JPH08311098A - New peptides and interleukin 6 antagonistic agent containing the peptide - Google Patents

Abstract

PURPOSE: To obtain a peptide or its derivative composed of relatively small number of constituent amino acids, exhibiting antagonistic action on IL-6 and capable of inhibiting the development of the activity and to provide an IL-6 antagonistic agent containing the peptide. CONSTITUTION: The objective peptide is a pentapeptide expressed by general formula (I): X-A-B-D-Y (X is hydrogen, an amino-protecting group, an amino acid residue or a peptide residue consisting of 2-10 amino acids bonded with each other; Y is hydroxyl, amino, a carboxyl-protecting group, an amino acid residue or a peptide residue consisting of 2-5 amino acids bonded with each other; A is Arg residue or other amino acid residue optionally having a protective group on guanidino group; D is Arg residue optionally having a protective group on guanidino group; B is Leu residue or other amino acid residue including non-natural amino acid and optionally having protective group on the side chain functional group of the amino acid). The present invention also relates to an interleukin-6 antagonistic agent containing the pentapeptide.

Description

Detailed Description of the Invention

[0001]

The present invention relates to novel peptides having antagonism for interleukin 6 or pharmaceutically acceptable salts thereof, and the above peptides or salts thereof together with a pharmaceutically acceptable carrier. The present invention relates to a novel interleukin 6 antagonist that inhibits the activity expression of 6.

[0002]

2. Description of the Related Art Due to the cloning of cDNA and determination of amino acid sequence of B cell differentiation factor (BCDF / BSF-2) which induces B cell antibody production, the B cell differentiation factor has been Interferon β ₂ (I
FNβ ₂ ), hybridoma / plasma cytoma growth factor (HPGF), and hepatocyte stimulating factor (HSF) were found to be the same substances, and these substances are collectively referred to as interleukin 6 (hereinafter abbreviated as IL-6). I will call it.

As mentioned above, IL-6 is extremely effective in the biological defense mechanism against inflammation and infection, such as enhancing antibody production as a B cell differentiation factor and inducing acute phase protein synthesis as a hepatocyte stimulating factor. Although it is an important protein that plays an important function, on the other hand, the I
Diseases believed to be due to L-6 overproduction are also problematic. For example, Medical Immunology (Medical Im
munology) Vol. 15, pp. 195-201 (1988)
Have reported the relationship between IL-6 and autoimmune diseases. Also, Hyundai Kagaku Special Edition 18 "Cytokine" Immune Response and Cell Proliferation / Differentiation Factors, edited by Toshiaki Osawa, 71-
Page 85 (1990, published by Tokyo Kagaku Dojin) has IL-
Examples of diseases in which 6 is involved as a causative agent include various autoimmune diseases such as rheumatoid arthritis, intraatrial myxoma, Castleman's disease, myeloma, Rennel T lymphoma, and mesangial proliferative nephritis.

As described above, IL-6 is considered to be a causative agent of various autoimmune diseases. Therefore, for the purpose of treating these diseases, studies on the active expression site of the IL-6 molecule, and this active expression site. Acts antagonistically against
Development of substances that inhibit the action of IL-6 is ongoing.

According to a research report on the active expression site of IL-6 molecule, for example, Just PJ Brakennhoff et al., N of IL-6 molecule which was conventionally considered to be a signal peptide.
The terminal peptide residue consisting of 28 amino acids is IL-
It is reported that it is not necessary for the expression of 6 activity (The Jour.
nal of Immunology, 143, 1175-82, 1
989). In addition, A. Kruttgen et al. Reported that the third amino acid from the C-terminal of IL-6 is essential for activity expression (FEBS Letters, 273, pp. 95-98, 19).
90 years). Furthermore, C. Nishimura et al.
When analyzed by R, when IL-6 binds to its receptor, it is predicted that the portion near the C-terminal plays an important role in binding (FEBS Letters, 281, 167 to 169,
1991). However, the activity expression site of IL-6 has not been identified in any of the reports, and a substance having an antagonistic effect on the activity expression has not been found yet.

[0006] Further, as a substance which is considered to act antagonistically on the active expression site of IL-6 and inhibit the action of IL-6, for example, BSF-2 (the same substance as IL-6)
A polypeptide in which a plurality of amino acids have been deleted from the N-terminal and C-terminal of the protein has been disclosed (JP-A-2-188600).
Issue). However, the substance disclosed in the above publication is a peptide having a total number of amino acids of 20 or more,
In order to produce such a long-chain peptide, it is necessary to make full use of a plurality of chemical synthesis methods, the operation is complicated, and there are many technically difficult problems, which is not practical. .

[0007]

The present invention has been made in view of the above problems, and its object is to provide a peptide having a relatively small number of constituent amino acids or a derivative thereof to IL-6. Another object of the present invention is to provide a peptide or a pharmaceutically acceptable salt thereof capable of antagonistically acting and inhibiting the expression of its activity, and an IL-6 antagonist containing the same.

[0008]

The present invention provides a compound of the general formula (I): X-A-B-D-Y (wherein X is hydrogen, an amino-protecting group, an amino acid residue or 2 to 10 amino acids). Represents a peptide residue to which Y is bonded, Y represents a hydroxyl group, an amino group, a carboxyl protecting group, an amino acid residue or a peptide residue to which 2 to 5 amino acids are bonded, and A represents a guanidino group having a protecting group. Is an arginine residue or an amino acid residue other than that, D is an arginine residue that may have a protecting group on the guanidino group, B is a leucine residue or any other amino acid including a non-natural amino acid A side chain functional group of the amino acid may have a protecting group.) Or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof. Carrier There is provided a interleukin 6 antagonist, characterized in that it comprises. The present invention also has the general formula (II): EFGH-Arg-NH.
₂ (wherein E, F and H are amino acid residues which may have a protecting group in the functional group, and G is an arginine residue which may have a protecting group in the guanidino group or other The present invention provides an interleukin-6 antagonist, characterized by comprising a pentapeptide represented by amino acid residue) or a pharmaceutically acceptable salt thereof, and further containing a pharmaceutically acceptable carrier therewith. To do.

In this specification, when an abbreviation for an amino acid residue, a protecting group, a solvent or the like is used, it is in accordance with the provisions of IUPAC and ICB or the common symbols in the field. Table-1 to Table-3 show these conventional symbols. In addition, the amino acid sequence in the peptide is described so that the N-terminal amino acid residue is located on the left side and the C-terminal amino acid sequence is located on the right side according to a conventional description method.

[0010]

[Table 1]

[0011]

[Table 2]

[0012]

[Table 3]

In the general formula (I) of the present invention, X is hydrogen, an amino protecting group, an amino acid residue or a peptide residue having 2 to 10 amino acids bonded thereto. As the amino protecting group, an alkoxycarbonyl group which may have a substituent such as a Boc group, a trichloroethoxycarbonyl group and a t-amyloxycarbonyl group; a substituent such as a cyclopentyloxycarbonyl group and a cyclohexyloxycarbonyl group. Optionally a cycloalkoxycarbonyl group;
Z group, p-methoxybenzyloxycarbonyl group, Cl
An aralkyloxycarbonyl group which may have a substituent such as a -Z group, a Br-Z group, a p-nitrobenzyloxycarbonyl group, an adamantyloxycarbonyl group, an Fmoc group; an acetyl group, a trifluoroacetyl group, a formyl group, Examples thereof include an acyl group which may have a substituent such as a propionyl group, a phenylacetyl group, a phenylpropionyl group, a benzenesulfonyl group and a Tos group. Moreover, Phe or Tyr can be illustrated as an amino acid residue. Furthermore, as peptide residues,
H-Trp-Asn-Ser-Ser-Phe-Tyr
-, H-Asn-Ser-Ser-Phe-Tyr-,
H-Ser-Ser-Phe-Tyr-, H-Ser-
Phe-Tyr- and H-Phe-Tyr- can be illustrated.

In the general formula (I), Y is a hydroxyl group,
Amino group, carboxyl protecting group, amino acid residue or 2
Is a peptide residue with ~ 5 amino acids attached. As the carboxyl protecting group, a mono- or di (lower) alkylamino group such as methylamino group or ethylamino group; an arylalkylamino group which may have a substituent such as benzylamino group or phenethylamino group; anilide Group or allylamino group which may have a substituent such as naphthylamino group; lower alkoxy group such as methoxy group and ethoxy group; aryloxy group such as phenyloxy group or naphthyloxy group; aryl such as benzyloxy group (lower ) Alkoxy group; a heterocyclic (lower) alkoxy group and the like can be exemplified. Moreover, Gly and Phe can be illustrated as an amino acid residue. further,
The peptide residue, can be exemplified -Phe-Glu-NH _2.

In the general formula (I), A represents an Arg residue which may have a protecting group in the guanidino group or an amino acid residue other than that, and D represents Arg which may have a protecting group in the guanidino group. It is a residue. Examples of these protecting groups include a nitro group; an aralkyloxycarbonyl group such as a Boc group, a Z group or an adamantyloxycarbonyl group; an arylsulfonyl group which may have a substituent such as a Tos group; and a trityl group. You can In the general formula (I), when A is an amino acid residue, Tyr, Lys, His, T
rp, Pro, Ala, Leu, Asn, Ser, As
Amino acid residues such as p, Glu and Met can be exemplified.

B is a Leu residue or another amino acid residue containing an unnatural amino acid, and the side chain functional group of these amino acids may be protected by a suitable protecting group containing Cl ₂ -Bzl. . Specific amino acid residues including unnatural amino acids used herein include D-type amino acids, Trp, Ala, Ile, Asn, Tyr,
Phe, Ser, Lys, Arg, His, Glu, M
et, Gly, Pro, Asp, norleucine, ter
Examples thereof include residues of natural or unnatural amino acids such as t-leucine, cyclohexylalanine, phenylglycine, homophenylalanine, β-alanine, homoserine, penicillamine, and naphthylalanine.

On the other hand, in the general formula (II), E, F and H represent amino acid residues, and G represents an arginine residue which may have a protecting group in the guanidino group or an amino acid residue other than that. . The amino acids of E, F and H are Ala, Arg, Asn, Asp, Gln and Gl.
u, Gly, His, Ile, Leu, Lys, Me
t, Phe, Pro, Ser, Thr, Trp, Ty
It can be arbitrarily selected from r and Val.

In synthesizing the peptides used in the present invention, a method usually used in peptide synthesis, for example, a solid phase synthesis method or a liquid phase synthesis method can be used. Specifically, for example, it may be carried out according to the method described in Haruaki Yajima (published by Hirokawa Shoten, 1991), supervised by "Development of Pharmaceuticals Vol. 14, Peptide Synthesis". As the solid phase synthesis method, for example, an amino acid corresponding to the C-terminal of the peptide to be synthesized is bound to a support which is insoluble in an organic solvent, and the α-amino group and side chain functional group are protected with an appropriate protecting group. By alternately repeating the reaction of condensing the prepared amino acids one amino acid at a time from the C-terminal to the N-terminal and the reaction of removing the protective group of the α-amino group of the amino acid or peptide bound on the resin, A method of extending the peptide chain is used. The solid phase peptide synthesis method is roughly classified into the Boc method and the Fmoc method depending on the type of protective group used.

After synthesizing the target peptide in this manner,
Deprotection reaction and cleavage of the peptide chain from the support are carried out.
For the cleavage reaction with the peptide chain, hydrogen fluoride or trifluoromethanesulfonic acid was used in the Boc method, and Fmoc was used.
In the method, it is appropriate to use TFA. For example, in the Boc method, the above-mentioned protected peptide resin is treated in hydrogen fluoride in the presence of anisole to remove the protective group and cleave from the support to recover the peptide. The crude peptide is obtained by freeze-drying this. On the other hand, in the Fmoc method, it is possible to perform the deprotection reaction and the cleavage reaction of the peptide chain from the support in TFA by the same operations as described above.

The crude peptide thus obtained is subjected to high performance liquid chromatography (hereinafter abbreviated as HPLC) for separation and purification. The elution may be carried out under optimum conditions using a water-acetonitrile solvent that is usually used for protein purification. A fraction corresponding to the obtained chromatographic peak is collected and freeze-dried. The purified peptide fraction thus purified is identified by molecular weight analysis by mass spectrum analysis, amino acid composition analysis, amino acid sequence analysis, or the like.

The pharmaceutically acceptable salts of the above peptides used in the present invention are physiologically acceptable salts, for example, hydrochloric acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid. , Maleic acid, fumaric acid, oxalic acid, malic acid, citric acid, oleic acid,
Salts with inorganic or organic acids such as palmitic acid; salts with alkali metals or alkaline earth metals such as sodium, potassium and calcium; with organic bases such as triethylamine, diethanolamine, t-butylamine, benzylamine, dicyclohexylamine and arginine And the like. Salts of these peptides can be prepared using a usual salt formation reaction.

The above-mentioned peptides used in the present invention and pharmaceutically acceptable salts thereof (hereinafter, these may be simply represented by peptides) are human IL-6 receptors or their signal transduction proteins. As a result of acting antagonistically on gp130, it is considered that it is possible to inhibit the expression of various activities. Therefore, the peptides of the present invention are useful as therapeutic agents for various diseases such as autoimmune diseases which are considered to be caused by overproduction of IL-6. Such autoimmune diseases include SLE (systemic lupus erythematosus), rheumatoid arthritis, scleroderma (progressive systemic sclerosis), polyarteritis nodosa, Behcet's disease (enteric type, vascular type) as collagen diseases. , Nerve type, oral cavity, skin, eyes, vulva, joint, epididymis, lung, kidney), psoriasis, Wegener's granuloma, aortitis syndrome, Sjogren's syndrome, autoimmune orchiditis, etc.,
As renal disease, type I diabetes (isletitis), nephrotic syndrome, glomerulonephritis (glomerulosclerosis), interstitial nephritis, Goodpasture's syndrome, diabetic nephropathy, hemolytic uremic, ischemic acute renal failure, chronic Examples include renal failure and suppression of intraglomerular thrombosis. In addition, primary mucosal edema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, early menopause, male infertility, myasthenia gravis, juvenile diabetes, pemphigus vulgaris, pemphigoid,
Sympathetic ophthalmitis, uveitis, multiple scleritis, hemolytic anemia, thrombocytopenia, primary biliary cirrhosis, active chronic hepatitis, spontaneous cirrhosis, ulcerative colitis, disc Erythematous lupus erythematosus, dermatomyositis, Hashimoto thyroiditis, Addison's disease, Crohn's disease and the like are also included.

The above peptides or salts thereof used in the present invention are capsules, microcapsules, tablets, granules, powders, troches, pills, ointments, suppositories, injections, suspensions, syrups. It can be administered in the form of a conventional pharmaceutical preparation such as a drug, emulsion, liquid, enteric coating, spray, inhalation, eye drop, nasal drop and the like. The administration route can be various routes including oral, subcutaneous, intramuscular, intravenous, vaginal, rectal, buccal (including buccal and sublingual), transdermal, nasal mucosa and the like.

The above peptides can be prepared by a conventional method using a pharmaceutically acceptable carrier. Such carriers include, for example, sucrose, starch, mannitol, sorbit, lactose,
Glucose, cellulose, talc, calcium phosphate,
Excipients such as calcium carbonate; binders such as cellulose, methyl cellulose, hydroxymethyl cellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch; starch, carboxymethyl cellulose, hydroxypropyl starch, sodium bicarbonate, etc. Disintegrating agents such as calcium phosphate and calcium citrate; lubricants such as magnesium stearate, aerosil, talc and sodium lauryl sulfate; flavoring agents such as citric acid, menthol, glycine, orange powder; sodium benzoate, bisulfite, etc. Preservatives such as sodium, methylparaben, propylparaben; stabilizers such as citric acid, sodium citrate, acetic acid; methylcellulose, polyvinylpyrrolide, etc. , Suspending agents aluminum stearate; e.g. dispersants such as hydroxypropylmethylcellulose; such as diluents such as water; e.g. cacao butter,
Examples include various organic or inorganic carriers conventionally used for formulation, such as base waxes such as white petrolatum and polyethylene glycol.

The dose of the above-mentioned drug of the present invention is an amount capable of exerting a sufficient therapeutic (or prophylactic) effect depending on the degree of symptoms, the general condition of the patient, age, weight, etc., and the administration route and dosage form. The amount of the peptides as the active ingredient is usually 0.01 μg to 2 g / kg / day in an adult in the case of oral administration.
Day, preferably 0.1 μg to 200 mg / kg /
Is the day. In addition, it is usually 0.01 per adult
μg to 200 mg / kg, preferably 0.1 μg
~ 100 mg / kg. In the case of parenteral administration, it is usually 0.001 μg to 1 g per day in adults.
/ Kg / day, preferably 0.01 μg to 200 m
g / kg. In addition, it is usually 0.001 μg to 500 mg / kg, preferably 0.01 μg to 100 mg / kg in an adult.

[0026]

The present invention will be described in more detail with reference to the following examples, but the following examples do not limit the present invention.
It is within the technical scope of the present invention to carry out the invention without departing from the spirits of the preceding and the following.

Example 1 Compound (1): H-Trp-Asn-Se
Synthesis of r-Ser-Phe-Tyr-Arg-Leu-Arg-NH ₂ ) Compound (1)
Was synthesized using a peptide synthesizer ABI430A manufactured by Applied Biosystems by a solid phase synthesis method generally called Boc method. The resin used is normal Bo
p-methylbenzhydrylamine resin (MBHA resin, NH ₂ group content: 0.5) used when synthesizing a peptide in which the C-terminal is an amide group in the solid-phase synthesis method by method c.
7 meq / g, purchased from Peptide Institute Inc.). The amount of resin used is 877 mg (corresponding to 0.5 mmol). The amino acids used in this example are Boc
Is an L-amino acid in which the amino group is protected with
An amount equivalent to 4 times the equivalent amount to the resin (this time the resin is 0.5 mmol equivalent, so the protected amino acid is 2 mmo
1) was used. Table 4 shows the protected amino acids specifically used and their amounts. The compound was synthesized by subjecting the above resin to a condensation reaction of corresponding amino acids in the direction from the C-terminal to the N-terminal of the desired peptide. In the present invention, the binding of the protected amino acid to the resin is carried out by the double coupling method (B) when the amino acids to be condensed are Asn, Gln and Arg, and in the case of other protected amino acids, the single coupling method is used. (A) was used. The obtained compound was subjected to HPLC analysis and amino acid analysis. The conditions of HPLC analysis, the amino acid analysis method, and the solid-phase synthesis method are shown below. The peptides obtained in each of Examples 1 to 86 are referred to as Compound (1) to Compound (86), and their structural formulas are shown in Table-5 and Table-6.

(Analysis conditions by HPLC) TS on the column
Kgel ODS-120T (0.46cmφ x 25c
m) (manufactured by Tosoh Corporation) was used, and a mixed solution of acetonitrile and a 0.1% (W / W) TFA aqueous solution was used as an eluent at a flow rate of 1.0 ml / min. A UV detector was used as a detector, and the measurement was performed at a wavelength of 214 nm.

(Measurement of Amino Acid) After acid-decomposing each compound with 6N hydrochloric acid (110 ° C., 24 hours), the amino acid composition of each compound was analyzed by Hitachi amino acid analyzer (L-8500).

(Solid Phase Synthesis Method) (A) When Performed by Single Coupling Method a) Stir in a DCM solution of 33% TFA for 80 seconds. b) Further stir for 18.5 minutes in 50% TFA in DCM. c) Further wash with DCM three times. d) Further, stirring is repeated twice for 1 minute with a DMF solution of 10% DIEA. e) Further, wash 5 times with DMF. f) Using DCC as the condensation reagent, the condensation reaction of the protected amino acid is performed. g) Wash 5 times with DCM. (B) When performing by double coupling method a) Stir in a DCM solution of 33% TFA for 80 seconds. b) Further stir for 18.5 minutes in 50% TFA in DCM. c) Further wash with DCM three times. d) Further, stirring is repeated twice for 1 minute with a DMF solution of 10% DIEA. e) Further, wash 5 times with DMF. f) Using DCC-HOBt as the condensation reagent, the first condensation reaction of the protected amino acid is performed. g) Wash 3 times with DCM. h) Then stir for 1 minute with a solution of 10% DIEA in DMF. i) Further, wash once with DMF. j) Further wash with DCM three times. k) Using DCC-HOBt as the condensation reagent, the second condensation reaction of the protected amino acid is performed. l) Wash once with DMF. m) Then wash 5 times with DCM.

The compound (1) was synthesized on the resin by sequentially condensing the protected amino acid on the resin from the C terminus by the above solid phase synthesis method. Then, the Boc group which is the N-terminal protecting group of the compound (1) was removed using TFA. The obtained peptide resin was placed on a glass filter, washed with DCM and then vacuum dried to obtain 1.49 g of a protected peptide resin. Cleavage of peptides from this protected peptide resin is Low-Hig.
According to the h method. That is, this protected peptide resin 830
mg using a hydrogen fluoride reactor (manufactured by Peptide Institute Co., Ltd.), hydrogen fluoride 9 ml, anisole 1.0 m
in a mixed solution of 1 and 0.1 ml of ethyl methyl sulfide at 0 ° C.
Treated for 1 hour. After the hydrogen fluoride and the added reagent in the mixed solution were distilled off under reduced pressure, the residue was treated again in a mixed solution of 8 ml of hydrogen fluoride, 0.8 ml of anisole and 0.08 ml of ethanedithiol at 0 ° C. for 1 hour. After the hydrogen fluoride and the added reagent in the mixed solution were distilled off under reduced pressure, the residue was thoroughly washed on a glass filter with diethyl ether and dried under reduced pressure. Next, the obtained residue is extracted with a 0.1 M acetic acid aqueous solution, and the extract is lyophilized to give compound (1).
230 mg of a crude product containing 100 mg of this
High performance liquid chromatography for preparative separation (column: "TSKgel ODS120-T" manufactured by Tosoh Corporation,
2.54 cmφ × 30 cm) and eluted by a linear gradient method with a mixed solvent of acetonitrile and a 0.1% (W / W) TFA aqueous solution, and a fraction containing the compound (1) is fractionated to obtain 32 mg of a purified product. Got The purified product thus obtained was dissolved in a 50% (W / W) acetic acid solution and passed through a cation exchange resin Amberlite IRA-410 (manufactured by Organo Corporation) to give an acetate salt of compound (1) 25
mg was obtained. The results of HPLC measurement of compound (1) and the results of confirmation by amino acid analysis are shown in Table-13 and Table-1, respectively.
4 shows. Here, in Table 13, A shows the mixing ratio of acetonitrile and a 0.1% (W / W) TFA aqueous solution at the start of elution, and B shows the mixing ratio of the eluate 30 minutes after the start of elution. The mixing ratio of the eluate was changed linearly.

Example 2 Compound (2): H-Asn-Ser-Se
using protected amino acids described in the synthesis) Table -7 r-Phe-Tyr-Arg -Leu-Arg-NH 2, was synthesized compound (2) by the Boc method. As the treatment of the protected peptide, all the protected amino acids were condensed with the resin, and then the Boc group, which is the N-terminal protective group of the target peptide, was removed using TFA. The obtained peptide resin was placed on a glass filter, washed with DCM, and dried in vacuum to obtain 1.32 g of a protected peptide resin. 457 mg of this protected peptide resin was used in a hydrogen fluoride reactor (manufactured by Peptide Institute Co., Ltd.)
0 in a mixed solution of 9 ml of hydrogen fluoride and 1.0 ml of anisole
It was treated at ° C for 1 hour. After the hydrogen fluoride and the added reagent in the mixed solution were distilled off under reduced pressure, the residue was thoroughly washed on a glass filter with diethyl ether and dried under reduced pressure. The residue thus obtained was extracted with a 0.1 M acetic acid aqueous solution, and the extract was lyophilized to give compound (2).
98 mg of a crude product containing was obtained. Of these, 90 mg of the crude product was subjected to preparative HPLC (column: "TSO""TS
Kgel ODS120-T ", 2.54 cmφ x 30
cm) with acetonitrile and 0.1% (W / W) T
Elution was carried out by a linear gradient method using a mixed solvent of an FA aqueous solution, a fraction containing the compound (2) was collected, and the purified product 32
mg was obtained. The obtained purified product was dissolved in a 50% (W / W) acetic acid solution and the cation exchange resin Amberlite was used.
By passing it through IRA-410 (manufactured by Organo Corporation), 28 mg of the acetate salt of compound (2) was obtained.

(Examples 3 to 10) Compounds (3) to (10) having the structural formulas shown in Table 5 were synthesized in the same manner as in Example 2 and protected amino acids shown in Table 7 were used. I went. In the synthesis of compound (6), since the C-terminal amino acid is a free carboxylic acid, Boc-Ar
g (Tos) -PAM resin was used instead of MBHA resin.

Example 11: Compound (11): H-Arg-Le
Synthesis of u-Arg-Gly-NH ₂ ) The compound (11) was synthesized by using a simple peptide synthesizer (Kokusan Chemical Co., Ltd., trademark “Cook-san”), which is generally called Fmoc method. I went there. Since the resin used in this example has an amide group at the C-terminal, an amide resin for Fmoc (Kokusan Kagaku Co., Ltd.)
Purchased (using a resin amount corresponding to an amino group content of 0.3 mmol).

Synthesis of compound (11) by Fmoc method a) The above resin is stirred with 6 ml of DMF for 1 minute. Repeat this 3 times. b) Furthermore, 20% (W / W) piperidine in DMF solution 6
Stir with ml for 3 minutes. Repeat this 3 times. c) Furthermore, 20% (W / W) piperidine in DMF solution 6
Stir with ml for 15 minutes. d) Further, stir with 6 ml of DCM for 1 minute. Repeat this twice. e) Further, it is stirred with 6 ml of DMF for 1 minute. Repeat this twice. f) Further, it is stirred with 6 ml of methanol for 1 minute. g) Further stir with 6 ml DCM for 1 min. Repeat this twice. h) Further, stir with 6 ml of NMP for 1 minute. Repeat this 3 times. i) Next, Fmoc-Gly-OH (357 mg, 1.
NMP of protected amino acid having a composition of 2 mmol)
(2.6 ml) solution, HOBt (1.2 mmol) and TBTU (1.2 mmol) in DMF solution (2.4 m
l) and DIEA (2.4 mmol) in NMP solution (1.0 ml) are added, and the mixture is stirred at room temperature for 7 minutes. In addition,
All amino acids used below are added in an amount 4 times (1.2 mmol) the content of amino groups contained in the resin. j) The solution obtained in i) above is added to the resin that has undergone steps a) to h) above, and then stirred for 30 minutes. k) The above resin is washed by stirring with 6 ml of methanol for 1 minute. l) Further, the above resin is washed by stirring with 6 ml of NMP for 1 minute. Repeat this 3 times. m) Further, the above resin is washed by stirring with 6 ml of DCM for 1 minute. Repeat this 5 times. In this way, Fm
oc-Gly is bound. o) In the above i), instead of Fmoc-Gly-OH, Fmoc-Arg (Pmc) -OH (795 mg,
A protected amino acid having the above composition is bonded to the resin by preparing a solution in the same manner as i) except that 1.2 mmol) is used, and then performing steps j) to m). p) Fmoc-Leu-OH (424 mg, 1.2 mmo instead of Fmoc-Gly-OH in i) above.
A solution is prepared in the same manner as in i) except that l) is used, and then the steps j) to m) are performed to bind the protected amino acid having the above composition to the resin. q) Instead of Fmoc-Gly-OH in i) above, Fmoc-Arg (Pmc) -OH (795 mg,
A protected amino acid having the above composition is bonded to the resin by preparing a solution in the same manner as i) except that 1.2 mmol) is used, and then performing steps j) to m).

After all the protected amino acids constituting the compound (11) were bound to the resin, they were vacuum dried to give 8
40 mg of protected peptide resin was obtained. Of these, 430
mg of protected peptide resin with 5% phenol in TFA
The desired peptide was cleaved from the resin by treating with a solution (15 ml), and at the same time, unnecessary side chain protecting groups were removed. The insoluble matter in the reaction solution was removed by a glass filter, diethyl ether was added to the filtrate, the obtained precipitate was collected by a glass filter, and vacuum dried to obtain 70 mg of a crude product containing the compound (11). . 40 mg of the obtained crude product was subjected to preparative HPLC (column: "T" manufactured by Tosoh Corporation).
SKgel ODS120-T ", 2.54cmφ x 3
0 cmL) and 0.1% (W /
W) Elution was performed by a linear gradient method using a mixed solvent of an aqueous TFA solution, and a fraction containing the compound (11) was collected to obtain 25 mg of a purified product. This was dissolved in a 50% (W / W) acetic acid solution and the cation exchange resin Amberlite I was added.
By passing through RA-410 (manufactured by Organo Corporation), 18 mg of an acetate salt of compound (11) was obtained.

(Examples 12 to 16) Compounds (12) to (16) shown in Table 5 were synthesized in the same manner as in Example 11 using the protected amino acids shown in Table 7.

Example 17: Compound (17): H-Arg-Le
Synthesis of u-Arg-NHCH ₂ C ₆ H ₅ ) Fmoc-Arg (Pmc)
-OH (663 mg, 1 mmol), TBTU (321
mg, 1 mmol), HOBt (135 mg, 1 mmo
l), DIEA (129 mg, 1 mmol) in DMF
It was dissolved in (10 ml) and stirred. Benzylamine (107 mg, 1 mmol) was added to the solution, and the mixture was stirred at room temperature for 5 hours. Then, the solvent was distilled off, and the residue was extracted with ethyl acetate. The ethyl acetate solution was washed with water, 10% citric acid aqueous solution, and cooled 5% aqueous sodium hydrogen carbonate solution and then dried, and ethyl acetate was distilled off to remove Fmoc-Arg (Pmc) -NHC.
520 mg of H ₂ C ₆ H ₅ was obtained. Fmoc-Arg (Pm
c) -NHCH ₂ C ₆ H ₅ (490 mg, 0.65 mmo
l) was dissolved in a DMF solution (4 ml) containing 20% piperidine and stirred for 1 hour at room temperature. The solvent was distilled off under reduced pressure,
Fmoc-Leu (248 mg, 0.7 mmo)
l), TBTU (225 mg, 0.7 mmol), HO
Bt (95 mg, 0.7 mmol) and DIEA (91
mg, 0.7 mmol) in DMF (10 ml) was added, and the mixture was stirred for 15 hours at room temperature. The solvent was distilled off, and the residue was extracted with ethyl acetate. The ethyl acetate solution was washed with water, 10% citric acid aqueous solution, and cooled 5% sodium bicarbonate aqueous solution, and then dried, and ethyl acetate was distilled off to remove Fmoc-Leu-Arg.
490 mg of (Pmc) -NHCH ₂ C ₆ H ₅ was obtained. Fm
oc-Leu-Arg (Pmc) -NHCH 2 C 6 H
₅ (450 mg, 0.52 mmol) was dissolved in a DMF solution (4 ml) containing 20% piperidine and stirred for 1 hour at room temperature. The solvent was distilled off under reduced pressure, and Fmoc-Ar was added thereto.
g (Pmc) -OH (365 mg, 0.55 mmo
l), TBTU (177 mg, 0.55 mmol), H
OBt (75 mg, 0.55 mmol) and DIEA
A solution of (78 mg, 0.55 mmol) in DMF (8 ml) was added, and the mixture was stirred at room temperature for 15 hours. The solvent was distilled off, and the residue was extracted with ethyl acetate. The ethyl acetate solution was added to water, 10
% Aqueous citric acid solution, washed with cooled 5% aqueous sodium hydrogen carbonate solution, and then dried, and ethyl acetate is distilled off to remove Fmoc-Arg (P
mc) -Leu-Arg (Pmc) -NHCH 2 C 6 H 5
Of 700 mg was obtained. Fmoc-Arg (Pmc) -Le
_{u-Arg (Pmc) -NHCH 2} C 6 H 5 (600m
g) was dissolved in a DMF solution (4 ml) containing 20% piperidine and stirred for 1 hour at room temperature. The solvent was distilled off under reduced pressure,
A TFA solution (10 ml) containing 5% phenol was added thereto and stirred for 1 hour. The TFA solution was poured into diethyl ether, and the deposited precipitate (120 mg) was collected by filtration. The precipitate was purified by HPLC as in Example 11,
36 mg of the desired peptide (Compound 17) was obtained.

Example 18 Compound (18): H-Arg-Le
Synthesis of u-Arg-NHCH ₂ CH ₂ C ₆ H ₅ ) The reaction was performed in the same manner as in Example 17 except that phenylamine was used instead of benzylamine, to obtain 25 mg of compound (18).

Example 19: Compound (19): H-Arg-Le
u-Arg (NO ₂₎ Synthesis of _{-OCH 2 C 6 H 5) Fmoc} -A of the starting materials
_{rg (Pmc) -NHCH 2 C 6} H 5 the Boc-Arg
18 mg of compound (19) was obtained by reacting in the same manner as in Example 17 except that BOC group was removed by treating with TFA solution instead of (NO ₂ ) -OCH ₂ C ₆ H ₅ .

Example 20: Compound (20): (CH ₃ ) ₃ CC
Synthesis of O-Arg-Leu-Arg-NHCH ₂ C ₆ H _{5 In} the same manner as in Example 17, Fmoc-Arg (P
mc) -Leu-Arg (Pmc) -NHCH 2 C 6 H 5
After synthesis of Fmoc-Arg (Pmc) -Leu-Ar
20 g (Pmc) -NHCH ₂ C ₆ H ₅ (750 mg)
Dissolve in a DMF solution containing 5% piperidine (5 ml) and
Stir at room temperature for hours. After distilling off the solvent, the precipitate obtained by precipitating with isopropyl ether was dissolved in DMF,
Pivalic acid (70 mg), TBTU, HOBt, DIE
0.7 mmol equivalent amount of each A was added, and the mixture was stirred at room temperature for 2 hours. The solvent was distilled off under reduced pressure, and the residue was extracted with ethyl acetate. The ethyl acetate solution was washed with water, 10% citric acid aqueous solution, and cooled 5% aqueous sodium hydrogen carbonate solution, and then dried. After distilling off ethyl acetate, the TFA solution containing 5% phenol (10 m
1) was added and stirred for 1 hour. The TFA solution was poured into diethyl ether, and the deposited precipitate (320 mg) was collected by filtration. This precipitate was purified by HPLC as in Example 11 to obtain 56 mg of compound (20).

Example 21: Compound (21): (CH ₃ ) ₃ CC
O-Arg-Leu-Arg- NHCH Synthesis of ₂ CH ₂ C ₆ H ₅₎ is reacted in the same manner as in Example 18, Fmoc-Arg (Pmc) -Leu
After synthesizing —Arg (Pmc) -NHCH ₂ CH ₂ C ₆ H ₅ ,
Reacting pivalic acid as described in Example 20,
18 mg of compound (21) was obtained.

Example 22: Compound (22): (CH ₃ ) ₃ CC
O-Arg-Leu-Arg ( NO 2) Synthesis of -OCH ₂ C ₆ H ₅₎ is reacted in the same manner as in Example 19, Fmoc-Arg (Pmc) -Le
u-Arg (NO ₂₎ after synthesizing a -OCH ₂ C ₆ H _5, is reacted in the same manner as pivalic acid as described in Example 20, compound (22) was obtained 21 mg.

Example 23: Compound (23): Z-Arg-Le
Synthesis of u-Arg-NHCH ₂ C ₆ H ₅ ) Fmoc-Arg (Pmc) -Leu-Arg (P
mc) -NHCH ₂ C ₆ H ₅ after the piperidine treatment (250 mg), was reacted with Z-OSu (50 mg) in DMF, was treated in the same manner as in Example 20, compound (23) 18
mg was obtained.

Example 24: Compound (24): Z-Arg-Le
_{u-Arg-NHCH 2 CH 2} C 6 Synthesis of H ₅₎ was obtained in the same manner as in Example 21 Fmoc-Arg (Pmc) -Leu -Arg
(Pmc) -NHCH ₂ CH ₂ C ₆ H ₅ (210 mg) was treated with piperidine, and then reacted with Z-OSu (50 mg) in DMF, treated in the same manner as in Example 20, to give compound (24).
21 mg was obtained.

Example 25: Compound (25): Z-Arg-Le
_{u-Arg (NO 2) -OCH} 2 C 6 Synthesis of H ₅₎ Example 22 In the same manner the resulting Fmoc-Arg (Pmc) -Leu- Arg
(NO ₂ ) -OCH ₂ C ₆ H ₅ (260 mg) was treated with piperidine, reacted with Z-OSu (60 mg) in DMF, and treated in the same manner as in Example 20 to give compound (25) 18 m.
g was obtained.

(Examples 26-32) Compound (26)-
The synthesis of (32) was performed in the same manner as in the method described in Example 11 to obtain the target compounds (26) to (32).

(Examples 33 to 45) Compound (33) to
The synthesis of (45) was performed in the same manner as in the method described in Example 2 to obtain the target compound. However, only when synthesizing the compound (41), the reaction using hydrogen fluoride for cleaving the peptide from the protected peptide resin was described in Example 1.
-High method.

(Examples 46 to 86) Compound (46) to
The synthesis of (86) was performed in the same manner as in the method described in Example 11 to obtain the target compounds (46) to (86).

[0050]

[Table 4]

[0051]

[Table 5]

[0052]

[Table 6]

[0053]

[Table 7]

[0054]

[Table 8]

[0055]

[Table 9]

[0056]

[Table 10]

[0057]

[Table 11]

[0058]

[Table 12]

[0059]

[Table 13]

[0060]

[Table 14]

[0061]

[Table 15]

Test Example: IL-6 Inhibition Test by IL-6 Using Human SKW6.4 Cells The IL-6 antagonistic action of the peptides of the present invention on human recombinant IL-6 (hrIL-6) is examined. Therefore, as the test substance, two kinds of compounds (17) and (20) among the compounds synthesized in the above examples were used, and the final concentration of the compound was 50.
It was appropriately diluted to 0 μg / ml. Human SKW6.4 cells were used as IgM antibody-producing cells, 1 × 10 ⁴ of the above cells were added to each well of a 96-well microplate, and the cells were mixed with the test substance and an appropriate amount of hrIL-6 in a carbon dioxide incubator 37 Culturing was carried out at 4 ° C for 4 days.
After the culture was completed, the culture supernatant obtained by centrifugation at 1,200 rpm for 5 minutes was collected, and the amount of IgM antibody contained in the supernatant was quantified by the ELISA method. In addition, as a control test, Ig was added in the same manner as above without adding the test substance.
The amount of M antibody was quantified. The IgM antibody production inhibition rate by the addition of the above compound was calculated by the following formula. The measurement results are shown in Table-16.

[0063]

[Equation 1]

[0064]

[Table 16]

As is clear from Table-16, the compound (1
Both 7) and (20) were found to significantly inhibit IgM antibody production.

[0066]

INDUSTRIAL APPLICABILITY According to the present invention, peptides capable of antagonistically acting on IL-6 and inhibiting the expression of its activity are provided. Therefore, various peptides considered to be caused by IL-6 are provided. It is useful as a therapeutic drug for autoimmune diseases.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masaaki Ito 1365 Shinya, Aboshi-ku, Himeji-shi, Hyogo Prefecture (72) Inventor Hachiro Senoo 12-1 Sengokuhigashi-cho, Kadoma-shi, Osaka (72) Nobuo Seki Nobuo Seki, Ibaraki-shi, Osaka 2-12-28-303

Claims (16)

[Claims] 1. General formula (I): X-A-B-D-Y (wherein X represents hydrogen, an amino protecting group, an amino acid residue or a peptide residue having 2 to 10 amino acids bonded thereto. .
Y represents a hydroxyl group, an amino group, a carboxyl protecting group, an amino acid residue or a peptide residue having 2 to 5 amino acids bonded. A represents an arginine residue which may have a protecting group on the guanidino group or an amino acid residue other than that. D is an arginine residue which may have a protecting group on the guanidino group. B is a leucine residue or another amino acid residue containing a non-natural amino acid, and the side chain functional group of the amino acid may have a protecting group. ) Or a pharmaceutically acceptable salt thereof. 2. The peptide or pharmaceutically acceptable according to claim 1, wherein A and D in the general formula (I) are both arginine residues which may have a protecting group on the guanidino group. Ruso salt. 3. The peptide or pharmaceutically acceptable salt thereof according to claim 2, wherein A and D in the general formula (I) are both arginine residues and B is a leucine residue. . 4. The general formula (II): EF-G-H-Ar.
g-NH ₂ (In the formula, E, F and H are amino acid residues which may have a protecting group in the functional group, and G is an arginine residue which may have a protecting group in the guanidino group, or Pentapeptides represented by other amino acid residues) or a pharmaceutically acceptable salt thereof. 5. In the general formula (II), E is asparagine,
Selected from the group of amino acid residues of tyrosine, proline, leucine, serine, lysine, arginine, histidine, tryptophan, alanine, methionine, aspartic acid or glutamic acid, and amino acid residues having a protecting group on the functional group of these residues. One of
The pentapeptides according to claim 4, wherein F is a tyrosine residue, G is an arginine residue, and H is a leucine residue, or a pharmaceutically acceptable salt thereof. 6. E in the general formula (II) is a phenylalanine residue, F is leucine, phenylalanine, serine, arginine, histidine, tryptophan, aspartic acid, glutamic acid, methionine, proline, alanine,
It is any one selected from the group of each amino acid residue of lysine, glycine or asparagine and an amino acid residue having a protective group on the functional group of these residues, G is an arginine residue, and H is a leucine residue. The pentapeptides according to claim 4 or a pharmaceutically acceptable salt thereof. 7. In the general formula (II), E is a phenylalanine residue, F is a tyrosine residue, G is tyrosine, lysine, histidine, tryptophan, proline, alanine, leucine, asparagine, serine, aspartic acid, glutamic acid or methionine. 5. The amino acid residue according to claim 1, which is selected from the group consisting of amino acid residues having a protecting group for the functional group of these residues, and H is a leucine residue. Pentapeptides or pharmaceutically acceptable salts thereof. 8. In the general formula (II), E is a phenylalanine residue, F is a tyrosine residue, G is an arginine residue, H is tryptophan, alanine, isoleucine, asparagine, tyrosine, phenylalanine, serine, lysine, arginine, It is any one selected from the group of each amino acid residue of histidine, glutamic acid, methionine, glycine, proline or aspartic acid and an amino acid residue having a protective group for a functional group in these residues. Item 5. The pentapeptide according to Item 4, or a pharmaceutically acceptable salt thereof. 9. General formula (I): X-A-B-D-Y (wherein X represents hydrogen, an amino-protecting group, an amino acid residue or a peptide residue having 2 to 10 amino acids bonded thereto. .
Y represents a hydroxyl group, an amino group, a carboxyl protecting group, an amino acid residue or a peptide residue having 2 to 5 amino acids bonded. A represents an arginine residue which may have a protecting group on the guanidino group or an amino acid residue other than that. D is an arginine residue which may have a protecting group on the guanidino group. B is a leucine residue or another amino acid residue containing a non-natural amino acid, and the side chain functional group of the amino acid may have a protecting group. ) An interleukin 6 antagonist, which comprises a pharmaceutically acceptable carrier together with the peptide represented by the formula (1) or a pharmaceutically acceptable salt thereof. 10. The interleukin 6 antagonist according to claim 9, wherein both A and D in the general formula (I) are arginine residues which may have a protecting group on the guanidino group. 11. The interleukin-6 antagonist according to claim 10, wherein A and D in the general formula (I) are both arginine residues and B is a leucine residue. 12. A compound represented by the general formula (II): E-F-G-H-A.
rg-NH ₂ (In the formula, E, F and H are amino acid residues which may have a protecting group in the functional group, and G is an arginine residue which may have a protecting group in the guanidino group or An interleukin 6 antagonist, which comprises a pharmaceutically acceptable carrier together with a pentapeptide represented by any other amino acid residue) or a pharmaceutically acceptable salt thereof. 13. In the general formula (II), E is asparagine, tyrosine, proline, leucine, serine, lysine,
Arginine, histidine, tryptophan, alanine,
It is any one selected from the group consisting of amino acid residues of methionine, aspartic acid, or glutamic acid and amino acid residues having a protective group in the functional group of these residues, F is a tyrosine residue, and G is an arginine residue. The interleukin 6 antagonist according to claim 12, wherein the group H is a leucine residue. 14. E in the general formula (II) is a phenylalanine residue, F is leucine, phenylalanine, serine,
Any selected from the group of amino acid residues of arginine, histidine, tryptophan, aspartic acid, glutamic acid, methionine, proline, alanine, lysine, glycine or asparagine, and amino acid residues having a protective group on the functional group of these residues. Or only one, G
Is an arginine residue, and H is a leucine residue, The interleukin 6 antagonist according to claim 12, wherein 15. In the general formula (II), E is a phenylalanine residue, F is a tyrosine residue, G is tyrosine, lysine,
Any one selected from the group consisting of histidine, tryptophan, proline, alanine, leucine, asparagine, serine, aspartic acid, glutamic acid or methionine amino acid residues, and amino acid residues having a protective group for the functional group of these residues. And H is a leucine residue. 13. The interleukin 6 antagonist according to claim 12, wherein H is a leucine residue. 16. In the general formula (II), E is a phenylalanine residue, F is a tyrosine residue, G is an arginine residue, and H.
Is tryptophan, alanine, isoleucine, asparagine, tyrosine, phenylalanine, serine, lysine,
Arginine, histidine, glutamic acid, methionine,
13. The interleukin according to claim 12, wherein the interleukin is any one selected from the group consisting of amino acid residues of glycine, proline or aspartic acid and amino acid residues having a protective group in the functional group of these residues. 6 antagonists.