Enantioselective Release of 5-Fluorouracil from N-(2-Hydroxypropyl

Mar 30, 1995 - 5-fluorouracil (5-FU) were synthesized by a new facilitated synthetic route and studiedfor theirability to release free 5-FU in the pre...
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Bioconjugate Chem. 1995, 6, 483-492

483

Enantioselective Release of 5-Fluorouracil from N-(2-Hydroxypropyl)methacrylamide-BasedCopolymers via Lysosomal Enzymes David P u t n a m and Jindfich KopeEek* Departments of Pharmaceutics and Pharmaceutical Chemistry/CCCD and of Bioengineering, University of Utah, Salt Lake City, Utah 84112. Received March 30, 1995@

Water soluble copolymers based on N42-hydroxypropy1)methacrylamide(HPMA) containing oligopeptide side chains terminated in an a-substituted glycine derivative of the anticancer compound 5-fluorouracil (5-FU)were synthesized by a new facilitated synthetic route and studied for their ability to release free 5-FU in the presence of lysosomal enzyme preparations. In addition, the properties of the low molecular weight a-substituted glycine derivatives were studied in the presence of lysosomal enzyme preparations and leucine aminopeptidase. The results revealed that (1)the stereochemistry (L vs D) of the a-substituted glycine derivative, (2) the hydrophobicity (Ala vs Leu) of the penultimate amino acid residue relative to the a-substituted glycine derivative, and (3) the total length of the oligopeptide sequence spacer (tetrapeptide vs hexapeptide) terminated in the a-substituted glycine derivative and the polymer carrier all directly influence the enzymatically catalyzed release of free 5-FU.

INTRODUCTION

Numerous polymeric carriers of anticancer compounds have been synthesized and used for targeted drug delivery (for review see ref 1). Ringsdorf(2)first reported a clear representation of the potential of polymers as targetable lysosomotropic drug carriers. This landmark paper describes the necessity of site-specific liberation of the active compound from the polymer carrier in order to maximize the efficacy of the conjugate. In recent years polymer drug conjugates were created for the delivery of the anticancer compound, 5-fluorouracil (5-FUI1(3-7). However, these conjugates permitted hydrolysis of the drug from the polymer carrier in the blood circulation and, therefore, reduced the sitespecific release of the drug in vivo. The attachment of drug molecules to polymer carriers limits the cellular entry of the drug to the process of endocytosis, and the endocytosed polymer-drug conjugate ultimately resides within the lysosomal compartment of the cell. Therefore, stabilization of the connection between the drug and polymer while in the bloodstream with a spacer that is hydrolyzed by lysosomal enzymes would greatly improve the targeting potential of polymer conjugates containing 5-FU. To this end, we have synthesized copolymers based on N-(2-hydroxypropyl)methacrylamide(HPMA) that contain oligopeptide side chains terminated with an a-substituted glycine derivative of 5-FU. The oligopeptide sequences are tailor-made to be stable in the

* Corresponding author. E-mail: [email protected]. utah.edu; telephone: (801) 581-7211; fax: (801) 581-7848. Abstract published in Advance ACS Abstracts, July 1,1995. Abbreviations: Amino acid abbreviations are those recommended by IUPAC-IUB; cf. (1972) J.Biol. Chem. 247,977. Bz, benzoyl; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; 5-FU, 5-fluorouracil; GSH, glutathione; HPMA, N-(2-hydroxypropyl)methacrylamide;MA, methacryloyl; NAP, p-nitroanilide; ONp, p-nitrophenoxy; P, HPMA copolymer backbone; P-Gly-Phe-ONp, copolymer of HPMA and N-methacryloylglycylphenylalanine p-nitrophenyl ester; P-Gly-Phe-Leu-GlyONp, copolymer of HPMA and N-methacryloylglycylphenylalanylleucylglycinep-nitrophenyl ester; PQ, primaquine; TMS, tetramethylsilane. @

bloodstream (81, susceptible to enzymes within the lysosomal compartment of the cell (91,and able to produce free 5-FU and not amino acid derivatives thereof. The release mechanism of 5-FU from the glycine a-carbon stems from the inherent instability of a-substituted glycines. Substitution at the a-carbon of glycine with a good leaving group, such as the secondary amine of 5-FU, results in an intrinsically unstable glycine derivative that spontaneously decomposes into the a-substituent and a glyoxylate according to Scheme 1 (20). However, acylation of the glycyl amino group, for example, through formation of a peptide bond, stabilizes the a-substituted glycine derivative. Therefore, addition of a stabilizing amino acid results in a stable a-substituted glycine drug derivative. Removal of the stabilizing amino acid, for example, through enzymatically catalyzed hydrolysis by aminopeptidases within the lysosomal compartment of the target cell, will create the unstable glycine drug derivative and subsequently result in the formation of the free drug. a-Substituted glycine derivatives of 5-fluorouracilwere first synthesized to study the potential of glycine derivatives for antimicrobial agent delivery (IO). More recently, a-substituted glycine derivatives of 5-fluorouracil were synthesized to study their potential as anticancer agent prodrugs (22). The purposes of this work were as follows: (1)to describe a new facilitated synthetic route for these a-substituted glycine derivatives of 5-fluorouracil, (2)to study the effects of the stereochemistry of the a-substituted glycine derivatives upon the enzymatically catalyzed release of 5-FU, (3) to study the effects of the hydrophobicity of the stabilizing amino acid upon the enzymatically catalyzed release of 5-FU from low molecular weight a-substituted glycine derivatives, and (4) to study the effect of side chain length and composition upon lysosomal enzyme catalyzed release of 5-FU from HPMAbased copolymers containing oligopeptide side chains terminated in the a-substituted glycine derivative of 5-FU.

1043-1802/95/2906-0483$09.00/00 1995 American Chemical Society

484 Bioconjugate Chem., Vol. 6,No. 4, 1995

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mmol), and 4 fi molecular sieves (8 g) were added with stirring. The mixture was allowed to reflux until the reaction was complete (approximately 2 h) as determined by TLC (mobile phase, ethyl acetate). The reaction apparatus was cooled to room temperature with stirring and filtered through Celite and the filtrate stirred with 20% aqueous citric acid (100 mL) for 10 min. The mixture was separated, and the organic layer was washed twice with 10% aqueous NaCl(75 mL each), dried W Unstable glycine derivative over MgS04, and reduced to a yellowish residue in vacuo. Pure product was obtained by recrystalization from ethyl acetatehexane. Yield: 1.91 g (54.3%); mp 127-129 "C; TLC (silica gel; ethyl acetate) showed one spot, Rf = 0.5. lH NMR (200 MHz, CDC13, ppm); 1.4 (overlapping d, 3H, diastereomeric alanyl CH,); 2.1 (s, 3H, acetyl CH3); 3.8 (s, 3H, methyl ester CH3); 4.3 (br m, l H , alanyl CH); 5.1 (8,l H , benzylic CH2); 6.4 (d, l H , glycyl CH); 7.35 (s, 5H, Stable glycine derivative aromatic H). Anal. Calcd for C16HzoNz07: C, 54.54; H, 5.68; N, 7.95. Found: C, 54.72; H, 5.77; N, 7.93. aAcylation of the glycyl a-amino group occupies the free amino electrons and stabilizes the bond between the glycine N-(Carbobenzyloxy)-~-alanyl-2-(5-fluorouracil1 a-carbon and the N1 of the 5-FU (modified from ref 10). yl)-L,D-glycine Methyl Ester (2). According to the method reported by Kingsbury (IO),acetate 1 (337.9 mg, EXPERIMENTAL PROCEDURES 0.96 mmol), 5-fluorouracil (119.7 mg, 0.92 mmol), and triethylamine (128.2 pL, 0.92 mmol) were stirred in dry Chemicals and General Methods. Melting points DMF (2 mL) for 20 h. The DMF was removed by (uncorrected) were determined on a n Electrothermal rotoevaporation in vacuo to a thick yellowish residue. The digital melting point apparatus. IH NMR spectra were residue was dissolved in ethyl acetate (20 mL) and recorded on a Bruker instrument (200 or 500 MHz) using extracted once with water (20 mL). The organic layer 1.0%v/v TMS as a n internal standard. U V spectra were was isolated and the water layer extracted with ethyl obtained either from a Perkin Elmer Lambda 19 specacetate (2 x 20 mL). The ethyl acetate extracts were trophotometer or a Perkin Elmer 7 spectrophotometer. combined and washed with water (2 x 20 mL), dried over TLC was performed using an aluminum backed silica gel MgS04, and evaporated in vacuo. The product was 250 pm layer from Whatman (Kent, England). HPLC isolated by silica gel chromatography (60-200 mesh) with was performed using a Dionex pumping system equipped a mobile phase of dichloromethane/methanol (98:2). with either an analytical Zorbax (4.6 x 150 mm) or Yield: 160 mg (40%);mp 113.5-114.5 "C (viscous liquid); a preparative Cl8 Whatman Partisil 10 ODs-3 column TLC (silica gel; dichloromethane/methanol/formicacid, with a UV Linear UVIS 204 detector and Axxiom data 9 5 5 1 ) showed one spot, R f = 0.25. 'H NMR (200 MHz, processing software. Polarimetry measurements were CDCl3, ppm): 1.4 (overlapping d, 3H, diastereomeric made on a Jasco DIP-370 digital polarimeter. General alanyl CH3); 3.75 (s, 3H, methyl ester CH3); 4.5 (9, l H , elemental analysis was performed by Atlantic Microlabs alanyl CH); 5.1 (s, 2H, benzyl CH2); 5.95 (m, l H , glycyl (Norcross, GA). Fluorine trace analysis was performed CHI; 7.3 (s, 5H, aromatic HI; 7.7 (overlapping d, l H , by Galbraith Laboratories (Knoxville,TN). CBZ-Ala-Serpyrimidine CHI. Anal. Calcd for C I E H I ~ N ~ O ~C, FI: OMe, porcine microsomal leucine aminopeptidase (EC 51.18; H, 4.50; N, 13.27. Found: C, 51.00; H, 4.57; N, 3.4.11.21, Leu-NAP, Bz-Phe-Val-Arg-NAP, reduced glu13.21. tathione (GSH), and Triton X-100 were purchased from N-(Carbobenzyloxy)-~-alanyl-2-(5-fluorouracil-lCBZ-Leu-Ser-OMe was Sigma (St. Louis, MO, USA). yl)-L,D-glycine(3). The methyl ester of 2 was removed purchased from Bachem Bioscience Inc. (Philadelphia, by treatment with sodium hydroxide. Compound 2 (54.2 PA, USA). Palladiudcharcoal, cyclohexene, lead tetmg, 0.128 mmol) was dissolved in the smallest amount raacetate, and 5-fluorouracil were purchased from Aldof methanol, to which 0.5 M NaOH (2.71 mL) was added, rich (Milwaukee, WI, USA). Dialysis tubing (Spectrapor, stirred for 1min, cooled in an ice bath, and then acidified molecular weight cutoE 6000-8000) and Gelman Acrowith stirring to pH = 2.0 with 2 N HCI. The methanol disc LC 13 PVDF filters were purchased from Baxter was removed by rotoevaporation in vacuo, the aqueous Scientific Products (Mcgaw Park, IL) All other chemicals solution was extracted with ethyl acetate (3 x 5 mL), the were of reagent grade or better. ethyl acetate extracts were combined and dried with N-(2-Hydroxypropyl)methacrylamide(HPh4.A)(121,MAsodium sulfate, and the ethyl acetate was removed by Gly-Phe-ONp (131, P-Gly-Phe-ONp (141, and P-Gly-Pherotoevaporation in vacuo. Yield: 40 mg (76%); mp 170 Leu-Gly-ONp (15)were synthesized as previously re"C (viscous liquid); TLC (silica gel; chlorofordmethanol, ported. Lysosomal enzymes were isolated in the form of 1:l) one spot, Rf = 0.5. lH NMR (200 MHz, DMSO-&, tritosomes according to the method of Trouet (16).The ppm): 1.3(overlapping d, 3H, diastereomeric alanyl CH3); term tritosomes is used to signify the use of Triton WR4.3 (br m, l H , alanyl CHI; 5.1 (s, 2H, benzyl CH2); 6.1 1339 to alter the density of the lysosomal compartment (d, l H , glycyl CH); 7.3 (s, 5H, aromatic HI; 7.8 (overlapand facilitate their isolation by centrifugation. The ping d, l H , pyrimidine CHI. program SCIENTIST was purchased from Micromath, Separation of L,L and L,D Diastereomers of 3 (3a Salt Lake City, UT. N-(Carbobenzyloxy)-~-alanyl-~,~-2-acetoxyglyand 3b). The diastereomers of 3 were separated by preparative reverse phase high pressure liquid chromacine Methyl Ester (1). Compound 1 was prepared Whatman Partisill0 ODS-3 column according to the procedure described by Steglich (17). Dry tography using a ethyl acetate (100 mL) was added to a dried 250 mL eluted with isocratic 0.1% acetic acid (pH 3.2Ymethanol three-neck round bottom flask through a rubber septum (7525) a t a rate of 2 m u m i n and detected a t 254 nm. using a glass syringe and needle under dry Nz. CBZFractions of 3 (30 mg each) were dissolved in water Ala-Ser-OMe (3.24 g, 10 mmol), Pb(OAcI4 (6.65 g, 15 containing the least amount of methanol (less than 2%) Scheme 1. The Stabilization of a Glycine Derivatized with 5-FU"

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Enantioselective 5-FU Release from Polymers

Bioconjugate Chem., Vol. 6,No. 4, 1995 485

0.9 (m, 6H, leucyl CHs); 1.4 (br m, 2H, leucyl CHZ);1.6 and injected into the HPLC system, and the peaks located (br m, l H , leucyl CHI; 3.7 (s, 3H, methyl ester CH3); 4.15 between 90-105 min and 106-140 min were collected. (q, l H , leucyl a-CHI; 5.0 (8, 2H, benzylic CHd; 6.4 (d, The acetic acid and methanol were removed in uacuo, and l H , glycyl CHI; 7.3 (s,5H, aromatic H); 8.0 (overlapping the products were obtained by lyophilization. d, l H , pyrimidine CHI. Anal. Calcd: C, 54.31; H, 5.38; First peak (Sa):Anal. Calcd for C17H17N407F*Hz0:C, N, 12.06. Found: C, 54.08; H, 5.48; N, 11.94. 47.88; H, 4.46; N, 13.14. Found: C, 48.07; H, 4.32; N, N-(Carbobenzyloxy)-~-leucyl-2-(5-fluorouracil113.08. Second peak (3b): Anal. Calcd for C17H17N4y1)-L,D-glycine(7). The synthetic intermediate, 7, was 07F-HzO: C, 47.88; H, 4.46; N, 13.14. Found: C, 47.62; formed by the removal of the methyl ester of 6 by H, 4.13; N, 12.95. All other analysis correlated to treatment with NaOH. The compound 6 (550 mg, 1.18 compound 3. "01) was dissolved in the smallest amount of methanol ~-Alanyl-2-(5-fluorouracil-l-yl)-~-glycine (4a)and (0.75 mL), to which 0.4 N NaOH (25.2 mL) was slowly ~-Alanyl-2-(5-fluorouracil-l-yl)-~-glycine (4b). The added with stirring. The solution was stirred for 1min, CBZ deprotection of Sa and 3b to produce 4a and 4b was cooled in a n ice bath, and brought to pH 2.0 with 5 N conducted according to the procedure described by KingsHC1 and the methanol removed by rotoevaporation. The bury (IO). Compounds Sa or 3b (71.4 mg, 0.175 mmol), cloudy reaction mixture was extracted with ethyl acetate palladiudcharcoal 10% (75 mg), and cyclohexene (0.125 (3 x 25 mL), whereupon the aqueous solution turned mL) were combined in anhydrous methanol (8.75 mL) clear. The ethyl acetate extracts were combined and and refluxed with stirring for 20 min. The reaction dried with anhydrous sodium sulfate, the solvent was mixture was filtered while hot through Celite, the filtrate removed by rotoevaporation, and the product was dried solvent removed by rotoevaporation, and the product in uacuo. Yield: 360 mg (67.8%). lN NMR (200 MHz, dried in uucuo. Yields: (Sa)42 mg (88%); (3b)44 mg DMSO-&, ppm): 0.85 (m, 6H, leucyl CHd; 1.4 (m, 2H, (92%). To ensure purity, the products were isolated using leucyl CHd; 1.6 (m, l H , leucyl CHI; 4.1 (br q, l H , leucyl preparative reverse phase HPLC. The samples were a-CHI; 5.0 (s, 2H, benzyl CHZ);6.25 (d, l H , glycyl CHI; dissolved in distilled water (0.75 mL) and isolated from 7.25 (s,5H, aromatic H); 8.0 (d, l H , pyrimidine CHI. The a Cl8 Whatman Partisil 10 ODs-3 column eluted with intermediate was used directly for the synthesis of 8. distilled water a t a rate of 2 m u m i n with detection at 254 nm, followed by lyophilization. ~-Leucyl-2-(S-fluo~uracil-l-yl)-qDglycine (8). The CBZ deprotection of 7 to produce 8 was conducted (4a)[aIz5~ = +136.18" (C = 0.073, HzO); UV (HzO)Am= according to the procedure described by Kingsbury (IO). = 268 nm, E = 7450. 'H NMR (200 MHz, DMSO-&, Compound 7 (360 mg, 0.8 mmol), palladidcharcoal 10% ppm): 1.25 (d, 3H, alanyl CH3); 3.95 (q, l H , alanyl CHI; (343 mg), and cyclohexene (0.57 mL) were combined in 5.9 (8,l H , glycyl CHI; 7.8 (d, l H , pyrimidine CHI. Anal. anhydrous methanol (40 mL) and refluxed with stirring Calcd for C ~ H I I N ~ O ~ F - HC, Z O36.98; : H, 4.45; N, 19.17. for 20 min. The reaction mixture was filtered while hot Found: C, 37.07; H, 4.48; N, 19.07. through Celite, the filtrate solvent removed by rotoevapo(4b)[aIz5~ = -117.68" (C = 0.064, HzO);W (HzO)I m a x ration, and the product dried in vacuo. Yield: 220 mg = 268 nm, E = 7450. IH NMR (200 MHz, DMSO-&, (87%). The diastereomers were purified directly from the ppm): 1.3 (d, 3H, alanyl CH3); 4.0 (q, l H , alanyl CHI; product and then analyzed. 5.9 (s, lH, glycyl CHI; 7.85 (d, l H , pyrimidine CHI. Anal. Separation of L,L and L,D Diastereomers of 8 (8a Calcd for CgHllN405F.HzO: C, 36.98; H, 4.45; N, 19.17. and 8b). The diastereomers of compound 8 were sepaFound: C, 37.04; H, 4.51; N, 19.07. rated by preparative reverse phase chromatography. N-(Carbobenzyloxy)-~-leucyl-~,~-2-acetoxyglyCompound 8 (50 mg) was dissolved in distilled water (1 cine Methyl Ester (5). Compound 5 was synthesized mL) and eluted from a preparative Cle Whatman Partisil according to the procedure described by Steglich ( I 7). The 10 ODs-3 column with a 0.1% acetic acid (pH = 3.2) procedure and chemical concentrations used were identimobile phase with detection a t 254 nm. The peaks cal to that described for compound (1). Yield: 2.888 g eluting from 80 to 100 min and from 150 to 180 min were (73%);mp 99.5-101.5 'C. TLC (silica gel; ethyl acetate) collected. showed one spot, Rf = 0.6. lNMR (200 MHz, CDCl3, (8a)The first peak collection weighed 10 mg following ppm): 0.95 (m, 6H, leucyl CH3); 1.6 (br m, 2H, leucyl lyophilization: [a125~ = +94.4" (c = 0.036, HzO); W CHZ); 1.7 (br m, leucyl CH); 2.1 (s, 3H, acetyl CH3); 3.8 (HzO) I,, = 270 nm, E = 7340. 'H NMR (500 MHz, (s, 3H, methyl ester CH3); 4.2 (br, l H , leucyl a-CHI; 5.1 DMSO-&, ppm): 0.95 (m, 6H, leucyl CH3); 1.5 (m, 2H, (s, 2H, benzylic CHZ); 6.4 (overlapping d, l H , glycyl CH); leucyl CHZ);1.65 (m, l H , leucyl CHI; 3.6 (m, l H , leucyl 7.35 (s,5H, aromatic HI. Anal. Calcd: C, 57.87; H, 6.59; a-CHI; 5.8 (s, lH, glycyl CHI; 7.8 (d, l H , pyrimidine CHI, N, 7.11. Found: C, 57.97; H, 6.64; N, 7.11. mlz = 316. N-(Carbobenzyloxy)-~-leucyl-2-(5-fluorouracil1(8b)The second peak collection weighed 25 mg followyl)-L,D-glycine Methyl Ester (6). According to the ing lyophilization: [aIz5~ = -70.6' (c = 0.034, HzO);W method reported by Kingsbury (IO), acetate 5 (1.514 g, (HzO) Imax= 270 nm, E = 7560. IH NMR (500 MHz, 3.84 mmol), 5-FU (0.478 g, 3.68 mmol), and triethylamine DMSO-&, ppm): 0.95 (m, 6H, leucyl CH3); 1.4 (m, 2H, (512.9 pL, 3.68 mmol) were stirred in dry DMF (8 mL) leucyl CHZ);1.6 (m, l H , leucyl CHI; 3.6 (m, l H , leucyl for 20 h. The DMF was removed by rotoevaporation in a-CHI; 5.8 (8, l H , glycyl CHI; 7.8 (d, l H , pyrimidine CHI, uacuo to a thick yellowish residue. The residue was mlz = 316. dissolved in ethyl acetate (80 mL) and extracted once Synthesis of P-Gly-Phe-Ala-Gly-a(S-FU) (9). Cowith water (80 mL). The organic layer was isolated and polymer 9 was synthesized from the polymer precursor the water layer extracted with ethyl acetate (2 x 80 mL). P-Gly-Phe-ONp (4.1 mol % ONp, 0.27 mmol of ONplg of The ethyl acetate extracts were combined and washed with water (2 x 40 mL), dried over MgS04, and evapopolymer) and the diastereomeric mixture of 4. The polymer precursor (210 mg, 0.0567 mmol ONp), 19.43 mg rated in uucuo. The pure product was isolated by silica of 4 (0.079 mmol), and 9.02 pL of ethylmorpholine (0.079 gel chromatography (60-200 mesh) with a mobile phase mmol) were combined in 0.8 mL of anhydrous DMSO and of dichloromethanelmethanol (98:2). Yield: 550 mg stirred for 24 h. Aminopropanol (25 pL) was added to (30.8%); mp 103.5-105.5 "C (viscous liquid); TLC (silica hydrolyze any unreacted ONp groups and the polymer gel; dichloromethandmethanoYfonnicacid, 95:5:1) showed immediately precipitated into a n excess of acetone. The one spot, Rf = 0.4. lH NMR (200 MHz, DMSO-&, ppm):

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polymer was isolated by filtration, thoroughly dried, Synthesis of P-Gly-Phe-Leu-Gly-Leu-Gly-a(L,L)dissolved in distilled water, and dialyzed (Spectrapor, (5-FU) (12a). Copolymer 1221was synthesized from the molecular weight cutoff: 6000-8000) against distilled polymer precursor P-Gly-Phe-Leu-Gly;ONp (5.7 mol % water for 3 days. The 5-FU-containing polymer was GNp, 0.34 mmol of ONp/g of polymer, M , = 16 800, M,/ isolated by lyophilization and the 5-FU content quantiM,, = 1.51) and 8a. The polymer precursor (126 mg, tated by UV spectroscopy and elemental analysis. UV: 0.043 mmol of ONp), 20 mg of 8a (0.0532 mmol), and 1.97 mol % of 5-FU containing comonomer (using E = 13.52 pL of ethylmorpholine (0.106 mmol, note: 2 equiv 7450) which corresponds to 7642 g of polymer/mol of due to salt form of 8a) were combined in 0.4 mL of 5-FU. Anal. Calcd: F, 0.248. Found: F, 0.27. anhydrous DMSO and stirred for 24 h. Aminopropanol Synthesis of P-Gly-Phe-Leu-Gly-Ala-Gly-a(5-FU)(25 pL) was added to hydrolyze unreacted ONp groups and the polymer immediately precipitated into an excess (10). Copolymer 10 was synthesized from the polymer of acetone. The polymer was isolated by filtration, precursor P-Gly-Phe-Leu-Gly:ONp (4.8 mol% ONp, 0.29 dissolved in distilled water, and dialyzed (Spectrapor, mmol of ONp/g of polymer, M , = 20 000, Mw/i@,,= 1.3) molecular weight cutoff: 6000-8000) against distilled and the diastereomeric mixture of 4. The polymer water for 3 days. The 5-FU-containing polymer was precursor (121 mg, 0.0351 mmol of ONp), 14.4 mg of 4 isolated by lyophilization and the 5-FU content quanti(0.0526 mmol), and 6.7 pL of ethylmorpholine (0.0526 tated by UV spectroscopy and elemental analysis. UV: mmol) were combined in 0.6 mL of anhydrous DMF and 1.25 mol % of 5-FU containing comonomer ( E = 7340) stirred for 24 h. Aminopropanol (25 pL) was added to which corresponds to 11841 g of polymer/mol of 5-FU. hydrolyze any unreacted ONp groups and the polymer Anal. Calcd: F, 0.16. Found: F, 0.18. immediately precipitated into an excess of acetone. The Enzyme Activity Assay. Enzyme activities were polymer was isolated by filtration, thorougly dried, determined spectrophotometrically and are expressed in dissolved in distilled water, and dialyzed (Spectrapor, terms of units. One unit of enzyme will hydrolyze 1pmol molecular weight cutoff: 6000-8000) against distilled of NAp/min from its substrate (either Leu-NAp for water for 3 days. The 5-FU-containing polymer was aminopeptidase activity, or Bz-Phe-Val-Arg-NApfor enisolated by lyophilization, and the 5-FU content was dopeptidase activity). quantitated by UV spectroscopy and elemental analysis. UV: 2.95 mol % of 5-FU containing comonomer (using E The endopeptidase activity of tritosome preparations = 7450) which corresponds to 5417 g of polymer/mol of was determined by following the liberation of NAP 5-FU. Anal. Calcd: F, 0.35. Found: F, 0.32. spectrophotometrically a t 410 nm ( E = 8600 M-I) (19) from the substrate Bz-Phe-Val-Arg-NAp in a 1 mL Synthesis of P-Gly-Phe-Leu-Gly-a(5-FU) (11). Cosample containing 0.84 mL of citrate/phosphate buffer polymer 11 was synthesized from the polymer precursor (citric acid 21.6 mM, NaZHP04 56.8 mM, EDTA 0.784 P-Gly-Phe-ONp (4.1 mol % ONp, 0.27 mmol of ONp/g of mM, pH 5.5), 0.020 mL of Triton X-100 solution (10% in polymer) and a diastereomeric mixture of 8. The polymer buffer), 0.020 mL of glutathione solution (0.25 M in precursor (200 mg, 0.0532 mmol of ONp), 21.01 mg of 8 buffer), 0.020 mL of Bz-Phe-Val-Arg-NAp solution (11.2 (0.0665 mmol), and 8.5 pL of ethylmorpholine (0.0665 mM in DMSO), and 0.1 mL of tritosome preparation. mmol) were combined in 0.6 mL of anhydrous DMSO and The aminopeptidase activity of tritosome preparations stirred for 24 h. Aminopropanol (25 pL) was added to was determined by following the liberation of NAp hydrolyze any unreacted ONp groups and the polymer spectrophotometrically a t 410 nm from the substrate Leuimmediately precipitated into a n excess of acetone. The NAP in a 1 mL sample containing 0.827 mL of citrate/ polymer was isolated by filtration, thoroughly dried, phosphate buffer, 0.020 mL of Triton X-100 solution (10% dissolved in distilled water, and dialyzed (Spectrapor, in buffer), 0.020 mL of glutathione solution (0.25 M in molecular weight cutoff: 6000-8000) against distilled buffer), 0.033 mL of Leu-NAp (24 mM in DMSO), and water for 3 days. The 5-FU-containing polymer was 0.1 mL of tritosome preparation. isolated by lyophilization and the 5-FU content quantitated by UV spectroscopy and elemental analysis. UV: The aminopeptidase activity of leucine aminopeptidase 1.59 mol % of 5-FU-containing comonomer (using E = was determined by following the liberation of NAp average of L,L and L,D diastereomer = 7450) which spectrophotometrically a t 405 nm ( E = 9800 M-I) (19) corresponds to 9432 g of polymer/mol of 5-FU. Anal. from the substrate Leu-NAp in a 1mL sample containing Calcd: F, 0.20. Found: F, 0.27. 0.867 mL of phosphate buffer (KHzP04 50 mM, pH 7.2), Synthesis of P-Gly-Phe-Leu-Gly-Leu-Gly-a(5-FU) 0.033 mL of Leu-NAp (24 mM in DMSO), and 0.1 mL of leucine aminopeptidase stock solution in buffer. (12). Copolymer 12 was synthesized from the polymer HPLC Analysis. 5-FU, 4a, 4b, 8a, and 8b were precursor P-Gly-Phe-Leu-Gly:ONp (4.8 mol-% QNp, 0.29 analyzed by HPLC using a Zorbax C18 (4.6 x 150 mm) mmol of ONp/g of polymer, M , = 20 000, M,/M, = 1.3) analytical column with mobile phase: 0.1% acetic acid, and a diastereomeric mixture of 8. The polymer precurflow rate: 0.5 mL/min, UV detection: 254 nm, and sor (171 mg, 0.0496 mmol of ONp), 20.37 mg of 8 (0.0645 injection volume: 25 pL. All samples were filtered mmol), and 8.2 pL of ethylmorpholine (0.0645 mmol) were through a 0.2-pm filter (Gelman Acrodisc LC 13 PVDF) combined in 0.5 mL of anhydrous DMSO and stirred for prior to HPLC analysis. Quantitation was conducted 24 h. Aminopropanol(25 pL) was added to hydrolyze any according to standard curves of the compounds. unreacted ONp groups and the polymer immediately precipitated into a n excess of acetone. The polymer was Enzymatically Catalyzed Release of 5-FUfrom 4a isolated by filtration, thoroughly dried, dissolved in and 8a. Enzymatically Catalyzed Release by Leucine distilled water, and dialyzed (Spectrapor, molecular Aminopeptidase. Enzymatic hydrolysis of 4a and 8a for weight cutoff: 6000-8000) against distilled water for 3 the determination of Michaelis-Menten parameters was days. The 5-FU-containing polymer was isolated by conducted in 1mL volumes with phosphate buffer (KH2lyophilization and the 5-FU content quantitated by UV PO4 50 mM, pH = 7.2) a t 37 "C. Stock solutions of 4a spectroscopy and elemental analysis. UV: 2.74 mol % (2.68 x M) and 8a (2.79 x M) were prepared of 5-FU containing comonomer (using E = average of L,L in the buffer, aliquots of which were used to produce and L,D diastereomer = 7450) which corresponds to 5841 M. concentrations ranging from 1 x 10-5 to 1 x g of polymer/mol of 5-FU. Anal. Calcd: F, 0.325. Leucine aminopeptidase (6.02 x unit) was added Found: F, 0.32. from a stock solution. The reaction was halted by the

Bioconjugate Chert., Vol. 6,No. 4, 1995 487

Enantioselective5-FU Release from Polymers

addition of concentrated HC1. 5-FU formation was determined by HPLC. In all samples the percent of cleavage of the starting material was limited to below 10%. All samples were run in triplicate, and MichaelisMenten parameters (Vmmand KM)were determined using Lineweaver-Burk plot analysis. Enzymatically Catalyzed Release by Tritosomes. Enzymatic hydrolysis of 8a for the determination of Michaelis-Menten parameters was conducted in l mL volumes with citratehodium phosphate buffer (citric acid 21.6 mM, NazHP04 56.8 mM, EDTA 0.784 mM, pH 5.5) containing 0.020 mL of Triton X-100 solution (10% in buffer) and 0.02 mL of glutathione solution (0.25 M in buffer) a t 37 "C. A stock solution of 8a (2.02 x M) was prepared in the buffer, aliquots of which were used to produce the concentrations ranging from 5.05 x to 1.01 x M. Tritosomes (0.1 mL), corresponding to 1.28 x unit of tritosome aminopeptidase activity, were added. The reaction was halted by immediate sample freezing. Samples were thawed immediately prior to analysis of 5-FU formation as determined by HPLC. In all samples the percent of cleavage of the starting material was limited to below 10%. All samples were run in triplicate and Michaelis-Menten parameters (V,,, and K M )were determined using Lineweaver-Burk plot analysis. The lysosomal enzyme catalyzed release of 5-FU from 8a over 24 h was determined by incubating 1mL samples containing 0.8 pmol of Sa (0.16 mL of a 5 x M stock solution), 0.7 mL of citratehodium phosphate buffer (citric acid 21.6 mM, NazHP04 56.8 mM, EDTA 0.784 mM, pH 5.5), 0.02 mL of Triton X-100 (10% in buffer), 0.02 mL of glutathione (0.25 M in buffer), and 0.1 mL of tritosome preparation (2.53 x unit of aminopeptidase activity). Triplicate samples were taken a t 1,6,12, and 24 h, frozen immediately, and then thawed immediately prior to analysis by HPLC. Enzymatically Catalyzed Release of 5-FU from Polymers. Percent Release Profiles. The percentage of 5-FU and 5-FU derivatives released from polymers 9- 12 after 24 h was determined by incubating the quantity of each polymer to equal 1pmol of 5-FU in each sample, i.e., polymers 9, 7.6 mg; 10, 5.4 mg; 11, 9.4 mg; 12, 5.8 mg, in 1 mL volumes containing citrate/sodium phosphate buffer (citric acid 21.6 mM, NazHP04 56.8 mM, EDTA 0.784 mM, pH 5.5),0.02 mL of Triton X-100 (10% in buffer), 0.02 mL of glutathione (0.25 M in buffer), and 0.1 mL of tritosome preparation (1.28 x unit of aminopeptidase activity, 1.53 x unit of endopeptidase activity). The samples were incubated in a shaking water bath a t 37 "C. Following 24 h, each sample was eluted with 1 mL fractions of water through a PD-10 column to separate the high and low molecular weight components. Fractions 7-15 containing the low molecular weight compounds were isolated, lyophilized, and reconstituted to a volume of 2 mL with distilled water. The reconstituted fractions were analyzed by HPLC. Time Release Profiles. The time release profiles of 5-FU and 8a and 8b from polymers 12 and 12a were determined by incubating the quantity of each polymer to equal 0.35 and 0.3 pmol of 5-FU respectively, Le., polymer 12, 2.04 mg, and polymer 12a, 3.55 mg, in 0.25 mL volumes containing citratehodium phosphate buffer (citric acid 21.6 mM, Na2HP04 56.8 mM, EDTA 0.784 mM, pH 5.5), 0.08 mL of Triton X-100 (2.5% in buffer), 0.08 mL of glutathione (0.0625 M in buffer), and 0.025 unit of amimL of tritosome preparation (6.33 x unit of endopeptidase nopeptidase activity, 1.44 x activity). Samples were taken a t 1, 6, 12, and 24 h and immediately frozen. Each sample was thawed immedi-

Table 1. Quick Reference for Compound Abbreviation Identification compd no.

compound n a m e

1

N-(carbobenzyloxy)-~-alanyl-~,~-2-acetoxyglycine methyl ester 2 N-(carbobenzyloxy)-~-alanyl-2-(5-fluorouraci11-y1)-L,D-glycine methyl ester 3a N-(carbobenzyloxy)-~-alanyl-2-(5-fluorouracill-yl)-~-glycine 3 b N-(carbobenzyloxy)-~-alanyl-2-(5-fluorouracill-yl)-D-glycine 4a ~-alanyl-2-(5-fluorouracil-l-yl)-~-glycine 4 b L-alanyl-2-(5-flUOrOUraCil-l-yl)-D-glyCine 5 N-(carbobenzyloxy)-~-leucyl-~,~-2-acetoxyglycine methyl ester 6 N-(carbobenzyloxy)-L-leucyl-2-(5-fluorouracil-l-yl)-L,D-glycine methyl ester N-(carbobenzyloxy)-~-leucyl-2-(5-fluorouracill-yl)-L,D-glycine 7 8a ~-leucyl-2-(5-fluorouracil-l-yl)-~-glycine

8b L-leUCyl-2-(5-flUOrOUraCil-l-yl)-D-glyCine 9 P-Gly-Phe-Ala-Gly-a(5-FU) 10 11 12 12a

P-Gly-Phe-Leu-Gly-Ala-Gly-a(5-FU) P-Gly-Phe-Leu-Gly-a(5-FU)

P-Gly-Phe-Leu-Gly-Leu-Gly-a(5-FU) P-Gly-Phe-Leu-Gly-Leu-Gly-a(~,~)(5-FU)

ately prior to elution with 1 mL water fractions from a PD-10 column. Fractions 7-15 which contained the low molecular weight components were collected for each sample, lyophilized, reconstituted to a volume of 2 mL, and analyzed by HPLC. All samples were run in triplicate. Kinetic Mathematical Modeling. The data resulting from the degradation of polymer 12a in the presence of lysosomal enzymes were modeled to estimate the kinetic rate constants associated with the formation of 8a and 5-FU. The release process can be modeled according to an irreversible degradation pattern of A goes to B goes to C assuming pseudo first order kinetics. This mathematical model can be described by the following set of differential equations: kl

A-B-C

kz

d[Al dt

-= -k,[A]

-d[B1 - K,[Al- K,[Bl

dt

-d[C1 - k,[Bl dt

The data obtained for the enzymatically catalyzed release of 8a and 5-FU from polymer 12a (Figure 4) were used to estimate the values of kl and kz. The analytical solutions to the differential equations were used in the program SCIENTIST, and the data were fit by the method of least squares. RESULTS AND DISCUSSION

Synthesis. A quick reference for the compound abbreviations used in this text is given in Table 1. Previous investigators of a-substituted glycine derivatives of 5-FU have utilized the formation of a hydroxyglycine amino acid derivative as a precursor to acylated glycine intermediates such as 1and 5. The new synthetic route described herein bypasses this intermediate through the direct conversion of serine residues to the acylated glycine intermediate. Comparison of the two synthetic routes is shown in Scheme 2.

Putnam and KopeEek

488 Bioconjugafe Chem., Vol. 6,No. 4, 1995

Scheme 2. Comparison of Old (A) and New (B) Methods of Synthesizing a-Substituted Glycine Derivatives"

PROTECTING GROUP

O

+

H

f 9H?HR HO-C-CH-CH-C- Ot

I

I I ? ?

H

PROT-O-C-CH-CH-C-+

R

P

R

CB+ NH-CH-C-

I

PH

P f FH2 17 +CH3 CBZ-NH-CH-GNH-CH-C-

I

PROT

AR+ C PROT

H-C-C-

Bl

Pf

P R CBZ- NH- CH- GNH

PHf

NH-CH-C- W P R O T

Pb(0AC)d

R

(>-C-CH3

CBZ-NH-CH-C-NH-CH-C-

I f

O-CH3

j J5F O

H

if

PGCH3

P R

I f

CBZ- NH- CH-C+NH-CH-C-*

I

a

PROT

0

The new method provides a much faster synthetic pathway.

An excellent discussion of the pitfalls associated with the earlier synthetic route is given in ref 11. The principle drawbacks of the earlier synthetic route are long reaction times, modification of the 5-FU chemical structure, and the formation of diketopiperazines by internal cyclization during CBZ deprotection using cyclohexene as a hydrogen-transfer agent. To minimize the formation of these side products, the authors used 1,4-cyclohexadiene as the hydrogen-transfer agent during CBZ deprotection and also formed a salt at the a-amine through the addition of methanolic acetic acid. However, using the new synthetic route, none of the described side products were detected even though cyclohexene was used as the hydrogen-transfer agent. This may be due to the removal of the methyl ester carboxyl protecting group prior to CBZ deprotection. Dipeptides containing amino groups and esterified carboxyl groups are known to undergo diketopiperazine formation (28); therefore, removal of the carboxyl group prior to CBZ deprotection dramatically decreases the potential of diketopiperazine formation.

The fast removal of the methyl ester protecting group by 0.4 N NaOH a t room temperature was unexpected. Methyl ester removal from amino acids using a similar NaOH concentration usually requires 2 h of reaction time (20). The removal of the methyl ester from the 5-FUcontaining dipeptides may be due to a n intramolecular catalysis mechanism induced by the 5-FU (21). This short time of contact with NaOH is fortuitous since 5-FU may be degraded following prolonged exposure to basic medium (22). Removal of the methyl ester also increased the aqueous stability of the 5-FU-containing dipeptides (data not shown). The formation of free 5-FU due to chemical hydrolysis was greatly increased in the methyl ester-containing compound relative to the free carboxyl compound. The synthetic route used for the a-substituted glycine derivatives of 5-FU produced an asymmetric carbon a t the 5-FU attachment carbon, resulting in diastereomers of compounds 4 and 8 designated as 4a (L,L), 4b (L,D), 8a (L,L), and 8b (L,D) as assigned by their optical rotations and susceptibility to enzymatic hydrolysis. The isolation

Bioconjugate Chem., Vol. 6, No. 4, 1995 489

Enantioselective5-FU Release from Polymers OS

1

0-41

1.25

f

1.00

-

0.3

0

10 time (h)

20

Figure 1. Release of 5-FU from 8a by the aminopeptidase activity of tritosomes. Open triangle: 5-FU; closed triangle: 4a, 4b, 8b, and controls. Table 2. Michaelis-Menten Kinetic Parameters for the Cleavage of 4a and 8a with Tritosome Preparations and Leucine Aminopeptidase compd

8a 4a 8a

(mWmin) K, (mM) kcat (min-’) Tritosomesa 3.08 x f 8.75 x 1.05 f 3.45 x lo-’ 0.000294 Leucine Aminopeptidase* 9.66 x loT2 f 4.2 x 3.63 f 1.66 0.026 4.61 x f 2.0 x 3.64 f 1.81 0.013

0

’

time (h)

20

Figure 3. Incubation of polymer 12 with tritosomes over 24 h. Open circle: 8b; closed square: 8a; open triangle: 5-FU; closed circle: amount of 5-FU left on polymer (as calculated from released components). 1.25 +

V,

1.00

4

a Substrates 4a, 4b, and 8b were not cleaved. * Substrates 4b and 8b were not cleaved.

1

loo

a

’

time (h) 2 o

Figure 4. Incubation of polymer 12a with tritosomes over 24 h. Closed square: 8a; open triangle: 5-FU; closed circle: amount of 5-FU left on polymer (as calculated from released components).

al 404

i 201 0

0

4 controls 9 (5-FU)

9 (4) 11 (5-FU) 11 (8)

b loo] 80

Figure 2. (a)Incubation of polymers 9 and 11 with tritosomes for 24 h. This graph shows the inability of the tritosomes to cleave the tetrapeptide side chain sequence. In parentheses are the released components from the polymers. (b) Incubation of polymers 10 and 12 with tritosomes for 24 h. This bar graph shows the ability of tritosomes to cleave the hexapeptide side chain sequence, and also the inability of tritosomes to remove the Ala stabilizing amino acid. In parentheses are the released components from the polymers.

of 4a and 4b was accomplished by separation using preparative HPLC of compound 3 into 3a and 3b followed by CBZ deprotection. The separation of 8 into 8a and 8b was performed by direct preparative HPLC of compound 8. Enzymatically Catalyzed Release of 6-FU from Low Molecular Weight a-Substituted Glycine Derivatives. The enzymatically catalyzed release of 5-FU from compounds 4a, 4b, 8a, and 8b was studied using two enzyme preparations: (1) leucine aminopeptidase,

isolated from porcine kidney microsomes; and (2) lysosomal enzymes isolated in the form of tritosomes from rat liver. Leucine aminopeptidase, a n enzyme that the polymer-bound 5-FU should not encounter in vivo, was used in order to study the feasibility of a-substituted glycine derivatives of 5-FU as potential prodrug compounds. Tritosomes, which contain a variety of hydrolytic enzymes, were used to mimic the enzyme population that the polymer-bound 5-FU glycine derivatives would encounter in vivo. Initial experiments using diastereomeric mixtures of 4 and 8 showed that leucine aminopeptidase was able to catalyze the production of 5-FU from both 4 and 8. However, subsequent experiments with diastereomeric mixtures of 4 and 8 with tritosome preparations showed that only 8 was enzymatically converted to free 5-FU. The active site of leucine aminopeptidase preferentially binds compounds with hydrophobic residues in the SI’ position, which may account for these results (23). The tritosome preparation results also correspond well with recently published data in this field (24). In this reference the kinetic rate constants for the cleavage of Ala and Leu from peptide derivatives of the antileishmania1 drug, primaquine (PQ), by lysosomal aminopeptidases were calculated. Their results indicate that lysosomal aminopeptidases have higher specificity for Leu-containing compounds than for Ala-containing compounds. However, in their experiments the Ala-containing compound was cleaved by the lysosomal aminopeptidase activity, whereas in our experiments, the Ala was not cleaved. These results can be explained in that the active site of the lysosomal aminopeptidase must accommodate the drug compound (25). In the case of the experiments outlined in ref 24, Ala was the second amino acid from PQ and Leu was directly attached to the drug,

490 Bioconjugate Chem., Vol. 6,No. 4, 1995

Putnam and KoDeEek

Scheme 3. Illustration of the Stages of Release for Glycine Derivatives of 5-FU and the Chemical Structure of the Illustrated Copolymer

t

Gly-Phe-Leu-Gly-Leu-Gly-(5-FU)

1

(endopeptidase)

Gly-Phe-Leu-Gly

+

Leu-Gly(5-FU)

I

(aminopeptidase)

t

Leu + [Gly-(5-FU)] Where

5

is the HPMA copolymer backbone.

The copolymer structure is shown below.

CH 3

t:

-CH2

a

c=o I

YH CH - C H 2-H

0CH 3 'CH 3

I

C=O I

NH I

y

2

c=o I

NH

I

OcH 'CH 3

CH - C H 2-H

I ?=O

1:-Q0 C=O I OH

F

whereas in our experiments, the Ala and Leu are both attached directly to the glycine 5-FU derivative. Therefore, it is feasible that in our case, 5-FU negatively influences the interactions between the aminopeptidase and the dipeptide that only the Leu-containing dipeptide can overcome. Literature regarding the subsite interactions of the principle lysosomal aminopeptidase, cathepsin H, supports this explanation. The S1 subsite of cathepsin H is a large hydrophobic pocket that requires a large amino acid side chain residue to achieve optimal interaction (26). Comparison of the KMvalues for cathepsin H toward amino acyl-P-naphthylamide substrates shows a 10-fold increase in KM for the alanine-containing substrate (1.0 mM) compared to the leucine-containing substrate (0.093 mM) (27). This substantial increase in KMdemonstrates the influence of the hydrophobicity and/ or amino acid side chain size of the P1 residue occupying the S1 subsite. In addition, the cleavage of kemptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly, by cathepsin H proceeds through the first three amino acids and ceases a t the Ala residue, further exemplifying the poor aminopeptidase activity of cathepsin H toward the Ala residue (27).

A salient point regarding the effect of stereochemistry upon the enzymatically catalyzed release of 5-FU is that only for the L,L configuration can the stabilizing amino acid be cleaved. This is not a suprising result considering that L is the configuration of amino acids in the body and that enzymes can be very stereoselective within their active sites. For this reason, the enzymatically catalyzed release of 5-FU from these a-substituted glycine derivatives is termed enantioselective since only one stereochemical configuration is able to undergo catalysis. These 5-FU derivatives provide an excellent example for the importance of stereochemically pure compounds for use in biological systems. Although each configuration contains the same chemical species, the spatial arrangement of those species is vitally important to the bioactivity of the compounds. The liberation of 5-FU via tritosomes from 8a over 24 h compared to that from 4a, 4b, and 8b is shown in Figure 1. The kinetic constants derived for compounds 4a and 8a are summarized in Table 2. Enzymatically Catalyzed Release of 5-FU from HPMA-Based Copolymers Containing a-Substituted Glycine Derivatives of 5-FU. The percent release of 5-FU and 5-FU derivatives over 24 h from polymers 9-12 was conducted to screen for those polymers that warranted more intensive investigation. The results shown in Figure 2 show that both of the tetrapeptide side chain-containing polymers, 9 and 11, did not release 5-FU or 5-FU derivatives in the presence of tritosomes whereas both the hexapeptide side chain-containing polymers, 10 and 12, released 5-FU and/or 5-FU derivatives. Previous work by our group and collaborators (for example, ref 29) focused on the use of tetrapeptide spacers for the release of the anticancer compound, adriamycin. The HPhlA-based copolymers in those studies contained side chains consisting of Gly-Phe-Leu-Gly with the adriamycin attached via an amide bond to the carboxyl end of the glycine. The side chains of these copolymers were designed to optimally interact with the subsites of the lysosomal enzyme, cathepsin B. At the time these copolymers were created, the X-ray structure of cathepsin B was not known, and the specificity of the enzyme was determined by traditional mapping of the enzyme's active site (9, 30).One of the results of these studies was that the Pz residue on the copolymer side chain should be hydrophobic. Leu in this position adequately fulfills this requirement. The X-ray structure of cathepsin B suggests that the PZside chain interacts with Ala200 and Ala173 of the active site cleft floor (31). In addition, the side chain of the PI residue must project out of the active site cleft and electrostatically interact with Glu 122. This requirement is avoided with the use of Gly in this position since the synthesis of copolymers containing ionizable side chains is difficult. These copolymers freely released adriamycin in the presence of tritosomes. However, the copolymers 9 and 11containing tetrapeptide side chains did not release 5-FU. These results can be explained with respect to the interactions of the copolymer side chain residues with the active site subsites in cathepsin B. The 5-FU-containing tetrapeptides in copolymers 9 and 11 are terminated in an a-substituted glycine 5-FU derivative and not with a glycyl derivative containing the drug bound via an amide bond a t the carboxyl end. In order to accommodate this substitution into the enzyme active site, the enzyme subsites must align closer to the polymer backbone which is energetically unfavorable. Additionally, comparing the release products from polymers 10 and 12, polymer 10 produced only 5-FU derivatives, corresponding to compound 4, while polymer

Enantioselective 5-FU Release from Polymers

12 produced 5-FU derivatives, corresponding to compound 8, along with free 5-FU. The formation of free 5-FU from polymer 12 and not from polymer 10 corresponds well to the low molecular weight results discussed previously. The hexapeptide sequences of these polymers optimize the cathepsin B catalyzed release of the dipeptide derivatives 4 and 8 by providing additional substrate binding sites. If subsite Sz is occupied by the Leu residue closest to the polymer backbone, then the SI’position is occupied by a readily accepted Ala (4) or Leu ( 8 )residue. The Si subsite contains two binding functionalities. The side chains of His199, Ala176, Phel80, and Leu 181 and the benzene part of Trp221 form a shallow hydrophobic pocket for the P i residue side chain. In addition, the P i residue carboxylate group would be electrostatically attracted to the exposed imidazole ring of His111 to help fixate the substrate in the active site (31). Since the N1 position of 5-FU must be free in order for the 5-FU to be activated in vivo (321, from these results it was concluded that polymer 12 was the best candidate for detailed investigation. The time release profile of the enzymatically catalyzed release of 5-FU and 8a and 8b from polymer 12 via tritosomes is shown in Figure 3. The results show the formation of 8b increasing over time while the formation of 8a increases over the first hour and then decreases upon conversion to 5-FU presumably via the tritosome aminopeptidase activity. The conversion of 8a to 5-FU corresponds well t o the results obtained from the incubation of 8a alone with tritosomes (Figure 1)if one considers a nonproductive association of 8b with the aminopeptidases in the tritosomes. The logical extension of the results obtained from polymer 12 was to synthesize polymer 12a containing only the (L,L) configuration of the 5-FU glycine derivative. The time release profile results are shown in Figure 4. Again, the conversion of 8a to 5-FU correlates well to the results obtained with tritosomes with 8a alone. Since the formation of 5-FU from 12a requires two stages resulting from tritosome endopeptidase activity (rate constant, k J followed by tritosome aminopeptidase (rate constant, Kz) activity as shown in Scheme 3, a rate limiting step may be functional. Modeling the data shown in Figure 4 resulted in a good estimate of the rate constants for each step. The modeling gave values for k l = 1.24 h-I and kz = 0.025 h-l. Therefore, the rate limiting step for the release of 5-FU from polymer 12a can be expected to be the aminopeptidase catalyzed conversion of 8a to 5-FU. In conclusion, a new synthetic route to a-substituted glycine derivatives of the anticancer compound 5-FU was described. The degradation of the low molecular weight derivatives by lysosomal enzyme preparations is dependent upon the hydrophobicity andlor amino acid side chain size of the stabilizing amino acid, and upon the stereochemistry of the a-carbon of the derivatized glycine. Covalent attachment of these 5-FU derivatives to water soluble polymers containing oligopeptide side chains of varying lengths results in high molecular weight polymer conjugates stable to chemical hydrolysis. The degradation of these conjugates by lysosomal enzyme preparations directly depends upon the length and composition of the oligopeptide side chains. ACKNOWLEDGMENT

The authors would like to thank Drs. Ramesh Rathi and Pavla KopeEkovA and Mr. Jane-Guo Shiah for their kind assistance with this work. This research was supported in part by NIH Grants CA51578 and GM08393,

Bioconjugate Chem., Vol. 6, No. 4, 1995 491

and by an Advanced Predoctoral Fellowship from the Pharmaceutical Manufacturers Association. LITERATURE CITED

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