- Email: [email protected]
Synthesis and characterization of new ointment-like poly(ortho esters)
European Polymer Journal 38 (2002) 971–975 www.elsevier.com/locate/europolj
Synthesis and characterization of new ointment-like poly(ortho esters) Jiahui Yu a,*, Zhengrong Lu a, Hua Zheng b, Renxi Zhuo a a
Department of Chemistry, The Key Laboratory of Biomedical Polymers of the Ministry of Education of China, Wuhan University, Wuhan 430072, China b Department of Chemical Industry, Wuhan University of Science and Technology, Wuhan 430074, China Received 4 April 2001; received in revised form 17 August 2001; accepted 20 September 2001
Abstract Ointment-like poly(ortho esters) were synthesized for the first time from the reaction of 3,9-bis(methylene)-2,4,8,10tetraoxaspiro[5. 5] undecane with poly(ethylene glycol)-400, N,N-bis(2-hydroxyethyl)-n-hexadecylamine and N,N-bis (2-hydroxyethyl) palmitamide, respectively. The obtained polymers were characterized by 1 H NMR spectra, 13 C NMR spectra, elemental analyses, light scattering, and measurements of intrinsic viscosity. The influence of catalyst on the intrinsic viscosity of polymers was investigated. The 9-[(1,3-dihydroxy-2-propoxy) methyl] guanine controlled release profiles of hydrophobic ointment-like polymers such as polymer PII in vitro were also discussed. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ointment-like poly(ortho esters); Synthesis; Characterization; Controlled release
1. Introduction Poly(ortho esters) is a class of biodegradable polymer because of the acid-sensitive linkage in the polymer backbone, and their degradation rate can be readily controlled by the incorporation of acid or basic excipients into the polymer matrix [1]. Several types of biocompatible poly(ortho esters) have been prepared and extensively investigated as the carrier for drug delivery system [2]. However, less work has been down on ointment-like poly(ortho esters). As a hydrophobic, bioerodible ointment-like material, the applications of ointment-like poly(ortho esters) are the topical treatment of severe lesions such as burns and decubitus ulcers and for the treatment of periodortal disease. Another important application is its use as a matrix into which sensitive therapeutic agents can be incorporated without
*
Corresponding author. Address: Synica Chemicals, 476 Zhenbei Road, Shanghai 200062, China. E-mail address: [email protected] (J. Yu).
the use of solvents or elevated temperature. This is of particular interest in the delivery of sensitive protein and peptide drugs, which are easily denatured by loss of tertiary structure [2]. It is also possible to develop an injectable subcutaneous depots with the suitable viscosity ointment-like poly(ortho esters). In this article, the monomers containing long alkyl chain such as N,N-bis(2-hydroethyl)-n-hexadecylamine (BHHA) and N,N-bis(2-hydroxyethyl) palmitamide (BHPA) were synthesized. The ointment-like poly(ortho esters) were obtained from the copolymerization reaction of 3,9-bis(methylene)-2,4,8,10-tetraoxaspiro[5. 5] undecane (BMTU) with poly(ethylene glycol)-400 (PEG-400), BHHA, and BHPA (Fig. 1), respectively. All the polymers were characterized via 1 H NMR spectra, 13 C NMR spectra, elemental analyses, light scattering, and measurements of intrinsic viscosity. The influence on the intrinsic viscosity of polymer brought about by the introduction of a catalyst (1 106 mol of iodine per ml of pyridine) was investigated. 9-[(1,3-dihydroxy2-propoxy) methyl] guanine (ganciclovir) was loaded into the hydrophobic ointment-like poly(ortho esters)
0014-3057/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 1 4 - 3 0 5 7 ( 0 1 ) 0 0 2 6 2 - 2
972
J. Yu et al. / European Polymer Journal 38 (2002) 971–975
Fig. 1. Reaction scheme for the synthesis of poly(ortho esters).
such as polymer PII for the investigation of the drug release profiles in vitro.
2. Experimental
washed with 5% Na2 CO3 aqueous solution (1 l 3), and dried twice with anhydrous CaCl2 . After filtration, 160 g of bromoacetaldehyde dimethyl acetal with a boiling point of 144–148 °C was obtained by distillation from 2 g of anhydrous K2 CO3 , and in a 81.5% yield (lit. Beil 1624, bp 148–150 °C).
2.1. Materials and methods Bromine was dried twice with concentrated sulfuric acid. Vinyl acetate and n-hexadecyl bromide were distilled before use. Methanol was dried by reflexing with magnesium/iodine, then distilled. Tert-butanol was distilled from sodium. p-Toluenesulfonic acid monohydrate and bisphenol A were recrystallized from water and toluene, respectively. PEG-400 was dried by azeotropic distillation with anhydrous toluene. Iodine was purified by sublimation. Pyridine, tetrahydrofuran, diglyme, and petroleum ether (boil point 60–90 °C) were kept over KOH for 48 h and distilled. Dichloromethane was dried with CaCl2 before distillation. N,N-bis(2-hydroxyethyl) amine was purified by vacuum distillation. 9-[(1,3-Dihydroxy-2-propoxy) methyl] guanine (ganciclovir) was recrystallized from water. IR spectra were measured on a Nicolet 170SX Fourier transform infrared spectrometer. 1 H NMR spectra and 13 C NMR spectra were recorded on Varian EM-360 spectrometer and Bruker ARX-500 spectrometer, respectively. Elemental analyses were conducted on a Carlo Erba 1106 elemental analyzer. Melting points were determined by a Yanaco micro-melting point apparatus. A Ubbelohde viscometer was used for the determination of the intrinsic viscosity of polymer at 30 °C in tetrahydrofuran. The measurements of weightaverage molecular weight were performed on a light scattering instrument (DAWNâ DSP, Wyatt Technology Co.). 2.2. Synthesis of bromoacetaldehyde dimethyl acetal 195 g (1.22 mol) of anhydrous bromine in 50 ml of dry dichloromethane was added dropwise to a solution of 105 g (1.22 mol) of vinyl acetate in 200 ml of dry dichloromethane previous cooled down to 10 °C. After the addition, the reaction was kept at 0 °C with vigorous stirring for 2 h. Then the mixture was poured into a large amount of anhydrous methanol (800 ml). After stirring for at least 60 h at room temperature, the solution was diluted with 3 l of water. The oil phase was
2.3. Synthesis of 3,9-bis(bromomethyl)-2,4,8,10-tetraoxaspiro[5. 5] undecane 168 g (0.994 mol) of bromoacetaldehyde dimethyl acetal, 160 g (0.45 mol) of pentaerythritol, 0.8 g of p-toluenesulfonic acid monohydrate, and 400 ml of anhydrous diglyme were added into a 1 l, three-necked flask equipped with a magnetic stirrer, immersion thermometer, and a Claisen distillation head connected to a vacuum system. The mixture was stirred at 110 °C for about 3 h. As the distillation of methanol subsided, the reaction temperature was incrementally increased to 150 °C, and the remainder of the methanol was collected. Then the reaction temperature was lowered to 110 °C, and a vacuum was gradually applied to remove residual methanol, excess bromoacetal, and a large amount of diglyme. The residue was added dropwise into 3 l of ice cold water with vigorous stirring. The solid was collected by filtration, washed thoroughly with ice cold water. After recrystallization from acetone, BMTU (135 g, mp ¼ 100–102 °C) was obtained in a 87% yield, and characterized with 1 H NMR and elemental analysis. 1 H NMR (CDCl3 , d ppm): 3.2–3.6 (m, 10H, BrCH2 CH , OCH2 ), 4.4–4.6 (m, 4H, BrCH2 ). Elem. anal.— calcd.: for C9 H14 O4 Br2 , C 31.21%, H 4.05%; found: C 31.31%, H 4.04%. 2.4. Synthesis of 3,9-bis(methyl)-2,4,8,10-tetraoxaspiro[5. 5] undecane 700 ml of anhydrous tert-butanol and 29.8 g (0.76 mol) fresh potassium were added into a 1 l, three-necked flask equipped with a magnetic stirrer and a reflux condenser under anhydrous argon atmosphere. The mixture was stirred at room temperature until the solid potassium was disappeared. Then 120 g (0.35 mol) of dry BMTU was charged into flask, and the reaction mixture was held at reflex temperature for about 12 h. The large amount of tert-butanol was removed by distillation at atmosphere pressure, while the remainder was distilled under gentle vacuum. The solid residue was
J. Yu et al. / European Polymer Journal 38 (2002) 971–975
dissolved with 600 ml of anhydrous petroleum ether (60– 90 °C) at reflex temperature. Then the reaction temperature was lowered to 40 °C, and the residue solid was removed by filtration. The filtrate was sealed in a flask and maintained in a freezer overnight. The crude product was isolated by filtration. After recrystallization twice from anhydrous petroleum ether (60–90 °C), 35 g of white solid (mp 54–55 °C) was obtained in a 55% yield (lit. mp 55–56 °C [4]). The product is very sensitive to water and acid, all manupulation must be performed in an anhydrous argon atmosphere. 2.5. Synthesis of N,N-bis(2-hydroxyethyl)-n-hexadecylamine N-hexadecyl bromide, N,N-bis(2-hydroethyl) amine, and anhydrous K2 CO3 at a molar ratio of 1:1.3:1.3 were added into 1 l, two-necked flask equipped with a magnetic stirrer and a reflex condenser. The reaction was carried at in anhydrous ethanol by reflexing for 24 h with vigorous stirring. After filtration, the filtrate was added into a large amount of ice cold water. The waxy solid was collected and washed thoroughly with cold water. The crystals of BHHA from petroleum ether (60– 90 °C) was obtained in a 60% yield (mp ¼ 44–45 °C, lit. 43 °C [4]). 2.6. Synthesis of N,N-bis(2-hydroxyethyl) palmitamide The solution of 27 g (0.1 mol) of palmitoyl chloride in dichloromethane was added into the solution of 21 g (0.2 mol) of N,N-bis(2-hydroxyethyl) amine in 50 ml of dichloromethane. The reaction mixture was stirred at 20 °C overnight, then at 50 °C for 2 h. After the solvent was distilled, the crude product was precipitated from 20 ml of 0.1 mol/l of dilute HCl. The precipitation was collected by filtration, washed thoroughly with water. BHPA (10.3 g) was eluted with ethyl acetate from 100– 200 mesh silica gel column (Rf ¼ 2:0–2.5). Yield: 30%, mp 69–70 °C. Elem. anal.—calcd.: for C20 H41 O3 N, C 69.97%, H 11.95%, N 4.08%; found: C 69.90%, H 11.84%, N 4.00%. IR(KBr, cm1 ): 3412m, 3296m (mO–H), 2920s (mC–H), 2649s (mCOC–H), 1619s (mC@O), 1477m (mC–N), 1058s (mC–O). 1 H NMR(CDCl3 , d ppm): 0.8–1.2 (m, 29H, CH3 (CH2 )13 ), 2.2–2.4 (t, 2H, CH2 CO), 3.4–3.8 (m, 8H, NCH2 CH2 O). 2.7. Syntheses of polymers Polymer PII was synthesized according to the following procedure: BMTU (2.86 g, 0.0155 mol) and equimolar of BHHA (5.11 g) were weighed into a 100 ml, two-necked flask in a dry argon atmosphere. Then 50 ml of anhydrous tetrahydrofuran was injected into the flask.
973
The mixture was stirred at room temperature overnight with or without iodine/pyridine as catalyst (1 ml of pyridine solution containing 1 106 mol of iodine [3]). 7.97 g of polymer labeled as PII was obtained by removing the solvent under vacuum. Complete reaction was confirmed by the absence of the hydroxyl band at 3470 cm1 and the absence of the acetal band at 1675 cm1 in the infrared spectrum. Polymer PII , Elem. anal.—calcd.: for C29 H55 O6 N, C 67.84%, H 10.72%, N 2.73%; found: C 67.43%, H 10.52%, N 2.44%. 1 H NMR(CDCl3 , d ppm): 0.8–1.4 (m, 31H, CH3 (CH2 )14 ), 2.1 (s, 6H, CCH3 ), 3.4–4.0 (m, 8H, CCH2 , 2H, NCH2 , 8H, NCH2 CH2 O). 13 C NMR(CDCl3 , d ppm): 111.43 (2C, spiro C–O), 62.91–61.29 (6C, OCH2 ), 56.02–54.16 (3C, NCH2 ), 31.86–14.07 (2C, CH3 , 16C, CH3 (CH2 )15 , 1C, spiro C–C). While diol monomers were PEG-400, BHPA and bisphenol A, the polymers labeled as PI , PIII and PIV were obtained, respectively (Fig. 1). Polymer PI , Elem. anal.—calcd.: for C26:36 H48:72 O13:68 , C 54.17%, H 8.34%; found: C 53.68%, H 8.40%. 1 H NMR(D2 O, d ppm): 2.1 (s, 6H, CCH3 ), 3.6–3.8 (m, 34.72H, OCH2 CH2 O), 4.1 (s, 8H, CCH2 O). 13 C NMR(D2 O, d ppm): 111.31 (2C, spiro C–O), 65.03–62.47 (4C, OCH2 , 17.36C, OCH2 CH2 O), 22.43 (2C, CH3 , 1C, spiro C–C). Polymer PIII , Elem. anal.—calcd.: for C29 H53 O7 N, C 66.03%, H 10.06%, N 2.66%; found: C 65.69%, H 10.05%, N 2.35%. 1 H NMR(CDCl3 , d ppm): 0.8–1.4 (m, 29H, CH3 (CH2 )13 ), 2.05 (s, 6H, CCH3 ), 2.2–2.4 (s, 2H, CH2 –C@O), 3.4–4.0 (m, 8H, CCH2 , 8H, OCH2 CH2 N). 13 C NMR(CDCl3 , d ppm): 175.48 (1C, C@O), 111.49 (2C, spiro C–O), 62.89–60.60 (6C, OCH2 ), 52.17–50.54 (2C, NCH2 ), 31.83–14.04 (2C, CH3 , 15C, CH3 (CH2 )14 , 1C, spiro C–C).
2.8. Drug loading and release from polymer PII Ointment-like polymer PII was selected for the study of drug controlled release profiles. A certain amount of polymer PII and 9-[(1,3-dihydroxy-2-propoxy) methyl] guanine (ganciclovir) were weighed into a 25 40 weighing bottle, and mixed thoroughly at room temperature. The contents of 9-[(1,3-dihydroxy-2-propoxy) methyl] guanine were 5 and 10 wt.%, respectively. After the drug release system standing for 36 h at room temperature, 5 ml of phosphate buffer solution (pH 7.4, 0.2 mol/l) was added into the weighing bottle. Drug release from the ointment was carried out at 37 °C with continuous shaking. At various times, the phosphate buffer solution was removed from the medium and 5 ml of fresh phosphate buffer solution was added to the weighing bottle. The ganciclovir concentration was determined by using a Shimadzu UV-160 spectrophotometer at 270 nm [5].
974
J. Yu et al. / European Polymer Journal 38 (2002) 971–975
Table 1 The intrinsic viscosity and molecular weight of poly(ortho esters) Polymer a
Diol ½g (g1 ml)b Mol. wt.c
PI
PII
PIII
PIV
PEG-400 15.65 (10.63) 193,000
BHHA 12.56 (11.16) 215,000
BHPA 11.72 (10.05) 176,000
Bisphenol A 12.75 (10.24) 9800
a
PEG-400: Poly(ethyl glycol)-400, BHPA: N,N-bis(2-hydroxyethyl) palmitamide, BHHA: N,N-bis(2-hydroethyl)-n-hexadecylamine. The data bracketed were the intrinsic viscosity of polymer without use of catalyst. c Determined by light scattering (DAWNâ DSP, Wyatt Technology Co.) in tetra-hydrofuran at 30 °C. b
3. Results and discussion 3.1. Syntheses, properties and characterization Fig. 1 shows the reaction scheme for the synthesis of poly(ortho esters). Polymer PI , PII and PIII were obtained from the reaction of BMTU with PEG-400, BHHA, and BHPA, respectively. In order to improve the hydrophilicity of polymer PI , PEG-400 was introduced to the polymer backbone. The introduction of a tertiary ammonium group to the polymer PI is to improve the acid sensitivity of poly(ortho esters) in storage. A long alkyl group was selected as a side group of polymer PII due to its good biocompatibility. All the poly(ortho esters) obtained from BMTU and PEG-400, BHHA or BHPA are ointment-like polymers. The poly(ortho esters) from BMTU and PEG-400 (PI ) is water soluble, and the poly(ortho esters) obtained from BMTU and BHHA (PII ) or BHPA (PIII ) are hydrophobic polymers which are ideal for the application as bioerodible drug releasing depots. The structures of polymer PI , PII and PIII were characterized by 1 H NMR spectra, 13 C NMR spectra and elemental analyses. A typical 13 C NMR spectrum of polymer PII is shown in Fig. 2. The region between 32.86 and 14.07 ppm showed the absorption peaks of –CH3 ,
Fig. 2. 13 C NMR spectrum of polymer PII in CDCl3 at room temperature.
CH3 (CH2 )15 and spiro C–C. The absorption peaks of NCH2 and OCH2 were appeared at 56.02–54.16 and 62.91–61.29 ppm, respectively. The absorption peaks at 111.43 ppm assigned to spiro C–O confirmed the expected structure of polymer PII . 3.2. The influence of catalyst on the intrinsic viscosity of polymers BMTU is a diketene acetal structure. Heller et al. reported that the pyridine solution of iodine was an effective catalyst for the copolymerization reaction of diols with BMTU [3]. However, we found that the presence of iodine/pyridine catalyst decreased the intrinsic viscosity of the polymer as compared with the corresponding reaction without the catalyst. This was also verified by the reaction of BMTU with bisphenol A (PIV ) (Table 1). 3.3. Drug release properties of polymer PII 9-[(1,3-dihydroxy-2-propoxy) methyl] guanine (ganciclovir) is a novel nucleoside analogue as antiherpetic agent. It was mainly used for the treatment of cytomegalovirus infections and AIDS [5]. Current therapy involves either massive doses of the antiviral drug ganciclovir through a catheter inserted into the chest with conse-
Fig. 3. Daily release of ganciclovir from polymer PII at pH 7.4 and 37 °C (drug load, (a) 10 wt.%, (b) 5 wt.%).
J. Yu et al. / European Polymer Journal 38 (2002) 971–975
quent side effects, or a daily injection of ganciclovir into the eye, an unpleasant and painful procedure. Fig. 3 shows the ganciclovir controlled release properties of polymer PII . The contents of ganciclovir were 5 wt.% (Fig. 3b) and 10 wt.% (Fig. 3a), respectively. Aside from an initial burst, which subsides after day 2, 3–2.1 mg/day (Fig. 3b) and 3.1–5.2 mg/day (Fig. 3a) constant release from day 4 to day 18 have been achieved. The detail results of ganciclovir controlled release properties of polymer PII will be reported soon. The toxicology of polymers and their degradation products will also be discussed in another paper.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China.
975
References [1] Heller J. Control of polymer surface erosion by the use of excipients. In: Chielini E, Giusti P, Migliaresi C, Nicolais L, editors. Polymers in Medicine II. New York, NY: Plenum Press; 1986. p. 357–68. [2] Heller J, Maa YF, Wuthrich P, Ng SY, Duncan R. Recent developments in the synthesis and utilization of poly(ortho esters). J Control Release 1991;16:3–14. [3] Heller J, Penhale DWH, Helwing RF. Preparation of poly(ortho esters) by the reaction of diketene acetals and polyols. J Polym Sci: Polym Lett Ed 1980;18:619– 24. [4] Marconi W, Martinelli A, Piozzi A. Synthesis and characterization of new amphiphilic polyurethane compositions. Eur Polym J 1995;31(2):131–4. [5] Martin JC, Dvorak CA, Smee DF, Matthews TR, Verheyden JPH. 9-[(1,3-Dihydroxy-2-propoxy) methyl] guanine: a new potent and selective antiherpes agent. J Med Chem 1983;26(5):759–61.
Synthesis and characterization of new ointment-like poly(ortho esters) Jiahui Yu a,*, Zhengrong Lu a, Hua Zheng b, Renxi Zhuo a a
Department of Chemistry, The Key Laboratory of Biomedical Polymers of the Ministry of Education of China, Wuhan University, Wuhan 430072, China b Department of Chemical Industry, Wuhan University of Science and Technology, Wuhan 430074, China Received 4 April 2001; received in revised form 17 August 2001; accepted 20 September 2001
Abstract Ointment-like poly(ortho esters) were synthesized for the first time from the reaction of 3,9-bis(methylene)-2,4,8,10tetraoxaspiro[5. 5] undecane with poly(ethylene glycol)-400, N,N-bis(2-hydroxyethyl)-n-hexadecylamine and N,N-bis (2-hydroxyethyl) palmitamide, respectively. The obtained polymers were characterized by 1 H NMR spectra, 13 C NMR spectra, elemental analyses, light scattering, and measurements of intrinsic viscosity. The influence of catalyst on the intrinsic viscosity of polymers was investigated. The 9-[(1,3-dihydroxy-2-propoxy) methyl] guanine controlled release profiles of hydrophobic ointment-like polymers such as polymer PII in vitro were also discussed. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ointment-like poly(ortho esters); Synthesis; Characterization; Controlled release
1. Introduction Poly(ortho esters) is a class of biodegradable polymer because of the acid-sensitive linkage in the polymer backbone, and their degradation rate can be readily controlled by the incorporation of acid or basic excipients into the polymer matrix [1]. Several types of biocompatible poly(ortho esters) have been prepared and extensively investigated as the carrier for drug delivery system [2]. However, less work has been down on ointment-like poly(ortho esters). As a hydrophobic, bioerodible ointment-like material, the applications of ointment-like poly(ortho esters) are the topical treatment of severe lesions such as burns and decubitus ulcers and for the treatment of periodortal disease. Another important application is its use as a matrix into which sensitive therapeutic agents can be incorporated without
*
Corresponding author. Address: Synica Chemicals, 476 Zhenbei Road, Shanghai 200062, China. E-mail address: [email protected] (J. Yu).
the use of solvents or elevated temperature. This is of particular interest in the delivery of sensitive protein and peptide drugs, which are easily denatured by loss of tertiary structure [2]. It is also possible to develop an injectable subcutaneous depots with the suitable viscosity ointment-like poly(ortho esters). In this article, the monomers containing long alkyl chain such as N,N-bis(2-hydroethyl)-n-hexadecylamine (BHHA) and N,N-bis(2-hydroxyethyl) palmitamide (BHPA) were synthesized. The ointment-like poly(ortho esters) were obtained from the copolymerization reaction of 3,9-bis(methylene)-2,4,8,10-tetraoxaspiro[5. 5] undecane (BMTU) with poly(ethylene glycol)-400 (PEG-400), BHHA, and BHPA (Fig. 1), respectively. All the polymers were characterized via 1 H NMR spectra, 13 C NMR spectra, elemental analyses, light scattering, and measurements of intrinsic viscosity. The influence on the intrinsic viscosity of polymer brought about by the introduction of a catalyst (1 106 mol of iodine per ml of pyridine) was investigated. 9-[(1,3-dihydroxy2-propoxy) methyl] guanine (ganciclovir) was loaded into the hydrophobic ointment-like poly(ortho esters)
0014-3057/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 1 4 - 3 0 5 7 ( 0 1 ) 0 0 2 6 2 - 2
972
J. Yu et al. / European Polymer Journal 38 (2002) 971–975
Fig. 1. Reaction scheme for the synthesis of poly(ortho esters).
such as polymer PII for the investigation of the drug release profiles in vitro.
2. Experimental
washed with 5% Na2 CO3 aqueous solution (1 l 3), and dried twice with anhydrous CaCl2 . After filtration, 160 g of bromoacetaldehyde dimethyl acetal with a boiling point of 144–148 °C was obtained by distillation from 2 g of anhydrous K2 CO3 , and in a 81.5% yield (lit. Beil 1624, bp 148–150 °C).
2.1. Materials and methods Bromine was dried twice with concentrated sulfuric acid. Vinyl acetate and n-hexadecyl bromide were distilled before use. Methanol was dried by reflexing with magnesium/iodine, then distilled. Tert-butanol was distilled from sodium. p-Toluenesulfonic acid monohydrate and bisphenol A were recrystallized from water and toluene, respectively. PEG-400 was dried by azeotropic distillation with anhydrous toluene. Iodine was purified by sublimation. Pyridine, tetrahydrofuran, diglyme, and petroleum ether (boil point 60–90 °C) were kept over KOH for 48 h and distilled. Dichloromethane was dried with CaCl2 before distillation. N,N-bis(2-hydroxyethyl) amine was purified by vacuum distillation. 9-[(1,3-Dihydroxy-2-propoxy) methyl] guanine (ganciclovir) was recrystallized from water. IR spectra were measured on a Nicolet 170SX Fourier transform infrared spectrometer. 1 H NMR spectra and 13 C NMR spectra were recorded on Varian EM-360 spectrometer and Bruker ARX-500 spectrometer, respectively. Elemental analyses were conducted on a Carlo Erba 1106 elemental analyzer. Melting points were determined by a Yanaco micro-melting point apparatus. A Ubbelohde viscometer was used for the determination of the intrinsic viscosity of polymer at 30 °C in tetrahydrofuran. The measurements of weightaverage molecular weight were performed on a light scattering instrument (DAWNâ DSP, Wyatt Technology Co.). 2.2. Synthesis of bromoacetaldehyde dimethyl acetal 195 g (1.22 mol) of anhydrous bromine in 50 ml of dry dichloromethane was added dropwise to a solution of 105 g (1.22 mol) of vinyl acetate in 200 ml of dry dichloromethane previous cooled down to 10 °C. After the addition, the reaction was kept at 0 °C with vigorous stirring for 2 h. Then the mixture was poured into a large amount of anhydrous methanol (800 ml). After stirring for at least 60 h at room temperature, the solution was diluted with 3 l of water. The oil phase was
2.3. Synthesis of 3,9-bis(bromomethyl)-2,4,8,10-tetraoxaspiro[5. 5] undecane 168 g (0.994 mol) of bromoacetaldehyde dimethyl acetal, 160 g (0.45 mol) of pentaerythritol, 0.8 g of p-toluenesulfonic acid monohydrate, and 400 ml of anhydrous diglyme were added into a 1 l, three-necked flask equipped with a magnetic stirrer, immersion thermometer, and a Claisen distillation head connected to a vacuum system. The mixture was stirred at 110 °C for about 3 h. As the distillation of methanol subsided, the reaction temperature was incrementally increased to 150 °C, and the remainder of the methanol was collected. Then the reaction temperature was lowered to 110 °C, and a vacuum was gradually applied to remove residual methanol, excess bromoacetal, and a large amount of diglyme. The residue was added dropwise into 3 l of ice cold water with vigorous stirring. The solid was collected by filtration, washed thoroughly with ice cold water. After recrystallization from acetone, BMTU (135 g, mp ¼ 100–102 °C) was obtained in a 87% yield, and characterized with 1 H NMR and elemental analysis. 1 H NMR (CDCl3 , d ppm): 3.2–3.6 (m, 10H, BrCH2 CH , OCH2 ), 4.4–4.6 (m, 4H, BrCH2 ). Elem. anal.— calcd.: for C9 H14 O4 Br2 , C 31.21%, H 4.05%; found: C 31.31%, H 4.04%. 2.4. Synthesis of 3,9-bis(methyl)-2,4,8,10-tetraoxaspiro[5. 5] undecane 700 ml of anhydrous tert-butanol and 29.8 g (0.76 mol) fresh potassium were added into a 1 l, three-necked flask equipped with a magnetic stirrer and a reflux condenser under anhydrous argon atmosphere. The mixture was stirred at room temperature until the solid potassium was disappeared. Then 120 g (0.35 mol) of dry BMTU was charged into flask, and the reaction mixture was held at reflex temperature for about 12 h. The large amount of tert-butanol was removed by distillation at atmosphere pressure, while the remainder was distilled under gentle vacuum. The solid residue was
J. Yu et al. / European Polymer Journal 38 (2002) 971–975
dissolved with 600 ml of anhydrous petroleum ether (60– 90 °C) at reflex temperature. Then the reaction temperature was lowered to 40 °C, and the residue solid was removed by filtration. The filtrate was sealed in a flask and maintained in a freezer overnight. The crude product was isolated by filtration. After recrystallization twice from anhydrous petroleum ether (60–90 °C), 35 g of white solid (mp 54–55 °C) was obtained in a 55% yield (lit. mp 55–56 °C [4]). The product is very sensitive to water and acid, all manupulation must be performed in an anhydrous argon atmosphere. 2.5. Synthesis of N,N-bis(2-hydroxyethyl)-n-hexadecylamine N-hexadecyl bromide, N,N-bis(2-hydroethyl) amine, and anhydrous K2 CO3 at a molar ratio of 1:1.3:1.3 were added into 1 l, two-necked flask equipped with a magnetic stirrer and a reflex condenser. The reaction was carried at in anhydrous ethanol by reflexing for 24 h with vigorous stirring. After filtration, the filtrate was added into a large amount of ice cold water. The waxy solid was collected and washed thoroughly with cold water. The crystals of BHHA from petroleum ether (60– 90 °C) was obtained in a 60% yield (mp ¼ 44–45 °C, lit. 43 °C [4]). 2.6. Synthesis of N,N-bis(2-hydroxyethyl) palmitamide The solution of 27 g (0.1 mol) of palmitoyl chloride in dichloromethane was added into the solution of 21 g (0.2 mol) of N,N-bis(2-hydroxyethyl) amine in 50 ml of dichloromethane. The reaction mixture was stirred at 20 °C overnight, then at 50 °C for 2 h. After the solvent was distilled, the crude product was precipitated from 20 ml of 0.1 mol/l of dilute HCl. The precipitation was collected by filtration, washed thoroughly with water. BHPA (10.3 g) was eluted with ethyl acetate from 100– 200 mesh silica gel column (Rf ¼ 2:0–2.5). Yield: 30%, mp 69–70 °C. Elem. anal.—calcd.: for C20 H41 O3 N, C 69.97%, H 11.95%, N 4.08%; found: C 69.90%, H 11.84%, N 4.00%. IR(KBr, cm1 ): 3412m, 3296m (mO–H), 2920s (mC–H), 2649s (mCOC–H), 1619s (mC@O), 1477m (mC–N), 1058s (mC–O). 1 H NMR(CDCl3 , d ppm): 0.8–1.2 (m, 29H, CH3 (CH2 )13 ), 2.2–2.4 (t, 2H, CH2 CO), 3.4–3.8 (m, 8H, NCH2 CH2 O). 2.7. Syntheses of polymers Polymer PII was synthesized according to the following procedure: BMTU (2.86 g, 0.0155 mol) and equimolar of BHHA (5.11 g) were weighed into a 100 ml, two-necked flask in a dry argon atmosphere. Then 50 ml of anhydrous tetrahydrofuran was injected into the flask.
973
The mixture was stirred at room temperature overnight with or without iodine/pyridine as catalyst (1 ml of pyridine solution containing 1 106 mol of iodine [3]). 7.97 g of polymer labeled as PII was obtained by removing the solvent under vacuum. Complete reaction was confirmed by the absence of the hydroxyl band at 3470 cm1 and the absence of the acetal band at 1675 cm1 in the infrared spectrum. Polymer PII , Elem. anal.—calcd.: for C29 H55 O6 N, C 67.84%, H 10.72%, N 2.73%; found: C 67.43%, H 10.52%, N 2.44%. 1 H NMR(CDCl3 , d ppm): 0.8–1.4 (m, 31H, CH3 (CH2 )14 ), 2.1 (s, 6H, CCH3 ), 3.4–4.0 (m, 8H, CCH2 , 2H, NCH2 , 8H, NCH2 CH2 O). 13 C NMR(CDCl3 , d ppm): 111.43 (2C, spiro C–O), 62.91–61.29 (6C, OCH2 ), 56.02–54.16 (3C, NCH2 ), 31.86–14.07 (2C, CH3 , 16C, CH3 (CH2 )15 , 1C, spiro C–C). While diol monomers were PEG-400, BHPA and bisphenol A, the polymers labeled as PI , PIII and PIV were obtained, respectively (Fig. 1). Polymer PI , Elem. anal.—calcd.: for C26:36 H48:72 O13:68 , C 54.17%, H 8.34%; found: C 53.68%, H 8.40%. 1 H NMR(D2 O, d ppm): 2.1 (s, 6H, CCH3 ), 3.6–3.8 (m, 34.72H, OCH2 CH2 O), 4.1 (s, 8H, CCH2 O). 13 C NMR(D2 O, d ppm): 111.31 (2C, spiro C–O), 65.03–62.47 (4C, OCH2 , 17.36C, OCH2 CH2 O), 22.43 (2C, CH3 , 1C, spiro C–C). Polymer PIII , Elem. anal.—calcd.: for C29 H53 O7 N, C 66.03%, H 10.06%, N 2.66%; found: C 65.69%, H 10.05%, N 2.35%. 1 H NMR(CDCl3 , d ppm): 0.8–1.4 (m, 29H, CH3 (CH2 )13 ), 2.05 (s, 6H, CCH3 ), 2.2–2.4 (s, 2H, CH2 –C@O), 3.4–4.0 (m, 8H, CCH2 , 8H, OCH2 CH2 N). 13 C NMR(CDCl3 , d ppm): 175.48 (1C, C@O), 111.49 (2C, spiro C–O), 62.89–60.60 (6C, OCH2 ), 52.17–50.54 (2C, NCH2 ), 31.83–14.04 (2C, CH3 , 15C, CH3 (CH2 )14 , 1C, spiro C–C).
2.8. Drug loading and release from polymer PII Ointment-like polymer PII was selected for the study of drug controlled release profiles. A certain amount of polymer PII and 9-[(1,3-dihydroxy-2-propoxy) methyl] guanine (ganciclovir) were weighed into a 25 40 weighing bottle, and mixed thoroughly at room temperature. The contents of 9-[(1,3-dihydroxy-2-propoxy) methyl] guanine were 5 and 10 wt.%, respectively. After the drug release system standing for 36 h at room temperature, 5 ml of phosphate buffer solution (pH 7.4, 0.2 mol/l) was added into the weighing bottle. Drug release from the ointment was carried out at 37 °C with continuous shaking. At various times, the phosphate buffer solution was removed from the medium and 5 ml of fresh phosphate buffer solution was added to the weighing bottle. The ganciclovir concentration was determined by using a Shimadzu UV-160 spectrophotometer at 270 nm [5].
974
J. Yu et al. / European Polymer Journal 38 (2002) 971–975
Table 1 The intrinsic viscosity and molecular weight of poly(ortho esters) Polymer a
Diol ½g (g1 ml)b Mol. wt.c
PI
PII
PIII
PIV
PEG-400 15.65 (10.63) 193,000
BHHA 12.56 (11.16) 215,000
BHPA 11.72 (10.05) 176,000
Bisphenol A 12.75 (10.24) 9800
a
PEG-400: Poly(ethyl glycol)-400, BHPA: N,N-bis(2-hydroxyethyl) palmitamide, BHHA: N,N-bis(2-hydroethyl)-n-hexadecylamine. The data bracketed were the intrinsic viscosity of polymer without use of catalyst. c Determined by light scattering (DAWNâ DSP, Wyatt Technology Co.) in tetra-hydrofuran at 30 °C. b
3. Results and discussion 3.1. Syntheses, properties and characterization Fig. 1 shows the reaction scheme for the synthesis of poly(ortho esters). Polymer PI , PII and PIII were obtained from the reaction of BMTU with PEG-400, BHHA, and BHPA, respectively. In order to improve the hydrophilicity of polymer PI , PEG-400 was introduced to the polymer backbone. The introduction of a tertiary ammonium group to the polymer PI is to improve the acid sensitivity of poly(ortho esters) in storage. A long alkyl group was selected as a side group of polymer PII due to its good biocompatibility. All the poly(ortho esters) obtained from BMTU and PEG-400, BHHA or BHPA are ointment-like polymers. The poly(ortho esters) from BMTU and PEG-400 (PI ) is water soluble, and the poly(ortho esters) obtained from BMTU and BHHA (PII ) or BHPA (PIII ) are hydrophobic polymers which are ideal for the application as bioerodible drug releasing depots. The structures of polymer PI , PII and PIII were characterized by 1 H NMR spectra, 13 C NMR spectra and elemental analyses. A typical 13 C NMR spectrum of polymer PII is shown in Fig. 2. The region between 32.86 and 14.07 ppm showed the absorption peaks of –CH3 ,
Fig. 2. 13 C NMR spectrum of polymer PII in CDCl3 at room temperature.
CH3 (CH2 )15 and spiro C–C. The absorption peaks of NCH2 and OCH2 were appeared at 56.02–54.16 and 62.91–61.29 ppm, respectively. The absorption peaks at 111.43 ppm assigned to spiro C–O confirmed the expected structure of polymer PII . 3.2. The influence of catalyst on the intrinsic viscosity of polymers BMTU is a diketene acetal structure. Heller et al. reported that the pyridine solution of iodine was an effective catalyst for the copolymerization reaction of diols with BMTU [3]. However, we found that the presence of iodine/pyridine catalyst decreased the intrinsic viscosity of the polymer as compared with the corresponding reaction without the catalyst. This was also verified by the reaction of BMTU with bisphenol A (PIV ) (Table 1). 3.3. Drug release properties of polymer PII 9-[(1,3-dihydroxy-2-propoxy) methyl] guanine (ganciclovir) is a novel nucleoside analogue as antiherpetic agent. It was mainly used for the treatment of cytomegalovirus infections and AIDS [5]. Current therapy involves either massive doses of the antiviral drug ganciclovir through a catheter inserted into the chest with conse-
Fig. 3. Daily release of ganciclovir from polymer PII at pH 7.4 and 37 °C (drug load, (a) 10 wt.%, (b) 5 wt.%).
J. Yu et al. / European Polymer Journal 38 (2002) 971–975
quent side effects, or a daily injection of ganciclovir into the eye, an unpleasant and painful procedure. Fig. 3 shows the ganciclovir controlled release properties of polymer PII . The contents of ganciclovir were 5 wt.% (Fig. 3b) and 10 wt.% (Fig. 3a), respectively. Aside from an initial burst, which subsides after day 2, 3–2.1 mg/day (Fig. 3b) and 3.1–5.2 mg/day (Fig. 3a) constant release from day 4 to day 18 have been achieved. The detail results of ganciclovir controlled release properties of polymer PII will be reported soon. The toxicology of polymers and their degradation products will also be discussed in another paper.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China.
975
References [1] Heller J. Control of polymer surface erosion by the use of excipients. In: Chielini E, Giusti P, Migliaresi C, Nicolais L, editors. Polymers in Medicine II. New York, NY: Plenum Press; 1986. p. 357–68. [2] Heller J, Maa YF, Wuthrich P, Ng SY, Duncan R. Recent developments in the synthesis and utilization of poly(ortho esters). J Control Release 1991;16:3–14. [3] Heller J, Penhale DWH, Helwing RF. Preparation of poly(ortho esters) by the reaction of diketene acetals and polyols. J Polym Sci: Polym Lett Ed 1980;18:619– 24. [4] Marconi W, Martinelli A, Piozzi A. Synthesis and characterization of new amphiphilic polyurethane compositions. Eur Polym J 1995;31(2):131–4. [5] Martin JC, Dvorak CA, Smee DF, Matthews TR, Verheyden JPH. 9-[(1,3-Dihydroxy-2-propoxy) methyl] guanine: a new potent and selective antiherpes agent. J Med Chem 1983;26(5):759–61.