JPH11269178A - Production of pyridinium compound, and compound therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain the subject compound useful as a diagnostic reagent for complications of diabetes and kindney failure by hydrolyzing a specific compound. SOLUTION: This pyridinium compound shown by formula III is obtained by hydrolyzing a compound shown by formula I (Ac is an acyl; and R is a lower alkyl) in a solvent (e.g. methanol) under an alkaline condition with, e.g. lithium methoxide, to produce a compound shown by formula II, and by hydrolyzing, under heating as required, the produced compound under and an acidic condition with, e.g. 6N hydrochloric acid, preferably without isolating it. The compound shown by formula I is obtained by reacting glucose penta-acetate with a cysteine derivative shown by formula IV in the presence of tin tetrachloride to produce a glucose-cysteine derivative shown by formula V, and by eliminating an Fmoc protective group from the derivative. Fmoc in the compound shown by formula IV has a protective group having a structure shown by formula VI.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a pyridinium compound and a compound therefor.

[0002]

2. Description of the Related Art As is well known, in a Maillard reaction between a sugar and a protein amine, a Schiff base is first formed by a non-enzymatic reaction (glycation) between the sugar and the protein amine, and an Amadori rearrangement from this Schiff base. The reaction forms the early Maillard reaction product, ketoamine, in a relatively short period of time, and goes through various stages to produce the later Maillard reaction late product.

[0003] The pre-products of the Maillard reaction include:
Clinically, glycated hemoglobin (HbAlc) and glycated albumin (fructosamine) are used as indicators of glycemic control.

[0004] On the other hand, late products of the Maillard reaction are produced by slow-turning proteins in the living body such as collagen, nerve myelin and the lens, and are known to increase due to long-term hyperglycemia (diabetes). As a hypothesis explaining why the characteristic change, persistent hyperglycemia, causes chronic complications specific to diabetes, this late product of the Maillard reaction has been noted for its origin and its production It has been suggested that inhibition may prevent the onset and progress of complications.

[0005] The possibility of diagnosing complications by measuring the concentration of the late product of the Maillard reaction in blood, urine and tissues is expected.

However, the knowledge obtained from the method for measuring the late product of the Maillard reaction developed so far indicates that there is a close causal relationship between the increase in the amount of the late product of the Maillard reaction in vivo and various tissue disorders. However, the chemical structure of major late Maillard reaction products in vivo is still unknown.

[0007] In view of the above-mentioned current state of the Maillard reaction late product as described above, some of the inventors of the present invention have developed a structure of an in vivo Maillard reaction late product having a close causal relationship with various tissue disorders. As a result of continued research for the purpose of identification, a new in vivo Maillard reaction late product was discovered, and further research was continued to obtain the compound represented by the formula,

Embedded image It was confirmed that the compound is a pyridinium compound having a structure represented by the following formula, and was further found to be useful as a diagnostic reagent for complications associated with diabetes or renal failure, and filed a patent application.

[0008]

As described above, since the pyridinium compound represented by the formula (2) is useful as a diagnostic reagent for complications associated with diabetes or renal failure, a predetermined amount of the compound is used. Efficient production is required, but such a production method of the pyridinium compound represented by the formula (2) has not been known.

[0009]

SUMMARY OF THE INVENTION The present invention has been made in view of the state of the prior art as described above.

Embedded image (Where Ac represents an acyl group and R represents a lower alkyl group), wherein the compound represented by the formula (2) is hydrolyzed.

Embedded image A method for producing a pyridinium compound represented by the formula:

The present invention also relates to formula (1)

Embedded image (Where Ac represents an acyl group and R represents a lower alkyl group), and hydrolyzes the compound represented by the formula (3)

Embedded image Wherein the compound represented by the formula (2) is further hydrolyzed:

Embedded image A method for producing a pyridinium compound represented by the formula:

The present invention further provides the following formula (4):

Embedded image Wherein the compound represented by the formula is hydrolyzed:

Embedded image A method for producing a pyridinium compound represented by the formula:

The present invention further relates to the following formula (1):

Embedded image (Wherein Ac represents an acyl group and R represents a lower alkyl group). A compound for producing a pyridinium compound, which is represented by the formula:

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

The pyridinium compound (2), which is the target compound of the production method of the present invention, is a late product of the Maillard reaction, for example, proteins such as albumin, lysozyme, casein, ribonuclease and globulin,
It is produced by a non-enzymatic reaction with carbonyl compounds such as glucose, diacetyl, glyoxal and fructose.

The pyridinium compound (2) is hydrolyzed by dialyzing the reaction solution of a system in which rabbit erythrocytes and glucose are reacted at 37 ° C. for 2 weeks for 48 hours, followed by ODS-5.
High-performance liquid chromatography with a column detects at an excitation wavelength of 340 nm and a fluorescence wavelength of 402 nm,
The amount of production increases in proportion to the concentration of glucose and the reaction time as well as the browning of the reaction solution, and its production is inhibited by aminoguanidine, a Maillard reaction inhibitor,
It is also produced similarly in a system of bovine serum albumin and glucose.

The structure of the pyridinium compound (2) is
After hydrolyzing the late Maillard reaction product generated by the reaction of bovine serum albumin and glucose, fractionation by high performance liquid chromatography using fluorescence wavelength,
The molecular weight was first determined to be 212 by measuring using FAB-mass spectrum using a mixture of glycerol and nitrobenzyl alcohol as a matrix.

Next, by utilizing the high-resolution mass spectrum, the composition formula of the pyridinium compound (2) is determined to be C ₉ H ₁₀ O ₃ N ₁ S _1, and NM including two dimensions is further determined.
As a result of the R analysis, the late product of the Maillard reaction is

Embedded image 8-hydroxy-5-methyldihydrothiazolo [3,2-a] pyridinium-3-carboxylate represented by the following formula:

The pyridinium compound (2) has already been isolated as a hydrolyzate having fluorescence from bovine liver.

Thus, the present invention is to produce the above pyridinium compound (2) as follows.

That is, in the manufacturing method of the present invention, the formula (1)

Embedded image The compound of the present invention represented by is used.

In the above formula (1), Ac represents an acyl group such as an acetyl group, and R represents a lower alkyl group such as a methyl group, an ethyl group, a propyl group or a butyl group.

The compound of the present invention is described in Elofsson, M;
se B,; Kihlberg, J. TetrahedronLett, 1991, 32, 76
According to the method described in 13, glucose pentaacetate and formula (5)

Embedded image With a cysteine derivative represented by the following formula (6):

Embedded image Is obtained, and then Fmoc in this glucose-cysteine derivative is obtained.
It can be obtained by removing the protecting group.

The Fmo in the compound of the above formula (5)
c represents a protecting group having the following structure.

Embedded image

In the production method of the present invention, the compound (1)
Is hydrolyzed to obtain the above pyridinium compound (2).
It can be carried out by heating, if necessary, under acidic conditions with N hydrochloric acid or the like.

The method for producing the pyridinium compound (2) is also reported in Acta, Chem, Scand., 23, 371, 1969, but it involves a relatively long process. As described above, a method for producing the pyridinium compound (2) in one step is not known.

The pyridinium compound (2) is obtained by hydrolyzing the compound represented by the formula (1) and temporarily converting the compound represented by the formula (3)

Embedded image Can also be obtained by hydrolyzing this compound (3).

In the above method, the former hydrolysis is carried out by
For example, the reaction can be carried out in a solvent such as methanol under alkaline conditions with iridium methoxide or the like, and the latter hydrolysis can be carried out by heating as necessary under acidic conditions with 6N hydrochloric acid or the like.

Further, the pyridinium compound (2) is
Equation (4)

Embedded image Can also be obtained by hydrolyzing the compound represented by

The hydrolysis in the above method is, for example, 6
It can be carried out by heating, if necessary, under acidic conditions with N hydrochloric acid or the like.

The compound represented by the above formula (4) is prepared by H. Mas
can be synthesized by the method of aki, A. Imahori (JP. 09067226 A2), and the compound represented by the formula (4) is obtained by changing the sugar moiety in the compound represented by the formula (3) to galactose. Things.

Incidentally, the physicochemical data of the pyridinium compound (2) obtained by the production method of the present invention agreed with the values described in the literature.

The pyridinium compound (2) obtained by the production method of the present invention is produced in large quantities in a diabetic condition and deposited on tissues such as kidney, tendon, skin and nerve.
Since it is excreted in large amounts in urine as the renal function decreases, it can be applied as a biomarker of diabetic pathology.

[0033]

EXAMPLES The present invention will be described below in detail with reference to examples, but these examples do not limit the present invention in any way.

Example 1 N- (9-Fluorenylmethoxycarbonyl) -S-triphenylmethyl-L
Synthesis of -cysteine methyl ester To a mixture of 9 ml of methanol and 30 ml of benzene, 1.
77 g (3 mmol) of N- (9-Fluorenylmethoxycarbony
l) -S-triphenylmethyl-L-cysteine was dissolved, and 5.5 ml (4.8 m) of trimethylsilyldiazomethane was added to this solution.
mol) and stirred at room temperature for 15 minutes. The solvent was distilled off from the reaction solution under reduced pressure to obtain a crude methyl ester, which was used without purification. ¹ HMMR (270 MHz, CDCl ₃ , TMS): δ
2.66 (d, J = 5.3 Hz, 2H), 3.71
(S, 3H), 4.23 (t, J = 6.8 Hz, 1
H) 4.30-4.39 (m, 3H), 5.23
(D, J = 8.3 Hz, 1H), 7.18 to 7.32
(M, 11H), 7.38 to 7.41 (m, 8H),
7.60 (d, J = 6.3 Hz, 2H), 7,76
(D, J = 7.3 Hz, 2H)

N- (9-Fluorenylmethoxycarbonyl) -L-cyste
ine methyl ester [compound of formula (5) where R = CH ₃ ]
Synthesis of crude N- (9-Fluorenylmethox
ycarbonyl) -S-triphenylmethyl-L-cysteine methyl est
To the solution in which er was dissolved, 419 mg (3.6 mmol) of triethylsilane and 1.5 ml of trifluoroacetic acid were added, and the mixed solution was stirred at room temperature for 1 hour, and the solvent was distilled off.
The residue was purified by silica gel column chromatography with hexane / ethyl acetate = 4: 1 → 2: 1 to give 1.04 g.
(97%) of the desired compound was obtained. ¹ HMMR (270 MHz, CDCl ₃ , TMS): δ
1.36 (t, J = 9.1 Hz, 1H), 2.98-
3.02 (m, 2H), 3.80 (s, 3H), 4.2
4 (t, J = 6.8 Hz, 1H), 4.44 (d, J =
7.9 Hz, 2H), 4.67 (dt, J = 7.4,
3.8 Hz, 1 H), 5.68 (d, J = 7.4 Hz,
1H), 7.28-7.44 (m, 4H), 7.61
(D, J = 7.3 Hz, 2H), 7.77 (d, J =
7.3 Hz, 2H) ¹³ C NMR (67.8 MHz, CDCl ₃ ) δ27.
0,47.1,52.8,55.2,67.0,12
0.0, 124.98, 125.01, 127.0, 1
27.7, 141.3, 143.6, 143.7, 15
5.6,170.4

N- (9-Fluorenylmethoxycarbonyl) -S- (2,3,
4,6-tetra-O-acetyl-bD-glucopyranosyl) -L-cysteine
Synthesis of methyl ester [Compound of formula (6) where Ac = COCH ₃ , R = CH ₃ ] 1,2,3,4,6-penta-O-acetyl-bD-glucopyranose1.56
g (4 mmol) and N- (9-Fluorenylmethoxycarbonyl) -L
0.48 ml of tin tetrachloride (4.1 m) was added to a solution of 1.00 g (2.8 mmol) of -cysteine methyl ester [compound of R = CH ₃ in formula (5)] in 20 ml of dichloromethane.
mol) was added. Stir the mixed solution at room temperature for 1 hour,
Furthermore, 1,2,3,4,6-penta-O-acetyl-bD-glucopyranose1.
56 g (4 mmol) and 0.48 ml of tin tetrachloride (4.
1 mmol) was added. After stirring at room temperature for 1 hour, the reaction solution was diluted with dichloromethane, washed with 10% hydrochloric acid, water and brine, dried over magnesium sulfate, and the solvent was distilled off.
The residue was subjected to silica gel column chromatography (hexane /
Refined with ethyl acetate = 3: 2), 1.69 g (88%)
The desired compound was obtained. ¹ HMMR (270 MHz, CDCl ₃ , TMS): δ
2.01 (s, 3H), 2.02 (s, 6H), 2.0
4 (s, 3H), 2.99 (dd, J = 14.2, 6.
8 Hz, 1 H), 3.26 (dd, J = 14.2, 4.
0 Hz, 1 H), 3.67 (dt, J = 9.9, 4.0)
Hz, 1H), 3.75 (s, 3H), 4.09-4.
15 (m, 2H), 4.24 (bt, J = 9.9, 9.
4,1H), 4.36 to 4.61 (m, 4H), 5.0
1 (dd, J = 9.9, 9.4 Hz, 1H), 5.06
(Dd, J = 9.9, 9.4, 1H), 5.23 (t,
J = 9.4 Hz, 1H), 5,93 (d, J = 7.9H)
z, 1H), 7.28-7.34 (m, 4H), 7.5
9 to 7.61 (m, 2H), 7.76 (d, J = 6.9)
Hz, 2H) ¹³ C NMR (67.8 MHz, CDCl ₃ ) δ 20.
4, 20.5, 31.8, 47.0, 52.6, 53.
8, 61.9, 66.8, 68.1, 69.5, 74.
3, 75.9, 82.9, 119.9, 124.85,
124.91, 127.0, 127.6, 141.1,
143.58, 143.61, 155.7, 169.
2,169.3,169.9,170.4,170.7

S- (2,3,4,6-tetra-O-acetyl-bD-glucopyr
anosyl) -L-cysteine methyl ester [A in formula (1)
c = COCH ₃ , R = CH ₃ Compound] N- (9-Fluorenylmethoxycarbony) in DMF (10 ml)
l) -S- (2,3,4,6-tetra-O-acetyl-bD-glucopyranosyl) -L
-cysteine methyl ester 390mg (0.57mmo
l) was treated with 20% piperidine for 1 hour at room temperature. After evaporating the solvent from the mixed solution under reduced pressure, the mixture was diluted with a mixed solution of ethyl acetate and ether 1: 1. The extract was washed with water and brine, dried over magnesium sulfate, the solvent was distilled off, and the residue was subjected to silica gel column chromatography (chloroform / chloroform).
Methanol = 40: 1) refined, 243 mg (91%)
The desired compound was obtained. ¹ HMMR (270 MHz, CDCl ₃ , TMS): δ
1.77 (bs, 2H), 2.01 (s, 3H), 2.
03 (s, 3H), 2.07 (s, 3H), 2.09
(S, 3H), 2.83 (dd, J = 13.9, 7.9)
Hz, 1H), 3.17 (dd, J = 13.9, 4.3)
Hz, 1H), 3.69 to 3.75 (m, 2H), 3.
75 (s, 3H), 4.16 (dd, J = 12.5,
2.6 Hz, 1 H), 4.24 (dd, J = 12.5,
4.6 Hz, 1 H), 4.59 (d, J = 9.9 Hz,
1H), 5.04 (t, J = 9.6 Hz, 1H), 5,
09 (t, J = 9.6 Hz, 1H), 5.23 (t, J
= 9.4 Hz, 2H) ¹³ C NMR (67.8 MHz, CDCl ₃ ) δ 20.
5, 20.6, 34.8, 52.3, 54.7, 61.
9, 68.1, 69.6, 73.7, 75.9, 83.
5,169.4,170.1,170.6,173.9

8-hydroxy-5-methyldihydrothiazolo [3,2-
a] Synthesis of pyridinium-3-carboxylate [compound (2)] S- (2,3,4,6-tetra-O-acetyl-bD-gluc
opyranosyl) -L-cysteine methyl ester [in the formula (1), a compound of Ac = COCH3, R = CH3]
After performing gas phase hydrolysis at 110 ° C. for 24 hours with N hydrochloric acid, the mixture was filtered, and 3% acetonitrile / 0.1% trifluoroacetic acid was flowed at a flow rate of 2 ml / min using an ODS-HG-5 column. A peak having an elution time of 10 minutes corresponding to the target compound was collected by high performance liquid chromatography at an excitation wavelength of 340 nm and a fluorescence wavelength of 402 nm. As a result of repeating this, S- (2,3,4,6-tetra-Oa
cetyl-bD-glucopyranosyl-L-cysteine methyl ester
[Compound of formula (1) where Ac = COCH ₃ , R = CH ₃ ] 22.
From 5 mg, 0.9 mg of the purified compound (2) was obtained (yield: 4.35%).

Example 2 Synthesis of 3-S- (bD-glucopyranosyl) -L-cysteine [Compound (3)] 200 mg (0.3 mmol) of S- (2,3,4,6-tetra-Oa
cetyl-bD-glucopyranosyl) -L-cysteine methyl ester
1 m of [compound of formula (1) where Ac = COCH ₃ , R = CH ₃ ]
1 methanol solution to 0.1 ml of methanol in 0.4 ml.
N sodium methoxide was added dropwise at 4 ° C.
Stirred for hours. Amberlyst 15 was added to neutralize, then filtered and the filtrate was concentrated. 93 mg of the residue was dissolved in a small amount of methanol, and methylene chloride was added to obtain 12 mg of a solid. The mother liquor was concentrated, and methanol
Treatment with methylene chloride gave 27 mg of fixation. By combining both solids, 39 mg of the target compound was obtained in a yield of 28%. ¹ H of obtained compound
MMR is described in the literature (Monsigny, MLP; Delay, D .; Vaculi
k, M. Carbohydr. Res. 1977, 59, 589).

3-S- (bD-glucopyranosyl) -L-cysteine
[Compound (3)] 1.5 mg was hydrolyzed and post-treated in the same manner as in Example 1, and from Compound (3) 30 mg, purified compound (2) 2.5 mg (11.23% yield). I got

Example 3 SD-galactopyranosyl-L-cysteine [Compound (4)]
1.5 mg was hydrolyzed and post-treated in the same manner as in Example 1 to obtain 2.4 mg of purified compound (2) (yield 10.82%) from 30 mg of compound (3). Compound (4) was synthesized according to the method described in the above-mentioned literature.

Claims (5)

[Claims] (1) Formula (1) (Wherein Ac represents an acyl group and R represents a lower alkyl group), wherein the compound represented by the formula (2) is hydrolyzed. A method for producing a pyridinium compound represented by the formula: 2. Formula (1) (Where Ac represents an acyl group and R represents a lower alkyl group), and the compound represented by the formula (3) is hydrolyzed. Wherein the compound represented by the formula (1) is further hydrolyzed: A method for producing a pyridinium compound represented by the formula: 3. The method for producing a pyridinium compound according to claim 2, which is carried out without isolating the compound represented by the formula (3). 4. Formula (4) Wherein the compound represented by the formula is hydrolyzed. A method for producing a pyridinium compound represented by the formula: 5. The formula (1) (In the formula, Ac represents an acyl group and R represents a lower alkyl group.) A compound for producing a pyridinium compound, characterized in that: