ESTERS)

CROSS-REFERENCE TO RELATED APPLICATION S

This application claims priority from United States Provisional Application Number 62/01 1 ,917, filed June 13, 2014 and from United States Provisional Application Number 62/035,236, filed August 8, 2014, which applications are herein incorporated by reference.

BACKGROUND OF THE INVENTION

Green chemistry aims to mitigate the environmental, health, and economic concerns associated with traditional chemical processes by increasing reaction efficiencies, reducing waste, using innocuous materials, and developing biodegradable products. Poly(anhydride- esters) (PAEs) are biodegradable surface-eroding polymers that represent such biodegradable products, exhibiting a controlled, near zero-order release of naturally occurring bioactives. Current synthesis of bioactive-based PAEs use excessive solvents, hazardous purification chemicals, and multistep reactions. Therefore, improved and/or less-toxic methodologies for preparing PAEs are needed, as well as intermediates that are useful in the preparation of such polymers.

SUMMARY OF THE INVENTION

Accordingly, described herein are synthetic methodologies that implement principles of green chemistry. For example, certain embodiments of the invention provide a method comprising treating a hydroxy-carboxylic acid compound:

with a compound of formula (I):

O O

Y ^{A A} Y (I)

in the absence of a solvent, to provide a diacid of formula (II):

wherein R is a linker molecule; each Y is independently a leaving group; and wherein X is a residue of a biologically active compound.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Scheme illustrating biodegradation of SA-based PAEs; each unit is composed of two salicylic acid moieties connected by a linker molecule.

Figure 2. A. Schemes illustrating one-pot synthesis of SA-PAEs. B. H-NMR of SA- PAE synthesized using one-pot synthesis.

Figure 3. FTIR Top: SA (adipic) Diacid with C=0 ester (1727 cm ^"1 ) and acid (1690 cm ^"1 ) via traditional synthesis. Bottom: SA (adipic) PAE C=0 anhydride and ester (1780- 1710 cm ^"1 ) via one-pot method.

Figure 4. SA-traditional, SA-OP, and modified SA-OP cytocompatibility in cell culture media at 0.1 , 0.01, and 0.001 mg/mL after 24, 48, and 72 h (for each group, the bar representing 24h is on the left, the bar representing 48 h is in the middle and the bar representing 72 h is on the right).

DETAILED DESCRIPTION

As described herein, synthetic methods that implement the principles of green chemistry have been established for the generation of PAEs. Examples of such methods are presented in Examples 1 and 2, which describe the synthesis of SA-PAEs using a one-pot methodology. Specifically, using a stoichiometric amount of pyridine, two moieties of salicylic acid (SA) were chemically conjugated through adipoyl chloride under solvent-free conditions to acquire SA diacid. SA diacid was subsequently activated and polymerized under high vacuum and heat to generate SA-based poly(anhydride-esters) via a one-pot reaction. This one-pot reaction methodology greatly reduced reaction time (i.e., twice as fast) while increasing efficiency (i.e., higher molecular weight Mw). Additionally, polymers were characterized to ensure that the greener methodology did not detrimentally impact

physicochemical and thermal properties. This one-pot methodology offers a means for synthesizing poly(anhydride-esters) for various biomedical applications while overcoming traditional polymer synthesis limitations such as toxic solvent entrapment and long reaction time from starting material to polymer product. While these Examples describe the generation of SA-PAEs, this methodology may be applied to the synthesis of PAEs comprising other compounds, e.g., other hydroxy-carboxylic acid compounds. Accordingly, certain embodiments of the invention provide a method comprising treating a hydroxy-carboxylic acid compound:

with a compound of formula (I):

O O Y R Y (I)

in the absence of a solvent, to provide a diacid of formula (II):

wherein R is a linker molecule; wherein each Y is independently a leaving group; and wherein X is a residue of a hydroxy-carboxylic acid compound.

It should be understood that in formula II, when X is the "residue of a hydroxy- carboxylic acid compound" X represents the structure of the compound other than the oxy group (-0-) and the carboxy group (-COOH) shown attached to X in formula (II). Hydroxy- carboxylic acid compounds that may be used in the methods described herein generally have a relatively low molecular weight of approximately 1,000 daltons or less (e.g., about 900 daltons, 800 daltons, 700 daltons, 600 daltons, 500 daltons, 400 daltons, 300 daltons, 200 daltons, 100 daltons, 50 daltons, etc.). Additionally, while these compounds must contain within their molecular structure at least one carboxylic acid group and one hydroxy group, the compound may also comprise other functional groups.

Certain other embodiments of the invention provide a method comprising treating a hydroxy-carboxylic acid compound:

with a compound of formula (I):

O O Y R Y (I)

in the absence of a solvent, to provide a diacid of formula (II):

wherein R is a linker molecule; wherein each Y is independently a leaving group; and wherein X is a residue of a biologically active compound.

In certain embodiments, the method further comprises treating the diacid of formula (II) with an acid anhydride (e.g., acetic anhydride) and heat to provide the corresponding polymer.

In certain embodiments, the method further comprises treating the diacid of formula (II) with an acid anhydride (e.g., acetic anhydride), vacuum and heat to provide the corresponding polymer.

In certain embodiments, the diacid of formula (II) is treated with 2 equivalents of acetic anhydride.

In certain embodiments, the diacid of formula (II) is treated with 3 equivalents of acetic anhydride.

In certain embodiments, the method further comprises treating the hydroxy-carboxylic acid compound with a compound of formula (I) in the presence of a base.

In certain embodiments, the base is pyridine, 2,4,6-collidine or poly(4-vinylpyridine).

In certain embodiments, the method further comprises washing the corresponding polymer with water or cyclopentyl methyl ether (CPME).

Certain embodiments of the present invention provide additional processes and intermediates disclosed herein that are useful for preparing PAEs (see, e.g. the Examples).

Leaving Group (Y)

As described herein, each Y is independently a leaving group. In certain

embodiments, each Y is independently a leaving group selected from a halogen, alkylsulfonyl or arylsulfonyl leaving group. In certain embodiments, each Y is independently a leaving group selected from chloro, bromo, iodo, mesylate and tosylate.

In certain embodiments, each Y is chloro. Accordingly, in certain embodiments, the compound of formula (I) is a compound of formula (la): Biologically Active Compounds

The methods of the invention are useful for preparing polymers from biologically active compounds that have at least one carboxylic acid group and one hydroxy group. It should be understood that in formula II, when X is the "residue of a biologically active compound," X represents the structure of the biologically active compound other than the oxy group (-0-) and the carboxy group (-COOH) shown attached to X in formula (II).

Biologically active compounds that may be used in the methods described herein generally have a relatively low molecular weight of approximately 1 ,000 daltons or less (e.g., about 900 daltons, 800 daltons, 700 daltons, 600 daltons, 500 daltons, 400 daltons, 300 daltons, 200 daltons, 100 daltons, 50 daltons, etc.). Additionally, these compounds must contain within their molecular structure at least one carboxylic acid group and one hydroxy group; however, the hydroxy-carboxylic acid compound can also comprise other functional groups.

The term "biologically active compound" includes therapeutic compounds that provide a therapeutically desirable effect when administered to an animal (e.g., a mammal, such as a human). Therapeutic compounds that can be incorporated into the polymers of the invention include suitably functionalized analgesics, anesthetics, anticancer, anti-Parkinson's agents, anti-infectives, antiacne agents, antibiotics, antioxidants, antimicrobials,

anticholinergics, anticoagulants, anticonvulsants, antidiabetic agents, antidyskinetics, antifibrotic agents, antifibrotics, antifungal agents, antiglaucoma agents, anti-inflammatory agents, antineoplastics, antiosteoporotics, antipagetics, antisporatics, antipyretics,

antiseptics/disinfectants, antithrombotics, bone resorption inhibitors, calcium regulators, cardioprotective agents, cardiovascular agents, central nervous system stimulants, cholinesterase inhibitors, contraceptives, deodorants, dopamine receptor agonists, erectile dysfunction agents, fertility agents, gastrointestinal agents, gout agents, hormones, hypnotics, immunomodulators, immunosuppressives, keratolyses, migraine agents, motion sickness agents, muscle relaxants, nucleoside analogs, obesity agents, ophthalmic agents, osteoporosis agents, parasympatholytics, parasympathomimetics, prostaglandins, psychotherapeutic agents, respiratory agents, sclerosing agents, sedatives, skin and mucous membrane agents, smoking cessation agents, sympatholytics, synthetic antibacterial agents, ultraviolet screening agents, urinary tract agents, vaginal agents, and vasodilators (see Physicians' Desk Reference, 55 ed., 2001 , Medical Economics Company, Inc., Montvale, New Jersey, pages 201-202).

Suitable examples of low molecular weight drugs with the required functional groups within their structure can be found in almost all classes of drugs including, but not limited to, antioxidants, analgesics, anesthetics, antiacne agents, antibiotics, synthetic antibacterial agents, antimicrobial anticholinergics, anticoagulants, antidyskinetics, antifibrotics, antifungal agents, antiglaucoma agents, anti-inflammatory agents, antineoplastics/anticancer, antiosteoporotics, antipagetics, anti-Parkinson's agents, antisporatics, antipyretics, antiseptics/disinfectants, antithrombotics, bone resorption inhibitors, calcium regulators, keratolytics, sclerosing agents and ultraviolet screening agents. Additional lists of therapeutic compounds can be found, for example, in: Physicians' Desk Reference, 55 ed., 2001, Medical Economics Company, Inc., Montvale, New Jersey; USPN Dictionary of USAN and International Drug Names, 2000, The United States Pharmacopeial Convention, Inc., Rockville, Maryland; and The Merck Index, 12 ed., 1996, Merck & Co., Inc.,

Whitehouse Station, New Jersey. One skilled in the art can readily select therapeutic compounds that possess the necessary functional groups for use in the methods described herein from these lists.

In certain embodiments, the biologically active compound is an antioxidant, an antimicrobial, an antifungal, an anticancer, an analgesic, an immunosuppressive or an antiinflammatory (e.g., a non-steroidal anti-inflammatory).

In certain embodiments, the biologically active compound is an antioxidant.

Examples of antioxidants suitable for use in the present invention include, but are not limited to, vanillic acid, syringic acid, coumaric acid, sinapic acid, and ferulic acid.

In certain embodiments, the biologically active compound is an antibacterial compound or an antimicrobial compound. Examples of antibacterial/antimicrobial compounds suitable for use in the present invention include, but are not limited to 4- sulfanilamidosalicylic acid, amoxicillin, apalcillin, aspoxicillin, biapenem, cefadroxil, cefamandole, cefatrizine, cefbuperazone, cefonicid, cefoperazone, cefpiramide, cefprozil, flomoxef, imipenem, meropenem, nadifloxacin, panipenem, salazosulfadimidine, sulfaloxic acid, teicoplanin, and the like.

In certain embodiments, the biologically active compound is an anti-neoplastic or anticancer compound. Examples of anti -neoplastic/anticancer compounds suitable for use in the present invention include, but are not limited to mycophenolic acid, podophyllinic acid 2- ethylhydrazide, ubenimex, and the like.

In certain embodiments, the biologically active compound is an immunosuppressive compound. Examples of immunosuppressive compounds suitable for use in the present invention include, but are not limited to mycophenolic acid, ubenimex and the like. In certain embodiments, the biologically active compound is an anti-inflammatory compound, such as an NSAID. Examples of anti-inflammatory compounds suitable for use in the present invention include, but are not limited to 3-amino-4-hydroxybutyric acid, fendosal, gentisic acid, mesalamine, olsalazine, oxaceprol, S-adenosylmethionine, sulfasalazinem salicylic acid, diflunisal, salsalate, 5 -aminosalicylic acid and the like.

In certain embodiments the anti-inflammatory is salicylic acid.

Linker Molecule (R)

In certain embodiments, each linker molecule (R) is selected from a branched aliphatic, linear aliphatic, and oxygen-containing linker molecule. In certain embodiments, the branched aliphatic, linear aliphatic, or oxygen-containing linker molecule comprises 1 to 15 carbon atoms.

In certain embodiments, R is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 1 to 25 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4) of the carbon atoms is optionally replaced by (-0-), (-NR _t -) or phenylene, and wherein the chain is optionally substituted on carbon with one or more (e.g. 1 , 2, 3, or 4) substituents selected from the group consisting of (Cj-C )alkoxy, (C ₃ -C ₆ )cycloalkyl, (Q- C ₆ )alkanoyl, (C]-C6)alkanoyloxy, (Ci-C ₆ )alkoxycarbonyl, (Ci-C )alkylthio, azido, cyano, nitro, halo, hydroxy, oxo, carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R] is independently selected from H or (C]-C ₆ )alkyl.

In certain embodiments, R is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 1 to 25 carbon atoms, wherein the chain is optionally substituted on carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected from the group consisting of (C _r C6)alkoxy, (C ₃ -C6)cycloalkyl, (Ci-C^alkanoyl, (Ci- C ₆ )alkanoyloxy, (C ₁ -C ₆ )alkoxycarbonyl, azido, cyano, nitro, halo, hydroxy, oxo, carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

In certain embodiments, R is a peptide.

In certain embodiments, R is an amino acid.

In certain embodiments, R is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 1 to 25 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4) of the carbon atoms is optionally replaced by (-0-), (-NRp) or phenylene, wherein each Rj is independently selected from H or (Ci-C6)alkyl.

In certain embodiments, R is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 3 to 15 carbon atoms, wherein one or more (e.g. 1 , 2, 3, or 4) of the carbon atoms is optionally replaced by (-0-), (-NR _r ) or phenylene, and wherein the chain is optionally substituted on carbon with one or more (e.g. 1 , 2, 3, or 4) substituents selected from the group consisting of (C ₁ -C6)alkoxy, (C3-C ₆ )cycloalkyl, (Q- C ₆ )alkanoyl, (C]-C6)alkanoyloxy, (Ci-C ₆ )alkoxycarbonyl, (Ci-C ₆ )alkylthio, azido, cyano, nitro, halo, hydroxy, oxo, carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each Ri is independently selected from H or (C ₁ -C ₆ )alkyl.

In certain embodiments, R is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 3 to 15 carbon atoms, wherein one or more (e.g. 1 , 2, 3, or 4) of the carbon atoms is optionally replaced by (-0-), (-NR ) or phenylene, wherein each Ri is independently selected from H or (C ₁ -C ₆ )alkyl.

In certain embodiments, R is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 3 to 15 carbon atoms.

In certain embodiments, R is a divalent, branched or unbranched, hydrocarbon chain, having from 3 to 15 carbon atoms.

In certain embodiments, R is a divalent, branched or unbranched, hydrocarbon chain, having from 2 to 10 carbon atoms (e.g., 6 to 10 carbon atoms).

In certain embodiments, R is a divalent hydrocarbon chain having 3, 4, 5, 6, 7, 8, or 9 carbon atoms.

In certain embodiments, R is a divalent hydrocarbon chain having 8 carbon atoms.

In certain embodiments, R is divalent hydrocarbon chain having 4 carbon atoms.

In certain embodiments, R is 1 ,4 phenylene or 1,3 phenylene.

In certain embodiments R is an adipic linker (-CH ₂ CH ₂ CH ₂ CH ₂ -).

In certain embodiments, R is a diglycolic linker (-CH ₂ OCH ₂ -).

In certain embodiments, R is a diethylmalonic linker (-CH ₂ C(Et) ₂ CH ₂ -).

Average Molecular Weight

In certain embodiments, poly(anhydride-esters) prepared in accordance with the methods of the present invention have an average molecular weight of about 1,000 daltons to about 100,000 daltons. In certain embodiments, the polymer has an average molecular weight of about 5,000 daltons to about 100,000 daltons. In certain embodiments, the polymer has an average molecular weight of about 5,000 daltons to about 50,000 daltons. In certain embodiments, the polymer has an average molecular weight of about 10,000 daltons to about 30,000 daltons. In certain embodiments, the polymer has an average molecular weight greater than about 20,000 daltons and less than about 30,000 daltons. Certain Specific Embodiments

Certain embodiments of the invention provide a method comprising treating salicylic acid with a compound of formula (la):

in the absence of a solvent, to provide a diacid of formula (Ha):

wherein R is a linker molecule.

In certain embodiments, the method further comprises treating the diacid of formula (lla) with an acid anhydride (e.g., acetic anhydride) and heat to provide the corresponding polymer.

In certain embodiments, the method further comprises treating the diacid of formula (lla) with an acid anhydride (e.g., acetic anhydride), vacuum and heat to provide the corresponding polymer.

The following non-limiting Examples set forth herein below illustrate certain aspects of the invention.

EXAMPLE 1

Synthesis of Bioactive Based Poly (anhydride-esters) via Green Solvent Free Chemistry:

One-Pot Polymer Synthesis

Abstract

SA-based PAEs were synthesized via a one-pot methodology that requires minimal purification and drastically reduces reaction time. For instance, SA-based PAEs were synthesized via solvent free methods by linking two SA moieties with an acyl chloride to form an SA diacid within minutes, which can be activated and subsequently polymerized under high vacuum and heat to form product. Products were then purified using nontoxic and environmentally safe solvents. Polymers and their precursors were then characterized to ensure the greener methodology did not have a detrimental influence on physiochemical and thermal properties. Similar methodologies were explored to synthesize and characterize FA- based PAEs. PAE Synthesis and Characterization

PAEs were synthesized using various green methodologies and characterized (see, Figure 2 A). Specifically. PAEs were characterized via Ή Nuclear Magnetic Resonance spectroscopy (Figure 2B), differential scanning calorimetry, gel permeation chromatography and Fourier transform infrared spectroscopy to elucidate that desired products were synthesized without the presence of impurities.

Solvent Free SA Diacid Synthesis. Eliminating the use of solvents significantly reduces reaction time, waste and leads to simpler purification. In certain embodiments, the reaction can be carried out using vortexing, which may involve the use of steel balls, or mechanical stirring (e.g., to overcome high viscosity).

Table 1.

Purification Methods. CPME is a safer alternative to acetone and hexanes due to its low toxicity and higher flash point. CPME has very low peroxide forming potential and is a reusable solvent. Water is the most ideal solvent due to its low toxicity, renewability and abundance.

Table 2.

One Pot Polymer Synthesis. A one pot polymer synthesis improves synthetic efficiency due to a reduced amount of steps and decreases reaction time significantly.

Additionally, synthesis involving the use of solvent can leave solvent trapped in the polymer matrix making it unsuitable for biological purposes. By switching to a one pot solvent free method, it can be ensured that no solvent impurities will be left behind.

Prior to performing one-pot synthesis, salicylic acid (SA) purity (98% purity acceptable) is assessed via 1H-NMR spectroscopy. Additionally, anhydrous pyridine, adipoyl chloride, and anhydrous acetic anhydride are confirmed to be colorless liquids. SA (1 , 1.00 g, 7.24 mmol) is then dissolved in anhydrous pyridine (1.17 mL, 14.5 mmol) in a 4 dram vial. Upon dissolution, the solution is transferred via syringe to an oven-dried, two-neck 100 mL round-bottom flask (RBF) equipped with a Teflon stirrer, stirrer adaptor, and rubber septum under nitrogen. The mechanical stirrer is set to 120 rpm after which adipoyl chloride (0.58 mL, 4.0 mmol) is added drop-wise manually. Following adipoyl chloride addition, the reaction is allowed to stir an additional 30 minutes. Acetic anhydride (1.71 mL, 18.1 mmol) is then added via syringe and the mixture heated to 75 °C with mechanical stirring (120 rpm). Upon complete dissolution, the reaction is allowed to stir an additional hour, after which the RBF is re-equipped with a vacuum adaptor and pressure reduced below 2 mmHg with continuous stirring. Following removal of excess acetic anhydride, the reaction is heated to 175°C and stirred until vitrification or constant viscosity (~ 3-4 h). Once constant viscosity is achieved, the reaction is cooled to room temperature, vacuum removed, and placed under nitrogen. The resulting crude polymer is dissolved in anhydrous DCM (10 mL) and precipitated over 400 mL chilled diethyl ether. Solvent is then decanted and polymer dried under vacuum at room temperature.

Table 3. Comparison of traditional and one-pot SA-PAE physicochemical and thermal ro erties.

*Averages compiled over a period of time. ^$ Averages of one pot polymerization

The PAEs described herein may be further characterized by investigating their drug release properties; these experiments may be performed using techniques and assays known in the art, for example [Erdmann, L. et al. Biomaterials, 2000, 21 , 1941 -1946].

Additionally, these methods described herein may be used to synthesize PAEs comprising other hydroxy-carboxylic acid compounds.

Finally, to further improve the green properties of these methods, pyridine could also be replaced in the base catalyzed reaction with other suitable reagents that are less toxic, renewable, and/or increase the synthetic efficiency, such as 2,4,6-collidine or poly(4- vinylpyridine). Conclusions

Polymers and polymer precursors were successfully synthesized using solvent free methods. SA-based PAEs were synthesized via a one-pot system. Polymer characterization revealed that greener synthetic methodology did not adversely influence thermal and physicochemical properties. Non-toxic purification techniques decreased the amount of solvent required and increased yield. By substituting traditional methods and green syntheses it was possible to increase efficiency and subsequently reduce the environmental impact while creating a safer work environment.

EXAMPLE 2

Exploring green methods through a one-pot salicylic acid-based poly (anhydride-ester) synthesis.

Salicylic acid-based poly(anhydride-esters), containing adipic-linkages, were prepared by a one-pot melt-condensation polymerization. One-pot polymer physicochemical and thermal properties were characterized and compared to salicylic acid-based poly(anhydride- esters) produced via traditional synthesis. The one-pot polymerization was found to drastically reduce reaction time while maintaining overall reaction yield. Furthermore, the one-pot polymer produced higher molecular polymer while still possessing similar thermal properties and polydispersity. In addition to increasing efficiency, the one-pot polymer was found to be a greener alternative, improving atom economy, minimizing solvent use and reducing waste.

As described herein, traditional SA-based PAE synthesis has been modified to increase efficiency while reducing raw material consumption and minimizing solvent use. SA Diacid synthesis, from SA and adipoyl chloride, and melt-condensation polymerization were performed sequentially in one-pot to drastically reduce reaction time (Scheme 1). The one-pot SA-based PAE (SA-OP) was characterized by proton (Ή) nuclear magnetic resonance (NMR) and Fourier-transform infrared resonance (FTIR) spectroscopies. Polymer weight-averaged M _w and polydispersity index were quantified by gel permeation

chromatography (GPC) while thermal properties were evaluated by thermogravimetric analysis (TGA) to obtain decomposition temperature (Ta) and differential scanning calorimetry (DSC) to acquire glass transition temperature (T _g ). Furthermore, SA-OP cytotoxicity tests were conducted to ensure this modified procedure did not adversely impact cytocompatibility.

Scheme 1.

Materials and Methods

Materials. 1 N hydrochloric acid (HC1) and polytetrafluoroethylene (PTFE) syringe filters were purchased from Fisher Scientific (Fair Lawn, NJ). All other chemicals were acquired from Sigma-Aldrich (Milwaukee, WI).

¹ H NMR and FTIR Spectroscopies. ^! H NMR spectroscopy was recorded on a Varian 400 MHz spectrometer by dissolving polymer or polymer precursor samples (-10 mg) in deuterated dimethyl sulfoxide (DMSO- ₆ ), which was also an internal reference. FTIR spectra were obtained using a Nicolet/ Avatar 360 spectrophotometer. Spectra were acquired by either grinding and pressing polymer precursors (1 wt. %) with potassium bromide (KBr) into discs or solvent casting polymers, via dichloromethane (DCM), onto sodium chloride (NaCl) plates.

Molecular Weight. GPC was utilized to determine polymer weight-averaged M _w and PDI. GPC consisted of a Waters (Milford, MA) system with a 1515 Isocratic HPLC pump, a 717plus autosampler, and a 2414 refractive index detector. Waters Breeze 3.20 software operating on an IBM ThinkCentre CPU was used for data processing and analysis. Polymers were solved in DCM (10 mg/mL), filtered through 0.45 PTFE syringe filters. Sample aliquots (10 μί,) were injected, and resolved, on a Jordi divinylbenzene mixed-bed GPC column (7.8 ^χ 300 mm, Alltech Associates, Deerfield, IL) at 25 °C, with DCM as the mobile phase at a flow rate of 1.0 mL/min. Molecular weights were calibrated relative to broad polystyrene standards (Polymer Source Inc., Dorval, Canada).

Thermal Analysis. TGA was performed to acquire polymer sample decomposition temperature (T _d ). TGA was conducted using a Perkin Elmer (Waltham, MA) TGA7 analyzer with TAC7/DX controller equipped with a Dell OptiPlex Gx 110 computer running on Perkin Elmer Pyris software. Polymer samples (~5 mg) were heated under nitrogen at a rate of 10°C/min from 25 - 400 °C. Tj was defined as the onset of decomposition, indicated by the beginning of a sharp slope on the thermogram.

DSC measurements were acquired using a Thermal Advantage (TA; New Castle, DE) DSC Q200 running on an IBM ThinkCentre computer equipped with TA Universal Analysis software for data acquisition and processing. Polymer glass transition temperature (T _g ) were procured. Samples (4-6 mg) were heated from -10 - 200 °C at a rate of 10 °C/min with a minimum of two cycles for each sample. The resulting data was analyzed using TA

Instruments Universal Analysis 2000 software.

Traditional Polymer Synthesis.

SA (1.00 g, 7.24 mmol) is dissolved in tetrahydrofuran (THF, 10 mL) under inert gas in a 50 mL round-bottomed flask (RBF). Pyridine (14.5 mmol) is added via syringe and the reaction magnetically stirred for 15 min at room temperature (RT). Adipoyl chloride (2, 3.80 mmol) is added dropwise to the reaction solution over 1 hr. After stirring an additional 2 hrs at RT, the reaction mixture is quenched over 70 mL Deionized (DI) H ₂ 0 and acidified to ~pH = 2, using concentrated HC1, in a 250 mL beaker. Crude diacid is isolated via vacuum filtration, washed 3 x 20 mL DI H ₂ 0, and allowed to air dry. Crude diacid is then dissolved in acetone (60 mL) with heating (40 °C) and reprecipitated in 5-fold excess hexanes with continued stirring and cooling to RT. Product is then isolated via vacuum filtration and dried in vacuum oven at 60 °C for >12 hrs.

SA (adipic) Diacid. Yield: 71.5% (performed in triplicate, white powder). Ή NMR (400 MHz, DMSO-4 : δ 7.95 (d, 2H, ArH), 7.65 (t, 2H, ArH), 7.40 (t, 2H, ArH), 7.20 (d, 2H, ArH), 2.65 (t, 4H, CH2), 1.75 (m, 4H, CH2). FTIR (KBr, cm ^"1 ): 1727 (C=0, ester), 1690 (C=0, acid).

SA Diacid (3, 0.97g) is stirred in excess acetic anhydride (-10 mL) at RT under inert gas in 50 mL RBF until suspension becomes a clear solution (6-12 hrs). Excess acetic anhydride is removed in vacuo to acquire activated monomer (4). Monomer (4, 2.52 mmol) is placed under vacuum (>2 Torr) and brought to 175 ^° C with active stirring (Teflon stirrer) at 120 rpm with overhead stirrer. Reaction proceeds until vitrification or polymer viscosity is attained (~ 3 hrs). Upon completion, reaction is cooled to RT, dissolved in 10 mL DCM, and precipitated in 400 mL chilled diethyl ether. Resulting polymer is isolated via decantation or vacuum filtration and dried under vacuum (> 12 hrs, RT).

SA (adipic) PAE. Yield: 61.8% (tan powder). ^] H NMR (400 MHz, DMSO- ₆ ): δ 8.15 (b, 2H, ArH), 7.75 (b, 2H, ArH), 7.40 (b, 4H, ArH), 2.60 (b, 4H, CH2), 1.60 (b, 4H, CH2). FTIR (NaCl, cm ^"1 ): 1781 , 1705 (C=0, anhydride), 1734 (C=0, ester). One-pot Polymer Synthesis. SA (1, 1.00 g, 7.24 mmol) is dissolved in anhydrous pyridine (1.17 mL, 14.5 mmol) in a 20 mL scintillation vial. Upon dissolution, the solution is transferred via syringe to an oven-dried, two-neck 100 mL RBF equipped with a Teflon stirrer, stirrer adaptor, and rubber septum under nitrogen. The mechanical stirrer is set to 120 rpm after which adipoyl chloride (0.58 mL, 3.98 mmol) is added drop- wise manually.

Following adipoyl chloride addition, the reaction is allowed to stir an additional 30 minutes after which acetic anhydride (1.71 mL, 1-8.1 mmol) is added via syringe and mixture heated to 75 °C. After complete dissolution, the RBF is equipped with a vacuum adaptor and pressure reduced below 2 mmHg with continuous stirring. Once excess acetic anhydride is completely removed, the reaction is heated to 175 ^° C and stirred until vitrification or constant viscosity (~ 3-4 h). Once achieved, the reaction is cooled to room temperature, vacuum removed, and placed under nitrogen. The crude polymer is dissolved in anhydrous DCM (10 mL) and precipitated over 400 mL chilled diethyl ether. Solvent is then decanted and polymer (4) dried under vacuum at room temperature. Polymer properties are presented in Table 4. Yield: 47.4 % (tan powder). 1H NMR (400 MHz, DMSO-i¾: δ 8.15 (b, 2H, ArH), 7.75 (b, 2H, ArH), 7.40 (b, 4H, ArH), 2.60 (b, 4H, CH2), 1.60 (b, 4H, CH2). FTIR (NaCl, cm ^"1 ): 1780-1710 (C=0 ester and anhydride)

Cytotoxicity Studies. In vitro cytotoxicity studies were conducted by culturing 3T3 mouse fibroblasts in cell media (Dulbecco's Modified Eagle Medium supplemented with 10% Fetal Bovine Serum, 1 % Penicillin Streptomycin) containing S A (adipic) PAEs from traditional (SA-traditional) and SA-OP synthesis. Polymers were sterilized under UV at λ = 254 nm for 900 s (Spectronics Corporation, Westbury, NY) prior to being dissolved in DMSO and subsequently diluted with cell media to reach concentrations of 0.1 mg/mL, 0.01 mg/mL and 0.001 mg/mL. Aliquots of cell media containing polymers were then distributed to allocated wells in a 96-well plate with 2000 cells/well and incubated at 37 °C. DMSO- containing cell media (1 %) was used as a negative control.

Cell viability was determined using CellTiter 96® Aqueous One Solution Cell Proliferation Assay. After 24 h, 48 h, and 72 h incubation with polymers, 20 iL of (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfop henyl)-2H-tetrazolium) (MTS) reagent was added to each well and further incubated for 4 h at 37 °C. Absorbance was then recorded with a microplate reader (Coulter, Boulevard Brea, CA) at 492 nm. Results and Discussion

Traditionally SA-based PAEs were synthesized via a two-step reaction pathway in which S A is first reacted with a diacyl chloride in THF with pyridine to facilitate SA Diacid synthesis (Schmeltzer R.C. ATJ, Uhrich K.E. Optimized Synthesis of Salicylate-based Poly(anhydride-esters). Polym Bull. 2003;49:441-8). Pyridine deprotonates SA's carboxylic acid and subsequently catalyzes O-acylation through an acyl-pyridinium ion. Though yields were near quantitative further purification was often necessary to acquire other SA Diacids (i.e., SA adipic Diacid). This was usually achieved through an acetone/hexanes

reprecipitation in which crude SA Diacid is first dissolved in acetone and then precipitated using five times the volume of hexanes. Additionally, prolonged vacuum drying with heat was frequently necessary to completely remove THF. In the absence of excessive drying, solvent would often persist after polymerization as indicated by Ή NMR. Following SA Diacid acquisition, acetic anhydride is used to activate the carboxylic acid functionalities. After activation excess acetic anhydride (~ 80 eq) would need to be removed in vacuo and the resulting monomer transferred prior to melt-condensation polymerization. These additional steps introduce opportunities in which the activated carboxylic acids could be hydrolyzed and subsequently result in a lower M _w polymer.

SA-OP was synthesized via a similar two-step synthetic method in a one-pot reaction. The near quantitative O-acylation of S A with adipoyl chloride was carried out in

stoichiometric amounts of pyridine to generate SA (adipic) Diacid as a white paste. Diacid was then activated with acetic anhydride at 75 °C prior to being placed under vacuum (> 2 Torr) and heated to 175 °C to acquire SA-OP. The one-pot method was conducted in triplicate to confirm reproducibility and its polymer properties and reaction efficiency compared to SA-traditional.

Table 4. Polymer properties and reaction efficiency of SA-OP vs SA-traditional.

* denotes averages compiled over 12 years whereas * denotes averages of SA-OP in triplicate. SA-OP exhibited comparable overall reaction yield while drastically decreasing the amount of time necessary to produce SA adipic PAE from SA (Table 4). Contributing to the enhanced reaction efficiency was the elimination of solvents during the synthesis (THF) and purification (acetone, hexanes) of SA adipic diacid intermediate. Reduced equivalents of acetic anhydride and removal of activated monomer isolation also improved efficiency. SA- OP displayed higher M _w than SA-traditional, which is likely attributed to the reduced exposure of the monomer with air, as well as similar T _g and PDI values.

During SA-OP it is possible that SA adipic diacid synthesis will not be quantitative and side-products (SA, adipic acid, mono-conjugated SA) may persist in small quantities. While pure monomer is desirable prior to polymerization, the side-products are all degradation products of SA adipic PAE. As SA-OP polymer is pure by ^! H NMR and FTIR spectroscopies (Figure 3), it is hypothesized that any side-products present prior to activation are either incorporated in the polymer anhydride-ester network or removed by fractionation following the polymerization. An additional concern was pyridine and pyridine HC1 removal following SA O-acylation. Pyridine and pyridine HC1 boil at 1 15 and 222-224 °C

respectively, and thus can be removed by the high temperature and low pressure of the polymerization process. Nonetheless, cytotoxicity studies (Figure 4) were conducted comparing SA-traditional, SA-OP, and a modified SA-OP in which pyridine is removed prior to activation using an acidic wash. All polymers were shown to be cytocompatible at 0.01 mg/mL and lower concentrations after 72 h. It is important to note that SA-traditional, SA- OP, and modified SA-OP did not display statistically different cytotoxicity at any

concentration or time point. Thus, it can be concluded that SA-OP does not contain sufficient levels of pyridine or pyridine HC1 to be detrimental to its cytocompatibility profile.

Conclusion

SA adipic PAEs were successfully synthesized using a one-pot melt-condensation polymerization method. SA-OP was found to be a more efficient synthetic method, drastically reducing reaction time while increasing weight-averaged M _w and maintaining thermal properties. Additionally, cytotoxicity studies revealed that SA adipic PAEs synthesized via the SA-OP methodology displayed no statistical difference from PAEs synthesized via the traditional method. Furthermore, similar polymerization yields were obtained for both methods following purification. Thus, this one-pot polymerization method offers a greener, more effective means of producing SA adipic PAEs, saving environmental and raw material costs. All publications cited herein are incorporated herein by reference. While in this application certain embodiments of invention have been described, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that certain of the details described herein may be varied without departing from the basic principles of the invention.

The use of the terms "a" and "an" and "the" and similar terms in the context of describing embodiments of invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. In addition to the order detailed herein, the methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate embodiments of invention and does not pose a limitation on the scope of the invention unless otherwise specifically recited in the claims. No language in the specification should be construed as indicating that any non-claimed element as essential to the practice of the invention.