FI88044C - PROCEDURE FOR THE PREPARATION OF THERAPEUTIC THERAPEUTIC CHARACTERISTICS - Google Patents

Description

1 88044

A process for the preparation of new therapeutically useful chestnutermine glycosides

Separated from application 883164 [3

The invention relates to the preparation of novel castamospermine glycosides of the formula I and their pharmaceutically acceptable salts 1 o / \ (I) H "O <) 1 5 ✓

OR '0R

wherein R, R 'and R "independently represent hydrogen or a glycosyl, O-methylglycosyl-20 or Ac-acylated glycosyl radical containing 1 to 3 hexose or pentose units, the glycosyl radical being attached via its 1-position, and Ac is benzoyl or C2-6 alkanoyl, in which case at least one but not more than two of the symbols R, R 'and R "denote hydrogen. These compounds are useful in the treatment of diacetoxins.

Chestnut spermine is an alkaloid isolated ·: ·; Castanospermum australe seeds, and its formula is; souraava: 30 a, "H.

OH OH

2 88044 This compound can be named systematically in several ways as follows: /lS-(lft.,6p,7p,8J!>,8ap)_7-octahydro-1,6,7,8-indolizine tetrol or (IS, 6S, 7R, 8R , 8aR) -1,6,7,8-tetrahydroxy-indolizine or 1,2,4,8-tetradeoxy-1,4,8-5 nitryl-L-glycero-D-galacto-octitol. The term chestnut sperm or the first systematic name is used in the description below.

Isolation of this compound and determination of its structure have been described by L.D. Hohenshutz et al., Phytochemistry, 10, 2011 (1981). As part of his research on chestnut spermine, Hohen shutz prepared chestnut spermine tetraacetate by the reaction of chestnut spermine with a very large excess of acetic anhydride, but the article does not mention any other esters of chestnut spermine.

Examples of C 2-6 alkanoyl groups (Ac) present in the compounds of formula I described above include acetyl, propionyl, butyryl, isobutyryl and hexanoyl.

Typical examples of glycosyl radicals 20 '> \ Ml «| 1 ilki>: iyy I I, · |. t I. i k I <> m y y I I, hicophilyl, i i bnuyl 1 I, 2-deoxyglycosyl, 3-O-methylglucosyl, cellobiosyl, maltobiosyl, maltotriosyl, cellotriosyl, arabinosyl and xylosyl. Particularly preferred are compounds wherein R is 1-glucosyl, 1-L-fucosyl or 1-cellobio [2]. Illustrative examples of salts include salts of inorganic acids such as hydrogen chloride, hydrogen bromide, sulfuric acid, phosphoric acid and the like; organic carboxylic acids such as acetic, propionic, glycolic, lactic, pyruvate, malonic, rosin, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymalein and dihydroxymalein, lnionio, phenyl, 4-aminobenzo, 4-hydroxy L-benzo, anthranil, cinnamon, salicyl, 4-aminosalyl, 2- salts of phenoxybenzoic, mandelic and similar acids; and organic sulfonic acids such as methane I.

3,88044 Salts of sulfonic acid and g-toluenesulfonic acid. Such salts may be prepared by conventional methods from the amine of formula I and the appropriate acid.

Compounds of formula I may be prepared by reacting castanospermine or esterified castanospermine with an appropriate Ac-acylated or O-benzylated glycosyl halide or acetamidate in an inert solvent, followed by catalytic hydrogenation with removal of the benzoyl groups, optionally with removal of Ac or to remove groups or other ester groups. The inert solvent used in the process may be a halogenated hydrocarbon such as methylene chloride. The glycosyl halide may be chloride or bromide; the preferred acetimidate is trichloroacetimidate

When a castanospermine ester is used, this prevents the reaction of all esterified hydroxide groups, so the reaction must take place in another, free hydroxide group. Once the castanospermine ester has been incorporated into a glycosyl derivative, any ester group, either in the glycoside or castanospermine core, can be removed by base hydrolysis, for example, with a strong base such as sodium methoxide in methanol. Such as: * ·. the hydrolysis is usually carried out before the catalytic debenzylation process described above.

! The compounds of formula I are useful in the treatment of diabetes. In particular, they can be used to prevent the development of excessive hyperglycemia, as can be observed under certain diabetic conditions when ingesting a glucose precursor. Thus, when carbohydrate is ingested as glucose or in forms such as maltose, sucrose or starch in food or beverage, the level of serum glucose concentration becomes high. In healthy individuals, this hyperglycemic condition rapidly restores the normal metabolism of the body and the rapid release and / or utilization of blood glucose. However, in diabetes, the patient's glucose tolerance is reduced and the unusually high serum Gluose level that develops remains high for an extended period of time. A similar reaction observed in humans can be observed in other animals, including livestock, poultry, pets, and laboratory animals. Such a condition can be described as postprandial hyperglycemia. One way to treat such a condition would be to administer certain agents that would prevent the conversion of sugar complexes to glucose and thus prevent the development of high glucose levels. In the present invention, it has been found that when high glucose levels are the result of hydrolysis of sugar complexes, administration of the present castanospermine derivatives prevents the onset of glucose formation in the blood and thus avoids the difficulties caused by prolonged high serum glucose levels.

The mechanism by which this result is achieved is as follows, although the benefit described above should not be limited to the details of this mechanism. Enzymes that catalyze the hydrolysis of complex carbohydrates convert unabsorbed carbohydrate into absorbable sugars. The rapid action of these enzymes in diabetes results in acute and undesirable elevations in serum glucose. The compounds of formula 1 are potential inhibitors of these enzymes and, when co-administered with a carbohydrate meal, prevent this type of deleterious hyperglycemic abnormality. however, it is desirable that the inhibition of these hydrolytic enzymes be limited to enzymes in the gut, as is the case with the present compounds. and thus causes metabolic problems.The first enzyme I described above, 88044 mi, is intestinal sucrase, while the second enzyme is intracellular lysosomal glucosidase, and the activity of the compounds of formula I against these enzymes was tested by the following methods. la.

5 Intestinal sucrose

Sucrase was isolated from rat intestine as a crude homogenate according to the Kolinska salt extraction method / TBiochem. Biosphys. Acta, 284, 235 (1984) .7. The incubation mixtures contained 10 μl of enzyme preparation and an additional 10 test compounds and were incubated for 1 hour, after which 6.6 μmol of sucrose in 100 μl of 0.1 M sodium maleate, pH 5.9 was added. The mixtures were incubated for an additional 30 min and then heat inactivated at 80-100 ° C for 3 minutes. The denatured protein was removed by centrifugation. The glucose released was determined with glucoseide hydrogenase reagents (Seragen Diagnostics). Lysosomal Δί-glucosidase Lysosomal β-glucosidase was isolated and partially purified from rat liver according to the dissous fractionating ammonium sulfate method / Anal.

Biochem., 116, 35 (1981) J. The enzyme was incubated with the test compound for 1 hour at 37 ° C before the addition of p-nitrophenyl-CX-D-glucoside in 0.6 ml of 0.1 M sodium acetate, 25 mM potassium chloride, pH 4.2. The mixtures were incubated for an additional 30 min. At 37 ° C and then heat-activated at 90 ° C. The denatured protein was removed by centrifugation. To the supernatant fraction was added 1 ml of 0.1 M sodium carbonate and the released nitrophenol • was determined by absorption at 410 nm.

The results obtained are shown in Table I

6 88044

Table I

IC50 5 Castanospermine Intestinal Lysosomal Derivative Sucrase <X-glucosidase _____ (M) ____ (M) __ - 8 - 4 8 - # - D-glucopyranoside 5 x 10 3 x 10 10 From the above results, it can be seen that the IC of the compound of formula I The Q-value is lower for intestinal sucrase than for lysosomal cK-glucosidase.

The compounds of formula I were further tested in 15 sucrose stress tests to determine their ability to inhibit serum glucose elevation. The method can be presented as follows.

Swiss ICR mice fasted for 18-20 hours were orally administered test compound-20 plus 2.0 g / kg sucrose. After 30 minutes of sucrose administration, the animals were sacrificed and their serum glucose concentration was determined. The amount of test compound required to significantly inhibit serum glucose elevation was determined by comparison to the serum glucose concentration of 25 animals administered sucrose alone. During the test, mice were orally administered the substance twice. The initial dose was a test compound or solvent. After 1, 2 or 3 hours, mice were dosed with 2.0 g / kg sucrose. After an additional 30 minutes of sucrose administration, the animals were sacrificed and their glucose concentration determined.

The activity of the test compound was demonstrated by a significant difference in serum glucose concentration compared to the corresponding ____ control. The activity observed is shown in Table II.

7 88044

Table II

Castanospermine- Effective Dose Derivative of Procedure_ (mg / kg) _Duration_ 5 8-Oil-D-Glucopyranoside 1 hour

The compounds of formula I are administered to the mammal in a suitable amount in an amount effective to inhibit postprandial hyperglycemia. The preferred route of administration is oral.

The effective amount of a compound, i.e., an amount sufficient to inhibit postprandial hyperlycemia, depends on many factors, such as the size, species, and age of the subject being treated, the particular compound or pharmaceutically acceptable salt used, the frequency of administration, the severity of the condition, and the time of administration. In general, the compounds should be administered orally in doses of 0.1 to 50 mpk, with a dose of 0.5 mpk being preferred to a dose of 20 mpk. In particular, the compounds of formula I should be administered to humans in single divided doses containing from 35 to 350 mg of active substance and administered three times daily with a meal. The active ingredient is preferably included in a composition comprising a pharmaceutical carrier and 5 to 90% by weight of a compound of formula I or a pharmaceutically acceptable salt thereof. The term pharmaceutical carrier /:. refers to known pharmaceutical excipients which are useful in formulating pharmaceutically active ingredients. . 30 dense compounds for internal administration and which are substantially non-toxic and non-sensitizing under the conditions of use. The compositions may generally be prepared by known techniques for the manufacture of tablets, capsules, elixirs, syrups, emulsions, dispersions and dispersible and effervescent tablets, and may contain suitable excipients known to be useful in preparing the desired type of composition. Suitable pharmaceutical carriers and formulation techniques can be found in works in the art, such as Reminfton's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

The following examples illustrate the invention.

Example 1 To 1.7 g of / IS- (ia, 6f>, 70 *., 8β, 8ap) -7-octahydro-1,6,7,8-10 indolizinetrol-6,7-dibenzoate in 60 ml of methyl -enylene chloride solution under nitrogen and cooled to -30 ° C was added with stirring 3.8 g of 2,3,4,6-tetra-O- (phenylmethyl) - (XD-glucopyranosyltrichloroacetimidate / prepared according to the procedure of RR Schmidt and J. Michelin). According to Method 15, Angew. Chem. Int. Ed. Engl., 19, 731-32 (1980), 7. This was followed by the dropwise addition of 1.4 g of boron trifluoroether, which was kept at -10 ° C for 72 hours and then washed successively. 60 ml of aqueous sodium bicarbonate solution and 60 ml of brine The organic 20 extracts were concentrated in vacuo to give a thick syrup.TLC analysis (silica gel, 4: 6 ethyl acetate: hexane) showed the formation of less polar material with R 0.31 The mixture was fractionated by preparative high pressure chromatography: silica gel, eluent 4: 6 ethyl acetate: hexane) to give 810 mg (21%) of the protected final glucoside and 670 mg (39%) of the recovered diester starting material. Recrystallization of the final product from ____: ether / methanol gave colorless 6,7- • 30-di-O-benzoyl-8-O- (2,3,4,6-tetra-O- (phenylmethyl) -OC-D-glucopyranosyl) - / Is- (1α, 6,7,7,8,8,8) -7-octahydro-1,6,7,8-indolizine tetrol crystals melting at about 145-147 ° C. 13 C NMR (DMSO-d 6) δ 5165.0 (2 x ':' '+' ·; PhC = 0), 95.9 (Cj,). MS (CI-CH 4) 920 (MH).

9 88044

Example 2

The protected final glucoside product of Example 1 (460 mg) was added with stirring to 20 ml of methanol solution under nitrogen. After addition of five tip-5 pairs of sodium methoxide solution (4.4 M in methanol), the mixture was stirred for 18 hours at room temperature. The mixture was evaporated to dryness and the residue was dissolved in 50 ml of ethyl acetate. The solution was washed twice with brine (20 ml) and evaporated in vacuo to give a syrup. The syrupy residue was taken up in 8 ml of glacial acetic acid containing 100 mg of 10% Pd / C and hydrogenated on a Parr apparatus (3.5 atmospheres of hydrogen) for 72 hours. The catalyst was filtered and 100 mg of fresh 10% Pd / C was added. The mixture was further hydrogenated (1.0 atmospheric hydrogen pressure) at 70 ° C for six hours. After filtration to remove the catalyst, the filtrate was evaporated to dryness and the thick residue was redissolved in 15 ml of water. The aqueous solution was washed twice with 50 mL of ether, concentrated in vacuo to about 3 mL, and applied to an anion-20 exchange column (Bio-Rad AG-1-X8, 200-400 mesh, OH form, 11 cm x 2.5 cm ID). . The column was eluted with distilled water and 8 ml fractions were collected. Fractions containing unprotected glucoside were combined and lyophilized to give a white solid powder which was dissolved in methanol and powder. immediately with acetone to give colorless 8-O- (α-D-glucopyranosyl) -β- (1α, 6β, 7cL, 8β, 8α, 7-octahydro-1,6,7,8-indolizine tetrol crystals), melting at about 208-210 ° C (147 mg, 83% yield) NMR * ··: 30 (D2) 51.7 (m, 1H), 2.0-2.4 (m, 4H) 3.0-3.2 (m, 2H), 3.4 (t, 1H), 3.5-3.9 (m, 8H), 4.4 (m, 1H, CH 2), 5.4

(3, 1H, J., 3.9 Hz, e., - H, anomeric proton). MS

1 '1 + 1 + (Cl-CH 4) 352 (MH), 334 (MH -H 2 O). The structural formula of this compound is as follows: 10 88044 HO ^ HO-CH2 \ / VH r ~ "5 ...... <) OH J \

OH OH

10

Example 3

If the procedures of Examples 1 and 2 are repeated starting with castanospermine and the appropriate glycosyl trichloroacetimidate (obtained as in Example 1), the following final products are obtained: 8-O-β-D-glucopyranosyl) -Z1S- (Ια, 6β, 7α, 8β, 8aji) -7-octahydro-1,6,7,8-indolizine tetrol.

8-O- (β-D-galactopyranosyl) -A- (Id, 6 (5,7, x, 8a, 8a) -7-octahydro-1,6,7,8-indolizine tetrol.

20 8-O- (α-D-galactopyranosyl) -fis- (Ια, 6 | ΐ>, 7a, 8β, 8aβ) 7-octahydro-1,6,7,8-indolizine tetrol.

8-O- (6-deoxy-β-L-galactopyranosyl) -N 5 - (Id, 6β, 7 (X, 8β, 8aP) -7-octahydro-1,6,7,8-indolizine tetrol).

8-O- (6-deoxy- (X-L-galactopyranosyl) -? 1S- (10-, 25 6β, 70c, 8β, 8aP> L7-octahydro-1,6,7,8-indolizine tetrol).

8-O- (1-D-ribofuranosyl) - [1S- (let, 6β, 7α, 8 | 2>, 8ap>) .7-Octahydro-1,6,7,8-indolizine tetrol.

8-O-Zcx-D-ribofuranosyl) -β 1S- (Ια, 6β>, 7d, 8β>, 8aiji) -7-octahydro-1,6,7,8-indolizine.

’. 30-O-O6-D-glucopyranosyl- (1-M) -O- [? -D-glucopyranosyl] -7S (1a, 6β, 7β, 8β, 8α) 7-octahydro-1,6 , 7,8-indolyticinetretrol.

8-O- (β-D-glucopyranosyl- (1-M) -O-β-D-glucopyranosyl-7β- (10c, 6β, 7 <χ, 8β, 8af?>) 7-octahydro -1,6,7,8-indo- · .- 35 lysine tetrol.

I: ,, 88044 8-O- [β-D-glucopyranosyl- (1-> 4) -β-α-D-glucopyranosyl] -AS (1α, 6, 7 <X, 8β>, 8aj3> ) 7-Octahydro-1,6,7,8-indolizine tetrol.

8-O- [β-D-glucopyranosyl- (1-> 4) -O- [α] -D-glucopyrra-?-Nosyl] -A- (1α, 6β, 8β, 8β, 8α |?> ) -7-octahydro-1,6,7,8-indolizine tetrol.

Example 4

To a solution of 5.2 g / "IS- (1 (X, 6E, 7ct, 8β, 8nJ3) -7-octahydro-1,6,7,8-indolizine tetrol-1,6,8-tri-10 benzoate In 150 ml of methylene chloride under nitrogen and cooled to -20 ° C, 8.5 g of 2,3,4,6-tetra-O- (phenylmethyl) -O-D-glucopyranoyltrichloroacetimidate were added with stirring. Add dropwise 0 ml of boron trifluoroether and keep the mixture at -10 ° C for 96 hours After warming to room temperature, the mixture was washed successively with 200 ml of aqueous sodium bicarbonate solution and 200 ml of brine The organic phase was dried over magnesium sulfate and evaporated in vacuo to dryness. layer chromatography (silica gel, 1: 3 ethyl acetate: hexane) showed less polar material to form, = 0.44 The mixture was fractionated by preparative medium pressure liquid chromatography (silica gel, 1: 3 ethyl acetate: hexane as eluent) to give 5.7 g. g (54%) 1, 6,8-tri-O-benzoyl-7-O-; ** (2,3,4,6-tetra-O- (phenylmethyl) -Ot-D-glucopyranosyl) -; · * As- (10C, 6β>, 70t, 8?>, 8aji) -7-octahydro-1,6,7,8-indolizine-.:. ,. 13 ...: tetrol as a thick colorless syrup. C NMR (CDCl 3) λ max δ $ 166.0, 165.9, 165.5 (3 x PhC = O), 99.4 (δ, J 13 C-1H = λ 0 168 11). MS (CI-CH 4) 1024 (MH +), 902 (MH + - PhCO 2 H).

Example 5 '· To a suspension containing 5.6 g of 1,6,8-tri-O-benzoyl-7-O- (2,3-4,6-tetra-O- (phenylmethyl) -Ot-D-glucopyranosyl ) - As- (ία, 6β, 7a, 8β, 8a) 7-octahydro-12 88044 1,6,7,8-indolizine tetrol in 100 ml of methanol, 30 drops of sodium ethoxide solution (4.4 M in methanol) were added, and the mixture was stirred at room temperature for 18 hours. To the mixture were added 3 g of potassium hydroxide and 5 ml of water, and the resulting solution was heated under reflux for three days. The mixture was cooled and evaporated to dryness in vacuo. The residue was dissolved in a mixture of 50 ml of ethyl acetate and 10 ml of water. After separation of the phases, the aqueous layer was extracted twice with 30 ml of ethyl acetate. The organic extracts were combined and evaporated to a yellowish, sticky residue. Recrystallization of the final product from ether gave 3.42 g (88%) of colorless, downy 7-O- (2,3,4,6-tetra-O- (phenylmethyl) -α.-D-glucopyranosyl-15-yl) -E (lot, 6) 3,1 (X, 8f3>, 8aE) -7-octahydro-1,6,7,8-indolizine nitetrol crystals melting at about 119-120 ° C. 1 H NMR (CDCl 3) δ δ - 2.5 (m, 5H), 2.8 - 4.0 (m, 12H), 4.1 - 5.0 (m, 12H), 7.0 - 7 .4 (m, 20H, aryl).

Example 6 The final product from Example 16 (250 mg) was dissolved in 4 ml of glacial acetic acid and 50 mg of 10% Pd / C was added. The mixture was hydrogenated (1.0 atmospheric hydrogen pressure) at 55 ° C for four hours. After removal of the carbon catalyst, the acetic acid solution was evaporated to dryness in vacuo. The resulting residue was redissolved in 10 ml (1: 1) of methanol-water solution and stirred for one hour with 2.0 g of anion exchange resin ZBio-Rad AG1-X8 (200-400; _ · mesh, OH - form) _7. The aqueous solution was evaporated to dryness to give a white solid powder which was dissolved in methanol and triturated with acetone to give 92 mg of colorless 7-O- (α-D-glucopyranosyl) -Z1S- '·' '(Ια, 6β, 7tt, 8β, 8aj5) J-octahydro-1,6,7,8-indolecinetetrol crystals, melting with decomposition at about 184-187 ° C. MS (CI-CH 4) 352 (MH +), 334 (MH + -H 2 O).

Claims (2)

A process for the preparation of new therapeutically useful castanospermine glycosides of the formula I 3 and their pharmaceutically acceptable salts "wherein R, R" and R "'independently of one another mean hydrogen or a glycosyl, O-methylglycosyl or Ac-acylated glycosyl radical containing 1 to 3 hexose or pentose units, the glycosyl-20 radical being attached via its 1-position, and Ac is benzoyl or a C 2-6 alkanoyl, at least one, but not more than two of the symbols R, R 'and R' 'denote hydrogen, characterized in that the castanospermine and the esterified castanospermine are reacted with a suitable .25 van Ae acylated or O-benzylated glycosyl halide or acetamidate in an inert solvent, followed by catalytic hydrogenation is carried out to remove benzyl groups or, if appropriate, treatment with a base to remove Ac groups or other Esters; to remove groups 30. A process according to claim 1 for 8-O- (α-D-glucopyranosyl) -β- (1β, 6C, 7β, 8B, 8aB) .7-octahydro-1,6,7,8- for the preparation of indolizine tetrol, characterized in that / Ts, (M, 6 Q, 7dw 8β, 8afi) γ-octa-35 hydro-1,6,7,8-indolizine tetrol-6,7-dibenzoate 14 88044 is reacted with 2.3 With 4,6-tetra-O- (phenylmethyl) -cK-D-ylucopyranosyl trichloroacetimidate, followed by treatment with a base and then hydrogenation using palladium on carbon. i is 88044