JPS6136760B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for producing the therapeutically useful decapeptide ceruletide (see US Pat. No. 3,472,832) and its salts.

Using symbols commonly used in peptide chemistry, the decapeptide of the present invention is represented by the following formula ().

That is, pyroglutamyl-glutaminyl-aspartyl-tyrosyl (O-sulfate)-threonyl-glucyl-tryptopyl-methionyl-aspartyl-phenylalaninamide. The phenolic hydroxyl group of the tyrosine residue of the peptide is esterified with a sulfate group,
All of the constituent amino acids, except for glycine, are L-type.

The previous synthesis method used a protected decapeptide in which the hydroxyl group of the threonine residue was acetylated.Formula (): in an anhydrous medium and then saponifying the acetyl groups. This method requires that the sulfation conditions are in anhydrous medium and that due to the saponification step, besides other unstructured impurities, there are several by-products, mainly polysulfated, deacidated and oxidized by-products. gave. All these impurities are
It could only be removed by large-scale countercurrent distribution methods, which inevitably led to losses in time and yield.

According to the novel method according to the invention, the unprotected decapeptide formula (): is sulfated in an aqueous medium and does not require a final saponification step. Under these milder conditions,
Ceruletide is obtained in high yield (>95% conversion) and in high purity (approximately 90% by HPLC).
Further purification can be carried out by countercurrent distribution methods.

The unprotected decapeptide () is obtained by various synthetic methods which not only avoid the risk of racemization but also reduce the risk of decomposition of tryptophan by repeated strong acid treatments. The synthesis of this decapeptide consists of the appropriate sequential condensation of protected amino acids or peptide fragments. Coupling allows the resulting decapeptide to contain 10 of the desired amino acids.
It is carried out to have an arrangement of molecules. Since the amino acids and peptide fragments that are condensed by methods known per se also in the field of peptide chemistry have amino and carboxyl groups that do not take part in the formation of the peptide chain, but can take part in the reaction, These are blocked with protecting groups that can be removed by hydrolysis, saponification, or hydrolysis. For example, the following protecting groups can be used to protect the amino group.

Carbobenzyloxy (benzyloxycarbonyl), tertiarybutoxy (t-butyloxycarbonyl), trityl (triphelmethyl), formyl or trifluoracetyl.

For the protection of carboxyl groups, the following protecting groups can be used, for example.

Methyl, ethyl, t-butyl or benzyl.

The formation of a peptide bond between an amino group on one molecule and a carboxyl group on another molecule can be achieved using mixed acid anhydrides, acids, p-nitrophenyl, 2,4,5-
by active acyl derivatives such as trichlorophenyl or N-hydroxysuccinimidyl ester, or by condensing agents alone such as dicyclohexylcarbodiimide or 1-cyclohexyl-3-morpholinylcarbodiimide, or by 1
This can be carried out by direct condensation of a free amino group and a free carboxyl group in the presence of -hydroxybenzotriazole, N-hydroxysuccinimide, 8-hydroxyquinoline, or the like. The condensation can be carried out in solvents such as N,N-dilower alkylformamides, lower aliphatic nitriles, pyridine or tetrahydrofuran, for example dimethylformamide, acetonitrile, pyridine or tetrahydrofuran. The reaction temperature ranges from -20°C to room temperature. Reaction times are generally 6 to 120 hours.

The sulfation (a characteristic step of the invention) is carried out with sulfation agent B: SO ₃ in an aqueous medium. B: is a tertiary base, such as pyridine, substituted pyridine, trimethyl-, triethyl or tri-lower alkylamine, N-lower alkylmorpholine,
These include dimethylaniline, imidazole, substituted imidazole, and quinoline.

Decapeptide () is an organic or inorganic base,
Or, by adding both, it can be dissolved in water at pH 9.5 to 10.5. Organic bases are those mentioned above, inorganic bases are NaOH, KOH, Na ₂ CO ₃ , K ₂ CO ₃
etc. The sulfating agent is added neat or dissolved in a water-miscible organic solvent such as dimethylformamide, acetone, acetonitrile, etc. The pH of the reaction solution was adjusted to PH8.5 by adding the base mentioned above.
12.5. The reaction time is generally 2 to
It is 120 hours.

This synthesis involves sulfation and work-up of the decapeptide () as described in Example 4. The decapeptide () is obtained by coupling a hexapeptide () and a tetrapeptide () by the azide method as described in Example 3.
Both intermediates () and () are synthesized by stepwise extension according to Examples 1 and 2, respectively.

The above synthetic method can be expressed as follows using symbols commonly used in peptide chemistry.

The following examples illustrate the invention without limiting it.

Example 1 Production of intermediates ().

H-Glu-Gln-Asp-Tyr-Thr-Gly-
NHNH ₂ HCl in 300 ml of anhydrous tetrahydrofuran
To a solution of 17.5 g of Boc-Gly-OH, 10.99 ml of N-methylmorpholine and 9.60 ml of chloroformic acid ethyl ester
ml are added sequentially at -15°C.

After stirring for 2 minutes at this temperature, 20.3 g of _NH2 -NHZ (Z=benzyloxycarbonyl) in 200 ml of anhydrous dimethylformamide containing 10.99 ml of N-methylmorpholine are added. The reaction mixture is stirred at -10°C for 2 hours, after which the temperature is raised from -10°C to 0°C over 2 hours. The solvent was removed under reduced pressure and the residue was taken up in ethyl acetate and diluted with IN HCl, 5% at 0°C.
Wash sequentially with aqueous NaHCO ₃ and then with saturated aqueous NaCl until neutral. When the solvent was distilled off and the residue was crystallized from petroleum ether, Boc-Gly-NH-NH-
29.1 g (yield 90%) of Z() is obtained. mp84
-87°C 29.1 g of Boc-Gly-NH-NH-Z () is dissolved in 300 ml of 99% formic acid and left at room temperature for 6 hours. The solvent was distilled off under reduced pressure at 30°C and the oily residue was crystallized from diethyl ether to give H-Gly-NH-NH-Z.
23.0 g (95% yield) of HCOOH() is obtained.
mp135−136℃ Via 18.75 g of Boc-Thr-OH and ethoxyformic anhydride, 23.0 g of H-Gly-NH-NH-Z.HCOOH () as described for the preparation of compound ().
Boc-Thr-Gly-NH-NH-Z ()
Obtain 32.3 g (89% yield). mp70−73℃〔α〕 ²⁰ _D
= -3.6° (Cl, DMF) 32.3 g of Boc-Thr-Gly-NH-NH-Z () is treated with 350 ml of trifluoroacetic acid at 0°C for 30 minutes. The solvent was distilled off under reduced pressure at 0°C, and the oily residue was crystallized from ether to give H-Thr-Gly-NH-
33.0 g (yield 99%) of NHZ.CF ₃ COOH () is obtained.

mp140−145℃〔α〕 ²⁰ _D = +8.9゜(C ₁ ,
MeOH) 21.2 g of Boc-Tyr-OH was converted to H-Thr-Gly-NH-NH-Z.CF ₃ COOH via ethoxyformic anhydride as described for the preparation of compound ().
Condenses with ()33g. Crystallized from ethyl acetate-petroleum ether, Boc-Tyr-Thr-Gly-NH-NH
-39.0 g (yield 88%) of Z() is obtained.

mp138−142℃ [α] ²⁰ _D = −2.3゜(C ₁ ,
DMF) Boc−Tyr−Thr−Gly−NH−NH−Z()
When 39.0 is hydrolyzed with trifluoroacetic acid as described for the preparation of compound (), H-Tyr
−Thr−Gly−NH−NH−Z・_CF3COOH (XI)
39.9 g (100% yield) is obtained.

mp145−150℃ [α] ²⁰ _D = +11.8゜(C ₁ ,
DMF) Boc-Asp(OB ₂ l)-OH15.5g was converted into H-Tyr-Thr-Gly-NH- by a conventional method via ethoxyformic anhydride.
Condensate with 39.9 g of NH-Z.CF ₃ COOH (XI). Crystallization from ethyl acetate gives Boc−Asp(OB ₂ l)
-Tyr-Thr-Gly-NH-NH-Z (XII) 48.9g
(Yield 93%) is obtained.

mp134−138℃ [α] ²⁰ _D = −11.2 (C ₁ ,
DMF) Boc−Asp(OB ₂ l)−Tyr−Thr−Gly−NH−
Hydrolysis of 48.9 g of NH-Z(XII) with trifluoroacetic acid in a conventional manner yields H-Asp(OB ₂ l)-Tyr-
Thr−Gly−NH−NH−Z・_CF3COOH ()
Gives 49.2g (99% yield). mp135−138℃
[α] ²⁰ _D = +13.7° (C ₁ , DMF) in a similar manner via ethoxyformic anhydride
Boc−Gln−OH15.0g and H−Asp(OB ₂ l)−Tyr−
Thr−Gly−NH−NH−Z・_CF3COOH ()
Condensation with 49.2g Boc after crystallization from methanol
−Gln−Asp(OB ₂ l)−Tyr−Thr−Gly−NH−
This gives 49.5 g (88% yield) of NH-Z(XI).

mp200−203℃ 〔α〕 ²⁰ _D = −15.8゜(C ₁ ,
DMF) When treated with trifluoroacetic acid in a conventional manner, Boc
−Gln−Asp(OB ₂ l)−Tyr−Thr−Gly−NH−
49.5g of NH-Z(XI) is H-Gln-Asp
(OB ₂ l) −Tyr−Thr−Gly−NH−NH−Z・
Gives 50.2 g (100% yield) of CF ₃ COOH ().

mp160−162℃ [α] ²⁰ _D = +2.0 (C ₁ ,
DMF) After crystallization from methanol, condensation with 50.2g via ethoxyformic anhydride is carried out by a conventional method. Gives 45.5g (91% yield).

mp220−225℃ (decomposition) [α] ²⁰ _D = −13.3° (C ₁ , DMF) 45.5 g was dissolved in 550 ml of dimethylformamide containing 2 equivalents of HCl and added in portions within 12 hours.
Hydrogenate in an open vessel with a stream of hydrogen in the presence of 14 g of % palladium on charcoal.

The catalyst is filtered off and washed thoroughly with dimethylformamide. When the oily residue is crystallized from absolute ethanol, 34.1 g (yield 94%) is obtained.

mp145-150℃ (decomposition) [α] ²⁰ _D = -21.8゜ (C ₁ , DMF) Example 2 Production of intermediate () H-Trp-Met-Asp-Phe-NH ₂ () Boc-Asp (OB ₂ l) -OH23.3g and H-Phe-NH ₂
20.0 g via ethoxyformic anhydride as described for the preparation of compound ().
After crystallization from ethyl acetate, Boc−Asp
(OB ₂ l)-Phe-NH ₂ () 39.9g (yield 85%)
get.

mp141−142℃ [α] ²⁰ _D = −32.7゜(C ₁ ,
DMF) Boc- dissolved in 180ml of dimethylformamide
39.9 g of Asp( _OB2l )-Phe- _NH2 () is hydrogenated at atmospheric pressure in the presence of 29 g of 10% palladium on charcoal. When crystallized from isopropanol, Boc−Asp−
29.0 g (yield 90%) of Phe-NH ₂ () is obtained.

mp196-198 [α] ²⁰ _D = 50.0° (C ₁ , DMF) 29.0 g of Boc-Asp-Phe-NH ₂ () are dissolved in 300 ml of 1.33N dry HCl in glacial acetic acid (HCl/AcOH). After 25 minutes at room temperature, the solution was evaporated under reduced pressure and the residue was crystallized from ether to give H-Asp-Phe-
24.2 g (100% yield) of NH ₂ .HCl (XI) is obtained.

mp100-150℃ (decomposition) [α] ²⁰ _D = +23.9゜ (C ₁ , AcOH95%) Boc by conventional method via ethoxyformic anhydride
-Met-OH19.1g and H-Asp-Phe- _NH2.CHl
(24.2 g) and crystallization from ethyl acetate yielded Boc-Met-Asp-Phe- _NH2 ()
mp194−198℃, [α] ²⁰ _D = +5.4゜(C ₁ ,
AcOH95%) 36.0g (yield 92%) is obtained.

Boc−Met−Asp−Phe−NH ₂ () Same as described for the preparation of 36.0 g of compound ()
Hydrolysis with HCl/AcOH results in H-Met-Asp
−Phe−NH ₂ .HCl(XI), mp191−192℃,
[α] ²⁰ _D = +5.4° (C ₁ , AcOH95%) 31.5 g (yield
Give 100%).

Via ethoxyformic anhydride by a conventional method,
Boc-Trp-OH21.5g and H-Met-Asp-Phe-
After condensation with 31.5 g of _NH2.CHl (XI) and crystallization from ethyl acetate, 44.2 g (yield 90%) of Boc-Trp
−Met−Asp−Phe−NH ₂ (XII), mp199−200
℃, [α] ²⁰ _D = -34.5° (C ₁ , DMF) is obtained.

Boc−Trp−Met−Asp−Phe−NH ₂ (XII)
Dissolve 44.2g in 450ml of 99% formic acid. After 5 hours at room temperature, the solvent was distilled off under reduced pressure at 30°C, and the residue was crystallized from isopropanol to give 33.7g (yield 89%) of H-
Trp−Met−Asp−Phe−NH ₂ (), mp205−
207°C, [α] ²⁰ _D = -25.6° (C ₁ , DMF-HMPT
1:1) is obtained.

Example 3 Production of intermediate () 21.2 g in 160 ml of anhydrous dimethylformamide Dry 2.44N in tetrahydrofuran to a solution of
24ml of HCl solution and 3.5ml of n-butylnitrile -
Add sequentially at 25°C. After stirring at this temperature for 25 minutes, 13.1 ml of N-methylmorpholine was added, followed by 14.8 g of H-Trp-Met-Asp-Phe-NH ₂ ().
is added as a solution in a mixture of 120 ml of anhydrous dimethylformamide and 40 ml of hexamethylphosphoramide containing 2.8 ml of N-methylmorpholine.

The reaction mixture is left at -10°C for 4 hours. The product is precipitated by evaporating the volatile solvent under reduced pressure and diluting with cold aqueous citric acid. After crystallization from methanol, 25.1 g (yield 79%) of this crude product was obtained.
of mp201−206°C (decomposition) [α] ²⁰ _D = −28.9° (C ₁ ,
DMF).

Example 4 Production of target compound () 12.7g are dissolved in water by adding IN NaOH until PH10. Add 5.72 g of pyridine sulfur trioxide complex little by little over 5 hours while stirring and maintain the pH between 9 and 11 with IN NaOH. After neutralizing the alkaline solution with solid _CO2 , the solvent was evaporated to dryness under reduced pressure and the residue was taken up in dimethylformamide.
Insoluble inorganic salts are removed by filtration. The filtrate is again evaporated under reduced pressure to an oily residue. The product is dissolved in 400 ml of dimethylformamide-water (1:1) mixture and treated with Dowex 50W (H ⁺ ) for 5 minutes at 0° C. with stirring. The mixture is filtered and made alkaline with diethylamine. The solvent was removed under reduced pressure to a small amount, and ether was added to precipitate 13.5 g of the desired product. After desalting through a column of Sephadex G10, 12.2 g (79% yield) of is given as the dimethylamine salt. [α] ²⁰ _D = -22
゜ (C ₁ , DMF).

Claims (1)

[Claims] 1 Formula (): The formula () obtained by coupling hexaptide with a tetrapeptide of the formula (): H-Trp-Met-Asp-Phe- _NH2 by a conventional method: The decapeptide, namely pyroglutamyl-glutaminyl-aspartyl-tyrosyl-threonyl-glycyl-tryptopyl-methionyl-aspartyl-phenylalanine amide (all constituent amino acids are in the L form except glycine), was prepared in an aqueous medium at pH 8. .5 to 12.5, formula B: SO ₃ (where B: is from the group consisting of pyridine, substituted pyridine, tri-lower alkylamine, N-lower alkylmorpholine, N,N-dimethylaniline, imidazole, substituted imidazole, quinoline); a sulfating agent (which is a selected tertiary base) and 2
Formula () characterized by reacting for 120 hours and isolating the product thus obtained by a conventional method: Ceruletide, which is a decapeptide of (the phenolic hydroxyl group of tyrosine is esterified with a sulfate group) and its salts with pharmaceutically acceptable bases. 2 Formula (): decapeptide by the addition of an organic base selected from the group consisting of pyridine, substituted pyridine, tri-lower alkylamine, N-lower alkylmorpholine, N,N-dimethylaniline, imidazole, substituted imidazole, quinoline, or by addition of an alkali metal The formula B A sulfating agent of :SO ₃ (B: has the above meaning) is added as it is or dissolved in a dipolar aprotic solvent, and one of the above bases is added to adjust the pH of the reaction solution to 8.5 to 12.5. The method according to claim 1, characterized in that the reaction is carried out while maintaining the range. 3. The method of claim 2, wherein said dipolar aprotic solvent is dimethylformamide, acetone or acetonitrile.