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Attachment of drugs to polyethylene glycols
Eur. Polvm. J. Vol. 19, No. 12, pp. 1177 1183, 1983 Printed in Great Britain. All rights reserved
Copyright
0014-3057/8353.00+0.00 1983 Pergamon Press Ltd
ATTACHMENT OF D R U G S TO POLYETHYLENE GLYCOLS S. ZALIPSKY, C. GILON and A. ZILKHA Department of Organic Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel (Received 13 December 1982)
Abstract--Polyethylene glycol (PEG) was used as a carrier polymer for the attachment, via end groups, of drugs such as penicillin V, aspirin, amphetamine, quinidine and atropine. For this purpose, methods were developed for the functionalization of PEG; PEG-NH2, PEG-COOH and PEG-NCO were prepared. Use was made of dicyclohexyl carbodiimide together with 4-dimethylamino pyridine or 1-hydroxybenzotriazole for the coupling reactions.
INTRODUCTION
glycols with one methoxy and one hydroxy end group (MPEG) of ~ = 750, 1900 and 5000 (Poly Sciences) were used. 4-Dimethylaminopyridine (DMAP), l-hydroxybenzotriazole (HOBT), dicyclohexylcarbodiimide (DCC)(distilled in vacuo), dibutyltin dilaurate, quinidine and atropine (Fluka) were used. L-Amphetamine (Aldrich), hexamethylene diisocyanate (HDI)(Bayer), Triton B (40°~oaqueous solution) and KOMe 0.1 M in MeOH/toluene (BDH) were used. DMF was distilled from P205 and then from ninhydrin. Chromatograph)" Packard 804 flame ionization GC instrument was used. N 2 was used as the carrier gas (flow rate 20 ml/min) and 6 ft of 1,/8in. glass tubing was used to contain the packing of 15% diethylene glycol succinate on Chromosorb P. Polygram Sil N-HR/u.v. 254 plates from Machery-Nagel Co. were used for thin layer chromatography (TLC).
Currently much interest is shown in the attachment of drugs to polymers as a means of increasing the duration of activity through slow release, or of targetdirecting drugs in the body [1-5]. In the present work we attached to polymers, drugs such as penicillin, aspirin, amphetamine, quinidine and atropine, We chose polyethylene glycol (PEG) as the carrier polymer because it is known to be non-toxic [f~8], non-antigenic and biocompatible, to be soluble in water and organic solvents and by itself to have solubilizing properties. It is not biodegradable [9] and is available in a wide range of molecular weights, Because of these properties, it is widely used in biochemistry and molecular biology, and is commercially used in pharmacological products, cosmetics and food. It is therefore suitable for use as a drug carrier in the body. Various drugs have been attached to PEG such as procaine [10], atropine [11] and various salicylates [12]. The polymer-drug conjugates showed longer duration of activity, due to slow release, Insulin as well as various enzymes have also been attached to P E G leading to products which retain their biological and enzymatic activity [13]. To attach the drugs to PEG, use was made of the
boxyl and isocyanate groups, Two approaches were used for the functionalization of PEG: (1)changing the terminal hydroxyl group, through a series of reactions, to a more active functional group; (2) reaction of P E G under controlled conditions with difunctional compounts so that one of its functional groups reacts with PEG and
MPEG-1900-CI [CHsO(CH2CH20)415CH2CH2CI ] MPEG-1900 (19g, 0.01 mol) was dissolved in toluene (100ml) and dried by azeotropic distillation. Dry pyridine (0.01 tool) was added and thionyl chloride (0.03mol), freshly distilled from quinoline, was added dropwise during 30 rain under reflux. The mixture was heated for 4 hr, cooled to room temperature, filtered from pyridine hydrochloride and the toluene was evaporated in t~acuo. The residue was dissolved in CH2CI 2, dried over anhydrous K2CO ~ and filtered. The filtrate was treated with alumina (50 g) (activated by heating at 120 for 2 hr) and precipitated by cold ether. The polymer was recrystallized from CH2Clz/ether: yield 18g (94°,~;); i.r. (nujol) 1110 (CH2--O--CH2), 663 (C--C1), no absorption for OH at 3300-3500cm ~. Analysis. Calcd for C86H~7~O425: C, 53.75: H, 9.02: CI, 1.85. Found: C, 53.73: H, 8.80: C1, 2.20. MPEG_I9OO_Ns[CHsO(CH2Ct_I20)4~sCH~CH, N d ~ To a solution of MPEG 1900-C1 (l.8g, 0.95mmol) in DMF (10ml), sodium azide (8 mmol) was added and the mixture was stirred at 120' for 2hr. The solution was cooled, filtered and the DMF evaporated in ~acuo. The residue was taken up in CH2CI:, cooled, filtered and precipitated by ether; yield 1.65 g (910~i); i.r. (nujol): 2108 (NO, 1110 (CHzOCH2) no absorption for CI at 663 cm J. Analysis. Calcd for C86HIv3NaO425 : C, 53.56: H, 8.98: N, 2.18. Found: C, 53.38: H, 8.74; N, 2.30.
the other remains active.
MPEG- 1900-NH2 [CH 30 (CH2 CH2 0 )4t ~CH2 CH2 NH2 ]
terminal hydroxyl groups. These can be reacted with drugs containing suitable groups such as carboxyl (esterification). But in order to make possible the attachment of drugs having other functional groups such as amino or hydroxyl, there was need for preparing P E G having other functional end-groups. Methods were therefore devised for converting the terminal hydroxyl groups of P E G into amino, car-
EXPERIMENTAL Materials Polyethylene glycols with two hydroxyl end groups (PEG) of JO'o= 1000, 2000 and 3000 (Fluka) and polyethylene
MPEG-1900-N~ (1.6 g) was dissolved in absolute ethanol (40ml), 10,"~i Pd/C (0.1g) added, and the mixture was hydrogenated in a Parr low pressure hydrogenation apparatus overnight. The catalyst was filtered, and the polymer precipitated by dry ether: yield 1.5 g (95°~i): i.r. (nujol): 3300
1177 EPJ 19/12 ~i
1178
S. ZALIPSKY et al.
(NH), l l l 0 (CH2OCH2), no absorption for N 3 at 2108cm ~, Analysis. Calcd for C86HI73NO42.5: C, 54.29; H, 9.20: N, 0.73, mol. wt 1899. Found: C, 54.14; H, 9.00; N, 0.91, mol. wt 1920, 1890 (titrations with 0.05 N HClO4/dioxane using thymol blue as indicator, and with 0.05 N HC1 using methyl red as indicator, respectively).
MPEG- 1900- COOH[CH 30 (CH2CH 20)41.5 CIt2 CH20COCH2CH2COOH ] (Method I) MPEG-1900 (9.5g, 5mmol) was dissolved in toluene (50ml) and dried by distilling off most of the toluene; succinic anhydride (20 g, 20mmol) was added and the mixture was stirred for 5 hr on an oil bath at 150°. The mixture was cooled, taken up in CH2C12 and the polymer precipitated by ether. The product was recrystallized twice from CH2C12/ether; yield 8.5 g (85~o); i.r. (nujol): 1735 (C=O), l l l 0 c m -t (CH,,OCH2); TLC (BuOH/AcOH/H20, 4:1:1) showed only one peak and no succinic anhydride, Analysis. Calcd for Co~HI78046.5 : C, 53.95; H, 8.89, mol. wt 2000. Found: C, 53.63; H, 8.95; tool. wt 2109, 1979 (titrations with 0.05N KOMe and 0.05N Triton B in pyridine, respectively, using thymol blue as indicator). PEG-3OOO-COOH (Method H) PEG-3000 (6 g, 4 mmol OH groups), succinic anhydride (5mmol), DMAP (4mmol) and triethylamine (4mmol) were dissolved in dioxane (30 ml) and left overnight at room temperature. The dioxane was evaporated in vacuo, the residue taken up in CC14, filtered, and precipitated by ether. It was recrystallized from CH2Cldether yield 6.4 g. Tests on the compound showed that it had the formula HOOCCH2CH2OCO(CH2CH20)67.8CO ) --~N CH2CH2COOH. Me2N
up in CC14, filtered from penicillin V residues, and precipitated by ether; yields 84-87%. TLC (methanol) of the product showed the absence of free penicillin V in the polymer. I . R (nujol): 1780 ( C : O , fl-lactam); 1735 ( C = O ester); 1680 (C-~-O, amide), 1600 (C6H5); l l l 0 c m (CH2OCH2). NMR (CCI4): & 3.6 (s, 4n (OCH2CH2)n); 1.5 (d, 6x, CMe~); 6.9-7.3 (m, 5x, C6H5) (x = 1 for MPEG derivatives; x = 2 for PEG difunctional derivatives). Analysis. Calcd for PEG-3000-penicillin V ester, CI67.6H305.1N4076.852: C, 54.88; H, 8.33; N, 1.53; S, 1.75. Found: C, 55.00; H, 8.31, N, 1.81; S, 1.85. Similar results were obtained with PEG-4000 and MPEG-1900.
PEG-esters of aspirin (general procedure) PEG (1 mmol OH) DCC (1.3 mmol) was added to a solution of aspirin (1.1 mmol) and DMAP (0.25 mmol) in CH2CI 2 (20 ml); the mixture was stirred for 3-4 hr at room temperature. The ppt of DCU was filtered and the filtrate evaporated to dryness. The residue was extracted by acetone, filtered and the polymer precipitated by ether; yields 70-85~o; i.r. (nujol): 1735 (C6H4COO), 1740 (C6H40C=O), no absorption at 3300-3500cm ~(OH): NMR(CCI4): &2.2 (s, 3x, CH3CO), 3.6 (s, 4n, (OCH2CH2)n); 6.75-8.2 (m, 4x, C6H4) x = 1 for MPEG, x = 2 for bifunctional PEG). Analysis. Caled for C62.6HI052029.3 (PEG-1000-aspirin): C, 56.74; H, 7.95. Found: C, 56.65; H, 8.08.
+ i.r, (nujol)1735 ( C = O ester), 1645 ( C ~ N ) , 1560 (COO-), l l l 0 c m -I (CH2OCH2). NMR (CC14): ~ 3.6 (s, 271, OCH,CH2); 3.0 (s, 6, N-Me); 2.5 (s, 8, --COCH2); 6.5-8.1 (m, 4, pyridine-H). Analysis. Calcd for CI43.6H281.1074.8.CTHIoN2: C, 54.38; H, 8.76; N, 0.84; mol. wt 3322. Found: C, 54.73; H, 8.71; N, 1.09; tool. wt 3240, 3294, 3533 (titrations with Triton B, KOCH 3 and 0.05 N HCIO 4 in dioxane using thymol blue as indicator [18]). PEG-2000-COOH was similarly prepared and gave the same results,
Reaction of MPEG-19OO-COOH with amphetamine--Formation of N-acyl urea derivative of MPEG-19OO-COOH (I) To a solution of MPEG-1900-COOH (1 g, 0.5 mmol) and amphetamine (1 mmol) in CH2C12 (20 ml), DCC (0.9 mmol) dissolved in a small volume of CH2C1., was added. The mixture was stirred overnight, filtered from a small amount of DCU, evaporated to dryness, extracted with acetone, precipitated by ether and recrystallized from CH2Cl2/ether; yield 0.95 g. The compound did not contain acid groups (titration with Triton B). NMR (CC14) &: 1-2 (m, 11, O II C6H H); 2.55 (s, 4, C CH2); 3.6 (s, 170, OCH2CH2), no peak in the aromatic region for amphetamine. I.R. (nujol): 1730 ( C = O ester), 1695 (C-~--O imide), 1655cm t ( C = O urea). Analysis. Calcd for Ct03H200N20465: C, 55.98; H, 9.06; N, 1.27. Found: C, 55.62; H, 8.85; N, 1.27. Amides of PEG-COOH and amphetamine (general procedure)
PEG-NCO (general method) A solution of PEG (dried by azeotropic distillation) was added dropwise during 2 hr under anhydrous conditions to hexamethylene diisocyanate (HDI) (5 times molar excess over the OH groups) and dibutyltin dilaurate (0.05 ml). The mixture was stirred for 10 hr, and the polymer precipitated by dry ether and further purified from toluene/ether. I.R. (nujol): 2250 (NCO); 1110 (CH2OCH2); 1715, 1535 (HNCOO), 3300cm -I (NH). N M R (EEl4): ~ 1.3-1.6 (m, CH2CH2CH2), 3.6 (s, OCH2). Molecular weights as determined by titration of NCO groups [36]: MPEG-750-HDI, 965 (required 918); MPEG-1900-HDI, 2160 (required 2068); MPEG 5000-HDI, 4944 (required 5168); PEG-2000-2HDI, 2408 (required 2336).
To a solution of PEG-COOH (0.5mmol COOH), l-amphetamine (1 mmol) and HOBT (0.5 mmol) in CH2C12 (20 ml), a solution of DCC (0.6 mmol) in a small volume of CH2CI 2 was added. The mixture was stirred overnight, filtered from DCU residues, and the polymer was precipitated by ether and recrystallized from absolute ethanol; yield 73-80~o; i.r. (nujol): 1730 ( C = O ester), 1645 ( C = O amide), 3290 (NH) and l l l 0 c m -t (CH2OCH2); NMR (CCl4):&7.2(s, 5x, C6Hs); 2.5 (s, 4x, O== CCH2);1.1(d, 3x, CHC_H_3),3.6 (s, 4n (OCH2CH2)n) (x = 1 for MPEG derivative, x = 2 for PEG derivative). Analysis. Calcd for CII6H214OsoN2 (PEG-2000-COOH amide with amphetamine): C, 57.19; H, 8.79; N, 1.15. Found: C, 56.96; H, 8.79; N, 1.06.
PEG-penicillin V esters (general procedure) K-salt of penicillin V (1.5 mmol) was dissolved in a small volume of water, cooled and acidified with 0.1 N HC1. The precipitated penicillin V was filtered, and dried immediately in vacuo over P20~ for 30min. It was added to a solution of PEG (1 mmol OH) and DMAP (0.25 mmol) in CH2C12 (20 ml), stirred and cooled (ice-water). DCC (1.3 mmol) was added and the mixture was stirred for 2 hr, filtered from precipitated dicyclohexyl urea (DCU) and evaporated. The residue was extracted with acetone, filtered from DCU residues and the product precipitated by ether It was taken
O-glycylquinidine (I11) To a solution of quinidine (1.62 g, 5 mmol), BOC-glycine (1.75 g, 10 mmol) and DMAP (0.61 g, 5 mmol) in CH2CI,, a solution of DCC (2 g, 10 mmol) in CH2CI 2 was added, and the mixture was stirred overnight. TLC (MeOH) then showed that all the quinidine has reacted, DCU was filtered and acetic acid (0.5 ml) was added to destroy excess DCC. After 10 min DCU was filtered, the solvent was evaporated and the residue taken up in acetone, filtered and evaporated. CH2C % (50 ml) was added to dissolve the residue and the solution was washed with NaHCO 3 and dried over MgSO 4.
Attachment of drugs to polyethylene glycols
1179
TLC (MeOH or BuOH-AcOH-H20 4:1:1) showed the titative yields. It is important, in carrying out a series presence of a single compound. The volume of the solution of reactions on polymers, that all reactions proceed was reduced to 5ml and trifluoroacetic acid (5ml) was quantitatively because of the inherent difficulties in added and left to stand at room temperature for 30rain. The separating reacted from unreacted polymer. The solution was evaporated, the residue taken up in CH,CI 2 relatively easy separation of low molecular weight washed with NaHCO~, dried and evaporated yielding a solid compound 1.3g(;72% ). I.R. (nujol): 1740 ( C = O ester) compounds from polymers makes it easy to use 3320 (NH2),1620cm ~ (C=C). NMR (CCID: 6 3.45 (s, 2, large excess of reagent and obtain quantitative conNCH2CO), 7.2 8.6 (m, 5-quinoline HI. versions. This course of reactions was investigated using as Coupling o[ O-glvcylquinidine (llI) to PEG (general pro- a typical compound, monofunctional PEG, namely cedure) MPEG-1900 [MeO(CH2CH20),,--OH,.471, = 1900]. PEG-COOH (0.5 mmol COOH), O-glycylquinidine The chlorination was carried out with thionyl chlo(0.58 mmol) and HOBT (0.57mmol) were dissolved in ride in toluene solution in the presence of pyridine. CHeCI: (20 ml), DCC (0.7 mmol) was added, and the mix- The product upon recrystallization gave a white solid ture stirred overnight. It was filtered and evaporated, extracted by ethyl acetate and filtered from DCU. The polymer with i.r. spectrum showing C - - C 1 absorption at was precipitated by ether and recrystallized from a small 669 c m - t and the absence of OH absorption in the volume of absolute ethanol, yield 82-85°0; i.r. (nujol): 1735 3300-3500 cm ~ region. Its elemental analysis con( C = O ester): 1670 ( C = O amide), 1620 (C=C), II 10cm t formed with the structure. (CH,OCH_,): NMR (CC14): a 3.6 (s, 4n (OCHeCH_,)~): The conversion of the chloride to the azide was 7.2 8.6 (m, 5x, H-quinoline) (x = 1 for MPEG derivative, investigated first using a low molecular weight cornx = 2 for PEG). TLC (BuOH-AcOH-H20, 4:1:1 and pound, namely EtOCH2CH~OCH2CH2Cl so that th e PrOH-20°~, NH 3 H_,O, 6:3:2) showed one spot, course of the reaction could be easily followed by Analysis. Calcd for CI~7sH3310788N6 (PEG-3000-COOH GC. When the reaction with sodium azide was caramide with Ill): C, 57.34: H, 8.43; N, 2.14. Found: C, 57.08: ried out under reflux in M e O H - H 2 0 (7:5), 96°0 of H, 8.11; N, 2.31. the chloride reacted yielding the azide after 4 2 h r PEG carbamates o[atropine (general procedure) reaction time. To shorten the time, the reaction was Freshly prepared PEG-NCO (0.5mmol NCO) was dis- carried out in a dipolar aprotic solvent, viz D M F at solved in dry toluene and atropine (0.6 mmol) and dibutyltin l l O . The reaction was much faster under these dilaurate (0.05 ml) were added. The solution was left over- conditions and 100'/'o substitution was achieved after night and the polymer precipitated by ether and rel hr (Fig. 1). Quantitative substitution was achieved crystallized from CH:Cl>:ether; yields 65-90o TLC after 30 min when the reaction was carried out at (PrOH-CHCI~, 1:1) (visualization by iodine vapour) 120". The long reaction time in M e O H - H 2 0 is due showed one spot and the absence of atropine: i.r. (nujol): to solvation of anions through hydrogen linkages 1665 1720 (wide C = O absorption ester-carbamate), 3330 which can lower the activity of the azide anion. D M F (NH), l l l 0 c m i (CHzOCH,); NMR (CCI4): a 3.6 (s, 4n, (OCH2CH:)°), 7.3 (s, 5x. C6H5); 1.3-1.6 (m, 8x, (CH2)4), leads to solvation of the cation and the azide anion 2.06 (s, 3x, --NCH~) (x = 1 for MPEG derivative, x = 2 for is freer and thus more active [15]. The azido derivaPEG derivative), tive of MPEG-1900 was prepared similarly in D M F Analysis. Calcd for Cjl ~H2090485N3 (MPEG-1900-NCO at 120 from the chloride. It showed the azide absorpcarbamate derivative of atropine): C, 56.47: H, 8.86; N, tion at 2108cm ~ and had the required elemental 1.78. Found: C. 56.62; H, 9.23; N, 1.83. analysis.
MPEG-19OO-COOH ester uith atropine MPEG-1900-COOH (1 g, 0.5mmol), atropine (0.63 retool) and HOBT (0.53 retool) were dissolved in CHeC12 (20ml) and a solution of DCC (0.7mmol) was added. The mixture was stirred overnight, filtered from DCU, evaporated, extracted with acetone and precipitated by ether. The product was recrystallized from absolute ethanol; yield 0.9 g (80°0); TLC (BuOH-AcOH-H20, 4:1:1 and PrOH-253~; N H 3 H20, 6:3:2) showed a single spot different from atropine. I.R. (nujol): 1735 C = O ester): l l l 0 c m - I (CH:OCH_~): NMR (CC14): ~53.6 (S, t70, OCH2CH2), 7.3 (s, 5, C~H~): 2.06 (s, 3, N CH3), 2.5 (s, 4, COCH2). Analysis. Calcd for CulTHI99NO48s: C, 56.48; H, 8.75; N, 0,62. Found: C, 56.09: H, 8,61; N, 1.0.
The amino derivative of M P E G was prepared by reduction of the azido derivative by Pd/C. Traces of sulphur from the preparation of the chloride with thionyl chloride may interfere with the catalytic reduction, and addition of another quantity of catalyst may be necessary. However, if a solution of the -g .'= 3oo _g o
~
150
RESULTS AND DISCUSSION
Preparation qf PEG having terminal amino groups Such polymers have been previously prepared via tosylation of PEG, reaction of the product with K-phthalimide and removing the phthaloyl group by hydrazine [14]. We decided to prepare them through the following series of reactions: PEG--OH-+PEG--CI-+PEG--N3-+PEG NH2 These are well-known reactions which can give quan-
._
n
co ~
r~ 40 6o Time {mini Fig. 1. Rate of disappearance of the chloride, EtOCH:CH2OCH_,CH2C1, in its reaction with NaN 3 in DMF. Experimental conditions. Chloride (0.025 tool), DMF (50ml), NaN~ (0.1 mol) heated at II0':--GC follow-up of the disappearance of the chloride. o
I eo
S. ZAL1PSKYet al.
1180
product is treated with alumina before the reduction, then traces of sulphur are removed, and no poisoning of the catalyst occurs. The reduction is carried out in alcohol but, since PEG especially of high molecular weight is not very soluble in alcohol at room ternperature, some precipitation of the polymer may occur. It can be prevented by addition of a small amount of chloroform or methylene chloride, but then the amine-HC1 derivative is formed. The amino compound is a white powder; acid titration with aqueous HCI, or anhydrous titration with perchloric acid, showed the correct molecular weight. The amino end group in PEG is more reactive than the OH group towards acylating agents, and allows the attachment of carboxylic groups of drugs through amide linkage.
Preparation of PEG having terminal isocyanato groups Monofunctional MPEG samples were used, and they were reacted with a large excess of hexamethylene diisocyanate (HDI) (5 tool/tool OH), so that polymers of the formula MeO(CH2 CH20)n CONH(CH2)6 NCO were formed. The reaction was carried out at room temperature and catalysed by dibutyltin dilaurate. At the end of the reaction, the PEG-NCO was purified from HDI and catalyst by repeated precipitation with ether in which PEG-NCO is insoluble. The NCO contents of the polymers were determined by titration; products of about 95~o purity were obtained. I.R. and N M R spectra were in accordance with the structure. These compounds react with moisture upon standing and their molecular weights start to increase, so they should be freshly used after being prepared. The reaction between PEG and HDI has been used previously [16] for the preparation of NCO-capped PEG, but it was carried out under more drastic conditions (reflux in benzene in the presence of pyridine).
~ oe ~ o t)I
0.4
d 03
I 90
o
~8o
2~o
360
Time (rain)
Fig. 2. Rate of reaction of PEG-2000 with succinic anhydride catalyzed by DMAP (C)) and DMAP + E%N (IN). Experimental conditions. PEG-2000 (2g, 2mmol OH), DMAP (2mmol), [E%N (2.5retool)], succinic anhydride (2.5 mmol) in dioxane, total volume 10 ml, temperature 25°. Follow-up of disappearance of acid groups by titration with Triton B (0.05 N in pyridine) [18].
Several PEG-succinates were prepared in this manner in the presence of D M A P and Et3 N. Analysis of the products PEG-2000 and PEG-3000 succinates, as well as i.r. and N M R spectra and anhydrous acid base titration, all pointed out to the formation of a salt of D M A P with the PEG-succinate. In i.r. besides the absorption at 1735 cm ~of the ester, there are peaks at 1560cm ~ ( C O 0 - ) and at 1645cm -t which belongs to C = I q of the resonance structure of D M A P cation. Me
\N / /
Me
Me
+j ~N I I
I I Preparation of PEG having carboxylic end groups H H The reaction of PEG with succinic anhydride [17] was used for the introduction of carboxylic end Anhydrous titration of the carboxylic or carboxylate groups. Thus MPEG-1900 was reacted with a large groups with KOCH3 or Triton B and that of amino excess of succinic anhydride; after 5 hr at 150°, the groups with perchloric acid in dioxane showed the polymer was precipitated by ether. The polymer did presence of 2 equivalents of carboxylic groups for 1 not contain any low molecular weight compounds amino group. Blank titration of D M A P in dioxane (TLC). I.R. spectrum showed an ester carbonyl ab- showed that it titrated as a mono amine; so that a sorption at 1735 cm -1. Anhydrous titration of the mono D M A P salt of PEG dissuccinate was obtained carboxylic groups with KOMe gave a molecular weight of 2109, and with Triton B in pyridine, 1979 (HOOC--CH2CH2CO(OCH2CH2),, (required 1990). OCOCH2CH2COOH. DMAP), This method is general for the preparation of PEG-succinates but it requires drastic conditions. We This conclusion was also supported by N M R evifound that it is possible to carry out the reaction dence (the ratio of the aromatic protons to the PEG). between PEG and succinic anhydride at room tern- It is interesting that a salt was formed with D M A P perature in the presence of 4-dimethylamino pyridine (pKa = 9.7) and not with E%N, p K a = 10.65) but this (DMAP) as catalyst. The rate of reaction was easily may be due to the volatility of the latter. This point followed by anhydrous titration of the reaction mix- was checked by adding excess E% N to succinic acid ture by Triton B in pyridine which titrates the in dioxane and evaporating to dryness. The residue anhydride as 2 equivalents [18]. The rate increased contained less than half of the Et3N required for still more if, besides the DMAP, an equivalent of making a full salt with the carboxylic groups, indiE%N was used (Fig. 2) and the reaction was over in cating the instability and decomposition of such salts 6 hr at room termperature, by loss of E%N.
Attachment of drugs to polyethylene glycols Penicillin V esters of PEG Several penicillin polymeric conjugates have been reported [5]. For example polymeric salts of penicillin G and penicillin V with a copolymer of vinylamine vinyl alcohol were prepared [19]; they were active against Staphylococcus 209R like the penicillins. These penicillins were also attached to this copolymer through amide linkage via a mixed anhydride derivative of the penicillin [20]. We used mild conditions for the preparation of penicillin V esters with PEG-3000, Peg-4000 and MPEG-1900, and utilizing dicyclohexyl carbodiimide (DCC) and D M A P for the coupling reaction. The esterification is carried out in one step and there is no need for prior activation of the reactants. The reaction was carried out below room temperature (exterhal cooling with ice water) to keep the reaction under control and avoid the appearance of a coloured product. The products obtained were pure (TLC). In NMR they showed the correct ratio between the integration of the polyether peak at 3 = 3.6 and the doublet of the two methyls of penicillin at 6 = 1.5.
118 I
ative of MPEG with dicyclohexyl urea (I). @__~
O ~ _ @ - --NH
O=C-CHu.CH2COIOCH z CH2 ), -Otde (I) The NMR spectrum of the compound showed a multiplet of cyclohexyl which was at the required ratio to the polymer peak. There was no aromatic peak for amphetamine. The i.r. spectrum showed, as required, three types of carbonyl peaks at 1730 cm (ester), 1695cm ~ (imide) and 1655cm ~ (amide). The compound did not titrate with Triton B indicating the absence of free carboxylic groups. The formation of I in preference to the coupling reaction is unexpected since DCC is known as a very good
coupling agent between amino and carboxylic acid groups. Formation of N-acyl urea is known only as a side-product [25]. In fact it was reported [26] that the coupling of PEG having carboxylic end groups The i.r. spectrum showed the presence of three types with glycine ethyl ester in water using a water-soluble of carbonyl groups, 1735cm ~ (ester), 1680cm ~ carbodiimide (amide) and 1780cm t (/~-lactam). This is evidence that the/t-lactam ring, essential for the antibacterial (EtN~C~---N --CH 2CH=CH 2N(Me)2" HCI) activity of penicillin, is not modified during the coupling reaction, proceeded smoothly at room temperature giving good yields of the amide. The difference between the two reaction paths may Aspirin esters q/ PEG be due to steric effects. The electrophilic carbon of the carbonyl of O-acyl isourea (|I) which is the reactive Aspirin (O-acetyl salicylic acid) is an important intermediate in the couplings mediated by dicycloanalgesic and antipyretic drug. Several studies have hexyl carbodiimide is more sterically hindered than been reported on the attachment of aspirin to poly- with the other carbodiimide. Further, PEG is a mers. Aspirin was attached to soluble starch [21] or random coil in organic solvents but in water is a helix to poly(vinyl alcohol) [22] by reaction of O-acetyl [27]. This will lead to steric hindrance from coiling of salicoyl chloride with the polymer. The polymers had the PEG around the active carbonyl groups in II and the same type of activity as aspirin but their toxicity will prevent the amine from attacking the carbonyl. was lower. Salicylates were also converted to their Thus the lifetime of II will be long enough to permit chlorocarbonate derivatives and attached to soluble O---,N migration leading to the formation of the starch, poly(vinyl alcohol) and PEG by carbonate N-acyl urea (I). With a helix conformation of PEG, linkage [12]. Similarly salicylate esters of oligo- it is expected that there would be less steric hinethylene glycols were prepared [23] by nucleophilic drance. attack of sodium salicylate on a p-toluene sulphonate derivative of the polymer. OCOR In the present work we prepared esters of aspirin 11 R"~,H~ R' N H - - C = N R" k ~ RCONHR'" with PEG using DCC and DMAP. Quantitative yields of pure products were obtained on carrying out (ll) the reaction for a few hours at room temperature. ~ R' N - - C O N H R " rearrangement
PEG derit~atives 0/ amphetamine Amphetamine is a CNS-stimulant and has various biological activities. In previous work [24] the methacryloyl derivative of amphetamine was prepared and polymerized. Amphetamine was also attached to starch through the chlorocarbonate derivative of the polysaccharide prepared by reaction of the starch with phosgene in pyridine [24]. We tried to attach amphetamine to M PEG-1900 COOH (mono succinate derivative) through amide linkage, by carrying out the reaction in methylene chloride using DCC as a coupling agent and utilizing excess amphetamine. The isolated product did not contain amphetamine and was the N-acyl urea deriv-
[
COR (I) R~PEG R,=R,,__C6H~ R':Et,
R":--CH2CH2CH2NMe 2
Steric factors are known to play a decisive role in the reaction of carbodiimides. For example it is known that O-acyl isourea derivative of amino acids having branching at the /3-carbon tend to give relatively large amounts of N-acyl urea so that it becomes the major product [28]. To overcome this problem we carried out the reaction between amphetamine and P E G - - C O O H in
1182
S. ZALIPSKYet al.
the presence of l-hydroxybenzotriazole (HOBT). It is known that HOBT reacts very quickly with O-acyl isourea (II) and thus prevents the formation of N-acyl urea [29]. The special high reactivity of the esters of HOBT formed in this reaction is due [25, 28] to participation of the heterocyclic nitrogen in the formation of a H-bond with the nucleophilic amine in the transition state, and to the fact that the oxybenzotriazole anion is a good leaving group (HOBT is a relatively strong acid (pKa = 4.0). o II R --C--O dlN~
/ RtNH2 -- H
5 I R--C--O / i ~
r
~
N H---
~/~J R' In fact the reaction between amphetamine and the carboxylic acids PEG-2000-COOH, PEG-3000COOH and MPEG-COOH in the presence of DCCHOBT led to the formation of the amides in good yield. All the products showed in the N M R the correct integration between the peak of polyethylene oxide and that of the aromatic amphetamine;i.r, and microanalysis were as required. Attachment of quinidine to PEG
Quinidine is a Chinchona alkaloid, important as an antiarhithmical agent. Some work has been carried out on the attachment of such alkaloids to polymers, Asymmetric or optically active resins that can be used in chromatographic columns for the separation of racemic compounds were prepared. Polymeric drugs were also prepared through polymerization of the O-methacryloyl derivative [30, 31]. The isomeric alkaloid quinine has been attached to polyisoprene and polybutadiene [32], or connected to cross-linked polystyrene-divinylbenzene for use as a chiral catalyst [33]. Since quinidine has a sterically hindered OH group and would be difficult to esterify with P E G - - C O O H , we used a spacer of glycine. N-tert. butoxycarbonylglycine (BOC-glycine) was reacted with quinidine in methylene chloride solution in the presence of DCC as coupling agent and DMAP as catalyst. After 12 hr, the reaction was over (TLC). The BOC protecting group was removed by treatment with trifluoroacetic acid. Thus the glycine ester of quinidine (Ill) was obtained.
tween PEG and quinidine. This product showed in i.r. two carbonyl absorptions at 1735 cm-~ (ester) and at 1670cm -~ (amide) besides characteristic absorptions of quinidine at 1620cm ~ and of PEG at ll l0 cm-~. N M R showed the correct ratio of the aromatic protons of quinidine to that of the polyether. Attachment o f atropine to PEG
Atropine is an anticholinergic compound and is important as an antidote for organophosphorus poisoning. We have previously attached atropine to short oligomers of PEG by a carbonate linkage [11] using the reaction of the chlorocarbonate derivative of PEG (DP 4-13) with the hydroxyl group of atropine. The highest molecular weight compound was the most active. In the present work we have attached atropine to PEG by a urethane linkage through a hexamethylene spacer. It is known [34,35] that polyurethane derivafives of PEG are suitable for medical use. They also suffer degradation in the living organism due to hydrolysis to the unstable carbamic acid and the alcohol. Thus the urethane linkage seems to offer a suitable system for the slow release of atropine. MPEG 750-NCO, MPEG 1900-NCO, MPEG 5000-NCO and PEG-2000-NCO were reacted with atropine in dry toluene in the presence of dibutyltin dilaurate as catalyst. All the NCO-polymers were freshly prepared before the reaction. The polymeric derivatives of atropine were precipitated by ether and checked for purity by TLC, i.r. and NMR. The products were soluble in water. Atropine was attached to PEG also through an ester linkage. Although MPEG 1900-COOH failed to react with atropine in the presence of DCC + DMAP as catalyst, it reacted well using DCC and HOBT as catalyst at room temperature yielding the required atropine derivative. REFERENCES H. Ringsdorf, J. Polym. Sci. Symp. 51, 135 0975). L. G. Donaruma, Progr. Polym. Sci. 4, 1 (1975). H. G, Batz, Adv. Polym. Sci. 23, 25 0977). A. Zaffaroni and P. Bonsen, Proc. Internat. Syrup. Polym. Drugs, p. 1. Academic Press, New York (1977). 5. C. H. Samour, Chemtech 494 (1978). 6. K. Soehrin~, K. Scriba. M. Frahm and G. Z611ner, Archs int. Pharmacodyn. Ther. 87, 301 (1951),
1. 2. 3. 4.
DCC+HOBT
(1Tr) The compounds was reacted with P E G - - C O O H (MPEG 1900-COOH, PEG-3000-COOH), using as coupling agent DCC + HOBT, giving product (IV) which has a spacer unit of N-succinoyl glycine be-
-
MeO~ (rv')
7. C. G. Hunter, D. E. Stevenson and P. L. Chambers, Fd Cosmet. Toxic. 5, 195 (1967). 8. H. F. Smyth, Jr, C. P. Carpenter and C. S. Well, J. Am. pharm. Ass. 44, 27 (1955).
Attachment of drugs to polyethylene glycols 9. G. M. Powell, Polyethylene glycol. Handbook of WaterSoluble Gums and Resins, (Edited by R. L. Davidson), Chap. 18. McGraw-Hill, New York (1980). 10. B. Z. Weiner and A. Zilkha, J. reed. Chem. 16, 573 (1973). [1. B. Z. Weiner, A. Zilkha, G. Porath and Y. Grunfeld, Eur. J. med. Chem.-Chim. Ther. 11, 525 (1976). 12. B. Z. Weiner, A. Havron and A. Zilkha, lsraelJ. Chem. 12, 863 (1974). 13. F. F. Davis, T. Van Es and N. C. Palczuk, Ger. Pat. 2, 433, 833 (1976); Chem. Abstr. 84, 146935 q (1976). 14. M. Mutter, Tetrahedron Lett. 31, 2839 (1978). 15. R. T. Morrison and R. N. Boyd, Organic Chemistrv, 2nd edn, p. 492. Allyn & Bacon, Boston, MA (1972). 16. F. Brandstetter, H. Schott and E. Bayer, Tetrahedron Lett. 31, 2705 (1974). 17. H. Inagaki and M. Tanaka, Makromolek. Chem. 74, 145 (1964). 18. A. Patchornik and S. E. Rogozinski, Analyt. Chem. 31, 985 (1959). 19. S. N. Ushakov, E. M. Lavrent'eva and L. I. Petrova, Vvsokomolek. Soedin. 282 (1964). 20. S. N. Ushakov and E. F. Paramin, Dokl. Akad. Nauk SSSR, 149, 334 (1963). 21. K. Kratzl and E. Kaufmann, Monatsch. Chem. 92, 371, 379 (1961). 22. V. A. Kropachev, E, M. Lavrent'eva, K. S. Podgorskaja and T. E. Semenova, Vysokomolek. Soedin. Sec. B, 11, 857 (1969).
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23. D. Tirrell, B. Bailey and O. Vogl, Proe. Intern. Symp. Polym. Drugs, p. 77. Academic Press, New York (1977). 24. B. Z. Weiner, M. Taham and A. Zilkha, J. reed. Chem. 15, 410 (1972). 25. M. Bodanszky, Y. S. Klausner and M. A. Ondetti, Peptide Synthesis, 2nd edn. Wiley, New York (1976). 26, G. P, Roger and G. M. Anantharmaiah, J. Am. chem. Soc. 101, 3394 (1979). 27. Macromolecular Science at Case Western Reserve University, Report for Acad. Year 1968/9 p. 5. 28. M. Bodanszky, The Peptides--Analysis, Synthesis, Biology, (Edited by E. Gross and J. Meienhofer), Vol. 1. Academic Press, New York (1979). 29. D. H. Rich and J. Singh The Peptides--Analysis, Svnthesis, Biology, (Edited by E. Gross and J. Meienhofer), Vol. 1. Academic Press, New York (1979). 30. E. Takesue, J. J. Hlavka and J. H. Boothe, U.S. Pat. 3, 356, 571 Dec. 5 (1967). 31. C. Pinazzi, J. C. Rabadeux and A. Pleurdeau, Makromolek. Chem. 179, 1699 (1978). 32. C. P. Pinazzi, A. Menil, J. C. Rabadeux and A. Pleurdeau, J. Pol.vm. Sci. Symp. 52, 1 (1975). 33. K. Hermann and H. Wynberg, Heh'. ehim. Acta 60, 2208 (1977). 34. T. E. Lipatova and R. A. Veslovsky, ~vsokomolek. Soedin. All, 1459 (1969). 35. T. E. Lipatova, J. PoO~m. Sci. Polvm. Svmp. 66, 239 (1979). 36. S. Siggia and J. G. Hanna, Analyt. Chem. 20, 1084 (1948).
Copyright
0014-3057/8353.00+0.00 1983 Pergamon Press Ltd
ATTACHMENT OF D R U G S TO POLYETHYLENE GLYCOLS S. ZALIPSKY, C. GILON and A. ZILKHA Department of Organic Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel (Received 13 December 1982)
Abstract--Polyethylene glycol (PEG) was used as a carrier polymer for the attachment, via end groups, of drugs such as penicillin V, aspirin, amphetamine, quinidine and atropine. For this purpose, methods were developed for the functionalization of PEG; PEG-NH2, PEG-COOH and PEG-NCO were prepared. Use was made of dicyclohexyl carbodiimide together with 4-dimethylamino pyridine or 1-hydroxybenzotriazole for the coupling reactions.
INTRODUCTION
glycols with one methoxy and one hydroxy end group (MPEG) of ~ = 750, 1900 and 5000 (Poly Sciences) were used. 4-Dimethylaminopyridine (DMAP), l-hydroxybenzotriazole (HOBT), dicyclohexylcarbodiimide (DCC)(distilled in vacuo), dibutyltin dilaurate, quinidine and atropine (Fluka) were used. L-Amphetamine (Aldrich), hexamethylene diisocyanate (HDI)(Bayer), Triton B (40°~oaqueous solution) and KOMe 0.1 M in MeOH/toluene (BDH) were used. DMF was distilled from P205 and then from ninhydrin. Chromatograph)" Packard 804 flame ionization GC instrument was used. N 2 was used as the carrier gas (flow rate 20 ml/min) and 6 ft of 1,/8in. glass tubing was used to contain the packing of 15% diethylene glycol succinate on Chromosorb P. Polygram Sil N-HR/u.v. 254 plates from Machery-Nagel Co. were used for thin layer chromatography (TLC).
Currently much interest is shown in the attachment of drugs to polymers as a means of increasing the duration of activity through slow release, or of targetdirecting drugs in the body [1-5]. In the present work we attached to polymers, drugs such as penicillin, aspirin, amphetamine, quinidine and atropine, We chose polyethylene glycol (PEG) as the carrier polymer because it is known to be non-toxic [f~8], non-antigenic and biocompatible, to be soluble in water and organic solvents and by itself to have solubilizing properties. It is not biodegradable [9] and is available in a wide range of molecular weights, Because of these properties, it is widely used in biochemistry and molecular biology, and is commercially used in pharmacological products, cosmetics and food. It is therefore suitable for use as a drug carrier in the body. Various drugs have been attached to PEG such as procaine [10], atropine [11] and various salicylates [12]. The polymer-drug conjugates showed longer duration of activity, due to slow release, Insulin as well as various enzymes have also been attached to P E G leading to products which retain their biological and enzymatic activity [13]. To attach the drugs to PEG, use was made of the
boxyl and isocyanate groups, Two approaches were used for the functionalization of PEG: (1)changing the terminal hydroxyl group, through a series of reactions, to a more active functional group; (2) reaction of P E G under controlled conditions with difunctional compounts so that one of its functional groups reacts with PEG and
MPEG-1900-CI [CHsO(CH2CH20)415CH2CH2CI ] MPEG-1900 (19g, 0.01 mol) was dissolved in toluene (100ml) and dried by azeotropic distillation. Dry pyridine (0.01 tool) was added and thionyl chloride (0.03mol), freshly distilled from quinoline, was added dropwise during 30 rain under reflux. The mixture was heated for 4 hr, cooled to room temperature, filtered from pyridine hydrochloride and the toluene was evaporated in t~acuo. The residue was dissolved in CH2CI 2, dried over anhydrous K2CO ~ and filtered. The filtrate was treated with alumina (50 g) (activated by heating at 120 for 2 hr) and precipitated by cold ether. The polymer was recrystallized from CH2Clz/ether: yield 18g (94°,~;); i.r. (nujol) 1110 (CH2--O--CH2), 663 (C--C1), no absorption for OH at 3300-3500cm ~. Analysis. Calcd for C86H~7~O425: C, 53.75: H, 9.02: CI, 1.85. Found: C, 53.73: H, 8.80: C1, 2.20. MPEG_I9OO_Ns[CHsO(CH2Ct_I20)4~sCH~CH, N d ~ To a solution of MPEG 1900-C1 (l.8g, 0.95mmol) in DMF (10ml), sodium azide (8 mmol) was added and the mixture was stirred at 120' for 2hr. The solution was cooled, filtered and the DMF evaporated in ~acuo. The residue was taken up in CH2CI:, cooled, filtered and precipitated by ether; yield 1.65 g (910~i); i.r. (nujol): 2108 (NO, 1110 (CHzOCH2) no absorption for CI at 663 cm J. Analysis. Calcd for C86HIv3NaO425 : C, 53.56: H, 8.98: N, 2.18. Found: C, 53.38: H, 8.74; N, 2.30.
the other remains active.
MPEG- 1900-NH2 [CH 30 (CH2 CH2 0 )4t ~CH2 CH2 NH2 ]
terminal hydroxyl groups. These can be reacted with drugs containing suitable groups such as carboxyl (esterification). But in order to make possible the attachment of drugs having other functional groups such as amino or hydroxyl, there was need for preparing P E G having other functional end-groups. Methods were therefore devised for converting the terminal hydroxyl groups of P E G into amino, car-
EXPERIMENTAL Materials Polyethylene glycols with two hydroxyl end groups (PEG) of JO'o= 1000, 2000 and 3000 (Fluka) and polyethylene
MPEG-1900-N~ (1.6 g) was dissolved in absolute ethanol (40ml), 10,"~i Pd/C (0.1g) added, and the mixture was hydrogenated in a Parr low pressure hydrogenation apparatus overnight. The catalyst was filtered, and the polymer precipitated by dry ether: yield 1.5 g (95°~i): i.r. (nujol): 3300
1177 EPJ 19/12 ~i
1178
S. ZALIPSKY et al.
(NH), l l l 0 (CH2OCH2), no absorption for N 3 at 2108cm ~, Analysis. Calcd for C86HI73NO42.5: C, 54.29; H, 9.20: N, 0.73, mol. wt 1899. Found: C, 54.14; H, 9.00; N, 0.91, mol. wt 1920, 1890 (titrations with 0.05 N HClO4/dioxane using thymol blue as indicator, and with 0.05 N HC1 using methyl red as indicator, respectively).
MPEG- 1900- COOH[CH 30 (CH2CH 20)41.5 CIt2 CH20COCH2CH2COOH ] (Method I) MPEG-1900 (9.5g, 5mmol) was dissolved in toluene (50ml) and dried by distilling off most of the toluene; succinic anhydride (20 g, 20mmol) was added and the mixture was stirred for 5 hr on an oil bath at 150°. The mixture was cooled, taken up in CH2C12 and the polymer precipitated by ether. The product was recrystallized twice from CH2C12/ether; yield 8.5 g (85~o); i.r. (nujol): 1735 (C=O), l l l 0 c m -t (CH,,OCH2); TLC (BuOH/AcOH/H20, 4:1:1) showed only one peak and no succinic anhydride, Analysis. Calcd for Co~HI78046.5 : C, 53.95; H, 8.89, mol. wt 2000. Found: C, 53.63; H, 8.95; tool. wt 2109, 1979 (titrations with 0.05N KOMe and 0.05N Triton B in pyridine, respectively, using thymol blue as indicator). PEG-3OOO-COOH (Method H) PEG-3000 (6 g, 4 mmol OH groups), succinic anhydride (5mmol), DMAP (4mmol) and triethylamine (4mmol) were dissolved in dioxane (30 ml) and left overnight at room temperature. The dioxane was evaporated in vacuo, the residue taken up in CC14, filtered, and precipitated by ether. It was recrystallized from CH2Cldether yield 6.4 g. Tests on the compound showed that it had the formula HOOCCH2CH2OCO(CH2CH20)67.8CO ) --~N CH2CH2COOH. Me2N
up in CC14, filtered from penicillin V residues, and precipitated by ether; yields 84-87%. TLC (methanol) of the product showed the absence of free penicillin V in the polymer. I . R (nujol): 1780 ( C : O , fl-lactam); 1735 ( C = O ester); 1680 (C-~-O, amide), 1600 (C6H5); l l l 0 c m (CH2OCH2). NMR (CCI4): & 3.6 (s, 4n (OCH2CH2)n); 1.5 (d, 6x, CMe~); 6.9-7.3 (m, 5x, C6H5) (x = 1 for MPEG derivatives; x = 2 for PEG difunctional derivatives). Analysis. Calcd for PEG-3000-penicillin V ester, CI67.6H305.1N4076.852: C, 54.88; H, 8.33; N, 1.53; S, 1.75. Found: C, 55.00; H, 8.31, N, 1.81; S, 1.85. Similar results were obtained with PEG-4000 and MPEG-1900.
PEG-esters of aspirin (general procedure) PEG (1 mmol OH) DCC (1.3 mmol) was added to a solution of aspirin (1.1 mmol) and DMAP (0.25 mmol) in CH2CI 2 (20 ml); the mixture was stirred for 3-4 hr at room temperature. The ppt of DCU was filtered and the filtrate evaporated to dryness. The residue was extracted by acetone, filtered and the polymer precipitated by ether; yields 70-85~o; i.r. (nujol): 1735 (C6H4COO), 1740 (C6H40C=O), no absorption at 3300-3500cm ~(OH): NMR(CCI4): &2.2 (s, 3x, CH3CO), 3.6 (s, 4n, (OCH2CH2)n); 6.75-8.2 (m, 4x, C6H4) x = 1 for MPEG, x = 2 for bifunctional PEG). Analysis. Caled for C62.6HI052029.3 (PEG-1000-aspirin): C, 56.74; H, 7.95. Found: C, 56.65; H, 8.08.
+ i.r, (nujol)1735 ( C = O ester), 1645 ( C ~ N ) , 1560 (COO-), l l l 0 c m -I (CH2OCH2). NMR (CC14): ~ 3.6 (s, 271, OCH,CH2); 3.0 (s, 6, N-Me); 2.5 (s, 8, --COCH2); 6.5-8.1 (m, 4, pyridine-H). Analysis. Calcd for CI43.6H281.1074.8.CTHIoN2: C, 54.38; H, 8.76; N, 0.84; mol. wt 3322. Found: C, 54.73; H, 8.71; N, 1.09; tool. wt 3240, 3294, 3533 (titrations with Triton B, KOCH 3 and 0.05 N HCIO 4 in dioxane using thymol blue as indicator [18]). PEG-2000-COOH was similarly prepared and gave the same results,
Reaction of MPEG-19OO-COOH with amphetamine--Formation of N-acyl urea derivative of MPEG-19OO-COOH (I) To a solution of MPEG-1900-COOH (1 g, 0.5 mmol) and amphetamine (1 mmol) in CH2C12 (20 ml), DCC (0.9 mmol) dissolved in a small volume of CH2C1., was added. The mixture was stirred overnight, filtered from a small amount of DCU, evaporated to dryness, extracted with acetone, precipitated by ether and recrystallized from CH2Cl2/ether; yield 0.95 g. The compound did not contain acid groups (titration with Triton B). NMR (CC14) &: 1-2 (m, 11, O II C6H H); 2.55 (s, 4, C CH2); 3.6 (s, 170, OCH2CH2), no peak in the aromatic region for amphetamine. I.R. (nujol): 1730 ( C = O ester), 1695 (C-~--O imide), 1655cm t ( C = O urea). Analysis. Calcd for Ct03H200N20465: C, 55.98; H, 9.06; N, 1.27. Found: C, 55.62; H, 8.85; N, 1.27. Amides of PEG-COOH and amphetamine (general procedure)
PEG-NCO (general method) A solution of PEG (dried by azeotropic distillation) was added dropwise during 2 hr under anhydrous conditions to hexamethylene diisocyanate (HDI) (5 times molar excess over the OH groups) and dibutyltin dilaurate (0.05 ml). The mixture was stirred for 10 hr, and the polymer precipitated by dry ether and further purified from toluene/ether. I.R. (nujol): 2250 (NCO); 1110 (CH2OCH2); 1715, 1535 (HNCOO), 3300cm -I (NH). N M R (EEl4): ~ 1.3-1.6 (m, CH2CH2CH2), 3.6 (s, OCH2). Molecular weights as determined by titration of NCO groups [36]: MPEG-750-HDI, 965 (required 918); MPEG-1900-HDI, 2160 (required 2068); MPEG 5000-HDI, 4944 (required 5168); PEG-2000-2HDI, 2408 (required 2336).
To a solution of PEG-COOH (0.5mmol COOH), l-amphetamine (1 mmol) and HOBT (0.5 mmol) in CH2C12 (20 ml), a solution of DCC (0.6 mmol) in a small volume of CH2CI 2 was added. The mixture was stirred overnight, filtered from DCU residues, and the polymer was precipitated by ether and recrystallized from absolute ethanol; yield 73-80~o; i.r. (nujol): 1730 ( C = O ester), 1645 ( C = O amide), 3290 (NH) and l l l 0 c m -t (CH2OCH2); NMR (CCl4):&7.2(s, 5x, C6Hs); 2.5 (s, 4x, O== CCH2);1.1(d, 3x, CHC_H_3),3.6 (s, 4n (OCH2CH2)n) (x = 1 for MPEG derivative, x = 2 for PEG derivative). Analysis. Calcd for CII6H214OsoN2 (PEG-2000-COOH amide with amphetamine): C, 57.19; H, 8.79; N, 1.15. Found: C, 56.96; H, 8.79; N, 1.06.
PEG-penicillin V esters (general procedure) K-salt of penicillin V (1.5 mmol) was dissolved in a small volume of water, cooled and acidified with 0.1 N HC1. The precipitated penicillin V was filtered, and dried immediately in vacuo over P20~ for 30min. It was added to a solution of PEG (1 mmol OH) and DMAP (0.25 mmol) in CH2C12 (20 ml), stirred and cooled (ice-water). DCC (1.3 mmol) was added and the mixture was stirred for 2 hr, filtered from precipitated dicyclohexyl urea (DCU) and evaporated. The residue was extracted with acetone, filtered from DCU residues and the product precipitated by ether It was taken
O-glycylquinidine (I11) To a solution of quinidine (1.62 g, 5 mmol), BOC-glycine (1.75 g, 10 mmol) and DMAP (0.61 g, 5 mmol) in CH2CI,, a solution of DCC (2 g, 10 mmol) in CH2CI 2 was added, and the mixture was stirred overnight. TLC (MeOH) then showed that all the quinidine has reacted, DCU was filtered and acetic acid (0.5 ml) was added to destroy excess DCC. After 10 min DCU was filtered, the solvent was evaporated and the residue taken up in acetone, filtered and evaporated. CH2C % (50 ml) was added to dissolve the residue and the solution was washed with NaHCO 3 and dried over MgSO 4.
Attachment of drugs to polyethylene glycols
1179
TLC (MeOH or BuOH-AcOH-H20 4:1:1) showed the titative yields. It is important, in carrying out a series presence of a single compound. The volume of the solution of reactions on polymers, that all reactions proceed was reduced to 5ml and trifluoroacetic acid (5ml) was quantitatively because of the inherent difficulties in added and left to stand at room temperature for 30rain. The separating reacted from unreacted polymer. The solution was evaporated, the residue taken up in CH,CI 2 relatively easy separation of low molecular weight washed with NaHCO~, dried and evaporated yielding a solid compound 1.3g(;72% ). I.R. (nujol): 1740 ( C = O ester) compounds from polymers makes it easy to use 3320 (NH2),1620cm ~ (C=C). NMR (CCID: 6 3.45 (s, 2, large excess of reagent and obtain quantitative conNCH2CO), 7.2 8.6 (m, 5-quinoline HI. versions. This course of reactions was investigated using as Coupling o[ O-glvcylquinidine (llI) to PEG (general pro- a typical compound, monofunctional PEG, namely cedure) MPEG-1900 [MeO(CH2CH20),,--OH,.471, = 1900]. PEG-COOH (0.5 mmol COOH), O-glycylquinidine The chlorination was carried out with thionyl chlo(0.58 mmol) and HOBT (0.57mmol) were dissolved in ride in toluene solution in the presence of pyridine. CHeCI: (20 ml), DCC (0.7 mmol) was added, and the mix- The product upon recrystallization gave a white solid ture stirred overnight. It was filtered and evaporated, extracted by ethyl acetate and filtered from DCU. The polymer with i.r. spectrum showing C - - C 1 absorption at was precipitated by ether and recrystallized from a small 669 c m - t and the absence of OH absorption in the volume of absolute ethanol, yield 82-85°0; i.r. (nujol): 1735 3300-3500 cm ~ region. Its elemental analysis con( C = O ester): 1670 ( C = O amide), 1620 (C=C), II 10cm t formed with the structure. (CH,OCH_,): NMR (CC14): a 3.6 (s, 4n (OCHeCH_,)~): The conversion of the chloride to the azide was 7.2 8.6 (m, 5x, H-quinoline) (x = 1 for MPEG derivative, investigated first using a low molecular weight cornx = 2 for PEG). TLC (BuOH-AcOH-H20, 4:1:1 and pound, namely EtOCH2CH~OCH2CH2Cl so that th e PrOH-20°~, NH 3 H_,O, 6:3:2) showed one spot, course of the reaction could be easily followed by Analysis. Calcd for CI~7sH3310788N6 (PEG-3000-COOH GC. When the reaction with sodium azide was caramide with Ill): C, 57.34: H, 8.43; N, 2.14. Found: C, 57.08: ried out under reflux in M e O H - H 2 0 (7:5), 96°0 of H, 8.11; N, 2.31. the chloride reacted yielding the azide after 4 2 h r PEG carbamates o[atropine (general procedure) reaction time. To shorten the time, the reaction was Freshly prepared PEG-NCO (0.5mmol NCO) was dis- carried out in a dipolar aprotic solvent, viz D M F at solved in dry toluene and atropine (0.6 mmol) and dibutyltin l l O . The reaction was much faster under these dilaurate (0.05 ml) were added. The solution was left over- conditions and 100'/'o substitution was achieved after night and the polymer precipitated by ether and rel hr (Fig. 1). Quantitative substitution was achieved crystallized from CH:Cl>:ether; yields 65-90o TLC after 30 min when the reaction was carried out at (PrOH-CHCI~, 1:1) (visualization by iodine vapour) 120". The long reaction time in M e O H - H 2 0 is due showed one spot and the absence of atropine: i.r. (nujol): to solvation of anions through hydrogen linkages 1665 1720 (wide C = O absorption ester-carbamate), 3330 which can lower the activity of the azide anion. D M F (NH), l l l 0 c m i (CHzOCH,); NMR (CCI4): a 3.6 (s, 4n, (OCH2CH:)°), 7.3 (s, 5x. C6H5); 1.3-1.6 (m, 8x, (CH2)4), leads to solvation of the cation and the azide anion 2.06 (s, 3x, --NCH~) (x = 1 for MPEG derivative, x = 2 for is freer and thus more active [15]. The azido derivaPEG derivative), tive of MPEG-1900 was prepared similarly in D M F Analysis. Calcd for Cjl ~H2090485N3 (MPEG-1900-NCO at 120 from the chloride. It showed the azide absorpcarbamate derivative of atropine): C, 56.47: H, 8.86; N, tion at 2108cm ~ and had the required elemental 1.78. Found: C. 56.62; H, 9.23; N, 1.83. analysis.
MPEG-19OO-COOH ester uith atropine MPEG-1900-COOH (1 g, 0.5mmol), atropine (0.63 retool) and HOBT (0.53 retool) were dissolved in CHeC12 (20ml) and a solution of DCC (0.7mmol) was added. The mixture was stirred overnight, filtered from DCU, evaporated, extracted with acetone and precipitated by ether. The product was recrystallized from absolute ethanol; yield 0.9 g (80°0); TLC (BuOH-AcOH-H20, 4:1:1 and PrOH-253~; N H 3 H20, 6:3:2) showed a single spot different from atropine. I.R. (nujol): 1735 C = O ester): l l l 0 c m - I (CH:OCH_~): NMR (CC14): ~53.6 (S, t70, OCH2CH2), 7.3 (s, 5, C~H~): 2.06 (s, 3, N CH3), 2.5 (s, 4, COCH2). Analysis. Calcd for CulTHI99NO48s: C, 56.48; H, 8.75; N, 0,62. Found: C, 56.09: H, 8,61; N, 1.0.
The amino derivative of M P E G was prepared by reduction of the azido derivative by Pd/C. Traces of sulphur from the preparation of the chloride with thionyl chloride may interfere with the catalytic reduction, and addition of another quantity of catalyst may be necessary. However, if a solution of the -g .'= 3oo _g o
~
150
RESULTS AND DISCUSSION
Preparation qf PEG having terminal amino groups Such polymers have been previously prepared via tosylation of PEG, reaction of the product with K-phthalimide and removing the phthaloyl group by hydrazine [14]. We decided to prepare them through the following series of reactions: PEG--OH-+PEG--CI-+PEG--N3-+PEG NH2 These are well-known reactions which can give quan-
._
n
co ~
r~ 40 6o Time {mini Fig. 1. Rate of disappearance of the chloride, EtOCH:CH2OCH_,CH2C1, in its reaction with NaN 3 in DMF. Experimental conditions. Chloride (0.025 tool), DMF (50ml), NaN~ (0.1 mol) heated at II0':--GC follow-up of the disappearance of the chloride. o
I eo
S. ZAL1PSKYet al.
1180
product is treated with alumina before the reduction, then traces of sulphur are removed, and no poisoning of the catalyst occurs. The reduction is carried out in alcohol but, since PEG especially of high molecular weight is not very soluble in alcohol at room ternperature, some precipitation of the polymer may occur. It can be prevented by addition of a small amount of chloroform or methylene chloride, but then the amine-HC1 derivative is formed. The amino compound is a white powder; acid titration with aqueous HCI, or anhydrous titration with perchloric acid, showed the correct molecular weight. The amino end group in PEG is more reactive than the OH group towards acylating agents, and allows the attachment of carboxylic groups of drugs through amide linkage.
Preparation of PEG having terminal isocyanato groups Monofunctional MPEG samples were used, and they were reacted with a large excess of hexamethylene diisocyanate (HDI) (5 tool/tool OH), so that polymers of the formula MeO(CH2 CH20)n CONH(CH2)6 NCO were formed. The reaction was carried out at room temperature and catalysed by dibutyltin dilaurate. At the end of the reaction, the PEG-NCO was purified from HDI and catalyst by repeated precipitation with ether in which PEG-NCO is insoluble. The NCO contents of the polymers were determined by titration; products of about 95~o purity were obtained. I.R. and N M R spectra were in accordance with the structure. These compounds react with moisture upon standing and their molecular weights start to increase, so they should be freshly used after being prepared. The reaction between PEG and HDI has been used previously [16] for the preparation of NCO-capped PEG, but it was carried out under more drastic conditions (reflux in benzene in the presence of pyridine).
~ oe ~ o t)I
0.4
d 03
I 90
o
~8o
2~o
360
Time (rain)
Fig. 2. Rate of reaction of PEG-2000 with succinic anhydride catalyzed by DMAP (C)) and DMAP + E%N (IN). Experimental conditions. PEG-2000 (2g, 2mmol OH), DMAP (2mmol), [E%N (2.5retool)], succinic anhydride (2.5 mmol) in dioxane, total volume 10 ml, temperature 25°. Follow-up of disappearance of acid groups by titration with Triton B (0.05 N in pyridine) [18].
Several PEG-succinates were prepared in this manner in the presence of D M A P and Et3 N. Analysis of the products PEG-2000 and PEG-3000 succinates, as well as i.r. and N M R spectra and anhydrous acid base titration, all pointed out to the formation of a salt of D M A P with the PEG-succinate. In i.r. besides the absorption at 1735 cm ~of the ester, there are peaks at 1560cm ~ ( C O 0 - ) and at 1645cm -t which belongs to C = I q of the resonance structure of D M A P cation. Me
\N / /
Me
Me
+j ~N I I
I I Preparation of PEG having carboxylic end groups H H The reaction of PEG with succinic anhydride [17] was used for the introduction of carboxylic end Anhydrous titration of the carboxylic or carboxylate groups. Thus MPEG-1900 was reacted with a large groups with KOCH3 or Triton B and that of amino excess of succinic anhydride; after 5 hr at 150°, the groups with perchloric acid in dioxane showed the polymer was precipitated by ether. The polymer did presence of 2 equivalents of carboxylic groups for 1 not contain any low molecular weight compounds amino group. Blank titration of D M A P in dioxane (TLC). I.R. spectrum showed an ester carbonyl ab- showed that it titrated as a mono amine; so that a sorption at 1735 cm -1. Anhydrous titration of the mono D M A P salt of PEG dissuccinate was obtained carboxylic groups with KOMe gave a molecular weight of 2109, and with Triton B in pyridine, 1979 (HOOC--CH2CH2CO(OCH2CH2),, (required 1990). OCOCH2CH2COOH. DMAP), This method is general for the preparation of PEG-succinates but it requires drastic conditions. We This conclusion was also supported by N M R evifound that it is possible to carry out the reaction dence (the ratio of the aromatic protons to the PEG). between PEG and succinic anhydride at room tern- It is interesting that a salt was formed with D M A P perature in the presence of 4-dimethylamino pyridine (pKa = 9.7) and not with E%N, p K a = 10.65) but this (DMAP) as catalyst. The rate of reaction was easily may be due to the volatility of the latter. This point followed by anhydrous titration of the reaction mix- was checked by adding excess E% N to succinic acid ture by Triton B in pyridine which titrates the in dioxane and evaporating to dryness. The residue anhydride as 2 equivalents [18]. The rate increased contained less than half of the Et3N required for still more if, besides the DMAP, an equivalent of making a full salt with the carboxylic groups, indiE%N was used (Fig. 2) and the reaction was over in cating the instability and decomposition of such salts 6 hr at room termperature, by loss of E%N.
Attachment of drugs to polyethylene glycols Penicillin V esters of PEG Several penicillin polymeric conjugates have been reported [5]. For example polymeric salts of penicillin G and penicillin V with a copolymer of vinylamine vinyl alcohol were prepared [19]; they were active against Staphylococcus 209R like the penicillins. These penicillins were also attached to this copolymer through amide linkage via a mixed anhydride derivative of the penicillin [20]. We used mild conditions for the preparation of penicillin V esters with PEG-3000, Peg-4000 and MPEG-1900, and utilizing dicyclohexyl carbodiimide (DCC) and D M A P for the coupling reaction. The esterification is carried out in one step and there is no need for prior activation of the reactants. The reaction was carried out below room temperature (exterhal cooling with ice water) to keep the reaction under control and avoid the appearance of a coloured product. The products obtained were pure (TLC). In NMR they showed the correct ratio between the integration of the polyether peak at 3 = 3.6 and the doublet of the two methyls of penicillin at 6 = 1.5.
118 I
ative of MPEG with dicyclohexyl urea (I). @__~
O ~ _ @ - --NH
O=C-CHu.CH2COIOCH z CH2 ), -Otde (I) The NMR spectrum of the compound showed a multiplet of cyclohexyl which was at the required ratio to the polymer peak. There was no aromatic peak for amphetamine. The i.r. spectrum showed, as required, three types of carbonyl peaks at 1730 cm (ester), 1695cm ~ (imide) and 1655cm ~ (amide). The compound did not titrate with Triton B indicating the absence of free carboxylic groups. The formation of I in preference to the coupling reaction is unexpected since DCC is known as a very good
coupling agent between amino and carboxylic acid groups. Formation of N-acyl urea is known only as a side-product [25]. In fact it was reported [26] that the coupling of PEG having carboxylic end groups The i.r. spectrum showed the presence of three types with glycine ethyl ester in water using a water-soluble of carbonyl groups, 1735cm ~ (ester), 1680cm ~ carbodiimide (amide) and 1780cm t (/~-lactam). This is evidence that the/t-lactam ring, essential for the antibacterial (EtN~C~---N --CH 2CH=CH 2N(Me)2" HCI) activity of penicillin, is not modified during the coupling reaction, proceeded smoothly at room temperature giving good yields of the amide. The difference between the two reaction paths may Aspirin esters q/ PEG be due to steric effects. The electrophilic carbon of the carbonyl of O-acyl isourea (|I) which is the reactive Aspirin (O-acetyl salicylic acid) is an important intermediate in the couplings mediated by dicycloanalgesic and antipyretic drug. Several studies have hexyl carbodiimide is more sterically hindered than been reported on the attachment of aspirin to poly- with the other carbodiimide. Further, PEG is a mers. Aspirin was attached to soluble starch [21] or random coil in organic solvents but in water is a helix to poly(vinyl alcohol) [22] by reaction of O-acetyl [27]. This will lead to steric hindrance from coiling of salicoyl chloride with the polymer. The polymers had the PEG around the active carbonyl groups in II and the same type of activity as aspirin but their toxicity will prevent the amine from attacking the carbonyl. was lower. Salicylates were also converted to their Thus the lifetime of II will be long enough to permit chlorocarbonate derivatives and attached to soluble O---,N migration leading to the formation of the starch, poly(vinyl alcohol) and PEG by carbonate N-acyl urea (I). With a helix conformation of PEG, linkage [12]. Similarly salicylate esters of oligo- it is expected that there would be less steric hinethylene glycols were prepared [23] by nucleophilic drance. attack of sodium salicylate on a p-toluene sulphonate derivative of the polymer. OCOR In the present work we prepared esters of aspirin 11 R"~,H~ R' N H - - C = N R" k ~ RCONHR'" with PEG using DCC and DMAP. Quantitative yields of pure products were obtained on carrying out (ll) the reaction for a few hours at room temperature. ~ R' N - - C O N H R " rearrangement
PEG derit~atives 0/ amphetamine Amphetamine is a CNS-stimulant and has various biological activities. In previous work [24] the methacryloyl derivative of amphetamine was prepared and polymerized. Amphetamine was also attached to starch through the chlorocarbonate derivative of the polysaccharide prepared by reaction of the starch with phosgene in pyridine [24]. We tried to attach amphetamine to M PEG-1900 COOH (mono succinate derivative) through amide linkage, by carrying out the reaction in methylene chloride using DCC as a coupling agent and utilizing excess amphetamine. The isolated product did not contain amphetamine and was the N-acyl urea deriv-
[
COR (I) R~PEG R,=R,,__C6H~ R':Et,
R":--CH2CH2CH2NMe 2
Steric factors are known to play a decisive role in the reaction of carbodiimides. For example it is known that O-acyl isourea derivative of amino acids having branching at the /3-carbon tend to give relatively large amounts of N-acyl urea so that it becomes the major product [28]. To overcome this problem we carried out the reaction between amphetamine and P E G - - C O O H in
1182
S. ZALIPSKYet al.
the presence of l-hydroxybenzotriazole (HOBT). It is known that HOBT reacts very quickly with O-acyl isourea (II) and thus prevents the formation of N-acyl urea [29]. The special high reactivity of the esters of HOBT formed in this reaction is due [25, 28] to participation of the heterocyclic nitrogen in the formation of a H-bond with the nucleophilic amine in the transition state, and to the fact that the oxybenzotriazole anion is a good leaving group (HOBT is a relatively strong acid (pKa = 4.0). o II R --C--O dlN~
/ RtNH2 -- H
5 I R--C--O / i ~
r
~
N H---
~/~J R' In fact the reaction between amphetamine and the carboxylic acids PEG-2000-COOH, PEG-3000COOH and MPEG-COOH in the presence of DCCHOBT led to the formation of the amides in good yield. All the products showed in the N M R the correct integration between the peak of polyethylene oxide and that of the aromatic amphetamine;i.r, and microanalysis were as required. Attachment of quinidine to PEG
Quinidine is a Chinchona alkaloid, important as an antiarhithmical agent. Some work has been carried out on the attachment of such alkaloids to polymers, Asymmetric or optically active resins that can be used in chromatographic columns for the separation of racemic compounds were prepared. Polymeric drugs were also prepared through polymerization of the O-methacryloyl derivative [30, 31]. The isomeric alkaloid quinine has been attached to polyisoprene and polybutadiene [32], or connected to cross-linked polystyrene-divinylbenzene for use as a chiral catalyst [33]. Since quinidine has a sterically hindered OH group and would be difficult to esterify with P E G - - C O O H , we used a spacer of glycine. N-tert. butoxycarbonylglycine (BOC-glycine) was reacted with quinidine in methylene chloride solution in the presence of DCC as coupling agent and DMAP as catalyst. After 12 hr, the reaction was over (TLC). The BOC protecting group was removed by treatment with trifluoroacetic acid. Thus the glycine ester of quinidine (Ill) was obtained.
tween PEG and quinidine. This product showed in i.r. two carbonyl absorptions at 1735 cm-~ (ester) and at 1670cm -~ (amide) besides characteristic absorptions of quinidine at 1620cm ~ and of PEG at ll l0 cm-~. N M R showed the correct ratio of the aromatic protons of quinidine to that of the polyether. Attachment o f atropine to PEG
Atropine is an anticholinergic compound and is important as an antidote for organophosphorus poisoning. We have previously attached atropine to short oligomers of PEG by a carbonate linkage [11] using the reaction of the chlorocarbonate derivative of PEG (DP 4-13) with the hydroxyl group of atropine. The highest molecular weight compound was the most active. In the present work we have attached atropine to PEG by a urethane linkage through a hexamethylene spacer. It is known [34,35] that polyurethane derivafives of PEG are suitable for medical use. They also suffer degradation in the living organism due to hydrolysis to the unstable carbamic acid and the alcohol. Thus the urethane linkage seems to offer a suitable system for the slow release of atropine. MPEG 750-NCO, MPEG 1900-NCO, MPEG 5000-NCO and PEG-2000-NCO were reacted with atropine in dry toluene in the presence of dibutyltin dilaurate as catalyst. All the NCO-polymers were freshly prepared before the reaction. The polymeric derivatives of atropine were precipitated by ether and checked for purity by TLC, i.r. and NMR. The products were soluble in water. Atropine was attached to PEG also through an ester linkage. Although MPEG 1900-COOH failed to react with atropine in the presence of DCC + DMAP as catalyst, it reacted well using DCC and HOBT as catalyst at room temperature yielding the required atropine derivative. REFERENCES H. Ringsdorf, J. Polym. Sci. Symp. 51, 135 0975). L. G. Donaruma, Progr. Polym. Sci. 4, 1 (1975). H. G, Batz, Adv. Polym. Sci. 23, 25 0977). A. Zaffaroni and P. Bonsen, Proc. Internat. Syrup. Polym. Drugs, p. 1. Academic Press, New York (1977). 5. C. H. Samour, Chemtech 494 (1978). 6. K. Soehrin~, K. Scriba. M. Frahm and G. Z611ner, Archs int. Pharmacodyn. Ther. 87, 301 (1951),
1. 2. 3. 4.
DCC+HOBT
(1Tr) The compounds was reacted with P E G - - C O O H (MPEG 1900-COOH, PEG-3000-COOH), using as coupling agent DCC + HOBT, giving product (IV) which has a spacer unit of N-succinoyl glycine be-
-
MeO~ (rv')
7. C. G. Hunter, D. E. Stevenson and P. L. Chambers, Fd Cosmet. Toxic. 5, 195 (1967). 8. H. F. Smyth, Jr, C. P. Carpenter and C. S. Well, J. Am. pharm. Ass. 44, 27 (1955).
Attachment of drugs to polyethylene glycols 9. G. M. Powell, Polyethylene glycol. Handbook of WaterSoluble Gums and Resins, (Edited by R. L. Davidson), Chap. 18. McGraw-Hill, New York (1980). 10. B. Z. Weiner and A. Zilkha, J. reed. Chem. 16, 573 (1973). [1. B. Z. Weiner, A. Zilkha, G. Porath and Y. Grunfeld, Eur. J. med. Chem.-Chim. Ther. 11, 525 (1976). 12. B. Z. Weiner, A. Havron and A. Zilkha, lsraelJ. Chem. 12, 863 (1974). 13. F. F. Davis, T. Van Es and N. C. Palczuk, Ger. Pat. 2, 433, 833 (1976); Chem. Abstr. 84, 146935 q (1976). 14. M. Mutter, Tetrahedron Lett. 31, 2839 (1978). 15. R. T. Morrison and R. N. Boyd, Organic Chemistrv, 2nd edn, p. 492. Allyn & Bacon, Boston, MA (1972). 16. F. Brandstetter, H. Schott and E. Bayer, Tetrahedron Lett. 31, 2705 (1974). 17. H. Inagaki and M. Tanaka, Makromolek. Chem. 74, 145 (1964). 18. A. Patchornik and S. E. Rogozinski, Analyt. Chem. 31, 985 (1959). 19. S. N. Ushakov, E. M. Lavrent'eva and L. I. Petrova, Vvsokomolek. Soedin. 282 (1964). 20. S. N. Ushakov and E. F. Paramin, Dokl. Akad. Nauk SSSR, 149, 334 (1963). 21. K. Kratzl and E. Kaufmann, Monatsch. Chem. 92, 371, 379 (1961). 22. V. A. Kropachev, E, M. Lavrent'eva, K. S. Podgorskaja and T. E. Semenova, Vysokomolek. Soedin. Sec. B, 11, 857 (1969).
1183
23. D. Tirrell, B. Bailey and O. Vogl, Proe. Intern. Symp. Polym. Drugs, p. 77. Academic Press, New York (1977). 24. B. Z. Weiner, M. Taham and A. Zilkha, J. reed. Chem. 15, 410 (1972). 25. M. Bodanszky, Y. S. Klausner and M. A. Ondetti, Peptide Synthesis, 2nd edn. Wiley, New York (1976). 26, G. P, Roger and G. M. Anantharmaiah, J. Am. chem. Soc. 101, 3394 (1979). 27. Macromolecular Science at Case Western Reserve University, Report for Acad. Year 1968/9 p. 5. 28. M. Bodanszky, The Peptides--Analysis, Synthesis, Biology, (Edited by E. Gross and J. Meienhofer), Vol. 1. Academic Press, New York (1979). 29. D. H. Rich and J. Singh The Peptides--Analysis, Svnthesis, Biology, (Edited by E. Gross and J. Meienhofer), Vol. 1. Academic Press, New York (1979). 30. E. Takesue, J. J. Hlavka and J. H. Boothe, U.S. Pat. 3, 356, 571 Dec. 5 (1967). 31. C. Pinazzi, J. C. Rabadeux and A. Pleurdeau, Makromolek. Chem. 179, 1699 (1978). 32. C. P. Pinazzi, A. Menil, J. C. Rabadeux and A. Pleurdeau, J. Pol.vm. Sci. Symp. 52, 1 (1975). 33. K. Hermann and H. Wynberg, Heh'. ehim. Acta 60, 2208 (1977). 34. T. E. Lipatova and R. A. Veslovsky, ~vsokomolek. Soedin. All, 1459 (1969). 35. T. E. Lipatova, J. PoO~m. Sci. Polvm. Svmp. 66, 239 (1979). 36. S. Siggia and J. G. Hanna, Analyt. Chem. 20, 1084 (1948).