JPH0446280B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to peptides and analgesics containing them as active ingredients.

Recent progress in research on bioactive peptides has been remarkable. Opioid peptides are one of them, and many studies have been conducted on endorphins, enkephalins, etc., which have morphine activity.

The present inventors conducted research on a series of such peptides and arrived at the present invention as part of the research.

That is, the gist of the present invention is the following general formula H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg
−Gln−Phe−Lys−Val−Val−Thr−Arg−Ser
−Gln−Glu−Asp−Pro−Asn−Ala−Tyr−E
-F-Glu-Leu-Phe-Asp-G-OH.

In the above formula, E is Ser or Tyr, F is Gly or
Glu and G represent Ala or Val. The configuration of the amino acid residue is D, L, or DL, but the L configuration is usually adopted.

The present invention will be explained in more detail below.

First, in the general formula, Tyr, Gly, Arg,
Gln, Phe, Lys, Val, Thr, Ser, Glu, Asp,
Pro, Asn, Ala, Leu, and Met represent tyrosine, glycine, arginine, glutamine, phenylalanine, lysine, valine, threonine, serine, glutamic acid, aspartic acid, proline, asparagine, alanine, leucine, and methionine, respectively.

Y represents a moiety bonded to the carbonyl of the C-terminal G.

The above peptides can be produced by appropriately selecting a method commonly used in peptide chemistry, and can be obtained by either a liquid phase method or a solid phase method.

As protecting groups for amino groups that do not participate in the reaction,
Examples include p-toluenesulfonyl group, benzyloxycarbonyl group, t-butyloxycarbonyl group, and phthaloyl group.

Further, examples of carboxyl protecting groups that do not participate in the reaction include alkyl esters such as methyl and ethyl. Furthermore, the carboxyl group to be reacted with the amino group can be chloride, hydrazide,
It is desirable to activate it by changing to azide, mixed anhydride with organic acid, thioester, cyanomethyl ester, etc.

A method in which a predetermined peptide chain is sequentially extended to the N-terminal side of a starting material by a solid phase method is preferably employed.

When using this method, it is preferred that the reactions in the repeated steps in each cycle (particularly in the second and subsequent cycles) be carried out in the presence of an active ester reagent such as N-hydroxysuccinimide or 1-oxypenzotriazole.

Removal of the protecting group from a peptide having a protecting group is carried out by a conventional method. For example, trifluoroacetic acid treatment, hydrolysis, reduction, etc.

Further, the obtained peptide can be purified using an ion exchange resin, various chromatography methods, and the like.

Furthermore, the peptides of the present invention can be made into acid addition salts of various commonly used inorganic acids and organic acids, and can also be made into acid addition salts of various commonly used inorganic acids and organic acids, and metal compounds such as zinc, nickel, and cobalt, and poly-L-glutamic acid and the like. It can be made into a complex compound with polyamino acids and the like.

The peptides of the present invention and the pharmaceutically acceptable acid addition salts or complexes described above are useful as analgesics for warm-blooded animals such as humans.

The dosage varies depending on the symptoms, the recipient, etc., but is usually 0.01 to 100 mg/Kg per day, and can be administered orally or parenterally in various dosage forms.

The formulation can be prepared by conventional methods, including excipients and
Stabilizers, emulsifiers, preservatives, etc. may be used.

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 unless the gist thereof is exceeded.

The Boc-amino acid used in the Examples is L-amino acid and is a product of Peptide Institute Co., Ltd. (Osaka). In addition, “Sephadex G-10” is
Pharmacia is a product of Huain Chemicals. Furthermore, N,N'-dicyclohexylcarbodiimide, chloromethylated polystyrene (divinylbenzene 1%, 200-400 mesh, Cl:
0.71 meq/g resin) and N-hydroxysuccinimide are products of Peptide Institute Co., Ltd.

Example 1 H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg
−Gln−Phe−Lys−Val−Val−Thr−Arg−
Ser−Gln−Glu−Asp−Pro−Asn−Ala−Tyr
−Ser−Gly−Glu−Leu−Phe−Asp−Ala−
Synthesis of OH Boc-Ala esterification of the resin is carried out as follows. Dissolved in dry Me ₂ SO (11 ml)
Add chloromethylated resin (2.50g, 0.71mequiv.Cl) to Boc-Ala (3.91mequiv) and further KO
Add -t-Bu (3.73 mequiv.). Shake the mixture vigorously and store the resulting slurry at 80°C for 1 hour.
Hold. The resin was then treated with Me ₂ SO, EtOH and
Wash _{with CH2Cl2} .

Peptide synthesis is carried out by a conventional solid phase method.

The synthesis is started by placing 2.50 g of Boc-Ala-OCH ₂ -resin into the reactor of a peptide synthesizer (Beckman 990B model), and amino acid sequences are sequentially synthesized. Removal of protecting groups was accomplished by treatment with 25% trifluoroacetic acid (TFA) in CH ₂ Cl ₂ for 30 min, followed by
Neutralized with 10% triethanolamine ( _Et3N ) _{in CH2Cl2} .

Sequential coupling of each amino acid (3.26 mmol) is made with dicyclohexylcarbodiimide (3.26 mmol) in CH ₂ Cl ₂ for 2 hours. The amount of solvent is 6.5 dicyclohexylcarbodiimide.
Other than ml, it is 20ml.

One cycle of synthesis consists of the following operations.

(1) Washing with CH ₂ Cl ₂ (1.5 min, 3 times) (2) Deprotection with 25% TFA/CH ₂ Cl ₂ (prewash for 1.5 min, then treatment for 30 min) (3) Washing with CH ₂ Cl ₂ Washing (1.5 minutes, 6 times) (4) Neutralization with 10% Et ₃ N/CH ₂ Cl ₂ (1.5 minutes, 3 times) (5) Washing with CH ₂ Cl ₂ (1.5 minutes, 6 times) (6) Boc-amino acid (3.26mequiv., in _{CH2Cl2 ,}
5 minutes) Treatment (7) Without filtration, add dicyclohexylcarboimide (3.26 mequiv.) in CH ₂ Cl ₂ and couple for 120 minutes.

(8) Washing with CH ₂ Cl ₂ (1.5 minutes, 6 times) (9) Repeat the steps from (4) onwards using 3.26 mequiv. of each Boc-amino acid and dicyclohexylcarbodiimide.

From the second cycle onwards, the reactions in the repeated steps are carried out in the presence of N-hydroxysuccinimide (6.52 mequiv.). Boc−
Amino acids are dissolved _{in CH2Cl2} . however,
Boc-Arg(Tos) is dissolved in dimethylformamide (DMF). Boc−Asn and Boc−
Gln is coupled as N-hydroxysuccinimide ester in DMF.

For this, Boc-Gln (3.26 mmol) and N-
Hydroxysuccinimide (6.52 mmol) in DMF
(20 ml), the mixture is placed in a reaction vessel and then dicyclohexylcarbodiimide (3.26 mmol) in CH ₂ Cl ₂ (6.5 ml) is added. The reaction vessel is kept under a nitrogen atmosphere during the synthesis to minimize oxidation by air.

A washing step is performed after each coupling reaction and the presence of unreacted free amino groups is monitored by the ninhydrin test.

The following protected amino acids are used as amino acids.

Boc-Ala, Boc-Asp (OBzl), Boc-Phe,
Boc−Leu−H ₂ O, Boc−Glu(OBzl), Boc−
Gly, Boc-Ser (Bzl), Boc-Tyr (Bzl), Boc-
Asn, Boc-Pro, Boc-Arg (Tos), Boc-Val,
Boc-Lys (ε-2-Cl-Z), Boc-Tyr (2,6
−Cl ₂ −Bzl), Boc−Gln.

After 15 cycles, 4.59 g of protected peptide was obtained,
Two-thirds of it is further provided for elongation of the peptide chain.

After completion of synthesis, protected peptide-resin (4.21g)
HF (30 ml) in the presence of anisole (8.0 ml)
Treated at 0°C for 60 minutes in a conventional manner, and then
HF is removed by a vacuum pump at 0°C. The resulting yellow resin is washed several times with ethyl acetate. Peptides are extracted with 1M acetic acid (150ml) and then lyophilized. The crude peptide (1.50 g) was applied directly to a "Sephadex G-10" column (2.5 x 136 cm) and eluted with 3M acetic acid. Each fraction (10g)
is monitored by a spectrophotometer at 280 nm.
Three peptide-containing fractions, namely, fraction (tube no. 25), (tube no.
26-29) and (tube No. 30-35), respectively, are pooled, evaporated under reduced pressure, and then freeze-dried. The fractions and contain 140 mg, 867 mg and 253 mg of the substance, respectively. These substances are subjected to thin layer chromatography (TLC) and reversed phase high performance liquid chromatography (reversed phase HPLC). The conditions for performing reverse phase HPLC are as follows.

Column: “Toryu Soda” Methylated C-18 (0.4 x 30cm) Solvent: 0.01N HCl/CH ₃ CN (75:25→68:32
Linear concentration gradient) Flow rate: 1.0 ml/min Injection amount: 20 μg peptide The target product is contained in the fraction.

Amino acid analysis Peptide hydrolyzate (6N HCl, 24 hours, 110
℃) using a Hitachi Model 835 amino acid analyzer. The amino acid composition is as follows.

Aep(3)3.02, Thr(1)0.99, Ser(2)1.92, Glu(4)4.15, Gly(3)3.10, Ala(2)2.07, Val(2)1.70, Leu(2)2.16, Tyr(2)2.13, Phe(3)2.97, Lys(1)1.23, Arg(3)2.82, Pro(1)0.88.

(Recovery rate approx. 91%) R _f 〓0.24, R _f 〓0.54 (TLC) Solvent system R _f 〓: 1-BuOH-AcOH-H ₂ O (4:
1:5) R _f 〓:1-BuOH-pyridine-AcOH
-H ₂ O (30:20:6:24) HPLC: As shown in Figure 1.

Example 2 H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg
−Gln−Phe−Lys−Val−Val−Thr−Arg−
Ser−Gln−Glu−Asp−Pro−Asn−Ala−Tyr
−Tyr−Glu−Glu−Leu−Phe−Asp−Val−
Synthesis of OH Boc-Val esterification of the resin is carried out as follows. Dissolved in dry Me ₂ SO (11 ml)
Add chloromethylated resin (2.50 g, 0.71 mequiv.Cl) to Boc-Val (4.45 mequiv), and then add KO
Add -t-Bu (3.73 mequiv.). Shake the mixture vigorously and store the resulting slurry at 80°C for 1 hour.
Hold. The resin was then treated with Me ₂ SO, EtOH and
Wash _{with CH2Cl2} .

Synthesis was started by placing 2.50 g of Boc-Val-OCH ₂ -resin in a reaction vessel of a peptide synthesizer (Beckman 990B model), and the amino acid sequence was sequentially extended by a conventional solid phase method. Removal of the protecting group is performed in CH ₂ Cl ₂
This is done by treatment with 25% trifluoroacetic acid (TFA) for 30 minutes, followed by neutralization with 10% ethanolamine (Et ₃ N) in CH ₂ Cl ₂ .

Sequential coupling of each amino acid (4.06 mmol) is made with dicyclohexylcarbodiimide (4.06 mmol) in CH ₂ Cl ₂ for 2 hours. The amount of solvent is 8.0 dicyclohexylcarbodiimide.
Other than ml, it is 20ml.

One cycle of synthesis is as in Example 1 except that 4.06 mequiv. of Boc-amino acid and dicyclohexylcarbodiimide are used.

From the second cycle onwards, the reactions in the repeated steps are carried out in the presence of N-hydroxysuccinimide (8.12mequiv.).

Boc amino acids are dissolved _{in CH2Cl2} . However, Boc-Arg (Tos) is dissolved in dimethylformamide (DMF). Boc−Asn and Boc−
Gln is coupled as N-hydroxysuccinimide ester. For this purpose, Boc−
Gln (4.06 mmol) and N-hydroxysuccinimide (8.12 mmol) were dissolved in DMF (20 ml) and the mixture was placed in a reaction vessel followed by CH ₂ Cl ₂ (8.12 ml).
dicyclohexylcarbodiimide inside (4.06m
mol). The reaction vessel is kept under a nitrogen atmosphere during the synthesis to minimize oxidation by air.

A washing step is performed after each coupling reaction and the presence of unreacted free amino groups is monitored by the ninhydrin test.

The protected amino acids used are the same as in Example 1.

After 15 cycles, 4.20 g of protected peptide was obtained,
Two-thirds of it is further provided for elongation of the peptide chain.

After completion of the synthesis, the protected peptide-resin (3.20 g) was treated with HF (30 ml) in the presence of anisole (7.0 ml) at 0°C for 60 minutes in a conventional manner, and then the HF was removed using a vacuum pump at 0°C. Ru. The resulting yellow resin is washed several times with ethyl acetate. The peptide is
Extracted with 1M acetic acid (150ml) and then lyophilized. Directly transfer the crude peptide (1.15 g) to a “Sephadex G-10” column (2.5 x 136 cm)
Elute with 3M acetic acid. Each fraction (10 g) is monitored spectrophotometrically at 280 nm. The three peptide-containing fractions, namely (tubes no. 21-25), (tubes no. 26-32) and (tubes no. 30-40), are each pooled, evaporated under reduced pressure and then lyophilized. The fractions and contain 180 mg, 688 mg and 137 mg of the substance, respectively. Thin layer chromatography (TLC) of these substances
and reversed-phase high performance liquid chromatography (reversed-phase
HPLC).

The object is included in the fraction.

Amino acid analysis (Do the same as in Example 1) Asp(3)3.24, Thr(1)0.99, Ser(1)0.99, Glu(5)5.33, Pro(1)0.98, Gly(2)2.14, Ala(1)0.88, Val(3)2.61, Len(2)2.09, Tyr(3)3.16, Phe(3)2.99, Lys(1)1.02, Arg(3)2.83.

(Recovery rate approximately 91%) R _f 〓0.24, R _f 〓0.37 (TLC) Solvent system: Same test example as in Example 1 (1) Binding test with opiate receptor using guinea pig ileal longitudinal muscle specimen Guinea pig ileal longitudinal muscle specimen The effect of suppressing the contraction response to electrical stimulation was investigated using the method of Kosterlitz et al.
et al.Br.J.Pharmacol (Practice Journal of Pharmacology), 39, 398,
1970).

That is, the specimen is suspended in Krebs solution at 33° C., and a mixed gas of 95% O ₂ +5% CO ₂ is bubbled through it.
Changes in contractile tension due to electrical stimulation (0.1Hz, 1.0msec pulse, 50V) are measured via a strain gauge.
recorded on a polygraph.

The test results for the peptide obtained in Example 1 are as follows.

Ic50 value: 2.73nM (2) Analgesic activity test by intraventricular administration in mice The intraventricular administration method is intracisternal administration (Takagi et al., Europ. J. Pharmacol., 56, 265,
1979), and the tail pinch method (Takagi et al., Jap.J.Pharmacol.
(Japanese Journal of Pharmacology, 16, 287, 1966).

Intracisternal administration method: Attach a 1/5 injection needle bent into a J-shape to a microsyringe, hold the unanesthetized mouse with one hand, and insert it into the back of the head, aiming at the cisterna magna.

Tail pinch method: The tail of the mouse is pinched with arterial forceps, and the bite response to the forceps is used as an indicator.

When 1 μg of the peptide obtained in Example 1 was administered, analgesic activity was observed in 4 out of 5 animals.

[Brief explanation of drawings]

Figure 1 shows the reverse phase of peptides according to the present invention.
This is an HPLC chromatogram.

Claims (1)

[Claims] 1 The following general formula H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg
−Gln−Phe−Lys−Val−Val−Thr−Arg−Ser
−Gln−Glu−Asp−Pro−Asn−Ala−Tyr−E
-F-Glu-Leu-Phe-Asp-G-OH [wherein, E is Ser or Tyr, F is Gly or Glu,
G represents Ala or Val. The configuration of the amino acid residue is D, L or DL. ] Peptides indicated by 2 In the general formula, E is Ser, F is Cly, and G is
The peptides according to claim 1, which are Ala.