Synthesis and Cytotoxicity of Salicylate-Based Poly(anhydride esters

Biomacromolecules 2005, 6, 359-367

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Synthesis and Cytotoxicity of Salicylate-Based Poly(anhydride esters) Robert C. Schmeltzer, Kristine E. Schmalenberg,† and Kathryn E. Uhrich* Department of Chemistry and Chemical Biology, Rutgers University, 610 Taylor Road, Piscataway, New Jersey 08854 Received August 6, 2004; Revised Manuscript Received September 21, 2004

This paper describes the synthesis and cytotoxicity of poly(anhydride esters) that are composed of several salicylate derivatives, including halogenated salicylates, aminosalicylates, salicylsalicylic acid, and thiolsalicylic acid. The incorporation of these nonsteroidal antiinflammatory drugs (NSAIDs) into a biodegradable polymer backbone yields drug-based polymers that have potential for a variety of applications. The poly(anhydride esters) were synthesized by melt condensation polymerization. The halogenated salicylate derivatives yielded the highest molecular polymers as well as the highest glass transition temperatures. All polymers displayed in vitro degradation lag times from 1 to 3 days, depending on the water solubility of the salicylate derivative. Cell viability and proliferation were determined with L929 fibroblast cells in serumcontaining medium to assess the polymer cytotoxicities, which varied as a function of the saliyclate chemistry. Cell morphology was normal for most of the polymers evaluated. Introduction Polyanhydrides have been investigated as a biomaterial for drug release and tissue engineering applications for more than two decades.1-3 Langer was the first to exploit the hydrolytically unstable polyanhydrides for sustained drug release,4-6 demonstrating that the hydrolytic instability of the anhydride bond can be manipulated by using aromatic and/or aliphatic anhydrides to release drug molecules at a predictable rate.7 The controlled, nonenzymatic degradation of the polyanhydrides makes them attractive for drug-delivery applications.8,9 For most applications, the drug is physically admixed with the polyanhydride by melting or dissolving the polymer. In fact, most degradable polymeric drug delivery systems in clinical use are physical mixtures of the release-controlling polymer and drug.8-12 Another significant advantage of the polyanhydrides is that these polymers degrade in vitro and in vivo to their acid counterparts as noncytotoxic products.5,6 Our laboratory previously reported the synthesis of poly(anhydride esters) (1) composed of salicylic acid (2) as novel degradable biomaterials.13-15 An important feature of homopolymer 1 is that it degrades into biocompatible compounds, salicylic acid (2) and sebacic acid (3), as outlined in Figure 1. Polymer 1 is unique in that the drug is chemically incorporated into the polymer backbone, not attached as a side group16-19 nor physically admixed, as described above. Therefore, drug release is directly dependent on the hydrolytic cleavage of the anhydride and ester bonds. Also, the polymeric incorporation of a drug can be more readily manipulated than the free drug to yield films by solvent casting, fibers by extrusion, or microspheres by emulsion* To whom correspondence should be addressed: Tel 732-445-0361; fax 732-445-7036; e-mail [email protected]. † Present address: Avon Products, Inc., Avon Place, Suffern, NY 10901.

precipitation methods. The device then can be implanted, ingested, or injected to reach the target site, where drug release occurs with hydrolytic degradation of the polymer backbone. Several advantages are attributable to the unique feature of polymer backbone-as-drug. First, a high amount of drug can be incorporated into the polymer structure; for example, approximately 62 wt % of polymer 1 is salicylic acid (2). By incorporating the drug into the polymeric backbone, problems encountered with drugs admixed into polymer matrixes, such as a burst release of drug, can be eliminated.20-22 Second, drug release correlates with the polymer composition, which is a function of the biocompatible linker molecule, such as sebacic acid (3) in polymer 1. Third, the linker molecules regulate the polymer properties (e.g., crystallinity and melting temperature) in addition to controlling drug release. A few examples of salicylate-based polymers exist, yet these materials are limited because they have low, therapeutically unfeasible concentrations of salicylate in a polyester backbone23 or are simply homopolymers of salicylic acid24,25 (i.e., no linker molecules) such that release of salicylate cannot be readily modified. Kricheldorf and others have incorporated salicylic acid into polymeric backbones but formed large macrocycles and/or oligomers.23-27 Kricheldorf et al. hypothesized that salicylic acid units tend to form hairpin conformations, thus cyclization is favored at high salicylic acid concentration, leading to the formation of macrocycles and low molecular weight polymers.23-27 In contrast, the formation of macrocycles and/or oligomers is limited when the salicylic acid is incorporated within the polymer backbone, likely due to increased chain flexibility. Many salicylate derivatives are low-cost drugs with diverse pharmacological applications and are classified as nonsteroidal antiinflammatory drugs (NSAIDs). On the basis of

10.1021/bm049544+ CCC: $30.25 © 2005 American Chemical Society Published on Web 11/26/2004

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Schmeltzer et al.

Figure 1. Hydrolytic degradation of poly(anhydride ester).

delivery of these drugs is achieved. This paper describes the synthesis, physicochemical characteristics, and cytotoxicity of seven salicylate-based poly(anhydride esters). Materials and Methods

Figure 2. Structure of salicylates with therapeutic relevance.

our initial success with salicylic acid, other salicylate derivatives were evaluated for inclusion into a polymeric backbone. The salicylates chosen have various uses and applications ranging from mild pain therapy to tuberculosis treatment, as detailed in Figure 2. Thiosalicylic acid (SH-SA) is used in the synthesis of a farnesyl derivative (FTS)28 that inhibits the growth of Colo 853 melanoma cells through a combination of cytostatic and proapoptotic effects. 5-Chlorosalicylic acid (5-Cl-SA) is a pharmacologically active moiety of the prodrugs Meseclazone29-32 and Seclazone33 used for pain mitigation. 4-Trifluoromethylsalicylic acid (4-CF3-SA) is a blood platelet antiaggregant with a more selective and specific action mechanism than that of acetylsalicylic acid (i.e., aspirin) for the treatment of thromboembolic diseases.34-38 4-Trifluoromethylsalicylic acid inhibits both platelet cyclooxygenase and c-AMP phosphodiesterase activity.39-43 Diflunisal (5FxSA) is 7-9 times more potent as an antiinflammatory agent and is appreciably less ulcerogenic than aspirin; it is also an effective antipyretic and analgesic drug.44-46 Salicylsalicylic acid (SA-SA) is simply the salicylate ester of salicylic acid and has similar antiinflammatory properties to salicylic acid.47 4-Aminosalicylic acid (4-ASA) was historically used as a systemic tuberculosis drug.48,49 Because of its short serum half-life, large oral doses (10-12 g per diem) are required49 which causes adverse side effects, such as gastrointestinal symptoms, hypersensitivity reactions, and renal failure. 48 Derivatives of 5-aminosalicylic acid (5-ASA) are currently used to treat inflammatory bowel disease (IBD).50-52 To be therapeutic, 5-aminosalicylic acid must reach the lower gastrointestinal (GI) tract, specifically the lower intestines, where its effect is topical.53,54 Our hypothesis is that by chemically incorporating these salicylate derivatives within a poly(anhydride ester) backbone, the drug release can be more effectively controlled, such that targeted and sustained

Solvents and reagents were purchased from Fisher (Pittsburgh, PA), and fine chemicals were obtained from Aldrich (Milwaukee, WI). Proton nuclear magnetic resonance (1H NMR) spectra were recorded on either a Varian 200 or 300 MHz spectrometer. Samples (5-10 mg) were dissolved in the appropriate deuterated solvent (CDCl3 or DMSO-d6), with the solvent used as the internal reference. Infrared (IR) spectra were measured on a Mattson Series spectrophotometer by solvent-casting samples onto a NaCl plate. Elemental analyses were provided by QTI (Whitehouse, NJ). Molecular weights (Mw) and polydispersity indices (PDI) were determined by gel-permeation chromatography (GPC) on a Perkin-Elmer (PE) LC system consisting of a series 200 refractive index detector, a series 200 pump, and an ISS 200 autosampler. A DEC Celebris 466 computer running PE TurboChrom 4 software was used for data collection and processing and to automate the analysis via PE-Nelson 900 interface and 600 link. Samples (5 mg/mL) were dissolved in tetrahydrofuran (THF) and filtered through 0.45 µm poly(tetrafluoroethylene) (PTFE) syringe filters (Whatman Inc., Clifton, NJ). Samples were resolved on a Jordi DVB mixedbed GPC column (7.8 × 300 mm) (Alltech Associates, Deerfield, IL) with THF at a flow rate of 0.5 mL/min. Molecular weights were calibrated relative to narrow molecular weight polystyrene standards (Polysciences, Dorval, Canada). Molecular weights (Mn) of THF-insoluble polymers were determined by intrinsic viscosity on a Cannon Ubbelhode viscometer (model 150-B236) in dimethyl sulfoxide (DMSO) at 23 °C. Thermal analyses were performed on a Perkin-Elmer system consisting of Pyris 1 DSC and thermogravimetric analysis (TGA) 7 analyzers with TAC 7/DX instrument controllers. Perkin-Elmer Pyris software was used for data collection and processing on a Dell OptiPlex GX110 computer. For DSC, samples (5 mg) were heated under dry nitrogen gas. Data were collected at heating and cooling rates of 10 °C/min with a two-cycle minimum. Glass-transition temperatures (Tgs) were calculated as half Cp extrapolated. For TGA, samples (10 mg) were heated under dry nitrogen gas. Data were collected at a heating rate of 20 °C/min. Decomposition temperatures were defined as the onset of decomposition. Melting points below 200 °C were determined on a Thomas-Hoover apparatus, whereas those above 200 °C were measured on the Pyris 1 DSC (see above). All cell manipulations were done under sterile conditions with a laminar flow hood and sterile techniques (e.g.,

Salicylate-Based Poly(anhydride esters)

sterilized materials and instruments). L929 mouse areolar/ adipose fibroblasts were obtained from M. Song of the Department of Biomedical Engineering (Rutgers University), then grown and manipulated in serum-containing medium. The medium consisted of 50 units of penicillin and streptomycin (Sigma, St. Louis, MO), 10% fetal bovine serum (J. T. Baker, Phillipsburg, NJ), and Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA). Salicylate-Based Diacid. In brief, the salicylate (7; 10 mmol) was dissolved in a solution of THF (15 mL) and pyridine (5 mL). Sebacoyl chloride (5 mmol) was added dropwise via syringe over 5 min to the stirring reaction mixture at room temperature. The reaction was stirred for 1 h and then poured over an ice-water slush (200 mL). After acidification to pH ∼ 2 with concentrated HCl, diacid (8) was isolated by vacuum filtration, washed with water (3 × 100 mL), and dried under vacuum. Any changes to the reaction conditions are noted below. 5-Chlorosalicylic Diacid (8a). Yield 95% (white powder); 1 H NMR (DMSO-d6) δ 7.90 (s, 2H, ArH), 7.71 (d, 2H, ArH), 7.25 (d, 2H, ArH), 2.55 (t, 4H, CH2), 1.62 (m, 4H, CH2), 1.35 (m, 8H, CH2); IR (NaCl, cm-1) 3400-2700 (COOH), 1753 (CdO, ester), 1687 (CdO, ester), 1098 (C-Cl, aryl chloride). Anal. Calcd: C, 56.39; H, 4.69. Found: C, 56.35; H, 4.69. Tm ) 177-181 °C. 5-(2,4-Difluorophenyl) Diacid (8b). Reaction was run at 0 °C. Yield 96% (white powder); 1H NMR (CDCl3) δ 8.25 (s, 2H, ArH), 7.65 (td, 2H, ArH), 7.41 (td, 2H, ArH), 7.22 (d, 2H, ArH), 6.95 (m, 4H, ArH), 2.65 (t, 4H, CH2), 1.80 (m, 4H, CH2), 1.45 (m, 8H, CH2); IR (NaCl, cm-1) 34002700 (COOH), 1754 (CdO, ester), 1658 (CdO, ester), 1139 (C-F, aryl fluoride), 1104 (C-F, aryl fluoride). Anal. Calcd: C, 64.88; H, 4.50. Found: C, 64.78; H, 4.54. Tm ) 162-165 °C. 4-Acetamidosalicylic Acid (7c). 4-Aminosalicylic acid (3.1 g, 20 mmol) was dissolved in pyridine (16 mL). Acetyl chloride (1.7 g, 22 mmol) was added dropwise via syringe over 5 min to the stirring reaction solution at room temperature. The reaction mixture was heated to 100 °C for 2 h. The reaction was cooled to room temperature and poured over an ice-water slush (200 mL). After acidification to pH ∼ 2 with concentrated HCl, the 4-acetamidosalicylic acid (7c) was isolated by vacuum filtration, washed with water (3 × 100 mL), and air-dried. Yield 87% (beige powder); 1H NMR (DMSO-d6) δ 10.25 (s, 1H, ArOH), 7.72 (d, 1H, ArH), 7.37 (s, 1H, ArH), 7.05 (d, 1H, ArH), 2.09 (s, 3H, CH3). IR (NaCl, cm-1) 3350-2650 (NH, OH, COOH), 1680 (CdO, acid, amide I), 1660 (NH, amide II). Anal. Calcd: C, 55.38; H, 4.62. Found: C, 55.16; H, 4.67. Tm ) 235 °C. 4-Acetamidosalicylic Diacid (8c). N,N-Dimethylformamide (DMF) was used as solvent and the reaction was run at 0 °C. Yield 98% (beige powder); 1H NMR (DMSO-d6) d 10.22 (s, 2H, ArNH), 7.82 (d, 2H, ArH), 7.50 (s, 2H, ArH), 7.38 (d, 2H, ArH), 2.40 (t, 4H, CH2), 2.02 (s, 6H, CH3), 1.60 (m, 4H, CH2), 1.30 (m, 8H, CH2); IR (NaCl, cm-1) 3400-2750 (COOH), 3340 (NH), 1765 (CdO, ester), 1700 (CdO, ester), 1680 (CdO, amide I), 1620 (NH, amide II). Anal. Calcd: C, 60.43; H, 5.76. Found: C, 59.95; H, 5.65. Tm ) 184-186 °C.

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5-Acetamidosalicylic Acid (7d). 5-Aminosalicylic acid (5.0 g, 32 mmol) was suspended in acetic anhydride (120 mL). The mixture was stirred at room temperature for 36 h, followed by the removal of acetic anhydride by rotary evaporation. NaOH (1.3 g, 32 mmol) and water (175 mL) were added to the residue, and the mixture was stirred for 2 h at room temperature. The reaction was poured over an icewater slush (200 mL). After acidification to pH ∼ 2 with concentrated HCl, the 5-acetamidosalicylic acid (7d) was isolated by vacuum filtration, washed with water (3 × 100 mL), and air-dried. Yield 85% (lavender powder); 1H NMR (DMSO-d6) δ 9.90 (s, 1H, ArOH), 8.10 (s, 1H, ArH), 7.65 (d, 1H, ArH), 6.92 (d, 1H, ArH), 2.02 (s, 3H, CH3); IR (NaCl, cm-1) 3400-2600 (NH, OH, COOH), 1665 (CdO, acid, amide I), 1625 (NH, amide II). Anal. Calcd: C, 55.38; H, 4.62. Found: C, 55.15; H, 5.03. Tm ) 226 °C. 5-Acetamidosalicylic Diacid (8d). Reaction was run at 0 °C. Yield 96% (pale lavender powder); 1H NMR (DMSOd6) d 10.15 (s, 2H, ArNH), 8.15 (s, 2H, ArH), 7.82 (d, 2H, ArH), 7.12 (d, 2H, ArH), 2.58 (t, 4H, CH2), 2.08 (s, 6H, CH3), 1.65 (m, 4H, CH2), 1.38 (m, 8H, CH2); IR (NaCl, cm-1) 3400-2600 (COOH), 3370 (NH), 1710 (CdO, ester), 1700 (CdO, ester), 1650 (CdO, amide I), 1610 (NH, amide II). Anal. Calcd: C, 60.43; H, 5.76. Found: C, 60.34; H, 5.84. Tm ) 205-206 °C. 4-Trifluoromethylsalicylic Diacid (8e). Yield 86% (white powder); 1H NMR (DMSO-d6) δ 8.15 (d, 2H, ArH), 7.80 (d, 2H, ArH), 7.45 (s, 2H, ArH), 2.55 (t, 4H, CH2), 1.64 (m, 4H, CH2), 1.35 (m, 8H, CH2); IR (NaCl, cm-1) 3400-2800 (COOH), 1771 (CdO, ester), 1706 (CdO, ester), 13301135 (C-F, alkyl fluoride). Anal. Calcd: C, 53.99; H, 4.15. Found: C, 54.32; H, 4.25. Tm ) 130-132 °C. Thiosalicylic Diacid (8f). Yield 96% (white powder); 1H NMR (DMSO-d6) δ 7.90 (d, 2H, ArH), 7.75 (m, 6H, ArH), 2.65 (t, 4H, CH2), 1.60 (m, 4H, CH2), 1.30 (m, 8H, CH2); IR (NaCl, cm-1) 3600-3100 (COOH), 1641 (CdO, ester), 700-600 (C-S, sulfide). Anal. Calcd: C, 60.78; H,5.48. Found: C, 60.51; H, 5.51. Tm ) 140-142 °C. Salicylsalicylic Diacid (8g). NaH (20 mmol) was also used as base, and reaction was run at 0 °C. Yield 91% (white powder); 1H NMR (CDCl3) δ 8.20 (dd, 4H, ArH), 7.60 (t, 4H, ArH), 7.45 (td, 4H, ArH), 7.15 (t, 4H, ArH), 2.50 (t, 4H, CH2), 1.65 (m, 4H, CH2), 1.35 (m, 8H, CH2); IR (NaCl, cm-1) 3400-2700 (COOH), 1751 (CdO, ester), 1662 (Cd O, ester). Anal. Calcd: C, 66.88; H, 4.98. Found: C, 67.35; H, 5.40. Tm ) 145-148 °C. Monomer Preparation. The diacid (8) was activated into monomer by previously described methods.55,56 In brief, the diacid (8) (2 g) was added to an excess of acetic anhydride (50 mL) and then stirred at room temperature until a homogeneous solution was observed. The monomer (9) was isolated by removing excess acetic anhydride under vacuum and then washed with diethyl ether (3 × 10 mL). 5-Chlorosalicylic Monomer (9a). Yield quantitative (pale yellow oil); 1H NMR (DMSO-d6) δ 7.95 (s, 2H, ArH), 7.75 (d, 2H, ArH), 7.30 (d, 2H, ArH), 2.57 (t, 4H, CH2), 2.23 (s, 6H, CH3), 1.63 (m, 4H, CH2), 1.35 (m, 8H, CH2); IR (NaCl,

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cm-1) 1805 (CdO, anhydride), 1762 (CdO, ester), 1710 (CdO, anhydride), 1101 (C-Cl, aryl chloride); Td ) 325 °C. 5-(2,4-Difluorophenyl) Monomer (9b). Yield quantitative (white solid); 1H NMR (CDCl3) δ 8.10 (s, 2H, ArH), 7.75 (td, 2H, ArH), 7.40 (td, 2H, ArH), 7.24 (d, 2H, ArH), 6.96 (m, 4H, ArH), 2.70 (t, 4H, CH2), 2.32 (s, 6H, CH3), 1.81 (m, 4H, CH2), 1.45 (m, 8H, CH2); IR (NaCl, cm-1) 1796 (CdO, anhydride), 1761 (CdO, ester), 1698 (CdO, anhydride), 1201 (C-F, aryl fluoride), 1142 (C-F, aryl fluoride); Td ) 386 °C. 4-Acetamidosalicylic Monomer (9c). Yield quantitative (beige solid); 1H NMR (DMSO-d6) δ 10.52 (s, 2H, ArNH), 8.00 (d, 2H, ArH), 7.65 (s, 2H, ArH), 2.60 (t, 4H, CH2), 2.30 (s, 6H, CH3), 2.10 (s, 6H, CH3), 1.60 (m, 4H, CH2), 1.35 (br, 8H, CH2); IR (NaCl, cm-1) 3345 (NH), 1805 (Cd O, anhydride), 1770 (CdO, ester), 1710 (CdO, anhydride), 1690 (CdO, amide I); Td ) 207 °C. 5-Acetamidosalicylic Monomer (9d). Yield quantitative (pale lavender solid); 1H NMR (DMSO-d6) δ 8.08 (s, 1H, ArH), 7.75 d, 2H, ArH), 7.58 (d, 2H, ArH), 2.58 (t, 4H, CH2), 2.35 (s, 6H, CH3), 2.20 (s, 6H, CH3), 1.68 (m, 4H, CH2), 1.35 (br, 8H, CH2); IR (NaCl, cm-1) 3360 (NH), 1815 (CdO, anhydride), 1765 (CdO, ester), 1715 (CdO, anhydride); Td ) 252 °C. 4-Trifluoromethylsalicylic Monomer (9e). Yield quantitative (9e; pale yellow oil); 1H NMR (DMSO-d6) δ 8.20 (d, 2H, ArH), 7.80 (d, 2H, ArH), 7.65 (s, 2H, ArH), 2.65 (t, 4H, CH2), 2.25 (s, 6H, CH3), 1.71 (m, 4H, CH2), 1.35 (m, 8H, CH2); IR (NaCl, cm-1) 1816 (CdO, anhydride), 1770 (CdO, ester), 1743 (CdO, anhydride), 1330-1135 (C-F, akyl fluoride); Td ) 358 °C. Thiosalicylic Monomer (9f). Yield quantitative (pale yellow oil); 1H NMR (DMSO-d6) δ 8.05 (d, 2H, ArH), 7.65 (m, 6H, ArH), 2.75 (t, 4H, CH2), 2.25 (s, 6H, CH3), 1.65 (m, 4H, CH2), 1.30 (m, 8H, CH2); IR (NaCl, cm-1) 1810 (CdO, anhydride), 1737 (CdO, ester), 1706 (CdO, anhydride), 700-600 (C-S, sulfide); Td ) 336 °C. Salicylsalicylic Monomer (9g). Yield quantitative (white solid); 1H NMR (CDCl3) δ 8.20 (dd, 4H, ArH), 7.65 (t, 4H, ArH), 7.40 (td, 4H, ArH), 7.20 (t, 4H, ArH), 2.52 (t, 4H, CH2), 2.25 (s, 6H, CH3), 1.65 (m, 4H, CH2), 1.35 (m, 8H, CH2); IR (NaCl, cm-1) 1797 (CdO, anhydride), 1749 (Cd O, ester), 1662 (CdO, ester), 1703 (CdO, anhydride); Td ) 288 °C. Polymerization. Monomer (9) (1 g) was placed in a twonecked round-bottom flask equipped with an overhead stirrer, which was heated to 180 °C by use of a temperature controller (Cole Parmer) in a silicone oil bath under high vacuum (