JPH0244315B2 - SUPAGARINN155HOSUFUEETOOYOBISONOSEIZOHO - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the formula [) N-[4-(3-aminopropyl)aminobutyl]-2-[(s)-7-guanidino-3-
This invention relates to phosphohydroxyhebutanamide]-2-hydroxyethanamide (hereinafter referred to as spagarin-15-phosphate) and its production method, and the compound of the present invention has a remarkable anticancer effect and is used as an anticancer agent. It is useful. In a systematic study of anticancer drugs, the present inventors discovered a novel substance named Spergualin in the culture solution of Bacillus laterosvorus BMG162-aF2 (Feikoken Bacterial Serial No. 5230) belonging to the genus Bacillus. discovered the cancer-fighting antibiotic BMG162-aF2 (Journal of Antibiotics, Vol. 34,
1619 and 1622, 1981). The chemical structure of sparigalin is the following formula [] The configuration at position 15 is S, and the configuration at position 11 has not yet been determined (Journal of Antibiotics, Vol. 34, p. 1622).
(1981). A compound with this structural formula was also synthesized by the condensation of acid amide and glyoxylsplumidine, and the resulting epimer was separated into natural (-)-spargarin and non-natural (+)-spargarin (dial).・Of Antibiotics, 34
Volume, p. 1625). As a result of various studies on derivatives of spagarin, the present inventors discovered that the above-mentioned phosphoric acid ester of spagarin has an excellent anticancer effect, and completed the present invention. The spagarin phosphate ester of the present invention is obtained by protecting the amino group at the 1-position, the imino group at the 4-position, and the hydroxyl group at the 11-position of naturally or synthetically obtained spagarin, and then converting the hydroxyl group at the 15-position into a phosphoric acid ester, Subsequently, the protecting group is removed, or one of the raw materials in the synthesis of spagarin, (S)-7-guanidino-3-hydroxyhebutanamide, is reacted with phosphoric acid to form the following formula: It can be obtained by obtaining a phosphoric acid ester represented by and then condensing it with N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroethaneamide, which is the other raw material for spagarin, by a conventional method. can. As the protecting groups for protecting the amino group at the 1st position, the imino group at the 4th position, and the hydroxyl group at the 11th position in the above method, amino protecting groups commonly used in conventional peptide synthesis etc. can be used. Since the compound [] is extremely unstable to alkalis and acids, an aralkyloxycarbonyl group such as a benzyloxycarbonyl group or a paramethoxybenzyloxycarnyl group, which can be easily eliminated by conventional hydrolysis, is preferred. To introduce these amino protecting groups into spagarin, it is advantageous to use known methods, such as the active ester method. Generally, these methods do not react with the guanidine group of spagarin. The compound in the formula [] of the present invention is in the (-) form (having the same optical rotation as natural spagarin)
The compound protects the amino group at position 1 and the imino group at position 4 of (-)-spargarin, then protects the hydroxyl group at position 11 without isomerizing it, and then protects it without phosphoric acid esterification. It can be obtained by phosphoric acid esterification. As a protecting group that can be introduced without isomerizing the hydroxyl group at position 11, a tetrahydropyranyl group is preferable. In order to selectively tetrahydropyranylate the hydroxyl group at position 11 without isomerizing it, anhydrous organic solvent, preferably 1-3 in anhydrous dimethylformamide,
0.1 to 3 equivalents of 2,3-dihydro-4H-pyran
The reaction is carried out in the presence of an equivalent, preferably 0.2 to 1 equivalent, of an acid catalyst such as para-toluenesulfonic acid at room temperature for 2 to 10 hours. Preferably 2
Selective tetrahydropyranylation without isomerizing the hydroxyl group at position 11 by reacting an equivalent amount of 2,3-dihydro-4H-pyran in the presence of 0.5 equivalents of para-toluenesulfonic acid for 7 hours. Can be done. This compound has the following general formula [] (In the formula, R ₁ represents a protected amino group and R ₂ represents a protecting group.) For example, the configuration at position 11 of (-)-1-N,4-bis-(benzyloxycarbonyl)-11-0-tetrahydropyranyl spagarin obtained by this method is due to amino protection by its hydrolysis. It was confirmed that no isomerization had occurred since (-)-spargarin with high optical purity was recovered by deprotection of the group and elimination of the tetrahydropyranyl group by weak acid hydrolysis described below.
Note that the tetrahydropyranyl group is a mixture of α-form and β-form. For phosphoric acid esterification, spagarin, which has been protected by removing the hydroxyl group at position 15 as described above, is heated in an organic solvent such as anhydrous pyridine for 1 hour under ice cooling.
It is reacted with ~3 equivalents of a phosphorochlorinate with a protected hydroxyl group such as diphenylphosphorochloridate to obtain a compound in which the hydroxyl group at position 15 is converted to diphenyl phosphoric acid ester. The reaction time is completed in 2-6 hours. Next, this compound is catalytically reduced in a single or mixed solvent such as methanol, dioxane, or water using palladium, platinum, or the like as a catalyst by a conventional method, whereby the phenyl group, which is a phosphoric acid group-protecting group, is eliminated and 15 - Synthesis of phosphate is achieved. At this time, if the other protecting group is an aralkyloxycarbonyl group, it is simultaneously deprotected. In the case of protecting groups other than the aralkyloxycarbonyl group, the compound can be obtained by subsequently removing them. For example, 11
-0-Tetrahydropyranyl group is removed by adding 2 equivalents of the acid addition salt of 11-0-tetrahydropyranyl spagarin obtained by removing the phosphate group protecting group.
Add about 0.1 equivalent of para-toluenesulfonic acid,
It is easily desorbed by stirring under ice cooling. A reaction time of 5-7 hours is sufficient. Since the spagarin-15-phosphate according to the present invention is unstable in the free base state, it is preferred to form any non-toxic acid addition salt by adding a pharmaceutically acceptable acid by conventional methods. As acid addition salts, salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, and boric acid, and organic acids such as acetic acid, citric acid, tartaric acid, and glutaric acid are used. In the compound of the present invention (-)-spargarin-15
- Phosphates have particularly excellent anticancer effects. The anticancer effect of (-)-spargarin-15-phosphate is shown below. Anticancer effect of (-)-spargarin-15-phosphate on murine leukemia L-1210 Mouse leukemia L1210 cells were used in 1 group of 8 mice.
10.5 were inoculated intraperitoneally, and then, from that day onwards, a sample dissolved in physiological saline was intraperitoneally administered once a day for 9 consecutive days.The animals were reared and observed for 60 days to determine the survival rate [T /C×100=(average survival days of treated group/average survival days of untreated group)×100] was calculated. The average survival time of the control group (no administration group) was 7.6 days.
It was ~8.9 days.

【table】

[Table] Next, the present invention will be explained by examples. Example 1 Synthesis of (-)-spargarin-15-phosphate (a) (-)-1-N,4-bis-(benzyloxycarbonyl)spargarin: 3.0 g (5.85 mmol) of trihydrochloride of (-)-spargarin. ) in 30 ml of methanol, add 7.2 ml (17.6 mmol) of triethylamine, then add a solution of 3.21 g (12.9 mmol) of N-benzyloxycarbonyloxysuccinimide dissolved in 8 ml of dioxane, and stir at room temperature for 3 hours. did. The reaction solution was concentrated to dryness, dissolved in 50 ml of 0.1 M sodium chloride, and adjusted to pH 6.5 with 2 N hydrochloric acid.
The mixture was applied to a column of CM-Sephadex C-25 (200 ml) equilibrated with 0.1 M sodium chloride, followed by gradient elution with 0.1 M to 0.5 M sodium chloride (1 L each) (20 ml fractions).
Fractions 34-80 were combined, concentrated to dryness, and extracted three times with 10 ml of methanol. This extract was applied to a column of Sephadex LH-20 (200ml), and 90%
It was eluted and desalted with methanol/water (fractionated into 2 ml fractions). Fractions 51-63 were combined and concentrated to dryness, and 3.8 g of (-)-1-N,4-bis-(benzyloxycarbonyl) spagarin hydrochloride was obtained in the form of a colorless syrup.
was obtained (yield 91%). [α] ²¹ _D −11° (cl, water). Elemental analysis value: 54.95%, H7.25%, N13.83%,
Cl.06%. Theoretical value of C ₃₃ H ₄₉ N ₇ O ₈・1/2H ₂ O: C55.26
%, H7.17%, N13.67%, Cl4.94%. (b) (-)-1-N,4-bis-(benzyloxycarbonyl)-11-0-tetrahydropyranyl spagarin: (-)-1-N,4-bis obtained in the previous section (a) 3.45 g (4.81 mmol) of -(benzyloxycarbonyl)spargarin hydrochloride was dissolved in 30 ml of anhydrous H,N-ditylformamide, and 0.63 ml (9.75 mmol) of 2,3-dihydro-4H-pyran and para-toluenesulfonic acid (H ₂ 464 ml (2.44 mmol) of O) was added and stirred at room temperature for 7 hours. After the reaction, 0.33 ml (2.44 mmol) of triethylamine was added and the mixture was concentrated to dryness. This was mixed with chloroform-methanol-pyridine-50% acetic acid water (240:40:4:
Silica gel C-200 developed with the mixture of 1),
300g) by column chromatography (fractionated into 20ml portions). Fractions 66-78 were combined and concentrated to dryness to give a colorless syrup-like (-)-1-N-
4-bis-(benzyloxycarbonyl)-11-
0-tetrahydronyl spagarin acetate 1.07g
was obtained (yield 26%) [α] ²² _B −13° (cl, methanol). Elemental analysis value: C56.65%, H7.76%,
N11.75%. Theoretical values _{of C38H57N7O9}・_CH3COOH・3 _{/ 2H2O} : C56・99%, H7.65%, _N11.63 %. fraction
85-92 were combined and concentrated to dryness to give (-)-1-N,4
362 mg (yield 10%) of -bis-(benzylcarbonyl)spargarin acetate was recovered. (c) (-)-1-N,4-bis-(benzyloxycarbonyl-11-0-tetrahydropyranyl spagarin-15-diphenyl phosphate: previous section)
663 mg (0.786 mmol) of (-)-1-N,4-bis-(benzyloxycarbonyl)-11-0-tetrahydropyranylspargarin acetate obtained in (b) was dissolved in 10 ml of anhydrous pyridine and cooled on ice. 0.34 ml (1.63 mmol) of lower diphenyl phosphorocridate was added dropwise and stirred for 4 hours. 0.5ml after reaction
of water was added and concentrated to dryness. This was dissolved in 20 ml of ethyl acetate, washed with 20 ml of water, dehydrated with anhydrous sodium sulfate, and concentrated to dryness. This was purified by column chromatography on silica gel (Wako Gel C-200, 550 g) developed with a mixture of chloroform-methanol-pyridine-50% aqueous acetic acid (320:40:4:1) (fractionated into 10 ml portions).
Fractions 30-91 were combined and concentrated to dryness to obtain 437 mg of the acetate of the surface compound in the form of a colorless syrup. Yield 51
% (d) (-)-11-0-tetrahydropyranyl spagarin-15-phosphate: (-)-1-N,4-bis-(benzyloxycarbonyl)-11- obtained in the previous section (c) 435 mg (0.415 mmol) of 0-tetrahydrospergarin-15-diphenylphosphate acetate was dissolved in 10 ml of 80% methanol water, and 30% of platinum oxide was added as a catalyst.
mg was added thereto, and the mixture was stirred at room temperature for 7 hours in a hydrogen stream.
The catalyst was removed and the liquid was concentrated to dryness. this
Dissolve in 20ml of 0.1M sodium chloride and dilute with lH hydrochloric acid.
The pH was adjusted to 6.4 and equilibrated with 0.1M sodium chloride.
CM-Sephadex C-25 (Sweden,
The mixture was applied to a 20 ml (manufactured by Pharmacia) column, followed by gradient elution with 0.1 M to 1.0 M sodium chloride (100 ml each) (fractionated into 2 ml portions). Fractions 36-44 were combined, concentrated to dryness, and extracted three times with 5 ml of methanol. This extract was applied to a column of Sephadex LH-20- (100 ml) and desalted by elution with 90% methanol water (1 ml).
fraction). Fractions 38-45 were combined and concentrated to dryness.
Colorless syrupy surface compound trihydrochloride 128mg
I got it. Yield 48% (e) (-)-Spargarin-15-phosphate: (-)-11-0 tetrahydropyranyl spagarin-15-phosphate trihydrochloride obtained in the previous section (d)
Dissolve 112 mg (0.175 mmol) in 5 ml of water,
Paratoluenesulfonic acid (H ₂ O) 3.3 mg under ice cooling
(0.017 mmol) was added and stirred for 8 hours. After the reaction, adjust the pH to 6.5 with 1M aqueous ammonia, add 20ml of 0.4M sodium chloride, and equilibrate with 0.4M sodium chloride.
(20ml), followed by 0.4M−1.0M
Gradient elution with sodium chloride (100 ml each) was performed (2 ml fractions). Fraction 46−
59 were combined, concentrated to dryness, and diluted with 5 ml of methanol.
Extracted twice. This extract is used as Cephadex LH.
-20 (100 ml) column and eluted with 90% methanol (fractionated into 1 ml portions). Fractions 41-52 were combined and concentrated to dryness, resulting in a colorless syrupy (-)-
67.7 mg of spagarin-15-phosphate trihydrochloride monosodium salt 1/2 hydrate was obtained. Yield 62% (-)-Spargarin-15-phosphate trihydrochloride monosodium salt 1/2 hydrate is colorless syrup-like.
No clear melting point can be determined. [α] ²² _D −8.2 (cl, water)
The elemental analysis values are C32.85%, H6.86%,
C ₁₇ H ₃₇ N ₂ O ₇ P at N15.7%, Cl16.87% P5.20%
Theoretical value of 3HCl・Na・1/2H ₂ O (C32.73%, H6.62
%, N15.72%, Cl17.05%, P4.96%). The thin layer chromatography using Shizerika gel mentioned above showed Rf0.08. Example 2 Synthesis of (±) spagarin-15-phosphate (a) (S)-7-guanidino-3-phosphohydroxyheptanamide: Mix 3.45 ml of 85% phosphoric acid and 4.46 g of phosphorus pentoxide, and add ( 1.0 g (4.193 mmol) of S)-7-guanidino-3-hydroxyhebetanamide hydrochloride was added little by little, mixed well, and stored in a phosphorus pentoxide desiccator for 3 days. ice water 50
ml, neutralized with 10N caustic soda, and concentrated under reduced pressure. The obtained oil was transferred to a milk needle and stirred with 50 ml of methanol, the resulting white crystals were further washed with 50 ml of methanol, and the methanol extract was concentrated to dryness to obtain 1.69 g of a residue. Dissolve in 10ml of water,
The mixture was applied to a column (inner diameter 20 mm) filled with 80 ml of CM-Sephadex C-25, Na ^{+ ,} and eluted with water to collect the Sakaguchi reaction positive fraction. After concentration to dryness, it was extracted three times with 5 ml of methanol, and the methanol extract was transferred to a tower (inner diameter 20
mm), expanded with methanol, collected the positive Sakaguchi reaction, Hannes reagent, and positive fractions, concentrated under reduced pressure, and obtained a syrup-like (S)-7-guanidino-
3-phosphohydroxyhebutanamide, 204mg
I got it. (yield 17%), [α] ²⁵ _D +4.6° (cl, H ₂ O) PMR. (D ₂ O) δ: 1.3-1.7 (CH ₂ × 3), 245 (2
−CH ₂ , d, J=6Hz) 3.07 (NCH ₂ , t,
J=7.0Hz) 4.3 (CH, m) IR (KBr) 3360, 3170, 2930, 2850, 1660,
1440, 1400, 1165, 1070, 1020, 955, 900cm
^-1 7-guanidino-3-phosphohydroxy-hebutanamide 204 mg (0.723 mmol), N-[4
-(3-aminopropyl)aminobutyl]-2,
2-dihydroxyethanamide dihydrochloride 253mg
(0.867 mmol), 114 mg (0.867 mmol) of glutanic acid, and 0.145 ml (8.1 mmol) of water were mixed and heated at 60°C for 24 hours. Add 5 ml of water to the reaction solution and add CM-Sephadex C-25, Na ⁺ 150
ml in a column (inner diameter 20 mm), then each
Gradient elution with 1.5 lM NaCl of water was performed. (17ml fraction). The Sakaguchi reaction-positive, ninhydrin-positive, and Hannes-positive fractions eluted in fractions 54 to 67 were collected and concentrated to dryness.
Extracted twice. The methanol extract was poured into a tower (inner diameter 20 mm) filled with 150 ml of Cephadex LH-20 and developed with methanol. The active fractions were collected and concentrated to dryness to give N-[4-3-aminopropyl]
aminobutyl]-2-[(S)-7-guanidino-
3-phosphohydroxyhebutanamide]-2-
White powder of hydroxyethanamide hydrochloride 128
I got mg. (Yield 34%) [α] ²⁵ _D +5.1° (cl, H ₂ O) PMR (D ₂ O) δ: 1.3-1.8 (CH ₂ × ₅ ), 2.0
( _CH2 ), 2.55 ( _CH2 , d, J=6Hz) 2.8-3.4
(NCH ₃ × 5), 4.3 (CH, m) 5.40 (CH) IR (KBr) 3380, 2930, 2850, 1660, 1530,
1460, 1160, 1075 ^{, 965cm -1}

Claims (1)

[Claims] 1 formula Spagarin-15-phosphate and its salts 2 represented by the formula After protecting the amino group at the 1-position, the imino group at the 4-position, and the hydroxyl group at the 11-position of spagarin, 15
The following formula is characterized in that the hydroxyl group at position is phosphoric acid esterified, and then the protecting group is removed. A method for producing spagarin-15-phosphate represented by