# Patent

## Title
Biodegradable polyacetals and methods

## Abstract

Condensation polymerization methods are used to prepare various biodegradable polyacetals of the general formula (I). Such polymers are useful for variety of drug, biomolecule and imaging agent delivery applications.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to biodegradable polyacetals and methods for making them. These polymers are useful for a variety of drug, biomolecule and imaging agent delivery applications.
2. Description of the Related Art
A variety of systems have been used for the delivery of drugs, biomolecules, and imaging agents. For example, such systems include capsules, liposomes, microparticles, nanoparticles, and polymers. Polymers are often classified as being either biodegradable or nonbiodegradable.
A variety of polyester-based biodegradable systems have been characterized and studied. Polylactic acid (PLA), polyglycolic acid (PGA) and their copolymers polylactic-co-glycolic acid (PLGA) are some of the most well-characterized biomaterials with regard to design and performance for drug-delivery applications. See Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S. and Shakeshelf, K. M. “Polymeric Systems for Controlled Drug Release.” Chem. Rev. 1999, 99, 3181-3198 and Panyam J, Labhasetwar V. “Biodegradable nanoparticles for drug and gene delivery to cells and tissue.” Adv Drug Deliv Rev. 2003, 55, 329-47. Biodegradable systems based on polyorthoesters have also been investigated. See Heller, J.; Barr, J.; Ng, S. Y.; Abdellauoi, K. S. and Gurny, R. “Poly(ortho esters): synthesis, characterization, properties and uses.” Adv. Drug Del. Rev. 2002, 54, 1015-1039. Polyanhydride systems have also been investigated. Such polyanhydrides are typically biocompatible and may degrade in vivo into relatively non-toxic compounds that are eliminated from the body as metabolites. See Kumar, N.; Langer, R. S. and Domb, A. J. “Polyanhydrides: an overview.” Adv. Drug Del. Rev. 2002, 54, 889-91.
Amino acid-based polymers have also been considered as a potential source of new biomaterials. Poly-amino acids having good biocompatibility have been investigated to deliver low molecular-weight compounds. A relatively small number of polyglutamic acid and copolymers have been identified as candidate materials for drug delivery. See Bourke, S. L. and Kohn, J. “Polymers derived from the amino acid L-tyrosine: polycarbonates, polyarylates and copolymers with poly(ethylene glycol).” Adv. Drug Del. Rev., 2003, 55, 447-466.
Acid-sensitive polymers containing acetal linkages have been reported, see Tomlinson, R. et al., “Pendent Chain Functionalized Polyacetals That Display pH-Dependent Degradation: A Platform for the Development of Novel Polymer Therapeutics,” Macromolecules 35, 473-480 (2002); Tomlinson R, Heller J, Brocchini S, Duncan R. Polyacetal-doxorubicin conjugates designed for pH-dependent degradation. Bioconjug Chem. 2003 November-December; 14(6):1096-106; and Murthy, N., Thng, Y. X., Schuck, S., Xu, M. C. & Fréchet, J. M. J., “A Novel Strategy for Encapsulation and Release of Proteins: Hydrogels and Microgels with Acid-Labile Acetal Cross-Linkers,” J. Am. Chem. Soc. 124, 12398-12399 (2002).
SUMMARY OF THE INVENTION
An embodiment provides a polymer comprising a recurring unit of the formula (I):




wherein:
X1 and X2 are each independently selected from the group consisting of single bond, —O—, —NR1—, and —(CH2)c—CR1R2—;
Y is selected from the group consisting of N, C1 to C20 alkyl and C6-C20 aryl;
Z is selected from the group consisting of —(CH2)m—C(═O)OR3, —(CH2)m—C(═O)SR3, —(CH2)m—C(═O)NR3R4, —(CH2)m—NR3R4, —(CH2)m—NH—C(═O)—R3, —NH—C(═O)—(CH2)m—C(═O)—NR3R4, —NH—C(═O)—(CH2)m—C(═O)—OA, —(CH2)m—NHR5, —NH—C(═O)—(CH2)m—W, and —NH—C(═O)—(CH2)m-Het;
R1, R2 and R5 are each independently selected from the group consisting of hydrogen, C1 to C20 alkyl, and C6-C20 aryl;
R3 and R4 are each independently selected from the group consisting of W, hydrogen, C1 to C20 alkyl, and C6-C20 aryl;
Het represents a heterocyclic ring;
W is selected from the group consisting of lactose, galactose, mannose, transferrin, antibody, antibody fragment, peptide, and imaging agent;
A is selected from the group consisting of succinimyl, H, and alkali metal;
a, b, c, and m are each independently zero or an integer in the range of 1 to 3; and
n is an integer in the range of about 3 to about 10,000.
Another embodiment provides a method for making a polymer comprising a recurring unit of the formula (I), the method comprising reacting a compound of the formula (XII) with a compound of the formula (XIII):




Another embodiment provides a method for making a polymer comprising a recurring unit of the formula (I), the method comprising reacting a compound of the formula (X) with a compound of the formula (XI):




Another embodiment provides a compound of the formula (X):




Another embodiment provides a method for making a compound of the formula (X) comprising reacting N,N′disuccinimidyl carbonate with 3,9-bis(3-aminopropyl)-2,4,8,10-tetraoxaspiro [5,5]undecane.
These and other embodiments are described in greater detail below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a reaction scheme illustrating an embodiment of a method for making a polymer comprising a recurring unit of the formula (I) that comprises reacting a compound of the formula (X) with a compound of the formula (XI).
FIG. 2 is a reaction scheme illustrating an embodiment of a general method for making polymers that comprise a recurring unit of the formula (I).
FIG. 3 is a reaction scheme illustrating an embodiment of a method for making a compound of the formula (X).
FIG. 4 is a reaction scheme illustrating an embodiment of a method of preparing a polymer of the formula (I) using a compound of the formula (X).
FIG. 5 is a reaction scheme illustrating an embodiment of a method of preparing a polymer of the formula (I).
FIG. 6 is a reaction scheme illustrating an embodiment of a method of preparing a polymer of the formula (I).
FIG. 7 is a reaction scheme illustrating an embodiment of a method of preparing a polymer of the formula (I).
FIG. 8 is a reaction scheme illustrating an embodiment of a method of preparing a polymer of the formula (I).
FIG. 9 is a reaction scheme illustrating an embodiment of a method of preparing a polymer of the formula (I).
FIG. 10 is a reaction scheme illustrating an embodiment of a method of preparing a polymer of the formula (I).



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
An embodiment provides a polymer comprising a recurring unit of the formula (I):




wherein:
X1 and X2 are each independently selected from the group consisting of single bond, —O—, —NR1—, and —(CH2)c—CR1R2—;
Y is selected from the group consisting of N, C1 to C20 alkyl and C6-C20 aryl;
Z is selected from the group consisting of —(CH2)m—C(═O)OR3, —(CH2)m—C(═O)SR3, —(CH2)m—C(═O)NR3R4, —(CH2)m—NR3R4, —(CH2)m—NH—C(═O)—R3, —NH—C(═O)—(CH2)m—C(═O)—NR3R4, —NH—C(═O)—(CH2)m—C(═O)—OA, —(CH2)m—NHR5, —NH—C(═O)—(CH2)m—W, and —NH—C(═O)—(CH2)m-Het;
R1, R2 and R5 are each independently selected from the group consisting of hydrogen, C1 to C20 alkyl, and C6-C20 aryl;
R3 and R4 are each independently selected from the group consisting of W, hydrogen, C1 to C20 alkyl, and C6-C20 aryl;
Het represents a heterocyclic ring;
W is selected from the group consisting of lactose, galactose, mannose, transferrin, antibody, antibody fragment, peptide, and imaging agent;
A is selected from the group consisting of succinimyl, H, and alkali metal;
a, b, c, and m are each independently zero or an integer in the range of 1 to 3; and
n is an integer in the range of about 3 to about 10,000.
The symbols X1, X2, Y, Z, R1-R5, W, A, a, b, c, m, and n, as used elsewhere herein, have the same meaning as specified above, unless otherwise stated. Examples of polymers comprising a recurring unit of the formula (I) include polymers comprising one or more recurring units of the formulas (II) to (IX):




The recurring unit of the formula (II) is an example of a recurring unit of the formula (I) in which Z is —NH—C(═O)—(CH2)m—W and W is an imaging agent. The recurring unit of the formula (III) is an example of a recurring unit of the formula (I) in which Z is —NH—C(═O)—(CH2)m-Het.
Polymers comprising a recurring unit of the formula (I) may be homopolymers or copolymers comprising two or more different recurring units of the formula (I), e.g., at least two recurring units of the formulas (I) to (IX). For example, polymers comprising a recurring unit of the formula (I) may comprise at least two recurring units selected from the group consisting of a recurring unit of the formula (VII), a recurring unit of the formula (VIII), and a recurring unit of the formula (IX), wherein each n is individually in the range of about 3 to about 5,000. Polymers comprising a recurring unit of the formula (I) may be copolymers that comprise other recurring units that are not of the formula (I).
Polymers comprising a recurring unit of the formula (I) may be prepared in various ways. For example, FIG. 1 illustrates a method for making a polymer comprising a recurring unit of the formula (I) that comprises reacting a compound of the formula (X) with a compound of the formula (XI):




A general reaction scheme for making polymers that comprise a recurring unit of the formula (I) is shown in FIG. 2. Reaction conditions suitable for carrying out the polymerizations illustrated in FIGS. 1, 2, and 6 are described in the Examples below. Those skilled in the art will understand that the reaction conditions may be varied to produce a wide variety of polymers comprising a recurring unit of the formula (I), including various homopolymers and copolymers. Polymers that comprise a recurring unit of the formula (I) may also be prepared by reacting a compound of the formula (XII) with a compound of the formula (XIII) under reaction conditions similar to those illustrated in FIGS. 1, 2, and 6 and described in the Examples below:




An embodiment provides a compound of the formula (XIV):




The compound of the formula (X) is an example of a compound of the formula (XIV). Compounds of the formula (XIV) are useful for making polymers of the formula (I) and may be prepared in the general manner described in the Examples below for the preparation of the compound of the formula (X).
EXAMPLES
Solvents and reagents were purchased from commercial sources and used without further purification. All amounts and reaction times described below are approximate unless otherwise stated. Molecular weights (MW) are weight average and were determined by aqueous gel permeation chromatography (GPC) using polyethylene glycol standards. 1H and 13C data were measured at room temperature on a 400 MHz (100 MHz for 13C) in CDCl3, D2O or DMSO-d6.
Example 1
A compound of the formula (X) was prepared according to the general scheme illustrated in FIG. 3 as follows: To a stirred solution of 1 (5.17 g, 20 mmol) in DMF (40 mL), a solution of 2 (1.11 g, 4.0 mmol) in DMF (40 mL) was added dropwise under argon. The resulting mixture was stirred for 3 h at room temperature. The completeness of reaction was confirmed by TLC (100% ethyl acetate, Rf=0.38, stained with Molybate/cerium ammonium solution in H2SO4).
The DMF mixture was concentrated down by rotary evaporation. Water was added, and the product compound of the formula (X) was extracted with dichloromethane from water. The organic phase was dried over Na2SO4. Dichloromethane was removed by rotary evaporation and compound (X) was obtained (1.90 g, 84%) as a sticky pale yellow gel.
1H NMR (CDCl3, 400 MHz): 5.88, 4.47, 3.54, 3.24, 3.08, 2.78, 1.67. 13C NMR (CDCl3): δ 170.1, 151.4, 102.1, 70.6, 70.1, 41.9, 32.3, 31.7, 25.6, 23.5.
Example 2
Additional compound (X) was prepared as described in Example 1, except that acetone was used in place of DMF and the yield was 68%.
Example 3
A polymerization was conducted using a compound of the formula (X) to prepare a polymer of the formula (I) according to the scheme illustrated in FIG. 4 as follows: To a stirred dichloromethane (DCM) solution (30 mL) of the compound (X) (0.34 g, 0.61 mmol), was added 4 (80 mg, 0.61 mmol, Aldrich Chemical Co.) in DCM (30 mL) under argon. Immediately, a white solid precipitated from the DCM mixture. The resulting mixture was stirred at room temperature under argon. The white precipitate was filtered out by a gravity filtration and the impure crude white solid (0.41 g) was dried in vacuum. The 0.41 g of crude white solid was redissolved in DMSO and excess acetone was added to induce precipitation and the solution mixture was stirred overnight. The residue was filtered and placed under high vacuum to yield 0.17 g of pale yellow oil as the product 5.
13C NMR (DMSO): δ 158.8, 102.1. Observation of characteristic urea carbonyl peak at 158.8 ppm in 13C NMR and acetal peak at 102.1 ppm.
Examples 4-5
Additional polymerizations were conducted in a manner similar to Example 3 to prepare polymers 8 and 9 of the formula (I) according to the scheme illustrated in FIG. 5.
Examples 6-10
Polymers 12, 13, and 14 of the formula (I) were prepared according to the scheme illustrated in FIG. 6 as follows: A solution of 10 (1.5 g, 3.2 mmol) in acetone (25 mL) was added into a solution of 1 (0.87 g, 3.2 mmol) in acetone (10 mL). White precipitate formed after a few minutes. The reaction mixture was stirred for 15 minutes. The solution was decanted and the residue was washed with more acetone, then dried under vacuum to provide 12 (0.87 g, 1.67 mmol). The MW of 12 is 45,417 daltons with PDI of 1.48. Similar reactions were carried out in DCM and DMF and produced additional amounts of 12 having molecular weights of 45,645 daltons (PDI 1.42) and 58,066 daltons (PDI 1.37), respectively. A similar reaction was carried in acetone to produce 13 having a MW of about 10,000-12,000 daltons.
The CBZ protecting group of 12 was removed by hydrogenation with. catalytic 10% palladium/carbon under 1 atm hydrogen gas. Caution was taken because Pd/C is highly flammable when flammable solvents are near, including conducting the reaction under an inert atmosphere. 12 (4.0 g) was added into a 500-mL flask equipped with a stirring bar. Pd/C (10%, 0.5 g) was added into the flask. The flask was purged with argon. Deoxygenated methanol (150 mL) was added into the flask. Hydrogen gas (1 atm) was introduced and the mixture was stirred under 1 atm hydrogen gas for 1 day. The insoluble residue was filtered. The filtrate was concentrated by rotary evaporation and dried under vacuum to produce 14 (3.0 g). 14 may also be obtained from 13 by using 20% piperidine in DMF.
Example 11
Polymer 16 of the formula (I) was prepared according to the scheme illustrated in FIG. 7 as follows: 14 (2.7 g) was dissolved in dimethylacetamide (20 mL). Glutaric anhydride (15, 1.70 g) was added into the mixture and stirred for 1 day. The reaction was quenched with saturated sodium bicarbonate. Acetone was added to induce precipitation. The residue was filtered and redissolved in water. The product was dialyzed overnight using a cellulosic semi-permeable membrane (molecular weight cutoff=3,500 daltons). The product 16 (0.75 g) was obtained after the water was removed by rotary evaporation.
Example 12
Polymer 17 of the formula (I) was prepared according to the scheme illustrated in FIG. 8 as follows: 16 (1.0 g) was dissolved in water (10 mL). EDC (0.18 g) was added, and NHS (0.11 g) was added. The reaction was stirred for 30 min. The resulting solution was placed on a Sephadex-G25 chromatography column with water as eluent. The product 17 was obtained from the column fractions after freeze-drying.
Example 13-15
Polymers 18-20 of the formula (I) were prepared according to the scheme illustrated in FIG. 9 as follows: Polyacetal 17 (50 mg) was dissolved in PBS (3 mL). Galactosamine-HCl (50 mg) was added into the mixture and stirred for 30 min. The solution was placed on the Sephadex-G25 chromatography column with water as eluent. The product 18 was obtained after freeze-drying the column fractions. Polymers 19 and 20 were obtained similarly.
Examples 16-17
Polymer 22 and 23 of the formula (I) were prepared according to the scheme illustrated in FIG. 10 as follows: Polymer 14 (30 mg) was dissolved in PBS (2 mL). Triethylamine (1 mL) was added in to the polymer solution. A solution of DOTA-NHS ester (100 mg, Macrocyclics, Inc) in PBS (1 mL) was added into the mixture. The mixture was stirred for 15 minutes. Water was removed by rotary evaporation. The crude residue was redissolved with distilled water (2 mL). Polymer 22 was purified by sephadex-G25 gel filtration. Polymer 22 (15 mg) was obtained after freeze-dried.
Polymer 22 (15 mg) was dissolved in PBS (1 mL). A solution of Gd(III)-Cl3 (1 eq) in water (1 mL) was added and stirred for 15 minutes. Polymer 23 was purified by sephadex-G25 gel filtration. Polymer 23 (10 mg) was obtained after freeze-dried.

## Claims

1. A polymer comprising a recurring unit of the formula (I):



wherein:
X1 and X2 are each independently selected from the group consisting of single bond, —O—, —NR1—, and —(CH2)c—CR1R2—;
Y is selected from the group consisting of N, C1 to C20 alkyl and C6-C20 aryl;
Z is selected from the group consisting of —(CH2)m—C(═O)OR3, —(CH2)m—C(═O)SR3, —(CH2)m—C(═O)NR3R4, —(CH2)m—NR3R4, —(CH2)m—NH—C(═O)—R3, —NH—C(═O)—(CH2)m—C(═O)—NR3R4, —NH—C(═O)—(CH2)m—C(═O)—OA, —(CH2)m—NHR5, —NH—C(═O)—(CH2)m—W, and —NH—C(═O)—(CH2)m-Het;
R1, R2 and R5 are each independently selected from the group consisting of hydrogen, C1 to C20 alkyl, and C6-C20 aryl;
R3 and R4 are each independently selected from the group consisting of W, hydrogen, C1 to C20 alkyl, and C6-C20 aryl;
Het represents a heterocyclic ring;
W is an imaging agent;
A is selected from the group consisting of succinimyl, H, and alkali metal;
a, b, c, and m are each independently zero or an integer in the range of 1 to 3; and
n is an integer in the range of about 3 to about 10,000.

2. The polymer of claim 1 of the formula (II):

3. The polymer of claim 1 of the formula (III):

4. The polymer of claim 1 of the formula (IV):

5. The polymer of claim 1 of the formula (V):

6. The polymer of claim 1 that is a copolymer comprising two or more different recurring units of the formula (I).

7. The polymer of claim 6 comprising at least two recurring units selected from the group consisting of a recurring unit of the formula (VII), a recurring unit of the formula(VIII), and a recurring unit of the formula (IX); wherein each n is individually in the range of about 3 to about 5,000:

8. A method of making the polymer of claim 1 comprising reacting a compound of the formula (X) with a compound of the formula (XI):

9. A method of making the polymer of claim 1, comprising reacting a compound of the formula (XII) with a compound of the formula (XIII):