JPH01104098A - Novel calcitonin derivative and salt thereof - Google Patents

Abstract

NEW MATERIAL:A compound (salt) wherein amino acid residue in 1-position of calcitonin is a cyclic amino acid residue expressed by the formula (X is S or methylene; n is 0 or 1) and amino acid residue in 7-position is a cysteine residue which may be substituted by a protecting group. USE:A remedy for osteoporosis, bone Behcet disease, hypercalcemia, etc. PREPARATION:For example, proline which is C end amino acid of calcitonin derivative is linked to benzhydrinamine resin and protected amino acid is successively linked from C end side according to amino acid sequence of the calcitonin derivative to provide a protected peptide resin, which is then treated with HF, etc., and elimination from peptide resin and removal of protecting group other than mercapto protecting group is carried out to provide the novel calcitonin.

Description

[Detailed description of the invention] [Object of the invention] (Industrial application field) The present invention has the effect of lowering serum Ca' concentration,
The present invention relates to a novel calcitonin derivative and its salt useful as a therapeutic agent for osseous diseases, Beget's disease of bone, hypercalcemia, etc.

(Prior Art and its Problems) Calcitonin (hereinafter referred to as rGTJ) is a peptide hormone that is present in the thyroid gland of various foramina such as humans, or in the carp body of fish, stomates, and birds. This hormone shows an antagonistic effect to the parathyroid hormone,
It is known that it acts on bones and the like to lower the Caz+ concentration in the blood. To date, CT has been performed on humans, cows, pigs, sheep, rats, chickens, salmon, and eels.
has been extracted and purified, and its amino acid sequence has been revealed. All of these animal-derived proteins are polypeptides consisting of 32 amino acids, and the cysteine residues at positions 1 and 7 form a disulfide bond, and the carboxyl terminal (hereinafter referred to as the "C-terminus") is a polypeptide consisting of 32 amino acids. They have in common that they are amides.

These CTs are expected to be used as therapeutic agents for phrenosis, Beget's disease of bone, or hypercalcemia. Because of the possibility of mimicking deterioration, its use as a medicine has been extremely limited.

As a means to solve this problem, cystine residue Ts in CT
Derivatives (hereinafter referred to as 1.7 rAsu- CTJ) was synthesized. This derivative has high CT activity and exhibits high stability in solution, so it was useful as a pharmaceutical.

1.7 However, since Asu-CT contains Asu, its synthesis is complicated, that is, it can only be synthesized by the liquid phase method in peptide chemistry, and the solid phase method, which is a simple and rapid synthesis method, cannot be applied. There is a problem.

Therefore, the present inventors conducted intensive research with the aim of obtaining a derivative that has high activity, high stability, and can be synthesized by solid-phase methods. The present inventors have discovered that the above object can be achieved by using a specific cyclic amino acid residue, and have completed the present invention.

The abbreviations and meanings of the abbreviations used in this specification are as follows.

1. Regarding amino acids Ala: Alanine, Arg: Arginine, Asn:
Asparagine, Asp: aspartic acid, Cys nigistein, Gln: glutamine, Glu: glutamonougly, Gly nigericin, His: histidine, Ile
: Isoleucine, Leu: Leucine, Lys: Lysine, Met: Methionine, Phenylalanine,
Pronibroline, Ser: serine, Dhr: threonine, Trp:)ributophane, Tyr: tyrosine,
Val: Valine, 91s: Cystine, Oct: 3-
Oxo-5-carboxyperhydro-1,4-thiazine, Kpc:2-keto-piperidine-localponugly, C
ys(SO3H) Nidistic acid, pGlu: Pyroglutamic acid Each may indicate the corresponding amino acid residue.

2. About protecting groups Boa: t-butyloxycarbonyl, Fmoc
: 9-fluorenylmethyloxycarbonyl, Buj
: t-butyl, Bzl: benzyl, Cl24zl: 2
, 8-dibenzyl, Z: benzyloxycarbonyl, CI-Z: 2-chlorobenzyloxycarbonyl, NP! 19: 3-nitropyridine sulfenyl, 0Bzl: benzyl ester, 0But: t-butyl ester, 0cHex dichlorohexyl ester,
Tos nidosyl, Br-Z: 2-bromobenzyloxycarbonyl, Mtr: 4-methoxy-2,3,8-
Trimethylbenzenesulfonyl, For: formyl, A
cII: acetamidomethyl, M-Bzl: 4-methoxybenzyl, 4CH3.Bzl: 4-methylbenzyl, Trt: ) lythyl, 5BuE: t-butylmercapto, CA: carbamoylmethyl, CM: carboxymethyl, AE niaminoethyl 3, reagent About OCC dicyclohexylcarbodiimide, HOBt
: 1-Hydroxybenzotriazole, DingT Nidithiothreitol, DCN Nidichloromethane, rJMF Nidimethylformamide, DMSO Nidimethylsulfoxide, MeOH: Methanol, TEA Nitriethylamine, TFA Nitrifluoroacetic acid, HF: Hydrogen fluoride, H
CI: hydrogen chloride or hydrochloric acid, CH3CN niacetonitrile, dirrs-HCI nidris (hydroxymethyl)
Aminomethane hydrochloride [Structure of the invention] (Means for solving the problems) The present invention provides that the amino acid residue at position 1 of calcitonin has the following formula (I): (wherein, X represents a sulfur atom or a methylene group) A cyclic amino acid residue represented by the following formula: n represents 0 or 1), wherein the amino acid residue at position 7 is a cysteine residue which may be substituted with an appropriate protecting group. This invention relates to derivatives and salts thereof.

Calcitonin refers to natural calcitonin consisting of 32 amino acid residues extracted and purified from organs of humans, cows, pigs, sheep, rats, chickens, salmon, eels, etc., and synthetic calcitonin (e.g., JP-A-81-474138). Publication No. 81-233899, U.S. Patent No. 4,8
No. 16,012, No. 4,497,732, i4,
No. 804,238, wS4,805,515,
Calcitonin described in Publications No. 4,805,514, No. 4,804,237, and No. 4.604,238)
and calcitonin derivatives).

The cyclic amino acid residue at position 1 is particularly Oct, Kpc,
, Cbl are preferred.

The cysteine residue, which is the amino acid residue at position 7, may be substituted with an appropriate protecting group.

The protecting group for cysteine residues may be any mercapto group that is commonly used in peptide chemistry.
Examples include z I group, Acm group, ↑ri group, or GA group, 0M group, and AE group introduced by reaction of a mercapto group with a modifier. In particular, Ac1m group and GA group are preferred.

Amino acid residues other than positions 1 and 7 are not limited as long as they can constitute calcitonin, and include, for example, the following.

2nd place: Ala, Set or cry 3rd place: Ser or ASn 4th place: Leu 5th place: 5er 6th place: Thr 8th place: Val or Net 8th place: Leu 10th place: Gl! or 5er 11th place: Lys, Thr or Ala 12th place: L
eu or Tyr 13th position: Ser, Thr or Trp 14th position: G
ln, L7g or Arg15th position: Glu, A
sp or Asn 16th position: Leu or Phe 17th position: His or Asn 18th position: Lys or Asn 18th position: Leu, Phe or Tyr 20th position: O
ln or old S 21st: Thr or Arg 22nd: Tyr or Phe 23rd: Pro or 5ar 24th: Arg, Gln or cry 25th: T
hr or Net 26th place: Asp, Asn, Ser, Gl
7 or Ala 27th position: Val, Thr, Il
e or Phe 28th position: cry 28th position: Ala, Ser, Val or Pr.

30th position: cry or Glu 31st position: Thr, Val or AIa Among these amino acid residues, combinations of amino acid residues constituting chicken calcitonin, eel calcitonin, and salmon calcitonin are particularly preferred.

Specific examples of the calcitonin derivatives of the present invention are as follows.

Oct-A I a-Ser-Leu-9e r-
Thr-Cys-Val-Leu-G17-
L,'s -Leu-5er-Gln-Glu-Leu
-11is-Lys-Leu-Gln-Thr-Tyr
-P r o-A rg-Th r-Asp-Va l
-G I y-A 1a-(71y-Th r-P
ro-NO3[(Oct', Cyg(GA)'
)-CCTIAca+ 0ct-AIa-9sr-Leu-9er-Thr-C
ys-Val-LeuJ12=Lys-Leu=Ser
-Gln-Glu-Leu-old 5-Lys-Leu-G
ln-Thr-Tyr-Pro-A tg-Thr
-Asp-Va 1J17-A 1a-G 17-Th
r-Pro-NO3[(Oct', Cys(A
ca+)' )-CC↑Icm 0c t-Ser r-Asn(eu-5e r-Th
r-Cys-Val-Leu-G17-L1
s -Leu-Ser-G In-G Iu-Leu
-H1s-Lys-Leu-G In-Th r-Ty
r-Pro ro-A rg-Th r-Asp-Va
1-Gly-A 1a-(i 1y-Th r-P
ro -NO3[(Oct', C2sC25(A'
)-ECTJl, OCT represents chicken calcitonin.

The calcitonin derivative of the present invention may be in the form of a mineral acid such as hydrochloric acid, sulfuric acid, phosphoric acid, or an organic acid such as acetic acid or citric acid; It may be in the form of a metal salt such as sodium, potassium, or calcium, or a salt with an aiM group such as ammonia, dicyclohexylamine, or pyridine.

The calciton-4 derivative of the present invention can be synthesized by a solid phase method or a liquid phase method commonly used for peptide synthesis. This synthesis can be explained, for example, by Haruaki Yajima and Shunpei Sakakibara, Complete Japanese Biochemistry, Biochemistry Experiment Course (I): "Chemistry of Proteins"
Volume 4, published by Tokyo Kagaku Dojin (1117?) A solid phase method is preferred as a method for synthesizing calcitonin derivatives.

Hereinafter, a case will be described in which the calcitonin derivative of the present invention is synthesized by a solid phase method.

First, the C-terminal amino acid of the desired calcitonin derivative, ie, Pro, is bound to an insoluble resin. Next, protected amino acids are sequentially bound from the C-terminal side according to the amino acid sequence of the derivative to obtain a protected peptide IM fat. The insoluble resin may be any of those known in the art, such as + (F''c defemizable colomethyl 4M resin, hydroxymethyl resin, benzhydrylamine resin CBOA resin), 4-(oxymethyl)phenoxymethyl resin, 4-(
Examples include aminomethyl) phenoxymethyl resin, and BIIA resin or 4-(aminomethyl)phenoxymethyl resin is particularly preferred since it directly provides an amide by cleavage between the resin and the peptide chain.

"Protected amino acid" is an amino acid whose functional group is protected with a protecting group by a known method.When synthesizing the calcitonin derivative of the present invention, various protected amino acids are used as follows. It is preferable to select one of the protecting groups shown below. First, the protecting group for the α-amino group of an amino acid is Boc or Fmoc. The hydroxyl protecting groups of Set and Thr are Bu' and Bzl. T7r
The protecting group for the hydroxyl group of is C12・Bzl, But or may not be protected.The protecting group for the ε-amino group of Lys is Z, CI-Z, Boa,
Np's.

Glu, the protecting group for the carboxyl group of Asp is OB
z I, 0But, '0cHai. Protecting groups for the guanidino group of Arg are Tos, NO2, and Mtr. The protecting group for the imidazolyl group of former S is Tas, F
It is moc. The protecting group for Net may be oxygen, but it is preferable not to protect it. The protecting group for the indolyl group of Trp may be For or it may not be protected. The protecting group for the mercapto group of Cpm is Bzl,
M-Bzl, 4CH3・Bzl, Ace, T
rt, Np's, But, and SBu'. Protecting groups for mercapto groups include CM, CA, and AM, which are introduced by the reaction of a mercapto group with a modifier as described below.
Although there are protecting groups such as, it is not preferable to use in the solid phase method, it is necessary to select an appropriate protecting group according to the synthesis conditions of the peptide.

Cyclic amino acids such as Oct, Kpc, etc. corresponding to the cyclic amino acid residue represented by formula (I) can be synthesized according to known methods. This synthesis is described in, for example, Japanese Patent Publication No. 41-20827; J.

(: hromatogr, 294 (1984),
413; Bull, Chera.

Sac, Japan, 3B (1983), 920
; It can be carried out according to the method described in JP-A-52-118485 and the like. In the case of Oct, it is synthesized, for example, according to the method shown below.

Cysteine or a salt thereof is suspended in water, and a carboxymethylating agent such as monoiodoacetamide or monobromoacetamide is added. Suitable crucibles such as sodium hydroxide, sodium phosphate, sodium carbonate, potassium carbonate,
Inorganic bases such as ammonium hydroxide, or TEA
, an organic base such as dicyclohexylamine is added, and the mixed solution is adjusted to pH 8 to 10, preferably pH 7 to 9. Reaction temperature is 20 to 60°C, preferably room temperature to 5G"C, and reaction time is 0.1 to 1. (between Mtr, preferably 0.5~
The reaction is carried out for 5 hours to carbamoylmethylate the mercapto group of cysteine. Subsequently, the reaction solution was placed in a sealed tube at a reaction temperature of 80 to 150°C, preferably 8o to 120°C.
Reaction time 0.5-50 hours, preferably 1-10:11
React with fl to obtain Oct. The obtained Oct is purified using methods such as recrystallization and various chromatography methods.

Attachment of protected amino acids can be accomplished by conventional condensation methods, e.g. DC
C method, active ester method, mixed or symmetrical acid anhydride method,
The condensation reaction can be carried out according to the carbonyldiimidazole method, the DCC-HOBt method, the diphenylphosphoryl azide method, etc., but the DCC method, the DCC-HOBt method, and the symmetrical acid anhydride method are preferred.
The reaction is carried out in an organic solvent such as NF, chloroform, [1NSO, benzene, etc.] or a mixed solvent thereof. DCM
, DMF, or a mixed solvent thereof, the protecting group removal reagent for the α-amine group is T
FA/DON, HCI/dioxane, piperidine 10
MF #- is used and is appropriately selected depending on the type of the protecting group. In addition, the degree of progress of the condensation reaction at each stage of the synthesis is determined by the method of E, Kaiser et al. [Anal, Bioche et al.
m, 34.595 (1970) 1).

In the manner described above, a protected peptide resin having a desired amino acid sequence is obtained, and specific examples thereof are shown below.

HA protected peptide resin is a reagent that removes the peptide from the resin and also removes protecting groups other than mercapto protecting groups.
For example, by treating with IF, TFA, etc. (M-terminal deprotection reaction), the desired calcitonin derivative is obtained.

2) In addition, when a protecting group that can be removed under the final deprotection conditions described above is used as the protecting group for the mercapto group of Cys at position 7, C
The mercapto group of ys gives a free peptide. This peptide itself is also included in the calcitonin derivative of the present invention, but by reacting this peptide with a modifier in a buffer to protect the mercapto group, the calcitonin derivative of the present invention in which Cps at position 7 is protected is obtained. can be obtained. Any modifier may be used as long as it can react with a mercapto group and introduce a protecting group.6 For example, monoiodoacetic acid, monobromoacetic acid, monoiodoacetic acid, ethyleneimine, acrylonitrile, Alkylating or 7-arylating agents such as vinylpyridine, 5.5
Examples include asymmetric disulfide forming agents such as °-dithiobis-(2-nitro)benzoic acid or 2,2'-dipyridyl disulfide, toethylmaleimide, 2-nitro-5-thiocia-7benzoic acid, and the like. Modification reactions are usually carried out at pH 2.
-11, preferably in a buffer with a pH of 8-8. The 11i solution used in this reaction is a known one,
For example, citric acid-sodium citrate, acetic acid-sodium acetate, phosphoric acid, imidazole-hydrochloric acid, Tris-
IC+, boric acid, jetanolamine-hydrochloric acid, glycine-sodium hydride, and the like. Conditions for the modification reaction vary depending on the type of modifier; for example, when monoiodoacetamide is used, it is usually p) I G ~ 10
Various buffer solutions are used. in this case.

Monoiodoacetamide usually has a mercapto group of 0.1
-10 times equivalent, preferably 1 to 2 times equivalent, reaction temperature is usually 0-SO℃, preferably 20 to 40゛0, and 1 to 15 hours is usually 0.1 to 10 hours, preferably It is 0.5 to 2 hours.

3) Furthermore, among the calcitonin derivatives of the present invention, a peptide in which the 7-min residue at position 1 is Oct may be synthesized by the method shown below, that is, desorption under the final deprotection conditions described above. Possible protecting groups, e.g. M-Bzl, 4C
A protected peptide resin is synthesized by using H3·Bzl-protected (ys in place of Oct. A specific example of this protected peptide resin is shown below.

+1-Gys-9er-A, 5n-Leu-Ser-T
hr-Cys-Val-Leu-Gly-Lys-Le
u-9er-Gln-Glu-Leu-old 5-L7s-
Leu-Gln-Pro-NH-BHA This protected peptide resin undergoes final deprotection according to the method described above to obtain a peptide with mm of mercapto groups. The mercapto group of this peptide is protected by carbamoylmethyl (OA) using a modifier such as monoiodoacetamide or monobromoacetamide. The desired peptide is obtained by heating the obtained CA-modified peptide in a buffer solution. The buffer solution used in such a reaction is a known one as mentioned above, and its pH is:
Usually 2 to 1O1, preferably 3 to 7. In addition, the concentration of the peptide is usually 0.1 to 100 ILH, preferably 0.5 to 10 IJ,M, and the reaction temperature is usually 30 to 100 ILH.
150℃, preferably 60~! 00°C, and the reaction time is usually 0.1 to 100 hours, preferably 0.5 to 50 hours.
It's time.

4) Furthermore, among the calcitonin derivatives of the present invention, Cys in which the amino acid residue at position 7 is protected with carbamoylmethyl
The peptide may be synthesized by the method shown below.

Natural calcitonin is treated with an appropriate reducing agent in a basic aqueous solution to reduce the disulfide bonds within one molecule and bring the mercapto group to mM.Next, the desired peptide is obtained in the same manner as in 3) above. . As the basic aqueous solution used in this reduction reaction, various buffer solutions having a pH of 7 to 13 are used. As such a buffer solution, the same one as mentioned above is used. As the reducing agent, all reducing agents commonly used in organic synthesis can be used. For example, thiols such as TT, thioglycolic acid, 2-mercaptoethanol, sodium borohydride, lithium aluminum hydride, zinc, etc. For example, when DTT is used, the amount used is usually 0.1 to 100 times, preferably 1 to 10 times the equivalent of the peptide, and the reaction temperature is 1 to 1.
The temperature is usually 1 to 80°C, preferably 20 to 40°C, and the reaction time is usually 1 to 24 hours, preferably 3 to 5 hours. In the modification reaction, a modifier such as monoiodoacetamide or monobromoacetamide is usually added in an amount of 0.1 to 100 times, preferably 1 to 2 times the amount of the reducing agent.

The peptide thus obtained can be prepared by conventional methods for peptides,
For example, isolation and purification can be carried out by extraction, recrystallization, various chromatography (gel filtration, ion exchange, distribution, adsorption, reversed phase), electrophoresis, self-flow distribution, etc. HPLC) is the most effective method.

(Examples of the Invention) Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the scope of the present invention in any way.

Example! 0ct-Ala-Ser-Leu-Ser-Dinghr-C
ys-Val-Leu-Gly-Lys-Leu -9
e r-Gin-G Iu-Leu-His-Lys-
Leu-Gln-Thr-Tyr-Pro
-A rg-Th r-Asp-Va 1-Gly-
A 1a-017-Thr-Pro-NII2(
TI, (Oat', Cys(OA)')-
(1) (1) Synthesis of 3-oxo-5-carboxyperhydro-1,4-thiazine L-cystine hydrochloride salt hydrate 1.75 g (10 mm
ol) in 50 ml of water and diluted with 1M sodium carbonate.
Adjusted to H8.0. Under a nitrogen stream, 1.85 g (10 mm+ol) of monoiodoacetamide and 1M thorium carbonate were added alternately while maintaining the pH at 8.0. After reacting at room temperature for 1 hour, the reaction was further carried out at 100° 0 for 2 hours in a sealed tube.

After adding 4N IC+ to the reaction solution and adjusting the pH to 3.25.

It was concentrated to dryness. The residue was extracted with hot ethanol, and after cooling,
The crystals were collected and recrystallized from hot ethanol. Yield 5θ
Qmg (34,8%), melting point 182-184℃, Fe
b Mass spectrometry [M-old+=! 62 (2) B) Introduction of proline into IA resin 81 (Av
Fat A (Peptide Research New Product, divinylbenzene 2%, 1
00-200 mesh, amino group equivalent 0.81 meq/
g) 2 g was placed in a reaction vessel for peptide solid-phase synthesis (manufactured by Vega), treated with 30 of each of the following solvents in sequence, and filtered after each treatment.

DCHC 3 times, 2 minutes each) Meal ((3 times, i minutes each) ・DCM (3 times, 2 minutes each) 10% TEA/DCN solution (5 minutes, 10 minutes each, once) Then, BHA#M fat B dissolved in DCM 15o+i
The mixture was stirred for 2 minutes with 0.32 g (1.5 ma+ou) of oc-Pro.

DCM 1 of DCG 0.31g (1.5ma+o view)
5 Add IIJL solution.

The reaction mixture stirred for 120 min was filtered and Boc-P
The ro-resin was washed and filtered with 30 tJl of each of the following solvents.

DCM (3 times, 2 minutes each) MeOH (3 times, 2 minutes each) DON (3 times, 2 minutes each) In addition, 1.34 g (12,211+moJL) of 1-7 cetyl imidazole was added to Baa-Pro-m fat. DON
3G+m solution was added and acetylation was carried out over 12 hours. As a result, unreacted amino groups in the BHA resin become f*I? I have been treated.

In the following explanation, the N-terminus of proline is the starting point for the reaction of the protected amino acid.

(3) The total amount of Boc-Pro-411 fat obtained in step (2) of introducing threonine at position 31 was DO
Washed 3 times with N, 3 times with MeOH, 3 times with DCM for 2 minutes each and filtered. A 50% DCM solution (solvent; DCM) was added to this resin at ra9. Add and stir for 5 minutes,
Filtered. Further, stirred for 30 minutes under the same TFA solution,
The Baa group was eliminated. The obtained resin was sequentially treated with 30+all of each of the following solvents in the same manner as in (2), and filtered after each treatment.

DCM (3 times, 2 minutes each) MeOll (3 times, 2 minutes each) DON (3 times, 2 minutes each) IO%TEA/DON solution (5 minutes, 10 minutes each)D
CM (3rrfJ, 2 min each) The Pro-resin was then treated with Boc-Thr (Bzl) 0.
Added 48g (1.5mmafL) of DCM 15IIft solution and stirred for 2 minutes. Next, added DCGO, 31g of DON 15mJl solution and stirred for 240 minutes.
After the reaction, it was washed 3 times with RoCN, 3 times with HeOH, and 3 times with rJCM for 2 minutes each, and filtered.

The amount of the resin was collected and the ninhydrin test was confirmed to be negative. Then, a part of the resin was collected,
When the amount of C-terminal amino acid bond was measured, it was 0.1
It was 511,110 sentences and 7g (4) 30-1st place each 7
Similar to the introduction of Minogon (3), Boc-Thr (Bzl)-
Protected amino acids corresponding to each constituent amino acid from position 30 to position 1 of CT shown by formula (II) were sequentially coupled to Pro-vA fat. and its usage amount.

As described in (3), the coupling reaction was performed twice with the same amount of protected amino acid, and the coupling reaction time was 120 minutes for the first time and 300 minutes for the second time. Here, L
ys is Boc-L7s (CI
・Used as Z). Boa-Leu-H2O and Oct are poorly soluble in 00M single solvent, so Hoe-Le
U-H2O was dissolved in a mixed solvent of F and DCN (1:4), and Oct was dissolved in DMF. Furthermore,
DMF was used instead of DCM as a washing solvent during Oct coupling. 14th and 20th place (7) Gllll
In the introduction of HO8t0.23g and Boc-G
lr+0.37g was simultaneously added to the reaction vessel.

After introducing the amino acid at position 1, the Boa group removal operation was performed in the same manner as in (2). The resin peptide was dried under reduced pressure for a day and night to obtain a dried lipophilic peptide.

(5) Decomposition with HF Weigh a portion (200 mg) of the dried resin PETIDE, place it in a reaction vessel for IF decomposition (made of Teflon), add 1.0+++jL of anisole, and leave it overnight to swell the resin. Ta. The reaction vessel containing the PIl stirrer was attached to an HF decomposition device (manufactured by Peptide Kenkyushin) and placed in a dry ice-ethanol bath, and 10 mJl of HF was introduced into the reaction vessel. The slurry was stirred in a water bath for 1 hour at <O'O. HF was gradually distilled off under reduced pressure. After 3 hours, the reaction vessel was removed, and the resin peptide decomposition product was taken out from the reaction vessel using diethyl ether and washed with diethyl ether. The resin peptide decomposition product was added to 20 mJL of 2M acetic acid to dissolve the deprotected peptide.

(6) Carbamoyl methylation of mercapto group Use this solution! Vtf was adjusted to 5 and pH was adjusted to 8.0 with aqueous ammonia (
730.8 mg of DT was added to the mixture, and the mixture was stirred at 37°C for 4 hours. In this reduction operation, the dimer that may be generated by oxidation of the mercapto group returns to the monomer. Next, 37.0 mg of monoiodoacetic acid was added, and the reaction was carried out at 37° C. for 30 minutes in the dark.

This reaction solution was adsorbed on a column packed with octadecylsilyl silica (ODS column, φ2X 30sm), and after washing with water, the peptide was eluted with a 60% acetonitrile solution. The eluate was lyophilized and crude (Oct', C
ys(GA)')-CO728, 8 mg was obtained.

(7) Crude (Oct', (ys(GA)')-O
Purification of CT Obtained crude (Oct', 'Gys(C
A) F) -COT was dissolved in 1M acetic acid (5mg/all
) L, purified by concentration gradient high performance liquid chromatography. The column is Chemco Pack 0DS-H (manufactured by Kemco).
φ10mwX 250m■), the eluent was water (too)-to%TFA (1) as liquid A and water (too) as liquid B.
40)-Acetonitrile (80)-10% TFA (1)
Elution was carried out using a linear concentration gradient from solution A to solution B. Here, the number in parentheses is the volume ratio of the solvent. (Oc.
Both fractions corresponding to t', 02s(CA)')-OCT were separated and freeze-dried to obtain 2.8 mg of white powder. Biological activity of white powder produced by IJ4
When measured according to the method described in Publication No. 3500, the biological activity of natural COT was 4500 MRCU/mg, whereas it was 54QOMRCU/rrrg. The amino acid analysis values are shown in Table 2.

Table 2 (Oct", C:ys(OA)')-
COT's Amino Acid Composition Book Leu was calculated by converting it to 5.00.

11 ^sn and Asp Gln and Glu 2 Kyomoto Q(: and Cys (CA) is hydrolyzed and detected as Cys (CM).

The obtained white powder was analyzed by high performance liquid chromatography.

Measurement conditions ■: Column: Chemco Pack 0-mouth 5-H (φ4.8
s+m
100:0:1) Solution B (water niacetonitrile: 10% TFA = 40:8
Linear concentration gradient elution from solution A to solution B (30 minutes) using 0:1) Measurement wavelength: 210 nm As a result, a single strong absorption based on the peptide was observed at a retention time of 24.7 minutes, which is the cause of the present invention. (Oct'.

It was Cys(OA)')-COT.

Next, 15 µg of crystal was dissolved in 5 ounces of a 1% ammonium bicarbonate aqueous solution (P) containing trypsin sPg, and trypsin digestion was performed at 37°C for 2 hours.

When this digest was analyzed under the same conditions as measurement conditions ① above, retention times were 11.2 minutes, 1.9 minutes, 13.0 minutes, and 18 minutes.
．． A peptide-based peak was detected at 8 minutes. The results of Feb mass spectrometry of these peptides are shown in Table 3, resulting in (Oat', Cyg(OA)')-OCT
As expected, it was cleaved at the G-terminus of Lys and Arg to give four peptide fragments, each of which had a mass number ([8481]) consistent with the theoretical value.

Table 3 (Oct', Cys(CA)?)-C
Fabjij amount analysis of trypsin-digested products of OT (in the table,
T, , T2,? , ,T, are each (Oct
When the tryptic digest of Cys(CA)')-CCT was subjected to reverse-phase HPLC under the conditions of ) In addition, a stability test in Motoichi's solution was conducted as follows.

(Oct', Gys(CA)')-COT was dissolved in 0.1 M sodium citrate buffer (pHEl, o) to give a concentration of IQpLg/1afL. 1 mjl of this solution was left in a sealed tube for 0.24 hours at 80 mm.It was analyzed by reverse phase HPLC using measurement conditions:
ct', Gys(CA)')-OCT elution peak area A was determined. Similarly, heat treated tea A-
(7) (Oct',' CysC
The elution peak area B of CA)')-COT was determined, and the ratio (%) of A to B was used as an index of stability. Similarly, stability tests were conducted on 0CT1.7 and Asu CCT, and the results are summarized in Table 4.

Table 4 (Octl, Cys(GA)')-OC
T, AL crystal 7COT, CCT stability test A ■ Oct-A 1a-Ser-Leu-9s r-T
h r-(4s'-Va l -Leu-G I 7-
Lys-Leu-9er-Gln-Glu-Leu-old 5-Lys-Leu-Gln-Thr-T2r-P r
o-A rg-Th r-Asp-Val-Gl
7-A 1a-G l 2-Th r-P ro-N)
Synthesis of 12(II) (2) L7s-Leu-Ser-Gln-Glu-Lsu-old 5-Lys-Lau-Gln-Thr-T7 r-Pr
o-Arg-Thr-Asp-Val-CI! −
200 ILg of A 1a-C1y-Thr-Pro -H2 was weighed, and 0.2M Trix-11
CJI Il buffer solution (CpH 82, containing 0.2% EDTA) was dissolved in 150 IL. In this solution, DTT 1
100 l of the same buffer in which 851 L g of monoiodoacetamide was dissolved was added under a nitrogen stream and reacted for 5 hours at 37°0.
25jLl of NaOH was added, and the mixture was allowed to react for 30 minutes at 37°C in the dark. Add IN AcOH and adjust pH to 2~
Adjusted to 3.

This mixture was purified using reverse phase npt,c, and the main peak was fractionated and lyophilized to obtain a peptide having the following structure.

)1-Cys-Ala-Ser-Leu-Ser-Th
r-Cys-Val-Leu-Gly-Lys-Leu
-Ser-Gln-Glu-Leu-Old 5-Lys-L
eu-Gln-Thr-1Tyr-Pr o-Arg
-Th r-Asp-Va 1-Gl 2-A 1a-
Gly-Thr-Pro-N)+2 Reverse phase 1 (PLC conditions are as follows.

Column: Chemco Pack 0-mouth 5-H (φ4.8
m+*X 150+wm) flow rate; 1. Eluent: Linear concentration gradient elution from solution A to solution B (30 minutes) using solution A (water niacetonitrile = 100:0) and solution B (water niacetonitrile - 80:4G). Measurement wavelength; 21Qn intestine This peptide was added to 0.2M sodium citrate buffer (p
H8,0) 10 ILg/li concentration),
80" 0.0 Reaction solution left in a sealed tube for 24 hours was subjected to reverse phase H
It was purified using PLC (conditions are the same as measurement conditions ①), the main peak was fractionated, and lyophilized to obtain 78.8 gg of white powder. The results of reverse-phase HPLC assay, amino acid analysis, and Fab purchasing amount analysis of the trypsin-digested product of this white powder are shown in Example 1 (Oct", Cys(CA)'
) - consistent with CCT. In addition, the results of amino acid analysis and Fab mass spectrometry of the carboxypeptidase Y-digested product of the N-terminal fragment peptide obtained by tryptic digestion of the present peptide are also shown in Example 1 (Oct”
, Cys(CA))-OCT, and this peptide corresponds to (Octl, Cys(CA)′)-OCT.
It turned out to be. The biological activity of the wood peptide was measured according to the method shown in Example 1 and was found to be equivalent to that obtained in Example 1.

Example 3 Ac+n Oct-Al a-9e r-Leu-9e r-
Thr-Cys-Va1-Leu-GI! −
Lys -Leu-SerJln-Glu-Leu-
)1is-Lys-LeuJIn-Thr-77r-P
ro-Arg-Thr-Asp-Val-Gly-Al
a-Gly-Dinghr-Pro-NH2[(Oct',
Cys(Act)')-CCD]cy) In the introduction of the amino acid at the 7th position of the synthesis, Bac-Cys(4GH3
・Boa-07s (Act) 0.
Solid-phase synthesis was performed under the same conditions as in Example 1 except that 44 g (1.5 mmol) was used to obtain a protected peptide resin.

A portion (200 mg) of the protected peptide resin was HF-digested (as in Example 1). The obtained acetic acid solution of the peptide was directly freeze-dried to obtain crude (Oct', Cpg(
^c+x) ' )-OCT 31. Obtained an OB. This peptide was purified in the same manner as in Examples 1 to (8), and (
Oct', Cys(Acm)')-OCT
The biological activity of the obtained 3.7 mg peptide was measured in the same manner as in Example 1, and it was found that the biological activity of natural CTT (7) was 4500 MRCU/sg.
It was 500 MRCU/wg.

Example 4 Acm Oct-Ser-Agn-Leu-9er-↑hr
-Cys-Val-Leu-G17-Lys-Leu
-9e r-Gln-G Iu-Leu-His-Ly
s-Leu-G In-Th r-T7 r -P r
o-A rg-Th r-Asp-Va 1-Gly
-A 1a-G Iy-Th r-Pro -NO3
[(Oct', C7sC75(A')-ECTb
7) In the introduction of amino acids at synthetic positions 2, 3, and 7, Hoe-Ser (
Bzl) 0.44 g, Boc-Asn 0.35 g and HOBt O, instead of Boc-5er (Bzl),
23g of Baa-Cys(40H3-Bzl) f)
Solid-phase synthesis was performed under the same conditions as in Example 1, except that 0.44 g of Bac-Cys (Acm) was used instead, to obtain a protected peptide resin. A part of the protected peptide resin was subjected to HF decomposition in the same manner as in Example 1, and the obtained crude peptide was purified in the same manner as in Example 1-(7).
ct", Cys(Act)')-ECT obtained, the biological activity of the peptide obtained was measured in the same manner as in Example 1, and the biological activity of the natural ECT was 4500 MR.
5400 MRCU/g compared to CU/mg
Met.

Example 5 Synthesis was carried out in the same manner as in Example 1, except that after the HF decomposition treatment, the operation to protect the mercapto group, that is, the monoiodoacetamide treatment was not performed.
' , C7s' )-COT The stability and biological activity of the obtained peptide were measured in the same manner as in Example 1.
) - (approximately equivalent to GET).

Example 6 Introducing the amino acid at position 1, Kp was used instead of Oct.
Synthesis was carried out in the same manner as in Example 1 except that 0.21 g (1.5 mmol) of c was used, and (Kpc'',
Cys(CA)')-1ll: When the stability and biological activity of the 0 peptide obtained from CT were measured, both were found to be (Oat', Cys(CA)')-0 of Example 1.
It was almost equivalent to 0 guns.

Example 7 In the introduction of amino acid at position 1, pG was used instead of Oct.
Except using lu O, 19 g (1,5 mmal),
Synthesis was carried out in the same manner as in Example 1, and (pG1u'
.

When the stability and biological activity of the 0 peptide for which Cys(GA)'>-CCT was measured, both were almost equivalent to (Oatl, Cys(OA)')-OCT of Example 1.

The methods for producing the compounds described so far or in each example can be modified in many respects. For example, as for the protected amino acids to be used, many amino acids with different protecting groups are commercially available. These commercially available protected amino acids can be used with appropriate modifications, and all of the resulting peptides are equivalent to the calcitonin derivatives of the present invention.

Comparative Example Calcitonin derivative of the present invention and A″1u7CCT and C
A comparison of activity with CT is shown in the table below.

1) Y, 5ako, M, 5hibata et al.
, PeptideChea+1stry In2.
189(19135)2) M, Hon+ma,
N., Watanabe et al., J.

Biochem,, 100.4513 (198f1
) [Effects of the Invention] According to the present invention, calcitonin derivatives having high activity and high stability can be stably supplied at low cost. Further, the calcitonin derivative of the present invention can be used as an experimental reagent, and furthermore, can be used as a diagnostic agent by establishing an assay system using it, and can also be used as a medicine or veterinary drug based on its biological activity.

Procedural amendment February 16, 1988 Kunio Ogawa, Commissioner of the Patent Office 1. Indication of the case 1988 Patent Application No. 252592 2. Name of the invention Novel calcitonin derivatives and salts thereof 3. Person making the amendment Case and Intermediate Patent applicant name (6051 Mitsubishi Yuka Co., Ltd. 4, Agent 5, Date of amendment order Voluntary 6, Subject of amendment Detailed explanation of the invention in the specification Procedural amendment document August 1983 8 Director General of the Japan Patent Office Yoshi 1) Takeshi Moon 1, Indication of the case, Patent Application No. 252592 of 1988, 2, Title of the invention, Novel calcitonin derivatives and salts thereof 3, Person making the amendment, Relationship to the case, Name of patent applicant (805) ) Mitsubishi Yuka Co., Ltd. 4, Agent 5, Date of amendment order Voluntary action 6, Subject of amendment Column 7 for detailed explanation of the invention in the specification, Contents of amendment (1) r on page 4, line 14 of the specification cylinder C313(SO,H
l Nidistic acid," is deleted.

(2) Insert "rNO□: nitro group" after "roxycarbonyl" on page 5, line 7 of the specification cylinder.

(3) “U.S. Patent No. 4.616.
No. 012'' is amended to read ``U.S. Patent No. 4.622.387''.

(4) "rTri group" at the bottom of page 7 or line 65 of the specification
Trt group”.

(5) "C-end" in the bottom line of page 11 of the specification and Table 1 on page 27 is corrected to "C-terminus."

(6) "Carboxymethyl" in lines 9-10 on page 14 of the specification is corrected to "rcarbamoylmethyl".

(7) On page 16 of the specification, line 10 from the bottom, "the protected peptide resin" is amended to "of this protected peptide resin."

(8) r on page 19 of the specification, line 8 from the bottom (CAI J
Delete.

(9) In the third line of page 20 of the specification, amend "r of the present invention" to "as described in 3)."

(10) Delete "as stated in (3)" on page 16, line 14 of the specification.

(11) r in the remarks column corresponding to columns 20 and 14 of the amino acid position in Table 1 on page 27 and on page 26 of the specification tube, line 4.
Correct HOBtJ to r)IOBtJ.

(12) Delete "... was performed for the amino acid at position 1" on page 26 of the specification, lines 6-7.

(13) Correct "Liquid B (water niacetonitrile = 60:40) J" on page 37, line 1 of the specification cylinder to "Liquid B (water niacetonitrile = 40:60) J.

(14) "Example 1" on page 38 of the specification, lines 6-5 from the bottom
- (6) Correct J to "Example 1-17) J.

(15) rC in the peptide name column of the table on page 42 of the specification
Correct TTJ with r CCTJ.

Claims (1)

[Claims] The amino acid residue at position 1 of calcitonin has the following formula: ▲ Numerical formula, chemical formula, table, etc. ▼ (I) (In the formula, X represents a sulfur atom or a methylene group; n is 0
Or represents 1. ) Calcitonin derivatives and salts thereof, characterized in that the amino acid residue at position 7 is a cysteine residue which may be substituted with an appropriate protecting group.