JP5117064B2 - Phosphorus compounds and antitumor agents - Google Patents

Description

  The present invention relates to a phosphorus-containing compound and an antitumor agent containing the same.

Amino sugar and thio sugar, which are heteroatom derivatives of sugar, exist in nature as natural products and have physiological activities such as antibacterial activity and blood glucose concentration increasing action. In contrast, sugar heteroatom derivatives (phosphorus sugars) having a phosphorus atom in the hemiacetal ring of sugar do not exist in nature, and due to the difficulty in synthesis, some synthetic examples and herbicides Only reports (see, for example, Patent Documents 1 to 4) are known.
On the other hand, as a compound having a carbon atom-phosphorus atom bond, fosfomycin having antiviral activity, cyclophosphamide having antitumor activity, and the like are known. Further, as sugar compounds having a carbon atom-phosphorus atom bond, 5-deoxy-1,5-C- (ethylphosphinylidene) -β-D-glucose derivative is known to have an anticancer action although it is weak. It has been.
Japanese Patent Laid-Open No. 3-11089 Japanese Patent Laid-Open No. 5-339281 Japanese Patent Laid-Open No. 9-241282 JP 2004-91435 A

Patent Document 1 describes that phosphosaccharides are expected to have important physiological activities such as anticancer activity, but the structure of specific compounds exhibiting physiological activities and their physiological activities are described. Neither listed nor suggested.
An object of the present invention is to provide a novel phosphorus-containing compound and an antitumor agent containing a phosphorus-containing compound having antitumor activity, and to achieve the object.

Specific means for solving the above problems are as follows.
That is, the present invention is a phosphorus-containing compound represented by the following general formula (I).

In the formula, R ¹ represents a hydrogen atom, a hydroxy group, an alkyl group, an aryl group, an alkoxy group or an aryloxy group, R ² and R ³ each represent a halogen atom, and R ⁴ and R ⁶ are each independently It represents a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, a hydroxyalkyl group, an aryl group, an acyloxy group or an amino group, R ⁵ represents a c androgenic atom.

R ¹ in the general formula (I) is preferably an aryl group. Also, ^{R 2} and ^{R 3} in the general formula (I), arbitrary preferred that a bromine atom.

  An antitumor agent comprising a phosphorus-containing compound represented by the following general formula (II) or (III).

In the formula, R ¹¹ represents a hydrogen atom, a hydroxy group, an alkyl group, an aryl group, an alkoxy group or an aryloxy group, and R ¹² and R ¹³ each independently represent a hydrogen atom, a halogen atom, a hydroxy group, an alkoxy group, Represents an aryloxy group, an acyloxy group, a hydroxyalkyl group or an amino group, and R ¹⁴ and R ¹⁶ each independently represent a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, a hydroxyalkyl group, an aryl group, an acyloxy group or an amino group. the ^{stands, R 15} represents a c androgenic original child.

In the general formula (II) or (III), R ¹¹ is preferably an aryl group. Further, the in the general formula (II) or (III), R ¹² and R ¹³ are each independently a hydrogen atom, preferred that a halogen atom or a hydroxy group arbitrarily.

  According to the present invention, it is possible to provide a novel phosphorus-containing compound and an antitumor agent comprising a phosphorus-containing compound having antitumor activity.

  The phosphorus-containing compound of the present invention is a phosphosaccharide compound represented by the following general formula (I). In the present invention, the phosphosaccharide means a phosphoderivative of sugar, particularly a compound containing a phosphorus atom in the hemiacetal ring of saccharide.

In the formula, R ¹ represents a hydrogen atom, a hydroxy group, an alkyl group, an aryl group, an alkoxy group or an aryloxy group, R ² and R ³ each represent a halogen atom, and R ⁴ and R ⁶ are each independently Represents a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, a hydroxyalkyl group, an aryl group, an acyloxy group or an amino group, and R ⁵ represents a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an aryl group, an acyloxy group or Represents an amino group.

R ¹ represents a hydrogen atom, a hydroxy group, an alkyl group, an aryl group, an alkoxy group or an aryloxy group, and the alkyl group, aryl group, alkoxy group and aryloxy group may further have a substituent. Good.
Examples of the substituent include a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), alkyl group, alkoxy group, nitro group, amino group, hydroxy group, amide group, azide group, cyano group, formyl. Group, carboxy group, acyloxy group, alkylthio group and the like.
In the present invention, R ¹ is an alkyl group having 1 to 12 carbon atoms, an aryl group having 6 to 12 carbon atoms, an alkoxy group having 1 to 12 carbon atoms, or an aryloxy group having 6 to 12 carbon atoms from the viewpoint of antitumor activity. It is preferable that it is a C1-C12 alkyl group or a C6-C12 aryl group, and it is still more preferable that it is a C6-C12 aryl group.

R ² and R ³ each independently represent a halogen atom. In the present invention, from the viewpoint of antitumor activity, the halogen atom is preferably a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, more preferably a chlorine atom or a bromine atom, and a bromine atom. Is more preferable. R ² and R ³ may be different halogen atoms, but are preferably the same halogen atom, more preferably R ² and R ³ are both bromine atoms.
In the present invention, the configuration of R ² and R ³ is not particularly limited, and may be any configuration.

R ⁴ and R ⁶ each independently represent a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, a hydroxyalkyl group, an aryl group, an acyloxy group, or an amino group, and the alkyl group, alkoxy group, hydroxyalkyl group, aryl group The group, the acyloxy group and the amino group may further have a substituent. Examples of the substituent include the substituents exemplified in the description of R ¹ .
In the present invention, R ⁴ and R ⁶ are preferably a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, or an aryl group having 6 to 12 carbon atoms, and are a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. It is more preferable.

R ⁵ represents a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an aryl group, an acyloxy group or an amino group, and the alkyl group, alkoxy group, aryl group, acyloxy group and amino group further have a substituent. It may be. Examples of the substituent include the substituents exemplified in the description of R ¹ .
In the present invention, R ⁵ is preferably a hydrogen atom, a halogen atom (fluorine atom, chlorine atom, bromine atom or iodine atom), an alkyl group having 1 to 12 carbon atoms or an aryl group having 6 to 12 carbon atoms, A hydrogen atom, a halogen atom (fluorine atom, chlorine atom or bromine atom) or an alkyl group having 1 to 3 carbon atoms is more preferable, and a hydrogen atom or a halogen atom (chlorine atom or bromine atom) is more preferable.

In the present invention, R ¹ is an alkyl group or an aryl group, R ² and R ³ are a chlorine atom or a bromine atom, and R ⁴ and R ⁶ are each independently a hydrogen atom, an alkyl group or an aryl group. R ⁵ is preferably a hydrogen atom or a halogen atom, R ¹ is an aryl group, R ² and R ³ are a chlorine atom or a bromine atom, and R ⁴ and R ⁶ are respectively More preferably, it is independently a hydrogen atom or an alkyl group, and R ⁵ is a hydrogen atom or a halogen atom.

In the present invention, the phosphate compound represented by the general formula (I) can be synthesized, for example, as follows.
A phosphosaccharide compound having a double bond represented by the following general formula (IV) can be obtained by the method described in JP-A-5-339281 or a method obtained by appropriately changing the method. A phosphoric acid compound represented by the general formula (I) can be synthesized by adding a halogen molecule to the phosphoric acid compound having a double bond represented by the general formula (IV) by a known method. it can.

In the formula, R ²¹ represents a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, an aryl group, or an aryloxy group, and R ²² and R ²³ represent a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, a hydroxyalkyl group, respectively. Represents an aryl group, an acyloxy group or an amino group, R ²⁴ represents a hydrogen atom, a halogen atom, a hydroxyl group, an alkyl group, an alkoxy group, an aryl group, an acyloxy group or an amino group, and R ²⁵ represents a hydrogen atom, a halogen atom or an alkyl group. Represents a group, an alkoxy group, an aryl group, an acyloxy group or an amino group.

  Hereinafter, as specific examples of the phosphosaccharide compound represented by the general formula (I) of the present invention, phosphosaccharide compounds (I-1) to (I-20) are shown, but the present invention is limited to these. is not.

  The phosphosaccharide compound represented by the general formula (I) has, for example, specific antitumor activity as described later, and thus is useful as a therapeutic agent for cancer, for example.

  The antitumor agent of the present invention is characterized by containing a saccharide compound represented by the following general formula (II) or (III). The antitumor agent of the present invention can comprise known additives such as surfactants and excipients in addition to the above-mentioned phosphosaccharide compound.

In the formula, R ¹¹ represents a hydrogen atom, a hydroxy group, an alkyl group, an aryl group, an alkoxy group or an aryloxy group, and R ¹² and R ¹³ each independently represent a hydrogen atom, a halogen atom, a hydroxy group, an alkoxy group, Represents an aryloxy group, an acyloxy group, a hydroxyalkyl group or an amino group, and R ¹⁴ and R ¹⁶ each independently represent a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, a hydroxyalkyl group, an aryl group, an acyloxy group or an amino group. R ¹⁵ represents a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an aryl group, an acyloxy group or an amino group.

R ¹¹ represents a hydrogen atom, a hydroxy group, an alkyl group, an aryl group, an alkoxy group or an aryloxy group, and the alkyl group, aryl group, alkoxy group and aryloxy group may further have a substituent. Good. Examples of the substituent include the substituents exemplified in the description of R ¹ .
In the present invention, R ¹¹ is an alkyl group having 1 to 12 carbon atoms, an aryl group having 6 to 12 carbon atoms, an alkoxy group having 1 to 12 carbon atoms or an aryloxy group having 6 to 12 carbon atoms from the viewpoint of antitumor activity. It is preferable that it is a C1-C12 alkyl group or a C6-C12 aryl group, and it is still more preferable that it is a C6-C12 aryl group.

R ¹² and R ¹³ each independently represent a hydrogen atom, a halogen atom, a hydroxy group, an alkoxy group, an aryloxy group, an acyloxy group, a hydroxyalkyl group, or an amino group, and are a hydrogen atom, a halogen atom, or a hydroxy group Is preferred.
In the present invention, at least one of R ¹² and R ¹³ is preferably a halogen atom.
Examples of the halogen atom in R ¹² and R ¹³ include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom. From the viewpoint of antitumor activity, a fluorine atom, a chlorine atom or a bromine atom is preferable, and a chlorine atom Or it is more preferable that it is a bromine atom, and it is still more preferable that it is a bromine atom.
In the present invention, the configuration of R ¹² and R ¹³ is not particularly limited, and may be any configuration.

R ¹⁴ and R ¹⁶ each independently represent a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, a hydroxyalkyl group, an aryl group, an acyloxy group, or an amino group, and the alkyl group, alkoxy group, hydroxyalkyl group, aryl group The group, acyloxy group or amino group may further have a substituent. Examples of the substituent include the substituents exemplified in the description of R ¹ .
In the present invention, R ¹⁴ and R ¹⁶ are preferably a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, or an aryl group having 6 to 12 carbon atoms, and are a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. It is more preferable. In particular, R ¹⁴ is particularly preferably an alkyl group having 1 to 3 carbon atoms.

R ¹⁵ represents a hydrogen atom, a halogen atom, an alkyl group or an aryl group, and the alkyl group and aryl group may further have a substituent. Examples of the substituent include the substituents exemplified in the description of R ¹ .
In the present invention, R ¹⁵ is preferably a hydrogen atom, a halogen atom (a fluorine atom, a chlorine atom, a bromine atom or an iodine atom), an alkyl group having 1 to 12 carbon atoms or an aryl group having 6 to 12 carbon atoms, A hydrogen atom, a halogen atom (fluorine atom, chlorine atom or bromine atom) or an alkyl group having 1 to 3 carbon atoms is more preferable, and a hydrogen atom or a halogen atom (chlorine atom or bromine atom) is more preferable.

In the present invention, in the phosphoric acid compound represented by the general formula (II), R ¹¹ is an aryl group, R ¹² and R ¹³ are each independently a halogen atom or a hydroxy group, and R ¹⁴ and R ¹⁶ is independently a hydrogen atom, an alkyl group or an aryl group, R ¹⁵ is preferably a hydrogen atom or a halogen atom, R ¹¹ is an aryl group, and R ¹² and R ¹³ are each independently A halogen atom (chlorine atom or bromine atom) or a hydroxy group, R ¹⁴ is an alkyl group having 1 to 3 carbon atoms, R ¹⁶ is a hydrogen atom or an alkyl group, and R ¹⁵ is a hydrogen atom or a halogen atom. More preferably, it is an atom (a chlorine atom or a bromine atom).

Further, in the phosphosaccharide compound represented by the general formula (III), R ¹¹ is an aryl group, R ¹² is a hydrogen atom, and R ¹⁴ and R ¹⁶ are each independently a hydrogen atom, an alkyl group or It is an aryl group, R ¹⁵ is preferably a hydrogen atom or a halogen atom, R ¹¹ is an aryl group, R ¹² is a hydrogen atom, and R ¹⁴ is an alkyl group having 1 to 3 carbon atoms. More preferably, R ¹⁶ is a hydrogen atom or an alkyl group, and R ¹⁵ is a hydrogen atom or a halogen atom (chlorine atom or bromine atom).

  Specific examples of the phosphosaccharide compound represented by the general formula (II) or (III) in the present invention include the phosphosaccharide compounds (I-1) to (I-20) and the phosphosaccharide compounds described below. (II-1) to (II-12) and (III-1) to (III-8) can be mentioned, but the present invention is not limited to these.

  The phosphosaccharide compound represented by the general formula (II) or (III) in the present invention can be synthesized, for example, by a known method using the phosphosaccharide compound represented by the general formula (IV) as an intermediate. .

  The phosphosaccharide compound represented by the general formula (II) or (III) in the present invention has an extremely high selectivity of acting only on tumor cells and not acting on normal cells. Therefore, it is expected that side effects are extremely low when the saccharide compound represented by the general formula (II) or (III) is used as an antitumor agent. For example, human chronic myelogenous leukemia cell K562 exhibits a lethal effect at a low concentration, whereas leukocytes collected from healthy individuals do not exhibit a lethal effect even at a high concentration. . Therefore, the antitumor agent containing the phosphosaccharide compound has extremely high selectivity. It also acts effectively on human acute monocytic leukemia cells U937 at low concentrations. Therefore, it is a drug with a very high antitumor activity and a broad spectrum, and is extremely useful as a therapeutic agent for cancer.

  Although the mechanism of action of the phosphosaccharide compound on tumor cells has not yet been clarified, for example, it is presumed as follows. That is, the phosphosaccharide compound is a kind of molecular target compound, and is considered to act on the cell cycle regulation mechanism of tumor cells and exert a lethal action on tumor cells.

  The antitumor agent of the present invention is effective not only for various leukocyte cancers such as chronic myelogenous leukemia and adult acute myeloid leukemia, but also for solid cancers such as digestive organs, nerves, muscles, lungs and skin. The antitumor agent of the present invention is a new type of antitumor agent having a wide spectrum and side effects are suppressed in addition to the high activity and selectivity described above.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, “%” is on a molar basis.

Example 1
Synthesis of 2,3,4-tribromo-3-methyl-1-phenylphosphorane 1-oxide

Under an argon atmosphere, 2.4 mmol of 3-methyl-1-phenyl-2-phospholene 1-oxide was dissolved in 25 ml of chloroform, 2.2 mmol of N-bromosuccinimide (NBS) and α, α′-azobisisobutyro Nitrile (AIBN) 0.20 mmol was added and stirred at 70 ° C. for 6 hours under reflux. After cooling, it was diluted with chloroform, and the precipitated succinimide was removed by filtration. The filtrate was washed successively with 10% aqueous sodium sulfite solution, water and brine and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol 20/1). The yield of 60% was obtained by using 4-bromo-1-phenyl-2-phospholene 1-oxide as a semisolid. I got it.
2.0 mmol of 4-bromo-1-phenyl-2-phospholene 1-oxide obtained above was dissolved in 15 ml of carbon tetrachloride and heated to 75 ° C. or higher. A solution obtained by dissolving 2.0 mmol of bromine in carbon tetrachloride was added thereto. After heating at 80 ° C. for 8 hours, the mixture was diluted with 30 ml of chloroform, washed successively with 10% aqueous sodium sulfite solution, water and brine, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol 25/1), and 2,3,4-tribromo-1-phenyl-2-phosphorane 1-oxide as a solid substance 69% The yield was obtained.
Melting point 142-144 ° C
¹ H-NMR (CDCl ₃ ): δ 2.14 (s, 3H, CH ₃ ), 2.78-3.14 (m, 2H, H-5), 4.26 (d, J _PH = 1.8 Hz , 1H, 2-H), 4.67-4.78 (m, 1H, 4-H), 7.57-7.78 (m, 5H, Ph)
¹³ C-NMR (CDCl ₃ ): δ 23.60, 25.06, 38.86, 54.65, 56.50, 129.05, 130.09, 131.65, 136.67
³¹ P-NMR (CDCl ₃ ; H ₃ PO ₄ ): δ30.55
MS (m / z): 431.86 (M ⁺ , + H)

(Example 2)
Synthesis of 2,3-dibromo-1-phenylphosphorane 1-oxide

1-Phenyl-2-phospholene 0.12 g of 1-oxide was dissolved in a mixed solvent of water / THF (water = 12.0 mL, THF = 3.0 mL), and 0.2 mL of bromine (excess amount) was added on an ice bath. The mixture was stirred at room temperature for 2 days. Next, 10 mL of a saturated aqueous sodium sulfite solution was added and stirred for a while to confirm that the bromine color had disappeared. Thereafter, the layers were separated, and the aqueous layer was extracted with 3 × 10 mL of chloroform. Subsequently, the organic phase was washed with 20 mL of a saturated aqueous sodium bicarbonate solution and then 20 mL of a saturated aqueous sodium chloride solution, and then the organic layer was dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the residue was purified by silica gel chromatography (chloroform / methanol = 20/1).
As a result, an oily compound 2,3-dibromo-3-hydroxy-1-phenylphosphorane 1-oxide (0.14 g, 0.42 mmol, y.64%) as an objective compound was obtained.
Melting point 139.94 ° C., boiling point 197.48 ° C.,
Rf = 0.56 (CHCl ₃ : MeOH = 20: 1),
MS m / z 338.38 (MH ^<+> ):
IR (KBr): 1187 cm ⁻¹ (P═O), 748 cm ⁻¹ , 1241 cm ⁻¹ (C-Br)
¹ H-NMR (CDCl ₃ , 300 MHz): δ (ppm); 2.30-2.82 (m, 4H, H-4, 5), 4.28-4.31 (m, 2H, H-2) 3), 7.50-7.88 (m, 5H, Ph)
Moreover, 2-bromo-3-hydroxy-1-phenylphosphorane 1-oxide (0.01 g, 2.25 × 10 ⁻³ mmol, y. 3.5%) as white crystals was obtained as a by-product.
Melting point 150.31-171.17 ° C.,
Rf = 0.16 (CHCl ₃ : MeOH = 20: 1),
MS m / z 276.47 (MH ^<+> )
IR (KBr): 1064 cm ⁻¹ (P═O), 732 cm ⁻¹ , 1195 cm ⁻¹ (C—Br), 3239 cm ⁻¹ (OH)
¹ H-NMR (CDCl ₃ , 300 MHz): δ (ppm); 1.70 (s, 1H, OH), 2.24-2.35 (m, 2H, H-5), 2.53-3. 02 (m, 2H, H-4), 4.09 (d, 1H, H-2, J = 7.8 Hz), 4.60-4.64 (m, 1H, H-3), 7.54 -7.88 (m, 5H, Ph)

(Example 3)
Synthesis of 2,3-dibromo-3-methyl-1-phenylphosphorane 1-oxide

0.66 g of 3-methyl-1-phenyl-2-phospholene 1-oxide was dissolved in a mixed solvent of water and chloroform (water = 20.0 mL, chloroform = 5.0 mL), and bromine 1.0 mL (excess) The mixture was stirred at room temperature for 2 days. Next, 10 mL of a saturated aqueous sodium sulfite solution was added and stirred for a while to confirm that the bromine color had disappeared. Thereafter, the layers were separated, and the aqueous layer was extracted with 4 × 15 mL of chloroform. Subsequently, the organic phase was washed with 25 mL of a saturated aqueous solution of sodium bicarbonate and 25 mL of a saturated aqueous solution of sodium chloride, and then dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the residue was purified by silica gel chromatography (chloroform / methanol = 20/1).
This gave 0.34 g (y. 29%) of 2,3-dibromo-3-methyl-1-phenylphosphorane 1-oxide as an oily compound.
In addition, 0.40 g (y. 41%) of 2-bromo-3-methyl-3-hydroxy-1-phenylphosphorane 1-oxide was obtained as white crystals.

<2-Bromo-3-methyl-3-hydroxy-1-phenylphosphorane 1-oxide>
Melting point 159.63 ° C.,
Rf = 0.28 (CHCl ₃ : MeOH = 20: 1),
MS m / z 289.36 (MH ^<+> )
IR (KBr): 1064 cm ⁻¹ (P═O), 748 cm ⁻¹ , 1265 cm ⁻¹ (C—Br), 3232 cm ⁻¹ (OH)
¹ H-NMR (CDCl ₃ ): δ 1.66 (s, 3H, CH ₃ ), 2.12-2.33 (m, 2H, H-4), 2.39-2.54 (m, 2H, H-5), 4.19 (s, 1H, C-2), 5.87 (s, 1H, OH), 7.26-7.83 (m, 5H, Ph)
<2,3-Dibromo-3-methyl-1-phenylphosphorane 1-oxide>
Melting point 189.20 ° C., boiling point 280.24 ° C.,
Rf = 0.42 (CHCl ₃ : MeOH = 20: 1),
MS m / z 353.20 (MH ^<+> )
IR (KBr): 1126 cm ⁻¹ (P═O), 748 cm ⁻¹ , 1396 cm ⁻¹ (C-Br)
¹ H-NMR (CDCl ₃ ): δ 1.67 (s, 3H, CH ₃ ), 2.36-2.46 (m, 2H, H-4), 2.97-3.02 (m, 2H, H-5), 4.09-4.31 (dds, 1H, C-2), 7.51-7.70 (m, 5H, Ph)

(Reference example)
Synthesis of 2,3-epoxy-3-methyl-1-phenylphosphorane 1-oxide

  After dissolving 2.88 g of 2-bromo-3-hydroxy-3-methyl-1-phenylphosphorane 1-oxide in 3.0 mL of chloroform, 20 mL of an aqueous potassium hydroxide solution (0.5 mol / L) was added, Stir at 40 ° C. for 1 hour. After the reaction, the solution was extracted with 3 × 15 mL of chloroform, and then the organic phase was washed with 25 mL of saturated aqueous sodium hydrogen carbonate solution and 25 mL of saturated aqueous sodium chloride solution in that order, and then the organic layer was dried over anhydrous sodium sulfate. The residue was purified by silica gel chromatography (ethyl acetate / methanol = 20/1). This gave 2.67 g (y. 48%) of erythro-2,3-epoxy-3-methyl-1-phenylphosphorane 1-oxide to threo-2,3-epoxy-3-methyl-1-phenylphospho 0.66 g (y. 11%) of run 1-oxide was obtained.

<Erythro body>
Rf = 0.43 (AcOEt: MeOH = 20: 1),
MS m / z 209.47 (MH ^<+> )
^{IR (KBr): 1180cm -1 (} P = O), 1064cm -1, 1257cm -1 (C-O-C)
1H-NMR (CDCl ₃ ): δ 1.94-2.11 (m, 2H, H-4), 2.54-2.58 (m, 2H, H-5), 3.38-3.48 ( dd, 1H, J2, P = 29.1 Hz, J2, 3 = 3.0 Hz, H-2), 3.83-3.85 (m, 1H, H-3), 7.51-7.75 ( m, 5H, Ph)
<Threo body>
Rf = 0.43 (AcOEt: MeOH = 20: 1),
MS m / z 209.47 (MH ^<+> )
^{IR (KBr): 1180cm -1 (} P = O), 1064cm -1, 1257cm -1 (C-O-C)
¹ H-NMR (CDCl ₃ ): δ (ppm); 1.94-2.11 (m, 2H, H-4), 2.54-2.58 (m, 2H, H-5), 3. 39-3.49 (d, 1H, H-2, J = 28.8 Hz) 7.51-7.80 (m, 5H, Ph)

-Antitumor activity-
(MTT assay)
The antitumor activity of phosphosaccharide compounds was evaluated using the MTT method in the following procedure.
Using the compounds (C-1), (C-2) and (E-1) to (E-4) shown below as the phosphosaccharide compound, the phosphosaccharide compound is dissolved in DMSO to prepare an assay sample solution. According to the following antitumor activity protocol, in vitro evaluation was performed by the MTT method.

1. Cells in the logarithmic growth phase were counted to a concentration of 5000 cells / well, and 50 μl was seeded in each well of a 96-well microtiter plate.
2. Pre-cultured for 24 hours in a carbon dioxide incubator.
3. The assay sample solution was adjusted to the desired concentration in the culture solution, and 50 μl was added to each well.
4). The cells were cultured for 48 hours in a carbon dioxide incubator.
5. 10 μl of MTT solution (5 mg / ml) was added to each well.
6). The color reaction was performed in a carbon dioxide incubator for 1 to 4 hours.
7). Absorbance at 550 nm was measured using a microplate reader.

  Since the absorbance by the MTT method depends on the number of living cells, in the measurement of anti-tumor activity, it means that the smaller the absorbance, the more the tumor cells are killed and thus the stronger the anti-tumor activity.

FIG. 1 is a graph using human chronic myeloid leukemia cell K562, the concentration of the phosphosaccharide compound added on the horizontal axis, and the vertical axis representing the absorbance corresponding to the number of living cells representing the antitumor activity. Show.
FIG. 2 (A) shows the results of using blood collected from AML patients with acute myeloid leukemia (tumor cells are found at a rate of 40%), and FIG. 2 (B) was collected from healthy individuals. The result using blood is shown.

  Next, 2,3-dibromo-3-methyl-1-phenylphosphorane 1-oxide (dibromo form, E-1 above) synthesized in Example 3 was subjected to high performance liquid chromatography (column: Wakosil 5SIL, φ4. 6 mm × 250 mm) and separation and purification using chloroform: methanol = 40: 1 as an eluent, four stereoisomers having different retention times (retention times, RT) were obtained. The nuclear magnetic resonance spectrum data for each stereoisomer is shown in Table 1 below. Moreover, the antitumor activity about each stereoisomer was evaluated by MTT method. The results are shown in FIG.

  FIG. 3 (A) shows the results of administration to human chronic myeloid leukemia cells K562 and human acute monocytic leukemia cells U937 as a mixture of dibromo stereoisomers. FIG. 3B shows a mixture of dibromo stereoisomers (Complex) and stereoisomers having different retention times (retention times (minutes): 8.2, 9.1, 9.9, 11.5). ) Shows the results of each administration.

Next, regarding the antitumor activity of 2,3,4-tribromo-3-methyl-1-phenylphosphorane 1-oxide (tribromo form, E-4 above) synthesized in Example 1, using the MTT method, Comparison was made with the dibromo compound and the antitumor agent AMN107 (imanitiv derivative). The results are shown in FIGS. 4 and 5, respectively.
FIG. 4 is a graph showing the antitumor activity of dibromo (E-1) and tribromo (E-4) against human chronic myeloid leukemia cell K562.
FIG. 5 (A) is a graph showing the antitumor activity of tribromo (E-4) and AMN107 (imatinib derivative) against human chronic myeloid leukemia cell K562, and FIG. 5 (B) is a human acute monocytic cell. FIG. 5C is a graph showing the antitumor activity of tribromo derivative (E-4) and AMN107 (imanitiv derivative) against leukemia cell U937, and FIG. 5 (C) shows tribromo derivative (E-4) against human promyelocytic leukemia cell HL60. It is a graph which shows the antitumor activity of AMN107 (imanitiv derivative).

(Cell cycle analysis)
Cell cycle analysis can be performed by measuring the DNA content in individual cells after staining with a DNA-binding dye (propidium iodide; PI). In cell cycle analysis, cells are classified into G0 / G1 phase cells with diploid (2n) DNA, G2 + M phase cells with tetraploid (4n) DNA, and S phase cells in between. Is done. Apoptotic cells can be detected as cells having a lower DNA content than cells in the G0 / G1 phase.

For cell cycle analysis, human chronic myeloid leukemia cell K562 was used and incubated for 48 hours at a concentration of 50 μM of the above-mentioned phosphosaccharide compound (E-1) and 50 μM of AMN107 (imanitiv derivative). (EPICS XL, manufactured by Beckman Coulter) was used.
1. The cells with 1 × 10 ⁶ cells were washed with PBS, transferred to a 1.5 ml tube, and centrifuged at 2500 rpm for 5 minutes in a centrifuge.
2. The supernatant was removed, 500 μl of cold ethanol was added, and the mixture was fixed at 4 ° C. for 4 hours or more.
3. Centrifugation was performed at 2500 rpm for 5 minutes, and the supernatant was removed.
4). 200 μl of PBS was added, and the mixture was centrifuged again at 2500 rpm for 5 minutes to remove ethanol.
5. The supernatant was removed, the cell pellet was suspended in 40 μl of phosphate-citrate buffer, and kept at room temperature for 30 minutes or more with occasional stirring to extract the fragmented DNA.
The supernatant was removed by centrifugation at 2500 rpm for 5 minutes. The cell pellet was resuspended in 100 μl of PBS, 1 μl of RNase A solution was added, and incubated at 37 ° C. for 30 minutes.
7. Centrifugation was performed at 2500 rpm for 5 minutes, and the supernatant was removed.
8). The cell pellet was suspended in 1 ml of PBS, 50 μl of PI solution (5 mg / ml) was added and mixed, and stained in the dark for 30 minutes.
9. After passing through a nylon mesh (50 μm), the DNA amount of each cell was measured by flow cytometry, and histogram analysis was performed using a cell cycle analysis program.
The results are shown in FIG.

(Evaluation using tumor cells)
The antitumor activity of a phosphosaccharide compound was evaluated using human-derived gastric cancer cells MKN45 and human melanin-deficient black tumor cells C32TG according to the following procedure.
1. Tumor cells in the logarithmic growth phase were counted to a concentration of 10,000 cells / well, and 500 μl was seeded in each well of a 24-well plate.
2. The cells were cultured for about 12 hours in a carbon dioxide incubator, and tumor cells were fixed in each well.
3. The culture solution was removed from each well, and 500 μl of a culture solution having a desired phosphosaccharide compound concentration was added. A culture solution having a desired phosphosaccharide compound concentration was prepared by dissolving the evaluation target compound in DMSO (dimethyl sulfoxide) in advance.
4). The cells were cultured for a predetermined time (1 to 48 hours) in a carbon dioxide incubator, and the morphological changes of the tumor cells were observed with an optical microscope. Observation with an optical microscope was performed with tumor cells alive at 100 × or 200 × magnification using a phase contrast lens without staining.
FIG. 7 shows the results using human-derived gastric cancer cells MKN45, and FIG. 8 shows the results using human melanin-deficient black tumor cells C32TG.

The left side of FIG. 7 is a photomicrograph after culturing for 48 hours using DMSO as a control, and the right side of FIG. 7 is a state after adding the above dibromo compound (E-1) as a phosphosaccharide compound at a concentration of 200 μM and culturing for 48 hours. It is a micrograph.
FIG. 8 (A) is a photomicrograph after culturing for 24 hours using DMSO as a control, and FIG. 8 (B) is the phosphosaccharide compound added with the tribromo compound (E-4) at a concentration of 100 μM. It is a microscope picture after culturing for 24 hours. The magnification is 100 times.

FIG. 1 shows that the phosphosaccharide compound represented by the above general formula (II) or (III) exhibits good antitumor activity. Further, FIG. 2 shows that the phosphosaccharide compound represented by the general formula (II) or (III) selectively acts on tumor cells.
FIG. 3 shows that the dibromo compound exhibits good antitumor activity. Moreover, it turns out that the antitumor activity of the dibromo body which has a specific three-dimensional structure is especially strong.

FIG. 4 shows that the tribromo compound (E-4) has stronger antitumor activity than the dibromo compound (E-1).
FIG. 5 shows that the tribromo derivative (E-4) exhibits a strong antitumor activity comparable to that of AMN107 (imantib derivative) against human chronic myeloid leukemia cell K562. It can also be seen that human acute monocytic leukemia cell U937 and human promyelocytic leukemia cell HL60 exhibit much stronger antitumor activity than AMN107 (imanitiv derivative).

From FIG. 6, it is presumed that the phosphosaccharide compound represented by the general formula (II) or (III) exerts an anticancer action by apoptosis or by stopping the cell cycle.
From FIG. 7, it can be seen that gastric cancer cells are actively dividing in the control, so that the cells are crowded over the entire sample surface. In contrast, in the sample after administration of the phosphosaccharide compound, gastric cancer cells are killed, and it can be seen that the dead cells shrink and decrease.
From FIG. 8, it can be seen that human melanin-deficient black tumor cells C32TG are actively dividing in the control, and thus the cells are crowded over the entire sample surface. In contrast, in the sample after administration of the phosphosaccharide compound, human melanin-deficient black tumor cells C32TG are killed, and it can be seen that the dead cells are reduced and decreased.

  From the above, according to the phosphosaccharide compound represented by the general formula (II) or (III), an antitumor agent having selective and good antitumor activity can be provided.

It is a graph which shows the antitumor activity of a phosphosaccharide compound. It is a graph which shows the specific antitumor activity of a phosphosaccharide compound. It is a graph which shows the anti-tumor activity of a dibromo body. It is a graph which shows the anti-tumor activity which compared the tribromo body and the dibromo body. It is a graph which shows the antitumor activity with respect to K562, U937, and HL60 which compared the tribromo body and AMN107 (imanitiv derivative). It is a graph which shows the result of a cell cycle analysis. It is a microscope picture which shows the antitumor activity with respect to the gastric cancer cell MKN45 of a dibromo body. It is a microscope picture which shows the antitumor activity with respect to the human melanin deficient black tumor cell C32TG of a tribromo body.

Claims (5)

A phosphorus-containing compound represented by the following general formula (I).

[Wherein, R ¹ represents a hydrogen atom, a hydroxy group, an alkyl group, an aryl group, an alkoxy group or an aryloxy group, R ² and R ³ each represent a halogen atom, and R ⁴ and R ⁶ each represent independently represent a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, a hydroxyalkyl group, an aryl group, an acyloxy group or an amino group, R ⁵ represents a c androgenic atom. ] The phosphorus-containing compound according to claim 1, wherein R ¹ in the general formula (I) is an aryl group, and R ² and R ³ are bromine atoms. An antitumor agent comprising a phosphorus-containing compound represented by the following general formula (II) or (III).

[Wherein R ¹¹ represents a hydrogen atom, a hydroxy group, an alkyl group, an aryl group, an alkoxy group or an aryloxy group, and R ¹² and R ¹³ each independently represent a hydrogen atom, a halogen atom, a hydroxy group or an alkoxy group. Represents an aryloxy group, an acyloxy group, a hydroxyalkyl group or an amino group, and R ¹⁴ and R ¹⁶ each independently represent a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, a hydroxyalkyl group, an aryl group, an acyloxy group or an amino group. represents a ^{group, R 15} represents a c androgenic atom. ] The antitumor agent according to claim 3 , wherein R ¹¹ in the general formula (II) or (III) is an aryl group. The antitumor agent according to claim 3 or 4 , wherein R ¹² and R ¹³ in the general formula (II) or (III) are each independently a hydrogen atom, a halogen atom or a hydroxy group. .