JPH10101695A - 2-position-substituted enkephalin derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a novel subject derivative which comprises an enkephalin derivative substituted the glycine at the second position from the N-terminus with methylserine and is useful as an enkephalin-like medicine having analgesic action with weak dependency and resistance to enzymatic decomposition. SOLUTION: This novel compound is a novel 2-substituted enkephalin derivative of a pentapeptide represented by the formula (X is Leu, Met) in which the glycine at the second position from the N-terminus is substituted with (S)- or (R)-2-methylserine. This pentapeptide has weak dependency, resists to enzymatic decomposition and has analgesic action and is useful as an enkephalin-like medicine, for example, as an analgesic, sedative, narcotic or narcotic auxiliary or for studies on the understanding of the bondage between enkephalin and its receptor or on the neuropharmacology. This 2-substituted enkephalin derivative is obtained by selectively synthesizing O-acyl derivative by utilizing the solid phase peptide synthesis technique using the Fmoc-amino acids followed by rearrangement of the O-acyl to the N-acyl.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a 2-substituted enkephalin derivative having an analgesic action and the like. More specifically, the present invention relates to (S)-or (R) -2-methylserine which is the second glycine from the N-terminal of enkephalin. The present invention relates to a 2-substituted enkephalin derivative which is expected to contribute to understanding of the binding mode between enkephalin and receptor, research of neuropharmacology, etc.

[0002]

2. Description of the Related Art Enkephalin is a pentapeptide represented by the following formula: Tyr-Gly-Gly-Phe-X (where X represents Leu or Met), and is one of endogenous opioid peptides. is there. It is found in the pituitary gland, hypothalamus, small intestine, blood, etc. and has a powerful analgesic action.

[0003] Receptors on which endogenous opioid peptides act are classified into three types, μ-, δ- and κ-receptors, and enkephalin acts non-selectively on μ- and δ-receptors. It is known.

By the way, since morphine acting on the μ-receptor has dependence, that is, narcotics, research on synthesis of receptor-selective enkephalin derivatives having low dependence has been actively conducted. And δ−
A cyclic peptide enkephalin (2-D-penicillamine, 5-D-penicillamine) (DPDPE) has been reported as an enkephalin derivative having acquired receptor selectivity.

Another major problem with enkephalins is that they are susceptible to enzymatic degradation and easily deactivated because they are peptides. As a solution to this, N-methylated or D-amino acids, or enkephalin ([A
ib ² ] -enkephalin) is known to be used.

However, at present, no enkephalin derivative has yet been obtained which simultaneously solves both the problems of reduced dependence and enzymatic degradation.

[0007]

Accordingly, an object of the present invention is to provide an enkephalin derivative which simultaneously solves both the problems of reduced dependence and enzymatic degradation.

[0008]

Means for Solving the Problems The present inventors have synthesized several enkephalin derivatives and examined their physical properties. As a result, the second glycine from the N-terminal of enkephalin was replaced with (S)-or (R). The present inventors have found that the above problem can be solved by substituting -2-methylserine, and completed the present invention.

[0009] That is, the present invention relates to the enkephalin N
It is intended to provide a 2-substituted enkephalin derivative in which the second glycine from the terminal is substituted with methylserine.
The present invention also provides a medicine, a measurement reagent and a measurement kit containing the enkephalin derivative as an active ingredient.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The 2-substituted enkephalin derivative of the present invention can be synthesized according to a known peptide synthesis method. A preferred method is a solid phase peptide using an Fmoc-amino acid as described below. A method of selectively synthesizing an O-acyl compound using a synthesis method and transferring the O-acyl compound to an N-acyl compound is exemplified.

[0011]

Embedded image

For the production of the 2-substituted enkephalin derivative of the present invention, [(S) -2-MeSer ² ] -Leu
-The following is a specific description using enkephalin as an example. First, N-α-9-fluorenylmethoxycarbonyl-L-leucine-O-resin p-alkoxybenzyl alcohol resin (Fmoc-Leu-A
lko-resin) to remove the Fmoc group under conditions such as piperidine in dichloromethane.
Condensation with Fmoc-Phe-OH under the reaction conditions of yl-oxy-tris (dimethylamine) phosphonium hexafluorophosphate (BOP), 1-hydroxybenzotriazole (HOBT) and triethylamine. The condensate is sequentially removed under the same conditions to remove Fmoc.
, Condensation with Fmoc-Gly-OH, removal of Fmoc,
The condensation with Boc- (S) -2-MeSer-OH is repeated.

Next, the hydroxyl group of the condensed 2-methylserine was added to Boc-Tyr (tBu)-in dichloromethane under the conditions of N, N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP).
OH is condensed and [(S) -2-MeSer (Boc-T
yr (tBu) -O-)]-Gly-Phe-Leu-
Synthesize Alko-resin. Trifluoroacetic acid (T
FA), the resin and the Boc group were removed, and the mixture was treated with a 10% aqueous ammonium hydrogencarbonate solution to effect acyl transfer, whereby the desired [(S) -2-MeSer ² ] -Leu was obtained.
-Enkephalin is obtained.

Further, among the compounds of the present invention, [(R)-
2-MeSer ² ] -Leu-enkephalin was synthesized by using Boc- (S) -2-MeSer-OH as a raw material.
Can be carried out in the same manner as described above, except that is used. Furthermore, [(S) -2-MeSer ² ] -Met-enkephalin and [(R) -2-MeSer ² ] -Met-
Enkephalin is an N-α-9-fluorenylmethoxycarbonyl-L-leucine-O-resin p-alkoxybenzyl alcohol resin (Fmoc-Leu-Al
ko-resin) was converted to N-α-9-fluorenylmethoxycarbonyl-L-methionine-O-resin p-alkoxybenzyl alcohol resin (Fmoc-Met-Al)
ko-resin), and can be produced by the above method.

The thus obtained enkephalin derivative of the present invention has a selective binding property to the δ-receptor, and its confirmation can be carried out by the following test method. That is, a cell in which each of μ-, δ-, and κ-receptors is specifically expressed in CHO cells is incubated with various concentrations of the enkephalin derivative of the present invention and a receptor-specific ligand for a certain period of time, and their competition is performed. From the reaction, the selective binding property of the enkephalin derivative of the present invention can be determined.

In this test method, the receptor-specific ligand includes [tyrosyl-2,6- ³ H (N)]-
Tyr-D-Ala-Gly-N-methyl-Phe-G
ly-ol ([ ³ H] DAMGO), [tyrosyl-2,6
- ³ H (N)] - enkephalin (2-D-penicillamine ^{5-D-penicillamine) ([3 H] DPDPE)} ,
[Tyrosyl-2,6- ³ H (N)]-(5a, 7a, 8b)
-(+)-N-methyl-N- [7- (1-pyrrolidinyl) -1-oxaspiro [4.5] dec-8-yl]-
Benzene acetamide ([ ³ H] U69593) or the like is used.
After filtration through an F / C filter, the amount of the label on the filter was measured by a scintillation counter.

The fact that the enkephalin derivative of the present invention is an opioid receptor agonist can be confirmed by measuring the intracellular cAMP concentration.

The 2-substituted enkephalin derivative of the present invention has a relatively low μ-receptor selectivity compared to enkephalin, and thus has a low dependence on analgesics.
It can be used as a medicine such as a sedative, an anesthetic, and an anesthetic aid. For use as a medicament, the dose and usage may be substantially the same as enkephalin or [Aib ² ] -enkephalin.

[0019]

Next, the present invention will be described in more detail with reference to examples and test examples, but the present invention is not limited to these examples.

Example 1 Synthesis of [(S) -2-MeSer ² ] -Leu-enkephalin: By solid-phase synthesis, [(S)-
[2-MeSer ² ] -Leu-enkephalin was synthesized. That is, 0.5 mmol per 1 g in advance
Fmoc- into which leucine, a C-terminal amino acid, is introduced
Leu-Alko resin 400mg (0.2mmol)
Into a manual peptide synthesis reaction vessel and add dichloromethane
Washed three times with 10 ml. 20% piperidine-
10 ml of a dichloromethane solution was added, and the mixture was stirred at room temperature for 30 minutes to remove Fmoc. The reaction solution was washed three times each with 10 ml of dichloromethane and 10 ml of DMF.

Next, N, N'-dimethylformamide (D
MF) 10 ml, and Fmoc-Phe-OH
232 mg (0.60 mmol), BOP 265 mg
(0.60 mmol), HOBt 92 mg (0.60 m
mol) and 0.084 ml of Et ₃ N (0.60 mmol)
l) were added in sequence, and the mixture was stirred at room temperature for 1.5 hours to carry out a condensation reaction. The end of the reaction was checked with ninhydrin.

The obtained reaction solution was washed three times each with 10 ml of DMF and 10 ml of dichloromethane. After removing Fmoc in the same manner as described above, 178 mg of Fmoc-Gly-OH (0.60 mm
ol), and after removing Fmoc again, Boc-L
-2-MeSer-OH 132mg (0.60mmo
l).

Further, 10 ml of dichloromethane was added,
The hydroxyl group of the condensed Boc-L-2-MeSer is
c-Tyr (tBu) -OH 202 mg (0.60 mm
ol) with 124 mg (0.60 mmol) of DCC (N, N'-dicyclohexylcarbodiimide) and DMAP
(4-dimethylaminopyridine) 37 mg (0.30 m
mol) at room temperature for 4 hours for condensation.

The reaction solution after the condensation was washed three times each with 10 ml of DMF and 10 ml of dichloromethane, and the resin was collected by filtration. The obtained resin was transferred to a 50 ml eggplant type flask, and 5 ml of a 95% TFA aqueous solution was added.
Stirred at room temperature for 2 hours. The reaction solution was filtered with a small amount of TFA, and 300 ml of ether was added to the filtrate. The resulting precipitate was collected by filtration, dried under reduced pressure, dissolved in a 10% aqueous ammonium hydrogen carbonate solution, and stirred at room temperature for 8 hours.
The reaction solution was freeze-dried to obtain 105 mg of a crude product. The product was further purified by HPLC to give the title compound (65 mg, 0.1
01 mmol).

[Α] ^D : 95.0 ° (c 0.325, M
eOH) HR-FAB MS (as _{C 30 H 42 N 5 0 8} ): Real measured value; m / z 600.3038 [M + H] + calculated value; m / z 600.3033

EXAMPLE 2 Synthesis of [(R) -2-MeSer ² ] -Leu-Enkephalin: [(S) -2-MeSer ² ]-of Example 1
By a method similar to the synthesis of Leu-enkephalin,
55 mg (0.099 mmol) of [(R) -2-MeSer ² ] -Leu-enkephalin were obtained.

[Α] ^D : + 228 ° (c 0.10, Me
OH) HR-FAB MS (as _{C 30 H 42 N 5 0 8} ): Real measured value; m / z 600.3041 [M + H] + calculated value; m / z 600.3033.

Example 3 The procedure of Example 1 or 2 was repeated except that Fmoc-Met-Alko resin was used as a starting material.
[(S) -2-MeSer ² ] -Met-enkephalin or [(R) -2-MeSer ² ] -Met-enkephalin is obtained.

Test Example 1 Receptor binding test: CHO cells expressing δ-, μ-, and κ-receptors (CHO / DOPR, CHO / MO)
PR, CHO / KOPR) were cultured in 175 cm ² plastic culture flasks until cell density was high. The CHO / DOPR, CHO / MOPR and CHO / KOPR used were those provided by Professor Kimito Sato of the Faculty of Pharmaceutical Sciences, Kyoto University.
The cells were cultured in Ham's F-12 medium (GibcoBR).
L), 10% fetal bovine serum (GibcoBRL), 10 mM
HEPES (pH 7.4), 100 units / ml penicillin (GibcoBRL), 100 mg / ml streptomycin (GibcoBRL) and 200 mg / ml
G418 (geneticin; GibcoBRL) was added and 5%
Incubation was performed by incubating in a 37 ° C. incubator filled with a CO ₂ -containing solution.

The cultured CHO cells were washed once with 20 ml of Dulbecco's phosphate-buffered saline (PBS) and then scraped off with a cell scraper. Cells were 10m in 50mM Tris-HCl buffer (pH 7.4).
The suspension was suspended in a buffer (buffer A) containing M MgCl ₂ and 1 mM EDTA, and disrupted with a polytron homogenizer. This lysate was centrifuged at 3,000 g for 20 minutes at 4 ° C., and the precipitate was suspended in buffer A and stored at −80 ° C.
The cell membrane fraction was used.

Next, 30 mg of the cell membrane fraction prepared as described above, and 1 nM [ ³ H] DPDPE of the enkephalin derivative prepared in Example 1 or 2 at each concentration were used.
0 Ci / mmol; DuPont-New England Nuclear), 1 nM [ ³ H] DAMGO (48.9 Ci / mm
ol; DuPont-New England Nuclear) and 2 nM
[ ³ H] U-69,593 (47.5 Ci / mmol; DuP
ont-New England Nuclear), and the binding reaction was performed in buffer A at 25 ° C. for 60 minutes.

To the reaction mixture was added 2.5 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4) to stop the binding reaction, and the Whatman GF / C filter treated with 0.1% polyethyleneimine was used. (Made by Whatman)
It was immediately filtered and washed five times with 2.5 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4). The filter was transferred to a vial containing 10 ml of Scintisol EX-H (manufactured by Dojindo Laboratories), and the radioactivity on the filter was measured with a scintillation counter.

When measuring non-specific binding, δ-, μ
-, Κ-receptor, 1 mM DPDPE
The binding reaction was performed in the presence of 1 mM DAMGO (Funakoshi) and 2 mM U-69,593 (Funakoshi) (Funakoshi).

The results are shown in Table 1 below. The enkephalin derivative obtained in Example 2 showed affinity for the μ- and δ-receptors in a receptor binding experiment,
-Showed very strong affinity for the receptor. In contrast, no affinity was shown for the κ-receptor. On the other hand, the enkephalin derivative obtained in Example 1 showed only weak affinity for any receptor.

[0035]

[Table 1]

Test Example 2 Measurement of intracellular cAMP concentration: CHO expressing δ-receptor
1 × 10 ⁵ cells (CHO / DOPR) were cultured on a 24-well cell culture plate under the above conditions for one day. The cultured cells were washed once with 0.5 ml of Hanks solution (Hanks buffer) supplemented with 20 mM HEPES (pH 7.4), and thereafter, Hanks supplemented with 1 mM 3-isobutyl-1-methylxanthine (IBMX; manufactured by Funakoshi). A buffer solution was added and reacted at 37 ° C. for 10 minutes.
Forskolin (Wako Pure Chemical) and 1m
MIBMX and DPDPE or the enkephalin derivatives of Examples 1 or 2 were added. After the reaction was continued for another 10 minutes, the reaction was stopped by adding 0.5 ml of 10% ice-cold trichloroacetic acid.

The cell culture plate was chilled on ice for 15 minutes and frozen. After thawing, the reaction solution was transferred to a glass test tube and centrifuged at 2,000 rpm for 5 minutes at 4 ° C. 1 ml of cold water-saturated diethyl ether was added to the supernatant, followed by thorough stirring. After removing the ether layer, the same washing operation was repeated. Heat at 40 ° C. for 20 minutes to remove residual ether. After that, cA manufactured by Amersham
Intracellular cA using MP quantification kit (RPN225)
The amount of MP was measured.

The results are shown in Table 2 below.
Since the MP concentration was decreased by the addition of the ligand peptide, it was confirmed that the above enkephalin derivative was an agonist of enkephalin. In addition, in the degradation test using liver homogenate, the 2-substituted enkephalin derivative of the present invention showed better results than Leu-enkephalin.

[0039]

[Table 2]

[0040]

As shown in Test Example 1, [(S) -2-MeSer ² ] -Leu-enkephalin obtained in Example 1 has only a weak affinity for any receptor in the competitive reaction. Not shown. On the other hand, [(R) -2-MeSer ² ]-obtained in Example ²
Leu-enkephalin showed affinity for μ- and δ-receptors, but no affinity for κ-receptors.

Further, as shown in Test Example 2, the enkephalin derivative of Example 1 was found to react with δ-receptor by Leu
-Showed a stronger affinity than enkephalin but [Ai
b ² ] -Leu-enkephalin showed only weaker affinity. On the other hand, the enkephalin derivative of Example 2 is 5% of [Aib ² ] -Leu-enkephalin.
It showed a strong affinity of 0 times or more.

Further, from the change in the intracellular cAMP concentration, it was confirmed that the above-mentioned enkephalin derivative was an agonist of enkephalin. Since Met-enkephalin has the same activity as Leu-enkephalin, the 2-substituted Met-enkephalin is also a 2-substituted Leu.
-Determined to have activity similar to enkephalin.

Since a great difference in affinity is recognized due to the difference in the stereoposition of the hydroxyl group, the spatial arrangement of the hydrophobic and hydrophilic sites of the amino acid residues of the receptor can be estimated. That is, [(R)-
It can be inferred that the side chain of the receptor site where the hydroxyl group of methylserine of 2-MeSer ² ] -Leu-enkephalin is located is rich in hydrophilicity, and that the methyl group side has a residue rich in hydrophobicity.

[0044]

The 2-position-substituted enkephalin derivative of the present invention can be used as an analgesic, sedative, anesthetic or anesthetic adjuvant as an enkephalin-like drug having a weak dependence and having analgesic activity. In addition, conversion of the hydroxyl group as a pharmaceutical intermediate leads to the development of a more effective pharmaceutical. that's all

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 14/70 A61K 37/02 AAH (72) Inventor Yasushi Shigesato 2-6-10 Noda, Fukushima-ku, Fukushima-ku, Osaka-shi, Osaka Prefecture 202 Glorry Hill Noda

Claims (4)

[Claims] 1. The method according to claim 2, wherein the second glycine from the N-terminal of enkephalin is substituted by methylserine.
Position-substituted enkephalin derivatives. 2. The method according to claim 2, wherein the 2-substituted enkephalin derivative is
^{[(S) -2-MeSer 2} ] -Leu- enkephalin or ^{[(R) -2-MeSer 2} ] -Leu- 2 -substituted enkephalin derivative according first claims is enkephalin. 3. A medicament comprising the 2-position substituted enkephalin derivative according to claim 1 or 2 as an active ingredient. 4. The medicament according to claim 3, which is an analgesic, a sedative, an anesthetic or an anesthetic aid.