JPH09221500A - Calcitonin analogue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new calcitonin analogue having a specific amino acid sequence, having lowering action of blood calcium ion concentration lowering action, having high activity, hardly having adverse effect and useful as a therapeutic agent, etc., for osteoporosis, neoplastic calcemia and Paget's disease. SOLUTION: This calcitonin analogue (salt) is represented by the formula [(n) is an integer of 3-7] and has action for lowering blood calcium ion concentration and high activity and a few adverse effect and useful as a therapeutic agent for osteoporosis, neoplastic calcemia and Paget's disease. The calcitonin analogue is obtained by using 4-(2,4-dimethoxyphenyl-aminomethyl)- phenoxyacetamide-4-methylbenzhydrylamine-bound polystyrene beads as a solid phase carrier and successively binding amino acids whose α-amino groups and side chain functional groups are protected according to amino acid sequence of peptide from C end side by a peptide solid phase synthetic method, carrying out cleavage of peptide chain from a carrier and removal of a protective group and cyclizing a part in the molecule.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel calcitonin analog having an action of lowering blood calcium ion concentration.

[0002]

BACKGROUND OF THE INVENTION Known natural calcitonin is eel, salmon, chicken, pig, human, bovine,
The origins of sheep, rats, etc. are known, all 32
It is composed of individual amino acids, and these calcitonin originally possessed by humans and animals are called natural calcitonin.

On the other hand, JP-A-63-2767698, JP-A-63-284198, JP-A-63-287800, J. Bi.
ochem.Vol.159 P125 (1986), Endocrinology Vol.117 P8
0 (1987), J. Biochem. Vol. 162 P399 (1987), etc.
Analogs of native calcitonin have been disclosed. This analog of natural calcitonin is obtained by substituting one or more amino acids of natural calcitonin with other amino acids (substitute), one or more amino acids of natural calcitonin deleted (deleted), natural. Examples include those in which one or more amino acids are added between or at the ends of type calcitonin (adducts), those in which two or more of substitution, deletion, and addition are combined. Two or more amino acids having these substitutions, deletions, and additions may be continuous in the peptide structure or may be separated. Hereinafter, these non-natural calcitonin are referred to as calcitonin analogs.

Although natural human calcitonin is originally possessed by humans, it has a low biological activity on humans. On the other hand, natural calcitonin of non-human animals such as salmon, eel, and chicken has high biological activity on humans, and is expected as a therapeutic drug for osteoporosis, neoplastic hypercalcemia, Paget's disease and the like. Analogs of eel calcitonin have been put to practical use as therapeutics.
However, natural calcitonin of non-human animals or its analogs have side effects such as strong vomiting action and digestive tract dysfunction action on humans and antigenicity to humans. Human calcitonin is extremely weak with or without such side effects. It is known that this problem has been solved by using a calcitonin analogue consisting of a hybrid of human calcitonin and non-human animal calcitonin (hereinafter, hybrid calcitonin) (WO91 / 15511, Endo).
crinology, Vol. 131, 2885 (1992)).

For example, a hybrid type represented by the following structural formula (Formula 3) using the sequence from the amino terminus of human calcitonin to the 16th position and the sequence from the 17th to 32nd position from the amino terminus of salmon calcitonin Calcitonin retains the same level of blood calcium ion lowering effect as salmon calcitonin, but does not show cross-activity with human-anti-salmon calcitonin antibody, and no weight suppression activity which is a side effect of salmon calcitonin is observed in animals. It has been known.

[0006]

Embedded image

However, it is presumed that the calcitonin is unstable in a solution because the disulfide bond by the 1st and 7th cysteines counted from the amino terminus of the calcitonin is still present, and the physiological activity may be lowered. There is.

[0008]

Therefore, the inventors of the present invention have conducted diligent studies for the purpose of obtaining a novel calcitonin analog that solves the above problems based on the structure of the hybrid calcitonin as described above. As a result, the first and 7th amino acids of the hybrid calcitonin counted from the amino terminus.
2-aminosuberic acid [HO
_{CO- (CH 2) 5 -CH (} NH 2) -COOH] , such as the α- amino-.alpha., .omega.
By using alkanedicarboxylic acid [HOCO- (CH ₂ ) _n- CH (NH ₂ ) -COOH], the side chain carboxyl group of this amino acid and the amino-terminal amino acid, that is, the amino group of glycine are used to achieve the above purpose. Found to achieve. The present invention is the following invention relating to the novel calcitonin analog thus obtained.

A calcitonin analog represented by the following structural formula (Chemical Formula 4, wherein n is an integer of 3 to 7) or a pharmaceutically acceptable salt thereof.

[0010]

Embedded image

A particularly preferred calcitonin analog of the present invention is a calcitonin analog represented by the following structural formula (Formula 5) or a pharmaceutically acceptable salt thereof.

[0012]

Embedded image

In the above two formulas, the notation of amino acids is the usual three letter notation except aminosuberic acid. For example, Arg for arginine, Asn for asparagine, Asp.
Is aspartic acid, Gly is glycine, Gln is glutamine, His is histidine, Leu is leucine, and Lys.
Represents lysine, Met represents methionine, Phe represents phenylalanine, Pro represents proline, Ser represents serine, Thr represents threonine, and Tyr represents tyrosine. In addition, () indicates a substitutable amino acid, and “death” indicates that it can be deleted. Furthermore, the position number is represented by the position number corresponding to the natural calcitonin. n is preferably 5.

The pharmaceutically acceptable salt of the calcitonin analog of the present invention includes salts of the above calcitonin analog with an acid or a base. Examples of the acid include hydrochloric acid, sulfuric acid, phosphoric acid, other inorganic acids, formic acid, acetic acid, propionic acid, lactic acid, oxalic acid, succinic acid, tartaric acid, benzoic acid, alkanesulfonic acid, toluenesulfonic acid, and other organic acids. is there. Examples of the base include alkali metal compound bases such as alkali metal hydroxide capable of forming an alkali metal salt.

In synthesizing the calcitonin analog of the present invention, a solid phase peptide synthesis method using a polymer resin, a peptide liquid phase synthesis method which is a general organic synthesis method, or a combination thereof is used to sequentially produce amino acids from the carboxyl terminal. Can be used, or a peptide solid phase synthesis method of a peptide fragment of a dipeptide or more cleaved at an arbitrary position, a peptide liquid phase method, or a method of performing sequential condensation by combining them can be used.

Resins used for solid phase peptide synthesis include benzhydrylamine resin, 4- (2,4-dimethoxyphenyl-aminomethyl) -phenoxyacetamide-aminomethyl resin, 4- (2,4-dimethoxyphenyl-amino). Methyl) -phenoxyacetamide-4-methylbenzhydrylamine resin and 4- (2,4-dimethoxyphenyl-aminomethyl) -phenoxy resin can be used.

As the amino acid, an amino acid derivative having a functional group protected may be used. Examples of α-amino protecting groups for amino acids include benzyloxycarbonyl group (Z), 9-
Fluorenylmethyloxycarbonyl group (Fmoc),
A tertiary butyloxycarbonyl group (Boc), a formyl group (HCO), an acetyl group (Ac), etc. can be used. As an α-carboxyl protecting group for amino acids, a benzyl group (B
zl), tertiary butyl group (tBu), methyl group (Me),
An ethyl group (Et), a phenacyl group (Pac), etc. can be used. In addition, the inside of () shows the symbol of the group usually used, and it is the same also in the following groups.

Further, as the amino acid side chain functional group protecting group,
Benzyl group (Bzl), p-toluene sulfonyl group (To
s), p-nitrophenoxy group (ONp), benzhydryl group (Bzh), acetamide group (Acm), tert-butyl group (tBu), tert-butyloxycarbonyl group (B)
oc), cyclohexyl group (cHex), benzyloxymethyl group (Bom), third butoxymethyl group (Bu
m), tert-butylthio group (tButhio), dinitrophenyl group (Dnp), 9-fluorenylmethyloxycarbonyl group (Fmoc), 2,2,5,7,8-pentamethylchroman-6-sulfonyl group ( Pmc), a trityl group (Trt), etc. can be used. One or more of these protecting groups can be arbitrarily selected and used depending on the purpose.

For the elongation reaction of the peptide chain, that is, the sequential addition reaction of amino acids or amino acid derivatives, the dehydration condensation method using carbodiimide or the active ester method can be used.

In the dehydration condensation method, as a condensing agent, dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (WSC), 2- (1H-benzotriazol-1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), benzotriazol-1-yloxy-tris (dimethylamino) -phosphonium hexafluorophosphate (BOP) and the like can be used. In the active ester method, N-hydroxysuccinimide ester (HONSu), pentafluorophenyl ester (OPfp), dihydroxybenztriazine ester (ODhbt) and the like can be used. In addition, the abbreviations of commonly used compounds are shown in parentheses.

As the reaction solvent, N, N-dimethylformamide, N, N-dimethylacetamide, tetrahydrofuran, chloroform, methylene chloride, ethyl acetate,
1,4-dioxane, dimethyl sulfoxide, N-methylpyrrolidone, pyridine, water and the like can be used.

As a releasing agent for the functional group-protecting group of amino acid or peptide, depending on the purpose, hydrogen fluoride, trifluoroacetic acid, trifluoromethanesulfonic acid, ammonia /
Methanol, hydrogen bromide / acetic acid, hydrogen / palladium on carbon,
Mercury acetate, acetic acid / zinc powder, alkali / water / methanol, etc. can be used.

Various types of chromatography such as reverse phase chromatography, normal phase chromatography, ion exchange chromatography, gel filtration and recrystallization can be used during the synthesis or as the final purification method.

[0024]

EXAMPLES The present invention will be specifically described below with reference to examples of the present invention, but the present invention is not limited to these examples.

Example 1 Synthesis of Acetate of Calcitonin Analogue Represented by Structural Formula (Chemical Formula 6) by Peptide Solid Phase Synthesis Method

[0026]

[Chemical 6]

(1) Synthesis of a polypeptide derivative represented by the following structural formula (Formula 7).

[0028]

Embedded image

As the resin, 4- (2,4-dimethoxyphenyl-aminomethyl) -phenoxyacetamide-
What crosslinked polystyrene beads with 4-methylbenzhydrylamine (hereinafter referred to as solid phase resin) was used. 1 g of the resin
Was packed in a column of an automatic synthesizer, and amino acids were sequentially added under the following conditions. DMF means N, N-dimethylformamide (the same applies hereinafter).

1) Reaction: room temperature 30 minutes Solvent: DMF, 2) Washing: room temperature 10 minutes Solvent: DMF, 3) Fmoc removal: room temperature 10 minutes Solvent: DMF, 20 (v
/ V)% piperidine-containing, 4) Washing: room temperature 10 minutes, solvent; DMF. 1) to 4) were sequentially repeated, and 31 amino acids were sequentially added. The amino acid derivatives used are shown in Tables 1 and 2 below.

[0031]

[Table 1]

[0032]

[Table 2]

After adding 31 amino acid derivatives, the amino-terminal Fmoc was eliminated by the above-mentioned Fmoc elimination method. After the reaction was completed, the obtained solid phase resin to which the polypeptide was bound was washed with DMF to obtain the desired product.

(2) Synthesis of a polypeptide derivative represented by the following structural formula (Formula 8).

[0035]

Embedded image

The solid phase resin bound with the polypeptide obtained in (1) was suspended in 10 ml of DMF. HBT to this
U1.5g, N-hydroxybenzotriazole 0.6
g and N, N-diisopropylethylamine 1.4
After adding ml, the mixture was reacted at room temperature for 3 days under nitrogen. After the reaction was completed, the solid phase resin to which the polypeptide was bound was washed with DMF to obtain the desired product.

(3) Synthesis of objective calcitonin analog acetate. The solid phase resin to which the polypeptide obtained in (2) was bound was washed again with methanol, and then dried under vacuum at room temperature for 24 hours. This is trifluoroacetic acid (hereinafter referred to as TFA) 1
It was suspended in 0 ml for 1 hour. The suspension was filtered through a glass filter, and the obtained TFA solution was diluted with diethyl ether 70
ml was added, and the precipitated crude polypeptide was collected by filtration to obtain about 600 mg of the crude target product. The crude target product was dissolved in 10 ml of water and purified by reverse phase chromatography to obtain about 35 mg of the target product in the TFA salt form. This was dissolved in a 5 (v / v)% acetic acid aqueous solution, and 28 mg of an acetate-type polypeptide, which was the object of this example, was obtained using a strongly basic ion exchange resin.

Amino acid analysis value: Asx 2.96 (3), Thr 5.49
(7), Ser 1.16 (2), Glx 2.00 (2), Gly 3.94 (4), Met 0.
54 (1), Aminosuberic acid 0.99 (1), Leu 2.90 (3), Tyr
1.84 (2), Phe 0.97 (1), Lys 0.96 (1) His 0.99 (1), A
rg 0.95 (1), Pro 1.74 (2) (amino acid analysis: about 8 subjects
μg was hydrolyzed with 150 μl of 6N hydrochloric acid at 140 ° C. for 4 hours, and then dissolved in 100 μl of 0.02N hydrochloric acid solution, 40 μl of which was dissolved in an amino acid analyzer (Hitachi L-8500).
Was analyzed. ) Mass spectrum analysis: 3440.0 (calculated value 3440.
6)

Example 2 Synthesis of Acetate of Calcitonin Analogue Same as in Example 1 by Peptide Liquid Phase Synthesis Method

(1) Synthesis of a polypeptide derivative represented by the following structural formula (Formula 9).

[0041]

Embedded image

7.36 g of the polypeptide derivative represented by the following structural formula (Formula 10) was dissolved in 120 ml of DMF,
Next, 2.6 g of a polypeptide derivative represented by the following structural formula (Formula 11) and N-hydroxysuccinimide 0.38
g, 0.95 g of HBTU and 0.43 ml of N, N-diisopropylethylamine were added, and the mixture was stirred overnight at room temperature.

[0043]

Embedded image

[0044]

Embedded image

After distilling off the reaction solvent, pure water 1 was added to the residue.
00 ml was added and suspended. This was filtered with a glass filter and dried to obtain 9.92 g of the desired product. The retention ratio (R _f ) by thin layer chromatography (TLC) was 0.52 (TLC solvent: chloroform 80 ml, methanol 20 ml mixed solvent).

(2) Synthesis of target polypeptide acetate. 9.92 g of the polypeptide derivative represented by the following structural formula (Formula 12) obtained in (1) above was added to 100 ml of TFA, and the mixture was stirred at room temperature for 1 hour.

[0047]

Embedded image

Diethyl ether 700 was added to the reaction solution after 1 hour.
ml was added and the mixture was stirred for 30 minutes. The generated precipitate was collected by filtration with a glass filter and dried to obtain 7.35 g of a crude target product. This was dissolved in 10 ml of water and purified by reverse phase chromatography to obtain 200 mg of the target product in the TFA salt form. This was dissolved in 2 ml of 5 (v / v)% acetic acid solvent, and 180 mg of the target product of the acetate salt type was prepared using a strongly basic ion exchange resin.
I got

Amino acid analysis value: Asx 2.99 (3), Thr 5.56
(7), Ser 1.20 (2), Glx 2.00 (2), Gly 4.03 (4), Met 0.
55 (1), Aminosuberic acid 1.01 (1), Leu 2.96 (3), Tyr
1.87 (2), Phe 0.99 (1), Lys 0.99 (1) His 1.03 (1), Ar
g 0.99 (1), Pro 1.83 (2) (amino acid analysis: same amino acid analysis method as in Example 1 was used) Mass spectrum analysis: 3440.0 (calculated value: 344)
0.6)

[Example 3: Measurement of biological activity] The polypeptide obtained in Example 1 was dissolved in 10 mM acetate buffer (pH 4.2) containing 0.1% BSA (bovine serum albumin) to prepare T47D cells. CAMP using (human breast cancer cells)
The activity was determined by quantitative measurement (Amersham: cAMP measurement kit). The results are shown in Table 3. In the table, “calcitonin analog” represents the calcitonin analog acetate synthesized in Example 1, and “hybrid CT” represents the known hybrid calcitonin represented by the above chemical formula 3.

[0051]

[Table 3]

[0052]

INDUSTRIAL APPLICABILITY According to the present invention, a hybrid type calcitonin derivative which is stable in a solution, that is, while maintaining a blood calcium lowering activity comparable to that of salmon calcitonin,
It is possible to supply a calcitonin analogue having low side effects such as antigenicity and low body weight suppressing activity.

[Sequence list]

SEQ ID NO: 1 Sequence length: 31 Sequence type: Amino acid Sequence type: Synthetic peptide Sequence feature: 6-position Xaa; 2-aminosuberic acid. The amino group of 1-position Gly and the carboxyl group of 6-position Xaa cyclize with an amide bond. Carboxyl group of 31st Pro is amidated. Sequence: Gly Asn Leu Ser Thr Xaa Met Leu Gly Thr Tyr Thr Gln Asp Phe His 1 5 10 15 Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro 20 25 30

Claims (2)

[Claims] 1. A calcitonin analogue represented by the following structural formula (Chemical formula 1, wherein n is an integer of 3 to 7) or a pharmaceutically acceptable salt thereof. Embedded image 2. A calcitonin analog represented by the following structural formula (Formula 2) or a pharmaceutically acceptable salt thereof. Embedded image