JPH07101972A - Anti-tumor agent and its production - Google Patents

Abstract

PURPOSE:To inexpensively and safely obtain an anti-tumor agent which has the action to increase the carcinostatic action of cisplatin in addition to its own powerful action to inhibit the growth of cancer cells by preparing a saturated solution of a crude chloro-osmocenium salt and effecting the recrystallization of the salt. CONSTITUTION:The objective antitumor agent containing a chloro-osmocenium salt of the formula [Y is anion, preferably hexafluorophosphate ion (PF6<->), tetrafluoroborate ion or picrate ion] is obtained by preparing a saturated aqueous solution of a crude chloro-osmocenium salt and effecting the recrystallization.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel antitumor agent and a method for producing the same.

[0002]

2. Description of the Related Art Alkylating agents, nucleic acid metabolism-inhibiting agents, antibiotics, plant alkaloids and the like are used as chemotherapeutic agents for cancer, 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. It is known that the ferrocenium salt represented by the following formula (II) affects 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]

[Chemical 2]

[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, an action mechanism similar to that of ferrocenium salt is expected, and further, development of an antitumor agent which has a stronger cancer cell growth inhibitory action and is stable in an aqueous solution is desired.

[0006]

The present inventors have synthesized and tested a large number of Os-containing metallocenium salts in order to overcome the drawbacks of such conventional organometallic anticancer agents. As a result, the following formula (III)

[0007]

[Chemical 3]

[0008] 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 inhibitory agent for cancer cell growth. Based on this finding, the present invention has been completed. That is, the present invention provides: (1) an antitumor agent comprising a chloroosmosenium salt represented by the following formula (I) as an active ingredient,

[0009]

[Chemical 4]

[0010] (wherein, Y represents an anion.) (2) Y anions hexafluorophosphate ion (PF ₆ ^-), tetrafluoroborate ion, characterized in that it is a picrate ion (1) term (3) A saturated aqueous solution of a crude chloroosmosenium salt is prepared and recrystallized from this to obtain a purified chloroosmosenium salt represented by the formula (I) according to item (1). The present invention provides a method for producing a characteristic antitumor agent.

The anion for the chloroosmosenium cation represented by the above formula (I) used in the present invention 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 and the like are used. In addition, ammonium hexafluorophosphate, sodium tetrafluoroborate and the like are represented by the formula (I) of the present invention.
It was confirmed that the compound has almost no antitumor activity in the concentration range applied, and the cancer cell growth inhibitory action in the present invention is considered to be due to the chloroosmosenium 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]

[Chemical 5]

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 to 4 to 10 ° C to obtain purified chloroosmosenium salt as crystals. 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.

[0017]

[Function] In order to study the mechanism of action, the consumption rate of dissolved oxygen by cells in the culture medium was measured using an oxygen electrode. In the presence of the compound (1) included in the formula (I) (Y ⁻ is PF ₆ ⁻ ), the O ₂ consumption rate is 4% as compared with the control.
It had dropped to 7%. 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 is based on respiratory inhibition, which is different from the mechanism of action of cisplatin by binding to nucleic acid.

[0018]

The present invention will be described in more detail based on the following 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

Example 2 S180 mouse sarcoma cells, U937 human histiocytic lymph
Tumor 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) to 1.07 × 10^Five Suspended at a rate of 24 cells / ml, 24
Dispense 1.4 ml to each well of the well multiplate
did. 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 aqueous solution containing 15 mM concentration or pure water
Add 0.1 ml to each well and CO at 37 ℃₂ ink
After culturing for 72 hours in an incubator, count the number of cells by Coulter Cow
Measured. From the curve of cell number vs. sample concentration
Inhibits proliferation to 50% of control cell number
Sample concentration (IC₅₀ , ΜM) was determined. The results are shown in Table 1.
Summarized. For reference, the ferrocenium salt (of formula (II)
Compound, but X^- Is PF₆ ^-) And osmosenium salt (formula
The results for (III) compounds are also shown. From this table
As is apparent, compound (1) of formula (I) (formula (I),
However, Y^-Is PF₆ ^-) Is a ferro for all cells
Cenium salt (formula (II), where X^-Is PF₆ ^-) Or Osmo
IC smaller than selenium salt (formula (III))₅₀Showed and did
Therefore, it has a stronger cancer cell growth inhibitory effect.
Light In addition, according to the acute toxicity test,
The semi-lethal dose in mice is 50 mg / kg or less.
It was

[0020]

[Table 1]

Example 3 The cell growth inhibitory rate when coadministration of 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, the compound (1) of the present invention
Shows that it has an effect of proliferating the cancer cell growth inhibitory action of cisplatin by 2 to 3 times more.

[0022]

[Table 2]

Example 4 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, the same number of cells was treated with the compound (1) of the present invention (wherein Y ⁻ is PF ₆ ^{− in the} formula (I)) as an IC _50.
After incubating for 1 hour in a culture containing about 10 times the concentration (200 μM), the oxygen reduction rate was measured in the same manner as above, and it was 47% of that in the case where compound (1) was not given. Therefore, the compound (1) of the present invention has an inhibitory action on cell respiration. 10 μM of cisplatin (= I
A similar test was conducted using C ₅₀ × 5), but no significant respiratory inhibition was observed.

[0024]

INDUSTRIAL APPLICABILITY As described above, the antitumor agent of the present invention containing the purified chloroosmosenium salt of the formula (I) as an active ingredient has a strong inhibitory effect on the growth of cancer cells, It has a different mechanism of action and has the effect of enhancing the carcinostatic action of cisplatin. The antitumor agent of the present invention remarkably suppresses the growth of a wide range of tumor cells. Further, according to the production method of the present invention, an antitumor agent having such an excellent effect can be produced 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 (3)

[Claims] 1. An antitumor agent comprising a chloroosmosenium salt represented by the following formula (I) as an active ingredient. [Chemical 1] (In the formula, Y represents an anion.) 2. The anion of Y is hexafluorophosphine.
Toion (PF₆ ^- ), Tetrafluoroborate io
Or a picrate ion
Item 1. The antitumor agent according to item 1. 3. An antitumor characterized in that a saturated aqueous solution of a crude chloroosmosenium salt is prepared and then recrystallized to obtain the purified chloroosmosenium salt represented by the formula (I). Method of manufacturing agent.