NO893365L - CONNECTIONS. - Google Patents

Description

Connections

The present invention relates to new polystyrene ion exchange resins, processes for their preparation, useful intermediates in their preparation, pharmaceutical compositions containing them and their use in lowering plasma cholesterol levels in humans.

Coronary heart disease (CHD) is one of the most serious health problems in today's society. Epidemiological studies worldwide have shown that the frequency of CHD is linked to a number of independent risk factors, especially for example high concentrations of serum cholesterol (hypercholesterolaemia). Such adverse factors lead to atherosclerosis and eventually to severe cases of periodic claudication, cerebrovascular failure, thrombosis and cardiac arrest.

It is known that ion exchange polymers, especially polystyrene polymers can be used as sequestrants to bind unabsorbed bile acids and salts in the digestive tract and form complexes which are then excreted in the faeces. This secretion leads to a reduction in the amount of bile acids that return to the liver through enterohepatic circulation.

The synthesis of the replacement of bile acids from hepatic polysterol lowers hepatic cholesterol, regulates hepatic LDL receptors and consequently reduces plasma cholesterol concentrations. Such sequestrant polymers have been found to be useful in the treatment of hypercholesterolaemia, and it has now been shown that reduction of serum cholesterol by bile acid sequestrants has a beneficial effect in protecting against the onset of coronary heart disease.

The polystyrene polymers known to have such release activity are generally those having a di- or tri-lower alkyl ammonium group such as a trimethylammonium group. For example, GB 1286949 describes a series of macroporous polystyrene polymers with 5-20% crosslinking, and GB 1579490 describes a series of microporous polymers with 8-20% crosslinking. In addition, GB 2026501 describes a number of, among other things, polystyrene polymers which are claimed to have special water absorption capacities, i.e. 69-73% by weight of the polymer weight. In each of the foregoing, the polystyrene polymers have di- or tri-lower alkyl ammonium groups, especially a trimethyl ammonium group.

A particular agent based on a polystyrene polymer currently used to lower serum cholesterol levels in humans by sequestering bile acids in the digestive tract is cholestyramine (GB 929391). Cholestyramine is a cross-linked anion-exchange polystyrene polymer with an ionizable trimethyl ammonium group attached to the polymer backbone. However, the use of this agent is associated with a number of unwanted side effects, for example it is inedible and must be taken in large doses (up to 36 g per day) and in some cases causes bloating, constipation and other intestinal side effects. In addition, its ability to bind bile acids is insufficient with regard to the amount of polymer that needs to be used.

It is the aim of the present invention to provide compounds which overcome these disadvantages of this known excretory agent and to provide improved bile acid excretory agents which are useful for lowering cholesterol levels in humans.

The present invention therefore provides in a first aspect polystyrene polymers with the structure (I):

in which

R<1> is a saturated or unsaturated C6 to C20 alkyl group;

R<2> and R<3> are the same or different and are each C 1-4 alkyl;

X~ is a physiologically acceptable exercise;

a, b and c are numbers indicating the relative molar percentages of the units present in a statistical distribution in the polymer (b) is from approx. 0.5 to approx. 10 molar%, and (c) is from approx. 30 to approx. 99 mol%; and

n is a number indicating the number of repeating units in the polymer.

Suitably R<1> is a saturated or unsaturated C8 to C2nalkyl group. Even more preferably, R<1> is a saturated C6 to C20 alkyl group. Preferably, R<1> is a saturated C8 to C14 alkyl group; preferably a C 12 alkyl group, especially an unbranched C 12 alkyl group.

Conveniently, the groups R<2> and R<3> are the same or different and are each C 1-4 alkyl; preferably they are the same; preferably R<2> and R<3> are both methyl.

Appropriately, (b) is from approx. 0.5 to approx. 10 mol% of the polymer, preferably (b) is from approx. 1 to approx. 8 mol% of the polymer; preferably from approx. 1 to approx. 4 mol%.

Suitably, X~ is a physiologically acceptable counterion such as a sulfate, bicarbonate, carbonate, formate, acetate, sulfonate, propionate, malonate, succinate, malate, tartrate, citrate, maleate, fumarate, ascorbate, glucoronate, phosphate or halide, or the anions of an amino acid such as aspartic or glutamic acid. Even more preferably, X~ is a phosphate, sulfate or halide ion; preferably a halide ion, especially chloride.

n is a number indicating the number of repeating units in the polymer. Because of the three-dimensional cross-linking, exact numbers cannot be given for n, but in any case it must be greater than 1,000.

The polystyrene polymers in the present invention are also characterized by their total exchange capacity, i.e. the theoretical maximum capacity of the polymer if each counterion were to be replaced by bile acid. In this description, the total exchange capacity is defined as the number of milliequivalents of motion per gram dry weight polymer.

Appropriate total ion exchange capacities are in the range, for example when the counterion X~ is a halide ion such as chlorine, from 1.5 to 3.5 milliequivalents of Cl~ per grams of polymer. Preferred within this range are polymers with a total exchange capacity of between 2 and 3 milliequivalent Cl<->pr. grams of polymer.

In addition, it must be pointed out that the approximate molar percentages (a), (b) and (c) are calculated from the monomer mixture, or in some cases (c) from microanalytical data.

It must be noted that the term "bile acid" as used herein shall be understood to include bile acids, bile acid salts and conjugates thereof.

The polystyrene polymers in the present invention can be produced by methods analogous to those known in the field. The present invention therefore provides in a further aspect a method for producing the polystyrene polymers with structure (I) which comprises:

(a) reaction of a polymer with the structure (II)

wherein a, b, c and n are as described for structure (I) and Y is a group that can be replaced by an amine, with an amine of the structure R<1>R<2>R<3>N(in) whereinR <1>to R<3> are as described for structure (i);

(b) conversion of a compound of the structure (IV)

wherein a, b, c and n are as described for structure (I) and Z is NR<1>R<2>or NR<2>R<3>wherein R<1>toR<3> are as described for structure (I) , with a compound of structure R<4>Y (V) wherein R<4>is a C1-4 alkyl group when Z is NR^-R2 or a saturated or unsaturated C6-20 alkyl group when Z is NR<2>R<3>, and Y is a group that can be replaced by an amine.

The reaction between a polymer with structure (II) and an amine with structure (III) can be carried out in a suitable solvent at a higher temperature. Suitable solvents are, for example, a C1-4 alkanol, N-methylpyrrolidone, sulfolane, dimethylformamide, nitromethane or tetrahydrofuran. Preferably, the reaction is carried out in N-methylpyrrolidone at a temperature between approx. 50°C and 80°C for up to 24 hours until the reaction is complete.

The reaction between a polymer of structure (IV) and a compound of structure (V) can be carried out in a suitable inert solvent such as a C 1-4 alkanol, nitromethane, sulfolane, N-methylpyrrolidine, dimethylformamide or tetrahydrofuran at a higher temperature.

The intermediate polymers of structure (II) are available

in commerce or can be made from readily available materials by methods known to those skilled in the art. For example, polymers with structure (II) in which Y

er chlorine is produced by reacting chloromethylsterene, sterene and divinylbenzene in an aqueous suspension comprising polyvinyl alcohol in the presence of an initiator such as AIBN at a higher temperature.

Alternatively, the polymer intermediates with structure (II) can be prepared directly from polystyrene by analogous methods to those known in the field, for example when Y is chlorine by chloro-methylation of polystyrene.

Certain intermediates with structure (IV) are new and form

a further aspect of connection, namely connection with structure (IVA)

wherein a, b, c and n are as described for structure (I) and Z<A> is NR<1>R<2>or NR^-R<3>wherein R<1>toR<3> are as described for structure ( I) •

The polymer intermediate of structure (IV) can be prepared from the polymer of structure (II) by reaction with an amine of the structure R2NH in which R2 is R1R<2>or R<2>R<3> under the same or similar conditions as shown for the reaction of a compound with structure (II) and a compound with structure

(III).

Alternatively, the intermediate polymers of structure (IV) can be prepared by polymerizing styrene, divinylbenzene and a compound of structure (V)

where Z<a>s as defined in structure (IVA) under standard polymerization conditions. For example, polymerization can be carried out in an aqueous suspension comprising, for example, polyvinyl alcohol in the presence of a higher temperature initiator. Suitable initiators include, for example, AIBN.

The intermediate compounds of structure (V) can be prepared by reacting an amine of structure R2NH in which R2 is a R<1>R<2>or R^R<3> with a corresponding compound of structure (V) where Z<a>is a group that can be replaced by an amine.

The polystyrene polymers of structure (I) have been found to bind bile acids in in vitro experiments and in vivo animal models have been found to increase the amount of bile acids detectable in faeces. Especially when compared with known excretory agents such as cholestyramine, the polymers of structure (I) have surprisingly been found to have an unexpected profile of activity which is expected to provide advantages over the known compounds in lowering serum cholesterol levels in animals, especially humans. More specifically in in vitro experiments by comparison with cholestyramine are the compounds with structure

(I) has been found to bind comparable amounts of bile acid

per gram of polymer (at physiological concentrations of bile acids), and to bind the bile acid more strongly, that is to say, the bile acids have been found to dissociate more slowly from the compounds according to the invention. It is expected that compounds with such properties will be able to significantly lower plasma cholesterol levels at much lower doses than has hitherto been possible with known excretory agents (which are still given at up to 36 g per day).

As previously shown, removal of bile acids from the digestive tract in this manner is recognized to lower serum cholesterol levels and also to have a beneficial effect on protection against atherosclerosis and its attendant clinical conditions. The present invention therefore provides a further aspect of polystyrene polymers of structure (I) for use in therapy, in particular for lowering serum cholesterol levels in mammals including humans. In addition, the polymers with structure (I) are expected to be useful for protection against atherosclerosis and its sequelae, for example in the treatment of pruritus and diarrhoea.

In light of the foregoing, the present invention also provides a method for lowering serum cholesterol levels in mammals which consists of giving a mammal in need thereof an effective cholesterol-lowering amount of a polystyrene polymer of structure (I); and a method for protection against atherosclerosis.

When used in therapy in the method according to the invention, the polystyrene polymers with structure (I) are generally given in a pharmaceutical mixture.

In yet another aspect of the present invention, there is therefore provided a pharmaceutical mixture comprising a polystyrene polymer of structure (I) in conjunction with a pharmaceutically acceptable carrier.

The mixtures according to the present invention can be prepared by well-known techniques for professionals in the field of pharmacy and include all such that are known for formulating polystyrene polymers for human use.

The polymers are preferably given as formulations mixed with one or more common pharmaceutical excipients which are physically and chemically compatible with the polymer, which are non-toxic, are without harmful side effects, but which give the dosage form the right properties.

In general, for liquid formulations, water-based pharmaceutically acceptable carriers such as water itself or aqueous dilute ethanol, propylene glycol, polyethylene glycol or glycerol or sorbitol solutions are preferred.

Such formulations may also contain preservatives and flavoring and sweetening agents such as sucrose, fructose, invert sugar, cocoa, citric acid, ascorbic acid, fruit juices, etc. In general, carriers based on digestible oil or fat should be avoided or minimized as they contribute to the condition sought help when using the polymers. They are also subject to absorption on the polymers during prolonged contact, and the polymer's capacity to absorb bile acids after administration is thus reduced.

The polymers can also be prepared as "concentrates" for dilution prior to administration, and as suitable formulations for direct and oral administration. They can be given orally ad libitum on a relatively continuous basis, for example by dispersing the polymer in drinks or food.

Preferably, the polymer is given in tablet form or in gelatin capsules containing solid particulate polymer or a non-aqueous suspension of a solid polymer containing a suitable suspending agent. Suitable excipients for such formulations would be obvious to one skilled in the art and include, for example, for tablets and capsules lactose, microcrystalline cellulose, magnesium stearate, povidone, sodium starch glycolate and starches; and for suspensions in capsules polyethylene glycol, propylene glycol and colloidal silicon dioxide.

Preferably, the polymer is given in unit dosage form, each dosage preferably containing from 0.3 g to 1 g of polymer.

The daily dosage range for an adult patient can for example be a total daily dose of between 1 and 10 g, preferably 1-5 g, the compound being given 1 to 4 times a day depending on the size of the individual dosage units. Conveniently, the compound is administered over a period of continuous therapy of one month or more sufficient to achieve the required reduction in serum cholesterol levels.

In addition, the polymers in the present invention can be co-administered (together or in sequence) with other ingredients such as HMGCoA reductase inhibitors and other hypocholesterolemic agents, and other drugs for the treatment of cardiovascular diseases.

The following data and examples show the properties and production of the polymers according to the present invention. Temperatures are given in degrees Celsius. The exchange capacity of the ammonium-substituted polymers was determined by elemental analysis and/or potentiometric titration of chloride ion. The listed figures are expressed as milliequivalents of exchangeable chloride ion per gram dry polymer weight.

Chloromethylstyrene was used as a 60:40 m:p mixture and was washed free of inhibitor before use.

Divinylbenzene (DVB) was used as a 55% mixture with ethyl styrene and the weights given are for the mixture. Percent crosslinking is based on the percent of divinylbenzene (not the mixture) estimated to be in the monomer mixture. The mole percent of divinylbenzene (b) listed is based on the mole percent of DVB in the monomer mixture.

Example 1

Chloromethylstyrene (60:40 m.p. mixture) (26.7 g), styrene (22.4

g), divinylbenzene (0.91 g) and azobisisobutyronitrile (AIBN)

(0.5 g) was mixed to give a homogeneous solution and added to a

solution of poly(vinyl alcohol) (m.w. 125,000) (1.0 g) in distilled water (500 ml). The mixture was then stirred at 80°C under an atmosphere of nitrogen at such a rate that the monomers were kept in suspension. After 7 hours, the stirring was stopped and the mixture poured into distilled water. The resulting resin was washed by decantation with cold and hot water, filtered and washed with acetone, tetrahydrofuran and acetone, and dried to give a 1% cross-linked chloromethyl substituted polystyrene containing

3.3 m.equivalent Cl/g (36.6 g). This polymer was then sieved and the 53-106 µM fraction (15.8 g) used in further reactions.

This fraction of dechloromethylated polystyrene (3.3 g)

was slurried in N,N-dimethylformamide (DMF) (65 ml), N,N-dimethyloctylamine (4.96 g) added, and the mixture stirred at 65°C

for 24 hours. The polymer was then filtered off and washed with methanol and diethyl ether and dried under vacuum to give N,N-dimethyl-N,octylammoniummethyl substituted polystyrene, chloride salt, (4.35 g), (exchange capacity = 2.17 m.eq Cl/g , (b) = 0.97 mole percent.

Examples 2-5

Chloromethylated polystyrenes containing 1% divinylbenzene as cross-linking agent and containing 2.58, 3.95, 4.91 and 5.96 m.equiv. Cl/g was prepared from chloromethylstyrene, styrene and divinylbenzene as in Example 1. The above polymers were then

reacted with N,N-dimethyloctylamine in DMF and worked up as in example 1 and gave N,N-dimethyl-N-octylammoniummethyl substituted polystyrene, chloride salt, with the following exchange capacities: -1.77, 2.34, 2.76, 3, 15 m.equivalent Cl~/ q and (b) values of 0.93, 1.00, 1.08 and 1.17 mole percent respectively (Examples 2-5).

Example 6

Chloromethylated polystyrene (1% cross-linked 4.91 m.eq Cl/g), (1 g) as used in Example 4, was slurried in DMF (25 ml), N,N-dimethyldecylamine (1.8 g) added, and the mixture stirred at 70°C for 24 hours. After working up with example 1, N,N-dimethyldecylammonium substituted polystyrene, chloride salt, was obtained as grey-white balls (1.83 g) (exchange capacity = 2.44 m.eq. Cl~/<?»

(b) = 1.08 mole percent).

Example 7

A 0.5% cross-linked chloromethylated polystyrene was prepared from chloromethylstyrene (31.29 g), styrene (18.25 g) and divinylbenzene (0.46 g) by the method described in Example 1. After sieving, 7, 52 g of particle size material

53-106 >jM containing 3.89 m.eq. Cl/g. This polymer (2.0

g) was slurried in DMF (60 ml), N,N-dimethyloctylamine (3.87 g) was added, and the mixture was stirred at 65°C for 24 hours. After working up as in example 1, N,N-dimethyl-N -octylammonium methyl substituted polystyrene, chloride salt, (3.0 g) (exchange capacity =2.23 m.eq. C\~/ q, (b) = 0.51 mole percent).

Example 8

A 2% cross-linked chloromethylated polystyrene was prepared

from chloromethylstyrene (32.7 g), styrene (16.99 g) and divinylbenzene (1.82 g) by the method described in example 1.

After sieving, 25.4 g of material with a particle size of 53-105 >µM containing 3.86 m.equiv. Cl/g obtained. This polymer (5 g) was reacted with N,N-dimethyloctylamine (5 g), DMF (80 ml) at 80°C for 48 hours and gave, after working up as in example 1, N,N-dimethyl-N-octylammonium methyl substituted polystyrene , chloride salt, (7.75 g) (exchange capacity = 2.51 m.eq. Cl"/g"(*>) = 0.98 mole percent).

Example 9- 16. 18- 21 and 25- 32

Commercially available 1% cross-linked chloromethylated polystyrene (3.72 m.eq. Cl"/cj or 4.15 m.eq. Cl/g, 200-400 mesh) ("Bio Rad S-Xl, chloromethylated") was used in these examples.

Example 9-13

N,N-dimethylhexylamine, N,N-dimethyloctylamine, N,N-dimethyldecylamine, N,N-dimethyldodecylamine and N,N-dimethyltetradecylamine were each reacted with the above-mentioned chloromethylated polystyrene (1% cross-linked, 3.72 m.eq. Cl~/ q) in DMF at 70°C and after working up as described in example 1 gave the corresponding N,N-dimethyl-N-alkylammonium methyl-substituted polystyrenes, chloride salts, with the following exchange capacities:- example 9, hexyl, 2.62 m.eq. Cl"/cj; example 10, octyl, 2.40 m.eq. Cl"/g; example 11, decyl, 2.21 m.eq. Cl~/q; example 12,

dodecyl, 2.11 m.eq. Cl~/g; example 13, tetradecyl, 2.02

m.eq. Cl~/g. For each of these examples (b) was 0.9 mole percent.

Example 13A

The chloromethyl polystyrene (33.0 kg) was washed twice with dichloromethane (2 x 34 0 L) and stirred with N-methylpyrrolidone (815 L) which had been flushed under an atmosphere of nitrogen for one hour at 50°C. The mixture was heated to 60-65°C and N,N-dimethyl-dodecylamine (81.6 kg) added. It was further heated to 65-70°C for 5 hours. The mixture was cooled to 55-60°C and the product isolated by centrifugation. The wet product was reslurried with a mixture of N-methylpyrrolidone (250 L) and dichloromethane (250 L), then dichloromethane (500 L) followed by a mixture of dichloromethane (450 L) and ethyl acetate (450 L). The product was then further washed with ethyl acetate (2 x 910 L) and vacuum dried for 24 hours at room temperature followed by 50°C for 72 hours to give N,N-dimethyl-N-dodecylammonium methyl substituted polystyrene chloride.

Example 14

Ethyl chloroformate (18.3 mL) was slowly added to a solution of heptanoic acid (20.0 g) and triethylamine (26.7 mL) in tetrahydrofuran (250 mL) kept at -10°C. The resulting mixture was stirred at 10°C for 0.75 h, cooled to 5°C and a 33% solution of dimethylamine in ethanol (41.6 mL) added dropwise. This mixture was then allowed to warm to room temperature and was stirred for 4 hours. The mixture was filtered and the filtrate evaporated to dryness. The residue was dissolved in chloroform (250 mL) and washed with 1% aqueous sodium carbonate solution (200 mL) and water, then dried and evaporated. The remaining oil was chromatographed on silica gel to give N,N-dimethylheptanamide (14.1 g) as a colorless oil.

This amide (13.5 g) was treated with lithium aluminum hydride (4.88 g) in dry tetrahydrofuran (250 ml) to give N,N-dimethylheptylamine (7.10 g) as a colorless oil after work-up.

Chloromethylated polystyrene (3.0 g, 3.72 m.eq. Cl~/q) was slurried in DMF, N,N-dimethylheptylamine (3.7 g) added and the mixture heated at 75°C for 24 hours. After workup as described in example 1, N,N-dimethyl-N-heptylammonium methyl-substituted polystyrene, chloride salt, was obtained as a grey-white resin (4.49 g), (exchange capacity = 2.50 m.eq. Cl~/q, (b) = 0.98 mole percent).

Example 15

N,N-dimethylnonylamine was prepared from nonanoic acid by the method described in example 14.

A slurry of chloromethylated polystyrene (3.0 g, 3.72 m.eq. C~l/g) in DMF (50 mL) was treated with N,N-dimethylnonylamine (4.5 g) and the mixture stirred at 70°C for 24 hours. Workup as described in Example 1 gave N,N-dimethyl-N-nonyl-ammonium methyl-substituted polystyrene, chloride salt (4.72 g, exchange capacity = 2.19 m.eq. Cl~/ q, (b) = 0.98 mole percent ).

Example 16

N,N-dimethylundectylamine was prepared from undecanoic acid by the method described in example 14.

A slurry of chloromethylated polystyrene (2.0 g, 3.72 m.eq. C~1/g) in DMF was treated with N,N-dimethylundecylamine (5.0 g) at 70°C for 28 hours. Workup as described in example gave N,N-dimethyl-N-undecylammonium methyl substituted polystyrene, chloride salt (3.38 g, exchange capacity = 2.05 m.eq. Cl~ /g, (b) = 0.98 mole percent).

Example 17

A 2% cross-linked, styrene vinylbenzene copolymer, chloromethylated to give a Cl content of 4.45 m.eq. Cl~/ q, (5 g) was treated with N,N-dimethyloctylamine (9.15 g) at 70°C for 27 hours. After work-up as described in example 1, N,N-dimethyl-N-octylammonio methyl substituted polystyrene, chloride salt, was obtained as yellow balls (8.26 g, exchange capacity = 2.5 m.eq. Cl~/g, (b) = 1.61 mole percent).

Example 18

Sodium borohydride (6.62 g) was added in portions to propionic acid (52.5 ml) cooled to 5°C, keeping the temperature below 20°C. N-methyloctylamine (5 g) was added dropwise while maintaining the temperature at 20°C. The reaction was stirred at 80°C for 3 hours, cooled and basified with 2N sodium hydroxide. The aqueous solution was extracted with dichloromethane (2 x 75 mL). The combined extracts were dried with anhydrous magnesium sulfate and evaporated to dryness. The resulting oil was purified by column chromatography to give N-methyl-N-propyloctylamine as a colorless oil (4.09 g, 63%).

To a suspension of chloromethylstyrenedivinylbenzene copolymer (1% w/w divinylbenzene, 4.15 m.eq. Cl~/ q (2 g) in DMF (50 mL)

the above amine was added, and the mixture was stirred at 70°c for 24 hours and, after washing and drying as in Example 1, gave the corresponding quaternized polymer as off-white resin balls (3.11 g) (exchange capacity = 2.26 m.eq Cl~ / q, (b) = 1 mole percent) .

Example 19

N-methyl-di-n-octylamine (14.26 g) was reacted with 1% cross-linked chloromethylpolystyrene (5 g, 18.6 mmol) in DMF (125 ml) at 70°C for 24 hours as after washing as in Example 1 gave the corresponding quaternized polymer as off-white resin spheres (9.05 g) (exchange capacity = 1.87 m.eq. Cl~/g (b) = 0.98 mole percent).

Example 20

N,N-diethyloctylamine (13.7 g) was reacted with 1% cross-linked chloromethylstyrene-styrenedivinylbenzene copolymer (10 g, 37.2 m eq Cl) in DMF (100 mL) at 80°C for 12 h. After work-up as in example 1, the corresponding quaternized polymer was isolated as off-white resin spheres (16.8 g) (exchange capacity = 2.09 m.eq. Cl~/<?>(b) = 0.98 mole percent).

Example 21

N,N-dibutyloctylamine was prepared from dibutylamine and octanoyl chloride by a similar procedure as in Example 14.

This amine (17 g) was reacted with 1% cross-linked chloromethylstyrene-styrene-divinylbenzene copolymer (10 g, 40.2 m eq Cl) in DMF (100 ml) at 80°C for 1 hour. Workup as in Example 1 gave the corresponding quaternized polymer as off-white resin balls (15.0 g) (exchange resin = 1.90 m.eq. Cl~/g, (b) = 1 mole percent).

Example 22

Chloromethylstyrene (50 g) in ethanol (100 ml) was added dropwise to 33% dimethylamine in i.m.s (500 ml) over 20 minutes and the temperature rose to 35°C. The reaction mixture was stirred at room temperature for 20 hours. 33% dimethylamine in i.m.s (50 mL) was added and the reaction mixture was heated at reflux for 30 minutes. The reaction mixture was cooled and evaporated to dryness. The resulting product was dissolved in dichloromethane and washed with 2N hydrochloric acid (2 x 500 ml). The combined extracts were basified with 2N sodium hydroxide and extracted with dichloromethane (2 x 200 mL). The combined organic extracts were washed with water, dried with anhydrous magnesium sulfate and evaporated to dryness. More inhibitor was added and the oil was purified by distillation to give 3(4)-(N,N-dimethylaminomethyl)-styrene as a colorless oil, bp 66°C at 0.1 Torr (37 g, 78%).

The above amine (31.17 g), styrene (17.93 g), divinylbenzene (0.87 g) and azobisisobutyronitrile (AIBN) (0.5 g) were polymerized as in Example 1. After 7 hours the mixture was poured into distilled water. The resin formed was washed by decantation with cold and hot water, filtered and washed with acetone, ether and finally water. Drying under reduced pressure gave a 1% by weight cross-linked N,N-dimethylamino methyl substituted polystyrene polymer containing 3.85 m.eq. N/g (11.01 g, 53-106 p , 22%).

The above cross-linked N,N-dimethylaminomethyl substituted polystyrene (5 g) was slurried in DMF (100 ml), 1-iododecane (15 g) was added and the reaction mixture was stirred at 70°C for 20 hours. The polymer was filtered off and washed with DMF and methanol. Anion exchange was performed by stirring the polymer in 2N HCl: MeOH (250 mL:250 mL) and leaving overnight. The resin

was then filtered off and washed with 2N HCl, water, methanol and ether. Finally dried under vacuum to give the corresponding quaternized polymer (4.26 g) (exchange capacity = 1.82 m.eq. Cl~/g»0.38 m.eq. I~/g, (b) = 0, 99 mole percent).

Example 23

To a mixture of N-methyldodecylamine (49.5 g) and anhydrous potassium carbonate (18.1 g) in ethanol (200 ml) was added chloromethylstyrene (20 g) in ethanol (75 ml) over 20 minutes. The reaction mixture was stirred at room temperature for 20 hours and heated at reflux for 45 minutes. The reaction mixture was evaporated to dryness and the residue was dissolved in 2N hydrochloric acid and extracted with chloroform (200 mL). The chloroform extract was washed with water, dried with anhydrous magnesium sulfate and evaporated to dryness. The oil was purified by chromatography to give 3(4)-(N-dodecyl-N-methylaminomethyl)styrene as an orange oil (27.5 g, 67%).

The above amine (11.46 g), styrene (3.22 g), divinylbenzene (0.33 g) and AIBN (0.2 g) were mixed to give a solution and added to a solution of polyvinyl alcohol (mw 125,000) (0.5

g) in distilled water (200 ml). The mixture was stirred at 80°C under an atmosphere of nitrogen at such a rate that the monomers

was kept in suspension. After 7 hours, the mixture was poured into distilled water. The resin was filtered off and washed with water. Product was transferred to a beaker and washed by stirring decantation with acetone (2 x 200 mL) and ether (2 x 200 mL). The resin was filtered off and dried under reduced pressure as

gave a 2 wt% crosslinked N-dodecyl-N-methylamino methyl substituted polystyrene resin (10 g, 66%).

c) The above 2% by weight cross-linked N-dodecyl-N-methylamino methyl substituted polystyrene (4 g) was slurried in

DMF (50 mL) and stirred for 1 hour. Methyl iodide (1.75 mL) was added and the reaction mixture was stirred at 60°C for 22 hours. Additional methyl iodide (1.75 mL) was added at 19 hours. The polymer was filtered off and washed as in Example 22 and after drying under vacuum gave the corresponding quaternized polymer (2.2 g, 106-212 p) (exchange capacity = 2.11 m.eq. Cl~/q, 0.065 m.eq. l~/ q, (b) = 1.99 mole percent).

Example 24

To a mixture of N-methyloctylamine (25 g) anhydrous potassium carbonate (13.3 g) in ethanol (150 ml) was added chloromethylstyrene (13.3 g) in ethanol (10 ml) over 15 minutes. The reaction mixture was stirred at room temperature for 24 hours, filtered and the filtrate was evaporated to dryness. The residue was dissolved in dichloromethane and washed with water. The organic layer was then dried with anhydrous magnesium sulfate and evaporated to dryness. The oil was purified by chromatography (using hexane:dichloromethane as eluent) to give 3(4)-(N-octyl-N-methylaminomethyl)styrene as an oil (11.5 g, 51%).

The above amine (11.02 g), styrene (3.61 g), distyrene (0.37 g) and AIBN (0.2 g) were polymerized as in Example 2 3 and after drying gave a 2 wt% crosslinked N-octyl-N-methylamino methyl substituted polystyrene resin (10.45 g, 70%).

The above N-methyl-N-octylamino methyl substituted polystyrene (7 g) was slurried in DMF (100 ml), methyl iodide (5.3 ml) was added and the reaction mixture was stirred at 60°C for 9 hours. Additional methyl iodide (2 mL) was added at 5 h. The polymer was filtered and washed as in Example 22 and after drying gave the corresponding quaternized polymer (5.6 g) (exchange capacity = 2.36 m.eq. Cl-/cj» 0.10 m.eq. I~/q, (b) = 1.99 mole percent).

Example 25

Diamyl ketone (12 g) was hydrogenated at 3.4 atmospheres in the presence of 10% Pd/C (1.5 g) and benzylamine (8.31 g) at room temperature for 6 hours. Additional catalyst (1.5 g) was added and the mixture was hydrogenated at 40°C for 5 hours. The reaction mixture was filtered and evaporated to dryness (combined with product from a previous 12 g hydrogenation).

The oil was purified by column chromatography to give 6-undecylaithiin

as an oil (9.1 g, 38%).

To 90% formic acid (11.9 ml) cooled to -5°C, the above amine (9.6 g) was added dropwise over 45 minutes while the temperature was kept below 5°C. 37% formaldehyde (10 mL) was then added to the cooled reaction mixture before heating under reflux for 6 hours. Concentrated hydrochloric acid (4 ml) was added and the reaction mixture was evaporated to dryness. The resulting oil was scraped under ethyl acetate to give a green solid. The solid was collected by filtration, but this waxy solid quickly turned to oil after drying. This oil was dissolved in water and basified with sodium bicarbonate. The aqueous phase was extracted with ethyl acetate. The extracts were washed with water, dried over anhydrous magnesium sulfate and evaporated to dryness. The oil was distilled at 0.1 torr/250°C

in a ball tube apparatus to give 6-(dimethylamino)undecane as a colorless oil (4.06 g, 36%).

The above amine (3 g) was reacted with 1% cross-linked chloromethylated polystyrene (2 g, 7.44 mmol) in DMF (50 mL) at 70 °C

for 20 hours and after washing and drying as in Example 1 gave the corresponding quaternized polymer as off-white resin spheres (2.96 g) (exchange capacity = 2.00 m.eq. Cl"/cj" (b) = 0.98 mole percent) .

Example 26

To a solution of potassium t-butoxide (34.68 g) in anhydrous diethyl ether (750 ml) under nitrogen was added 7-carboxyheptyltriphenylphosphonium bromide (75 g) and the mixture was heated at reflux for 2 hours. The diethyl ether was removed by distillation and 6-undecanone (40 g) was added over 10 minutes. After 30 minutes, anhydrous diethyl ether (200 mL) was added to aid stirring. The mixture was heated at reflux for 2 hours, cooled and acidified with 2N HCl. This aqueous solution was extracted with dichloromethane (300 mL). The organic extracts were combined and washed with 2N NaOH (2 x 200 mL). The aqueous extracts were acidified with 2N HCl and extracted with dichloromethane (2 x 200 mL). The organic extracts were combined, dried with anhydrous magnesium sulfate and evaporated to dryness. The resulting oil was triturated with light petroleum ether. The light brown precipitate was removed by filtration and the filtrate was evaporated to dryness. The oil was purified by chromatography to give 9-pentyl-8-tetradecenoic acid as an oil (13.94 g, 30.4%).

To a solution of the above acids (13.8 g) and triethylamine (7.8 ml) in dry tetrahydrofuran (200 ml) cooled to 5°C, ethyl chloroformate (5.4 ml) was added dropwise over 15 minutes. The reaction mixture was stirred at 10°C for 2 hours. 33% dimethylamine in methanol denatured alcohol (125 mL) was added while maintaining the temperature at 10°C. The reaction mixture was then stirred at room temperature for 4 hours and evaporated to dryness. The resulting oil was dissolved in diethyl ether and washed with 1N NaOH, water, and then dried with anhydrous magnesium sulfate and evaporated to dryness, (this was then combined with the product from a previous reaction of the above acid (10.4 g). The oil was purified by column chromatography (with dichloromethane as eluent) to give 9-pentyl -8-Tetradecenamide as a colored oil (14.91 g, 57%).

The above amide (14.6 g) in dry tetrahydrofuran was added dropwise to a suspension of lithium aluminum hydride (2.6 g) in dry tetrahydrofuran (150 ml) under a nitrogen atmosphere. The temperature rose to 38°C during the addition. The reaction mixture was stirred at room temperature for 1.5 hours and heated at reflux for 1 hour. The reaction mixture was cooled and water (2.6 mL), 15% aqueous sodium hydroxide (2.6 mL), water (7.8 mL) was added. The reaction mixture was stirred for 1 hour and then filtered. The filtrate was evaporated to dryness. The oil was purified by distillation to give 1-dimethylamino-9-pentyl-8-tetradecene as an oil (bp 0.01 torr, 161-168-C) (10.79 g, 77%).

The above amine (3 g) was reacted with 1% cross-linked chloromethylated polystyrene (1.5 g, 6.23 mmol) in DMF (60 ml) at 60°C for 24 hours and after washing and drying as in Example 1 gave the corresponding quaternized polymer as off-white resin spheres (3.18 g) (exchange capacity = 1.88 m.eq. Cl~/ q, (b) = 1 mole percent) .

Example 27

1-dimethylamino-9-pentyl-8-tetradecene (Example 26c) (7.5

g) was hydrogenated in ethanol (75 mL) at 3.06 atmospheres in the presence of 10% Pd/C (0.7 g) at room temperature for 5 h.

The reaction mixture was filtered and evaporated to dryness. The resulting oil was purified by distillation at 0.01 torr (in a bubble tube apparatus at 210°C) to give 1-dimethylamino-9-pentyl-tetradecane as a colorless oil (5.7 g, 76%).

The above amine (5.5 g) was reacted with 1% cross-linked chloromethylated polystyrene (3 g, 12.45 mmol) in DMF (100 mL) and methanol (20 mL) at 60 °C for 28 h as after washing and drying as in Example 1 it gave the corresponding quaternized polymer as off-white resin spheres (6.6 g) (exchange capacity = 1.81 m.eq. Cl~/q, (b) = 1 mole percent).

Example 28

Granyl bromide (20 g) in ethanol (50 ml) was added to 33% dimethylamine in methanol denatured alcohol (240 ml) and the mixture was heated at reflux for 8 hours. The reaction mixture was evaporated to dryness, mixed with 2N sodium hydroxide (50 mL)

and then extracted with diethyl ether (2 x 100 ml). The ether extracts were combined and washed with water (50 mL), then dried with anhydrous magnesium sulfate and evaporated to dryness. The resulting orange oil was purified by chromatography (with

a gradient CH2Cl2:MeOH system as eluent) which gave 1-dimethylamino-3,7-dimethyl-2,6-octadiene as an oil (12.1 g, 72%).

The above amine (3.76 g) was reacted with 1% cross-linked chloromethylated polystyrene (2 g, 8.3 mmol) in DMF at 70°C for 20 hours and after grafting and drying as in Example 1 gave the corresponding quaternized polymer as off-white resin spheres (3.1 g) (exchange capacity = 2.44 m.eq. Cl~/cj» (b) = 1 mole percent) .

Example 29

1-Dimethylamino-3,7-dimethyl-2,6-octadiene (Example 28a) (12 g) was hydrogenated in ethanol (100 mL) at 2.72 atmospheres in the presence of 10% Pd/C (1.2 g) at room temperature for 5 hours.

The reaction mixture was filtered and evaporated to dryness. The residue was dissolved in 2N sodium hydroxide and extracted with diethyl ether (2 x 100 ml). The ether extracts were combined and washed with water, dried with anhydrous magnesium sulfate and evaporated to dryness. The oil was purified by chromatography (10:1 dichloromethane:methanol as eluent) to give 1-dimethylamino-3,7-dimethyloctane as a wax (7.14 g, 60%).

b) The above amine (3 g) was reacted with 1% cross-linked chloromethylated polystyrene (2 g 8.3 mmol) in DMF (60 ml) at 70°C for 24 hours which after washing and drying as in example 1 gave the corresponding quaternized polymer as off-white resin spheres (3.24 g) (exchange capacity = 2.31 m.eq. Cl~/cj» (b) = 1 mole percent) .

Example 30

To 90% formic acid (24.2 ml) cooled to 10°C, isoamylamine (10 g) was added in portions while the temperature was kept below 20°C. 37% formaldehyde (20 mL) was added to the cooled reaction mixture. The reaction mixture was then heated at 70°C for 6 hours. The reaction mixture was cooled and concentrated sulfuric acid (2 molar equivalents) was added and the reaction mixture was evaporated to dryness. Isopropanol (25 ml) was added to the oil under cooling, and this was diluted with anhydrous diethyl ether. The resulting thick white precipitate was collected by filtration and washed with cold anhydrous diethyl ether. This was quickly dried under reduced pressure to give an oil. This was sucked dry at 0.01 torr for 24 hours to give 1-dimethylamino-3-methylbutane as the sulfate salt (11.1 g, 45%).

The above amine sulfate salt (4.5 g) was reacted with 1% cross-linked chloromethylated polystyrene (2 g, 7.44 mmol) and anhydrous potassium carbonate (4.1 g) in DMF (50 mL) at room temperature for 24 h above 60 °C in 20 hours. The product was filtered off and washed with DMF, methanol, water, 2N hydrochloric acid, water, methanol and ether, and after drying under reduced pressure gave the corresponding quaternized polymer as off-white resin balls (2.28 g, exchange capacity = 2.76 m.eq. .Cl~/g, (b) = 0/98 mole percent).

Example 31

3-Pentylamine (20 g) was added to 90% formic acid (48.4 mL), cooled to 0°C, while maintaining the temperature below 10°C, followed by 37% formaldehyde (40 mL). The reaction mixture was heated at reflux for 7 hours, cooled and concentrated hydrochloric acid (2 molar equivalents) was added. The reaction mixture was evaporated to dryness. Isopropanol (25 mL) was added, and the solution was cooled and diluted with anhydrous diethyl ether. The resulting cream-colored solid was collected by filtration and dried under reduced pressure to give 3-dimethylaminopentane-ethylpropane hydrochloride as a cream colored powder (mp 144-145°C) (26.1 g, 75%).

The above amine (9 g) was reacted with 1% cross-linked chloromethylated polystyrene (4 g, 16.6 mmol) and anhydrous potassium carbonate (8.2 g) as in Example 30(b) to give the corresponding quaternized polymer (5.12 g ) (exchange capacity = 2.30 m.eq. Cl_/g, (b) = 1 mole percent).

Example 32

N,N-Dimethylheptadecylamine (boiling point 117°C @ 0.15 mmHg) was prepared from dimethylamine and heptadecanoic acid by the method described in Example 14.

This amine (11.7 g) was reacted with 1% cross-linked chloromethylstyrene-styrene-divinylbenzene copolymer (5 g, 21 m eq Cl) in DMF (100 ml) at 80°C for 12 hours. Workup as in example 1 gave the corresponding quaternized polymer as off-white resin spheres (9.78 g) (exchange capacity = 1.93 m.eq. Cl~/g, (b) = 1 mole percent).

Example A

A liquid formulation for oral administration is prepared from the following:

Example B

A capsule formulation for oral administration is prepared by introducing the following into a soft gelatin capsule: Compound of Example 12 (500 mg), Aerosil 200 (5 mg), Magnesium Stearate (5 mg), Avicel PHI01 (40 mg), and optionally sodium starch glycolate (10 mg).

Example C

A food additive formulation, for example a pouch reconstitution or mixture with food, is prepared by introducing into a powder formulation the compound from example 12 (2500 mg), sodium carboxymethyl cellulose (50 mg), sucrose (2400 mg) and flavorings (50 mg).

Biological data

In the binding measurements, the polymers used contained 5 to 15% by weight H2O and all weights used in the measurements refer to the non-dried materials.

Equilibrium binding of bile acids (as sodium salts) to resins

The compounds from the examples (10 mg per tube) were incubated with a range of<14>C radiolabeled sodium taurochenodeoxycholate (TCDC) or sodium glycolate (GC) solutions (0.05 mM-24 mM) of known specific activity in Krebs Henseleit buffer (2.5 ml final volume). The resin was allowed to equilibrate with the bile acid salt solution for a period of three hours at 37°C in a shaken water bath. The resin was removed from the incubation medium by centrifugation (15 minutes at 3,000 g). The radioactivity of the supernatant was determined, and from this the free and bound amounts of bile acid salt were calculated. These were expressed as moles of bile acid salt bound per grams of resin.

Results

The compounds from examples 1-32 in a concentration of 5.7 mM GC were found to bind in the range from 0.63 to 1.24 mmol GC per gram weight per resin; and at a concentration of 5.7 mM TCDC was found to bind in the range from 0.53 to 1.34 mmol TCDC per gram weight of resin.

In vitro dissociation measurements.

In the procedure in Table 1, the test compound (20

mg) shaken in Krebs' buffer for 3 h in the presence of 5 mM sodium [<14>C] glycocholate (5 ml). In the procedure in Table 2, the test compound (150 ml) was equilibrated with 5 mM sodium glycocholate (30 ml) in the same buffer. The compound was separated by centrifugation, and the total bound determined by subtracting the amount in the supernatant from the total bile acid used. Dissociation was measured either by continuous perfusion of the preloaded compound with Krebs' buffer and determination of the radioactivity in the perfusate (Table 1), or by resuspending the compound in Krebs' buffer, shaking and sampling the mixture through a filter at several time points up to 20 minutes. Radioactivity and consequently bile acid that had dissociated was determined in the filtrate (Table 2).

Claims (9)

1. Polystyrene polymers with structure (I) in which R<1> is a saturated or unsaturated C6 to C20 alkyl group; R<2> and R<3> are the same or different and are each C1_4<a> alkyl; X- is a physiologically acceptable exercise; a, b and c are numbers indicating the relative molar percentages of the unit that exists in a static distribution in the polymer, (b) is from approx. 0.5 to approx. 10 mole percent, and (c) is from about. 30 to approx. 99 mole percent; and n is a number indicating the number of repeating units in the polymer. 2. Polystyrene polymer according to claim 1 wherein R<1> is a saturated Cq to <C>14 alkyl group, and R<2> and R<3> are both methyl. 3. Polystyrene polymer according to claim 2 in which (b) is from approx. 1 to approx. 4 mole percent of the polymer. 4. Polystyrene polymer according to claim 1 which is N,N,dimethyl-N-nonylammoniomethyl-substituted polystyrene, chloride salt N,N,dimethyl-N-octylammoniomethyl-substituted polystyrene, chloride salt N,N,dimethyl-N-decylammoniomethyl-substituted polystyrene, chloride salt N,N,dimethyl-N-undecylammoniomethyl-substituted polystyrene, chloride salt or N,N,dimethyl-N-dodecylammoniomethyl-substituted polystyrene, chloride salt. 5. Process for producing a polystyrene polymer according to each of claims 1 to 4, which comprises (a) reaction of a polymer with structure (II) wherein a, b, c and n are as described in claim (I) and Y is a group that can be replaced by an amine, with an amine of structure R<1>R<2>R<3>N (in) whereinR <1> to R<3> are as described for structure (I); or (b) conversion of a compound of structure (IV) wherein a, b, c and n are as described for structure (I) and Z is NR<1>R<2> or NR<2>R<3> wherein R<1> to R<3> are as described for structure (I) with a compound of structure R <4> Y (V) in which R <4> is a C 1_4 alkyl or a saturated or unsaturated C 6 _2 o alkyl group and Y is a group which can be replaced by an amine. 6. Pharmaceutical mixture comprising a polystyrene polymer with structure (I) according to claim 1 in connection with a pharmaceutically acceptable carrier. 7. Polystyrene polymer according to claim 1 for use as a therapeutic agent. 8. Polystyrene polymer according to claim 1 for use in lowering serum cholesterol concentrations. 9. Polystyrene polymer with structure (IVA) in which a, b, c and n are as described for structure (I) and Z<A> is N r <I> r <2> orNR<2>R<3> in which R<1> and R<2> are as described for structure (I) in claim 1.