Synthesis of macromolecular Mitomycin C derivatives

journal of

corm&.l.ld ELSEVIER

Journal of Controlled

Release 32 ( 1994) 129-I 37

Synthesis of macromolecular

Mitomycin C derivatives

Anne De Marre, Heidi Soyez, Etienne Schacht* Received

10 January

1994; accepted 2 May 1994

Abstract A series of polymeric prodrugs of Mitomycin C (MMC) were prepared by coupling the drug via a tri- or tetrapeptide spacer onto poly- [‘N- (2-hydroxyethyl) -L-glutamine] (PHEG). In the first step MMC was linked to the peptide spacer. For preparing the oligopeptide-MMC derivatives N-Fmoc protected oligopeptide was activated as the pentafluorophenyl ester. After coupling with MMC the N-protective group can be removed under mild conditions. The spacer-MMC derivatives were finally coupled to 4-nitrophenyl chloroformate activated PHEG. Keword.s:

Synthesis:

Mitomycin

C: Prodrug

1. Introduction The success of cancer chemotherapy has been limited due to a lack of cell selectivity of the cytostatic drugs. They interfere not only with tumor cells but also with healthy cells. This leads to severe toxic effects and seriously limits the efficacy of cancer chemotherapy. One way to improve the efficiency of antitumor drugs is by attachment to macromolecular carriers. This alters the body distribution, limits uptake to the pinocytotic route [ 1 ] and affords a mechanism for the concentration of the drug in the target cells. Both natural and synthetic polymers have been proposed as potential carriers for a variety of anticancer drugs [ 2-51. Among the literature is a vast amount of work by Sezaki and co-workers who prepared dextran derivatives of Mitomycin C (MMC) in an attempt to optimize the pharmacokinetics and the efficacy of the drug [ 6101. In their approach, a spacer group,i.e. 5-amino caproic acid, was first attached to the polymeric carrier. *Corresponding

author.

016%3659/94/$07.00

0 1994 Elsevier

.SSDlOl68-3659(94)00049-Z

Science B.V. All rights reserved

Finally, MMC was coupled to the resulting dextran derivative. In such a reaction sequence characterization of the final reaction product may not be so straightforward since most polymer analogous reactions are not quantitative. Moreover, spectroscopic analysis of polymer derivatives is more complex than that of low molecular weight analogues. Therefore, we decided to work out an alternative strategy for the preparation of well-characterized polymeric derivatives of MMC whereby a spacer group is first coupled with MMC. This conjugate which can be well purified and characterized is then coupled onto a suitable activated polymeric carrier. For this work poly-[ “N-( 2-hydroxyethyl)-L-glutamine] (PHEG) was selected as an alternative for dextran. In a previous paper we have demonstrated that dextran has a limited biodegradability which is further reduced by chemical modification [ Ill. Moreover, reaction of dextran with chloroformate does lead to different types of carbonate ester moieties, due to the presence of vicinal dial groups in the polysaccharide [ 121. On the other hand, it has been demonstrated that

130

A. De Mum

et trl. / Joumtrl

of Controlled

Releuse 32 (1994) 129-137

motherapy is limited by the manifestation of severe bone marrow depression and gastrointestinal damage

[251. One approach to overcome these side-effects and to enhance the therapeutic efficiency of the drug is to link the antitumor drug to a polymeric carrier. These polymeric drug conjugates can only be absorbed by pinocytosis. 2.1. Synthesis of PHEG

2

1

Fig. I. Structural roformate 2.

3

formula of PHEG 1 and activation

of 1 with chlo-

poly- [‘N- (2-hydroxyethyl) -L-glutamine] (PHEG) is a good substrate for lysosomal enzymes. The polymer is non toxic, non immunogenic and possesses primary hydroxyl side groups [ 13-l 81 which can easily be converted into reactive carbonate esters by reaction with chloroformate 2 (Fig. 1) [ 191. Peptides of variable composition were selected as spacers between the carrier and the drug. It has been shown that liver lysosomal enzymes can release drugs from certain peptidyl side-chains in polymeric drug conjugates [ 20-231. Based on the available literature data gly-phe-gly, gly-phe-leu-gly, gly-phe-leu, glyphe-phe, gly-gly-phe, gly-gly-phe-leu, gly-phe-ala-leu and ala-leu-ala-leu-MMC derivatives were prepared. In a last step these amine containing peptide-MMC moieties were conjugated to the chloroformate activated carrier.

2. Results and discussion MMC (4 Fig. 2) is an antitumor antibiotic which was first isolated by Wakaki [ 241. It belongs to the class of alkylating cytostatics. Although MMC has been found to have a broad spectrum of activity against several solid tumors of different organs, its use in che0 CH2

H2N I

I

R?i

0

I

0

t

NH~CH~E I CH2

0 NH~CH~I! + ”

NH2-

hH2

(CH2)

2-OH *+

6

Lo 6

I CH*

”

AH2 Lo I NH-

cn**

+

(CH2)

,,,i OCH3 N

H3C

fl

-O-C-NH2

In this study PHEG is selected as macromolecular drug carrier. This synthetic poly-aminoacid is a neutral, water-soluble and biocompatible polymer that was first proposed as a plasma expander by Neri [ 131. PHEG is usually prepared by aminolysis of poly-( y-benzylglutamate) with 2-aminoethanol (Fig. 3). However, extensive chain scission due to aminolysis of the amide linkages of the polymer backbone has always been observed [ 26-281. This limits the preparation of welldefined PHEG derivatives with controlled molecular weight. Feijen [ 291 reported the use of the bifunctional catalyst 2-hydroxypyridine in the aminolysis of polybenzylglutamate with 3-aminopropanol. Application of catalyst proved being succesful both with respect to yield and molecular weight. However, aminolysis occured at 60°C and main-chain cleavage was still significant. In a previous study [30] we have demonstrated that the application of different amounts of 2-hydroxypyridine greatly influences the rate of the aminolysis reaction and the molecular weight of the obtained PHEG, both at ambient temperature and at 40°C. The reaction at 40°C in presence of aSfold molar excess of catalyst leads to complete conversion of sidechain ester groups into 2-hydroxyethyl groups within 24 hours. Moreover, chain scission due to aminolysis of main-chain amide bonds was minimal under these conditions. This indicates that the described method is

NH

Fig. 2. Structural formula of MMC 4

1

5

Fig. 3. Aminolysis

of 5 with 2-aminoethanol6.

2 OH

A. De Marre et al. /Jotrmal

uf Controlled Release 32 (1994) 129-137

useful for the prep~ation of PHEG derivatives with well-defined molecular weights which can be used for biomedical purposes. 2.2. Synthesis of peptide-MMC

derivatives

In this work MMC is linked to PHEG via a peptide spacer. Peptides that are substrates to lysosomal thioldependent proteinases were selected in order to prepare polymeric derivatives that could be used for lysosomotropic drug delivery. Following oli~opeptides were selected: glycyl-phenylalanyl-glycine (gly-phe-gly), glycyl-phenylalanyl-leucyl-glycine (gly-phe-leu-gly), glycyl-phenylalanyl-leucine (gly-phe-leu), glycyl-phenylalanyl-phenylalanine (gly-phe-phe), glycyl-glycyl-phenylalanine (gty-gly-phe), glycylglycyl-phenylalanyl-leucine (gly-gly-phe-leu), glycyl-phenylalanyl-alanyl-leucine (gly-phe-ala-leu) and alanyl-leucyl-alanyl-leucine (ala-lcu-ala-leu). A strategy was worked out for the preparation of these polymeric-peptide-MMC derivatives which minimizes degradation of MMC during derivatization. First, MMC was coupled to a peptide spacer (Fig. 4), finally these oligopeptide-MMC derivatives were coupled with the polymeric carrier (Fig. 9). The peptide-MMC derivatives (gly-phe-giy, glyphe-leu-gly, gly-phe-leu, gly-phe-phe, gly-gly-phe, gly-gly-phe-leu, gly-phe-ala-leu and ala-leu-ala-leu) were synthetized following the strategy presented in Fig. 4. (a) Frnoc protection of the peptides In order to couple MMC to the peptide spacer, blocking of the a-amino function of the peplide is necessary. Since MMC is unstable under acidic and alcaline conditions and is easily reduced, the choice of the aminoprotecting group is rather limited. The 9-AuorenylmethyloxycarbonyI (Fmoc) group, proposed by Carpino and Han [ 3 1 ] for the protection of the a-amino function in peptide synthesis, was

131

+ NH:, -

Qly-pht-glyCOO”

9

a

I

v =I

1;

cn* -

0 -

FI NH

c-

-

giy-phs-gty-

cmx

10

Fig. 5. Fmoc-protection of peptide 9.

selected. The Fmoc protection has been applied, and with considerable success, in solid-phase peptide synthesis and in stepwise chain lengthening of peptides in solution [ 32-331. A particular advantage of this protecting group is that it can be removed by basic reagents under mild conditions, e.g. under influence of a secondary or tertiary amine. The protecting group can be introduced by reaction of the peptide with 9-fluorenyloxycarbonylchlo~de (Fmoc-Cl) in aqueous/organic medium as shown in Fig. 5 for gly-phe-gly as an example. Complete conversion was verified by 1H NMR spectroscopy. (6) Preparation of the pentajkorophenyl ester of the Fmoc-peptides Active esters can be used for acylation of amino acids in peptide chain leng~ening. Pentafluorophenyl esters are often applied in solid-phase peptide synthesis. Spectacular acceleration of acylation is observed using these active esters [ 341. Therefore, we selected pentafluorophenyl esters of peptides for the coupling of the Fmoc protected oligopeptides with MMC. The reactive esters were readily prepared by DCC coupling between Fmoc-peptide and pentafluoro-

Fig. 4. Reaction sequence for the synthesis of the ~eptide-MMC derivatives.

132

A. De Morre et al. /Jofournul c~Cmtfmlled

I

(d) ~eprot~ct~o~ of the F~oc-p~pt~de-~~~d~riv~t~ve Removal of the Fmoc protecting group occurs by treatment with a base. /3 Elimination process leads to the formation of dibenzofulvene, carbondioxide and the free amine-containing moiety. In this work triethylamine was used for the deprotection of the peptideMMC derivatives. After isolation of the reaction product complete removal of the protecting group could be verified by NMR analysis. The above results demonstrate the feasibility of the novel strategy for the preparation of well characterized peptide-MMC derivatives.

DCC

F

F

12

Fig. 6. Esterification

of 10 with penta~uorophenoi

Release 32 (1994) 129-137

Il.

phenol as presented in Fig. 6 for gly-phe-gly as an example. The IR spectrum of these derivatives showed a significant absorption at 1790 cm- ’ from the aromatic ester. Identification was also possible by TLC in CHClJMeOH (9i 1) , Rf values of the different Fmocpeptide esters are given in Table 2.

(c) Preparation of the F~~loc-p~pt~de-~~~ de$~vat~ves Low molecular lipophilic prodrugs of MMC reported in literature were prepared by acylation of the aziridine nitrogen, using N-hydroxysuccinimide esters [ 35 ] or acid chlorides [ 361. In this study reactive esters of the protected peptides and MMC results in the acylation of the aziridine nitrogen of MMC (Fig. 7). After purification of the reaction product by column chromatography,‘H NMR analysis proved complete conversion. By comparing the integrations one could con&de that MMC (CH,: 1.7 ppm) , Fmoc (aromatic protons: 7.3-7.8 ppm) and peptide (see Table 1) were present in a 1/ I/ 1 ratio. It should be noted that during the preparation of the Fmoc-gly-phe-phe-MMC derivative racemization had occured. The LL and DL-phe isomers were separated by column chromatography and identified using a CC analysis method based on L-phe and D-phe standards. Both conjugates were identified using the following procedure: following acid hydrolysis of the peptide, the corresponding amino acids were derivatized by esterification of the carboxyl group with isopropanol and acylation of the amino function with trifluoroacitic anhydride. Finaily, the products were analyzed and identified by GC.

2.3. 4-N~trophenyl chlor~f~r~ate activation of PHEG PHEG is a hydroxyl containing polymer. These hydroxyl groups should be activated to allow coupling of amine containing peptide-MMC moieties. The 4nitrophenyl chloroformate activation was used [ 191. Reaction of this chloroformate and polymer results in conversion of the hydroxyl groups into reactive carbonate esters (Fig. 1) . The degree of carbonate substitution was determined by UV analysis (402 nm) after alcaline hydrolysis and expressed as the amount of Table I ‘H NMR data from the diffeent Fmoc-protected Peptide

Signal

peptide derivatives 6 (ppm)

gly-phe-gly

CH, (phe)

gly-phe-leu

aromat. H (phe) 2XCH3 (leu)

gly-phe-phe

CHZ (phe) aromat. H ( phe) 2XCH, (phe) aromat. H (phe)

gly-gly-phe

CH2(phe)

gly-phe-leu-gly

aromat. H (phe) 2xCH, (leu)

gly-gly-phe-leu

aromat. H (phe) 2XCH3 (leu)

CHZWe)

CH2We) gly-phe-ala-leu

ala-ieu-ala-leu

aromat. H (phe) 2xCH, (leu)

2.1-J 7.1-7.2 0.9 2.7-3 7.2 2.7-3.1 7.1-1.2 2.8-3. I 7. i-7.2 0.8 2.1-3 1.2 0.8-0.9 2.7.-3. I 7.1-7.2 0.8-0.9

Cl& (ala)

I .2

CH, tphef

2.7-3.1 7.2 0.x-0.9

aromat. H (phe) 4XCH3 (leu) ~xCH, (ala)

1.2

A. De Marre

et al. /.kxtmai

c~C~~ntr&ed

Release 32 (1994) 129-137

133

carbonate esters per 100 repeat units in the polymer

Table 2 Rf values of the pentafluorophenyl peptides (TLC

esters of the different

was run in CHCi,/MeOH

91

Fmoc-

Peptide

Rf

gly-phe-gly

0.65

gly-phe-leu-gly

0.7

gly-phe-ieu

0.64

gfy-phe-phe

0.75

gkgiy-phe giy-gly-phe-teu

0.65

gly-phe-ala-leu

0.59

ala-leu-ala-leu

0.63

Fmoc-

(mol%). In a previous study we have demonstrated that the content of 4-nitrophenyl carbonate groups reaches a maximum value after 4 hours of reaction at 0°C. Furthermore, the effect of the added amount of chloroformate on the activation degree was investigated. It was demonstrated that the carbonate content is proportional to the amount of chloroformate added. Thus, the degree of activation of the polymer can be controlled by adjusting the amount of chloroformate added (Fig. 8) _Further, the coupling of activated PHEG with amines is quantitative for model amines as well as for drug and targeting moieties with a terminal amino group. Therefore, the 4-nitrophenyl chloroformate activation is selected for the activation of the hydroxyl groups of PHEG, in order to introduce the prepared peptideMMC derivatives into the polymeric carrier.

If

0.62

NH -

.&-ph.~-g,~

-

F

F

F

F

&o 4

12

2.4. C~l~~litlgof the di~re~fpe~tide-TIC derivatives to the polymeric carrier Fmoc- NH - guy-phe-gty - 8

Activated polymer (4 mol%) and peptide-MMC derivative were reacted for 48 hours in the dark (Fig. 9). The reaction product was isolated by precipitation in ether/ethanol and further purification by preparative CPC (Sephadex G25) with water as eluent and freezedrying. The MMC content in the polymer conjugates was determined by UV spectrometry at 364 nm. Complete conversion of the reactive carbonate esters into urethane-bound peptide-MMC derivative was observed. The molar and weight percent of MMC substitution in the different PHEG-peptide-MMC conjugates are given in Table 3.

13

Fig. 7. Coupling

of MMC

4 with reactive ester X2.

30 7

-

25 I

/ .*’

15 ,/’ >’

_ 10 t

,/ ,/

5 + ! /

3

/’ ,’

I

0 EL----.._. 0 0.5

(1

)/ 1

1.5

2

Mole ratio 2 / 1 - unit Fig. 8. Effect of the amount of 2 added on the degree of activation

Fig. 9. Coupling of peptide-MMCdLkative

of 3.

3.

14 with activated PHEG

114

A. De Mm-w et ui. /Journal

Table 3 Molar and weight percent substitution PHEG-peptide-MMC conjugates

of Contmiled

of MMC in the different

Peptide

MOP%

WV%

gly-phe-gly gly-phe-leu-gly gly-phe-leu gly-phe-phe

1.7 2.2 2.9 3.2

3 3.9 5.1 5.5

gly-gly-phe

3A

5.9

giy-gly-phe-leu gly-phe-ala-leu ala-leu-ala-leu

3 3.6 2.9

5.2 6 5

3. Experimental

part

3. I. Materials MMC was a kind gift of Kyowa Hakko Ltd (Tokyo, Japan). All aminoacids, peptides and Fmoc-Cl were obtained from Bachem Chem. Co. (Bubendorf, Switzerland). All amino acid and peptide derivatives were L-configuration. 4-nitrophenyl chloroformate was obtained from Merck (Darmstadt, Germany). All other chemicals were purchased from Janssen Chimica (Beerse, Belgium).

Synthesis of PHEG PHEG can be prepared by aminolysis of poly-ybenzyl-L-glutamate 5 with 2-amino-l-ethanol& PBLG was prepared using standard procedure: the N-carboxyanhydride (NCA) of y-benzyl-L-glutamate was prepared by the method of Daly [ 371. The NCA was polymerized at room temperature in dioxane in the presence of triethylamine as the initiator [ 381. After polymerization for 3 days, 5 was precipitated in excess ether, filtered and dried. Aminolysis of 5 with 2hydroxypyridine 7 as a catalyst (ratio catalyst/ester was in DMF groups=S/l), carried out (dimethyl-formamide) as a solvent [ 301. 1 g (4.57 mmol) of 5 and 2. I7 g (22.8 mmol) of 2-hydroxypyridine were dissolved in 20 ml DMF, 2-aminoethanol was added dropwise over 1 hour (5.57 ml, 9 1.3 mmol). The reaction mixture was stirred at 40°C for 24 h. PHEG was precipitated in excess diethylether/ethanol (4/l v/v), filtered and dialyzed against water for 2 days. After freeze-drying pure PHEG was obtained as

Release 32 (1994) 129-137

a white powder. The molecular weight of 1 was determined by gel-permeationchromatography (GPC) with water as eluent (TSK-G3~OSW, G2~SW) using dextran standards (MW = 88500, 33 100 and 10000). The number-and weight-average molecular weights were Mw = 16500 and Mn = 13000. Preparation of peptide-MMC derivatives All pept~de-MMC derivatives were prepared using the same strategy. As an example is given the synthesis of gly-phe-gly-MMC. (a) Fmoc protection of the peptide 9 0.2 g of gly-phe-gly 9 (0.7025 mmol) was dissolved in 5 ml of a 10% solution of Na,CO, in water/dioxane mixture (vol. ratio 21’1) and cooled in an ice bath. Then 0.182 g of Fmoc-Cl 8 (0.7025 mmol), dissolved in I ml dioxane, was added. The reaction mixture was stirred for 2 h at room temperature and added to 50 ml of water. After extraction with ether (20 ml) the aqueous layer was acidified with cont. HCI. The white precipitate was extracted with ethylacetate. The combined ethylacetate fractions were dried on magnesium sulphate. After evaporation of the solvent the protected peptide 10 was obtained as a solid (85%). The product was ch~acterized by’H NMR spectroscopy (360 MHz) in DMSO-d6 (dimethyl sulfoxide): 6=2.9 ppm : H, CHZ-phe, S= 3.2 ppm : Hn CH,-phe, S = 3.7 6= 3.9 6=4.2 ppm : CH,-gly, ppm : CHZ-gly, ppm : CH-Fmoc, 6 = 4.35 ppm : CH,-Fmoc, 6= 4.65 ppm CH-phe, 6 = 7.2 ppm : arom. protons phe, 6= 7.37.8 ppm : arom. protons Fmoc. By comparing the integrations of the signals at 7.37.8 ppm (aromatic protons of Fmoc) and the signals of the different aminoacids in the peptide eg. 7.1-7.2 ppm (aromatic protons of phe) complete protection could be confirmed. Typical yield of the reaction: 7& 80%. Fmoc-gly-phe-leu-gly, gly-phe-leu and gly-phe-phe were obtained following the same reaction procedure. This method could not be used for the protection of gly-gly-phe since it was very difficult to extract the product from the acidified aqueous phase. Therefore, Fmoc-gly-gly-phe was prepared by reacting equimolar amounts of Fmoc-glycine pentafluorophenyl ester and gly-phe in a 1/ 1 mixture of THF( tetrahydrofuran) / ethylacetate. The protected tripeptide could be isolated after reaction overnight and precipitation in a l/l mix-

ture of diethylether/hexane. Complete conversion was observed by TLC (~H~l~/Me~H 911, ninhydrin detention of free peptide) _ All protected peptides were characterized by NMR spectroscopy in DMSO-d6 by comparing the integrations of the signals of the aromatic protons of Fmoc (7.3-7.8 ppm) and these of the different aminoacids in the peptide as given in Table 1.

(b) Synthesis of the pe~ta~l~orophenyl ester of the Fmoc-peptide 10 0.18 g of 10 (0.355 mmol) and 0.078 g of pentafluoropheno1 11 (0.425 mmol) were dissolved in 3 ml dry THF. After cooling to O*C 0.073 g of dicyclohexylcarbodiimide (DCC) (0.355 mmol) was added. The reaction was stirred for 2 h at 0°C and overnight at room temperature. Complete conversion of Fmoc-peptide into reactive ester derivative was followed by TLC. After filtration of dicyclohexylurea (DCU) and concentration of the solution, the reaction product 12 was obtained by precipitation in a 1I1 mixture of diethyletherlhexane. All other peptides were converted intoreactiveesters following the same procedure. The reactive esters were characterized by IR spectroscopy (aromatic ester: 1790 cm-‘) and TLC (etuent: CHCl~/MeOH 911) Rf values are given in Table 2. Typical yield of the reaction: 80-900/o. (c) Coupling of MMC to the reactive ester 12 0.18 g of 12 (0.267 mmol) and 0.089 g of MMC 4 (0.267 mmol) were dissolved in 5 ml DMF and 0.1 ml of pyridine was added. After 48 h reaction in the dark at room temperature, the solvent was evaporated under vaccuum (temperature not exceeding 30-4O”C) and the residue was purified by coIumn chromatography on silica f eluent: ~HCl~/MeOH 911). The selected fraction was dried over MgS04. After removal of the solvent the Fmoc-gly-phe-gly-MMC derivative 13 was finally obtained as a blue solid. ‘H NMR in MeOD-d4: S= 1.7 ppm:CH,3-MMC, 6= 2.9 ppm : H,-phe, 6 = 3.1 ppm : Hn-phe + OCH,MMC, S = 3.4-3.65 ppm: H2,H3’,H 1, H9-MM~, S- 3.65-3.8 6=3.85-4.1 ppm : CH2-gly, ppm : CH,-gly + H lo-MMC, S= 4.2 ppm : CH-Fmoc, S = 4.35 ppm : CH,-Fmoc, 6 = 4.45 ppm : H3-MMC, Sz4.65 ppm: CH-phe, 6=4.75 ppm:HlO’-MMC,

6= 7.15 ppm : arom.protons phe, 6=7.25-7.X ppm : arom.protons Fmoc. The same method was applied for the other Fmocpeptide-MMC derivatives. All derivatives were characterized by’H NMR analysis in MeOD-d4. The integrations of the most important signals of MMC (CH,: 1.7 ppm), Fmoc (aromatic protons: 7.3-7.8 ppm) and signals from the different aminoacids in the peptide were compared and complete conversion was observed. Typical yield of the reaction: 60-70%. The purity of Fmoc-protected peptide-MMC derivatives was further tested by reversed phase HPLC (Bondapak C18, eluent: 80120 CH~CN/phosphate buffer pH= 7.4 O.O2M, rate: I mflmin, detection: UV : 364 run), all product appeared as one single peak in the chromatogram. During the preparation of Fmoc-gly-phe-phe-MMC racemization occured. The LL and DL-phe isomers were separated by column chromatography on silica (eluent: CHClJMeOH 95/5) and identified by GC analysis. TLC of the products was run in CHCl~/MeOH 9515 and the product with Rf = 0.16 contained 44%Dphe and 56% L-phe, that with Rf = 0. I contained L-phe for more than 95%. (d) Remo~~a~ of the Fmoc protecting group 0.05 g of 13 was dissolved in 1 ml DMF, 0.1 ml of triethylamine (TEA) was added. After reaction for 3 h at roomtemperature the solvent was evaporated under vaccuum (temperature not exceeding 30-4O’C). The residue was dissolved in 3 ml MeOH and the solution was filtered. The amine containing peptide-MM~ conjugate 14 was finally obtained after evaporation of the solvent. After NMR analysis in MeOD-d4 no signals from Fmoc could be detected all other signals had the same chemical shift as discribed above for 14.

4~n~tropheny~chloroformate activation of PHEG 0.25 g of 1 (1.45 mmol units) and 16 mg of 4dimethylaminopyridine (0.13 mmol) were dissolved in 6.25 ml of a NMP( N-methylpyrrolidone) /pyridine solution (vol. ratio 4/ 1), 0.176 g of chloroformate 2 (0.87 mmol) was added at 0°C. After 4 h reaction at 0°C the reaction mixture was precipitated in an anhydrous diethylether/ethanol mixture (vol. ratio 21 I ). A white precipitate was collected and washed repeatedly with the same mixture. The product 3 was finally dried. The carbonate content was determined by UV analysis

136

A. De Murre et al. /Journal

after alcaline hydrolysis in NaOH EM= 18400 1molI’ cm-‘) [ 191.

( pM =402

of Conrrolled nm,

Preparation of the polymeric-peptide-MMMMC conjugates 0.2 g of 3 (0.05 mm01 reactive groups, 4 mol% activation) and 30 mg of gly-phe-gly-MMC 14 (0.05 mmol) were dissolved in a 15 ml NMP/pyridine solution (vol. ratio 4/ 1), After 48 h of reaction in the dark the conjugate was separated by precipitation in an anhydrous diethylether/ethanol mixture (vol. ratio 2/l ). The product was washed and dried. Finally the conjugate 15 was purified by preparative GPC (Sephadex G25) with water as eluent and freeze-drying. The degree of MMC substitution in the conjugates was determined by UV analysis in water ( pM = 364 nm, e,=22000 I mall’ cm-‘). The molar and weight percentages of MMC substitution in the different polymeric-peptide-MMC conjugates are shown in Table 3.

4. Conclusion This paper describes a procedure for the preparation of macromolecular peptide-MMC derivatives. The present method allowes the coupling of MMC with oligopeptide spacer under mild conditions without degrading the mitomycin structure. In a next step these peptide-drug moieties are conjugated to the polymeric carrier leading to well characterized polymeric derivatives. The influence of the oligopeptide spacer on the hydrolytic and enzymatic stability of the polymeric conjugates as well as the in vitro and in vivo antitumor activity of these conjugates will be reported in a subsequent paper.

Acknowledgement This research has been supported by the Institute for Encouragement of Research in Industy and Agriculture (I.W.O.N.L.) The Nationaal Fonds voor Wetenschappelijk Onderzoek and the Concerted Action Programme on A~tit~mor Drug Delivery by the European C~ini~z~ni~.Special thanks to KJ~OWUHukko Co. for the supply of MMC.

Release 32 (1994) 129-137

References I I] C. De Duve, T. De Barsy, B. Pode, A. Trouet, P. Tulkens, F. Van Hoof, Lysosomotropic agents, Biochem. Pharmacol. 23, 19742495253 1. 121 H. Ringsdorf, Structure properties of pharmacologically active polymers, J. Polym. Sci. Symp. 51, 1975135153. ]3] J. Kopecek, R. Duncan, Poly-[N-(2hydroxypropyl)methacryl amide ] macromolecules i2r, drug carrier systems, in: L. Illum. S. Davis (Eds.), Polymers in Controlled Drug Delivery, Wright, Bristol, ( 1987) p, l52- 170. 141 L. Molteni In Drug Carriers in Biology and Medicine (Gregoriadis G.,ed.) ~107, Academic Press, London ( 1979). ]5] A. Trouet, D. Deprez-De Cameneere. C. De Duve, Chemotherapy through lysosomes with DNA-daunomycin complex, Nature New Biol. 239, ( 1972) I 10-l 12. ] 6] T. Kojimn, M. Hashida, S. Muranishi, H. Se&i, Mitomycin C-dextran conjugate: a novel high molecular weight prodrug of Mitomycin C, J. Pharm. Pharmacol. 32 ( 1980) 30-34. [ 7 1A. Kato, Y. Takakura, M. Hashida, H. Sezaki, Physicochemical and antitumor characteristics of high molecular weight prodrugs of Mitomycin C, Chem. Pharm. Bull. 30 (1982) 295 l-2957. 181 S. Matsumoto, Y. Arase. Y. Takakura, M. Hashida, H. Sezaki, Plasma disposition and in viva and in vitro antitumor activities of Mitomycin C-dextran conjugates in relation to its mode of action, Chem. Pharm. Bull. 33 ( 1985) 2941-2947. ]9] Y. Takakura, A. Tnkagi. M. Hashida. H. Sezaki, Disposition and tumor localization of Mitomycin C-dextran conjugates in mice, Ph,arm. Res. 4 ( 1987) 293-300. I1K. Nishida, C. Tonegawa, S. Nakane. Y. Takakura, M. Hashida, H. Se&i, Effect of electric charge on the hepatic uptake of macromolecules in the rat liver, hit. J. Pharm. 65 ( 1990) 717. I R. Vercauteren, D. Bruneel, E. Schacht, Effect of the chemical modification of dextran on the degradation by dextranase, J. Bioact. Compat. Polym. 5 (1990) 4-15. [ 121 F. Vandoorne, R. Vercauteren, D. Permentier, E. Schacht, Reinvestigation of the 4nitrophenyl chloroformate activation of dextran. Evidence for the formation of different types of carbonate moieties, M~romol. Chem. 186 ( 1985 ) 2455-2460. 1131 A. Gerola, G. Antoni, F. Benvenuti, F. &cola, P. Net?, In: Shock: biomedical, pharmacoIogic~ and clinical aspects, Plenum Press, N.Y. (1970). 1141 H. Dinckinson, A. Hiltner, Biodegradation of poly-a-amino acid hydrogels II. In vitro. J. Biomat. Res. IS ( 198 I ) 591-603. I 151T. Hayashi, Y. Tabata, A. Nakajima, Degradation of poly-cyamino acids in vitro II. Biodegradation of copolymers consisting of N-hydroxyalkyl-L-glutamine and L-glutamic acid, Rep. Progr. Polym. Phys. Jap. 26. (1983) 591-594. [ 161 F. Rypacek, V. Saudek, J. Pytela, V. Skarda, J. Drobnik. Polyglutamines-Versatile biodegradable carriers, Makromol. Chem. Suppl. 9 (1985) 129-135. 1171 J. Pytela, V. Saudek, J. Drobnik,F. Rypacek, Poly(N-hydroxyalkylglutamines) IV. Enzynlatic degmdation of N(2-hydroxy ethyl)-L~~lutan~ine holnopolymers and copolymers, J. Contr. Release 10 (1989) 17-25.

ctfControNed Releuse

A. De Marre et ul. /Journal

I I8 I J. Pytela, R. Kotva, M. Metalova, F. Rypacek, Degradation of N-( ~-hydroxyethyI)-L-glutaminc and L-glutamic acid homopolymers and copolymers I2(1990)241-246.

by papain, Int. J. Biol. Macromol,

I 191A. De Marre, E. Schacht, carbonate

Preparation

of 4nitrophenyl

esters of poly-[N-(2-hydroxyethyl)-L-glutamine]

and coupling

with bioactive

agents,

Makromol.

Chem.,

193

( 1992) 3023-3030. 1201 R. Duncan,

P. Rejmanova.

uptake and intracellular methacrylamide (IYSI)

J. Kopecek,

degradation

copolymers,

J. Lloyd,

Pinocytic

of N-(2-hydroxypropyl)

Biochim.

Biophys.

Acta, 678

143-150.

121i V. Subr, J. Kopccek. R. Duncan, Degmdation of oligopeptide sequences

connecting

poly-( N-( 2.hydroxypropyl)nlethac~I

alnide)chains

by lysosomal

Compat. Pal..

1 (1986) 133-146.

1221 K. Ulbrich, E. Zacharieva, Polymer-bound

cysteine

proteinases.

J. Bioact.

J. Kopecek,

I. Hume, R. Duncan,

ofsarcolysin

and their antitumor

derivatives

activity against mouse and human leukemia Makromol. Chem., 188 (1987) 2497-2509.

in

vitro,

I23 I V. Subr, J. Strohalm, K. Ulbrich, R. Duncan, I. Hume, Polymers containing

enzymatically

spacer stuctrureon

degradable

bonds,

poly-(N-(2-hydroxypropyl)methac~Iamide carriers in vitro and antitumor activity Control. Release. 18 ( 1992) 123-132. 1241 ST.

X11. Effect of

the rate ofdaunomycinandadriamycin

from

copolymer drug measured in viva, J.

Crooke.

In Mitomycin

C: Current

Developments,

S.K. Carter,

S.T. Crooke,

Status

and New

Academic

Press

(1979). 125 I S. Carter. S. Crooke, In Mitomycin C: Current Status and New Developments Academic Press, New York ( 1979). ] 26 ] N. Lupu-Lotan,

A. Jaron. A. Berger, M. Sela, Conformational

changes in the nonionizable

watersoluble

synthetic polypeptide

poly-N-(3-hydroxypropyl)-L-glutamine, (1965) 625-655. 1271 K. Okita,

A.Tenmoto,

synthetic polypeptides hydroxy 738.

H. Fuji&

Biopolymers, Solution

properties

3 of

Vi. Helix-coil transition of poly-(N-3-

propyl)-L-glutamine,

Biopofymer.

9 ( 1970) 717-

32 (1994) 129-137

117

1281 D. Noskova, R. Kotva, F. Rypacek, PoIy-(N-hydraxyalkyl glutamines): 2. The effect of conformation and solvent on the kinetics of poly-y-alkyi glutamate aminolysis, Polymer, 29 (1988) 2072-2075. [29] W. van Heeswijk, G. Brinks, J. Feijen, Preprints of the International Symposium on Polymers in Medicine, Port0 Cervo (Italy, (1982)) 3169. 1301 A. De Marre, H. Soyez, J. Pytela, E. Schacht, Improved method for the preparation of poly-[ N(2-hydroxyethyl )-L-glutamine ] by aminolysis of poly-( y-benzylglutamate), Polymer, I1 (1994) 2443-2446. [ 3 I ] L. Carpino. G. Han, The 9-fluorenylmethoxycarbonyl aminoprotecting group, J. Org. Chem., 22 ( 1972) 3404-3409. j32] A. Bodanszky, M. Bodanszki, N. Chandramouli, J. Kwei, .I. Martinez, J. Tolle, Active esters of 9.~uorenylmethyloxy carbonyl amino acids and their application in the stepwise lengthening of a peptide chain, J. Org. Chem., 45 (1980) 7276. [33] L. Carpino, D. Sadat-AaIaee, H. Guang, R. DeSeIms, 9Auorenyl methyloxycarbonyl aminoacid fluorides. Convenient new peptide coupling reagents applicable to the FMOC/tertbuthyl strategy for solution and solid-phase synthesis, J. Am. Chem. Sot., I12 (1990) 9651-9652. [ 34 1L. Kisfaludy, J. Roberts, R. Johnson, G. Mayers, J. Kovacs, Synthesis of N-carbobenzoxyamino acid and peptide pentafluorophenyl esters as intermediates in peptide synthesis, J. Org. Chem., 35 ( 1970) 3563-3565. ] 35 ] Y. Tokunaga, T. Iwasa, J. Fuji&i, S. Sawai, A. Kagayarna. Liposomal sustained-release delivery system for intravenous injectionl. Physicoche~cal and biological properties of newly synthesized lipophilic derivatives of Mitomycin C, Chem. Pharm. Bull., 3 ( 1988) 3060-3069. [ 361 H. Sasaki, E. Mukai, M. Hashida, T. Kimura, H. Se&i, Development of lipophilic prodrugs of Mitornycin Cl. Synthesis and antitumor activity of la-Nsubstituted derivatives witharomatic pro-moiety, Int. J. Pharm., 15 ( 1983) 49-59. [ 37 ] W. Daly, D. Poche, Preparation of N-carboxyanhydrides of cyamino acids using bis( trichloromethyl)carbonate, Tetrahed. Len, 46 ( 1988) 5859-5862. [ 381 E. Blout, R. Carlson, Polypeptides.IIl. The synthesis of high molecular weight poly-y-benzyl-L-glutamates. J. Am. Chem. Sot., 78 (1956) 941-946.