JPH08301875A - Production of sodium gold thiomalate - Google Patents

Abstract

PURPOSE: To obtain in a single step a compound useful as an antirheumatic agent by reaction in an aqueous solution between thiomalic acid, sodium hydroxide and sodium chloroaurate. CONSTITUTION: First, a yellow transparent reaction liquor containing sodium gold thiomalate is obtained by reaction in an aqueous solution between (A) sodium chloroaurate, (B) the gold (III) in the component A is reduced into gold (I), and thiomalic acid acting as a ligand, and (C) sodium hydroxide, pref. at the molar ratio A/B/C of pref. (1:4:8). Secondly, the reaction liquor is added to ethanol to develop the sodium gold thiomalate as white precipitate which is then repeatedly precipitated pref. at least twice. Subsequently, the resultant precipitate is set to dryness and then dissolved in water and purified through an anion exchange resin column.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel method for producing gold sodium thiomalate.

[0002]

BACKGROUND ART Sodium gold thiomalate has a medicinal effect of suppressing inflammation of rheumatoid arthritis and delaying destruction of cartilage and bone, and is used as an antirheumatic agent.
Sodium gold malate (trade name "Ciozole", Shionogi Pharmaceutical Co., Ltd.), which is used clinically, has been conventionally produced by reacting gold halide with sodium thiomalate (US Pat. No. 1,994,213). The thing is C ₄ H ₃ AuNa ₂
It is a mixture of O ₄ S (molecular weight 390.08) and C ₄ H ₄ AuNaO ₄ S (molecular weight 368.10), and the abundance ratio of both is not fixed (12th Revised Japanese Pharmacopoeia Manual).

[0003]

However, since it is desirable that a pharmaceutical product has a constant standard, it is desired to develop a method for producing a single product of sodium gold thiomalate.

[0004]

[Means for Solving the Problems] The inventors of the present invention have been earnestly researching a method for producing sodium gold thiomalate. However, thiomalic acid, sodium chloride, and sodium chloroaurate (NaAuCl ₄ .2H ₂ O) were mixed in an aqueous solution. It was found that a single product of sodium gold thiomalate can be synthesized in one step by carrying out the reaction inside, and completed the present invention.

The structure of the sodium gold thiomalate obtained by the method of the present invention was analyzed and the biological activity thereof was examined. As a result, it was confirmed that it was used clinically and chemically and biologically. It was equivalent.

The present invention will be described in more detail below.

The process of the present invention comprises reacting thiomalic acid, sodium hydroxide and sodium chloroaurate in an aqueous solution. By this reaction, a reaction liquid containing yellow transparent sodium gold thiomalate is obtained. In this reaction, thiomalic acid was converted to gold (II
I) is reduced to gold (I) and acts as a ligand.

The molar ratio of sodium chloroaurate, thiomalic acid and sodium hydroxide is stoichiometrically preferably 1: 4: 8, and the yield is extremely reduced when excess thiomalic acid and sodium hydroxide are present. .

To isolate and purify sodium gold thiomalate from this reaction solution, for example, the following method is used.

When the above reaction solution is added to ethanol, sodium gold thiomalate is produced as a white precipitate. The precipitate is collected by filtration and dried by vacuum drying or the like.
In order to increase the purity, it is preferable that the reprecipitation with ethanol is performed twice or more. The obtained dried product contained thiomalate ion ([OOCCH (S) CH ₂ COO] ^3- ;
It may be referred to as A)), so that in order to remove it, the dried solid matter is dissolved in water and then applied to an anion exchange resin column (Cl ^-- form),
Elute with sodium chloride and collect and concentrate the fractions. The concentrate is then applied to a gel filtration column to remove sodium chloride, eluting with water and the fractions are collected and dried. By the above purification process, extremely high-purity sodium gold thiomalate can be obtained.

[0011]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.

Example 1 (Synthesis Method and Purification Method) 0.75 g (5 mmol) of thiomalic acid was dissolved in 10 ml of water, to which 0.40 g (10 mmol) of sodium hydroxide dissolved in 10 ml of water was added. Purple solution).

Sodium chloroaurate (NaAuCl ₄ .2H ₂ O) 0.50
g (1.25 mmol) was dissolved in 5 ml of water and added dropwise to the above purple solution while stirring, and stirred for 20 minutes to obtain a pale yellow transparent solution. The clear solution was filtered through Whatman # 2 fold filter paper, and then the filtrate was concentrated with an evaporator at 50 ° C. until it became slightly viscous. 250 ml of this concentrated solution was stirred
Was added dropwise to ethanol and the resulting white precipitate was filtered with a glass filter (Fine). The white precipitate was washed with 20 ml of ethanol and 20 ml of ether, and dried under vacuum at 50 ° C. overnight. Dissolve the dried precipitate in 10 ml of water and use Whatman # 2
It was filtered with a filter paper having a fold, and the filtrate was added dropwise to 250 ml of stirred ethanol. The obtained precipitate was collected with a membrane filter (Millipore FG 0.2μm) and ethanol 20m
After washing with 1 and 20 ml of ether and vacuum drying at 50 ° C. for one day, about 1 g of a white dry precipitate was obtained.

When this precipitate was analyzed by ¹ H NMR and FT-IR, it was a crude product containing a disulfide of thiomalate ion as a contaminant and had a high hygroscopic property. Therefore, purification was performed as follows.

The above crude product was dissolved in 5 ml of water and
an DE-52 anion exchange resin column (Cl ^-- form, diameter 1.5c
m × height 17 cm). The adsorbate was eluted with 1.0 M aqueous sodium chloride solution, and the fractions containing sodium gold thiomalate were collected with a fraction collector.
It was concentrated with an evaporator at ℃. In order to remove the sodium chloride mixed in the obtained concentrated liquid, this concentrated liquid was
Sephadex G-10 gel filtration column (diameter 1.5 cm x height 17 cm)
And eluted with water. The yellow eluate was collected by a fraction collector and dried under vacuum at 50 ° C. overnight. The final yield was 0.15 g (30% yield).

From the elemental analysis, thermal analysis (TG / DTA), 1 ^H NMR and ¹³ C NMR, the purified product obtained by the above production method was found to be a highly pure complex.

The complex was prepared stoichiometrically with a molar ratio of sodium chloroaurate: thiomalic acid: sodium hydroxide = 1: 4: 8. Sodium chloroaurate containing excess thiomalic acid and sodium hydroxide: Thiomalic acid:
Sodium hydroxide = 1: 5: 10 or 1: 8: 16
The yield of this complex was extremely low at the molar ratio of.

The complex is a yellow or pale yellow powder, is easily soluble only in water, is insoluble in all organic solvents, is unstable to light in an aqueous solution (decomposes when left at room temperature for several days), It was stable in the dark.

(Structural Analysis) Structural analysis of the present complex was carried out as follows.

(1) Total Element Analysis When measured by Mikroanalytisches Labor Pascher (Remagen, Germany), the results are shown in Table 1 below (unit: unit).
%).

[0021]

[Table 1]

The composition ratio of this complex is TMA: Au (I): Na ⁺ = 1:
When calculated assuming 1: 2 and 1.75 molecules of water attached, the calculated value is almost the same as the measured value. Therefore, the molecular formula is {Na ₂ [Au (OOCCH (S) CH ₂ COO)] 1.75H ₂ O}
_n (molecular weight 421.6n).

(2) FT-IR spectrum The results measured by the KBr method at room temperature using a Nicolet 510 FT-IR spectrometer are shown in (c) of FIG. For comparison, FT-IR spectra of thiomalic acid and sodium thiomalate are shown in (a) and (b), respectively.

The present complexes, the following significant absorption bands in the region of 1700～400Cm ^-1 were observed (both units of cm ^-1). Fifteen
83 (vs), 1403 (vs), 1311 (vw), 1265 (w), 1185 (w, br),
989 (w), 932 (w), 876 (m), 842 (vw), 693-558 (s, br).
The strong absorption at ~ 3450 cm ^-1 indicates the existence of water molecules, which supports the validity of the inclusion of water in the estimated molecular formula.

In the FT-IR spectrum (a) of thiomalic acid, S-
The H-based absorption at ~ 2560 cm ^-1 disappears in this complex (c), indicating the formation of an Au-S bond. Furthermore, the strong absorption at ~ 1690 cm ^-1 derived from the carbonyl group of thiomalic acid is similar to that of sodium thiomalate (b) at ~ 1590c even with this complex.
Since it is shifted to m ^-1 , it is considered that the Na ⁺ ion is attached to the carboxyl group.

The FT-IR spectrum of this complex was in agreement with the FT-IR spectrum of sodium gold thiomalate (Ciozole) clinically used (measured under the same conditions as above).

(3) Thermogravimetric analysis (TG) and differential thermal analysis (DTA) Simultaneously measuring TG and DTA using Seiko SSC 5000 TG / DTA 300 in air from 20 ° C to 500 ° C in 10 ° C increments. As a result, a loss of 6.93% was observed below 250 ° C, and the substance decomposed at 264 ° C. The weight loss of 6.93% is close to the theoretical value of 7.48% for 1.75 molecules of water, confirming the validity of the above estimated molecular formula.

(4) ¹ H NMR (399.65 MHz) and ¹³ C NMR (10
0.40MHz) In a tube with an outer diameter of 5mm at 25 ℃, JEOL JNM-EX 400 FT-NM
R spectrometer and JEOLEX-400 NMR data processing sys
It was measured using tem. DSS is used for the internal standard, and approximately 2% (w
/ v) in a D ₂ O solution at room temperature and 80 ° C. Moreover, in order to examine the presence or absence of polymerization, the measurement was performed in the presence of 0.5 M or 1.0 M NaCl. Further, to see the ligand exchange of the complex, room temperature, ¹ H NMR in D ₂ O solution, ¹³ C
NMR was measured in the presence of neutral or thiomalic acid ligands (2 or 3 equivalents). The system of the measured solution is the complex:
Thiomalic acid: NaOH = 0.05: 0.10: 0.25 (mmol) or 0.
It was a 0.55 ml D ₂ O solution containing 05: 0.15: 0.30 (mmol).

¹³ C NMR of this complex measured in D ₂ O at room temperature
The spectrum is shown in Fig. 2 (b) (* in the figure is the signal derived from the methylene group of DSS). This spectrum is
It was in agreement with the ¹³ C NMR spectrum of clinically used sodium gold thiomalate (Ciozole). For comparison, the ¹³ C NMR spectrum of thiomalic acid is shown in FIG. 2 (a). Comparing this complex with the ¹³ C NMR spectrum of the thiomalic acid ligand, this complex is shifted to the low magnetic field side, and the carbon resonances of methylene and methine are very close (5
0.0ppm and 49.7ppm), all ligands that are oligomeric but coordinated to gold (I) are equivalent, and the four simple signals in the spectrum indicate that the oxygen of the carboxyl group is a metal. The feature was that it was not coordinated.

¹ H NMR of this complex measured in D ₂ O at room temperature
The spectrum is shown in Fig. 3 (b) (* in the figure is the signal derived from the methylene group of DSS). This spectrum is
It was in agreement with the clinically used ¹ H NMR spectrum of sodium gold thiomalate (Ciozole). For comparison, the ¹ H NMR spectrum of thiomalic acid is shown in FIG. 3 (a). Comparing the ¹ H NMR spectra of this complex and the thiomalic acid ligand, the protons of methine were shifted to the low magnetic field side (0.3 ppm) and the protons of methylene were shifted to the high magnetic field side (0.1 ppm) in this complex. It was

Isab and Sadler reported that the thiolate exchange reaction easily occurred in an aqueous solution of gold disodium thiomalate (PJ Sadler; Struct. Bo.
nding., 1976, 29 , 171, AA Isab and PJ Sadle
r; J. Chem. Soc. Chem. Commun., 1976, 1051, AA
Isab and PJ Sadler; J. Chem. Soc. Dalton Tran
s., 1982, 135, AA Isab and PJ Sadler; J. Che
m. Soc. Dalton Trans., 1981, 1657). In fact, in the ¹ H NMR spectrum and ¹³ C NMR spectrum, apparent ligand exchange was carried out under the condition of pH ~ 7 in the presence of 2 or 3 equivalents of ligand to TMA-Au (I). There is. And, the chemical shift observed there depends on the amount of the added ligand (FIGS. 4 and 5). However, in the absence of ligand, ¹ H, ¹³ C of TMA-Au (I) at room temperature and 80 ° C.
When looking at the NMR spectrum, no chemical shift was observed (Fig. 2 (c) and Fig. 3 (c)). Report of ¹ H NMR measurement of gold disodium thiomalate in the presence of 0.5 M and 1.0 M NaCl (PJ Sadler; Struct. Bonding., 1976, 2
No evidence of polymerization as described in [ 9 , 171] [Fig. 2 (d) and (e), Fig. 3 (d)] was obtained.
And (e)]. From these results, it was considered that no new oligomer was formed or the ligand was dissociated in the aqueous solution. Therefore, it was found that this complex is an oligomer and stably exists in an aqueous solution.

(5) Molmass Measurement in Aqueous Solution The formula weight of the complex, that is, the molecular weight can be determined by multiplying the number of existing species in the aqueous solution by the molmass value. Further, the number of existing species in the solution in the equilibrium state is correlated with the degree of dissociation α (0 <α <1) of the weak electrolyte. α
Is the measured value of Na ⁺ at various Na concentrations (C _Na ) ([N
a ⁺ ]), and when the obtained [Na ⁺ ] plot for C _Na becomes a straight line, it is given as the slope.

The molmass of this complex in an aqueous solution is Mikroanal.
Using ytisches Labor Pascher (Remagen, Germany)
It was measured by the freezing point depression method. [Na ⁺ ] is measured by pH / Ion mete
r (Horiba F-23) with Na ⁺ ion selective electrode (Horiba 1512A-
10C) and a reference electrode (Horiba 2565A-10T)
Six points were taken in the range of _Na of 0.914 to 182.9 _mM , and the test was repeated 3 times.

In a control experiment, sodium chloride (molecular weight 58), which is considered to be a strong electrolyte (α = 1) in an aqueous solution, was used.
Using disodium succinate (molecular weight 162) as a standard substance, the molmass values were 29 and 59, respectively (experimental error ± 5%).

11.46 mg of this complex was dissolved in 0.397027 g of water, and mo
When the lmass value was measured, it was 330 (measurement error ± 5%). Moreover, when 6 points were set in the Na concentration range of 0.914 to 182.86 mM and Na ⁺ was measured using a Na ion selective electrode, a straight line with a correlation coefficient of 1.00 to 0.996 was drawn. The slope of this straight line, that is, α was 0.44 to 0.54. Therefore, the dissociation formula of this complex in water is

[0036]

Embedded image

When the dissociation degree is α, the number of moles of all molecular species existing in the aqueous solution can be calculated from this dissociation equation as (1 + 2nα)
Is. Therefore, the molecular weight can be shown as 421.6n = 330 (1 + 2nα). Substituting the value of α into this formula to calculate the value of n, n = 2.5 to 5.0, and the molecular weight of this complex is 1054 to 210.
8

On the other hand, if the dissociation formula of this complex is

Embedded image

Assuming that, the number of moles of all molecular species existing in water is (1 + nα), and the molecular weight is 421.6n = 330 (1 + n
α). Here, when the value of n is calculated by substituting α = 0.44 to 0.54, n = 1.19 to 1.36, which is unreasonable because the value is too small. That is, the compound obtained by this synthetic method was considered to be an oligomer.

Example 2 (Adhesion Factor Expression Inhibition Test) Although the mechanism of action of the gold preparation is not clear, recent findings indicate that ICAM-1 has an important function in cell-cell communication.
(intercelluar adhesion molecule-1), VCAM-1 (vascula
r cell adhesion molecule-1), ELAM-1 (endothelial le)
It is considered that the inflammatory reaction may be suppressed by suppressing the expression of cell adhesion molecules such as ukocyte adhesion molecule-1). Therefore, the biological activity of this complex
The inhibitory effect of cell adhesion molecule expression was used for in vitro studies.

Hydrocortisone, heparin, EGF, ECG
Human umbilical vein endothelial cells (HUVEC) were cultured in a 5% CO ₂ incubator (37 ° C.) to a saturated cell density using MCDB131 medium containing S and 2% FCS. After culturing, TNF-α
(5 ng / ml) and this complex were added and the culture was continued. TNF
-Trypsin-ED 24 hours after adding α and the complex
The cells were detached with TA solution, and mouse anti-CD54 (ICAM-
1) Monoclonal antibody, mouse anti-VCAM-1 monoclonal antibody or mouse anti-ELAM-1 monoclonal antibody was reacted on ice for 45 minutes. After the reaction, the cells were washed 3 times with DME medium containing 2% FCS, and then horseradish peroxidase-labeled goat anti-mouse IgG antibody was reacted with the cells for 45 minutes on ice. After the reaction, the cells were washed 3 times with DME medium containing 2% FCS, and FACS analysis was performed. In addition, the same test as above was carried out using sodium gold thiomalate (trade name: Ciosol, Shionogi Pharmaceutical Co., Ltd.) currently clinically used, and the biological activity of this complex was compared.

FIG. 6 shows the measurement results of the expression-suppressing activity of various adhesion molecules at a concentration of 100 μg / ml of the present complex and at a concentration of 100 μg / ml of Siozole. Neither drug showed ICAM-1 expression inhibitory activity, but both drugs showed similar levels of VCAM-1 and EL.
It showed the activity of suppressing the expression of AM-1. Therefore, this complex is
Also in terms of biological activity, it is judged to be equivalent to clinically used sodium gold thiomalate.

[0043]

Industrial Applicability According to the method of the present invention, it is possible to produce a single piece of sodium gold thiomalate. The complex produced by the method of the present invention is chemically and biologically equivalent to sodium gold thiomalate currently clinically used.

[Brief description of drawings]

FIG. 1 is an FT-IR spectrum of this complex measured by the KBr method. In the figure, (a) is thiomalic acid, (b) is thiomalic acid sodium salt (a solution containing TMA and sodium hydroxide equivalent to its 3 equivalents is dried), (C) is the spectrum of this complex, Shown respectively.

FIG. 2: ¹³ C NMR spectrum (D ₂ O solution, internal standard DS
S). (a) is thiomalic acid (room temperature), (b) is this complex (room temperature), (c) is this complex (80 ℃), (d) is this complex (0.5MN).
(aCl in the room temperature), (e) is the present complex (in the presence of 1.0M NaCl,
Room temperature). Note that * in the figure is a signal derived from the methylene group of DSS.

FIG. 3 ¹ H NMR spectrum (D ₂ O solution, internal standard DSS)
Is. (a) is thiomalic acid (room temperature), (b) is this complex (room temperature), (c) is this complex (80 ° C), (d) is this complex (0.5M NaCl).
In the presence of room temperature), (e) shows the present complex (in the presence of 1.0 M NaCl, room temperature). The symbols *, **, and * in the figure indicate signals derived from water, mixed acetonitrile, and methylene group of DSS, respectively.

FIG. 4 ¹³ C NMR spectrum (D ₂ O solution, internal standard DS
S, room temperature, neutral solution). (a) is thiomalic acid + NaOH
(2 equivalents), (b) shows this complex, (c) shows this complex + thiomalic acid (2 equivalents), (d) shows this complex + malic acid (3 equivalents), respectively. In the figure, * indicates a signal derived from the methylene group of DSS.

FIG. 5 ¹ H NMR spectrum (D ₂ O solution, internal standard DSS,
Room temperature, neutral solution). (a) is thiomalic acid + NaOH (2
Equivalent), (b) is this complex, (c) is this complex + thiomalic acid (2
(Equivalent) and (d) are the present complex and thiomalic acid (3 equivalents), respectively. In the figure, * and * are signals derived from methylene groups of water and DSS.

[Fig. 6] Fig. 6 is a graph showing the suppression of the expression of adhesion molecules by the present complex, as compared with that of shiozole, by the suppression effect of the expression of adhesion factors induced by TNF-α.

 ─────────────────────────────────────────────────── ─── Continued front page (72) Inventor Hiroyuki Ito 540 Narita, Odawara, Kanagawa Meiji Dairy Co., Ltd., Cell Engineering Center (72) Inventor, Sadatoshi Sakuma, 540 Narita, Odawara, Kanagawa Cell Engineering Co., Ltd. In the center (72) Kenji Nomiya Kenji Nomiya 390 No. 6 Furuki, Hadano City, Kanagawa Prefecture

Claims (2)

[Claims] 1. A process for producing gold sodium thiomalate, which comprises reacting thiomalic acid, sodium hydroxide and sodium chloroaurate in an aqueous solution. 2. The method for producing gold sodium thiomalate according to claim 1, wherein the molar ratio of sodium chloroaurate, thiomalic acid and sodium hydroxide in the reaction is 1: 4: 8.