AU624215B2 - Alkylated polyethylenimine derivatives, process for their preparation, their use as pharmaceuticals and pharmaceutical preparations - Google Patents

Abstract

The novel uncrosslinked and crosslinked alkylated polyethyleneimines can be used as hypolipidaemics due to their gallic acid-binding activity.

Description

6242 1 5 Form COMMONWEALTH OF AUSTRALIA PATIENTS ACT 1952-69 COMPLETE SPEC IFI CA-T ION (OR IGMAL) Class Int. Class Applicf'tlon Number: Lodged: #Gompleto Specification Lodged: Accepted: Published: Priority 0 a 0 a 0 *11Iated Art 0 0~ 0 0 0 'f~ame of Applicant H0i~CHST 0 o 00 0

AKTIENGESELLSCHAFT

A~ldress of Applicant 50 Bruningstrasse, D-6230 Frankfurt/Main 80, Federal Republ ic of Germany 0 Actual Inventor: Address for Service DWALTER HEITZ, THOMAS FISCHER, BELA KEREKJARTO WATERMARK PATENT TRADEMARK ATTORNEYS.

290 Burwood Road, Hawthorn, Victoria, Australia Complete Specification for the invention entitled: ALKYLATED POLYETHYLENIMINE DERIVATIVES, PROCESS FOR THEIR PREPARATION, THEIR USE AS PH4ARMACEUTICALS AND PHARMACEUTICAL PREPARATION, The following statement Is a full description of this lnvqntlon, Including the best method of performirig it known to HOECHST AKTIENGESELLSCHAFT HOE 89/F 018 Dr. D/rh Description Alkylated polyethylenimine derivatives, process for their preparation, their use as pharmaceuticals and phazmaceutical preparations The invention relates to alkylated polyethylenimine derivatives, to a process for their preparation, to pharmaceutical preparations based on these compounds and to their use as pharmaceuticals, in particular for lowering increased lipid levels.

4 It

I

Get# Insoluble basic, crosslinked polymers have been used for a considerable time for binding bile acid and are used Q0 0o therapeutically in the light of these properties.

0 00 0Chologenic diarrhea (for example after resection of the 15 ileum) and increased cholesterol blood levels are treated causally as the object of therapy. In vhe latter case, it is a matter of inter.vention in the enterohepatic circulation, the corresponding resynthesis of cholesterol in °ooo° the liver being provoked in place of the bile acid como'00o 20 ponent taken out of the circulation. Recourse is made to 00 0 the circulating LDL (low density lipoprotein) cholesterol 0 to meet the cholesterol need in the liver, the hepatic LDL receptors coming into effect in increased number. The acceleration of LDL catabolism thus caused has an effect 25 owing to the reduction of the atherogenic cholesterol content in the blood.

o 4 The ion exchangers used as pharmaceuticals have oither quaternary ammonium groups (such as colestyramine) or secondary or tertiary amine groups (such as colestipol) as active groups. The daily dose of colestyramine is expediently 12-24 g, and 32 g are recommended as the highest daily dose. 15-30 g is the recommended daily colestipol dose. Taste, odor and high dosage make patient compliance more difficult. The side effects go back to lack of selectivity (for example avitaminoses), which 2 even have to be considered in the dosage of medicaments given simultaneously, and also to bile acid depletion, which cause various gastrointestinal disturbances (constipation, steatorrhea) to a different degree. For both preparations, a therapeutic significance by combination with other hypolipidemic pharmaceuticals such as fibrates, HMG-CoA reductase inhibitors, probucol for example, M.N. CAYEN, Pharmac. Ther. 29, 187 (1985) anu 8th International Symposium on Atherosclerosis, Rome, Oct. 9-13, 1988, Abstracts p. 544, 608, 710) has been described, the effects obtained even making the therapy of severe hyperlipidemia possible. It therefore appears significant to find suitable substances with the given principle of action and without the disadvantages of the preparations presently used.

Th following features of the preparations mentioned and, [t~gin particular, of colestipol are r:egarded as worthy of improvement: 00 The high daily doses, which are to be put down to a relatively low binding rate at neutral pH in isotonic medium and the release (partial) c the adsorbed bile acid again.

0 6 2. The qualitative shift in the bile acid composition o of the bile with a decreasing tendency for chenodeoxycholic acid and the increasing risk of cholelithiasis associated with this.

3. The lack of a damping effect on the cholesterol metabolism of the intestinal bacteria.

4. The binding rate of vitamins and pharmaceuticals, which is too high, makes a need for substitution of these substances and for blood level controls necessary in some cases.

A further improvement can be obtained in the form -1 I I 3 for administration.

The removal of the deficiencies listed is surprisingly achieved by the use of high nolecular weight alkylated polyethylenimines. The non-absorbable macromolecules exhibit their action both in soluble form, corresponding to the non-crosslinked structure, and in the insoluble state as the crosslinked polymer.

Crosslinked polyethylenimines are described in US Patent 3,332,841. The crosslinking is carried out, inter alia, 10 by means of alkylene groups having 2 to 8 carbon atoms, the molecular weight of the starting polymers being 4 between 800 and 100,000. For the treatment of temporary *o hyperacidity of the stomach, 0.25 to 5 g are administered 1 per dosage unit, Neither the binding of bile acid nor a lipid-lowering activity of the crosslinked polyethylenimines associated with this is described, as without alkylabOon the polyethylenimines have no binding capacity or only an insignificant binding capacity compared to the o-o bile acids, depending on the type. Owing to the large potential charge density, provision can be made for sufficient binding capacity by means of alkylation and 0 o for affinity and binding specificity by means of the choice of substituents cf appropriate hydrophilic/hydrophobic character.

o o o4 a 225 The invention therefore relates to non-crosslinked and crosslinked alkylated polyethylenimines, wherein the starting polyethylenimine has a molecular weight of 10,000 to 10,000,000, the alkylating agent has the formula I R-X (I) in which X is chlorine, bromine, iodine, CH,-SO 2 or CH,3 ,-S02-O and R is a straight-chain or branched C-C 3 o-alkyl 4 radical which is optionally substituted by a monoor bicyclic saturated hydrocarbon having 5 to ring carbon atoms, or by a phenyl radical and, in the case of the crosslinked alkylated polyethylenimines, the crosslinking agent is an a,w.-dihaloalkane having 2-10 carbon atoms or a higher functionalized haloalkane having 2-10 carbon atoms.

The process for the preparation of the alkylated polyethylenimine derivatives according to the invention comprises alkylating a polyethylenimine having a molecular weight between 10,000 and 10,000,000 with an alkylating agent of the formula R-X, in which X and R have the meanings indicated, and, if desired, crosslinking with an a,w-dihaloalkane having 2-10 carbon atoms or a higher 15 functionalized haloalkane having 2-10 carbon atoms by methods customary in polymer chemistry.

The crosslinking can be carried out before or after the alkylation. Carrying out the crosslinking and the alkylation simultaneously is particularly preferred.

4 41 4 4a 44 4 a 0 6 0 44 So 0~oa a -4 004 4 20 Polyethylenimines having a molecular weight above 100,000 are preferably employed.

In the alkylating agents R-X, X is preferably chlorine or bromine.

R is preferably a primary alkyl radical. If the alkyl radicals are substituted by the ring systems mentioned, these are preferably arranged so that they are linked to the polyethylenimine via a spacer having 1 to 4 CH 2 groups. The cyclohexyl radical is particularly suitable as a monocyclic saturated substituent. A suitable bicyclic hydrocarbon radical is, for example, decalin. A particularly suitable alkylating agent, whose alkyl radical is substituted by phenyl, is benzyl bromide. A suitable alkylating agent without substituents in the alkyl radical is preferably butyl chloride,, 1 11 5 The alkylation can be carried out in several stages. In this way, the possibility exists of fixing different substituents to the same polymer.

The ratio of the alkylating agent employed to the amino groups of the polyethylenimine is 0.2:1 to 5:1, preferably 0.5:1 to 2:1.

By means of the reaction with alkylating agents, a part of the secondary amino groups in the chain are converted into tertiary and quaternary structures. The formation of S10 tertiary amino groups is preferred.

o a Suitable crosslinking agents are, for example, di- and trihaloalkanes, preferably a,w-dihaloalkanes such as, for ex&mple, 1,6-dibromo.!.wxne and 1,10-dibromodecane. The amount of the crosslinker is preferably 2-25 mol-%, relative to the alkylating agent employed.

The alkylated polyethylenimines according to the inveno. tion adsorb acids intrinsic to the body, in particular gallic acid. In the light of these properties, they are in a position to lower elevated cholesterol levels. The 0 0 alkylated polyethylenimines according to the invention have essentially more favorable bile acid-absorbing o° oa properties compared to colestipol, as is discernable from U0 0 o the experiments described below.

A

1. In vitro experiments 1.1 Adsorption batch containing individual gallic acids Experimental conditions: volume 10 ml;temperature: 37°, incubation in a shaking water bath, duration: 2 hours; medium: isotonic buffered physiological saline solution, pH 7.0; bile acid: 10-15 pmoles; adsorV jr compound according to the invention or comparison compound): 10-100 mg, I 6 t The gallic acid in equilibrium with the adsorbate is determined by means of enzymatic analysis. The methods via 3a-hydroxy- or 7a-hydroxysteroid dehydrogenases (EC 1.1.1.50 or EC 1.1.1.159) were carried out according to the description in Bergmeyer Bergmeyer, Methoden der enzymatischen Analyse (Methods of Enzymatic Analysis), 2nd edition (1970), p. 1824) or the product information to product No. H-9506 from SIGMA CHEMICAL Co. (St. Louis, USA). The proportion of bile acids bound was calculated from the difference between the control batches without adsorber and the complete batches. The experiments for the characterization of the adsorber were carried out with variation of the prestated bile acid concentration or the adsorber amount, and less frequently the incubation period, pH or ionic strength.

An alkylated crosslinked polyalkylenimine according to Example 2 showed a qualitatively better effect owing to stronger cholate binding than colestipol since, incubated with a 2 mM glycocholate solution, mg of colestipol adsorbed under 6 of the bile acid, on the other hand polyimine as in Example 2 absorbed 74 to 76 of the gallic acid.

1.2 Reversibility testing.

The adsorbate removed from the equilibrium in a batch as described under 1.1 was postincubated with fresh .bile acid-free medium and the bile acid released was determined as under 1.1.

In this experiment, it was possible to show that less bile acid was released again from adsorbates containing alkylated crosslinked polyethylenimine as in Example 2 since, from the adsorbate of 50 mg of colestipol with 2 mM of glycocholate, 57 of the bile acid was released again, on the other hand 1,1 0 25 00410001 0o 0 I.

I

10 54 f 000 e o o a 0 0 20 o 4 7 only 4 to 5 was released from the adsorbate of mg of compound according to Example 2.

1.3 Adsorption batch containing bile acid mixtures.

The conditions indicated under 1.1 were modified with respect to gallic acid such that 40 pnrles of the tauroconjugate of cholate, chenodeoxycholate, deoxycholate and lithocholate were brought into the batch simultaneously, 20 100'mg of the adsorber being used. The individual adsorption rate of the bile acid was determined by separation and determination by means of high pressure liquid chromatography Parris, Analyt. Biochem. 100 (1979) 260-263).

Under these experimental conditions, an alkylated polyethylenimine according to the invention as in Example 1 showed a larger binding rate, as 20 mg of colestipol bound 54 of a mixture of 4 tauroconjugated bili acids (each containing 4 mM), on the other hand polyimine as in Example 1 bound 83 In vivo experiments 0 0 0 a 0 Young male Wistar rats of a body weight of about 200 g were divided into groups of 6 animals and kept on standard feed. In each case samples of faeces were taken from the animals for analysis before the start of the experiment, and 1 and 2 weeks after the start of the experiment. If water-soluble, the adsorber was administered daily as a weakly acidic buffered solution at 100, 250 or 500 mg/kg of body weight for 14 days using the stomach tube; as the insoluble substance the adsorber, suspended with 1 of Tylose(R) (water-soluble cellulose ether) as the vehicle, was incorporated at 250 or 500 mg/kg of body weight daily for 14 days using the stomach tube.

In samples of faeces, the neutral steroids were extracted I 8 after homogenizing with chloroform/methanol 2:1 the extract was hydrolyzed and the hydrolyzate was extracted with diethyl ether/heptane 2:1 After evaporating the solvent, the sample was subjec, 'ld to gas chromatographic separation and analysis (H-CH. Curtius and W. BUrgi, Z. Klin. Chemie 4 (1966) 38 42).

The examinations of the samples of faeces showed the following: 0011 0 0O 01000 0 0* 0 00O~ 15 00( o 2.1 If che adsorber was a crosslinked alkylated polyethylenimine as in Example 2, a more rapid onset of action was determined since, after feeding 250 mg of adsorber/kg of body weight daily for 7 days to rats, the additional excretion of unconjugated bile acid in the faeces was 38 with colestipol, on the other hand it was 115 with the compound as in Example 2.

The effect of polyethylenimine is obtained with colestipol only after 14 days.

2.2 The desired inhibition of the bacterial conversion of cholesterol in the intestine, associated with an addition4 excretion of cholesterol, is caused to a larger degree by alkylated crosslinked polyethylenimine as in Example 2, since under identical experimental conditions, the cholesterol excretion in rat faeces by colestipol is increased by 28 and by 89 by a compound as in Example 2; at the same time, the coprostanol excretion owing to colestipol changes by +4 but by -30 owing to a compound as in Example 2.

0 o 0 0 20 o a 4o 0 D 25 From the experimental results it can be clearly discerned that: both the non-crosslinked and the crosslinked alkylated polyethylenimines show by means of in vitro adsorption enperiments that, compared to colestipol the quantity of the bile acid bound is increased by 50-60 (Examples 1 and 2) the binding of cholate is increased 10-12-fold -t I 9 (Example 2) the desorption rate is 10-15-fold lower from bile acid polymer adsorbates (Example 2).

In the rat experiments, it was shown that non-crosslinked polyethylenimines, like crosslinked alkylated polyethylenimines, given orally in the test range up to 500 mg/kg of body weight daily are tolerated without symptoms. It was possible to show advantages compared to identical doses of colestipol, in that a more rapid onset of action takes place, whereby o 0o the additional bile acid excretion was tripled o after one week'u use So the cholesterol elimination was increased three-fold the bacterial conversion of cholesterol in the °ooo 15 intestine was slowed down and in this way the ,o production and excretion of coprostanol was significantly 30 reduced (Example 2) The compounds according to the invention are suitable in 0000 the light of their properties for use as pharmaceuticals, 0 0 20 in particular for lowering increased lipid levels. The invention therefore also relates to the use as hypolipi- S0000? demic agent and pharmaceutical agent. In the pharmaceutical agents, polyethylenimines according to the invention csa also be present in the form of physiologi- 25 cally tolerated salts with acids.

A particular advantage is the use of crosslinked alkylated polyethylenimines. The crosslinked products can give off no substances into their environment. This is of significance for the development of a non-toxic material.

The dose to be administered daily is preferably 1.0 to 10.0 g, in particular 5 g. It can be divided into several individual doses.

The compounds according to the invention can be converted as such, or after addition of customary auxiliaries, into forms of preparation for oral administration, such as, for example, tablets, capsules, syrups, aqueous solutions, suspensions etc. In this connection, it may be expedient first to bring active compounds obtained in solid form to a desired particle size, for example, by fine grinding. Suitable auxiliaries are, for example, lactose, starch, gelatin, talc etc. The production of tablets is carried out, for example, by means of moist granulation and subsequent compression.

Moreover, the alkylated polyethylenixiines can also be incorporated into foodstuffs such as bread, fruit juice etc. or taken together with foodstuffs.

.The compounds according to the invention can also be ed in combination with other active compounds. Other active 15 cofpounds which are suitable are, for example, HMG-CoA Feductase inhibitors, vitamins, geriatric agents and antidiabetic agents.

The following examples are intended to illustrate the invention: Expaple 1 0 o 44.3 g of polyethylenimine (mol. wt. about 1000,000) are dissolved in 1.05 1 of H 2 0 in a 4 1 reaction flask; 191.4 g of butyl chloride are added and the mixture is 0 heated to reflux for 24 hours with vigorous stirring (500 rpm). A turbid viscous reaction mixture is formed.

1 1 of 2 N NaOH solution is added to this irtture after cooling and the batch is again brought to reflux for 24 hours. After cooling, two phases form. The organic phase is separated off and freed from the solvent in vacuo. The weight of solid is 75.4 g.

The product has a degree of alkylation of about 50 and, af£'er addition of equivalent amounts of acid, is watersoluble.

11 Example 2 44.3 g (1 mol) of polyethylenimine is dissolved in 1.05 1 of H 2 0 in a 4 1 reaction flask; 186.6 g of butyl chloride (2 mol) and 48.8 g (0.2 mol) of 1,6-dibromohexane are added and the mixture is heated to reflux for 24 hours with vigorous stirring (500 rpm). A turbid viscous reaction mixture is formed. 1 1 of 2 N NaOH solution is added to this mixture after cooling and the batch is brought to reflux again for 24 hours. After cooling the reaction mixture, the solid is filtered off with suction, washed until neutral and dried. The weight is 87.27 g.

The product is insoluble in water and can be swollen in S methanol.

The ratio of starting compound to alkylating agent and crosslinking agent can be varied within certain limits.

Under the reaction conditions indicated, products are then obtained having another degree of alkylation and crosslinking, o oa O.s0 Example 3 0 "20 The reaction mixture from 4.3 g of polypo hyljnitine, 100 ml of water and 29 3 g of 1-chloro-2-cyclohexylethane is heated to reflux for 24 hours, a yellowish turbid mixture being formed. After adding 100 ml of 2 N NaOH o solution, the mixture is heated to reflux again for 24 Ai 25 hours. The reaction mixture forms a two-phase system. The organic phase is separated off and freed from the solvent on a rotary evaporator, 15.34 g of a highly viscous material are obtained.

Example 4 A mixture of 88.6 g of polyethylenimine (50 percent in water; 1 mol), 186.6 g of n-=LutLyl chloride and 48.8 g of 1,6-dibromohexane in 2 1 of water is heated to ifin I lie 4 V 12 for 24 hours under 10 bar of nitrogen in a stirring or shaking autoclave. The protective gas is replaced by 7 bar of ammonia and the mixture is again heated to for 24 hours.

After cooling the reaction mixture, the precipitate formed is filtered off with suction and washed with water until neutral. The product is washed with methanol and eluted in a column using about 2.5 1 of methanol, about 1 of 2 N acetic acid, about 2 1 of -2 N ammonia water and finally about 2 1 of methanol. After filtering off with suction, the product is dried in vacuo at a maximum of 50 0

C.

Yield 78 g.

The product can be swollen in various solvents, but is 15 insoluble.

44444 44044 4444 0u 0 0 0 44

Claims (4)

1. A non-crosslinked or cro enimine, wherein the starl molecular weight of 10,000 1 agent has the formula I R in which X is chlorine, broi CH 3 SO 2 -O and L3 HOE 89/F 018 RE AS FOLLOWS: sslinked alkylated polyethyl- ting polyethylenimine has a to 10,000,000, the alkylating -X mine, iodine, CH 3 -S0 2 or oO0d V I c, o o) o 0 0 R is a straight-chain or branched Cl-C,,-alkyl radical which is optionally substituted by a mono- or bicyclic saturated hydrocarbon having 5 to ring carbon atoms, or by a phenyl radical and, in the case of the crosslinked alkylated polyethyl- enimines, the crosslinking agent is an a,w-dihaloalkane having 2-10 carbon atoms or a higher ,unctionalized haloalkane having 2-10 carbon atoms.

2. A process for the preparation of non-crosslinked or crosslinked alkylated polyethylenimine derivatives, which comprises alkylating a polyethylenimine having a molec- ular weight between 10,000 and 10,000,000 with an alkyl- atinq agent of the formula R-X, in which X and R have the meanings indicated and, if desired, crosslinking with an a,w-dihaloalkane having 2-10 carbon atoms or higher functionalized haloalkane having 2-10 carbon atoms by methods customary in polymer chemistry. The process as claimed in claim 2, wherein one or more of the following measures are observed: a) polyethylenimines having a molecular weight above 100,000 are employed, b) in the alkylating agents R-X, X is chlorine or bromine, c) in the alkylating agents, R is a primary alkyl radical which is optionally substituted by a cyclo- A- .x I Oi I 14 I hexyl, decalin or phenyl radical, where these substituents are arranged such that they are linked to the polyethylenimine via a spacer having 1 to 4 CH 2 groups, d) the ratio of the alkylating agent employed to the amino groups of the polyethylenimine is 0.5:1 to 2:1, e) the crosslinking agent is 1,6-dibromohexane or 1,10- dibromodecane, f) alkylation and crosslinking are carried out simultaneously.

4. A pharmaceutical preparation which contains a compound as claimed in claim 1 or its physiologically tolerated salt with an acid. A method for the production of a pharmaceutical preparation, which comprises converting a compound as claimed in claim 1 into a suitable form for administration. I o1 o r 0 0; a o r Z. 1lt2 IIA UT Ji fflf-1Fi~fV TP PIIhi~i n~i~m I hypolipidemic agent.

7. The use of compounds as claimed in cla i 1 as an additive in foodstuffs and fruit juice DATED this 18th day of ary 1990. HOECHST AKTIENGFE LSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS "THE ATRIUM" 290 BURWOOD ROAD -It-lHORN. VIC. 3122. I AY/J t I ~1 i- 6. A method of hypolipidemic treatment comprising administering to a patient suffering hyperlipidemia, an effective amount of a compound as claimed in claim 1. 7. A method of rendering foodstuffs and fruit juices hypolipidemic comprising adding to them in an effective quantity a compound as claimed in claim 1. DATED this 3rd day of February 1992 HOECHST AKTIENGELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA oo I I at a a t 40 0 ee 0 0 0 t 0 0 00 «0 0 ioa t aoo 0 0