JPH08208676A - Production of chloroosomocenium slat - Google Patents

Abstract

PURPOSE: To inexpensively and safely obtain purified chloroosomocenium salt useful as an antitumor agent stable in an aqueous solution, having strongly inhibitory action on proliferation of cancerous cells, by preparing a saturated aqueous solution of a crude chloroosomocenium salt and recrystallizing chloroosomocenium salt from the aqueous solution. CONSTITUTION: This purified chloroosomocenium salt of the formula (Y is an anion) is obtained by preparing a saturated aqueous solution of a crude chloroosomocenium salt and recrystallizing the aqueous solution. Hexafluorophosphate ions or tetrafluoroborate ions are used as the anion. The chloroosomocenium salt, for example, is obtained by oxidizing osomocene on the market with ferric chloride in the presence of hydrochloric acid and precipitating the purified chloroosomocenium by adding a salt of a proper anion.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a chloroosmosenium salt.

[0002]

2. Description of the Related Art Alkylating agents, nucleic acid metabolism inhibitors, antibiotics, plant alkaloids and the like are used as cancer chemotherapeutic agents, and many new antitumor agents are being developed year by year. However, there are very few cases where a complete cure can be achieved with only chemotherapy except for a few cancer types, and the appearance of a better antitumor agent is desired. Among conventional anticancer agents, platinum complexes represented by cisplatin (cis-dichloroamineplatinum) may be effective for solid tumors in which other agents do not show effective effects, and thus platinum and other metal element-containing compounds. Antitumor agents are being actively searched for. However, no compound has been found that outperforms cisplatin. On the other hand, the appearance of cisplatin-resistant cells is also a problem. The mechanism of action of cisplatin is intracellular D
It is considered to be a modification of NA, but from the viewpoint of complementing cisplatin and damaging cisplatin-resistant cells, it is important to develop a metal-containing antitumor agent having a different mechanism of action from cisplatin. The ferrocenium salt represented by the following formula (II) is known to affect the electron transport system in cells (Carney et al., J. Am. Chem. Soc., 1
06 , 2565 (1984)) Growth suppression at cell level (Koep
f et al., J. Cancer Res. Clin. Oncol., 108 , 336 (198
4)) and its effectiveness as an antitumor agent has been revealed (Koepf et al., DE 3404443A1). In particular, synergistic effects in co-administration with cisplatin have been noted (Neuse et al., Appl. Organomet. Chem., 4 , 19).
(1990)). Formula (II)

[0003]

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[0004] (wherein, X ^- is hexafluorophosphate ion (PF ₆ ^-), tetrafluoroborate ion,
Anions such as picrate and tetrachloroferrate. )

[0005]

However, the efficacy of this ferrocenium salt is not satisfactory in actual treatment. Further, the aqueous solution is unstable at neutral and gradually decomposes to form a precipitate. Therefore, a compound having the same mechanism of action as that of ferrocenium salt is expected, and further, a compound having a stronger cancer cell growth inhibitory action and more stable in an aqueous solution and usable as an antitumor agent is desired.

[0006]

In view of such circumstances, the present inventors have synthesized and tested a large number of Os-containing metallocenium salts. As a result, the following formula (III)

[0007]

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Although the osmosenium salt itself showed a weak inhibitory effect on cancer cells, a chloroosmosenium salt was obtained as a purified product by recrystallization from an aqueous solution, which is a very strong inhibitor for cancer cell growth. Based on this finding, the present invention has been completed. That is, the present invention comprises (1) preparing a saturated aqueous solution of a crude chloroosmosenium salt, and recrystallizing the saturated aqueous solution to obtain a purified chloroosmosenium salt represented by the following formula (I). Salt production method

[0009]

[Chemical 4]

(Wherein Y represents an anion).

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The above formula (I) used in the present invention
The anion for the chloroosmosenium cation represented by is not particularly limited as long as it is an anion sufficient to neutralize the cation and is pharmaceutically acceptable,
In addition to the above hexafluorophosphate ion, tetrafluoroborate ion, picrate ion or the like is used. It has been confirmed that ammonium hexafluorophosphate, sodium tetrafluoroborate and the like have almost no antitumor activity in the concentration range in which the compound of the formula (I) of the present invention is applied, and the effect of suppressing cancer cell growth is chloroosmose. It is thought that this is due to the nickel cation.

The compound of the present invention can be synthesized by oxidizing commercially available osmosene with ferric chloride in the presence of hydrochloric acid, and adding a salt of a suitable anion to precipitate the compound. Conventionally, the manufacturing method itself of an osmosenium salt is known (Smith
Et al., Inorg. Chem., 26 , 2882 (1987)) uses an organic solvent for purification by recrystallization, and the structure of the product has not been clarified hitherto, and a method for producing a chloroosmosenium salt according to the present invention is disclosed. Is not known. The production method of the present invention is characterized in that water is used as a recrystallization solvent. The synthetic reaction of the chloroosmosenium salt represented by the formula (I) according to the present invention can be represented by the following formula.

[0013]

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The oxidant is not limited to ferric chloride, but if hydrochloric acid is present, iron alum (FeNH ₄ (SO ₄ ) ₂ .12H
₂ O) may be used. The osmosenium salt formed precipitates as a crude product, which is usually used in the present invention at a saturated concentration (5 mg) at a temperature of 40 to 60 ° C.
/ ml to 10 mg / ml), the insoluble matter is filtered off, and the filtrate is left to cool at 4 to 10 ° C to obtain crystals as a purified chloroosmosenium salt. In the method of the present invention, by using water as a solvent, high-purity crystals can be obtained, which is advantageous in that they are inexpensive and safe.

The compound represented by the above formula (I) exhibits an excellent antitumor action. For example, this chloroosmosenium hexafluorophosphate (Y ^{− in the} formula (I) is P
F ₆ ^-) is as shown in the experimental examples described later, strongly inhibits the growth of a variety of cancer cells. Comparing this activity by IC ₅₀ ,
In any cell, the compound of formula (I) shows a smaller value than the ferrocenium salt (formula (II), where X ⁻ is PF ₆ ⁻ ), and is more potent, but especially U of human cancer cells.
It is 3 to 5 times stronger than 937 and COLO320. Furthermore, when co-administered with cisplatin, it has the effect of dramatically increasing the carcinostatic effect of 0.3 μM cisplatin, which is not effective by itself. Thus, in addition to having a strong anti-cancer effect, the aqueous solution is stable at room temperature for several tens of hours, and does not cause gradual precipitation unlike the ferrocenium salt (formula (II)).

The compounds of formula (I) may be prepared for administration in any conventional manner by analogy with other antitumor agents such as cisplatin. Therefore, the present invention also comprises at least one compound represented by the formula (I) or a pharmaceutical composition thereof, which is suitable as a medicine for human body.
Such a composition is provided for use in a conventional manner with any required pharmaceutical carrier or excipient.

In order to study the mechanism of action, the rate of consumption of dissolved oxygen by the cells in the culture medium was measured using an oxygen electrode. Compound (1) (Y ⁻ is P is included in the formula (I)
F ₆ ^- In the presence of) the O ₂ consumption rate was reduced to 47% compared to the control. On the other hand, the compound (1) was mixed with adenosine or guanosine in a heavy aqueous solution and the NMR spectrum was measured, but the coordination to these nucleic acid bases was hardly observed. Therefore, it is speculated that the cancer cell growth inhibitory action may be based on respiratory inhibition,
This point is different from the mechanism of action of cisplatin by binding to nucleic acid.

[0018]

Next, the present invention will be described in more detail with reference to examples. Example 1 966 mg of commercially available osmosene was dissolved in 70 ml of benzene, and 42 ml of 4N hydrochloric acid and 5.74 g of second chloride were dissolved in this solution.
Iron (hexahydrate) was added, and the mixture was vigorously stirred at room temperature for 24 hours.
After adding 35 ml of water to dissolve the insoluble matter, the benzene layer was removed. Then, the water layer is added with 50 ml of benzene for 2 times.
Washed twice. This water layer was ice-cooled, 5 ml of water containing 2.33 g of ammonium hexafluorophosphate was added,
Stir well and let stand at 4 ° C. for 12 hours. The precipitate was collected by filtration, washed with a small amount of cold water, and dried under reduced pressure to obtain 1.474 g of a slightly reddish powder (yield 98%). This crude product 1.2
g was dissolved in 192 ml of water at 50 ° C., the insoluble matter was filtered off, and the mixture was ice-cooled to obtain 1.1 g of a red needle-shaped product of the compound (1) of formula (I) (Y ⁻ is PF ₆ ⁻ ). Yield from 89
%). Decomposition point 180 ° C or higher. Elemental analysis value: carbon, 24.
03%; hydrogen, 2.07%; chlorine, 6.96% (calculated value for molecular formula C ₁₀ H ₁₀ ClOsPF ₆ is carbon 23.9.
8%, 2.01% hydrogen, 7.08% chlorine). This crystal was subjected to X-ray crystallography to determine its structure, and its crystallographic data were as follows. Crystal form: triclinic Lattice constant: a = 6.589, b = 9.417, c = 1
0.382Å α = 84.71, β = 88.30, γ = 87.71 ° Z = 2, V = 640.7Å ³

Reference Example 1 S180 mouse sarcoma cells, U937 human histiocytic lymphoma cells, HL60 human promyelocytic leukemia cells and Colo
320 MEM containing 5% fetal bovine serum containing human colon adenocarcinoma cells
RPM containing medium (S180) or 10% fetal bovine serum
I1640 medium (U937, HL60, Colo32
0) suspended at 1.07 × 10 ⁵ cells / ml, and
1.4 ml was dispensed into each well of the well multiplate. Compound (1) obtained in Example 1 was added at 1.5 mM, 0.1 mM.
45 mM, 0.15 mM, 0.045 mM or 0.0
0.1 ml of an aqueous solution containing a concentration of 15 mM or 0.1 ml of pure water was added to each well, and after culturing for 72 hours in a CO ₂ incubator at 37 ° C, the number of cells was counted with a Coulter counter. From the curve of cell number vs. sample concentration, the sample concentration (IC ₅₀ , μM) at which the proliferation was suppressed was determined so as to be 50% of the control cell number. The results are summarized in Table 1. For reference, the results for the ferrocenium salt (compound of formula (II), where X ⁻ is PF ₆ ⁻ ) and the osmosenium salt (compound of formula (III)) are also shown. As is clear from this table, the compound (1) of formula (I) (formula (I),
However, Y ⁻ is PF ₆ ⁻ ) shows an IC ₅₀ smaller than that of ferrocenium salt (formula (II), X ⁻ is PF ₆ ⁻ ) or osmosenium salt (formula (III)) in any cell, and therefore, It can be seen that it has a stronger cancer cell growth inhibitory action. In addition, according to the acute toxicity test, the compound (1)
The semi-lethal dose in mice was 50 mg / kg or less.

[0020]

[Table 1]

Reference Example 2 The cell growth inhibitory rate when co-administered with 0.3 μM or 1.0 μM of cisplatin and 15 μM of the compound (1) of the formula (I) (Y ^{− in the} formula (I) is PF ₆ ⁻ ) is the same as above. Tested. The control was a case where neither cisplatin nor compound (1) was administered, and in Table 2, the number of cells at this time was 10
It is represented by 0. From this table, it is understood that the compound (1) obtained by the method of the present invention has an effect of proliferating the cancer cell growth inhibitory action of cisplatin by 2 to 3 times.

[0022]

[Table 2]

Reference Example 3 7 × 10 ⁶ U937 cells were added to 1 m of RPMI medium containing serum.
It was suspended in 1 and the rate of decrease of dissolved oxygen in the medium was measured at 30 ° C. with an oxygen electrode. On the other hand, in the same number of cells, the compound (1) obtained by the method of the present invention (wherein Y ⁻ is PF
₆ ^-) for one hour incubation in culture with approximately 10-fold concentration (200 [mu] M) of the IC _50, was measured in the same manner as the above oxygen reduction rate, 47% in the case which does not give the compound (1) there were. Therefore, the compound (1) of the present invention has an inhibitory action on cell respiration. A similar test was conducted using 10 μM of cisplatin (= IC ₅₀ × 5), but no significant respiratory inhibition was observed.

[0024]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, in addition to having a strong cancer cell growth inhibitory effect, it has a different mechanism of action from cisplatin and potentiates the antitumor effect of cisplatin. The purified chloroosmosenium salt can be manufactured inexpensively and safely.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shuichi Oka 1-3-1, East Tsukuba, Ibaraki Industrial Technology Institute of Biotechnology, Institute of Biotechnology (72) Inventor Hiroaki Okuno 1-3-1, East Tsukuba, Ibaraki Industrial Technology Institute of Industrial Science and Technology (72) Inventor Midori Goto 1-1, Higashi, Tsukuba, Ibaraki Prefecture Institute of Industrial Science and Technology, Institute of Materials Engineering

Claims (1)

[Claims] 1. A purified chloroosmosenium salt represented by the following formula (I) is obtained by preparing a saturated aqueous solution of a crude chloroosmosenium salt, and recrystallizing the saturated aqueous solution. Production method. Embedded image (In the formula, Y represents an anion.)