JPH0133480B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to therapeutically advantageous peptides. The peptide is represented by the following amino acid sequence. Y-Gly-Leu-Ala-X-(Gly)n (where X is an amidino-substituted α-amino acid
Arg or Can; Y is Leu; His-Leu; Ser-His-Leu; Ala-Ser-His-Leu; CH ₃ CO-Ala-Ser-His-Leu; Arg-Gln-His-Ala-Arg − Ala−Ser−His−Leu; Ala−Ala−Ala−Leu; Ala−Ala−Leu; Ala−Leu; Asn−Lys−Pro−Leu; CH ₃ CO−Leu; CH ₃ CO−His−Leu; CH ₃ CO-Ala-Leu; and CH ₃ CO-Ala-Ala-Ala-Leu;
is 0 or 1. ) The compound of the present invention has smooth muscle contracting action, histamine release action from mast cells, and vascular wall permeability increasing action, and is a useful therapeutic agent. Furthermore, as will be explained later, some of the compounds of the present invention have the opposite effect and are therefore useful therapeutic agents. That is, some of the compounds of the present invention are promoters or facilitators and others are antagonists or inhibitors. Some of these compounds are useful in treating cardiac arrhythmias. The carboxyl end of the peptide is an amidino-substituted alpha-amino acid such as arginine or canavuanine. As the amino acid directly bonded to arginine or canavanine, glycine or alanine is preferred. The third amino acid in the peptide is connected by its carboxyl group to the amino group of the second amino acid. The third amino acid is leucine, which is a hydrophobic amino acid. This amino acid is available in both L and D forms and is preferred because it contributes satisfactorily to the activity of the peptide. The type of fourth amino acid is not critical, but its presence is necessary for useful activity. Although most of the common 20 amino acids could be used, the highest degree of activity is obtained with glycine. The fifth hydrophobic amino acid is leucine. The presence of this leucine significantly increases the activity of the peptide. An increase in activity was observed as the peptide chain lengthened to 13 amino acids. However, the cost of synthesizing peptides with higher amino acid moieties is believed to be outweighed by the resulting modest performance improvement. ; Compounds of the invention in which the first amino acid is arginine or canavanine are promoters. If the arginine or canavanine is bonded by its carboxyl group to the amino group of the lower aliphatic amino acid glycine, the resulting compound of the invention is an inhibitor. Such compounds inhibit smooth muscle contraction, histamine release and vascular permeability at low dosage levels. Amino acids that are bound to arginine or canavanine are called inhibitory amino acids. The inhibiting amino acid in the compounds of the invention is glycine.
Glycine is easily available and can be introduced into the molecule without adding a new asymmetric center. In addition, when the peptide of the present invention replaces its constituent L-amino acid with a D-amino acid, for example, L-
Some compounds using D-arginine instead of arginine are resistant to the hydrolytic action of carboxypeptidase B and can therefore be used in the preparation of long-acting preparations. Due to their amphoteric nature, the compounds of the present invention may be used in pharmaceutically acceptable forms such as metal salts or acid addition salts. These salts have the advantage of being water soluble, making them particularly useful for parenteral administration. Metal salts, particularly alkali metal salts, are relatively stable and are often preferred over acid addition salts. Sodium salts are particularly preferred because they are easy to manufacture. Acids used in the preparation of acid addition salts of compounds of the invention are non-toxic anionic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, lactic acid, citric acid, tartaric acid, oxalic acid, succinic acid, maleic acid, etc. Contains acids, gluconic acid, sugar acids, etc. Overall, a wide variety of non-toxic derivatives of the polypeptides of the invention may be usefully utilized. For example, these compounds, especially those with glycine carboxyl groups, react with thionyl chloride to form acid chlorides, which in turn react with ammonia to form amides. Moreover, amines naturally form N-substituted amides. Compounds of the invention include peptides acylated with alkanoyl groups in which the free amino group is _CH3CO- . Compounds of the invention may be synthesized by a wide range of techniques currently available for the synthesis of simple and complex polypeptides and relatively low molecular weight proteins. Generally, these techniques involve stepwise synthesis, with sequential addition of amino acids to yield progressively larger molecules. The amino acids are bonded to each other by condensing the carboxyl group of one with the amino group of the other to form a peptide bond. In order to control this reaction, it is necessary to block the amino group of one amino acid and the carboxyl group of the other amino acid. The capping group must be selected so that it can be easily removed without adversely affecting the polypeptide by hydrolysis or racemization of the peptide bond formed. Certain amino acids, such as serine, have additional functional groups such as hydroxyl groups.
It is usually necessary to seal such functional groups with an easily removable sealing agent so as not to interfere with the peptide bond forming condensation reaction. Many methods have been devised and many sealing agents have been devised in the prior art for polypeptide synthesis. Most of these methods can also be applied to polypeptides belonging to the invention. It would be unnecessary to explain the applications of all these methods. Suitable methods are described in the Examples. A preferred method for the synthesis of compounds of the invention is the Melifide method. In this method, amino acids are fixed to resin particles as ester bonds, and peptides are formed by sequential addition of protected amino acids to the growing chain. This method is well known and described in numerous publications, including B. D. Erickson and R. B. Merrifield in "The Proteins" by Neuras and Hill (1976), published by New York Academic Press. Solid-state peptide synthesis P.255~
527. For convenience in describing the present invention, conventional abbreviations for various amino acids will be used. Although this abbreviation is well known to those skilled in the art, for confirmation the main amino acids relevant to the present invention are listed below: Arginine Arg Alanine Ala Leucine Leu Glycine Gly Histidine His Serine Ser Glutamine Gln As mentioned above, this As promoters, the compounds of the invention enhance smooth muscle contraction, assist in histamine release, and increase vascular permeability. Accordingly, such compounds are capable of eliciting such a response in mammals lacking anti-inflammatory effects and are therapeutically useful. Therefore,
The compound can be used to promote the formation of defense mechanisms in the body against invasion by pathogenic bacteria and the like. The compounds of the present invention are also clinically effective as inhibitors of local or tissue accidental or extrinsic smooth muscle contraction, histamine release or increased vascular permeability. This can be used, for example, as a nasal aerosol for attacks of asthma or bronchial allergies. It can also be used to prevent symptoms such as shock associated with dengue fever. The compounds of the present invention are useful therapeutic agents in mammals, exerting stimulatory and inhibitory effects in very small amounts. The optimal dosage for a particular application will be determined by your physician or veterinarian. Dosage will vary from subject to subject and will depend on the patient's weight, the condition being treated, and other factors that can be assessed by a professional. The compounds may be administered in large amounts, such as 2 grams or more per day. Usually the active ingredient is approx.
Administered in dosage units of 250 mg or less. The number of units administered per day will vary depending on the condition of the case being treated. Although the compound may be used alone, it is usually administered with a pharmaceutically acceptable non-toxic carrier;
The proportion will be determined by the acceptability and chemical nature of the particular carrier, the chosen route of administration, and standard pharmaceutical practice. For intravenous or intramuscular administration, the compound may be used as a sterile solution containing other solutes, such as salts or glycose to impart isotonicity. For local administration,
The compounds may be used with a suitable carrier such as petrolatum or other inert semi-viscous substance. Also,
Coated dosage units for enteral administration may also be used as tablets or capsules containing excipients such as starch, lactose, clay, and the like. As the first amino acid, arginine or canavanine, which are amino di-substituted α-amino acids, are used, and among these, arginine is preferred. These are omega amidino groups [-C(=NH)-
Due to the presence of NH ₂ ], all arginine analogs can be considered. Embodiments of the invention are described below for purposes of illustration. Example 1 L-alanyl-L-seryl-L-histidyl-
L-leucyl-glycyl-L-leucyl-L-
Synthesis of alanyl-L-arginine Nα-tertiary butyroxycarbonyl-N ^g -(4
-Toluenesphonyl)-L-arginyl oxymethyl-(styrene-copolymerized 1% divinylbenzene) (). Cesium bicarbonate (0.43 g) was added to a solution of N -tert-butyroxycarbonyl-N ^g -(4-toluenesulfonyl)-L-arginine (1.3 g) in ethanol (15 ml) and water (1.35 ml). ) in water (1.35 ml) and adjust the pH to 7.1. The resulting solution was removed under reduced pressure.
Evaporate to dryness at 40°C. Benzene (25 ml) is added and the mixture is evaporated to dryness under the same conditions. This method is repeated four more times. The resulting solid was dried at a pressure of 1 Torr for 2.5 hours to form Boc-Arg (Tos).
The cesium salt of () is obtained as a white solid (1.7 g). Chloromethyl-(styrene-copolymerized 1% divinylbenzene) (3.5g; per 1g of polystyrene
Cl0.23 mmol) was swollen in N,N-dimethylformamide (DMF; 15 ml) and salt (1.7 g)
is dissolved in 10 ml of DMF. The mixture is maintained at 50° C. for 75 hours with mechanical stirring. A solution of the salt (3.7 g) in DMF (25 ml) is added and the mixture is maintained at 50° C. with stirring for 95 hours. Collect the resin onto a sintered glass funnel;
DMF (25 ml, 3 times), absolute ethanol (25 ml, 3 times)
2 times) and dichloromethane (25 ml, 3 times). After drying for 2 hours at a pressure of 1 torr, the resin (3.3 g) contained arginine per gram of polystyrene.
Contains 0.17 mmol. This means that 10mg of sample is
% of anisole for 30 min at 0 °C, and the acid-soluble substances were purified using 6NHCl.
This was confirmed as a result of amino acid analysis of the hydrolyzate obtained by hydrolysis at 110°C for 24 hours. N〓-Tertiary butyroxycarbonyl-L-alanyl-O〓-benzyl-L-seryl-N ^im (4-trienesulfonyl)-L-histidyl-L-leucyl-glycyl-L-leucyl-L-alanyl-
Preparation of N ^g -(4-trienesulfonyl)-L-arginyloxymethyl (styrene copolymerized 1% divinylbenzene) (). The BOc-Arg (Toc) resin (2.0 g; 0.34 mmol arginyl) is swollen in dichloromethane (40 ml) in a 75 ml glass reaction vessel with a sintered glass disk. Air or nitrogen pressure is applied to the vessel and/or vacuum is applied to the outlet below the glass disk to filter the resin until the lower portion of the solution is removed. A. The protection removal step is carried out by adding a 1:1 (V/V) solution of trifluoroacetic acid and dichloromethane (40 ml) to the vessel, stirring the mixture for 1 minute and filtering as before. Then 1:1 of trifluoroacetic acid and dichloromethane.
(V/V) solution is added again and the mixture is stirred and filtered for 30 minutes. B Washing step: Wash the resin in the same way with dichloromethane (40ml x 5, 1 minute each), 2-propanol (40ml
ml x 2, 1 minute each) and dichloromethane (40
This is done by sequentially washing with 5 mL of water for 1 minute each. C. The neutralization step is carried out by washing the resin with a 1:19 (V/V) mixture of ethyldiisopropylamine and dichloromethane (3 x 40 ml, 2 minutes each). D. Repeat wash step B. E. The coupling step involves adding to the resin a dichloromethane solution (10 ml) of the desired Boc amino acid (3 mmol per 1 mmol of initial arginine), stirring for 2 minutes, and dissolving the mixture in N,N ¹ -dicyclohexylcarbodiimide (1 mmol per 1 mmol of initial arginine) in dichloromethane. 1 mmol to 3 mmol) in dichloromethane solution (10 ml), mixed for 30 minutes,
It is done by passing. F Repeat wash step B. G Repeat neutralization step C. H Repeat wash step B. I Repeat coupling step E. J Repeat wash step B. Performing steps A through J performs one cycle of synthesis, whereby the protected amino acid residue is attached to the peptide. This cycle was first performed with Boc-L-alanine and then
Boc-L-leucine, Boc-glycine, Boc-L
-Leucine, Boc-L ^im -(4-toluenestylfonyl)-L-histidine, Boc-O〓-benzyl-
Performed using L-serine and Boc-L-alanine sequentially. However, Boc means N-tertiary butyroxycarbonyl. L-alanyl-L-seryl-L-histidyl-
Protection removal and purification of L-leucyl-glycyl-L-leucyl-L-alanyl-L-arginine (). Protected octapeptide resin (0.26
g) with 10% of anisole in liquid HF (6 ml)
Process at 0° C. for 1 hour. After evaporating the HF using an aspirator and then a vacuum pump, the resin was washed with ether (2 ml x 3) to remove the anisole and then with trifluoroacetic acid (2 ml x 5) to remove the peptide. After evaporating the trifluoroacetic acid, the residue was dissolved in a 5% aqueous acetic acid solution and lyophilized under vacuum to obtain a peptide.
200 mesh (BioRad) 2.5 x 100 cm column, and 1% acetic acid was introduced at a rate of 23 ml/hour. Ninhydrin positive main peak (175-300
ml) and freeze-dried under vacuum. Part of the residue (19
ml) 0.01MNaCl, _0.01MNaHPO4 (PH4.5; 1.5
ml) and adsorbed onto a 1 x 50 cm column of carboxymethyl cellulose (Serex CM, BioRad), and the solution was pumped at a rate of 30 ml per hour while changing the NaCl concentration linearly from 0.01 to 0.20 M. 100ml of solution, 0.01M NaH ₂ PO ₄ ; PH
4.5). The main peptide peak (130-170 ml) detected by ultraviolet absorption at 206 nm is collected and freeze-dried in vacuum. Salt and leucine residues in 1% acetic acid (2 ml)
Dissolve in solution and desalt on a 1.5 x 85 cm column of Biogel P-2 by pumping 15 ml of 1% acetic acid per hour. The main peptide peak (50-75 ml) detected by absorption at 206 nm is collected and lyophilized under vacuum to obtain pure octapeptide (10 ml). This material gave the expected amino acid analysis results (Table 1), and the amino acid chain was also found to be as expected by automated Edman degradation. The substance is tobutanol:water:pyridine;acetic acid (volume ratio 15:
12:10:3) showed one ninhydrin-positive spot (Vualin Rf value 0.64 vs. Rf value 0.73), and high-pressure electrophoresis showed one Pauly-positive spot (PH5. R _Arg 0.76 at 0 and R _Arg at PH6.4
0.58, where R _Arg is the value obtained by dividing the distance between the peptide and valin spot centers by the distance between the arginine and valin spot centers). The other peptides listed in Tables 1 and 2 were each prepared in substantially the same manner. (In Table 1, No. 1 peptide (Gly-Leu-Ala
-Arg) is a reference example. )

【table】

[Table] Regarding smooth muscle contractile activity, peptide Ala-Ser
−His−Leu−Gly−Leu−Ala−Arg−Gly is
Up to 50 μg did not show its promoting activity. This means that its promoting activity is 100 times lower compared to the corresponding peptide without glycine attached to arginine (enhancer). In the smooth muscle test, the peptide with glycine (inhibitor) was added prior to the addition of the corresponding promoter peptide without glycine. The results showed that 10 μg of the inhibitor inhibited the promoting activity by more than 50%. Additionally, 50 μg of inhibitor (5.7 x 10 ^-6 M)
completely inhibited the promoting activity. That is, the above test revealed that blocking the carboxyl group of arginine in the promoter peptide with glycine results in the promoter peptide exhibiting inhibitory activity.

Claims (1)

[Claims] 1. A peptide represented by the following amino acid sequence. Y-Gly-Leu-Ala-X-(Gly)n (where X is an amidino-substituted α-amino acid
Arg or Can; Y is Leu; His-Leu; Ser-His-Leu; Ala-Ser-His-Leu; CH ₃ CO-Ala-Ser-His-Leu; Arg-Gln-His-Ala-Arg − Ala−Ser−His−Leu; Ala−Ala−Ala−Leu; Ala−Ala−Leu; Ala−Leu; Asn−Lys−Pro−Leu; CH ₃ CO−Leu; CH ₃ CO−His−Leu; CH ₃ CO-Ala-Leu; and CH ₃ CO-Ala-Ala-Ala-Leu;
is 0 or 1. )