JPH0254823B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to spagarin-related nitrile compounds, which are new compounds useful as synthetic precursors of spagarin-related compounds, salts thereof, and methods for producing them. [Prior Art] Spagarin is a compound isolated by the present inventors from a culture solution of a spagarin-producing bacterium belonging to the genus Bacillus, and has the following structure. Spagarin exhibits a growth-inhibitory effect on Gram-positive and Gram-negative bacteria, and has shown remarkable therapeutic effects in therapeutic experiments against murine leukemia L-1210, murine leukemia FL-4, Ehrlitsuhigan, and sarcoma 180 (S-180). It is a compound that is expected to be used as an antitumor agent because it shows a life-prolonging effect (Japanese Patent Application Laid-Open No. 1983-1999).
(See No. 48957). It is also known that the above spagarin can be obtained by synthesis [The Journal of Antibiotics, Vol. 34,
See page 1625 (1981)]. In addition, as a result of searching for compounds related to spagarin, the present inventors found the following general formula: (In the formula, p represents an integer of 3 to 8, R ₃ is a hydrogen atom,
A compound represented by a lower alkyl group having 1 to 4 carbon atoms or a benzyl group which may have a hydroxyl group (Japanese Patent Application No. 185991/1983) or the following general formula: (In the formula, R ₄ is a hydrogen atom, a hydroxyl group, or a carbon number of 1 to 10
aliphatic acyloxy groups, R ₅ is α or ω-
1 hydrogen atom from α- or ω-amino group of amino acid
and residues obtained by removing the hydroxyl group from the carboxyl group,
However, excluding α-hydroxylglycine residues,
(represents an acid amide bond with the adjacent carbonyl group and amino group, and q represents an integer of 4 to 6)
It has been found that the compound represented by (Japanese Patent Application No. 57-151698) also exhibits excellent anticancer activity. Among these related compounds, the following general formula (): (In the formula, R ₆ is a hydrogen atom, CH ₂ OH, OCH ₃ or OH
and n is an integer of 6 to 8) shows particularly excellent anticancer activity. From these results, we have discovered a compound that is useful as a synthetic precursor and suitable for mass synthesis, leading to the completion of the present invention. When mass-synthesizing the spagarin analog compound represented by the above formula, spermidine (1,
The development of a method for selectively acylating only the N ¹⁰ primary amine of (5,10-triazadecane) provides an advantageous synthetic method. Until now, the methods for synthesizing these spagarin-related compounds are (1) The following formula obtained by decomposing spagarin obtained by the above-mentioned culture. A method using glyoxylspermidine expressed as a raw material (Japanese Patent Application Laid-Open No. 58-52263) (2) The following formula: 1,5-diprotected-1, represented by (in the formula, X ₁ and X ₂ are the same or different amino protecting groups)
Using 5,10-triazadecane as raw material, 1 of ^N10
A method is known in which a secondary amine is acylated and then deprotected (Japanese Patent Application Laid-Open No. 192347/1983). The compound of the formula is synthesized from spermidine by the following route. Method (1) uses spagarin obtained through culture as a raw material, and method (2) requires the synthesis of the compound of formula through several reaction steps using several amino protecting groups. This is not advantageous for mass synthesis. [Object of the Invention] An object of the present invention is to provide a spagarin-related nitrile compound useful as a synthetic precursor for a spagarin-related compound and a method for producing the same. [Structure of the Invention] To summarize the present invention, the first invention of the present invention is an invention of spagarin-related nitrile compounds and salts thereof, which have the following general formula: (In the formula, R ₁ represents a hydrogen atom, a hydroxy lower alkyl group, a lower alkoxy group, or a benzyloxy group, and n represents an integer of 6 to 8.) Further, the second invention of the present invention is an invention of a method for producing a spagarin-related nitrile compound represented by the above general formula or a salt thereof, which comprises the following general formula: (In the formula, R ₁ represents a hydrogen atom, a hydroxy lower alkyl group, a lower alkoxy group, or a benzyloxy group, and n represents an integer of 6 to 8) or a reactive derivative thereof, and the following formula: _H2N ( _CH2 ) _4NH ( _CH2 ) _2CN ...N-2-cyanoethylbutane-1,4 represented by []
- It is characterized by condensing with diamine. And, the third invention of the present invention has the following general formula a: (In the formula, _R2 is a hydrogen atom or a hydroxy lower alkyl group, and n is an integer of 6 to 8.) An invention of a method for producing a spagarin-related nitrile compound or a salt thereof, represented by the following general formula: (wherein n has the same meaning as above) or a reactive derivative thereof, and the following general formula: It is characterized by condensing with a compound represented by (wherein R ₂ has the same meaning as above). Therefore, the present inventors investigated N-2-cyanoethylbutane-1,4-diamine represented by the above general formula, which is easily obtained by condensing 1,4-diaminobutane and acrylonitrile [Journal of Medicinal Chemistry (J. Med.Chem) 2nd
Vol., p. 710 (1964)], and completed a synthetic route using the nitrile compound represented by the above general formula, which can be synthesized by acylating only the primary amine, as a synthetic precursor. By reducing -CN of the nitrile compound of formula _{to -CH2NH2} , the following general formula: A compound represented by the formula (wherein R ₁ and n are the same as in the above formula) can be obtained. A known reaction can be used to reduce -CN to _-CH2NH2 without acting on _the acid amide included in the formula. For example, a method of reducing with sodium borohydride in the presence of a salt of a transition metal such as cobalt chloride or nickel chloride [see Tetrahedron Letters, Vol. 52, p. 4555 (1969)] is preferred. R ₁ in the formula
In the case of a compound where is OCH ₂ C ₆ H ₅ , a spagarin analog compound represented by the general formula and in which R ₆ is OH can be easily obtained by hydrolyzing benzyl ether. In the general formula, when R ₁ represents a substituent other than a hydrogen atom, its configuration may be S-, R- or
Any SR-type may be used. Table 1 shows the structures and compound names of specific representative examples of the compounds of the general formula.

【table】

[Table] The compound of the general formula forms a salt with an acid, and the acid for forming the salt may be either an inorganic acid or an organic acid, and is not particularly limited, but hydrochloric acid is preferred for handling reasons. Table 2 shows the infrared absorption spectrum (measured as a KBr tablet), proton NMR spectrum (measured using TMS in deuterated methanol as a reference material), and optical rotation of a representative example of the hydrochloride.

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 8-[N-(7-guanidinoheptanoyl)-glycyl]-1-cyano-3,8-diazaoctane dihydrochloride 3.89 g (13.85 mmol) of N-(7-guanidinoheptanoyl)glycine hydrochloride 1.91 g (16.61 mmol) of N-hydroxysuccinimide was dissolved in 30 ml of dimethylformamide, and a solution of 3.43 g (16.61 mmol) of dicyclohexylcarbodiimide in 20 ml of dimethylformamide was added dropwise under ice cooling. After dropping, the solution was stirred at room temperature for 3 hours to remove the precipitated dicyclohexyl urea, and the solution was diluted with N-2-
Cyanoethylbutane-1,4-diamine 3.13g
(22.15 mmol) was added dropwise to a solution of 20 ml of dimethylformamide and stirred overnight at room temperature. The residual solid obtained by distilling off the solvent was dissolved in 300 ml of water, washed with ethyl acetate, and washed with CM-Sephadex C-25.
(Na type) (manufactured by Pharmacia) Pour into a 500 ml column, wash with water (500 ml), then add 2.5 ml of water and 2.5 ml of 1M NaCl.
Gradient elution was performed using Fractions containing the target product were collected and dried under reduced pressure, and methanol was added to the remaining solid to remove insoluble NaCl. This operation was repeated twice, and the resulting solution was applied to a 500 ml column of Sephadex LH-20 and eluted with methanol. Fractions containing the target product were collected and concentrated under reduced pressure to obtain 2.62 g of 8-[N-(7-guanidinoheptanoyl)glycyl]-1-cyano-3,8-diazaoctane dihydrochloride. mp111～
113°C, yield 43.0% Example 2 8-[N-(7-guanidinoheptanoyl)-L
-Ceryl]-1-cyano-3,8-diazaoctane dihydrochloride 7-guanidinoheptanoic acid hydrochloride 116.9g
(0.52 mol) and N-hydroxysuccinimide
72.2g (0.63mol) of dimethylformamide 500
dicyclohexylcarbodiimide dissolved in ml
129.4g (0.63mol) dimethylformamide 500
ml of the solution was added dropwise under ice-cooling, and after the dropwise addition, the solution was stirred overnight at room temperature. The precipitated dicyclohexyl urea was removed, and the liquid was diluted with 8-(L-seryl)-1-cyano-
A solution of 166.0 g (0.63 mol) of 3,8-diazaoctane hydrochloride in 500 ml of dimethylformamide was added dropwise and stirred overnight at room temperature. The residual solid obtained by distilling off the solvent was dissolved by adding 2 parts of water, washed with ethyl acetate, diluted to 5 with water, and applied to a column of CM-Sephadex C-25 (Na type) 2.
After washing with water (5), it was eluted with 0.1-0.2M NaCl.
Fractions containing the target product were collected and dried under reduced pressure, and the remaining solid was extracted with methanol. Methanol is distilled off from the methanol extract and the remaining solid is removed.
The column was applied to a column of Diaion HP-20 (manufactured by Mitsubishi Chemical Industries, Ltd.) 5 dissolved in 1M NaCl solution and adjusted with 1M NaCl, washed with 1M NaCl5, and eluted with water-methanol (8:2) 10. Fractions containing the target product are collected, dried under reduced pressure, and methanol is added to separate insoluble NaCl. This operation 2
Repeat several times and transfer the resulting solution to Sephadex LH.
It was applied to a -20 9 column and eluted with methanol. Fractions containing the target product were collected and concentrated under reduced pressure to obtain a syrupy form of 8-[N-(7-guanidinoheptanoyl)-L-seryl]-1-cyano-3,8-
151.9 g of diazaoctane dihydrochloride was obtained. [α] _D =
−14.8°, yield 61.8%. Example 3 (±)-8-[N-(7-guanidinoheptanoyl)-α-methoxyglycyl]-1-cyano-
3,8-diazaoctane dihydrochloride N-(7-guanidinoheptanoyl)-α-methoxyglycine hydrochloride 774 mg (2.49 mmol) and p
- Dissolve 346 mg (2.49 mmol) of nitrophenol in 8 ml of dimethylformamide, add dropwise a solution of 1.03 g (4.98 mmol) of dicyclohexylcarbodiimide in 2 ml of dimethylformamide, and at room temperature
Stirred for 3.5 hours. The reaction solution was added dropwise to a solution of 422 mg (2.99 mmol) of N-2-cyanoethylbutane-1,4-diamine in 2 ml of dimethylformamide, and the mixture was stirred at room temperature for 1 hour. Remove the precipitated dicyclohexyl urea, concentrate the liquid under reduced pressure, dissolve the resulting solid in 50 ml of water, and adjust the pH to 6.5.
After adjusting to , it was washed with ethyl acetate. CM− aqueous layer
Pour 300ml of Sephadex C-25 (Na type),
After washing with water (500 ml), gradient elution with 1.5 of water and 1.5 of 0.6M NaCl was performed. Fractions containing the target product were collected and dried under reduced pressure, and methanol was added to the remaining solid to remove insoluble NaCl. This operation was repeated twice, and the resulting solution was applied to a 300 ml column of Sephadex LH-20 and eluted with methanol. Fractions containing the target product were collected and concentrated under reduced pressure to form a syrupy (±)-8-[N-
933 mg of (7-guanidinoheptanoyl)-α-methoxyglycyl]-1-cyano-3,8-diazaoctane dihydrochloride was obtained. Yield 79.6%. Example 4 (-)-8-[N-(7-guanidinoheptanoyl)-α-methoxyglycyl]-1-cyano-
3,8-diazaoctane dihydrochloride (-)-N-(7-guanidinoheptanoyl)-
Dissolve 709 mg (2.28 mmol) of α-methoxyglycine hydrochloride in 10 ml of dimethylformamide and
810 mg (4.56 mmol) of 2-cyanoethylbutane-1,4-diamine hydrochloride [643.9 mg (4.56 mmol) of N-2-cyanoethylbutane-1,4-diamine was dissolved in 4.56 ml of 1N HCl and dried under reduced pressure. ] in dimethylformamide and N-
Hydroxysuccinimide 524 mg (4.56 mmol)
followed by dicyclohexylcarbodiimide
Drop a 5 ml solution of 941 mg of dimethylformamide,
Stir overnight at room temperature. The precipitated dicyclohexyl urea was removed, and the solution was added to 100 ml of water and washed with ethyl acetate. CM the aqueous layer as in Example 3.
-Purified using 400 ml of Cephadex C-25 (Na type) and 500 ml of Cephadex LH-20 columns to form syrupy (-)-8-[N-(7-guanidinoheptanoyl)-α-methoxyglycyl. ]
599 mg of -1-cyano-3,8-diazaoctane dihydrochloride was obtained. [α] _D = -33.9°, yield 55.9%. Example 5 (−)-8-[N-(7-guanidinoheptanoyl)-α-benzyloxyglycyl]-1-cyano-3,8-diazaoctane dihydrochloride (+)-N-(7-guanidino heptanoyl) −
α-benzyloxyglycine hydrochloride 240 mg (0.62
220 mg (1.24 mmol) of N-2-cyanoethylbutane-1,4-diamine hydrochloride in 5 ml of dimethylformamide and 143 mg (1.24 mmol) of N-hydroxysuccinimide were added, followed by A solution of 256 mg (1.24 mmol) of dicyclohexylcarbodiimide in 3 ml of dimethylformamide was added dropwise, and the mixture was stirred overnight at room temperature. The precipitated dicyclohexyl urea was removed, the liquid was concentrated under reduced pressure, the resulting residual solid was dissolved in 20 ml of water, and the solution was dissolved in the same manner as in Example 3.
It was purified using a 300 ml column of CM-Sephadex C-25 (Na type) and a 300 ml column of Sephadex LH-20, and a syrupy (-)-8-[N-(7
202 mg of -guanidinoheptanoyl)-α-benzyloxyglycine]-1-cyano-3,8-diazaoctane dihydrochloride was obtained. [α] _D = −20.8°, yield
59.5%. Example 6 (±)-8-[N-(9-guanidinononanoyl)
-α-methoxyglycyl]-1-cyano-3,
8-Diazaoctane dihydrochloride N-(9-guanidinononanoyl)-α-methoxyglycine hydrochloride 487 mg (1.50 mmol) and p-
209 mg (1.50 mmol) of nitrophenol was dissolved in 8 ml of dimethylformamide, a solution of 619 mg (3.00 mmol) of dicyclohexylcarbodiimide in 2 ml of dimethylformamide was added dropwise, and the mixture was stirred at room temperature.
Stirred for 3.5 hours. The reaction solution was added dropwise to a solution of 318 mg (2.25 mmol) of N-2-cyanoethylbutane-1,4-diamine in 2 ml of dimethylformamide, and the mixture was stirred at room temperature for 1 hour. Remove the precipitated dicyclohexyl urea, concentrate the liquid under reduced pressure, dissolve the resulting solid in 50 ml of water, and adjust the pH to 6.5.
After adjusting to , the mixture was washed with ethyl acetate. CM− aqueous layer
Pour 300ml of Sephadex C-25 (Na type),
After washing with water (500 ml), gradient elution with 1.5 of water and 1.5 of 0.6M NaCl was performed. Fractions containing the target product were collected and dried under reduced pressure, and methanol was added to the remaining solid to remove insoluble NaCl. This operation was repeated twice, and the resulting solution was applied to a 300 ml column of Sephadex LH-20 and eluted with methanol. Fractions containing the target product were collected and concentrated under reduced pressure to form a syrupy (±)-8-[N-
(9-guanidinononanoyl)-α-methoxyglycyl]-1-cyano-3,8-diazaoctane 2
519 mg of hydrochloride was obtained. Yield 71.4%. Reference Example 1 Reduction of 8-[N-(7-guanidinoheptanoyl)glycyl]-1-cyano-3,8-diazaoctane dihydrochloride 8-[N-(7-guanidinoheptanoyl)glycyl]-1-cyano -3,8-diazaoctane 2
Dissolve 2.52 g (5.71 mmol) of hydrochloride and 1.63 g (6.86 mmol) of cobalt chloride (CoCl ₂ 6H ₂ O) in 40 ml of methanol, and add sodium borohydride.
Gradually add 1.30 g (34.28 mmol) under ice cooling.
After stirring at room temperature for 1.5 hours, add 20 ml of water.
The pH was adjusted to 7.0 with 2N-HCl to remove the black precipitate. The remaining solid obtained by concentrating the liquid under reduced pressure was added to 30 ml of water.
Dissolve in CM-Sephadex C-25 (Na
Type) 500ml column, washed with water (500ml), and then
Gradient elution was performed with 2.5 and 1M NaCl2.5. Fractions containing the target product are collected, dried under reduced pressure, and methanol is added to the remaining solid to remove insoluble matter.
NaCl was removed. This operation was repeated twice, and the resulting solution was applied to a 500 ml column of Sephadex LH-20 and eluted with methanol. Fractions containing the target product were collected, concentrated under reduced pressure, and the residual solid (1.65 g) was mixed with methanol-ethanol (1:2).
The product was crystallized to obtain 1.43 g of 10-[N-(7-guanidinoheptanoyl)glycyl]-1,5,10-triazadecane trihydrochloride. Yield 52.0% mp178-180°C NMR (CH ₃ OH-d ₄ ): δ = 1.2-1.9 (CH ₂ ×6),
1.9 to 2.4 (CH ₂ × 2), 2.8 to 3.4 (NCH ₂ × 5),
3.80 (CH ₂ ) IR (KBr): ν (cm ^-1 ) = 3300, 3150, 2930, 1640,
1550, 1515, 1460, 1410, 1160 Reference example 2 8-[N-(7-guanidinoheptanoyl)-L
-Ceryl]-1-cyano-3,8-diazaoctane dihydrochloride reduction 8-[N-(7-guanidinoheptanoyl)-L
-Ceryl]-1-cyano-3,8-diazaoctane dihydrochloride 151.0g (0.32 mol) and cobalt chloride
Dissolve 92.2 g (0.39 mol) in 2.5 methanol,
97.8 g (2.58 mol) of sodium borohydride is gradually added under ice cooling. After stirring at room temperature for 1 hour, water 6 was added to adjust the pH to 7.0 and remove the black precipitate. The liquid was concentrated to half its volume, and a saturated aqueous solution of Na ₂ CO ₃ was added to adjust the pH to 8.5 to remove precipitated cobalt carbonate. After adjusting the pH of the liquid to 6.0,
Dilute to 50% with water and use CM-Sephadex C-
25 (Na type) 2 columns and washed with water (4)
Afterwards, it was eluted with 0.2M NaCl. Fractions containing the target product were collected and dried under reduced pressure, and methanol was added to the remaining solid to remove insoluble NaCl. Repeat this operation twice and use the liquid obtained as Sephadex LH.
The mixture was applied to a -20 9 column and eluted with methanol. Fractions containing the target product were collected and concentrated under reduced pressure. The residual solid obtained was crystallized from methanol (20 ml) and ethanol (100 ml) to give 10-[N-(7-guanidinoheptanoyl)-L-seryl]-1, 5,
74.9 g of 10-triazadecane trihydrochloride was obtained. yield
45.8%. mp145-146.5℃ NMR (CH ₃ OH-d ₄ ): δ = 1.2-1.9 (CH ₂ ×6),
1.9 to 2.5 (CH ₂ × 2), 2.9 to 3.4 (NCH ₂ × 5),
3.78 (CH ₂ ), 4.35 (CH) IR (KBr): ν (cm ^-1 ) = 3350, 3150, 2930, 1650,
1530, 1460, 1380, 1250, 1165, 1060 Reference example 3 (±)-8-[N-(7-guanidinoheptanoyl)-α-methoxyglycyl]-1-cyano-
Reduction of 3,8-diazaoctane dihydrochloride (±)-8-[N-(7-guanidinoheptanoyl)-α-methoxyglycyl]-1-cyano-3,
Dissolve 900 mg (1.91 mmol) of 8-diazaoctane dihydrochloride and 455 mg (1.91 mmol) of cobalt chloride in 12 ml of methanol and add sodium borohydride.
Gradually add 361 mg (9.55 mmol) under ice cooling.
After stirring at room temperature for 1.5 hours, post-treatment was carried out in the same manner as in Reference Example 1, and CM-Sephadex C-25 (Na
Type) 300ml and Sephadex LH-20 300ml
Purification using a column of white powder (±)-
10-[N-(7-guanidinohepetanoyl)-α-
391 mg of methoxyglycyl]-1,5,10-triazadecane trihydrochloride was obtained. Yield 40.0%. NMR (CH ₃ OH−d ₄ ): δ = 1.2 to 2.0 (CH ₂ ×6),
2.0 to 2.5 (CH ₂ × 2), 2.9 to 3.4 (NCH ₂ × 5),
3.37 (OCH ₃ ), 5.26 (CH) IR (KBr): ν (cm ^-1 ) = 3420, 2950, 1650, 1520,
1460, 1360, 1190, 1160, 1090 Reference example 4 (-)-8-[N-(7-guanidinoheptanoyl)-α-methoxyglycyl]-1-cyano-
Reduction of 3,8-diazaoctane dihydrochloride (-)-8-[N-(7-guanidinoheptanoyl)-α-methoxyglycyl]-1-cyano-3,
Dissolve 584 mg (1.24 mmol) of 8-diazaoctane dihydrochloride and 355 mg (1.49 mmol) of cobalt chloride in 13 ml of methanol, and add sodium borohydride.
Gradually add 282 mg (7.44 mmol) under ice cooling.
After stirring at room temperature for 1 hour, post-treatment was performed in the same manner as in Reference Example 1 to obtain CM-Sephadex C-25 (Na type).
It was purified using a 200 ml column and a 200 ml column of Cephadex LH-20, and a white powder (-)-10-
[N-(7-guanidinoheptanoyl)-α-methoxyglycyl]-1,5,10-triazadecane 3
405 mg of hydrochloride was obtained. Yield 63.9%. [α] _D = −29.4° (C = 1, H ₂ O) NMR (CH ₃ OH−d ₄ ): δ = 1.2 to 2.0 (CH ₂ × 6),
2.0 to 2.5 (CH ₂ × 2), 2.9 to 3.4 (NCH ₂ × 5),
3.37 (OCH ₃ ), 5.26 (CH) IR (KBr): ν (cm ^-1 ) = 3420, 2950, 1650, 1520,
1460, 1360, 1190, 1160, 1090 Reference example 5 (−)-8-[N-(7-guanidinoheptanoyl)-α-benzyloxyglycyl]-1-cyano-3,8-diazaoctane dihydrochloride Reduction (-)-8-[N-(7-guanidinoheptanoyl)-α-benzyloxyglycyl]-1-cyano-3,8-diazaoctane dihydrochloride 201 mg (0.37
mmol) and cobalt chloride 105 mg (0.44 mmol)
Dissolve in 4 ml of methanol, and gradually add 84 mg (2.21 mmol) of sodium borohydride under ice cooling. After stirring at room temperature for 2 hours, post-treatment was carried out in the same manner as in Reference Example 1, and CM-Sephadex C-25 (Na
(type) 300ml and a 300ml column of Sephadex LH-20 were used to purify the (-)-10 as a white powder.
64.8 mg of -[N-(7-guanidinoheptanoyl)-α-benzyloxyglycyl]-1,5,10-triazadecane trihydrochloride was obtained. Yield 30.0%. [α] _D −22.1° (C = 1, H ₂ O) NMR (CH ₃ OH-d ₄ ): δ = 1.2 to 2.0 (CH ₂ × 6),
2.0 to 2.5 (CH ₂ × 2), 2.8 to 3.4 (NCH ₂ × 5),
4.60 (CH ₂ ), 5.46 (CH), 7.30 (C ₆ H ₅ ) IR (KBr): ν (cm ^-1 ) = 3320, 3250, 3160, 2930,
1660, 1525, 1460, 1450, 1370, 1160, 1070,
1025, 745, 700 (-)-10-[N-(7-guanidinoheptanoyl)-α-benzyloxyglycyl]- obtained above
1,5,10-triazadecane trihydrochloride 63.2mg
(0.11 mmol) was dissolved in 1.5 ml of 1N acetic acid, 30 mg of palladium black was added, and under hydrogen pressure (9 Kg/cm ² ),
I stirred it for a day. Remove the catalyst and add 10ml of water to the liquid.
Add CM-Sephadex C-25 (Na type)
Pour into a 150 ml column, add 900 ml of water and 900 ml of 1M NaCl.
Gradient elution was performed using Fractions containing the target product were collected and dried under reduced pressure, and methanol was added to the remaining solid to remove insoluble NaCl.
This operation was repeated twice, and the resulting solution was applied to a 300 ml column of Sephadex LH-20 and eluted with methanol. Fractions containing the target product were collected and concentrated under reduced pressure to obtain a white powder (-)-10-[N-
30.7 mg of (7-guanidinoheptanoyl)-α-hydroxyglycyl]-1,5,10-triazadecane trihydrochloride was obtained. Yield 57.4%. [α] _D = −12.0° (C = 1, H ₂ O) NMR (CH ₃ OH−d ₄ ): δ = 1.2 to 2.0 (CH ₂ × 6),
2.24( _CH2 ), 2.30( _CH2 ), 2.9~3.4( _NCH2 ×
5), 5.56 (CH) IR (KBr): ν (cm ^-1 ) = 3400, 2950, 1655, 1525,
1460, 1360, 1160, 1120, 1080 Reference example 6 Synthesis of N-(7-guanidinoheptanoyl)glycine hydrochloride 7-guanidinoheptanoic hydrochloride 10.0g
(44.70 mmol) and 6.17 g (53.64 mmol) of N-hydroxysuccinimide were dissolved in 50 ml of dimethylformamide, and a solution of 11.06 g (53.64 mmol) of dicyclohexylcarbodiimide in 20 ml of dimethylformamide was added dropwise under ice cooling. After dropping, the solution was stirred at room temperature for 2 hours to remove precipitated dicyclohexyl urea, and the solution was added dropwise to a solution of 7.49 g (53.64 mmol) of glycine ethyl ester hydrochloride and 7.54 ml of triethylamine in 10 ml of dimethylformamide, and the mixture was stirred at room temperature overnight. . The residual solid obtained by distilling off the solution was dissolved in 0.5M NaCl and diluted with 0.5M NaCl.
800ml of Diaion HP-20 adjusted with NaCl
0.5M NaCl2.5, water 5,8
It was eluted with 2% methanol water. Fractions containing the target product are collected, concentrated under reduced pressure, and dissolved in a syrupy form of N.
11.68 g of -(7-guanidinoheptanoyl)glycine ethyl ester hydrochloride was obtained. Yield 70.8%. N-(7-guanidinoheptanoyl)glycine ethyl ester hydrochloride 4.28 g (13.85 mmol)
Dissolved in 20ml of 50% methanol water and diluted with 1N−
16.6 ml of NaCl was added under ice cooling, and the mixture was stirred at room temperature for 1 hour. After adjusting the pH to 7.0, it was concentrated under reduced pressure, and 10 ml of water was added to the resulting residual solid, the pH was adjusted to 2.0, it was dissolved, and the mixture was dried under reduced pressure. The obtained N-(7-guanidinoheptanoyl)glycine hydrochloride was used in the next reaction without being purified. Reference Example 7 Synthesis of 8-(L-seryl)-1-cyano-3,8-diazaoctane hydrochloride 598 g (2.5 mol) of carbobenzoxy-L-serine and 317 g of N-hydroxysuccinimide
(2.75 mol) was dissolved in 2.5 mol of dioxane, and 576 g of dicyclohexylcarbodiimide was cooled on ice.
A solution of (2.75 mol) in dioxane (2) was added dropwise. After dropping, the solution was stirred overnight at room temperature to remove the precipitated dicyclohexyl urea, and the solution was poured with N-2
-cyanoethylbutane-1,4-diamine 530g
(3.75 mol) was added dropwise to a dioxane 1 solution, and the mixture was stirred at room temperature overnight. Add 1.5 liters of water to the reaction solution, concentrate under reduced pressure, add 2.5 liters of a saturated aqueous solution of NaHCO ₃ to the obtained residual solid, and dilute with ethyl acetate (1.5 liters).
Extracted twice. The ethyl acetate layers were combined, washed twice with saturated brine, and dried over anhydrous Na ₂ SO ₄ . The residual solid obtained by distilling off the solvent (approximately 700 g) was diluted with 2.5 g of ethanol.
and ethanol saturated with hydrogen chloride gas was added until acidic. Take the precipitated crystals and
693 g of -(carbobenzoxy-L-seryl)-1-cyano-3,8-diazaoctane hydrochloride was obtained.
Yield 69.5%. mp151-153℃ [α] _D -4.6゜(C=1, CH ₃ OH) 8-(Carbobenzoxy-L-seryl) obtained above
-1-cyano-3,8-diazaoctane hydrochloride
Dissolve 350g (0.88mol) in methanol 4,
8.75 g of 10% palladium-carbon was added and stirred for 2 days in a hydrogen stream. The catalyst was removed and the liquid was concentrated under reduced pressure to obtain 233 g of syrupy 8-(L-seryl)-1-cyano-3,8-diazaoctane hydrochloride. It can be used as a raw material for the next reaction without purification. Reference example 8 (±)-N-(7-guanidinoheptanoyl)-
Synthesis of α-methoxyglycine hydrochloride 7-guanidinoheptanamide hydrochloride 10g
(44.9 mmol) was heated and dissolved in 150 ml of dimethylformamide, 4.55 g (49.4 mmol) of glyoxylic acid and 150 ml of acetone were added, and the mixture was refluxed in an oil bath at 90° C. for 3 hours. The residual solid obtained by distilling off the solvent was dissolved in water.
Dissolve in 100ml, CM-Sephadex C-25
(Na type) It was applied to a 1100ml column and eluted with water.
Collect fractions containing the target product, concentrate under reduced pressure,
9.36 g of N-(7-guanidinoheptanoyl)-α-hydroxyglycine was obtained. mp165-6℃ (decomposition), yield 80.1%. 9.36 g (36.0 mmol) of N-(7-guanidinoheptanoyl)-α-hydroxyglycine was dissolved in 300 ml of anhydrous methanol, 5 ml of concentrated sulfuric acid was added under ice cooling, and the mixture was stirred at room temperature for 3 hours. Concentrate the reaction solution to about 100ml under reduced pressure, and add 200ml of water.
and adjust the pH to 10.0 with 2N-NaOH.
After 1 hour, the pH was adjusted to 6.5 and the residue obtained by concentration under reduced pressure was converted into CM-Sephadex C-25 (Na type).
It was applied to a 1100ml column and eluted with water. Fractions containing the target product were collected, concentrated under reduced pressure, and (±)-N
7.90 g of -(7-guanidinoheptanoyl)-α-methoxyglycine was obtained. mp201~2℃ (decomposition) yield 80.0%. NMR (CH ₃ OH−d ₄ ): δ = 1.2 to 1.9 (CH ₂ ×4),
2.30 ( _CH2 ), 3.17 ( _NCH2 ), 3.32 ( _OCH3 ), 5.23
(CH) IR (KBr): ν (cm ^-1 ) = 3370, 3275, 2930, 1655,
1580, 1540, 1475, 1415, 1330, 1195, 1120,
1090, 985, 800 (±)-N-(7-guaninoheptanoyl)-α
-Methoxyglycine 7.80g (2.84 mmol) 1N
-Dissolved in 28.4ml of HCl and concentrated under reduced pressure to obtain a white powder (±)-N-(7-guanidinoheptanoyl)-
8.83g of α-methoxyglycine hydrochloride was obtained. Reference example 9 (-)-N-(7-guanidinoheptanoyl)-
Synthesis of α-methoxyglycine hydrochloride (±)-N-(7-guanidinoheptanoyl)-α-methoxyglycine hydrochloride obtained in Reference Example 8 6.58
(21.2 mmol) and 4.88 g (42.4 mmol) of N-hydroxysuccinimide are dissolved in 70 ml of dimethylformamide, and 4.87 g (21.2 mmol) of L-phenylalanine ethyl ester hydrochloride and 2.98 ml of triethylamine are added. Under ice cooling, a solution of 8.75 g (42.4 mmol) of dicyclohexylcarbodiimide in 30 ml of dimethylformamide was added dropwise, and after the addition, the mixture was stirred at room temperature for 4 hours. Precipitated dicyclohexyl urea was removed, and the liquid was concentrated under reduced pressure. The remaining solid obtained was dissolved in 300 ml of water, applied to a column of CM-Sephadex C-25 (Na type) 1, washed with water (1), and eluted with 0.2 M NaCl. did. The fractions containing the target product were collected and concentrated under reduced pressure.The resulting residue was adjusted with water.Diaion HP-20 1
column, 1.5% water, 22% methanol water
, 50% methanol water 4 were sequentially eluted. The eluate of 50% methanol water containing the target product was concentrated under reduced pressure to obtain 5.31 g of N-(7-guanidinoheptanoyl)-α-methoxyglycyl-L-phenylalanine ethyl ester hydrochloride. Yield 51.6%. NMR( _CH3OH − _d4 )δ: 1.17( _CH3 ), 1.1-1.9
(CH ₂ × 4), 2.27 (CH ₂ ), 2.9-3.4 (5H), 3.31
(OCH ₃ ), 4.11 (CH ₂ ), 5.30 (CH), 7.20 (C ₆ H ₅ ) N-(7-guanidinoheptanoyl)- obtained above
Dissolve 5.21 g (10.7 mmol) of α-methoxyglycyl-L-phenylalanine ethyl ester hydrochloride in 35 ml of methanol, and add 1N-NaOH under ice cooling.
Added 12.8ml and stirred for 1.5 hours. Adjust the pH to 6.5 and concentrate the residual solid (about 5.7 g) under reduced pressure.
0.05M sodium acetate-hydrochloric acid buffer (PH5.0) 100
ml, 10 mg of carboxypeptidase P (manufactured by Hinshuzo Co., Ltd.) was added, and the mixture was shaken at 30°C for 8 hours. The residue obtained by distilling off the solvent was
-20 4 column and eluted with water. Collect the fractions containing the target product, concentrate to about 30 ml, adjust the pH to 2.1, and add CM-Sephadex C.
-25 (Na type) was applied to a 500ml column and eluted with water. Fractions containing the target product were collected and concentrated under reduced pressure to give (-)-N-(7-guanidinoheptanoyl)-
1.16g of α-methoxyglycine was obtained. Yield 32.6
%. mp197-198℃ (decomposition) [α] _D -20.2゜ (C=1, H ₂ O) (-)-N-(7-guanidinoheptanoyl)-
1g (3.65 mmol) of α-methoxyglycine
By dissolving in 3.65 ml of 1N-HCl and concentrating under reduced pressure, (-)-N-(7-guanidinoheptanoyl)-α-methoxyglycine hydrochloride as a white powder was obtained.
Obtained 1.13g. Reference example 10 (+)-N-(7-guanidinoheptanoyl)-
Synthesis of α-benzyloxyglycine hydrochloride N-(7-guanidinoheptanoyl)-α-hydroxyglycine obtained in the same manner as Reference Example 8 6.94
g (26.7 mmol) to 130 ml of benzyl alcohol
and 6.5 ml of concentrated hydrochloric acid were added and stirred at 65°C for 5 hours. The reaction solution was added to a mixture of 130 ml of water and 130 ml of methanol, the pH was adjusted to 12.0 with 2N-NaOH, and the mixture was stirred at room temperature for 2 hours. The pH was adjusted to 7.0, methanol was distilled off under reduced pressure, the aqueous layer was separated, and the benzyl alcohol layer was further extracted twice with water. Combine the water layer,
After washing twice with ethyl acetate, it was concentrated to about 50 ml under reduced pressure, and CM-Sephadex C-25 (Na type)
It was applied to a 500ml column and eluted with water. Fractions containing the target product are collected, concentrated under reduced pressure, and (±)−
3.66 g of N-(7-guanidinoheptanoyl)-α-benzyloxyglycine was obtained. Yield 39.1%. mp197-199℃ (decomposition) NMR (CH ₃ OH-d ₄ ): δ = 1.2-1.8 (CH ₂ × 4),
2.25 ( _CH2 ), 3.04 ( _NCH2 ), 4.58 ( _CH2 ), 5.39
(CH), 7.28 (C ₆ H ₅ ) IR (KBr): ν (cm ^-1 ) = 3400, 3270, 3150, 2925,
1675, 1625, 1575, 1520, 1470, 1405, 1325,
1240, 1205, 1060, 975, 915, 800, 750, 700,
605 (±)-N-(7-guanidinoheptanoyl)-
(±)-N-(7-guanidinoheptanoyl)-α-benzyloxyglycine hydrochloride obtained by dissolving 1.64 g (4.68 mmol) of α-benzyloxyglycine in 4.68 ml of 1N HCl and drying it under reduced pressure. Dissolved in 75 ml of formamide, 4.08 g (9.36 mmol) of N-hydroxysuccinimide, 1.08 g of L-phenylalanine ethyl ester hydrochloride.
(4.68 mmol), triethylamine 0.65 ml (4.68
Then, under ice-cooling, a solution of 1.93 g (9.36 mmol) of dicyclohexylcarbodiimide in 10 ml of dimethylformamide was added dropwise. After the addition, the mixture was stirred at room temperature overnight to remove precipitated dicyclohexyl urea. The residue obtained by concentrating the liquid under reduced pressure was applied to a 500ml column of Diaion HP-20 prepared with water.
It was sequentially eluted with 1.5 ml of 75% methanol water. The eluate of 75% methanol water containing the target product was collected and concentrated under reduced pressure to obtain N-(7-guanidinoheptanoyl)-α.
1.04 g of -benzyloxyglycyl-L-phenylalanine ethyl ester hydrochloride was obtained. Yield 39.5
%. NMR ( _CH3OH - _d4 ): δ=1.19 and 1.22 ( _CH3 ),
1.1~2.0 ( _CH2 x 4), 2.28 ( _CH2 ), 3.0~3.4
(5H), 4.14 and 4.17 (CH ₂ ), 4.58 (CH ₂ ), 5.53
(CH), 7.22 (C ₆ H ₅ ), 7.33 (C ₆ H ₅ ) N-(7-guanidinoheptanoyl)-α-benzyloxyglycyl-L-phenylalanine ethyl ester hydrochloride 1.00 g (1.78 mmol) ) in 10 ml of methanol, and add 2.14 ml of 1N-NaOH under ice-cooling.
ml and stirred for 1.5 hours. Add 20ml of water,
After adjusting the pH to 7.0, the residual solid obtained by concentration under reduced pressure was
0.05M sodium acetate-hydrochloric acid buffer (PH5.0) 270
ml and 30 ml of methanol, add 10 mg of carboxypeptidase P (manufactured by Hin Shuzo Co., Ltd.), and incubate at 30°C.
It was shaken for 7 hours. After concentrating to about 50 ml, remove the precipitate and pour the liquid into a
column and eluted with water. Collect fractions containing the target product and extract (+)-N-(7-guanidinoheptanoyl)-α-benzyloxyglycine.
256.2 mg was obtained. Yield 41.1%. [α] _D +11.5° (C=1,
(CH ₃ OH) [Effects of the Invention] According to the present invention, there is no need for the synthesis and deprotection steps of the selective amino-protected form of spermidine that were required in conventional synthesis methods, and N, which is an extremely cheap raw material, is used. -2-cyanoethylbutane-1,4-
Spagarin analogs can be synthesized from diamines in a short process. Therefore, the present invention provides an economically advantageous method for producing spagarin-related compounds.

Claims (1)

[Claims] 1. The following general formula: (wherein, R ₁ represents a hydrogen atom, a hydroxy lower alkyl group, a lower alkoxy group, or a benzyloxy group, and n represents an integer of 6 to 8) and salts thereof. 2 General formula below: (In the formula, R ₁ represents a hydrogen atom, a hydroxy lower alkyl group, a lower alkoxy group, or a benzyloxy group, and n represents an integer of 6 to 8) or a reactive derivative thereof, and the following formula: _H2N ( _CH2 ) _4NH ( _CH2 ) _2CN ...N-2-cyanoethylbutane-1,4 represented by []
- The following general formula is characterized by condensing with diamine: A method for producing a spagarin-related nitrile compound or a salt thereof represented by the formula (wherein R ₁ and n have the same meanings as the above formula). 3 General formula below: (wherein n represents an integer of 6 to 8) or a reactive derivative thereof, and the following general formula: (In the formula, R ₂ represents a hydrogen atom or a hydroxy lower alkyl group.) The following general formula a is characterized by condensing with a compound represented by: A method for producing a spagarin-related nitrile compound or a salt thereof represented by the formula (wherein R ₂ and n are as defined above).