JP2003002878A - Method for preparing 2-aminophenoxazin-3-one derivative or 3-aminophenoxazin-2-one derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing a 2-aminophenoxazin-3-one derivartive or a 3-aminophenoxazin-2-one derivative, having antiotumor activities, which have been synthesized by using human orbovine hemoglobin as a raw material, but this method includes some problems due to expensive cost of the raw material. SOLUTION: The 2-aminophenoxazin-3-one derivative or 3-aminophenoxazin-2- one derivative can be prepared by reacting an o-aminophenol derivative represented by formula I [wherein, R1 and R2 are each selected independently from the group consisting of hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms and an acyloxy group having 1 to 4 carbon atoms] with an oxidizing agent having trivalent iron at pH 5 to 9.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a 2-aminophenoxazin-3-one derivative or a 3-aminophenoxazin-2-one derivative.

[0002]

Actinomycin D is a compound having a strong antitumor activity against tumors such as renal sarcoma. However, since actinomycin D has strong side effects,
At present, it is only used experimentally as an inhibitor of DNA-dependent RNA polymerase. Therefore, antitumor activity of 2-aminophenoxazin-3-one derivatives, which are the basic skeleton of actinomycin D, has been investigated (Honohashi et al., Potential Antitumor Phenoxazines,
Medical Research Review, (1991), Volume 11, 250-254
page).

2-Aminophenoxazin-3-one 7
A phenoxazine derivative substituted at the position, for example 2-amino-4,4a-dihydro-4a, 7-dimethyl-3H-
Phenoxazin-3-one (hereinafter referred to as "2-amino-PX
Z-3-one ”) is known to show a strong antitumor activity against human cancer cells (Japanese Patent Laid-Open No. 02-
193984). Further, 2-amino-PXZ-3-one can be used by dissolving it in a small amount of ethanol (for example, 99.9%) and then diluting it with distilled water (finally 0.5%). 2 mL of water containing 1 mL of ethanol
4 μg of 2-amino-PXZ-3-one dissolves).
Therefore, it is preferable from the viewpoint of preparation since it can be dissolved and used as an injection upon use. Furthermore, 2-amino-
PXZ-3-one, compared to actinomycin D,
It has the advantage of relatively few side effects.

A known method for synthesizing 2-amino-PXZ-3-one is to react 2-amino-5-methylphenol in the presence of human or bovine hemoglobin at 37 ° C. for 3 days. (Japanese Patent Laid-Open No. 02-1939)
84).

However, this synthetic method using human or bovine hemoglobin has the following drawbacks. That is, since a large amount of human or bovine blood is required for the synthesis reaction, the raw material cost is high, and the synthesis reaction time is 72 hours.
Time and long point, synthesized 2-aminophenoxazine-
The processing cost is high because the processing for purifying the 3-one derivative from human or bovine hemoglobin requires a long time of up to 2 weeks. Due to problems such as mad cow disease, bovine blood for pharmaceuticals is used. There is a problem that it is difficult to use for compound synthesis.

A phenoxazine derivative in which the 8-position of 3-aminophenoxazin-2-one is substituted, specifically 3-amino-1,4a-dihydro-4a, 8-dimethyl-2H-phenoxazine-2. As for "-one", similarly to the above-mentioned "2-amino-PXZ-3-one", a method of reacting 2-amino-4-methylphenol in the presence of human or bovine hemoglobin at 37 ° C is known. (Oxidativ
e Condensation of 2-Amino-4-methylphenol to Dihydr
ophenoxazinone Compound by Human Hemoglobin, Akio
Tomoda et. Al., J. Biochem, 110, 1004-1007,
(1991)).

[0007]

[Problems to be Solved by the Invention] Heretofore, in a method for synthesizing a 2-aminophenoxazin-3-one derivative or a 3-aminophenoxazin-2-one derivative, the reaction was carried out in the presence of human or bovine hemoglobin. There is no known method other than synthesis. Therefore, an object of the present invention is to provide a method that can chemically synthesize a 2-aminophenoxazin-3-one derivative or a 3-aminophenoxazin-2-one derivative at a low cost in a short period of time. That is, the present invention is as follows.

[0008]

2-Aminophenoxazin-3-one derivative or 3-aminophenoxazine-2
The -one derivative has the formula I

[Chemical 2] (In the formula I, R ₁ and R ₂ are each independently a group consisting of a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms and an acyloxy group having 1 to 4 carbon atoms. Can be produced by reacting an orthoaminophenol derivative represented by (1) to an oxidizing agent having ferric iron at pH 5-9.

Preferably, the reaction is carried out in an amount such that the molar ratio of the oxidizing agent to the orthoaminophenol derivative is 0.1 or more and less than 1.0. Preferably, the oxidizing agent having trivalent iron is hexacyanoferrate (III), and more preferably the hexacyanoferrate (III) is potassium ferricyanide. Preferably pH
Is 5-8, more preferably pH 6-7.5. Preferably the reaction is carried out in water or a water soluble solvent, preferably the water soluble solvent is methanol or ethanol. Preferably, in the formula I, R ₁ and R ₂ are orthoaminophenol derivatives selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group or an acetyl group, and more preferably in the formula I, R ₁ is An orthoaminophenol derivative in which CH ₃ and R ₂ are hydrogen atoms, or an orthoaminophenol derivative in which R ₂ is CH ₃ and R ₁ is a hydrogen atom.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a 2-aminophenoxazin-3-one derivative or a 3-aminophenoxazin-2-one derivative is synthesized using an orthoaminophenol derivative as a starting material.

Formula I

[Chemical 3] The compound represented by represents an orthoaminophenol derivative. Here, in the formula I, R ₁ and R ₂ each independently represent a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms and an acyloxy group having 1 to 4 carbon atoms. It is arbitrarily selected. For example, in the case of 2-amino-5-methylphenol, R ₁ is CH ₃ and R ₂ is a hydrogen atom, and in the case of 2-amino-4-methylphenol, R _{2 is}
Is CH ₃ and R ₁ is a hydrogen atom.

The oxidizing agent is preferably a drug having ferric iron therein. Preference is given to using hexacyanoferrate (III) (formula M ^I ₃ [Fe (CN) ₆ ], where M ^I represents a monovalent cation). Examples of preferred M ^I include (NH ₄ ) ₃ , 1/2 (K ₃ Na ₂ ), 1 /
2Mg ₃ , Mg (NH ₄ ), 1 / 2Ca ₃ , CaK, 1 /
2Sr ₃ , Sr (NH ₄ ), 1 / 2Ba ₃ , BaK, 1 /
2Zn ₃ , 1 / 2Cd ₃ , 1 / 2Cu ₃ , Ag ₃ , Ce, Z
r ₃ , 1 / 2Sn ₃ , 1 / 2Pb ₃ , Bi, 3 / 5Bi,
1/2 Hg ₃ is included. Particularly preferable is potassium hexacyanoferrate (III) (that is, potassium ferricyanide).

More specifically, the method for producing the 2-aminophenoxazin-3-one derivative or the 3-aminophenoxazin-2-one derivative according to the present invention is as follows. Hereinafter, a case where 2-amino-5-methylphenol is used as a starting material and potassium ferricyanide is used as an oxidizing agent will be described. However, the starting material and the oxidizing agent are not limited to these and are not limited. Nor is it understood by.

The above-mentioned 2-amino-5-methylphenol and potassium ferricyanide are reacted by the following method.
Since 2-amino-5-methylphenol is completely dissolved in the solvent, an aqueous hydrochloric acid solution is added. Subsequently, a sodium hydroxide aqueous solution is added to adjust the pH to 5-9.
Preferably pH 5-8, more preferably pH 6-7.
Adjust to 5. Water or a water-soluble solvent can be used as the solvent. Examples of the water-soluble solvent include methanol,
Ethanol, acetone, acetonitrile and the like can be used. Particularly preferably, the water-soluble solvent is methanol or ethanol.

In order to react 2-amino-5-methylphenol with potassium ferricyanide, it is preferable to carry out the reaction at a pH of 5-9. When the pH is less than 5, 2-aminophenoxazine-the target substance of the present application
Derivatives of 3-one cannot be obtained efficiently, and the synthesized compounds do not show antitumor activity. When the pH is higher than 9, many by-products are generated, which is not preferable. The pH is preferably 5 to 8, more preferably 6 to 7.5. Particularly, at pH 6 to 7, a derivative of 2-aminophenoxazin-3-one, which is a target substance, is synthesized with high purity, which is preferable.

While the solution containing 2-amino-5-methylphenol having the pH adjusted to 5 to 9 is well stirred and mixed, an aqueous potassium ferricyanide solution is dropped little by little into the solution to carry out the reaction. Immediately before dropping the potassium ferricyanide aqueous solution into the solution, the pH was adjusted to 5
It is preferable to adjust to -9. This is because after adjusting the pH, 2-amino-5- that was dissolved when left for a long time
Methylphenol easily precipitates, which is the target substance 2
The yield of the aminophenoxazin-3-one derivative and
This is because the purity decreases. After adjusting the pH, it is preferable to start dropwise addition of an aqueous potassium ferricyanide solution to the solution within 30 minutes, more preferably within 5 minutes.

The molar ratio of potassium ferricyanide to 1 mol of 2-amino-5-methylphenol is preferably 0.1 to less than 1.0 mol, more preferably 0.125.
˜0.8 mol, more preferably 0.25 to 0.5 mol. When the molar ratio of potassium ferricyanide to 2-amino-5-methylphenol is less than 0.1, the reaction is difficult to proceed, and when the molar ratio is 1.0 or more, that is, equimolar or more, tar-like water solubility This is because the compound is mainly produced and the yield of the phenoxazine compound, which is the target substance, deteriorates. Thus, 2-amino-5
Depending on the molar ratio of potassium ferricyanide to methylphenol, the yields of the product formed and the desired compound differ.

In the reaction solution to which the potassium ferricyanide aqueous solution is added, the starting material and the target substance are not decomposed and the reaction is carried out at a temperature at which the reaction efficiency is good, but preferably 4 to 37 ° C., more preferably 20 to 30 ° C. C., more preferably 23 to 27.degree. The reaction time is preferably 10 minutes to 2 hours, more preferably 30 minutes to 1
It can be time.

After the reaction, in order to purify the phenoxazine compound which is the target substance, it is preferable to perform the following purification operation.

The solvent in the reaction solution is removed by treating it with any distillation device such as a vacuum evaporator. Next, by adding methanol to the residue after the distillation, the phenoxazine compound as the target substance and 2-amino-5-methylphenol as the starting material are dissolved in methanol. On the other hand, potassium ferricyanide used as an oxidizing agent is insoluble in methanol and thus precipitates. The precipitate is settled by using the methanol solution using an arbitrary device such as a centrifuge. In this way, the supernatant containing the target substance and the starting material can be separated from the methanol-insoluble precipitate containing potassium ferricyanide.

Finally, in order to further purify the target substance, for example, Sephadex (registered trademark of Amersham Pharmacia Japan KK) LH-20, LH
It can be subjected to column chromatography using H-60 or the like. The carrier is preferably sufficiently swelled and equilibrated with methanol, ethanol, water and a mixed solvent thereof. Similarly, as the elution solvent, methanol, ethanol, water and a mixed solvent thereof can be used. Further, the target substance may be purified by subjecting it to column chromatography packed with normal-layer silica and reverse-layer silica. This purification operation may be performed by arbitrarily combining and repeating the above methods.

The target substance 2 thus obtained is 2
-Aminophenoxazin-3-one derivatives are ¹ H-N
Its structure can be confirmed by MR, ¹³ C-NMR, UV, IR and the like. As a result, the compound prepared according to the present invention, when compared with 2-amino-PXZ-3-one having antitumor activity obtained by reacting 2-amino-5-methylphenol with human or bovine hemoglobin,
UV and IR absorption patterns, ¹ H-NMR, ¹³ C-NM
The chemical shift values of R match.

The 2-aminophenoxazin-3-one derivative has the formula II

[Chemical 4] Is represented by (when R ₂ = H). Where R _{1 in} formula II
And R ₂ represent a functional group corresponding to the starting material orthoaminophenol or its derivative.

For example, when 2-amino-5-methylphenol is used as a starting material, 2-amino-5-methylphenol represented by the following formula III:
Amino-4,4a-dihydro-4a, 7-dimethyl-3
H-phenoxazin-3-one is synthesized by the production method of the present invention.

[Chemical 5]

The 3-aminophenoxazin-2-one derivative has the formula IV

[Chemical 6] (When R ₁ = H). Where R _{1 in} formula IV
And R ₂ represent a functional group corresponding to the ortho aminophenol derivative which is the starting material.

For example, when 2-amino-4-methylphenol is used as a starting material, 3-amino-1,4a-dihydro-4a, 8-dimethyl-2H- represented by the following formula V is shown.
Phenoxazin-2-one is synthesized by the production method of the present invention.

[Chemical 7]

The 2-aminophenoxazin-3-one derivative and the 3-aminophenoxazin-2-one derivative are
Since it easily decomposes when stored at room temperature, it should be stored in a solvent and frozen at -20 ° C or stored as a powder at -20 ° C.
It is preferable to store it in a frozen state. 2-Aminophenoxazin-3-one derivative and 3-aminophenoxazine-
The 2-one derivative can be converted into a small amount of ethanol (for example, 9-
After being dissolved in 9.9%), it can be diluted with physiological saline or distilled water before use.

Next, the antitumor activity of the 2-aminophenoxazin-3-one derivative and the 3-aminophenoxazin-2-one derivative shows the growth inhibitory effect (% survival rate) on HCT-116 human colon cancer cells. It can be assayed by measuring.

According to the above assay, the 2-aminophenoxazin-3-one derivatives and 3-produced according to the present invention
Aminophenoxazin-2-one derivatives have been shown to have high antitumor activity.

As described above, according to the manufacturing method of the present invention,
The 2-aminophenoxazin-3-one derivative and the 3-aminophenoxazin-2-one derivative can be produced inexpensively and in a short period of time.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0032]

Example 1 (A) Synthesis of 2-amino-4,4a-dihydro-4a, 7-dimethyl-3H-phenoxazin-3-one 246 mg of 2-amino-5-methylphenol (powder, Tokyo Kasei Co., Ltd.) was added to 20 mL of distilled water. Subsequently, 90 mL of a hydrochloric acid aqueous solution (0.1 N) was added to completely dissolve 2-amino-5-methylphenol. 90 mL of sodium hydroxide aqueous solution (0.1 N) was added dropwise to the solution little by little to neutralize to pH 7.0. Immediately stir and mix the solution with 0.16
5 wt% potassium ferricyanide aqueous solution (330 mg
200 mL of potassium ferricyanide (powder, manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise to the above solution.

After sufficiently stirring the aqueous solution to which potassium ferricyanide had been added dropwise, the mixture was allowed to stand at 25 ° C. for 30 minutes for reaction.

Subsequently, the solvent in the aqueous solution was reduced under reduced pressure.
It was removed by treatment with a vacuum evaporator. 1
00 mL of 99.9% methanol was added to the residue, the target brown substance was extracted, and then the suspension solution of methanol was centrifuged with a centrifuge (3,000 rpm, 10 minutes).
By doing so, the methanol solution of the supernatant was recovered.

Subsequently, the concentrated solution was treated with Seph as follows.
Columns packed with adex (Amersham Pharmacia Japan KK, registered trademark) LH-20 were subjected to column chromatography.

The carrier of Sephadex LH-20 was sufficiently swollen and equilibrated with a 50% aqueous ethanol solution in advance, and the column was packed in an open column having an inner diameter of 5 cm and a height of 30 cm.

The concentrated liquid was subjected to column chromatography on a column packed with LH-20 and then eluted with a 50% ethanol aqueous solution to fractionate. The yellowish brown fraction was collected, and the solvent of the fraction was distilled off under reduced pressure. Subsequently, the obtained fraction was dissolved in a methanol solution, and further Sephadex (registered trademark) L manufactured by Amersham Pharmacia Japan Co., Ltd.
A column packed with H-20 was subjected to column chromatography (inner column 2.5 cm, height 30 cm, open column, other conditions were the same as above), and a fraction having a yellowish brown color was collected. The solvent of was distilled off under reduced pressure.

The purity of the obtained compound in the fraction was confirmed by thin layer chromatography (reverse layer, Rf value 0.5, developing solvent chloroform: acetone = 4.
5: 1.5 (volume: volume)). As a result, 100 mg of dark brown powder (synthetic product I) was obtained. The compound I thus obtained was ¹ H-NMR (300 MHz, model number J
EOL JMNM-GX270, JEOL Ltd., ¹³ C-NMR (50.3 MHz, model number Varia)
n XL-200, manufactured by Varian Co., Ltd., UV (model U-2000, manufactured by Hitachi, Ltd.), IR (model IR-70)
0, manufactured by JASCO Corporation). The analysis value is as follows.

¹ H-NMR (270 MHz, DMSO-
d ₆ , 25 ° C.) δ (ppm): 1.10 (s, 3H, −
_{CH 3), 2.27 (s,} 3H, Ar-CH 3), 2.9
8 (d, 1H, J = 15.6Hz, -CH 2), 3.1
8 (d, 1H, J = 15.6Hz, -CH 2), 6.0
5 (s, 1H, = CH-), 6.36 (brs, 2H,
_{-NH 2), 6.71 (brs,} 1H, Ar-H),
6.79 (brd, 1H, J = 7.8Hz, Ar-
H), 7.09 (d, 1H, J = 7.8Hz, Ar-
H).

¹³ C-NMR (50.3 MHz, DMSO
−d ₆ , 25 ° C.) δ (ppm): 20.88 (q, −C
_{H 3), 21.97 (q,} Ar-CH 3), 49.18
_{(T, -CH 2), 70.86} (s, -C-O -), 1
05.86 (d, Ar-CH), 116.48 (d, =
CH-), 122.96 (d, Ar-CH), 125.
42 (d, Ar-CH), 132.63 (s, = C
-), 136.49 (s, Ar-C), 143.77
(S, Ar-C), 146.31 (s, Ar-C), 1
60.70 (s, N = C), 191.40 (s, C
O).

UV (λ ^MeOH _max ) (nm, (ε)): 2
26 (15.2), 272 (9.8), 403 (18.
6). In addition, 0.3 mg of the powder of the compound I was dissolved in 1 mL of methanol, 0.1 mL of this solution was collected, diluted with 2 mL of methanol, and then the absorption spectrum was measured. The chart is shown in FIG.

IR (λν ^KBr _max ) (cm ^-1 ): 340
0, 3330, 3180, 2930, 1965, 162
5, 1582, 1535, 1490, 1395, 137
0, 1310, 1290, 1270, 1230, 114
5, 1125, 1105, 1060, 880, 855,
840, 810.

¹ H-NMR, ¹³ C-NMR, UV, IR
From the various instrumental analysis values of, the compound I is 2-amino-4,
It was identified as 4a-dihydro-4a, 7-dimethyl-3H-phenoxazin-3-one by comparison with various known instrumental analysis values.

(B) Antitumor activity of Compound I The antitumor activity of Compound I was measured according to the following method.
HCT-116 human colon cancer cells were used and the cells (5 x
10 ² pieces) were pre-inoculated on a 60 mm dish containing Eagle's modified culture solution (Gibco, USA) and cultured. A solution (100 mM) of 24.2 mg of Synthetic I in 1 mL of dimethylsulfoxide was added to give final concentrations of Synthetic I of 25 μM, 50 μM and 100 μM.
Was added to the culture solution to start cell culture.
For comparison, cell culture was performed in the same manner without adding Compound I, and this was used as a control.

After culturing the cells at 37 ° C. for 2 weeks, the number of colonies formed was counted. The survival rate (%) of cancer cells was calculated from the number of colonies formed according to the following formula 1.

[0046]

[Formula 1] ((Number of colonies formed when Compound I is added) / (Number of colonies formed when Compound I is not added)) x 100

From FIG. 2, the survival rate when the synthetic product I was added was 30% and 50 μl when the concentration of the synthetic product I was 25 μM.
It was 1% for M and 0.1% or less for 100 μM.

Example 2 (A) Synthesis of 3-amino-1,4a-dihydro-4a, 8-dimethyl-2H-phenoxazin-2-one 246 mg of 2-amino-4-methylphenol (powder, Tokyo Kasei) Co., Ltd.) was added to 20 mL of distilled water. Subsequently, 90 mL of a hydrochloric acid aqueous solution (0.1 N) was added to completely dissolve 2-amino-4-methylphenol. 90 mL of sodium hydroxide aqueous solution (0.1 N) was added dropwise to the solution little by little to neutralize to pH 7.0. Immediately stir and mix the solution with 0.16
5 wt% potassium ferricyanide aqueous solution (330 mg
200 mL of potassium ferricyanide (powder, manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise to the above solution.

After sufficiently stirring the aqueous solution to which potassium ferricyanide was added dropwise, the mixture was allowed to stand at 25 ° C. for 30 minutes for reaction.

Subsequently, the solvent in the aqueous solution was reduced under reduced pressure.
It was removed by treatment with a vacuum evaporator. 1
By adding 00 mL of 99.9% methanol to the residue, extracting the target dark brown substance, and subsequently centrifuging the methanol suspension solution in a centrifuge (3000 rpm, 10 minutes), A clear methanol solution was recovered.

Subsequently, the concentrated solution was treated with Seph as described below.
Columns packed with adex (Amersham Pharmacia Japan KK, registered trademark) LH-20 were subjected to column chromatography.

The Sephadex LH-20 carrier was sufficiently swelled and equilibrated in advance with a 50% aqueous ethanol solution, and the column was packed in an open column having an inner diameter of 5 cm and a height of 30 cm.

The concentrated solution was subjected to column chromatography on a column packed with LH-20 and then eluted with a 50% aqueous ethanol solution to fractionate. A fraction having a dark green color was collected, and the solvent of the fraction was distilled off under reduced pressure. Subsequently, the obtained fraction was dissolved in a methanol solution, and further Sephadex (registered trademark) L manufactured by Amersham Pharmacia Japan Co., Ltd.
A column packed with H-20 was subjected to column chromatography (inner diameter 2.5 cm, height 30 cm, open column, other conditions were the same as above), and a fraction having a dark green color was collected. The solvent of was distilled off under reduced pressure.

The purity of the obtained compound in the fraction was confirmed by thin layer chromatography (reverse layer, Rf value 0.45, developing solvent chloroform: acetone = 4.
5: 1.5 (volume: volume)). As a result, 90 mg of dark brown powder (synthetic compound I) was obtained. The compound II thus obtained is ¹ H-NMR (270 MHz, model number J
EOL JMNM-GX270, JEOL Ltd., ¹³ C-NMR (50.3 MHz, model number Varia)
n XL-200, manufactured by Varian Co., Ltd., UV (model U-2000, manufactured by Hitachi, Ltd.), IR (model IR-70)
0, manufactured by JASCO Corporation). The analysis value is as follows.

¹ H-NMR (270 MHz, DMSO-
_{d 6, 25 ℃) δ (} ppm): 1.30 (s, 3H, -
_{CH 3), 2.78 (s,} 3H, Ar-CH 3), 3.2
0 (d, 1H, J = 14.9Hz, -CH 2), 3.4
0 (d, 1H, J = 14.9Hz, -CH 2), 5.8
2 (s, 1H, = CH-), 6.90 (m, 3H, Ar
-Hx3).

¹³ C-NMR (50.3 MHz, DMSO
−d ₆ , 25 ° C.) δ (ppm): 20.40 (q, Ar
_{-CH 3), 24.03 (q,} CH 3), 51.81
_{(T, -CH 2), 72.00} (s, -C-O -), 1
01.71 (d, = CH-), 117.00 (d, Ar
-CH), 116.44 (d, Ar-CH), 124.
72 (d, Ar-CH), 125.62 (s, Ar-
C), 132.16 (s, Ar-C), 139.08.
(S, Ar-C), 147.03 (s, = C-), 15
8.79 (s, N = C-), 193.66 (s, C
O).

UV (λ ^MeOH _max ) (nm, (ε)): 2
67 (11.12), 282 (9.08), 306 (s
h) (8.55), 348 (13.62). In addition, 0.
5 mg of the compound II powder was dissolved in 1 mL of methanol, 0.1 mL of this solution was collected, diluted with 2.2 mL of methanol, and then the absorption spectrum was measured.
The chart is shown in FIG.

IR (λν ^KBr _max ) (cm ^-1 ): 345
0, 3060, 2980, 2930, 1725, 162
0, 1590, 1550, 1460, 1430, 128
0, 1250, 1110, 1060, 950, 890,
810.

¹ H-NMR, ¹³ C-NMR, UV, IR
According to various instrumental analysis values of compound II, 3-amino-1,
It was identified as 4a-dihydro-4a, 8-dimethyl-2H-phenoxazin-2-one by comparison with various known instrumental analysis values.

(B) Antitumor activity of Compound II The antitumor activity of Compound II was measured by the same method as described in Example 1 (B). The result is shown in FIG.

From FIG. 4, the survival rate when the synthetic compound II was added was 18% and 50% when the concentration of the synthetic compound II was 25 μM.
It was 0.3% for M and 0.1% or less for 100 μM.

[0062]

According to the production method of the present invention, it is possible to produce a 2-aminophenoxazin-3-one derivative and a 3-aminophenoxazin-2-one derivative at low cost in a short period of time.

[Brief description of drawings]

1 is a UV spectrum of the compound I prepared in Example 1. FIG.

FIG. 2 Antitumor activity effect of the compound I produced in Example 1

FIG. 3 is a UV spectrum of the compound II produced in Example 2.

FIG. 4 Antitumor activity effect of the compound II produced in Example 2

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[Procedure amendment]

[Submission date] December 25, 2001 (2001.12.
25)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Correction content]

[Claims]

[Chemical 1] (Wherein R ₁ is arbitrarily selected from the group consisting of a lower alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms and an acyloxy group having 1 to 4 carbon atoms , and R ₂ is Hydrogen source
The orthoaminophenol derivative represented by
It is dissolved in an aqueous hydrochloric acid solution, and then the solution is reacted in the presence of an oxidizing agent having ferric iron at pH 5 to 9 , where the reaction is an ortho aminophenol derivative.
When the molar ratio of the oxidant to the above is 0.1 or more and less than 1.0
The method as described above, which is performed in a fixed amount .

[Chemical 2] (Here, in the formula I, R ₁ is a hydrogen atom, and R ₂ is a carbon number.
A lower alkyl group having 1 to 4 and an acyl group having 1 to 4 carbon atoms and
Arbitrarily selected from the group consisting of acyloxy groups having 1 to 4 carbon atoms
Orthoaminophenol derivative represented by
Dissolve in hydrochloric acid aqueous solution, then add the solution containing ferric iron
In the presence of an oxidizing agent at a pH of 5-9 , wherein the reaction is an ortho aminophenol derivative.
When the molar ratio of the oxidant to the above is 0.1 or more and less than 1.0
The method as described above, which is performed in a fixed amount.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction content]

[0008]

A method for preparing a 2-aminophenoxazin-3-one derivative, which comprises the formula I

[Chemical 3] (Wherein R ₁ is arbitrarily selected from the group consisting of a lower alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms and an acyloxy group having 1 to 4 carbon atoms , and R ₂ is Hydrogen source
The orthoaminophenol derivative represented by
It is dissolved in an aqueous solution of hydrochloric acid and subsequently the solution is reacted in the presence of an oxidizing agent having ferric iron at pH 5-9 , where
The reaction is the same as described above for the ortho aminophenol derivative.
When the molar ratio of the oxidizing agent is 0.1 or more and less than 1.0,
The derivative can be produced by tampering . Also, 3-aminophenoxazin-2-one derivatives
A method of manufacturing a compound of formula I

[Chemical 4] (Here, in the formula I, R ₁ is a hydrogen atom, and R ₂ is a carbon number.
A lower alkyl group having 1 to 4 and an acyl group having 1 to 4 carbon atoms and
Arbitrarily selected from the group consisting of acyloxy groups having 1 to 4 carbon atoms
Ortho aminophenol derivative represented by
A solution containing a hydrochloric acid aqueous solution is prepared, and then the solution is continuously added.
Reacted at pH 5-9 in the presence of an oxidant containing trivalent iron
Where the reaction is ortho-aminophenol-induced
The molar ratio of the oxidant to the body is 0.1 or more and less than 1.0
To produce the above derivative.
Rukoto is Ru can.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction content]

Preferably, the reaction has a molar ratio of the oxidizing agent to the orthoaminophenol derivative of 0.125.
It is performed in an amount of up to 0.8 mol . Preferably 3
The oxidizing agent having a valent iron is hexacyanoferrate (III), and more preferably hexacyanoferrate (III),
It is potassium ferricyanide. Preferably, the pH is 5
~ 8, more preferably at a pH of 6-7.5.
Preferably the reaction is carried out in water or a water soluble solvent, preferably the water soluble solvent is methanol or ethanol. Preferably, 2-aminophenoxazine-
In the case of the method for producing a 3-one derivative, in the formula I, R ₁ is
Selected from the group consisting of methyl group, ethyl group or acetyl group
And also 3-aminophenoxazin-2-one induction
In the case of the method for producing the body, in the formula I, R ₂ is a methyl group,
It is selected from the group consisting of tyl or acetyl groups.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction content]

Formula I

[Chemical 5 ] The compound represented by represents an orthoaminophenol derivative. 2-aminophenoxazin-3-one derivative produced
In the case of the production method, in the formula I, R ₁ is lower _one having 1 to 4 carbon atoms.
Alkyl group, C1-4 acyl group and C1-4
Optionally selected from the group consisting of acyloxy groups of R ₂
Is a hydrogen atom. For example, 2-amino-5-methylphenyl
In the case of an enol, R ₁ is a methyl group. Also, 3-a
Of the method for producing the minophenoxazin-2-one derivative
In the formula I, R ₁ is a hydrogen atom and R ₂ is a carbon atom of 1
~ 4 lower alkyl group, C1-4 acyl group and charcoal
Arbitrarily selected from the group consisting of acyloxy groups having a prime number of 1 to 4.
To be done. For example, of 2-amino-4-methylphenol
In this case, R ₂ is a methyl group .

[Procedure Amendment 5]

[Document name to be amended] Statement

[Name of item to be corrected] 0023

[Correction method] Change

[Correction content]

The 2-aminophenoxazin-3-one derivative has the formula II

[Chemical 6 ] Is represented by (when R ₂ = H). Where R _{1 in} formula II
And R ₂ represent a functional group corresponding to the starting material orthoaminophenol or its derivative.

[Procedure correction 6]

[Document name to be amended] Statement

[Name of item to be corrected] 0024

[Correction method] Change

[Correction content]

For example, when 2-amino-5-methylphenol is used as a starting material, 2-amino-5-methylphenol represented by the following formula III:
Amino-4,4a-dihydro-4a, 7-dimethyl-3
H-phenoxazin-3-one is synthesized by the production method of the present invention.

[Chemical 7 ]

[Procedure Amendment 7]

[Document name to be amended] Statement

[Name of item to be corrected] 0025

[Correction method] Change

[Correction content]

The 3-aminophenoxazin-2-one derivative has the formula IV

[Chemical 8 ] (When R ₁ = H). Where R _{1 in} formula IV
And R ₂ represent a functional group corresponding to the ortho aminophenol derivative which is the starting material.

[Procedure Amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

For example, when 2-amino-4-methylphenol is used as a starting material, 3-amino-1,4a-dihydro-4a, 8-dimethyl-2H- represented by the following formula V is shown.
Phenoxazin-2-one is synthesized by the production method of the present invention.

[Chemical 9 ]

[Procedure Amendment 9]

[Document name to be amended] Statement

[Correction target item name] 0054

[Correction method] Change

[Correction content]

The purity of the obtained compound in the fraction was confirmed by thin layer chromatography (reverse layer, Rf value 0.45, developing solvent chloroform: acetone = 4.
5: 1.5 (volume: volume)). As a result, 90 mg of dark brown powder (synthetic compound II ) was obtained. The compound II thus obtained is ¹ H-NMR (270 MHz, model number J
EOL JMNM-GX270, JEOL Ltd., ¹³ C-NMR (50.3 MHz, model number Varia)
n XL-200, manufactured by Varian Co., Ltd., UV (model U-2000, manufactured by Hitachi, Ltd.), IR (model IR-70)
0, manufactured by JASCO Corporation). The analysis value is as follows.

   ─────────────────────────────────────────────────── ─── Continued front page    (71) Applicant 501250186             Keiichi Tomoda             1-30 Sagimiya, Nakano-ku, Tokyo 4 (72) Inventor Tomoda ▲ Aki ▼ Husband             1-30 Sagimiya, Nakano-ku, Tokyo 4 (72) Inventor Ryoji Ishida             1037 Otononyama, Tenpaku-ku, Nagoya-shi, Aichi Gray             Suotoyama 202 (72) Inventor Sadao Arai             Kanagawa Prefecture Yokohama City Totsuka Ward Shinanomachi 515-1 South             4-405 (72) Inventor Keiichi Tomoda             1-30 Sagimiya, Nakano-ku, Tokyo 4 F-term (reference) 4C056 AA02 AB01 AC03 AD05 AE02                       AF01 EC01 ED08

Claims (9)

[Claims] 1. Formula I: (In the formula I, R ₁ and R ₂ are each independently a group consisting of a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms and an acyloxy group having 1 to 4 carbon atoms. Selected from the following) by reacting an ortho-aminophenol derivative represented by the formula (3) with an oxidizing agent having a trivalent iron at pH 5 to 9 A method for producing a 2-one derivative. 2. The method according to claim 1, wherein the reaction is carried out in an amount such that the molar ratio of the oxidizing agent to the orthoaminophenol derivative is 0.1 or more and less than 1.0. 3. The oxidant having trivalent iron is hexacyanoferrate (III) salt, as claimed in claim 1 or 2.
The method described in any one of. 4. The hexacyanoferrate (III) salt is potassium ferricyanide.
The method described in any one of. 5. The method according to any one of claims 1 to 4, wherein the reaction is carried out at a pH of 6 to 7.5. 6. The method according to claim 1, wherein the reaction is performed in water or a water-soluble solvent. 7. The method according to claim 6, wherein the water-soluble solvent is methanol or ethanol. 8. In the formula I, R ₁ and R ₂ are selected from the group consisting of hydrogen atom, methyl group, ethyl group or acetyl group. The method described. 9. In the formula I, R ₁ is CH ₃ and R ₂ is a hydrogen atom, or R ₂ is CH ₃ and R ₁ is a hydrogen atom. The method described in one.