JPH07278080A - Alanylalaninol derivative - Google Patents

Abstract

PURPOSE:To obtain an alanylalaninol derivative expressed by a specific formula, having in vitro proliferation inhibitory activity against mouse lymphatic leukemia cells, thus useful as an antineoplastic agent. CONSTITUTION:The objective derivative of formula I [R<1> and R<2> each is (substituted) aromatic group; R<3> and R<4> each is H, benzyloxycarbonyl, t- butyloxycarbonyl, a (substituted) aromatic group or an aromatic acyl, when one party of R<1> and R<2> is nonsubstituted phenyl, the other being a group except nonsubstituted phenyl]. This derivative is recommended to obtain, for example, by the following processes: an N-t-butoxycarbonyl-alanine derivative of formula II is reacted with an alaninol derivative of formula III in the presence of a condensation agent and an auxiliary thereof followed by reaction with e.g. an acid anhydride in the presence of a base to effect acylation followed by treatment with trifluoroacetic acid and then acylation.

Description

Detailed Description of the Invention

[0001]

The present invention relates to the following general formula (1)

[0002]

[Chemical 2]

(Where R¹ , R²Independently have substituents
Represents an optionally substituted aromatic group, R³, R ^FourIndependently, hydrogen
Atom, benzyloxycarbonyl group, t-butyloxy
Carbonyl group, or aliphatic which may have a substituent
Alternatively, it represents an aromatic acyl group. However, R¹ , R² of
When one is an unsubstituted phenyl group, the other is unsubstituted
It is a group other than a phenyl group. ) Represented by alanylara
The present invention relates to a ninor derivative. This compound
The mouse lymphocytic leukemia cells (P388)
As an antitumor agent, it has growth inhibitory activity in vitro.
Have uses.

[0004]

BACKGROUND OF THE INVENTION Many antitumor agents derived from natural substances such as adriamycin, mitomycin, vinca alkaloids, etc. are known, but there are still problems such as unsatisfactory therapeutic results and serious side effects. Further, even if a substance having a remarkable effect, such as taxol or halichondrin B, is found, the supply amount is insufficient, and it is the actual situation that there are many cases where the substance cannot be actually used, including in tests. On the other hand, in the medical field, there is always a demand for a drug that is more effective and has fewer side effects.

The following equation (2)

[0006]

[Chemical 3]

Phenylalanyl having a structure of phenylalaninol is Euphorbia f ( Euphorbia f)
ischeriana Steudel) (D. Uemura et al., Chem. Lett., 537,
1975.) is not known to have antitumor activity. Further, there is no known one having a substituent on the phenyl group.

[0008]

Therefore, an object of the present invention is to provide an antitumor substance having a novel basic structure.

[0009]

As a result of searching various compounds, the present inventors have found that a specific novel alanylalaninol derivative has an in vitro growth inhibitory activity against P388, and the present study was conducted. completed.

That is, the present invention has the general formula (1)

[0011]

[Chemical 4]

(Where R¹ , R²Independently have substituents
Represents an optionally substituted aromatic group, R³, R ^FourIndependently, hydrogen
Atom, benzyloxycarbonyl group, t-butyloxy
Carbonyl group, or aliphatic which may have a substituent
Alternatively, it represents an aromatic acyl group. However, R¹ , R² of
When one is an unsubstituted phenyl group, the other is unsubstituted
It is a group other than a phenyl group. ) Represented by alanylara
The present invention provides a ninor derivative.

In the general formula (1), examples of the aromatic group represented by R ¹ and R ² include a phenyl group, a naphthyl group and a furyl group. At least one of R ¹ and R ² has a substituent, and as the substituent, a lower alkyl group such as a methyl group and an ethyl group, a halogen atom such as a fluorine atom, a chlorine atom and a bromine atom, a methoxy group, an ethoxy group and the like. And lower alkoxy groups, cyano groups, nitro groups, amino groups, hydroxyl groups, carboxyl groups, formyl groups and the like.

In the general formula (1), the aliphatic acyl group represented by R ³ and R ⁴ is preferably a linear or branched aliphatic acyl group having 2 to 10 carbon atoms, and specifically, Examples thereof include an acetyl group, a propanoyl group, an isopropanoyl group, a glycyl group and an alanyl group. Examples of the aromatic acyl group include a benzoyl group which may have a substituent, a naphthoyl group, a furoyl group, a thenoyl group and a nicotinoyl group. As the substituent, a methyl group, a lower alkyl group such as an ethyl group, a fluorine atom, a chlorine atom, a halogen atom such as a bromine atom, a methoxy group, a lower alkoxy group such as an ethoxy group, a cyano group, a nitro group, an amino group, a hydroxyl group, Examples thereof include a carboxyl group and a formyl group.

The novel alanylalaninol derivative represented by the general formula (1) of the present invention is generally obtained as follows.

[0016]

[Chemical 5]

Nt-butoxycarbonyl (hereinafter referred to as BO
Abbreviated as C) -alanine derivative (3) and alaninol derivative (4), for example, dicyclohexylcarbodiimide (D
CC) and other condensing agents such as hydroxybenztriazole (H
It is allowed to act in the presence of a condensation auxiliary agent such as OBT) to obtain a BOC-alanylalaninol derivative (5). BOC-
The alanine derivative is the 2- (t-
Butoxycarbonyloxyimino) -2-phenylacetonitrile (BOCON) is allowed to act in the presence of a base, and the alaninol derivative is obtained by reducing the corresponding alanine derivative.

The hydroxyl group of the BOC-alanylalaninol derivative (5) is acylated by reacting, for example, an acid anhydride or an acid chloride in the presence of a base such as pyridine,
A BOC-alanylacylalaninol derivative (6) is obtained. This compound (6) is treated with trifluoroacetic acid (TFA) to remove the BOC group, and the resulting amino group is acylated by the above-mentioned method to obtain an alanylalaninol derivative (1).

Specific examples of the compound contained in the above general formula (1) in the present invention include:

[0020]

[Chemical 6]

And the like. Hereinafter, examples and test examples will be described in more detail, but the present invention is not limited thereto.

[0022]

【Example】

 Example 1 Synthesis of (7)

[0023]

[Chemical 7]

100 ml of water, 100 of 1,4-dioxane
ml, L-phenylalanine 25 g, 2- (t-butoxycarbonyloxyimino) -2-phenylacetonitrile (BOCON) 93.3 g, triethylamine 3
After stirring 1.5 ml for 3 hours at room temperature, 200 ml of water
Was added and the mixture was washed 3 times with 400 ml of ethyl acetate. 15%
After adjusting the pH of the aqueous layer to 3 with an aqueous citric acid solution, add ethyl acetate 4
It was extracted twice with 00 ml, and the extract was washed with 400 ml of water three times, dried over anhydrous sodium sulfate, and then concentrated (1
39.5 g of the crude product of 3) was obtained.

1.5 g of the crude product of (13), 1.1 g of parachlorophenylalaninol, 1.5 g of dicyclohexylcarbodiimide (DCC), 1.8 g of hydroxybenztriazole-water (HOBT-H ₂ O), dimethylformamide (DMF). ) 30 ml was stirred for 3.5 hours.
After distilling off DMF, it was dissolved in 200 ml of ethyl acetate, 200 ml of saturated sodium hydrogen carbonate aqueous solution, 200 ml of 2M citric acid aqueous solution, and 200 m of saturated sodium hydrogen carbonate aqueous solution.
1, washed with 200 ml of water. The extract was dried over anhydrous sodium sulfate and then concentrated to obtain 2.7 g of a crude product of (14) as a diastereomer mixture.

962.2 g of the crude product of (14), 7 ml of acetic anhydride and 14 ml of pyridine were stirred at room temperature for 16 hours. Concentration gave (15) 1.08 g. (15) 1.
10 ml of TFA was added to 04 g, and the mixture was stirred at room temperature for 15 minutes, and after confirming that the reaction was completed by thin layer chromatography (TL) C, the mixture was concentrated. Purified by silica gel column chromatography to give a set of diastereomers (1
6, high polarity product: 402.8 mg, low polarity product: 3
21.6 mg, mixture: 127.7 mg) was obtained.

83 m of the diastereomeric mixture of (16)
To g, benzoyl chloride (50 μl), sodium hydrogen carbonate (43.5 mg) and dichloromethane (10 ml) were added, and the mixture was stirred at room temperature for 60 minutes and at 50 ° C. for 30 minutes, concentrated, added with 20 ml of water and extracted twice with 30 ml of chloroform. Purification by silica gel column chromatography and TLC gave a set of diastereomers (7, highly polar product 31.1 mg, less polar product 16.3 mg).

Highly polar product ¹ H-NMR (CDCl ₃ ): δ7.72 (2H, dd, J = 7.9,2.0Hz), 7.20-7.5
3 (10H, m), 7.02 (2H, d, J = 8.5Hz), 6.74 (1H, br.d, J = 7.9H
z), 6.14 (1H, br.d, J = 9.1Hz), 4.80 (1H, dt, J = 7.3,6.1H
z), 4.29 (1H, m), 3.95 (2H, m), 3.11 (1H, dd, J = 6.7,15.0H
z), 3.08 (1H, dd, J = 7.8,15.0Hz), 2.66 (1H, dd, J = 6.7,14.
0Hz), 2.56 (1H, dd, J = 7.3,14.0Hz), 1.98 (3H, s).

Low polarity product ¹ H-NMR (CDCl ₃ ): δ6.97-7.72 (14H, m), 6.65 (1H, br.d, J =
7.9Hz), 5.97 (1H, br.d, J = 8.5Hz), 4.75 (1H, dt, J = 7.9,6.
1Hz), 4.32 (1H, m), 3.94 (1H, dd, J = 5.5,12.0Hz), 3.80 (1
H, dd, J = 4.8Hz), 3.11 (1H, dd, J = 6.0,13.0Hz), 3.05 (1H, d
d, J = 9.1,13.0Hz), 2.71 (2H, d, J = 6.7Hz), 2.02 (3H, s).

Example 2 Synthesis of (8)

[0031]

[Chemical 8]

Of (16) obtained in Example 1, 141.1 mg of the highly polar compound was added with 4 ml of acetic anhydride and 8 ml of pyridine, and the mixture was stirred at room temperature for 2 hours and then concentrated to give (8). 130.0 mg of one diastereomer was obtained.

¹ H-NMR (CDCl ₃ ): δ7.14 (7H, m), 6.98 (2H,
d, J = 8.5Hz), 6.04 (1H, br.d, J = 7.3Hz), 5.95 (1H, br.d, J =
7.3Hz), 4.56 (1H, dt, J = 9.1,6.1Hz), 4.25 (1H, m), 3.93
(2H, m), 3.00 (2H, m), 2.69 (1H, dd, J = 6.7,15.0Hz), 2.52
(1H, dd, J = 7.9,15.0Hz), 2.03 (3H, s), 1.97 (3H, s).

Of the (16) obtained in Example 1, 145.9 mg of the low polarity compound was treated in the same manner to obtain 137.3 mg of the other diastereomer of (8).

¹ H-NMR (CDCl ₃ ): δ7.16-7.33 (7H, m), 7.04
(2H, d, J = 8.5Hz), 5.98 (1H, br.d, J = 7.3Hz), 5.81 (1H, br.
d, J = 11.5Hz), 4.50 (1H, m), 4.27 (1H, m), 3.90 (1H, dd, J =
5.5,12.0Hz), 3.73 (1H, dd, J = 4.2,12.0Hz), 2.98 (2H, m),
2.70 (2H, d, J = 7.3Hz), 2.01 (3H, s), 1.95 (3H, s).

Example 3 Synthesis of (9)

[0037]

[Chemical 9]

1.644 g of the diastereomeric mixture of (14) obtained in Example 1, benzoyl chloride 0.
66 ml, pyridine 10 ml at room temperature for 16 hours at 75 ° C
After stirring for 1 hour, the mixture was concentrated, dissolved in 80 ml of ethyl acetate and washed with 100 ml of water, and the ethyl acetate layer was concentrated. Purification by silica gel column chromatography gave 1.426 g of (17). 10 ml of TFA is added to this 1.396 g
Was added, and the mixture was stirred at room temperature for 15 minutes, and after confirming that the reaction was completed by TLC, the mixture was concentrated. Purification by silica gel column chromatography gave 1.088 g of the diastereomeric mixture of (18).

Diastereomeric mixture 53 of (18)
4.5 mg, 213 μl of benzoyl chloride, and 20 ml of pyridine were stirred for 3 hours. 100 ml of water after concentration,
The mixture was extracted with 100 ml of ethyl acetate and the ethyl acetate layer was concentrated. Purified by silica gel column chromatography,
173.4 mg of the diastereomeric mixture of (9) was obtained.

¹ H-NMR (CDCl ₃ ): δ7.89-7.99 (2H, m), 7.66
-7.00 (13H, m), 5.59-5.78 (1H, m), 4.33-4.60 (2H, m), 3.
92-4.28 (2H, m), 2.86-3.23 (2H, m), 2.50-2.83 (2H, m).

Example 4 Synthesis of (10)

[0042]

[Chemical 10]

DL-Orthofluorophenylalanine 2
07.1mg, lithium aluminum hydride 12
8.2 mg was refluxed with 20 ml of diethyl ether for 20 hours. After cooling to room temperature, 5 ml of water and 10 ml of a 15% aqueous sodium hydroxide solution were added, and the mixture was extracted 3 times with 50 ml of ethyl acetate and concentrated to obtain 170.3 mg of a crude DL-orthofluorophenylalaninol (19). Crude 394.5mg of obtained in Example 1 (13), 159.6mg of (19), DCC440.5mg, HOBT- H 2
O 282.5 mg and DMF 20 ml were stirred for 7 hours,
After DMF was distilled off, the residue was dissolved in 20 ml of ethyl acetate and saturated aqueous sodium hydrogencarbonate solution 20 ml, 2M citric acid solution 20
ml, saturated aqueous sodium hydrogen carbonate solution 20 ml, water 20
Washed sequentially with ml. It was dried over anhydrous sodium sulfate and then concentrated to obtain a crude product of (20) as a diastereomer mixture.

To this (20), 7 ml of acetic anhydride and 14 ml of pyridine were added, and the mixture was stirred at room temperature for 4 hours, concentrated and purified by silica gel column chromatography.
375.3 mg of the diastereomer mixture of 1) was obtained.
Add 8 ml of TFA to this, stir for 30 minutes at room temperature, and
After confirming the completion of the reaction by LC, the mixture was concentrated. Benzoyl chloride (150 μl), pyridine (5 ml) and dichloromethane (20 ml) were added and the mixture was stirred at room temperature for 90 minutes, concentrated, added with water (50 ml), extracted twice with ethyl acetate (50 ml) and separated by silica gel column chromatography to obtain a set of diastereomers ( 10, high polarity product: 48.5m
g, low polarity product: 36.5 mg, mixture: 49.9 m
g) was obtained.

Highly polar product ¹ H-NMR (CDCl ₃ ): δ7.00-7.33 (15H, m), 6.23 (1H, br.d, J =
7.7Hz), 4.15 (1H, dt, J = 7.9,7.9Hz), 4.38 (1H, m), 4.02
(2H, d, J = 5.5Hz), 2.98 (2H, m), 2.74 (1H, m), 2.04 (3H,
s), 1.98 (3H, s,).

Low polarity product ¹ H-NMR (CDCl ₃ ): δ6.94-7.36 (15H, m), 5.78 (1H, br.d, J =
9.1Hz), 4.57 (1H, dt, J = 7.3,5.5 = Hz), 4.36 (1H, m,), 3.9
4 (1H, dd, J = 4.8,12.0Hz), 3.79 (1H, dd, J = 4.2,12.0Hz),
3.15 (1H, dd, J = 6.1,14.0Hz), 3.00 (1H, dd, J = 4.8,14.0H
z), 2.79 (2H, m), 2.03 (3H, s).

Example 5 Synthesis of (11)

[0048]

[Chemical 11]

DL-paranitrophenylalanine 51
3.0 mg, borane-THF complex (1M)
6.0 ml and 5 ml of THF were stirred, and after 90 minutes, 10 ml of borane-THF complex (1M) was added.
The stirring was continued for 2 hours. After adding 25 ml of water and 1 g of sodium hydroxide, the mixture was extracted 3 times with 50 ml of ethyl acetate and concentrated to obtain a crude product of DL-paranitrophenylalaninol (22). BOC-phenylalaninol (13) 60
4.0 mg, the total amount of the crude product of (22) obtained in the above reaction, DCC 591.0 mg, HOBT-H ₂ O 694.9.
After stirring for 8 hours with 20 mg of DMF and distilling off DMF, the residue was dissolved in 50 ml of ethyl acetate and washed successively with 50 ml of saturated sodium hydrogen carbonate aqueous solution, 50 ml of 2M citric acid aqueous solution, 50 ml of saturated sodium hydrogen carbonate aqueous solution and 50 ml of water. After drying over anhydrous sodium sulfate, concentrate (2
The crude product of 3) was obtained as a mixture of diastereomers. To this, 8 ml of acetic anhydride and 16 ml of pyridine were added, and the mixture was stirred at room temperature for 2 hours, concentrated and purified by silica gel column chromatography to obtain 257.0 mg of the diastereomer mixture of (24).

Diastereomeric mixture 27 of (24)
5.0 mg of TFA was added to 5 ml of the mixture, and the mixture was stirred at room temperature for 20 minutes, confirmed that the reaction was completed by TLC, concentrated, and then separated by silica gel column chromatography to obtain a set of diastereomers (25, highly polar). Product: 9.1m
g, low polarity product: 14.9 mg, mixture: 131.7.
mg) was obtained.

Diastereomer mixture 131.
To 7 mg, 50 μl of benzoyl chloride and 50 pyridine
μl and 10 ml of dichloromethane were added, and the mixture was stirred at room temperature for 160 minutes and concentrated. The concentrate was dissolved in benzene, and the remaining residue was recrystallized from pyridine (9.5 mg).
And the diastereomeric mixture of (11) was purified by 3.1.
mg was obtained.

¹ H-NMR (CDCl ₃ ): δ7.97 (2H, d, J = 8.5Hz),
7.24-7.71 (7H, m), 6.53 (1H, br.d, J = 6.7Hz), 6.21 (1H, b
rd, J = 8.5Hz), 4.73 (1H, dt, J = 8.5,7.3Hz), 4.41 (1H, m),
4.00 (1H, dd, J = 5.5,12.0Hz), 3.86 (1H, dd, J = 4.2,12.0H
z), 3.15 (2H, m), 2.98 (2H, m), 2.06 (3H, s).

Example 6 Synthesis of (12)

[0054]

[Chemical 12]

Of (25) obtained in Example 5, 4.6 mg of the highly polar compound was added to 3.5 ml of acetic anhydride and 7 of pyridine.
After adding ml, the mixture was stirred at room temperature for 1.5 hours and then concentrated to obtain the diastereomer (12) having a higher polarity of 2.9 m.
g was obtained.

¹ H-NMR (CDCl ₃ ): δ8.12 (2H, dd, J = 6.8,2.0H
z), 7.13-7.33 (7H, m), 6.18 (1H, br.d, J = 7.7Hz), 5.99 (1
H, br.d, J = 7.4Hz), 4.56 (1H, dt, J = 8.0,8.0,6.7Hz), 4.32
(1H, m), 4.04 (1H, dd, J = 4.0,12.0Hz), 3.92 (1H, dd, J = 4.
9,12.0Hz), 3.01 (2H, m), 2.86 (1H, dd, J = 6.8,14.0Hz),
2.70 (1H, dd, J = 7.0,12.0Hz), 2.05 (3H, s), 1.98 (3H, s).

Of the (25) obtained in Example 5, 7.5 mg of the low polarity compound was treated in the same manner to obtain 8.2 mg of the other diastereomer of (12).

¹ H-NMR (CDCl ₃ ): δ8.12 (2H, d, J = 9.1Hz),
7.15-7.43 (7H, m), 6.08 (1H, br.d, J = 10Hz), 5.99 (1H, br.
d, J = 7.9Hz), 4.52 (1H, dt, J = 7.9,6.1Hz), 4.37 (1H, m),
3.95 (1H, dd, J = 5.5,12.0Hz), 3.76 (1H, dd, J = 4.8,12.0H
z), 3.02 (2H, m), 2.85 (2H, d, J = 6.7Hz), 2.04 (3H, s), 1.
95 (3H, s).

Test Example 1 Mouse lymphocytic leukemia cells (P388) were treated with 2-hydroxyethyl disulfide 5 μM and kanamycin sulfate 100.
RPMI containing 10% fetal bovine serum supplemented with μg / ml
-1640 medium, add 1 × 10 ⁴ cells / ml
Prepared by adding the alanylalaninol derivative to a predetermined concentration, and adding a CO ₂ incubator (CO ₂ 5%,
The culture was performed at 100% humidity and 37 ° C for 4 days. The number of viable cells was measured by the MTT colorimetric method, and the 50% cell growth inhibitory concentration (IC ₅₀ ) was determined from the growth inhibition rate relative to the control group.
The results are shown in Table 1.

[0060]

[Table 1]

[0061]

INDUSTRIAL APPLICABILITY The substance of the present invention shows a growth inhibitory activity against P388 of cancer cells in vitro and is expected to be used as an antitumor agent. Moreover, since it can be easily synthesized, a large amount can be supplied if necessary.

Claims (1)

[Claims] 1. The following general formula (1):(In the formula, R¹ , R²May independently have a substituent.
Represents an aromatic group, R³, R ^FourIndependently, hydrogen atom, benz
Luoxycarbonyl group, t-butyloxycarbonyl
A group, or an aliphatic or aromatic group which may have a substituent.
Represents an aromatic acyl group. However, R¹ , R² One side is free
When it is a substituted phenyl group, the other is an unsubstituted phenyl group
It is a group other than. ) Invitation of alanyl alaninol represented by
conductor.