KR890004136B1 - Sialo cyclecholesterol and the preparation process thereof and medicine for treating diseases of nervous system - Google Patents

Abstract

A prepn. of a sialocyl cholesterol of formula (I) comprises reacting a cpd. of formula (II) with cholesterol in the presence of Koenigs- Konrr catalyst to produce a cpd. of formula (III), and then hydrolyzing the cpd. (III). In the formulas, Ac is acetyl; R1 and R2 are each -COOCH3 or (a); R3 and R4 are each -COONa or (a). The catalyst is HgBr2, Hg(CN)2, AgClO4 or trifluoromethane sulfonic acid. (I) has a neural spine growth action and differenciation induction capability. (I) is useful in the treatment of a nervous system disease.

Description

Sialoxyl cholesterol, a method for producing the same, and a therapeutic agent for neurological diseases consisting of sialosil cholesterol

The present invention relates to sialyl cholesterol, and more particularly, to sialyl cholesterol useful as a drug for treating various diseases caused by disorders of peripheral and central nerves.

Previously, sialic acid has been known to exist on the cell surface of animal origin or a large number of bacteria as sialo complexes, for example, glycoproteins, glycolipids, oligosaccharides or polysaccharides.

Recently, this compound has been of medical and pharmaceutical importance, including neuronal function, cancer, inflammation, immunity, viral infections, differentiation hormone receptors.

This compound has also been attracting attention as a unique active molecular sieve present on the cell surface.

However, the role of sialic acid in the sialo complex is not yet clear.

Moreover, this compound has been studied by many natural product organic chemists, and many kinds of derivatives have already been obtained.

However, derivatives having significant physiological activity have not yet been obtained.

In addition, the average life expectancy of human beings has been prolonged by various improvements in medical treatments such as hematopoietic organ tumors, various cancers and collagen diseases.

However, due to this improvement, the use of drugs such as, for example, corticosteroids, immunosuppressants, and the like has inevitably increased.

Thus, undesirable side effects such as a decrease in immunity occurred.

The present inventors paid attention to sialic acid which is an intrinsic component of a living body, and chemically transformed this sialic acid, thereby devoted to the study of an immunomodulator having virtually no side effects and having an adjustment function of an immune monitoring apparatus.

As a result, the present inventors have prepared sialosil cholesterol useful as a therapeutic agent for neurological diseases.

This is due to the neuralites of neuroblastoma cells (Neuro 2a) induced from mice when current sialosil cholesterol was added to the culture of neuro 2a and tested for its activity. ) Is based on the knowledge that promotes growth.

It is an object of the present invention to provide new sialosil cholesterol.

Another object of the present invention is to provide a new method for producing sialosil cholesterol.

Another object of the present invention is to provide a novel neurological disease treatment agent.

Incidentally, since the sialosil cholesterol of the present invention is present in the form of Na salt, the cholesterol is excellent in water solubility, thereby further expanding the use of the cholesterol.

The present invention relates to a compound having the following general formula (4) or (5).

General formula (4):

General formula (5):

The present invention further provides compound (1) having the general formula (1)

Reacted with cholesterol to produce a compound having formula (2) or formula (3),

It relates to a method for producing compound (4) or (5) by hydrolyzing compound (2) or (3).

Moreover, this invention relates to the neurological disease therapeutic agent containing compound (4) or (5).

The present invention will be described in detail as follows.

First, the method of preparing compound (4) or (5) is summarized in the following table.

Compound (1) to be used in the present invention can be obtained commercially easily as a known compound.

In this reaction, compound (1) is reacted with cholesterol for about 1 to 7 days at atmospheric pressure of about 20 to 25 ° C. in the presence of a Königshnor catalyst.

At this time, cholesterol is made to react with the molar ratio of about 1-5 mol of cholesterol with respect to 1 mol of compound (1).

Examples of the catalyst include emulsifying agent mercury, second mercury cyanide, silver chlorate, silver trifluoromethanesulfonic acid, silver trifluoroacetate and the like.

The amount of the catalyst is about 1.0 to 1.2 equivalents based on 1 equivalent of Compound (1).

As the solvent used in the present invention, acetonitrile, nitrobenzene, acetone, benzene, tetrahydrofuran, dichloromethane and the like are used.

Of these, benzene, dichlorochloromethane and tetra hydrofuran are preferred solvents.

In the present invention, a desiccant may be used.

As a desiccant, dry light or molecular sieve 4A can be used.

The obtained compounds (2) and (3) are separated and purified by conventional methods such as column chromatography.

These compounds are then hydrolyzed to replace their methoxycarbonyl groups with sodium carboxyl groups and their acetyl groups to hydrogen.

By this hydrolysis, compounds (4) and (5) are produced.

This hydrolysis is generally performed by a conventional method.

For example, this hydrolysis is carried out by treating the compounds in an alkaline solution having a concentration of about 1 to 3 N at about 15 to 15 ° C. for about 5 to 15 hours.

This compound can be eaten.

However, the compound is preferably administered by eye administration, inhalation, intramuscular injection, subcutaneous injection or intravenous injection.

The dosage will vary depending on the severity of the disease and the weight of the patient.

However, the amount is preferably 0.001 to 10 mg.

EXAMPLE

Next, this invention is demonstrated concretely according to the Example.

Example 1

Methyl 5-acetoamide-4, 7, 8, 9-tetra-0-acetyl-2-0- (5-cholsten-3-β-yl) -3.5-dideoxy-α-D-glycero- Synthesis of D-galacto-2-nonnupylopranosonate Dissolve 0.77 g (2 mmol) of sufficiently dried cholesterol in 10 ml of a solvent such as dried methylene chloride. After adding 0.5 g of molecular sieve 4A previously dried under high temperature vacuum to the said cholesterol solution, this mixture was stirred for 30 minutes-1 hour at room temperature in argon stream.

To this mixture, 1.02 g (2 mmol) of compound (1) was added, followed by 2 to 2.4 mmol of trifluoromethanesulfonic acid.

Subsequently, this mixture was reacted under stirring light at room temperature overnight.

The reaction solution was filtered through celite. The silver salt was removed with saturated brine. The mixture was dried over anhydrous sodium sulfate and the like.

The solvent was then streamed in vacuo to give a white solid. This white solid was put in colum chromatography (silica gel) to separate the compounds (2) and (3) and purified them.

0.56 g (33%) of compound (2) and 0.55 g (32%) of compound (3) were obtained.

Physical Properties of Compound (2): Mass (EI) m / z 860 (m + 1), 800 (M-59)

Elemental Analysis (%) C ₄₇ H ₇₃ O ₁₃ N

Calculated value: C; 65. 63, H; 8. 55, N; 1.63

Found: C; 65. 41, H; 8. 61, N; 1. 60

m. p. 113-115 ℃

¹ H NMR CDC1 ₃ δ (TMS) 400 MHz

0.669 3H, s, CH ₃ -18

0.985 3H, s, CH _3- 19

1.88. 3H, s, NAc

2.026, 2.031, 2.126, 2.145, 3Hx4, sx4, OAcx4

2.596 1H, dd, J = 5.2, 12.8 Hz, 2 'Heq

3.650 1H, m, H-3

3,790 3H, s, COOMe

4.02-4.09 2H, m, H-4 ', H-5'

4.166 1H, doublet of doublets, J = 5.8. 12.5 Hz, Ha-8 '

4.347 1H, dd, J = 2.5, 12.8 Hz, HB-8 '

4.854 1H, ddd, J = 5.2, 9.8, 12.OHz, H-3 '

5.205 1H, d, J = 10.1 Hz, NH

5.33-5.37 2H, m, H-6 ', 7'

Physical Properties of Compound (3):

Mass (EI) m / z 860 (M + 1), 800 (M-59)

Elemental Analysis (%) C ₄₇ H ₇₃ O ₁₃ N

Calculated value: C; 65.63, H; 8. 55, N; 1. 63

Found: C; 65. 89, H; 8. 58, N; 1.66

m. p 138-140 ℃

¹ H NMR CDC1 ₃ δ (TMS) 400 MHz

0.970 3H, s, CH ₃ -18

0.999 3H, s, CH ₃ -19

1.871 3H, s, NAc

2.021, 2.021, 2.077, 2.130, 3Hx4, sx3, OAcx4

2.525 1H, dd, J = 4.9, 13. 1 Hz, Heq-2 '

3.572 1H, m, H-3

3.798 3H, s, COOCH ₃

4.04-4.13 2H, m, H-4 ', 5'

4.146 1H, dd, J = 7.6, 12.5 Hz, Ha-8 '

4.888 1H, dd, J = 1.8, 12.5Hz, HB-8 '

5.07 1H, tt, J = 2.0, 8.2Hz, H-7 '

5.22-5.27 1H, m, H-3 '

5.34-5.38 2H, m, NH, H-6 '

Example 2

Synthesis of 5-acetoamide-2-0- (5-cholestan-3-β-yl) -3.5-dideoxy-α-D-glycero-D-galacto-2-nonnulpyranosanoic acid.

Compound (2) obtained in Example 1 was dissolved in 2 ml of methanol.

To this solution, 3 ml of 1N aqueous sodium hydroxide solution was added, followed by stirring at room temperature overnight.

After 2 ml of water was added to the solution, the solution was neutralized with Doexx 50 (H ⁺ ), a small amount of precipitate was filtered off, and the filtrate was dried under vacuum to give compound (4) (white powder). 31.4 mg (79.9%) were obtained.

Compound (3) was treated in the same manner as described above to obtain 30.0 mg (76.1%) of compound (5).

Physical Properties of Compound (4):

¹ H NMR CDC1 ₃ δ (TMS) 9 MHz

0.71 3H, s, CH ₃ -18

0.84 and 0.91 6H, CH ₃ -26 and CH ₃ -27

0.95 3H, d, J = 4.5 Hz, CH ₃ -21

1.00 3H, s, CH ₃ -19

2.01 3H, s, NAc

2.43 1H, dd, J = 4.5 and 12.6 Hz, 3-Heq

Physical Properties of Compound (5):

(TMS) 90MHz ¹ H NMR CD ₃ OD

(TMS) 90 MHz

0.71 3H, s, CH ₃ -18

0.86 and 0.92 6H, CH ₃ -26 and CH ₃ -27

0.95 3H, d, J = 4.5 Hz, CH ₃ -21

1.00 3H, s, CH ₃ -19

2.00 3H, s, NAc

2.39 1H, dd, J = 4.5 and 12.6 Hz, 3-Heq

Example 3

-일)-3.5-디데옥시-

-D-글리세로-D-갈락토-2-노뉼로피라노소네이트 인 화합물 (2) 500mg을 메탄올 100ml에 용해 시켰다.Methyl 5-acetoamide-4, 7, 8, 9 tetra-0-acetyl-2-0- (5-cholsten-3-

-Yl) -3.5-dideoxy-

-500 mg of -D-glycero-D-galacto-2-nonulopyranosonate phosphorus compound (2) was dissolved in 100 ml of methanol.

About 200 ml of 2N sodium hydroxide aqueous solution was dripped at this solution, stirring, it was made into about 0.2N sodium hydroxide / methanol solution, and it stirred at room temperature overnight, and the compound was saponified.

Next, the Douxex 50 W (H ⁺ ) resin was added to the urine with stirring. The resin was removed after adjusting the pH of the solution to acidic (about pH 4).

The solution is then dried in vacuo to give 5-acetoamide-2-0-acetyl- (5-cholen-3-β-yl) -3.5-dideoxy-α-glycero-D-galacto-2 A white powder of nonntopyranosonic acid was obtained. This powder was dissolved in 0.02N sodium hydroxide aqueous solution.

The obtained solution was passed through Dianon HP 20 resin collem chromatography, adsorption occurred on the resin, and the resin was washed with water.

-일)-3.5-디데옥시-

-D-글리세로-D-갈락토-2-노뉼로피라 노소네이트[화합물(4)]의 백색분말 37mg(91%)을 얻었다.Next, after eluting with a 75% aqueous methanol solution, methanol was removed by middle flow, and then freeze-dried to give sodium 5-acetoamide-2-0- (5-cholene-3-

-Yl) -3.5-dideoxy-

37 mg (91%) of a white powder of -D-glycero-D-galacto-2-nonulopyranosonate [Compound (4)] were obtained.

The physical properties of compound (4) were substantially the same as in Example 2.

Example 4

50 mg of Compound (2) was dissolved in 100 ml of methanol, and then 20 ml of 2N aqueous sodium hydroxide solution was added thereto.

The resulting solution was stirred overnight at room temperature to cause saponification.

DoWex 500 W (H ⁺ ) was added to the solution, followed by stirring the mixture. The pH of the mixture was adjusted to 7 to 8 degrees.

The mixture was suction filtered and washed with methanol to remove the resin.

The filtrate and washing methanol were collected and methanol was distilled off.

The precipitated white insoluble matter was filtered and freeze-dried to obtain 39 mg (96%) of white powder [Compound (4)].

Physical Properties of Compound (4):

Mass (FD) m / z 722 (M + Na), 700 (M + 1), 386, 336 and 314

Elemental Analysis (%) C ₃₈ H ₆₂ O ₉ NNa2H ₂ O

Calculated value: C; 61. 96, H; 8.42, N; 1.90

Found: C; 61. 92, H; 8.71, N; 2. 04

(TMS) 400MHz ¹ H NMR CD ₃ OD

(TMS) 400 MHz

0.704 3H, s, CH ₃ -18

0.870 and 0.885 3Hx2, d, J = 1.7 Hz, CH ₃ -26

CH ₃ -27

0.936 3H, d, J = 6.5 Hz, CH ₃ -21

0.992 3H, s, CH ₃ -19

2.010 3H, s, NAc

2.839 1H, dd, J = 4.2 and 12.0 Hz, 2'-Heq

5.332 1H, d, J = 5.5Hz, 6-H

(TMS) 100MHz ¹³ C NMR CD ₃ OD

(TMS) 100 MHz

175.9NAc

175.26 1'-COONa

142.87 C-5

122.59 C-6

102.57 C-1 '

70.50 C-3

41.00 C-2

Example 5

-일)-3.5-디데옥시-

-D-글리세로-D-갈락토-2-노뉼로피라노소네이트인 화합물(4)을 사용한 것 이외에는 실시예 3과 동일하게 처리하여, 나트륨 5-아세토아미드-2-0-(5-콜레스텐-3-

-일)-3.5-디데옥시-

-D-글리세로-D-갈락토-2-노뉼로피라노소네이트인 화합물(5) 36mg(88%)을 얻었다.Methyl 5-acetoamide-4, 7, 8, 9-tetra-0-acetyl-2-0- (5-cholester-3- instead of compound (2)

-Yl) -3.5-dideoxy-

The treatment was carried out in the same manner as in Example 3, except that Compound (4), which was -D-glycero-D-galacto-2-nonulopyranosonate, was used. Sodium 5-acetoamide-2-0- (5-chole Sten-3-

-Yl) -3.5-dideoxy-

36 mg (88%) of compound (5) which was -D-glycero-D-galacto-2-nonulopyranosonate was obtained.

The physical properties of compound (5) were substantially the same as in Example (2).

Example 6

40 mg (98%) of compound (5) was obtained in the same manner as in Example 4 except that compound (3) was used instead of compound (2).

Physical Properties of Compound (5):

Mass (FD) m / z 722 (M + Na), 700 (M + 1) and 386

Elemental Analysis (%) C ₃₈ H ₆₂ O ₉ NNaH ₂ O

Calculated value: C; 63. 52, H; 8.91, N; 1.95

Found: C; 63. 81, H; 9.25, N; 2. 13

(TMS) 400MHz ¹ H NMR CD ₃ OD

(TMS) 400 MHz

0.700 3H, s, CH ₃ -18

0.861 and 0.880 3Hx2, d, J = 1.5 Hz, CH ₃ -26

CH ₃ -27

0.928 3H, d, J = 6.5 Hz, CH ₃ -21

0.991 3H, s, CH ₃ -19

1.972 3H, s, NAc

2.482 1H, dd, J = 4.5 and 13.0 Hz, 2'-Heq

5.282 1H, d, J = 5.3 Hz, 6-H

(TMS) 100MHz ¹³ C NMR CD ₃ OD

(TMS) 100 MHz

176.95NAc

174.51 1'-COONa

143.08 C-5

122.46 C-6

101.37 C-1 '

72.37 C-3

43.82 C-2

Example 7

50 mg of compound (2) was dissolved in 100 ml of anhydrous methanol. To this solution, 0.02 ml of 28% sodium methylate solution was added, and then the solution was stirred at room temperature for about 1 hour to deacetylate.

Dowex 50Wx8 (H ⁺ ) resin was then added to this solution, and the solution was stirred and neutralized.

The solution was suction filtered, washed with methanol and then the resin was removed.

The filtrate and washing methanol were collected, and 20 ml of a 2N sodium hydroxide aqueous solution was added thereto, followed by stirring at room temperature overnight for saponification. Amberlite IRC-50 (H ⁺ ) resin was added to the saponified solution. The resulting solution was stirred and adjusted to pH 5-7 of this solution.

The solution was suction filtered, and then washed with methanol to remove the resin.

The filtrate and washing methanol were collected, dried in vacuo and the methanol was distilled off. A small amount of precipitated white insoluble matter was filtered off and then freeze-dried to obtain 38 ml (93%) of white powder [Compound (4)].

The physical properties of this compound were substantially the same as in Example 2.

Example 8

35 mg (86%) of Compound (5) was obtained in the same manner as in Example 7 except that Compound (3) was used instead of Compound (2). The physical properties of this compound were substantially the same as in Example 2.

Example 9

Injection composition

Into the ampoule, 2.5 mg of the compound of the present invention, 0.25 mg of sodium dihydrogen phosphate dihydrate, 3 mg of 12-water sodium hydrogen phosphate 12-water and distilled water for injection were introduced. An injection solution composition of 1 ml was prepared.

Example 10

Injection composition to dissolve before use

0.5 mg of the compound of the present invention was mixed with 1 ml of physiological saline, and the freeze-dried composition of the injectable drug was introduced into the vial, and 1 ml of distilled water for injection was added to the vial to dissolve the injectable composition.

Example 11

Composition of Eye Drops

Into a vial, 1 mg of the compound of the present invention, 52.5 mg of boric acid, 14.5 mg of service, an appropriate amount of benzalkonium chloride, and an eye drop dissolving solution were introduced to prepare an eye drop solution composition having a total volume of 5 ml.

Example 12

Inhalation Composition

The compound of the present invention was well decomposed with an agate grinder to give a fine powder having a particle diameter of 1 to 20 microns.

Lactose was added to this powder, it was ground and mixed with each other.

20 to 40 powdery medicines were prepared by finely pulverizing the mixture and adding the lactose little by little to the mixture.

20 to 40 mg of this powder is introduced into the capsule or folded into powder paper by a conventional method.

The capsule was used for powder aerosol. The folded powder was used for liquid aerosols. Experiments for confirming the activity of the compounds of the present invention that promote the proliferation of neurites are described below.

[Experiment 1]

Effects on the proliferation of neuroblastoma cells, Neuro 2a strain Neuro 2a consists of 90% Delbecco's Modified Eagle's Medium and 10% fetal bovine serum (FCS), penicillin G 100 units It was suspended on a medium containing / ml and 100 µg / ml of sulfated mycin sulfate and incubated in 37 ° C of an air containing carbon dioxide incubator containing 5% of carbon dioxide.

The vessel used was a polystyrene tray with a diameter of 60 mm. 1 to 2 × 10 ⁴ neuroblastoma cells were transplanted into each tray and incubated for 48 hours.

-1-3) Ga1Nac (

1-4)<NAc Neu-(

2-3)>Gal(

1-4)G1c(

1-1)-세라미드(이하"GM₁"이라 약칭함)FCS-containing culture was removed from the resulting cell culture (Cultue). Compounds (4) (Table 1), Compounds (5) (which contained 100% MEM, and the concentration of antibiotics were the same as those of the cultures prior to removal of FCS) were cultured without FCS. Table 2) Gal (

-1-3) Ga1Nac (

1-4) <NAc Neu- (

2-3)> Gal (

1-4) G1c (

1-1) -ceramide (hereinafter abbreviated as "GM ₁ ")

2-8) NAcNeu-(

2-3)Gal(

1-3) Ga1NAc (

1-4)-〈 NAcNeu(

2-8) NAcNeu-(

2-3)-Gal(

1-4)G1c(

-1-1)-세라미드(이하"GQ_1b"라 약칭함(제 4 표)인 시료를 각각 소정향 투여한 후에 배양을 계속하였다.(Table 3) and <NAcNeu (

2-8) NAcNeu- (

2-3) Gal (

1-3) Ga1NAc (

1-4)-<NAcNeu (

2-8) NAcNeu- (

2-3) -Gal (

1-4) G1c (

-1-1) -ceramide (hereinafter, abbreviated as "GQ _1b " (Table 4)) was administered after predetermined administration of each of the samples, the culture was continued.

24 and 48 hours after the administration of these agents, the increased number of living cells in the culture, the number of increased new light, the length of the new light, and the like were measured.

The experiment was carried out in three trays for each concentration. The data obtained are expressed as mean value + standard error (S.E).

result :

The minimum effective concentrations of Compound (4), GM, and GQ _1b 48 hours after administration of these agents to the cultures were 10 ng / ml, 10 µg / ml and 10 µg / ml, respectively. Taking into account the molecular weight of these materials, the activity of compound (4) was as high as 420 times that of GM ₁ and 270 times that of GQ _1b . The minimum effective concentration of compound (5) was 48 ng / ml 48 hours after drug administration, and the activity of compound (5) was as high as 42 times that of GM ₁ and 27 times that of GQ _1b .

In addition, GM ₁ and GQ _1b did not cause any activation when cultured for 24 hours.

However, compounds (4) and (5) showed activity when cultured for 24 hours using 10 ng / ml of these compounds.

These results clearly show that compounds (4) and (5) have a strong activity in the diet of neurites.

Acute Toxicity Test:

45 weeks old ddy male mice were subjected to an acute toxicity test by intravenous injection of the debris.

The results showed that LD ₅₀ 's of Compounds (4) and (5) were 93 mg / kg and 291 mg / kg, respectively.

[Table 1]

24 hours

48 hours

S. E.: Standard Error

U. C.: unmeasurable

* p <0.001, ** p <0.01, *** p <0.05

[Table 2]

24 hours

48 hours

S. E.: Standard Error

U. C.: unmeasurable

* p <0.001, ** p <0.01, *** p <0.05

[Table 3]

24 hours

48 hours

*** p <0.05

[Table 4]

24 hours

48 hours

U. C.: unmeasurable

* p <0.001,

Industrial utility:

Sialocil cholesterol of the present invention is particularly useful as a therapeutic agent for neurological diseases.

Claims (10)

Each of the following general formula (4) or (5) is sialoyl cholesterol:

or

(Where Ac is an acetyl group) Compound (1) having the formula (1)

Is reacted with cholesterol in the presence of a Königskror catalyst to give a compound of formula (A)

[Wherein one of R ¹ and R ² is -COOCH ₃ and the other is a group having the formula

The preparation method of sialosil cholesterol which has following General formula (B) by preparing and hydrolyzing this compound (A).

[Wherein any one of R ³ and R ⁴ is -COONa and the other is a group having the formula

The siolosyl compound according to claim 2, wherein the Königshnormae is selected from the group consisting of emulsifying agent 2 mercury, second mercury cyanide, perchloric acid, silver trifluoromethanesulfonic acid silver and trifluoro silver acetate. Method of producing cholesterol. The method of claim 3, wherein the reaction of Compound (1) with cholesterol is performed for about 1 to 7 days under normal pressure of about 20 to 25 ℃. The method for preparing sialosil cholesterol according to claim 2, wherein the cholesterol is used in an amount of about 1 to 5 mol per mol of the compound (1). 4. The process for producing sialosil cholesterol according to claim 3, wherein the Königshnor catalyst is used in an amount of about 1.0 to 1.2 equivalents based on 1 equivalent of Compound (1). The method of claim 2, wherein a solvent is used, and the solvent is selected from the group consisting of benzene, dichloromethane and tetrahydrofuran. The sial according to claim 2, wherein the compound (A) is treated with about 1 to 3N alkaline solution at about 15 to 25 DEG C for about 5 to 15 hours in order to cause hydrolysis of the compound (A). How to prepare rosyl cholesterol. Haga's general formula.

or

A therapeutic agent for neurological diseases, consisting of sialosil cholesterol (4) or (5) having (wherein Ac is an acetyl group). The therapeutic agent for neurological diseases according to claim 9, wherein the drug is in the form of eye drop, inhalation intravenous injection, intramuscular injection, or subcutaneous injection.