DE3241199C2 - - Google Patents

Description

The present invention relates to new α , α- trehalose-6,6'-diesters of medium-chain, aliphatic acids and pharmaceutical agents which contain the same as an active ingredient.

α , α- Trehalose is one of the so-called disaccharides, which are composed of two glucose molecules and are widespread in nature. Various derivatives of this compound have been synthesized and examined for their pharmacological activities. For example, it has been reported that a mixture of different diesters in which the aliphatic acids are attached at different positions show antitumor activity.

In the course of the synthesis of various 6,6'-aliphatic acid diesters of α , α- trehalose and investigations of their pharmacological effects, the inventors found that an α , α- trehalose-6,6'-diester was medium-chain, aliphatic Acids according to the following formula (I)

where n is an integer of 6 to 10, has excellent antitumor activity, excellent antibiotic activity and excellent activity in terms of delay-type hypersensitivity, and is useful for pharmaceuticals. The present invention is based on these studies.

It is accordingly the object of the invention to provide a new α , α- trehalose-6,6'-diester medium-chain, aliphatic acids according to the above formula (I). It is also an object of the invention to provide a pharmaceutical agent which contains an α , α- trehalose-6,6'-diester medium-chain, aliphatic acids according to the above formula (I) as the active ingredient.

The inventive α , α- trehalose-6,6'-diester of medium-chain, aliphatic acids (I) (hereinafter referred to simply as "trehalose-diester of medium-chain aliphatic acids") can be prepared, for example, according to the following reaction scheme. Then α , α- trehalose (II) is converted into 2,3,2 ', 3'-tetra-O-benzyl- α , α- trehalose (III) and this is then treated with a medium-chain, aliphatic acid (IV) (the The term "medium-chain" means here and below an aliphatic radical of medium chain length) or its active derivative to a new intermediate 2,3,2 ', 3'-tetra-O-benzyl- α , α- trehalose-6,6'- diester medium-chain, aliphatic acids (V) reacted, and this is further debenzylated using a reducing catalyst:

where Bn stands for a benzyl group and n has the meaning given above.

In the above process, the 2,3,2 ', 3'-tetra-O-benzyl- α , α- trehalose according to the formula (III) is prepared by reacting the trehalose according to the formula (II) with benzaldehyde in a conventional manner to form 4,6,4 ', 6'-di-O-benzylidene- α , α -trehalose and then this compound with benzyl chloride to form a 2,3,2', 3'-tetra-O-benzyl derivative and finally reacting the same with an acid.

The reaction between the compound (III) and the medium-chain, aliphatic acid (IV) or its active derivative under conventional conditions an esterification reaction occurs performed by using the medium-chain, aliphatic acid (IV) or its active derivative in a molar ratio of preferably 1.8 to 2.4 mol / mol the compound (III) in the presence of an acid binder, such as Pyridine and triethylamine.

The reaction is preferably carried out in the presence or absence a solvent between 0 ° C with ice cooling to 30 ° C for 1 to 24 hours. That included solvents used include, for example, methylene chloride, Chloroform, benzene, toluene, ether, tetrahydrofuran and dioxane.

The reducing debenzylation reaction that leads to Compound (V) obtained in this way is subjected, is carried out by means of a catalyst, preferably in an amount of 0.5 to 10 moles per 1 mole of compound (V) in a suitable solvent.

The reduction catalyst includes, for example, palladium black, Palladium-carbon and raney nickel. The ones used Solvents include, for example, alcohols, such as methanol, ethanol and isopropanol; halogenated compounds, such as chloroform and methylene chloride; as well as mixtures this solvent.

The pharmacological activities of the invention Trehalose diester of medium-chain, aliphatic acids (I) are shown below.

(1) Cell-killing activity of trehalose diester medium-chain, aliphatic acids

(a) Leukemia L-5178Y cells are used and adjusted in such a way that 10⁵ cells / ml in one RPMI 1640® medium containing 10% ox fetal serum are present. The samples are added to the medium up to a predetermined level Concentration applied. After 48 hours of cultivation in an incubator at 37 ° C under 5% CO₂ is the Number of living cells counted under a microscope and compared with a control group by the multiplication factors to determine. These are on a logarithm. probable. Paper applied, and the values for IC₅₀ are determined based on the curve thus obtained. The concentration for each sample is 25, 50, 100, 200 or 400 µg / ml.

The results are shown in Table 1.

Table 1

(b) In the same manner as in (a), the cell killing activities are carried out the trehalose diester medium-chain, aliphatic acids against cells of Leukemia L-1210 examined. The results are shown in Table 2.

Table 2

(2) anti-cancer activities of trehalose diesters of medium chain, aliphatic acids

Male std-ddy mice, 5 weeks old, are used namely six mice / group each. Cancer cells from Sarcoma 180 will be in the peritoneal cavity of each mouse transplanted, namely 5 × 10⁶ cells. Starting 24 h later, a test compound suspended in 30% Propylene glycol, in an amount of 150 mg / kg once per Administered intraperitoneally for 5 days continuously. 7 days after the cancer cells were transplanted Samples of the abdominal ascites are taken to determine the Amount of abdominal ascites and the cell ratio to determine. The tumor growth ratio (T / C in%) is determined using TPCV (total pack cell volume) (total cell volume), which is the product the amount of abdominal ascites and the cell ratio acts according to the following formula. The anti-cancer activity the test compound is rated. The results are listed in Table 3.

Table 3

As can be seen from the above description, the Trehalose diester of medium-chain, aliphatic acids has a remarkably stronger anti-cancer activity than the corresponding compounds with 6 to 22 carbon atoms.

(3) Antimicrobial Activity

The minimum inhibitory concentrations (MIC) of the invention Compounds are made against different bacteria examined. The results are shown in Table 4. The tests were carried out according to the MIC measurement method, that is approved by the Japan Society of Chemotherapy is. The Mueller-Hinton Medium® was used as the culture medium used. The test connections were in 25% DMSO to concentrations of 1 mg / ml and then dissolved with sterile, distilled Water diluted to the predetermined concentrations.

Results:

Table 4

(4) Effect on delay type hypersensitivity

Several groups of 6 week old female ICR mice, each group consisting of ten mice are provided, and they develop red blood cells subcutaneously of the sheep (SRBC), 0.05 ml / mouse in a concentration of 3 × 10⁹ cells / ml. The injection takes place in the sole of the foot of the right hind paw, whereby immediately thereafter the test compounds intraperitoneally injected * (day 0). 4 days later will be the same Apply amount of SRBC (0.05 ml / mouse) to the sole of each mouse injected subcutaneously on the left hind paw, and a further 24 h later the thickness of the left sole of the foot is determined by means of a Measuring circle measured.

Other groups that instead of the test compounds are physiological Saline solution administered should be considered Controls used, and again other groups that only subjected to the second injection used as a reference (base). The swell ratio the sole of the foot is obtained from the following equation:

where T, C and B each denote the thickness of the sole of the foot of the left hind paw of the test groups, the control groups and the reference groups, respectively. The results are shown in Table 5.

Table 5

Tabelle 6

Tabelle 7

Beispiel 5 Tabletten Auf herkömmliche Weise wird eine Tablette hergestellt, die die folgenden Bestandteile, in den jeweils angegebenen Mengen, enthält.mg Trehalose-diester mittelkettiger,
aliphatischer Säure (Decanoat)100 D-Mannit150 kristalline Cellulose 50 Stärke 28 Calciumcarboxymethylcellulose 16 Talkum  4 Magnesiumstearat  2 Gesamtmenge350 Beispiel 6 Kapseln Es wird auf herkömmliche Weise ein Granulat hergestellt, welches die folgenden Bestandteile in den jeweils angegebenen Mengen enthält. Das Granulat wird in Nr.-3-Kapseln abgefüllt. mg Trehalose-diester mittelkettiger,
aliphatischer Säure (Caprylat)25 kristalline Cellulose17 leichte, wasserfreie Kieselsäure 7 Magnesiumstearat 1 Beispiel 7 Injektionslösungen Es wird eine Injektionslösung hergestellt, indem man die folgenden Bestandteile jeweils in den angegebenen Mengen auf herkömmliche Weise formuliert. Trehalose-diester mittelkettiger,
aliphatischer Säure (Decanoat)25 mg mit Olivenöl aufgefüllt auf1 ml Beispiel 8 Suppositorien Die folgenden Bestandteile werden jeweils in den angegebenen Mengen geschmolzen und gerührt und anschließend geformt, um sie zu einem Stück Suppositorium zu verfestigen, wobei eine herkömmliche Verfahrensweise angewendet wird. mg Trehalose-diester mittelkettiger,
aliphatischer Säure (Laurat) 100 Kakaobutter1100 Gesamtmenge1200 & / UDF The trehalose diesters of medium-chain aliphatic acids according to the invention are almost non-toxic and thus represent a safe connection, which is also clear from the fact that, for example, sucrose esters of aliphatic acids, which are similar to the esters according to the invention, are permitted as food additives and can be widely used as harmless surfants in the field of foods, medicines or cosmetics. The compounds of the present invention can also be formulated into various types of preparations depending on the mode of application such as oral or non-oral administration. For example, orally administrable preparations such as tablets, capsules, powders, granules and liquids, as well as non-orally administrable formulations such as injection solutions for subcutaneous, intramuscular or intravenous injection, transfusions and suppositories, can be produced. The above-mentioned preparations can be prepared according to any known Process are produced. That is, the trehalose diester of medium-chain, aliphatic acids can be shaped into tablets, capsules, powders or granules by optionally combining the compound with excipients such as starch, lactose and mannitol; or binders such as sodium carboxymethyl cellulose and hydroxypropyl cellulose; Disintegrants such as crystalline cellulose and calcium carboxymethyl cellulose; Lubricants such as talc and magnesium stearate; Fluidity improvers such as light, anhydrous silica. Liquids and injections can also be prepared by dissolving the trehalose diester of medium chain aliphatic acids in vegetable oils to form an oil soluble solution for injection, or by conventionally dissolving or suspending the compound in water to form a syrup. In addition, suppositories can be prepared by placing the compound in a commonly used substrate, e.g. B. cocoa butter, synthetic oils and fats, dispersed and then solidified. Even if the amount in the application of the trehalose diester medium-chain aliphatic acids according to the invention depends on the degree of the disease, it is preferred in adults to use the compound in one Dose of 0.1 to 2000 mg / kg for oral administration and 0.05 to 1000 mg / kg for non-oral administration to be used, either all at once or in portions several times a day. In the following the invention is explained in more detail by means of examples . EXAMPLE 1 20 ml of pyridine and 20 ml of dichloromethane are added to 2.11 g of 2,3,2 ', 3'-tetra-O-benzyl- α , α-trehalose and the whole is dissolved. While cooling with ice, 1.26 g of decanoyl chloride are added dropwise at 0 ° C. with stirring. After stirring for 30 minutes, the temperature is raised to room temperature and stirring is continued for an additional hour. The reaction mixture thus obtained is poured into ice water and extracted with chloroform. This is then distilled off under reduced pressure. The residue is purified by silica gel column chromatography. 2.79 g of 2,3,2 ', 3'-tetra-O-benzyl-6,6'-di-O-decanoyl- α , α- trehalose are obtained as a colorless, oily product (yield 92%). Example 2 2.67 g of 2,3,2 ', 3'-tetra-O-benzyl-6,6'-di-O-decanoyl- α , α- trehalose, obtained in Example 1, are dissolved in 40 ml of chloroform and the solution is subjected to catalytic reduction for 1 hour. To this end, 1.00 g of palladium black is added as a catalyst and hydrogen gas is passed into the solution. The reaction solution thus obtained is filtered to separate the catalyst, and the filtrates are concentrated and concentrated to dryness under reduced pressure. The residue is recrystallized from isopropanol. 1.06 g of 6,6'-di-O-decanoyl- α , α- trehalose are obtained as colorless, needle-shaped crystals (62% yield). Example 3 The compounds listed in Table 6 below are obtained in the same manner as in Example 1. Table 6 also contains the compound obtained in Example 1. Example 4 The compounds listed in Table 7 below are obtained in the same manner as in Example 2. Table 7 also contains the compounds obtained in Example 2.

Table 6

Table 7

EXAMPLE 5 Tablets A tablet is prepared in a conventional manner which contains the following ingredients, in the amounts indicated in each case: mg trehalose diester medium-chain,
aliphatic acid (decanoate) 100 D-mannitol 150 crystalline cellulose 50 starch 28 calcium carboxymethyl cellulose 16 talc 4 magnesium stearate 2 total amount 350 Example 6 capsules Granules are conventionally prepared which contain the following ingredients in the amounts indicated. The granules are filled into No. 3 capsules. mg trehalose diester medium-chain,
aliphatic acid (caprylate) 25 crystalline cellulose 17 light, anhydrous silica 7 magnesium stearate 1 Example 7 Injection solutions An injection solution is prepared by formulating the following ingredients in the stated amounts in a conventional manner. Trehalose diester medium chain,
aliphatic acid (decanoate) 25 mg made up to 1 ml with olive oil Example 8 Suppositories The following ingredients are each melted and stirred in the amounts indicated, and then shaped to solidify into a piece of suppository using a conventional procedure. mg trehalose diester medium-chain,
aliphatic acid (laurate) 100 cocoa butter 1100 total amount 1200

Claims (2)

1. α , α -Trehalose-6,6'-diester medium-chain, aliphatic acids according to the following formula (I) where n is an integer from 6 to 10. 2. A pharmaceutical agent containing an α , α- trehalose-6,6'-diester according to claim 1 as the active ingredient.