CA1202622A - .alpha..alpha.-TREHALOSE-6,6'-MEDIUM CHAINED LENGHT ALIPHATIC ACID DIESTER AND A PHARMACEUTICAL AGENT CONTAINING THE SAME - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Disclosed herein is a novel .alpha.,.alpha.-trehalose-6,6'-medium chain length aliphatic acid diester represented by the following formula (I):

(I) in which n represents an integer from 6 to 10.
This compound is produced, for instance, by converting .alpha.,.alpha.-trehalose into 2,3,2',3'-tetra-o-benzyl-.alpha.,.alpha.-trehalose, and then reacting it with a medium chain length aliphatic acid or an active derivative thereof to form a novel intermediate 2,3,2',3'-tetra-o-benzyl-.alpha.,.alpha.-trehalose-6,6'- medium chain length aliphatic acid diester and further reductively de-benzylating it using a reduction catalyst. The compound according to the invention has an excellent antitumor activity, antibiotic activity and action for delayed-type hypersensitivity, and is useful for pharmaceuticals. Pharmaceutical agents containing an .alpha.,.alpha.-trehalose-6,6'-medium chain length aliphatic acid diester of the formula (I) as an effective ingredient are also disclosed.

Description

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BACKGROUND OF THE INVENTION
(i) Field of the Invention:
This invention relates to a novel a,~-trehalose-6,6'-medium chain len~th aliphatic acid diester and a ~h~ eu-tical agent containing the same as an effective ingredient.

(ii) Description of the Prior Art:
a,a-trehalose is a compound belonging to a so-called disaccharide composed of two glucose molecules and it is broadly distributed in nature. Various derivatives of this compound have been synthesized and pharmacological activities thereof have been examined. For instance, it has been reported that a mixture of various diesters in which the aliphatic acids are bonded at different positions shows an antitumor activity.

SUMMARY OF THE INVENTION
In the course of synthesizing various 6,6'-alipha-tic acid diesters of a,a-trehalose and studying their pharma-cological effects, the present inventors have found that an a,a-trehalose-6,6~ -medium chain length aliphatic acid diester represented by the following formula (I):
CH3(CH2)nC ~ O OH
~ ~ OH~ OOC(C~2) C~3 (I) HO
OH

~ 2 ~
r", 12Q;~

in which n represents an integer from 6 to 10 has an excellent antitumor activity, antibiotic activity and action for delayed-type hypersensitivity, and is useful for pharmaceuticals, and thus accomplished the present inventon.
Accordingly, it is an object of this invention to provide a novel ~,a-trehalose-6,6'-medium chain length aliphatic acid diester represented by the above formula (I).
Another object of this invention is to provide a pharmaceutical agent containing as an effective ingredient an a,a-trehalose~6,6-medium chain length aliphatic acid diester represented by the above formula (I).
DETAILED DESCRIPTION OF THE INVENTION
AND PREFERRED EMBODIMENTS
The a,~-trehalose-6-6'-medium chain length aliphatic acid diester (I) (hereinafter simply referred to as a trehalose medium chain length aliphatic acid diester) according to this invention can be produced, for instance, in accordance with the following reaction scheme, by converting a,a-trehalose (II) into 2,3,2',3'-tetra-o-benzyl-a,a-trehalose (III?, and then reacting it with a medium chain length aliphatic acid of formula (IV) or an active derivative thereof to form a novel intermediate

2,3,2',3'-tetra-o-benzyl-a,a-trehalose-6-6'-medium chain length aliphatic acid diester (v), and further r~
I

reductively de-benzylating it using a reduction catalyst: .
-- 3a -." ~1 .~
~j , :~2~ 2 ~Ol OH ~o- O~n OH , ~ ~ OH

OH ~ OBn (II) (III) C~3t~X2)nC~ OBn CH~(C~2)nCOOH ~ ~ 2 n 3 OBn (V) C~ (C~ ) C--L 0~
reduction catalyst ~ HO ~ ~OC(C~2)nC~3 OFI
(I) in which Bn represents a benzyl group and n has the same ?~n; ng a~ stated above.
In the above process, the 2,3,2',3'-tetra-o-benzyl~ trehalose represented by the formula (III) is prepared by reacting the trehalose represented by the formula (II) with benzaldehyde in a conventional manner to form 4,6,4',6'-di-o-benzylidene-a,~-trehalose and then reactlng it with benzylchloride to form a 2,3,2',3'-tetra-o-benzyl derivative and further reacting the same with an acid.
The reaction between the compound (III) and the lZ~26;2Z

medium chain length aliphatic acid (IV) or its active derivatives is carried out under the usual conditions of esterifying reaction by reacting the medium chain length aliphatic acid of formula (IV) or an active derivative thereof in a molar ratio, preferably of 1.8 - 2.4 moles per one mole of the compound (III) under the presence of an acid remover such as pyridine and triethylamine.
The reaction is preferably conducted under the presence or absence of a solvent at a temperature between 0C (under ice cooling) to 30C for 1 - 24 hours.
The solvent used herein includes, for example, methylene chloride, chloroform, benzene, toluene, ether, tetrahydrofuran and dioxane.
The reducing de-benzylating reaction for the thus obtained ~ ~ow-d (V) is carried out U,~uy1-the use of a catalyst, preferably, in 0.5 - 10 mol per one mol of the compound (V) in a suitable solvent.
The reduction catalyst includes, for example, palladium black, palladium-carbon and Raney nickel. The solvent used herein includes, for example, alcohols such as methanol, ethanol and isopropanol; halogenated compounds such as chloroform and methylene chloride, as well as a mixture of these solvents.
Ph~ ol~;c~l activities of the trPh~lose medium chain length aliphatic acid diester (I) according to L~

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this invention are shown below.
~1) Cell-killing activity of trehalose medium chain length aliphatic acid diester:
(a) Cells of Leukemia L-5178Y were used and conditioned to be 10 cells/ml in RPMI 1640 medium containing 10~ ox fetal serum, and specimens were applied into the medium to a predetermined concentration. After 48 hours' culture in an incubator at 37C under 5% CO2, the number of living cells were counted under a microscope and compared with a control group to determine the multiplication factors.
They were plotted on a logarithmic probability paper and values for IC50 were determined based on the curve thus obtained. The concentration for each specimen was 25, S0, 100, 200 and 400 ~g/ml respectively.
The results are shown in Table 1.

Table 1 Test compound (indicated by IC50 the number of (~g/ml) n in formula (I)) Compounds of 8 255 the Invention Comparative 14 >400 Compounds 20 >400 , .,~ ,~

(b) Cell-killing activities of the trehalose medium chain length aliphatic acid diester against the cells of Leukemia L-1210 were examined in the same manner as in (a). The results are as shown in Table 2.

Table 2 Test compound IC
(indicated by 50 the number of (~g/ml) n in formula (I)) Compounds of 8 150 the Invention -Comparative 14 >1000 Compounds 20 >1000 (2) Anticancer activities of trehalose medium chain length aliphatic acid diester: ~
Std-ddy male mice of 5-week age were used as six mice per group and cancer cells of sarcoma 180 were transplanted by 5 x 106 cells into the peritoneal cavity of each mouse. From after 24 hours, a test compound suspended in 30% propylene glycol was intraperitoneally administered in an amount of 150 mg/kg once per day con-tinuously for 5 days. 7 days after the transplantation of the cancer cells, abdominal ascites were sampled to determine the amount o'f abdominal ascites and th~ cell ratio. The tumor growth ratio (T/C ~%)) was determined . .
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by using TPCV (total pack cell volume) which is the prod-uct of the amount of abdominal ascites and the cell ratio from the formula specified below, and the anticancer activity of the test compound was judged. The results are shown in Table 3.

T/C(%) TPCV for treated group 100 TPCV for control group x Table 3 Test compound Tumor growth (indicated by rat the number of T/C (%) n in formula (I)) 6 9.1 Compounds of 8 1.9 the Invention 6.2 Comparative14 37.7 Compounds 20 55.8 Control group 100 As described above, the trehalose medium chain length aliphatic acid diester has an anticancer activity which is remarkably stronger than that of the correspond-ing compounds having 16 - 22 carbon atoms.

(3) Antimicrobial Activity:
The m; n;m~l inhibitory concentrations (MICl of the compounds of the invention were ex~m;~ed against various bacteria. The results are shown in Table 4.

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The tests were conducted according to the MIC measure-ment meth~d authorized by Japan Society of Chemotherapy and, for the culture medium, the Mueller-Hinton medium (Difco Laboratories, U.S.A.) was employed. The test . .
compounds were dissolved in 25~ DMSO to have the concent-ration of 1 mg/ml, thereafter diluted to the predesignat-ed concentration with sterile distilled water.

Results:
Table 4 MIC value (ug/ml) Test Compound;
present comparative indicated by the number of invention product n in formula (I) --~ 6 8 14 20 Bacterial Stralns - _ Bacillus subtilis ATCC 6633 50 12.5 >100 >100 Staphylococcus aureus ATCC 50 12.5 >100 >100 Staphylococcus epidermides 100 >100 >100 >100 Streptococcus faecalis var. 50 >100 >100 >100 gimogenes IFO 3939 Sarcina lutea ATCC 9341100 >100 >100 >100

(4) Action on Delayed-type Hypersensitivity:

Several groups of ICR 6 weeks old female mice, each group consisting o 10 mice, were provided and sub-. . ~

:~Z~26~2 cutaneously injected with sheep red blood cells (SRBC) of 3 x 109 cells/ml by 0.05 ml/mouse at the footpads of xight hind paw, after which, test compounds were immedi-ately injected intraperitoneally* (0 day). Four days thereafter, the same amount of SRBC (0.05 ml/mouse) was subcutaneously injected to each mouse at the footpad of the left hind paw and, after a further 24 hours, the thick-ness of the left foo~tpad was measured with slide calipers.
Other groups which were administered with physiological saline solution in place of test compounds ~ . ., were used as controls and, further other groups which were subjected to the 2nd injection alone were used as bases.
The swelling ratio of footpad was obtained from the following equation:

T - B
Swelling ratio (%) = x 100 C - B
in which T, C and B represent the thickness of the foot-pad of left hind paw of the test groups, control groups, and the base groups respectively.
The results are shown in Table 5.

Resul-ts:
Table 5 Test Compound; Thickness of footpad Swelling ratio indicated by the number , ,%~
of n in formula (I) ~mm) 6 3.71 - 126 Compounds of 8 the Invention 3 7 133 3.82 138 Comparative 14 3.61 109 Compounds 20 3.55 98 Control 3.56 100 * Dose: 100 mg/kg, Vehicle: normal saline + 1 drop of 'Tween 80' (trademark for polyoxyethylene (20) sorbitan monooleate - a nonionic surfactant).

The tr~h~lose medium chain length aliphatic acid diester according to this invention is an almost non-toxic and thus safe compound as apparent from the facts, for example, that sucrose aliphatic acid esters similar to the present compound are allowed as food additives and broad~ly used as non-deleterious surfactants in the field of foods, medicines, cosmetics or the like .
Further, the compound of this invention can be formed into various types of preparations depending on the manner of application such as oral or non-oral admin-istration, for example, oral preparations such as tablets, capsules, powder, granules and liquids, as well as non ~' ~ . ...

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-oral foxmulations such as injection solution for subcutaneous, intramuscular or intravenous injection, transfusion and suppositories and the like.
The above-mentioned preparations can be made according to any methods known per se. That is, the tr~h~lose m ~ um chaLn length aliphatic acid diester can be formed into tablets, capsules, powder or granules by .formulating the compound optionally in combination with excipients such as starch, lactose and mannitol; binders such as sodium carboxymethyl cellulose and hydroxypropyl cellulose; disintegrators such as crystalline cellulose and calcium carboxymethyl cellulose; lubricants such as talc and magnesium stearate; fluidity improvers such as light anhydrous silicic acid and the like. Further, liquids and injections can be prepared by dissolving the tr~hAlose medium chain length ~l;ph~t;c acid diester into vegetable oils or the like to form an oil-soluble injec-tion solution, or by dissolving or suspending the compound by a conventional manner into water or the like to form a syrup. Furthermore, suppositories can be prepared by dispersing the compound in ordinarily employed substrates, for example, cacao butter, synthetic oils and fats or the like and then solidifying them.
Although the amount for the application of the 3~ZS~;26%;~:

treh~l~se medium chain length aliphatic acid diester accord-ing to this invention varies depending on the degree o~
the disease, it is preferably administered for an adult in a dosage of 0.1 - 2000 mg/kg for oral administration and 0.05 - 1000 mg/kg for non-oral administration, all at once or portionwise for several times per one day.
This invention is further described by way of the following examples.

Example 1 To 2.11g of 2,3,2',3'-tetra-o-benzyl-~,-treha-lose were added pyridine 20 ml and dichloromethane 20 ml and dissolved, to which 1.26g of decanoyl chloride were added dropwise under stirring while ice cooled at 0C.
After 30 mins' stirring, the temperature was raised to room temperature and stirring was continued for further one hour. The reaction mixture thus obtained was.charged into ice-water and extracted with chloroform, which was then distilled off under reduced pressure. The residue was purified by way of silica gel column chromatography to obtain 2.79 g of 2,3,2',3'-tetra-o-benzyl-6,6'-di-o-decanoyl-~,~-trehalose as a colorless oily product (yield: 92~).

~ - 13 -6~%

Example 2 2O67g of 2,3,2',3'-tetra-o-benzyl-6,6' di-o--decanoyl~ trehalose obtained in Example 1 was dissolved in 40 ml of chloroform and the solution was subjected to catalytic reduction for one hour by adding l.OOg of palladium black as a catalyst and introducing hydrogen gas into the solution.
The reaction solution thus obtained was filtered to eliminate the catalyst and the filtrates were concen-trated and dried to solid under reduced pressure. The residue was recrystallized from isopropanol to obtain 1.06g of 6,6'-di-o-decanoyl-~,~-trehalose as colorless needle crystals (yield:62%).

Example 3 The compounds shown in Table 6 below were obtained in the same manner as in Example 1. The Table 6 also contains the compound obtained in Example 1.

Table 6 RCO3 ~ OBn OBn o~OOCR Bn ; Benzyl Rmp(C) [a]D (chloro ) IRmax cm NMR(60MHz, CDC13~ ~ppm form CH (CH ~ Oily + 82 3 1735 0.86 (6H), 1.26 (2QH), 2.25 3 2 6 Compound m - (4H, J=7Hz), 3.2-4.5 (12H), 5.15 (2H, J=3.5Hz), 4.65S(4Hj, 4.86 (4H), 7.0-7.5 (20H) CH3(CH2)8 Compound 1740 0.88 (6H), 1.25 (28H3, 2.30 (4H, J=7Hz), 3.2-4.5m(12H), 5.20d (2H, J=3.5Hz), 4.71s(4H), 4.92 (4H), 7.1-7.6 (20H) CH3(CH2)10 Compound 1740 0.88 (6H), 1.21 (36H), 2.23 d (4H, J=7Hz), 3.2-4.4 (12H), 5.11 (2H, J=305Hz), 4.62 ~4H), 4.81 (4H), 6~9-7O3 ~20H) z Example 4 The compounds shown in Table 7 were obtained in the same manner as in Example 2. The Table 7 also contains the compounds obtained in Example 2.

:

Table 7 RCOO OH
~ ~ ~ OOCR

OH OH
OH

R mp(C) [a]D (chloro ) IRVmar cm 1 NMR(60MHz, CDC13) ~ppm form CH (CH )170 - + 116.6 1735 0.90 (6H), 1.12 (20H), 2.28 6 1 3 2 6 171 (4H, J=7Hz), 3.8-5.1 (12H),

5.72d(2H, J=3.5Hz), 6.12s(6H) CH (CH )157 - - + 108.8 1740 0.88 (6H), 1.18 t28H), 2.32 d 3 2 8 163 (4H, J=7Hz), 3.8-5.2 (12H~, 5.78 (2H, J=3.5Hzj, 6.08s(6H) CH (CH ) 0 + 88.4 1730 0.88t(6H), 1.22 (36H), 2.30 d 3 2 1 164 (4H, J=7Hz), 3.8-5.1 (12H), 5.70 (2H, J=3.5Hz), 5.96s(6H) z~

Example 5: Tablet One tablet having the following ingredients each in the indicated amount was prepared in a conven-tional manner.
Tr~hAlose medium chain length aliphatic 100 mg acid diester (decanoate) D-mannitol 150 mg Crystalline cellulose 50 mg Starch 28 mg Calcium carboxymethyl cellulose 16 mg Talc 4 mg Magnesium stearate 2 mg Total amount 350 mg Example 6: Capsule Granules having the following ingredients each in the indicated amount were prepared in a conventional manner and they were filled in a number 3 capsule.
Tr~h~lo~e medium chain length aliphatic 25 mg acid diester (caprylate) Crystalline cellulose 17 mg Light anhydrous silicic acid7 mg Magnesium stearate 1 mg Example 7: Injection solution One pack of an injection solution was prepared ,~ ! -- 18 --

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from the following ingredients each in the indicated amount in a conventional Ann~r, Trehalose medium chain length Al;~hAt;~ 25 mg acid diester (decanoateJ
To make with olive oil into 1 ml Example 8: Suppository The following ingredients each in the indicated amount were melted and stirred and then molded to solid-ify into one piece of suppository in a conventional manner.

Tr~hAl~se medium chain length ~1-P~A~;~ 100 mg acid diester (laurate) Cacao butter 1100 mg Total amount 1200 mg

Claims (11)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing an .alpha.,.alpha.-trehalose--6,6'- medium chain length aliphatic acid diester represented by the following formula (I):

(I) in which n represents an integer from 6 to 10, which comprises the steps of (1) converting .alpha.,.alpha.-trehalose of formula (II) (II) into 2, 3, 2', 3'-tetra-o-benzyl-.alpha.,.alpha.-trehalose of formula (III):

(III) where Bn represents a benzyl group, then (2) reacting said 2, 3, 2', 3'-tetra-o-benzyl -.alpha.,.alpha.- trehalose with a medium chain length aliphatic acid of formula (IV) CH3(CH2)nCOOH
in which n represents an integer from 6 to 10, or an active derivative thereof, to form 2, 3, 2', 3'-tetra-o-benzyl-.alpha.,.alpha.-trehalose-middle chained aliphatic acid diester of formula (V) (V) and (3) then reductively de-benzylating said diester of formula (V) using a reduction catalyst to form the desired compound of formula (I).

2. A process according to claim 1 wherein the 2, 3, 2', 3' -tetra-o-benzyl -.alpha.,.alpha.-trehalose of formula (III) is prepared by reacting trehalose with benzaldehyde to form 4,6,4',6'-di-o-benzylidene--.alpha.,.alpha.-trehalose and then reacting it with benzyl chloride to form a 2, 3, 2', 3'-tetra-o-benzyl derivative, and further reacting the same with an acid.

3. A process according to claim 1 wherein the reaction between the 2, 3, 2', 3'-tetra-o-benzyl -.alpha.,.alpha.-trehalose of formula (III) and the medium chain length aliphatic acid of formula (IV) or active derivative thereof is effected using 1.8 to 2.4 moles of said acid per mole of the above-named compound of formula (III), in the presence of an acid remover.

4. An .alpha.,.alpha.-trehalose-6,6'- medium chain length aliphatic acid diester represented by the following formula (I):

(I) in which n represents an integer from 6 to 10, when prepared by the process of any of claims 1-3, or by an obvious chemical equivalent thereof.

5. A process in accordance with claim 1 wherein the reduction catalyst is selected from the group consisting of palladium black, palladium-carbon and Raney nickel.

6. A process in accordance with claim 1, 2 or 3 wherein the medium chain length aliphatic acid of formula (IV) is decanoic acid.

7. A process in accordance with claim 1, 2 or 3 wherein the active derivative of said medium chain length aliphatic acid is an acid chloride.

8. A process in accordance with claim 1, wherein step (2) is effected by reacting said 2, 3, 2', 3' -tetra-o-benzyl-.alpha.,.alpha.-trehalose with decanoyl chloride, thereby to obtain 2, 3, 2', 3' -tetra-o-benzyl-6,6'-di-o-decanoyl-.alpha.,.alpha.-trehalose, which is subsequently catalytically reduced employing palladium black as a catalyst.

9. A process in accordance with claim 2 wherein step (2) is effected by reacting said 2, 3, 2', 3' -tetra-o-benzyl-.alpha.,.alpha.-trehalose with decanoyl chloride, thereby to obtain 2, 3, 2', 3' -tetra-o-benzyl-6,6'-di-o-decanoyl-.alpha.,.alpha.-trehalose, which is subsequently catalytically reduced employing palladium black as a catalyst.

10. A process in accordance with claim 3 wherein step (2) is effected by reacting said 2, 3, 2', 3'-tetra-o-benzyl-.alpha.,.alpha.-trehalose with decanoyl chloride, thereby to obtain 2, 3, 2', 3' -tetra-o-benzyl-6,6'-di-o-decanoyl-.alpha.,.alpha.-trehalose, which is subsequently catalytically reduced employing palladium black as a catalyst.

11. 6,6'-di-o-decanoyl-.alpha.,.alpha.-trehalose, whenever prepared by the process of claim 8, 9 or 10 or by an obvious chemical equivalent thereof.