CS223417B1 - Polymere substances with antitumour activity and method of preparing the same - Google Patents

Description

The invention relates to polymeric substances having antitumor activity and to a process for their preparation.

Binding of biologically active substances to polymers by so-called spacer groups is known (Ringsdorf n., J. Polym. Sci. Polym. Symp. 51, 135-153, 1957, Kopecek J., Polymers in Medicine 7, 191-220, 1977 ) that allow for their convenient bonding and release. These include water-soluble copolymers containing oligopeptic chains (Ulbrich K., Zacharieva IE, Obererigner B., Kopecek J., Biomaterials, 1, 199, 1980) enzymatically degradable in vitro by papain (Ulbrich K., Zacharieva IE, Oberereigner B., Kopecek J., Biomaterials, 1, 199, 1980), chemotrypsin, trypsin (Ulbrich K., Strohalm J., Kopecek J., Macromol. Chem., In press.), Tritosome (Dunkan R., Lloyd Y , B., Kopecek J., Biochchem, Blophys, Res. Comim., 284-290, 94, 1, 1980). The structure of the polymer must be chosen such that the binding of the active ingredient is based on a specific amino acid for the enzyme that induces cleavage.

There are no data in the literature on the use of polymeric carriers of cytostatics of this type.

The drawbacks of these cytostatics are their toxicity, i.e., they are detrimental to proliferative cells such as bone marrow, lymphatic tissue, gastrointestinal tract, genitourinal epithelium and non-specificity of their effect.

SUMMARY OF THE INVENTION The present invention provides a polymeric compound having an antitumor activity based on copolymers of N- (2-hydroxypropyl) methacrylamide and methacrylamide of the general formula:

r- 1 ³ -CHfC- * I X -CHfC " 1 WHAT ---, 1 <co <| >

NH

AND

CH-OH

AND

CW, i

WHAT

AND

NH

I λ

SBfPE where x = 85 - 97 mol. y = 3-15 mol. why. in mol. M _w = 15-40,000, R is an enzymatically cleavable spacer, and —X — SBIPE is a low molecular weight antitumor agent bound to a carrier by a covalent bond.

The side spacer unit may preferably be glycine (Gly) or glycine-leucine- (Gly-Leu-j).

The substance of the process of the invention is characterized in that the polymeric carrier is dissolved in dry dimethylsulfoxide and, with stirring in the presence of triethylamine, NH 2 -X-SBIPE is added and reacted for 20 hours and the product isolated by precipitation in acetone: diethyl ether. (3: 1) followed by purification by doubly dissolving in methanol and precipitation into acetone: diethyl (3: 1).

When NH2-Phe-SBIPE is used, the present invention is first prepared by NH2-Phe-SBIPE by reacting sarcolysine isopropyl ester hydrochloride with benzyloxycarbonylphenylalanine (BOC-Phe-) in the presence of triethylamine. OH] at -10 to -5 ° C in tetrahydrofuran by DCCI, then the blocking benzyloxycarbonyl (BOC) is removed with a solution of HCl in methanol and the phenylalanylsarcolysin hydrochloride is isolated by filtration and added to the polymer solution in dry dimethylsulfoxide or dimethylformamide in the presence of triethylamine. and allowed to react at room temperature for about 20 hours and the product obtained is isolated by precipitation into acetone: diethyl ether (3: 1) followed by purification by doubly dissolving in methanol and precipitation into acetone: diethyl ether (3: 1).

Compounds with antitumor activity according to the invention have reduced toxicity, increased specificity and prolonged effect of action.

The problem is solved by covalently bonding a certain active cytostatic with a water soluble copolymer based on N- (2-hydroxypropyl) methacrylamide (HPMA) and methacrylamide with side chain extended oligopeptide units subject to enzymatic cleavage.

As active cytostatics used isopropylaminopropoxy opylester p di- (2-chloroethyl) -aminof methyl ester (sarcolysin - SBIPE] and a polymeric carrier ¹ copolymer of N- (2-hydroxypropylj methacrylamide (HPMA) and methacrylamide, in which the extended methacrylamide groups side chains containing units capable of enzymatic cleavage.

General formula of synthesized compounds with anticipated antitumor activity ίθ

P — NH — X — SBIPE, wherein P is a polymeric carrier prepared according to Scheme 1,

SCHEME 1 = x = 85 to 90 mol. %, y = 5 to 15 mol. %

M _w = 15-40,000, and X = O or -Phe-.

To monitor the effect of side chain length on antitumor activity, he was in one group of polymeric carriers

R = —NH — CH2—, ie - Gly— and in the other

R = -NH-CH2-NH-CH-CH2CH- (CI-13) z, i.e., &quot; Gly-Leu &quot;.

When R = —Gly—, X = O, it is used as a low molecular weight cytostatic

SBIPE and in the preparation of a compound having antitumor activity according to the invention of the general formula, one-step procedure is followed according to Scheme t 2, ie Ρ + NH 2 - X-SBIPE;

P — NH — X — SBIPE

__ CH _X 1 * -CH 2 -C 1 WHAT -CH-Ck * 1 J X

+ NH 4 X-SeiPE

CO I

NH

CH-OH

AND

CH "

CH,

CO I

NH

AND

CH ~

I ^from CH-OH I

CH _O

★ H0C ₆ H ^ N0 _£

NH

AND

SBIPE

SCHEME 2

In the case where R = -Gly-Leu- and X = = -NH-CH-CH 2 C 6 H 5, ie "Phe", the preparation of the antitumor compound is two-stage. In the first step, HCl-Phe-SBIPE (phenylalanylsarcosine hydrochloride) is prepared and reacted with the polymeric carrier in the second step.

1st degree. The formation of NH2-X-SBIPE (NH2-PHE-SBIPE) results from the reaction of benzyloxycarbonyl-phenylalanine (BOC-PHE-OH) with sarcosyl isopropyl ester hydrochloride in the presence of triethylamine. The reaction is carried out in tetrahydrofuran by DCC1 (dicyclohexylcarbodiimide). The solutions used are approximately 20% of the concentration. BOS - blockage is removed by 20 wt. HCl solution in methanol. The process follows scheme 3.

DCClttťC)

BOC-Pk.e-OH * HCl. NH ^CH —CH ^H ^N (CH ^CH CHClÍ)

COOCH (CH ₅ ) ₂

HCl (CH ₃ OH)

BOC ~ Ph.e-NH-C and CH-C _s H £ ^ N (CH ^ CH ^ Cl) _£ *

COOCH (CH3)

- _:: -piperazine-NH-CH-CH ^ H ^ N (CH ^ CH ^ Cl or HCl Ph ~ * -SBIPE COOCH (CH _{3) z} in tetrahydrofuran THF (C ₂ H ₅₎ N ₀

SCHEME 3

The reaction product HCl-Phe-SBIPE-phenylalanylsarboxylic acid hydrochloride was purified by recrystallization from ether.

II. degree. The polymeric carrier containing the active groups is dissolved in dry dimethylsulfoxide in an ONp: NH2 ratio of 1: 2 in the presence of an equivalent amount of triethylamine. The reaction proceeds with stirring at room temperature for 20 hours. The reaction product is isolated by precipitation into acetone: diethyl ether (3: 1) and reprecipitated from methanol to acetone: diethyl ether (3: 1). The product is dried at room temperature under vacuum to constant weight.

The following examples illustrate the invention without limiting it.

Example 1

The polymeric carrier is prepared by radical precipitation polymerization in acetone at a monomer concentration of 12.5% by weight. (90, 93, 95 wt.% HPMA and 10, 7, 5 wt.% Comonomer, 0.6 wt.% Azobis isobutyronitrile initiator in ampoules at 50 ° C, reaction time 48 hours. The polymer is filtered off and washed twice with acetone and Dry to constant weight under vacuum at room temperature.

Example 2

The intermediate NH 2 -Phe-SBIPE was prepared according to Scheme 3.

An approximately 20% solution of BOC-Phe-OH in dry tetrahydrofuran was prepared. In the presence of an equimolar amount of dicyclohexylcarbodiimide (DCCI) and sarcolysine isopropyl ester hydrochloride at -5 ^° C in the presence of triethylamine and under vigorous stirring, BOC-Phe-SBIPE was prepared. The blockade was removed by dissolving the prepared product (? 0% w / w solution) in methanol saturated with HCl with stirring at room temperature. The hydrochloride of the prepared product was isolated by filtration.

Example 3

P-NH-X-SBIPE was prepared by dissolving P in dimethylsulfoxide (20 wt% solution). HCl was added with stirring. NH2-X-SBIPE, ratio ONp: NH2 = 1: 2 and equimolar amount of triethylamine, calculated on HCl. After stirring at room temperature for 20 hours, the polymer compound was isolated by precipitation into ether: acetone (1: 3). Precipitation from methanol to ether: acetone (1: 3) twice. The prepared product was dried at room temperature to constant weight.

The cytostatic range is determined from the chlorine content.

A fraction with M _w = 24,000 was isolated by GPC chromatography for in vivo testing. UV spectroscopy showed that the polymer fraction did not contain free sarcolysin.

Claims (7)

SUBJECT CLAIMS 1. Polymeric substance having antitumor activity based on copolymers of N- (2-hydroxypropyl) -methacrylamide and methacrylamide of general formula r> CH, ³ -Cl-L-C- * 1 X -ch ₉ -c ~ 1 what ř ° J k 1> NH 1 R AND CH _Z WHAT 1 CH-OH Wherein x = 85 to 97 mol. %, y = 3-15 mol. percent by mol. M _w = 15 to 40,000, R is an enzymatically cleavable spacer, and —X — SBIPE is a low molecular weight antitumor agent bound to a carrier by a covalent bond. 2. A polymeric substance having antitumor activity according to claim 1, characterized in that R = &quot; Gly &quot; 3. A polymeric substance having antitumor activity according to claim 1, characterized in that, as a side spacer, R = —Gly — Leu—. 4. A polymeric substance having antitumor activity according to items 1 and 2, characterized in that: OF THE INVENTION X = 0, and sarcolysin isopropyl ester is used as a low molecular weight cytostatic. 5. Polymeric substance having antitumor activity according to items 1, 3 and 4, characterized in that X = “Phe”. 6. A process for the preparation of a polymer having antitumor activity according to claims 1 to 5, characterized in that the polymeric carrier is dissolved in dry dimethylsulfoxide and, with stirring in the presence of triethylamine, NHz-X-SBIPE is added and allowed to react for 20 hours. is isolated by precipitation into acetone: diethyl ether (3: 1j) followed by purification by doubly dissolving in methanol and precipitating into acetone: diethyl ether (3: 1). 7. A process for the preparation of a polymeric substance having antitumor activity according to rolls 1-6, characterized in that NH2 -Phe-SBIPE is first prepared by reacting sarcolysine isopropyl ester hydrochloride with benzyloxycarbonylphenylalanine (BOC-Phe) in the presence of triethylamine. —OA) at –10 to –5 ° C in tetrahydrofuran by DCCI, then the blocking benzyloxycarbonyl (BOC) is removed with a solution of HCl in methanol and the phenylalanylsarcolysin hydrochloride is isolated by filtration and added to the polymer solution in dry dimethylsulfoxide or dimethylformamide in the presence of stirring. of triethylamine and allowed to react at room temperature for about 20 hours, and the product obtained is isolated by precipitation into acetone-diethyl ether (3: 1) followed by purification by doubly dissolving in methanol and precipitation into acetone: diethyl ether (3: 1j).