JPS61243096A - Sialic acid derivative and production thereof - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (one of R<1> and R<2> is carboxyl or methoxycarbonyl and the other is group of formula II or III; R<3> is H or acetyl). EXAMPLE:5-Acetamido-2-O-acetyl-( 5-cholesten-3-beta-yl )-3,5-dideoxy-alpha-D-glycero-D- galacto-2-nonulopyranosonic acid. USE:Immuno-regulator. It activates suppressor T-cell and suppresses the production of immunoglobulin of B cell. PREPARATION:The compound of formula IV is made to react with cholesterol or 3'-acetyl-2'-deoxyuridine, and if necessary, the reaction product is hydrolyzed.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to immunologically active sialic acid derivatives and methods for their production. Sialic acid is present in sialocomplexes (IIIn2 glycolipids,
It is known to exist as oligosaccharides and polysaccharides.

In recent years, this compound has become important in medical and pharmaceutical fields such as neurological function, cancer, inflammation, immunity, viral infection, differentiation, and hormone receptors, and is attracting attention as a unique active molecule localized on the cell surface.

However, the role played by sialic acid in the sialo complex is still a matter of speculation.

This compound has been further studied by many natural product organic chemists and has already led to various simple derivatives. However, no significant physiologically active derivatives are known yet. The purpose of the invention is to obtain a new compound with excellent physiological activity.

Furthermore, in recent years, the diversification of treatments for malignant tumors of hematopoietic organs, various cancer diseases, collagen diseases, etc. has certainly had a life-extending effect. However, on the other hand, it is unavoidable that the frequency of use of drugs such as adrenocortical hormones and immunosuppressants will increase, and as a result, many side effects are occurring as well as a decline in the so-called immunity. The inventors have focused on sialic acid, which is a component unique to living organisms, and have conducted intensive research into immunomodulators that have fewer side effects and have the ability to adjust the immune monitoring mechanism through chemical modification.
As a result, we have arrived at a novel compound of the present invention that has so-called immunomodulatory effects that activate suppressor T cells and suppress immunoglobulin production by B cells.

That is, the present invention provides a sialic acid derivative represented by the general formula (wherein R1 and R2 are a carboxyl group or a methoxycarbonyl group, and R3 is hydrogen or an acetyl group) Regarding.

Further, the present invention is characterized in that the compound represented by the general formula [r] is reacted with cholesterol or 3''-acetyl-2''-deoxyuridine, and then hydrolyzed if necessary.
The present invention relates to a method for producing a Sial M derivative.

The sialic acid derivative of the formula (r) for -4 is produced as shown in the following chemical formula using cholesterol as an example.

The compound of formula (n) used in the present invention is a known compound and can be easily obtained commercially.

In the above production method, a compound of formula (n) is used.

Incidentally, as the above-mentioned catalyst, mercuric bromide, mercuric cyanide, silver perchlorate, silver trifluoromethanesulfonate, silver trifluoroacetate, etc. can be used.

The above catalyst is about 1.0 equivalent 1 to 1 equivalent of compound [■]
It is used in the range of ~1.2 equivalents.

Further, examples of the solvent include acetonitrile, nitromethane, acetone, benzene, tetrahydrofuran, dichloromethane, methylene chloride, and the like.

Among these, benzene, tetrahydrofuran, and dichloromethane are preferred solvents.

The reaction product thus obtained is isolated and purified by a conventional method such as column chromatography.

Furthermore, the above product can be hydrolyzed, if necessary, to convert methoxycarbonyl groups to carboxyl groups and acetyl groups to hydrogen. Hydrolysis is carried out by a conventional method, for example, using about 1 to 3N
This can be carried out by treatment with an alkaline solution of at a temperature of about 15 to 25° C. for about 5 to 15 hours.

In the present invention, the compounds represented by the -a formula (1) each have a remarkable immunomodulating effect.

The immunomodulatory effect could be confirmed by the following method.

Effect of ConA on mouse lung lymphocyte activation; T cells are non-specifically activated by ConA, but
The sialic acid derivative of the present invention was added to this reaction system, and its effect was investigated. That is, ConA and the compound of general formula CI) were added to lung lymphocytes (SPC) obtained from BALB/C mice, and the cells were cultured on a microplate at 37° C. for 20 hours while providing 5% CO 2 .

Tritium-labeled thymidine was added thereto, and after further culturing at 37°C for 10 hours, spcs were collected, and the amount of 3H1-thymidine incorporated into SPCs was measured using a scintillation counter.

The compound represented by general formula (1) was found to promote and enhance the uptake of 3H-thymidine, and had an enhancing effect on T cell activation by ConA.

Effect on immunoglobulin production by mouse lung lymphocytes: The sialic acid derivative of the present invention, which was shown to have a T cell activating effect in the above experiment, was further investigated for its effect on immunoglobulin production by measuring the number of plaque-forming cells. did. First, sheep red blood cells (SRB C) are added to SPC.
) and each compound of general formula (1) were added, and the mixture was heated at 37°C for 5 minutes.
Cultured for 1 day. 5RBC and complement were added again to the obtained sensitized SPC.

PFCs (plaque-forming cells) were counted after culturing at 37° C. for 3 to 12 hours in a Cunningham chamber.

A decrease in PFC was observed, and the cell viability was equivalent to that of the control standard, confirming an enhanced suppressive effect on immunoglobulin production.

The compounds of the present invention showed activity in the two aforementioned tests. In other words, it is thought that immunoglobulin production was suppressed by activation of suppressor T cells.

Conventionally, in autoimmune diseases such as collagen diseases, a decline in the function of suppressor T cells has been observed.

Therefore, the sialic acid derivative of the present invention having suppressive T cell activating activity is expected to be useful in clinical applications as an immunomodulator.

The present invention will be explained below using examples. These examples are merely illustrative of the invention and therefore, of course, are not intended to limit it.

Example 1 3.87 g (10 mnofe) of cholesterol was dissolved in 20 ml of dry benzene, and A g C1 was added to the solution.
0ao, 829 g (4 mmole) was added, and 2 g of dry light was further added, and the mixture was stirred at room temperature for 1.5 hours.

Next, 50 methylene chloride of the compound of formula (If)! The solution was added and allowed to react at room temperature for 7 days.

The reaction solution was passed through Celite, and the filtrate was dried under reduced pressure.

Dissolve the residue in ethyl acetate, wash thoroughly with saturated brine,
After drying with Na z SO 4 , ethyl acetate was distilled off under reduced pressure. 4.78 g of brown powder was obtained.

This product was separated and purified by column chromatography (silica gel), and 244 mg of the α-form of the title compound (16,
0%) and 253 mg (16.6%) of β-isomer were obtained.

Also, 106 mg (11° 2%).

Physical constant Mass (El) m/z 859 (M,
800 (M”-59) IR Sill, 1..172
0.1630 and 1520cm-''If N? fR
CDCf 3 δ (TMS) 90MFI20.68
3H,s, CHz-180,84 and 0.87 6
L CH, -26, CI! ! =270.90 3)
1. d, J=4.5Hz, CHt-210,9
93H,s, CH3-19 1,903H,s, NAc 2.03,2.12.2,15 12H,3s,
OAc X 42.60 1H,aa,,r,4.
5 and 12.6Hz, 3-Heq3.78 3
8. s, COOMe4.86 1H,m,
4-H 22° (α) o -8,02' (C=1.25, in methanol) Physical constant of β form Mass (FD) m/z 860 (M”+1
)IRν,,, 1720.1640 and 1520c
m-''HNMRCDCl s δ (TMS) 90
MHz0.6F 3) I, s, CH3-180,
83 and 0.87 68. CH3-26, CH3-
270,903H,d, J=4.5Hz, CH3-
211,003H,s, CH3-19 1,863H,s, NAc 2.00, 2.03.2.06 and 2. H12FI,
4s, OAcx42.53 LH, dd, J=4
．． 5 and 12.6Hz, 3-Heq3.78
3+1. S, COOMe [α) o −26
, 71° (C=0.97, in methanol) Example 2 The compound (α form) obtained in Example 1 was added to 2 ml of methanol.
3 ml of a caustic soda solution of IN was added thereto, and the mixture was stirred at room temperature overnight. Add 2ml of water to the reaction solution! Add
After neutralization with Dowex (Doi4e-χ) 50, a trace amount of precipitate was filtered off, and the filtrate was dried under reduced pressure. 31.4 mg (79.7%) of the title compound as a yellow powder was obtained.

In the same manner as above, from the β-form obtained in the practical side light, the β-form which is an isomer of the title compound is 30.0. mg (76,1%)
I got it.

Physical constant rt of the title compound? j', -X2750.1575cm-''H
NMR coczi δ (TMS) 90MHzO
,7I 3H,s, CH3-180,84 and 0.91 6H,C1,-26 and cu3-27
0.95 3H, d, J=4.5Hz, C
Hz-211,003H,S+ CHz-19 2,0138,s, NAc 2.43 11. dd, J=4.5 and 12.6H
z, 3-Heq [α) o -12,58 (C=0
．． 41, in methanol) Physical constant of β-form it? J/ , , 2870.1620 and 1
550cm-''HNMRCD*OD δ(TMS
) 90MHz0.71 3ff, s 0.86 and O,,926B, CH:l-26 and cu, -270,9538,d, J=4.5Hz,
CH2-211,003H,s, CH3-19 2,003FL s, NAc 2.39 1H,dd, J=4.5 and 12.6)
1z, 3-Heq [α)D -31,77 (C=0
．． 78 in methanol) Example 3 3′-0-acetyl-2′-deoxyuridine 0.5
40 g (2mM) of dry 7 was dissolved in 20ml of dry setonitrile, 0.721g (2mM) of mercuric bromide, 0.505g (2mM) of mercuric cyanide and 5411g of Drylite O were added and the solution was stirred at room temperature. Stirred for 1.5 hours. Next, 1.020 g (2 m
M) was added, and after reacting at room temperature for 48 hours, 0.510 g (1 mM) of the compound of formula (If) was added, and after reacting for 24 hours, 0.510 g (1 mM) of the compound of formula (n) was further added. After reacting for an additional 24 hours, the reaction solution was filtered through Celite, and the filtrate was dried under reduced pressure. 1.35 g of the obtained green powder was purified by chromatography. (200 g of silica gel.

Developing solvent CHCl s / M e OH = 20 /
1) Here, 0.43 g of 2.3-dehydro compound (22,
7%) and 1.50 g of the desired crude product were obtained. This product was purified by chromatography using a Rover column, and 0.288 g (19.4%) of the α-isomer and 0.28 g (19.4%) of the β-isomer of the title compound were obtained.
467 g (31.4%) were obtained.

Physical constant of α body Mass (El) m/z 743 (M”),
684(FT"-59)'HNMRCDCl s
δ(TMS) 300MHz1.897 30. s
, NAc 2.045-2.16215H,0AcX52.444
1H, dd, J=5.37.4.41Hz, 2'
-H2,558LH,dd, J=4.89,12.7
0Hz, 3'''-Fleq3.549 LH, dd
, J=2.45,10.75H2,6″-H3,82
43H,s, COOMe 4.934 1H,ddd, J=4.89,11.00
．． 11.0OHz, 4″-II5.243 LH, d
, J=17.09Hz, NHAc5.761 1L
d, J=8.30Hz, 5-H6,33B 1
H, dd, J=5.37, 8.79Hz, 1'-H
7,8OB LH,d, J=8.30Hz, 6-
H24,5° [α) o +4.2° (C=0.67, MeO
H) Physical constant of β body Mass (EI) m/z 743 (W”)
, 684(M"-59)'HNMI? CDCI
, δ(TMS) 300MHz1.912 3H,
s, NAc 2.029-2.169 15L OAc x 52.
297 1H, ddd, J4.30, 8.30+14.
77Hz, 2'42.399 1H, ddd, J=2.
20.5.86.14.77Hz, 2'-H2,483
1B, dd, J=4.89, 13.19Hz, 3
”-freq3.702 1H, dd, J=2.44
．． 7.33Hz, 6”-H3,8433H,s, C
OOMe 5.125 1H, ddd, J=4.89. IO, 51
, IO, 5fHz, 4'-H5,9431B, d,
J=8.3082.5-H6,28f 1B, dd
, J=5.86.8.30Hz, 1'-H6,45
81H, d, J=9.77Hz, NHAc6.59
4 1B, d, J=8.30Hz, 6-H25゜[α) o +1.8° (C=1.O
Ol MeOH) Example 4 3゛-〇-acetyl-2゛-deoxyuridine 1,03
g (3.8 mmole) in dry acetonitrile 1
00ml, 1.58g of silver perchlorate (7.0ml)
ale) and 1 g of Drylite were added, and the mixture was stirred at room temperature for 1.5 hours.

Next, 3.88 g (7.6 mmol
e) was added and reacted at room temperature for 48 hours. After the reaction, the reaction solution was filtered through Celite, and the filtrate was dried under reduced pressure.

The residue was dissolved in ethyl acetate, thoroughly washed with saturated brine, dried over sodium sulfate, and ethyl acetate was distilled off under reduced pressure. 4.14 g of light brown powder was obtained.

This was separated and purified using a silica gel Rover column to obtain 1.07 g (37.8%) of the α-form and 1.1 g of the β-form of the title compound.
9 g (42.0%) were obtained. The physical constants of the α-form and β-form were the same as those of Example 3.

Claims (2)

[Claims] (1) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, one of R_1 and R_2 is a carboxyl group or methoxycarbonyl group, and the other is a formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ or ▲There are mathematical formulas, chemical formulas, tables, etc.▼ A sialic acid derivative represented by the following (where R^3 is hydrogen or an acetyl group). (2) General formula ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ A general formula characterized by reacting a compound represented by the following with cholesterol or 3'-acetyl-2'-deoxyuridine, and then hydrolyzing it if necessary. ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] (In the formula, one of R_1 and R_2 is a carboxyl group or methoxycarbonyl group, and the other is a group represented by the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼ and R^3 is hydrogen or an acetyl group) A method for producing a sialic acid derivative represented by: