive heme) levels are decreased in tumor cells (4). For therapeutic applications, PpIX is activated using red light (typically at 635 nm), with mitochondria (5) believed to be key targets of singlet oxygen oxidative damage resulting in cell death mainly via apoptosis (6). PpIX levels return to normal after 24 h, following administra-

tion so skin photosensitization is relatively short-lived compared to many exogenous sensitizers (7). A major limitation of ALA-PDT is the low intracellular availability of ALA. Cellular uptake of ALA occurs via a relatively slow active transport mechanism (8, 9). Recently it has been shown that ALA esters, being more lipophilic than ALA (e.g., ALA hexyl ester), can quickly cross the cell membrane by diffusion and (10, 11), once inside the cell, are cleaved by nonspecific esterases (1118). Since higher intracellular ALA levels lead to higher PpIX concentrations, lipophilic ALA prodrug derivatives are more efficient at inducing PpIX photosensitization. A further improvement based on this strategy would be to design a prodrug that can deliver several ALA molecules into the cell, and selectivity of the prodrug for target cells would be an obvious advantage. Polymeric drug delivery systems present an attractive method for drug targeting, particularly in cancer chemotherapy (19). In the past, most of the polymers investigated as drug carriers have been polydisperse and either linear or highly branched. Recent advances in polymer chemistry now allow the synthesis of structurally defined, hyperbranched polymers (or dendrimers) which can be conjugated with drug molecules (20). The drug can either be reacted with a preformed dendrimer (21-23), in which case the exact loading of the drug cannot easily be controlled, or can be incorporated into the structure of the dendrimer during synthesis. The latter offers the opportunity for a polymeric prodrug which is structurally defined with a known size and drug loading. Here we describe the synthesis of a novel series of dendritic ALA ester prodrugs for use in PDT applications. MATERIALS AND METHODS

* E-mail: [email protected]. Tel: +44 (0) 1206 873821, Fax: +44 (0) 1206 872592. † University of Essex. ‡ University College London.

General Methods. 1H and 13C NMR spectra were recorded on a JEOL EX270 MHz spectrometer. Chemical shifts are quoted in ppm measured downfield relative to

10.1021/bc010027n CCC: $20.00 © 2001 American Chemical Society Published on Web 11/03/2001

5-Aminolevulinic Acid-Containing Dendrimers

TMS. Ultraviolet-visible spectra were recorded on a Unicam UV2 spectrophotometer in dichloromethane solution. Infrared spectra were recorded on a Nicolet FTIR spectrophotometer as thin films in dichloromethane. Mass spectra were obtained using either an EI, CI, FAB or electrospray technique with a NOBA matrix. Melting points were measured using a Kolfler hot-plate microscope. Analytical thin-layer chromatography was carried out using Merck silica gel 60, aluminum-backed silicacoated plates which were visualized using ultraviolet light (254 nm). Column chromatography was carried out using Prolabo silica gel (220-400 mesh flash grade). All compounds were purified to one spot by TLC and Rf values are quoted using the same solvent system as used for column chromatography unless otherwise stated. All reagents were purchased from Aldrich, Avocado, or Acros chemical companies and used without further purification. Dendrimer Synthesis. ALA was prepared by the method of Hart et al. (24). Compounds 2, 3, 4, 7, and 11 were prepared according to Ashton et al. (25). N-tert-Butyloxycarbonyl-5-aminolevulinic Acid (8, Boc-ALA). A solution of 5-aminolevulinic acid (0.42 g, 2.50 mmol) in water (5 mL) was adjusted to pH 8-10 with aqueous sodium hydroxide (0.1 N). Di-tert-butyl dicarbonate (1.16 g, 5.33 mmol) was dissolved in 1,4dioxane (5 mL) and added to the mixture which was stirred at room temperature for 18 h. The excess of ditert-butyl dicarbonate was removed by washing the mixture with diethyl ether (2 × 50 mL). Ethyl acetate (50 mL) was added, and the aqueous layer was acidified with dilute hydrochloric acid (1 N). Separation of the organic layer, followed by removal of the solvent, gave 0.35 g (60%) of Boc-ALA as a colorless oil; IR (film) 3445, 2765, 2242, 1721, 1389, 1364 cm-1; 1H NMR (270 MHz, CDCl3) δ 10.42 (br s, 1H), 5.25 (br s, 1H), 4.00 (s, 2H), 3.26 (m, 4H), 1.42 (s, 9H); 13C NMR (67.5 MHz, CDCl3) δ 204.47, 176.75, 155.89, 128.58; 80.10, 49.61, 33.88, 27.34; CI-MS m/z 232 (M+ + H). N-(Benzyloxycarbonyl)[tris(N-tert-butyloxycarbonyl-5-aminolevulinyloxymethyl)]methylamine (9). Compound 7 (0.31 g, 1.22 mmol) in DMF (10 mL) was added dropwise, over 50 min, to a mixture of Boc-ALA (8, 1.0 g, 4.30 mmol), DCC (0.82 g, 4.10 mmol), and DMAP (0.05 g, 0.42 mmol) in dichloromethane and DMF (15 mL, 2:1 v/v). The mixture was stirred at room temperature for 48 h, after which time the heavy precipitate was filtered off, and the solvents were evaporated in vacuo. The residue was purified by column chromatography on silica gel (EtOAc/hexane 7:3, Rf ) 0.46) to obtain 0.52 g (75%) of compound 9 as a colorless oil; IR (film) 3372, 2680, 2352, 1755, 1718, 1513, 1457, 1370 cm-1; 1H NMR (270 MHz, CDCl3) δ 7.41 (m, 5H), 5.86 (br s, 1H), 5.58 (br s, 3H), 5.13 (s, 2H), 4.47 (s, 6H), 4.05 (d, J ) 5.2 Hz, 6H), 2.76 (m, 6H), 2.09 (m, 6H), 1.37 (s, 27H); 13C NMR (67.5 MHz, CDCl3) δ 127.68, 127.86, 153.23, 155.17, 171.17, 172.33, 203.44; 127.54, 79.11, 61.85, 56.50, 48.92, 33.48, 27.64, 27.35, 26.90; FAB-MS m/z 917 (M+ + Na); FAB-HRMS calcd for C42H62N4O17Na (M+) 917.4008, found 917.3998. Tris(tert-butyloxycarbonyl-5-aminolevulinyloxymethyl)methylamine (1). The Z-protected amine 9 (0.53 g, 0.59 mmol) was dissolved in ethyl acetate and methanol (15 mL, 2:1 v/v). Palladium on carbon (0.17 g, 10%) was added, and the mixture was stirred under an atmosphere of hydrogen for 8 h at room temperature. The reaction mixture was filtered through Celite which was washed with a further amount of ethyl acetate. The solvents were evaporated in vacuo, and the residue was

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dried to obtain 0.44 g (97%) of compound 1 as a colorless oil; IR (film) 3375, 2654, 1760, 1718, 1370 cm-1; 1H NMR (270 MHz, CDCl3) δ 8.95 (br s, 2H), 5.54 (br s, 3H), 4.32 (s, 6H), 3.98 (d, J ) 4.3 Hz, 6H), 2.75 (s, 6H), 2.69 (m, 6H), 1.38 (s, 27H); 13C NMR (67.5 MHz, CDCl3) δ 205.56, 171.90, 155.62, 79.64, 65.36, 54.05, 49.99, 34.07, 28.15, 27.45; ES-MS m/z 761 (M+ + H); ES-HRMS calcd for C34H57N4O15 (M+ + H) 761.3820, found 761.3821. 1,3,5-Benzenetricarbamido-N,N′,N′′-tris(ethylpropionate) (10). A solution of 1,3,5-benzenetricarbonyl trichloride (3.10 g, 11.34 mmol) in dichloromethane and DMF (15 mL, 2:1 v/v) was slowly added, over a period of 2 h, to a stirred mixture of β-alanine ethyl ester hydrochloride (6.90 g, 45.0 mmol) and triethylamine (4.20 mL, 30.0 mmol) in dichloromethane and DMF (15 mL, 2:1 v/v) at 0 °C. Stirring was continued at the same temperature for 4 h and at room temperature for 16 h. The reaction mixture was then filtered, and the solvents were evaporated in vacuo. The resulting residue was dissolved in dichloromethane (50 mL) and washed successively with aqueous hydrochloric acid (50 mL, 2 N), saturated aqueous sodium hydrogen carbonate (50 mL), and water (20 mL), dried (MgSO4), and evaporated to obtain 6.20 g (61%) of the title compound as a white powder, mp 181183 °C; IR (film) 3419, 2276, 2340, 1710, 1650 cm-1; 1H NMR (270 MHz, CDCl3) δ 8.10 (s, 3H), 7.49 (s, 3H), 4.18 (q, J ) 4.0 Hz, 6H), 3.71 (m, 6H), 2.69 (m, 6H), 1.27 (t, J ) 4.0 Hz, 9H); 13C NMR (67.5 MHz, CDCl3) δ 173.00, 166.42, 135.36, 128.56, 61.30, 36.28, 34.26, 14.55; CI-MS m/z 508 (M+ + H); EI-HRMS calcd for C24H33N3O9 (M+) 507.2217, found 507.2228. 1,3,5-Benzenetricarbamido-N,N′,N′′-tripropionic Acid (5). Compound 10 (4.00 g, 7.92 mmol) was dissolved in methanol (15 mL), and the solution was cooled to 0 °C. Aqueous sodium hydroxide (70 mL, 2 N) was added, and the solution was left to stir for 3 h. The resultant precipitate was dissolved by the addition of water (7 mL), and the solution was neutralized with Amberlite IR-120 (H+ form) ion-exchange resin, filtered, evaporated, and dried thoroughly to afford 0.61 g (93%) of 5 as a white powder, mp 251-252 °C; IR (Nujol) 3455, 2237, 1689, 1631 cm-1; 1H NMR (270 MHz, d6-DMSO) δ 12.42 (br s, 3H), 8.91 (t, J ) 5 Hz, 3H), 8.51 (s, 3H), 3.68 (m, 6H), 2.63 (m, 6H); 13C NMR (67.5 MHz, d6-DMSO) δ 172.88, 165.49, 134.80, 128.51, 35.72, 33.61; CI-MS m/z 424 (M+ + H); CI-HRMS calcd for C18H22N3O9 (M+ + H) 424.1356, found 424.1368. N,N′-{N-[Tris(N-tert-butyloxycarbonyl-5-aminolevulinyloxymethyl)methyl]acetamido}terephthalamide (11). A solution of 1 (0.42 g, 0.58 mmol) in dichloromethane (10 mL) was added dropwise to a mixture of 3 (70 mg, 0.22 mmol), EDC (0.12 g, 0.50 mmol), and HOBt (74 mg, 0.50 mmol) in dichloromethane and DMF (15 mL, 2:1 v/v) at 0 °C under argon. The reaction mixture was allowed to stir for 48 h at room temperature. The solvents were then evaporated, and the residue was dissolved in ethyl acetate (30 mL) and washed with hydrochloric acid (2 × 20 mL, 2 N), saturated aqueous sodium hydrogen carbonate (2 × 20 mL), and water (2 × 20 mL). The solvent was evaporated to give an crude oily product which was subjected to column chromatography on silica gel (EtOAc/MeOH 9:1, Rf ) 0.55) to give 0.19 g (46%) of compound 11 as a white solid, mp 106 °C; IR (film) 3397, 2359, 2229, 2130, 1695, 1598, 1394 cm-1; UV (MeOH) 202, 241 nm; 1H NMR (270 MHz, CDCl3) δ 7.95 (s, 4H), 7.82 (br s, 2H), 6.93 (br s, 2H), 5.49 (br s, 6H), 4.44 (s, 12H), 4.07 (d, J ) 6.1 Hz, 4H), 4.01 (d, J ) 7.5 Hz, 12H), 2.88 (m, 12H), 2.61 (m, 12H), 1.43 (s, 54H); 13C NMR (67.5 MHz, CDCl3) δ 206.17,

982 Bioconjugate Chem., Vol. 12, No. 6, 2001

171.51, 166.60, 155.22, 135.86, 127.08, 79.45, 62.33, 59.88, 49.64, 43.45, 33.70, 28.84, 27.81, 27.53; FAB-MS m/z 1787 (M+ + Na). Anal. Calcd for C80H120N10O34: C, 54.4; H, 6.9; N, 7.9. Found: C, 53.9; H, 6.9; N, 7.7. N,N′-{N-[Tris(5-aminolevulinyloxymethyl)methyl]acetamido}terephthalamide‚6Trifluoroacetic Acid (12). Compound 11 (30 mg, 20 µmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid TFA (2 mL) was added, and the mixture was stirred at room temperature for 2 h. The solvents were evaporated under reduced pressure to yield an oily product which was crystallized from a mixture of methanol, ethyl acetate, and diethyl ether. 20 mg (63%) of compound 12 was obtained as a pale yellow solid; 1H NMR (270 MHz, CD3OD) δ 7.98 (s, 4H), 4.43 (s, 12H), 4.40 (s, 4H), 4.01 (s, 12H), 2.82 (m, 12H), 2.68 (m, 12H); 13C NMR (67.5 MHz, CD3OD) δ 200.89, 171.57, 169.66, 160.62, 135.76, 126.63, 76.51, 61.27, 58.81, 45.99, 42.25, 33.12, 29.17, 26.31. 1,3,5-Tris(N-{N-[tris(N-tert-butyloxycarbonyl-5aminolevulinyloxymethyl)methyl]acetamido}carbamido)benzene (13). A solution of 1 (0.50 g, 2.16 mmol) in dry dichloromethane (10 mL) was added dropwise to a stirred solution of 4 (0.23 g, 0.60 mmol), DCC (0.43 g, 2.14 mmol), and HOBt (0.28 g, 2.14 mmol) in dichloromethane and DMF (15 mL, 2:1 v/v) at 0 °C under argon. The reaction mixture was left to stir at room temperature for 4 days and then filtered, and the solvents were evaporated. The resulting residue was dissolved in ethyl acetate and washed with 5% hydrochloric acid (2 × 30 mL), saturated sodium hydrogen carbonate (30 mL), and water (2 × 30 mL). The organic layer was evaporated and the residue purified by column chromatography (EtOAc/MeOH 8:2, Rf ) 0.45) to obtain 0.61 g (40%) of 13 as a foamy solid, mp 115-116 °C; IR (film) 3366, 2955, 2365, 2137, 1693, 1678, 1519, 1364 cm-1; UV (MeOH) 209, 240 nm; 1H NMR (270 MHz, CDCl3) δ 8.81 (s, 3H), 8.22 (br s, 3H), 6.17 (br s, 3H), 6.12 (br s, 9H), 4.36 (s, 6H), 3.97 (s, 18H), 3.94 (d, J ) 6.5 Hz, 18H), 2.65 (m, 18H), 2.45 (m, 18H), 1.31 (s, 81H); 13C NMR (67.5 MHz, d6-acetone) δ 206.22, 172.74, 170.32, 156.88, 135.63, 129.98, 79.36, 63.16, 62.97, 59.10, 50.57, 44.39, 34.67, 28.96, 28.56; FAB-MS m/z 2632 (M+ + Na). Anal. Calcd for C117H177N15O51: C, 53.8; H, 6.8; N, 8.1. Found: C, 54.6; H, 7.4; N, 7.7. 1,3,5-Tris[N-{N-[tris(5-aminolevulinyloxymethyl)methyl]acetamido}carbamido)benzene‚9Trifluoroacetic Acid (14). Compound 13 (30 mg, 10 µmol) was dissolved in dichloromethane (10 mL). TFA (2 mL) was added to the solution, and the mixture was stirred at room temperature for 2 h. The solvents were evaporated under reduced pressure to yield an oily product which was crystallized from a mixture of methanol, ethyl acetate, and diethyl ether to obtain 2 mg (66%) of the TFA salt as a pale yellow solid; 1H NMR (270 MHz, CD3OD) δ 8.58 (s, 3H), 4.45 (s, 18H), 4.35 (s, 6H), 4.04 (d, J ) 4.5 Hz, 18H), 2.87 (m, 18H), 2.70 (m, 18H); 13C NMR (67.5 MHz, CD3OD) δ 206.22, 205.92, 172.74, 170.32, 156.88, 135.63, 129.98, 79.36, 63.16, 62.97, 59.10, 50.57, 44.39, 34.67. Tris{N-[tris(N-tert-butyloxycarbonyl-5-aminolevulinyloxymethyl)methyl]acetamido}amine (15). Nitrilotriacetic acid (6, 60 mg, 0.32 mmol) was dissolved in dichloromethane and DMF (20 mL, 2:1 v/v), and DCC (0.23 g, 1.10 mmol) and HOBt (0.15 g, 1.10 mmol) were added. The mixture was stirred under argon for 30 min at 0 °C, and then compound 1 (0.85 g, 1.14 mmol) was added dropwise over 50 min. The mixture was allowed to stir at room temperature for 3 days, the precipitate was filtered off, and the solvents were evaporated. The

Battah et al.

residue was dissolved in ethyl acetate (30 mL) and washed with hydrochloric acid (2 × 30 mL, 2 N), saturated sodium hydrogen carbonate (30 mL), and water (2 × 30 mL). The organic layer was evaporated to give an oily product which was subjected to flash chromatography (EtOAc/MeOH 49:1, Rf 0.6). The product was crystallized from a mixture of methanol, diethyl ether, and ethyl acetate giving 0.45 g (60%) of compound 15 as a foamy solid, mp 102-104 °C; IR (film) 3372, 2980, 2365, 1699, 1640, 1364 cm-1; UV (MeOH) 214 nm; 1H NMR (270 MHz, CDCl3) δ 6.39 (br s, 3H), 5.41 (br s, 9H), 4.37 (s, 18H), 4.04 (s, 18H), 2.75 (m, 18H), 2.65 (m, 18H), 2.51 (s, 6H), 1.44 (s, 81H); 13C NMR (67.5 MHz, CDCl3) δ 204.28, 171.51, 171.38, 155.37, 61.78, 57.64, 49.64, 33.91, 33.72, 29.41, 27.01, 27.16; FAB-MS m/z 2419 (M+). Anal. Calcd for C108H171N13O48: C, 53.6; H, 7.1; N, 7.5. Found: C, 53.9; H, 7.0; N, 7.6. Tris{N-[tris(5-aminolevulinyloxymethyl)methyl]acetamido}amine‚9Trifluoroacetic Acid (16). Compound 15 (0.34 g, 0.22 mmol) was dissolved in dichloromethane (15 mL), TFA (3 mL) was added, and the solution was stirred at room temperature for 2 h. The solvents were evaporated under reduced pressure to yield an oily product which was crystallized from a mixture of methanol, ethyl acetate, and diethyl ether to obtain 0.24 g (69%) of the TFA salt as a pale yellow solid; 1 H NMR (270 MHz, CD3OD) δ 4.31 (s, 18H), 3.97 (s, 18H), 2.80 (m, 18H), 2.63 (m, 18H), 2.54 (s, 6H); 13C NMR (67.5 MHz, CD3OD) δ 203.78, 203.32, 174.72, 173.66, 163.07, 162.54, 63.31, 59.05, 35.32, 30.37, 28.31. N-(Benzyloxycarbonyl)imino-3,3′-bis{N-[tris(Ntert-butyloxycarbonyl-5-aminolevulinyloxymethyl)methyl]propionamide} (18). Compound 2 (0.12 g, 0.43 mmol) was dissolved in dichloromethane and DMF (20 mL, 2:1 v/v). DCC (0.21 g, 0.96 mmol) and HOBt (0.14 g, 0,96 mmol) were added, and the mixture was allowed to stir for 30 min at 0 °C under argon. Compound 1 (0.80 g, 1.11 mmol) was added dropwise over 50 min. The reaction mixture was stirred at room temperature for 3 days, and then the solvents were evaporated. The residue was dissolved in ethyl acetate (20 mL) and washed with hydrochloric acid (2 × 25 mL, 2 N), saturated aqueous sodium hydrogen carbonate (25 mL), and water (2 × 25 mL). The organic layer was evaporated to give an oily product, which was subjected to flash chromatography on silica gel (EtOAc/MeOH 19:1, Rf 0.6) to yield 0.51 g (66%) of compound 18 as a colorless oil; IR (film) 3347, 2974, 2924, 1712, 1709, 1513, 1370 cm-1; UV (MeOH) 209, 240 nm; 1H NMR (270 MHz, CDCl3) δ 7.48 (m, 5H), 7.45 (br s, 2H), 6.34 (br s, 6H), 5.21 (s, 2H), 4.48 (s, 12H), 4.05 (s, 12H), 2.88 (m, 12H), 2.79 (m, 12H), 2.69 (m, 12H), 2.59 (m, 12H), 1.49 (s, 54H); 13C NMR (67.5 MHz, CDCl3) δ 203.740, 173.97, 170.79, 157.96, 134.44, 130.45, 129.89, 80.50, 74.58, 73.63, 67.76, 52.14, 51.82, 36.82, 35.87, 31.15, 30.86, 29.29, 23.69; ES-MS m/z 1803 (M+ + Na). Imino-3,3′-bis{N-[tris(N-tert-butyloxycarbonyl-5aminolevulinyloxymethyl)methyl]propionamide} (19). Compound 18 (0.50 g, 0.32 mmol) was dissolved in ethyl acetate and methanol (30 mL, 2:1 v/v). Palladium on carbon (0.17 g, 10%) was added, and the mixture was stirred under an atmosphere of hydrogen gas for 8 h at room temperature. The reaction mixture was filtered through Celite and washed with ethyl acetate. The solvents were evaporated, and the residue was dried to obtain 0.38 g (82%) of 19 as a colorless oil; IR (film) 3353, 2980, 2930, 2365, 1712, 1513, 1364, 1258, 1159, 1046, 860 cm-1; UV (MeOH) 208 nm; 1H NMR (270 MHz, CDCl3) δ 7.43 (br s, 2H), 6.30 (m, 7H), 4.09 (s, 12H), 4.05 (s, 12H), 2.89 (m, 12H), 2.72 (m, 12H), 2.70 (m, 12H), 2.67

5-Aminolevulinic Acid-Containing Dendrimers

(m, 12H), 1.49 (s, 45H); 13C NMR (67.5 MHz, CDCl3) δ 202.32, 176.84, 155.91, 80.06, 77.50, 60.42, 50.06, 34.03, 28.19, 27.58, 20.95, 14.07, 9.16; FAB-MS m/z 1647 (M+). 1,3,5-Tris[N-(N-bis{N-[tris(N-tert-butyloxycarbonyl-5-aminolevulinyloxymethyl)methyl]propionamido}acetamido)carbamido]benzene (20). Compound 19 (0.05 g, 0.11 mmol) was dissolved in dichloromethane and DMF (20 mL, 2:1 v/v), EDC (90 mg, 0.48 mmol) and HOBt (0.06 g, 0.48 mmol) were added, and the mixture was stirred for 30 min under argon at 0 °C. Compound 4 (0.77 g, 0.55 mmol) was dissolved in dichloromethane (5 mL) and added dropwise over 50 min. The mixture was stirred at room temperature for 3 days, and then the solvents were evaporated. The residue was dissolved in ethyl acetate and washed with hydrochloric acid (2 × 25 mL, 2 N), saturated aqueous sodium hydrogen carbonate (25 mL), and water (2 × 20 mL). The organic layer was evaporated to give an oily product which was purified by flash chromatography on silica gel (EtOAc/MeOH 3:1, Rf 0.35) yielding 0.37 g (54%) of compound 20 as a foamy white solid, mp 126-128 °C; IR (film) 3384, 2974, 2918, 2359, 1711, 1699, 1513, 1364 cm-1; 1H NMR (270 MHz, CDCl3) δ 7.28 (s, 3H), 6.39 (br s, 3H), 5.34 (br s, 6H), 5.06 (br s, 18H), 4.31 (m, 6H), 4.23 (s, 36H), 3.97 (s, 36H), 3.68 (m, 12H), 2.70 (m, 36H), 2.57 (m, 36H), 2.45 (m, 12H), 1.37 (s, 162H); 13C NMR (67.5 MHz, CDCl3) δ 203.56, 172.49, 171.61, 155.39, 134.31, 128.01, 79.46, 65.32, 62.30, 61.74, 59.66, 57.52, 49.61, 33.78, 33.72, 29.50, 27.80, 14.74. Anal. Calcd for C237H366N30O102: C, 54.0; H, 7.0; N, 8.0. Found: C, 54.1; H, 7.4; N, 8.1. 1,3,5-Tris[N-(N-bis{N-[tris(5-aminolevulinyloxymethyl)methyl]propionamido}acetamido)carbamido]benzene‚18Trifluoroacetic Acid (21). Compound 20 (0.28 g, 50 µmol) was dissolved in dichloromethane (20 mL), TFA (4 mL) was added, and the solution was stirred at room temperature for 2 h. The solvents were evaporated under reduced pressure to yield an oily product which was crystallized from a mixture of methanol, ethyl acetate, and diethyl ether to obtain 0.18 g (62%) of the TFA salt as a pale yellow solid; 1H NMR (270 MHz, CD3OD) δ 7.36 (s, 3 H), 4.40 (s, 36 H), 4.35 (m, 6 H), 4.03 (s, 36 H), 3.76 (m, 12 H), 2.89 (m, 36 H), 2.85 (m, 36 H), 2.69 (m, 12 H); 13C NMR (67.5 MHz, CD3OD) δ 204.22, 172.00, 155.70, 130.60, 128.00, 79.87, 65.51, 62.84, 58.05, 54.22, 50.17, 41.78, 34.59, 34.24, 28.30, 27.73, 27.60. 1,3,5-Tris[N-(N-bis{N-[tris(N-tert-butyloxycarbonyl-5-aminolevulinyloxymethyl)methyl]propionamido}propionamido)carbamido]benzene (22). Compound 5 (30 mg, 60 µmol) was dissolved in dichloromethane and DMF (15 mL, 2:1 v/v), EDC (40 mg, 0.20 mmol) and HOBt (30 mg, 0.20 mmol) were added, and the mixture was stirred under argon for 30 min at 0 °C. Compound 19 was dissolved in dichloromethane (0.39 g, 0.21 mmol) and added dropwise to the mixture over 50 min. The mixture was stirred at room temperature for 3 days, the heavy precipitate was filtered off, and the solvents were evaporated. The residue was dissolved in ethyl acetate (25 mL) and washed with hydrochloric acid (2 × 25 mL, 2 N), saturated aqueous hydrogen carbonate (25 mL), and water (2 × 25 mL). The organic layer was evaporated to give an oily product which was subjected to flash chromatography on silica gel (EtOAc/ MeOH 3:1, Rf 0.35). Crystallization of the product using a mixture of dichloromethane, diethyl ether, and ethyl acetate gave 0.22 g (63%) of compound 22 as a foamy solid, mp 129-128 °C; IR (film) 3384, 2980, 2930, 2359, 2253, 1706, 1699, 1507, 1370 cm-1; 1H NMR (270 MHz, d6-acetone) δ 8.46 (br s, 3H), 8.30 (s, 3H), 6.17 (br s, 6H), 5.62 (br s, 18H), 4.41 (s, 36H), 4.11 (m, 6H),

Bioconjugate Chem., Vol. 12, No. 6, 2001 983

4.03 (s, 36H), 3.73 (m, 12H), 2.73 (m, 36H), 2.70 (m, 6H), 2.66 (m, 36H), 2.63 (m, 12H), 1.44 (s, 162H); 13C NMR (67.5 MHz, CDCl3) δ 204.48, 172.06, 171.98, 168.92, 155.69, 128.46, 72.26, 65.79, 61.97, 60.06, 54.16, 50.12, 48.98, 34.18, 33.84, 28.24, 27.54, 25.54, 24.86. Anal. Calcd for C240H372N30O102: C, 54.3; H, 7.1; N, 7.9. Found: C, 54.2; H, 7.2; N, 7.6. 1,3,5-Tris[N-(N-bis{N-[tris(5-aminolevulinyloxymethyl)methyl]propionamido}propionamido)carbamido]benzene‚18Trifluoroacetic Acid (23). Compound 22 (0.31 g, 60 µmol) was dissolved in dichloromethane (20 mL), TFA (4 mL) was added, and the solution was stirred at room temperature for 2 h. The solvents were evaporated under reduced pressure to yield an oily product that was crystallized from a mixture of methanol, diethyl ether, and ethyl acetate to obtain 0.12 g (41%) of the TFA salt as a pale yellow solid; 1H NMR (270 MHz, CD3OD) δ 8.40 (s, 3H), 4.40 (s, 36H), 4.17 (m, 6H), 4.14 (s, 36H), 4.01 (m, 12H), 2.95 (m, 36H), 2.93 (m, 6H), 2.84 (m, 36H), 2.82 (m, 12H); 13C NMR (67.5 MHz, CDCl3) δ 203.71, 203.03, 173.76, 173.14, 163.02, 162.38, 132.67, 127.23, 63.12, 58.36, 51.80, 36.14, 35.27, 35.14, 34.67, 29.02, 28.34, 28.01. Cell Culture. PAM 212 cells (naturally transformed murine keratinocytes)(26) were grown monolayered in sterile, vented-capped, angled-necked cell culture flasks. The culture medium was RPMI 1640 medium containing L-glutamine (0.02 mM) and phenol red, supplemented with 10% foetal calf serum (FCS) (First Link), penicillin (500 units/mL), streptomycin (0.5 mg/mL), and HEPES buffer (0.01 M). Cells were maintained at 37 °C in a humidified 5% CO2 incubator until confluent. Approximately twice weekly, the cells were passaged. The cells were detached from the flasks with a solution of 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS), spun down in a centrifuge at 1500 rpm for 5 min, resuspended in fresh medium, and subcultured in new flasks. Chemicals and Solutions. ALA was purchased from Photocure (Norway) as the hydrochloride salt. ALA and dendrimers 16 and 21 were dissolved in FCS- and phenol red-free RPMI medium immediately prior to incubation (serum is known to cause release of PpIX from the cells, thus resulting in loss of the fluorescence signal(27) and phenol red would optically interfere with the detection of PpIX). Solutions were sterile filtered using a 0.2 µm syringe-tip filter before incubation. Incubation in the presence of ALA or an ALA-containing dendrimer was performed under subdued lighting. Fluorescence Spectroscopy. Fluorescence spectroscopy allows quantification of porphyrin yields over a period of time. Cells were seeded in gamma-sterilized 96-well plates at a density of 5 × 104 cells per well, 48 h prior to constant drug exposure, and solutions of ALA and ALA-containing dendrimers were freshly prepared and sterile-filtered. PpIX fluorescence was recorded at 2, 4, and 6 h after drug exposure. PpIX fluorescence was measured with a well plate fluorescence reader (PerkinElmer, UK) connected to a Perkin-Elmer LS 50B fluorescence spectrophotometer (Perkin-Elmer, UK) using 410 nm excitation and 635 nm emission with slit widths set to 10 nm both on the excitation and emission monochromators, and an internal 530 nm highpass filter used on the emission side; spectral scans were made between 550 and 750 nm to check for the presence of other porphyrins. Intensity calibrations were performed using rhodamine B embedded in a Perspex disk as a standard. Control wells containing RPMI alone were included. The experiments were performed in quadruplicate, and stan-

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Figure 1. Dendrimer building blocks.

Figure 2. Synthesis of ALA-containing dendrimer, 1.

dard deviations expressed as error bars. The respective controls were used for background subtraction of the spectrum, and equalization of the number of cells in the solution to reduce the error caused by scattering effects. Photocytotoxicity. Forty-eight hours prior to the experiment, 5 × 104 cells were seeded in 16 wells located in the middle of each 96-well platesthe irradiation zone produced by the laser is up to 6 cm in diameter and covers 16 wells (4 wells × 4 wells) on the well plate. Three different controls were prepared. Two controls consisted of cells incubated with RPMI, and only one of them was irradiated with light. In the third control, cells were incubated with the maximum concentration of each compound (2 mM) with no light irradiation to determine dark cell toxicity. The cells were incubated with ALA or an ALAcontaining dendrimer for 4 h at 37 °C in a humidified 5% CO2 atmosphere. After 4 h, the compounds were removed and replaced with 0.1 mL of RPMI containing 10% FCS (warmed at 37 °C). For illumination a diode laser was used (Diomed) emitting at a wavelength of 630 nm, for which the power output at the fiber tip was set at 1.13 W using a power meter (Gentech). The cells were irradiated in a 6 cm diameter area within a single field, providing simultaneous illumination of all wells. Exposure time was 125 s, providing an energy density of 5 J/cm2 (fluence rate: 40 mW/cm2). Cells were not rinsed before irradiation; loss of PpIX in the medium has been neglected since it does not affect cell survival (28). Once irradiated, the cells were placed into the incubator (37 °C in a humidified 5% CO2 atmosphere) and cell cytotoxicity was determined 18 h later using the MTT assay.

MTT Assay. Cell viability was tested by means of an MTT assay. This technique allows quantification of cell survival after cytotoxic insult by testing the enzymatic activity of the mitochondria. It is based on the reduction of a water-soluble tetrazonium salt to a purple, insoluble formazan derivative by mitochondrial enzyme dehydrogenases. This enzymatic function is only present in living, metabolically active cells. The optical density of the product was quantified by its absorption at 570 nm using a 96-well microplate reader (MR 700 Dynatech). A 0.1 mL volume of a solution of 3-[4,5-dimethylthiazolyl]-2,5diphenyltetrazolium bromide (MTT) in PBS (1 mg/mL) was added to each well, and the cells were incubated for 3 h at 37 °C in a humidified 5% CO2 atmosphere (29). Control studies showed no cytotoxicity induced by the dendrimers in the absence of light. Phototoxicity results are expressed as percentage of cell viability against control cells under the same conditions but in the absence of test compound.

Figure 3. Synthesis of core molecule, 5.

5-Aminolevulinic Acid-Containing Dendrimers

Bioconjugate Chem., Vol. 12, No. 6, 2001 985

Figure 4. Synthesis of the first-generation dendrimers. RESULTS AND DISCUSSION

Dendrimers are constructed by stepwise methods yielding structurally and topologically defined, monodisperse, unimolecular entities. There are two approaches to the synthesis of dendritic polymers based on either convergent or divergent synthesis. In the divergent approach, growth of the polymer begins at the center and proceeds radially outward in a series of coupling reactions, whereas in the convergent approach, growth begins at the chain ends. A disadvantage of using the divergent method is that, because the number of polymer chain ends increases geometrically at each coupling or activation process, it eventually becomes difficult to ensure that all chain ends have reacted at each stage of growth. A convergent growth strategy, similar to that of Ashton et al. (25), has therefore been adopted for the synthesis of the ALA-containing dendrimers. The dendrimers were assembled in segments: first the individual building blocks were synthesized, and then these dendrons were attached to a multipodent core unit in the final steps of dendrimer construction. Classical coupling chemistry using either DCC or EDC with HOBt was used to join the individual dendrons by amide bonds. The synthetic strategy adopted makes use of easily accessible building blocks (Figure 1). Dendron 1, which bears three Boc-protected ALA molecules, is the terminal dendron in all five dendrimers. Four different core molecules were used: the diglycyl amide of 1,4-benzenedicarboxylic acid (3), the triglycyl amide of 1,3,5-benzenetricarboxylic acid (4), the tri-β-alanyl amide of 1,3,5benzenetricarboxylic acid (5), and nitrilotriacetic acid (6). In the larger dendrimers, Z-protected 3,3′-iminodipropionic acid (2) was used as a branching unit. These building blocks, when combined in a suitable synthetic

sequence, gave rise to a series of dendrimers varying in size and ALA loading. The three primary hydroxyl groups of tris(hydroxymethyl)aminomethane (TRIS) allowed attachment of three ALA molecules through ester linkages, and the free amino group enabled further elaboration through the formation of an amide bond with either a branching unit or a core molecule. Therefore, Z-protection of TRIS using benzyl chloroformate gave 7 (25), which was then coupled with N-tert-butyloxycarbonyl-5-aminolevulinic acid (Boc-ALA, 8), giving rise to compound 9. Removal of the Z-protecting group by hydrogenation gave the terminal dendron 1 (Figure 2). The core molecules 3 and 4 were prepared by reacting the corresponding di- and tribenzoic acid chlorides and glycine methyl ester, followed by basic hydrolysis of the di- and triester products (25). Core molecule 5 was synthesized in an analogous fashion: 1,3,5-benzenetricarbonyl trichloride reacted with β-alanine ethyl ester to give the triester 10 and base-induced hydrolysis gave 5. Compound 6 is commercially available (Figure 3). Attempts to directly couple dendron 1 with 1,3,5benzenetricarboxylic acid failed due to steric congestion around the core. However, when 1 was reacted with the extended core 4, using DCC as the coupling reagent, the reaction proceeded smoothly to give dendrimer 13. Dendron 1 also reacted with core 3 to give dendrimer 11 which contains six ALA residues. The 9-ALA dendrimer 15, which has an aliphatic core, was synthesized by the reaction between dendron 1 and nitrilotriacetic acid in the presence of DCC and HOBt (Figure 4). Branching unit 2 was prepared by hydrolysis of the nitrile groups in 3,3′-iminodipropionitrile using barium hydroxide to give the corresponding dicarboxylic acid as

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Figure 5. Synthesis of the second-generation dendrimer, 21.

its hydrogen sulfate salt (17) (25). Z-Protection using benzyl chloroformate gave compound 2 (25). DCC coupling between dendron 1 and branching unit 2 gave dendron 18. Hydrogenation gave amine 19, however, using DCC as the coupling reagent, the reaction between this new 6-ALA dendron and core molecule 4 did not give the desired second generation dendrimer 20. When EDC was used for the amide coupling, 20 was obtained in good yield (Figure 5). The homologous dendrimer 22 was prepared by coupling dendron 20 with core molecule 5 (Figure 6). The Boc protecting groups were removed from dendrimers 11, 13, 15, 20, and 22 by treatment with trifluoroacetic acid, and the cationic dendrimers 12, 14, 16, 21, and 23, respectively, were isolated as their trifluoroacetate salts (Figures 5 and 6). The simplicity and reliability of this approach should permit an extension of this synthetic methodology to the synthesis of higher-generation dendrimers by the incorporation of additional branching units. Although higher molecular weight dendrimers would be required for enhanced permeability and retention in tumor tissue, we have evaluated the concept of using dendrimers as macromolecular carriers for the delivery of ALA to the tumorigenic cell line, PAM 212, in vitro. Figure 7(a) shows the PpIX fluorescence induced by ALA, the 9-ALA dendrimer 16, and the 18-ALA dendrimer 21 at 0.1 mM concentration in PAM 212 cells. The ALA-containing dendrimers are clearly able to pass through the cell membrane and release ALA intracellularly; however,

there is no direct correlation between the number of ALA molecules attached to the dendrimer and PpIX accumulation. The more than 2-fold greater efficacy of 21

Figure 6. The second-generation dendrimer, 23.

5-Aminolevulinic Acid-Containing Dendrimers

Figure 7. (a) Mean PpIX fluorescence induced by 0.1 mM ALA, dendrimer 16 or dendrimer 21. (b) Cell survival using 4 h incubation with 0.1 mM and 0.5 mM ALA, dendrimer 16 or dendrimer 21, after illumination with 5 J cm-2.

over 16 may be due to increased uptake of the larger dendrimer by endocytosis. Figure 7b shows the results of a PDT experiment in which the cells were illuminated 4 h after incubation with either ALA, dendrimer 16, or dendrimer 21. At the concentrations used, neither of the dendrimers induced cytotoxicity in the absence of light, and dendrimer 21 again shows greatest potency. These results demonstrate the potential of this type of multivalent ALA carrier for the delivery of ALA in PDT applications. ACKNOWLEDGMENT

We thank the University of Essex for a research studentship, the Wellcome Trust for the award of a vacation research studentship, and the EPSRC Mass Spectrometry Service, Swansea. The authors would also like to thank Dr. Ross Boyle and Dr. Keith Matthews for useful discussion before embarking on this project. LITERATURE CITED (1) Dougherty, T. J., Gomer, C. J., Henderson, B. W., Jori, G., Kessel, D., Korbelik, M., Moan, J., and Peng, Q. (1998) Photodynamic therapy. J. Natl. Cancer Inst. 90, 889-905. (2) Peng, Q., Berg, K., Moan, J., Kongshaug, M., and Nesland, J. M. (1997) 5-aminolevulinic acid-based photodynamic therapy: principles and experimental research. Photochem. Photobiol. 65, 235-251. (3) Navone, N. M., Polo, C. F., Frisardi, A. L., Andrade, N. E., and Batlle, A. M. D. C. (1990) Heme biosynthesis in human breast cancer - mimetic “in vitro” studies and some heme enzymatic activity levels. Int. J. Biochem. 22, 1407-1411. (4) Van Hillegersberg, R., Van Denberg, J. W. O., Kort, W. J., Terpstra, O. T., and Wilson, J. H. P. (1992) Selective accumulation of endogenously produced porphyrins in a liver metastasis model in rats. Gastroenterology 103, 647-651. (5) Ratcliffe, S. L., and Matthews, E. K. (1995) Modification of the photodynamic action of δ-aminolaevulinic acid (ALA) on rat pancreatoma cells by mitochondrial benzodiazepine receptor ligands. Br. J. Cancer 71, 300-305.

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