FI72716B - FOERFARANDE FOER FRAMSTAELLNING AV NYA TERAPEUTISKT ANVAENDBARA 2-HYDROXIMETYL-3,4,5-TRIHYDROXIPIPERIDINDERIVAT. - Google Patents

Description

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Process for the preparation of new therapeutically useful 2-hydroxymethyl-3,4,5-trihydroxypiperidine derivatives 5 Separated from application 782607

The present invention relates to a process for the preparation of new therapeutically useful 2-hydroxymethyl-3,4,5-trihydroxypiperidine derivatives of general formula I.

CH 2 OH / R

HO- / \ —H (I)

HO OH

Wherein R is a C 1-6 alkyl group or a C 2-8 alkenyl or C 2-8 alkynyl group optionally substituted by phenyl, nitrophenyl or halophenyl, and for the preparation of their salts and stereoisomers.

These new compounds of the formula I can be used as medicaments, in particular against diabetes, hyperlipemia and obesity, and to have an effect on the meat / fat ratio in animals.

The invention further relates to pharmaceutically acceptable salts of the compounds of formula I, such as chlorides, sulphates, acetates, carbonates and oxalates, and stereoisomers of the compounds.

It has been found that the novel compounds of the formula I are potent inhibitors of α-glucosidases, in particular disaccharidases. Therefore, the new compounds are valuable substances that affect many metabolic events.

Compared to 2-hydroxymethyl-3,4,5-trihydroxypiperidine known from DE-A-2 656 602, the new compounds have advantageous therapeutic properties.

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The method according to the invention is characterized by

that 1-deoxynojirimycin of general formula II

CH OH> ^ NH

5 fe) (I1 ’

H0 OH

is reacted with a reactive alkylating agent of general formula IV

Z-R. (IV) wherein R is as defined above and Z is a common, easily leaving group in the alkylating agents, for example a halide group, and if desired the compound of formula I obtained is converted into a salt and / or the compound of formula I obtained if desired is partitioned stereoisomeerikseen.

An example of the reaction between 1-deoxynojirimycin and an alkylating agent is the reaction with allyl bromide:

CH "OH

CH-OH <_ 20> -nh> “n-gh2-ch = ch2 HO- / \ + Br-CH2-CH = CH2 _HBr> H0 \ /

/ OH OH

OH 0H

The starting material of formula II is known and is obtained from either catalytic hydrogenation fermentation of nojirimycin / S. Inouye et. ai, Tetrahedron 23 (1968) 2125-21447 or by extraction from the bark of mulberry wood (DE application publication 2,656,602) or also completely synthetically. In a novel, preferred manner, 1-deoxynojirimycin can also be prepared by culturing Bacillaceae organisms in conventional fermentation vessels in standard nutrient solutions at a temperature in the range of about 15 ° C to about 80 ° C for 18 days by aerating, centrifuging the cells, and isolating the deoxy compound or culture medium. from the extract by standard 35 purification methods. (DE application publication 26 58 563.

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The reactive alkylating agents of formula IV are known or can be prepared by known methods. The reaction with a compound of formula II is carried out in an inert, organic solvent at a temperature from room temperature to boiling point and optionally with the addition of an acid scavenger.

The inhibitors according to the invention are suitable as medicaments for the following indications:

Precursor of diabetes, gastritis, constipation, gastrointestinal infections, flatulence, flatulence, hypertension, atherosclerosis and especially obesity, diabetes and hyper-lipoproteemia.

To broaden the spectrum of action, it may be advisable to combine them with glucoside hydrolase inhibitors, in which case these drugs complement each other. Combinations may include inhibitors of the invention with each other or in combination with known inhibitors. For example, it may be appropriate to combine the sucrase inhibitors of the invention with known amylase in-20 inhibitors.

In many cases, the combination of the inhibitors according to the invention and known oral antidiabetic drugs ((*> - cytotropic sulfonyl current derivatives and / or biguanides which act on blood sugar) as well as blood lipid-lowering drugs such as clibrate, nicotyric acid, colicyric acid, is also preferred.

The compounds may be used as pharmaceutical compositions undiluted, e.g. in powder or gelatin coating or in combination with a carrier.

The pharmaceutical preparations may contain varying amounts of inhibitor, e.g. 0.1-99.5%, together with a pharmaceutically acceptable, non-toxic, inert carrier which may contain one or more solid, semi-solid or liquid diluents or excipients, and / or 35 non-toxic, inert and pharmaceutically acceptable excipients. Such pharmaceutical preparations are preferred. __ 7271 6 ti in dosage units, i.e. as physically limited units containing a predetermined amount of inhibitor, corresponding to a portion or multiple of the dose required to produce the desired inhibitory effect. The dosage unit may contain 1, 2, 3, 4 or more unit doses or 1/2, 1/3 or 1/4 of the unit dose. The unit dose preferably contains an amount of active ingredient that achieves the desired inhibitory effect in drug delivery with a predetermined dosing regimen when administering one or more dosage units, with the full daily dose, half, third or quarter of the daily dose usually administered with all main and intermediate meals consumed throughout the day. Other therapeutic agents may also be taken. Although the dosage and dosing schedule will be carefully planned by one skilled in the art on a case-by-case basis, taking into account the age, weight and condition of the patient, the nature and severity of the disease, the dosage will usually be from about 1 to about 4 SIC / kg per day. In some cases a lower dose may be sufficient, and in some cases a higher dose.

For oral administration, solid and liquid dosage units can be used, e.g., powders, tablets, dra-ge, capsules, granules, suspensions, solutions, and the like.

Powders are prepared by comminuting the active ingredient to a suitable level and mixing it with a finely divided pharmaceutical carrier. Although an edible carbohydrate, e.g. starch, lactose, sucrose or glucose, which can also be used in this case, is normally used as the carrier, it is more preferable to use a non-metabolizable carbohydrate, e.g.

30 cellulose derivatives.

Sweeteners, flavors, preservatives, dispersants and colors may also be added.

Capsules can be prepared by filling the powder mixture described above into finished gelatin shells. A lubricant such as silica gel, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol may be added to the powder mixture prior to filling. A disintegrant or solubilizer, such as agar, calcium carbonate, or sodium carbonate, may also be included in the mixture to provide a more easily utilized form of inhibitor when taken into capsules.

Tablets are prepared, for example, by preparing a coarse or fine powder mixture with the addition of a lubricant and a disintegrant. This mixture is then formed into tablets. The powder mixture is prepared from a powder-10 rack in a suitable manner with the active ingredient, and by mixing with a diluent or carrier pressures as described above. It is also possible to add binders, such as carboxymethylcellulose, alginates, gelatin or polyvinylpyrrolidones, solubilizers, such as paraffin, a resorption agent, such as, for example, a quaternary salt and / or an absorbent, such as bentonite, kaolin or kaolin. . The powder mixture may be granulated together with solutions of a binder such as syrup, starch paste, acacia or cellulosic or polymeric materials. The product is then pressed through a large mesh screen. Alternatively, the powder mixture can be compressed into tablets on a tableting machine, and the resulting pieces of uneven quality are crushed to a suitable grain size. To prevent the resulting granules from sticking to the nozzles of the tableting machine, a lubricant such as stearic acid, stearate salt, talc or mineral oil may be added. This lubricated mixture is then compressed into tablets. The active ingredients can also be combined with acid-flowing, inert carriers and formed directly into tablets by omitting the granulation or crushing steps. The product may be coated with a clear or opaque coating, e.g. a shellac, sugar or polymer coating and a polished wax layer. Dyes may be added to such a coating to separate the various dosage units.

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Formulations for oral use, solutions, syrups, elixirs can be prepared in unit dosage form so that a certain amount of the preparation contains a certain amount of the active ingredient. The syrup may be prepared by dissolving the active ingredient in an aqueous solution containing suitable flavoring agents. Elixirs can be prepared using non-toxic, alcoholic carriers. Suspensions are prepared by dispersing the compound in a non-toxic carrier. Solvent mediators and emulsifiers may also be added, e.g. ethoxylated isostearyl alcohols and polyoxyethylene sorbitan esters, preservatives, flavoring agents, e.g. peppermint oil or saccharin.

Dosage instructions can be indicated on the package. Dosage can be further ensured by the use of sustained release formulations, e.g. by incorporating the active ingredient into a polymeric material, wax or the like.

In addition to the above-mentioned preparations, foods containing active ingredients, e.g. sugar, bread, potato products, fruit juice, beer, chocolate and other confectionery products, as well as preserves, such as marmalade, can be prepared to which a therapeutically effective amount of at least one inhibitor of the invention is added.

The nutrients containing the active ingredients according to the invention are suitable both as a dietary food for patients suffering from metabolic disorders and as a diet for healthy individuals to prevent metabolic disorders by means of food.

In addition, the inhibitors according to the invention have the ability to greatly reduce the proportion of unwanted fat formation in animals and to increase the proportion of lean fatty meat (lean meat). This is particularly important in the rearing and feeding of farm animals, especially pigs, but also in the rearing of other animals and pets. In addition, the use of inhibitors can achieve a significant rationalization of animal nutrition in terms of both time and quantity as well as quality. Because they have a certain effect of slowing down digestion, the residence time of nutrients in the gastrointestinal tract is prolonged, which contributes to ad libitum feeding savings. In addition, the use of the inhibitors according to the invention results in considerable savings in valuable protein feed in several cases.

Thus, in virtually all areas of animal nutrition, the active ingredients can be used as a fat-reducing agent and as a feed protein-regulating agent.

The efficacy of the active substances is then somewhat independent of the species and sex of the animal. Substances of particular value have been shown to exist in animal species which, at certain stages of life, tend to have a strong victory over ras-15.

Examples of animals in which inhibitors can be used to reduce fat content and / or save dietary protein include the following useful and pet animals: warm-blooded animals such as cattle, pigs, horses, sheep, goats, cats, dogs, rabbits, fur animals such as minks, chinchillas, other pets such as guinea pigs and hamsters, laboratory and zoo animals such as rats, mice, monkeys, etc. birds such as broilers, chickens, geese, ducks, turkeys, pigeons, parrots, and canaries, and cold-blooded birds animals such as fish, e.g., carp, and reptiles, e.g., snakes.

The amount of active ingredient to be administered to the animals required to achieve the desired effect may vary widely due to the beneficial properties of the active ingredient.

It is preferably about 0.5 mg to 2.5 g, especially 10 to 100 mg / kg of feed color. The duration of its consumption can vary from a few hours or days to several years. The appropriate amount of active ingredient and the appropriate period of ingestion depend closely on the purpose of the feeding. They depend on the species, age, sex, state of health and purpose of keeping the animal and can be easily prescribed by a person skilled in the art.

8,72716

The active compounds according to the invention are administered to animals by customary methods. The route of administration depends in particular on the species, behavior and general condition of the animal. Administration may be performed orally once daily or 5 twice daily at regular or irregular intervals. In most cases, it is most appropriate to administer the active ingredients orally, especially in accordance with the feeding and / or drinking rhythms of the animals.

The active ingredients can be in pure form or in preparations, in which case the preparation can be either as a premix, i.e. as a mixture with non-toxic, inert carriers, or also as part of a complete diet as a complementary feed or as a mixture of a compound feed administered alone. It is also possible to administer them as suitable preparations with drinking water.

The active ingredients may also be administered in suitable forms as preparations with other nutrients and active ingredients, eg mineral salts, trace elements, vitamins, proteins, energy-giving substances (eg starch, sugar, fats), colorings and / or flavorings or other with feed additives such as growth promoters. The active substances can be administered to the animals during or after their feeding.

Preferably, the active ingredients are administered orally with the feed or drinking water, in which case they are added to all or part of the feed and / or drinking water, if necessary.

The active ingredients can be added to the feed and / or drinking water by the usual methods simply as pure substances, preferably in finely divided form, or in the form of preparations mixed with edible, non-toxic carriers or also as a premix or concentrate.

The feed and / or drinking water may contain, for example, about 0.001-5.0% of active ingredient according to the invention, in particular 0.02-2.0% (w / w). The most preferred concentration of the active substance in the feed and / or drinking water depends in particular on the amount of feed and / or drinking water ingested by the animals and can be easily determined by any person skilled in the art.

The quality and composition of the feed do not then matter. Any of the usual commercial or special feed compositions can be used, which preferably contain the usual energy and protein substances, vitamins and minerals balanced in terms of systemic shaking. The feed may contain, for example, plant substances, e.g. oil cake, cereal groats, cereal by-products, but also hay, silage, beets and other fodder plants, as well as animal products such as meat and fish products, bone meal, fat, vitamins, e.g. A-, D , E, K and B complex vitamins as well as specific protein sources such as yeast as well as certain amino acids, minerals and trace elements such as phosphorus and iron, zinc, manganese, copper, cobalt, iodine and the like.

The premixes may preferably contain about 0.1-50%, especially 0.5-5.0% (w / w) e.g. N-methyl-1-deoxynojirimycin with various edible carriers and / or mineral salts. , e.g. with feed lime (calcium carbonate) and are prepared by standard mixing methods.

Compound feeds preferably contain 0.001-5.0%, in particular 0.02-2% (by weight) of, for example, N-methyl-1-deoxino-jirimycin in addition to the usual feed compound components, e.g. cereal meal, oil cake cake, animal protein, minerals, trace elements and vitamins. They can be prepared by conventional mixing methods.

The active ingredients in the premixtures and compound feeds can advantageously be protected against the effects of air, light and / or moisture by covering their surface with suitable substances, e.g. non-toxic waxes or gelatin.

The composition of a compound feed for birds containing the active ingredient according to the invention is, for example: 35,200 g of wheat, 340 g of maize, 360.3 g of soybean meal, 60 g of beef tallow, 15 g of dicalcium phosphate, 10 g of calcium carbonate, 72 g of carbonate, 4 g of iodinated common salt, 7.5 g of a vitamin-mineral mixture and 3.2 g of a premix of the active substance mixed thoroughly with 1 kg of feed.

The vitamin-mineral mixture contains: 5 6000 k.y. Vitamin A, 1000 k.y. Vitamin D 2, 10 mg vitamin E, 1 mg vitamin K 2, 3 mg riboflavin, 2 mg pyridoxine, 2C ug vitamin B 2, 5 mg calcium pantothenate, 30 mg nicotinic acid, 200 mg choline chloride, 200 mg MnSO4-H2O, 140 ZnSO4.7H2O 100 mg FeSO4.7H2O and 20 mg CuSO4.5H2O.

For example, the premix of active ingredient contains the desired amount of e.g. 1600 mg of N-methyl-2-deoxynojirimycin and in addition 1 g of DL-methionine in such an amount of soybean meal that 3.2 g of premix are obtained.

A compound feedingstuff for pigs containing an active substance of formula I is presented with the following composition: 630 g of feed grain meal (containing 20C g of maize, 150 g of barley, 150 g of oats and 130 g of wheat meal), 80 g of fishmeal, 60 g of soybean meal , 58.8 g tapioca flour, 38 g beer beer-20 vaa, 50 g vitamin-mineral mixture for pigs (composition e.g. same as for chickens) 30 g linseed cake flour, 30 g cornstarch flour, 10 g soybean oil, 10 g sugar cane molasses and 2 g premix of active substance (composition eg same as for chickens) mixed thoroughly with 1 kg 2 5 feed.

The compound feeds shown are preferably suitable for rearing and fattening chickens and pigs. However, they can also be used in the same or similar compositions for the rearing and fattening of other animals.

The inhibitors can be used alone or in different mixtures with each other.

In vitro sucrase inhibition assay

The sucrase inhibition assay makes it possible to determine the enzyme inhibitory activity of a substance by comparing the activity of a dissolved intestinal disaccharidase complex in the presence and absence of an inhibitor (so-called 100 l value). Substantially glucose-free sucrose (glucose <100 ppm) is then used as the substrate which determines the specificity of the inhibition test; the enzyme activity assay is based on the spectrophotometric assay of glucose released by Gluoside dehydrogenase and the cofactor nicotinamide-5 adenine dinucleotide.

A sucrase inhibitor unit (SIY) is defined as an inhibitory activity that reduces saccharolytic activity by one unit in a defined test batch (sucrase unit = SY); the sucrase unit is then defined as the enzyme activity which, under predetermined conditions, degrades one / imol of sucrose per minute and thus releases one yol of glucose, which is determined in the experiment, and fructose, which is not taken into account in the experiment.

The intestinal disaccharidase complex is obtained from porcine small intestinal mucosa by trypsin digestion, precipitation from 66% ethanol at -20 ° C, addition of 10 mM phosphate buffer (pH 7.0) to the precipitate, and finally dialysis against the same buffer.

10 μl of test solution prepared so that the extinction of the test batch is at least 10% but not more than 25% below 100%, add a dilution of 100 μl of intestinal disaccharidase complex in 0.1 M maleinate buffer-25 (pH 6.25) , and the mixture is preincubated for 10 min at 37 ° C. The dilution of the diazaccharidase complex must be adjusted to an activity of 0.1 SY / ml. The saccharolytic reaction is then initiated by adding 100 μl of a 0.4 M sucrose solution (SERVA 35579) in 0.1 M maleinate buffer at pH 6.25 and terminated after a 20 minute incubation period at 37 ° C by adding 1 ml of glucose dehydrogenase reagent. lyophilized glucose dehydrogenase-mutarotase mixture (MERCK 14053) and 33.17 mg / 5-nicotinamide adenine dinucleotide (free acid, BOEHRINGER, purity I) 250 3 in 5 ml of 0.5 m Tris buffer, pH 7.6.7. To detect glucose, the mixture is incubated for 30 minutes at 37 ° C, and glucose 12 7271 6 is determined with a photometer at a wavelength of 340 mm against a reagent test (containing enzyme but not) against sucrose.

The calculation of the inhibitory activity of inhibitors is complicated by the fact that even small changes in the test system, for example 5 small variations in the determinations of 100% values, affect the values of the test results in amounts that cannot be ignored. These difficulties can be avoided by performing each assay simultaneously with the standard; a sucrase-10 inhibitor of the formula C25H43 ° 18N with a specific inhibitory activity of 77,700 SIY / g is used as a standard, and the addition of 10 to 20 ng to the experiment provides an inhibition of the order of magnitude defined above. When the difference between the extinctions of the 100% value and the standard inhibited test batch extensions at 340 nm is known, the difference between the 100% value and the 15 inhibited batch extinction extinctions is obtained by calculating the specific inhibitor activity in grams of sucrase inhibition per gram (SIY). / g).

Specific sucrase inhibitory activity in vitro: 1-deoxynojirimycin 465,000 SIY / g N-methyl-1-deoxynojirimycin 2,330,000 SIY / g The valuable and surprising therapeutic properties of the compounds prepared by the method of the invention are also evident from the following therapeutic test results.

25 In the exercise experiment performed, rats were fed a diet zero with varying amounts of cooked starch and test substance. Blood glucose levels were measured 30 min after feeding and rats fed only boiled starch as well as rats fed 30 were used for comparison.

For deoxino-irimycin known from FR-A-2 336 941? H? 0H vr 7271 6 HO --- 1 --- N> h 13 HO - "5 The stress value obtained in the stress test was denoted by a relative value of 1. The table in the appendix shows how much better the The compounds prepared by the method of.

HO - ^ - H

OH

1 5 _R_ Coefficient CH3 18.9 n-C4H9 11.1 C2H5 24.4 n-C3H7> 16.7 20 iso ~ C4H9> 16.7 nC? H15 5.5 «2- © <11.1 CH2- ^ 3 <16.7 25 CH 2 -CH \ / 5.5 "-c5h, n-C6H13 3.8 n-C8H17 6.7 30 n-C9H19 <16.7" -c10H21 <16'7 n_c11H23 1 · 1 n-C12H25 <16.7 n-CuH29 <16.7 7271 6 14 R__Factor CH2- (p ^ -Cl <1.1 CH2-CH2-CH2 - ^^ 16.7 _ CH ~ -CH = CH0 11 , 1 CH2 “C = CH 2.2 <16.7 ch2 - <^) - NO, <16.7 10 CH2 ^ 0 <16.7 ^ 2 CH2CH = CH-CH3 47.8

Example 1: N-allyl-1-deoxynojirimycin HOCH

15 hoH) CH2 "CH = CH2

HO OH

A mixture of 5 g of 1-deoxynojirimycin, 30 ml of diethylformamide and 30 ml of water was stirred with Ag 2 O 20 (5 g) and allyl bromide (5 g) for 3 hours at room temperature. The silver salts were then filtered and the filtrate was evaporated to dryness on a rotary evaporator. The residue was crystallized from ethanol. Yield: 4.5 g of N-allyl-1-deoxynojirimycin, m.p. 131-132 ° C.

The following compounds were prepared in an analogous manner. Isolation and purification of the final products was optionally also performed by chromatography on a strongly acidic 0 ion exchanger (Form H).

N-proparyl-1-deoxynojirimycin

30 HOCfL

VHNI-CH -C = CH

Hoy)

HO OH

35 mp: 160 ° C (from acetone)

II

15 7271 6 N- (31,41-dichlorobenzyl) -1-deoxynojirimycin HOCH_ / ΤΛ.

5 HO OH

M.p .: 130-132 ° C

N- (p-nitrobenzyl) -1-deoxynojirimycin 10 H0CH2 / -Λ HO Λ ™ # ™ 2

HO OH

M.p .: 144-146 ° C

15 N- (m-nitrobenzyl) -1-deoxynojirimycin 20 Y_ / NO7

HO 0H

M.p .: 168-170 ° C.

Claims (2)

16 7271 6 A process for the preparation of novel therapeutically useful 2-hydroxymethyl-3,4,5-trihydroxypiperidine-5 derivatives of the general formula I CH0OH JR ^ Γ ~ Ν HO- / \ - H (I) 10> —c HO OH wherein R is a C 1-6 alkyl group or an alkenyl or C 1-6 alkynyl group optionally substituted by phenyl, nitrophenyl or halothenyl, and z-6 2-6 for the preparation of their salts and stereoisomers, characterized in that 1-deoxynojirimycin, having the general formula II CH 2 OH HO OH 25 is reacted with a reactive alkylating agent of general formula IV ZR (IV) 30 wherein R is as defined above and Z is a common, easily leaving group in the alkylating agents, for example a halide group, and, if desired, the resulting formula I The compound of formula I is converted into a salt and / or, if desired, the compound of formula I obtained is divided into its stereoisomer. li 17 7271 6 Process according to Claim 1, characterized in that a compound of the formula IV is used in which R is benzyl.