CALCITONIN GENE RELATED PEPTIDE DERIVATIVES

Abstract: A compound of the formula (I) wherein R is H or H-Ala-NH- and: A is Asp, B is Asp or Glu, C is Leu and D is Val; A is Asn, B is Gly, C is Phe and D is Gly; A is Asn, B is Asp or Glu, C is Leu and D is Gly; or A is Asn, B is Asp or Glu, C is Phe and D is Val; or a pharmaceutically acceptable salt thereof.

Description: This invention relates to novel human calcitonin gene related peptide (hereinafter designated as h-CGRP) derivatives, which are useful for the treatment of calcium metabolic disorders, cardiac disease and ulcers, and for the improvement of cerebral circulation. h-CGRP has an amino acid sequence of the formula wherein A³ is Asp or Asn, A²² is Val or Met, and A²⁵ is Asn or Ser, and is known to have a various biological proterties [Nature, 308( 19): 746-748 (1984), FEBS Letters, 183 (2): 403 (1985), Neuropeptides, 4 : 425-434 (1984) and Nature, 313 (3): 5456 (1984) ]. It is important in the medical field to synthesize h-CGRP derivatives having superior properties to that of h- CGRP, especially calcium and phosphate reducing activities. We have synthesized various derivatives of h-CGRP and have made comparative studies of their biological activities. As a result, we have found that they have stronger serum calcium reducing activity and serum phosphate reducing activity with a superior prolonged action, as compared with the known h-CGRP. The present invention provides a h-CGRP derivative of the formula (I): wherein R is H or H-Ala-NH- and: A is Asp, B is Asp or Glu, C is Leu and D is Val; A is Asn, B is Gly, C is Phe and D is Gly; A is Asn, B is Asp or Glu, C is Leu and D is Gly; or A is Asn, B is Asp or Glu, C is Phe and D is Val; or a pharmaceutically acceptable salt thereof. The present invention also provides a process for the preparation of a compound of formula (1) or a physiologically acceptable salt thereof, which process comprises (i) synthesising a peptide which has a chain structure of the formula wherein R, A, B, C and D are as defined above, Q₁ is hydrogen or a mercapto-protecting group and Q₂ is optional but, if present, is -NH₂ or together with the carboxyl group from Phe represents a protected amide group, and in which side-chain functional groups are optionally protected; (ii) when Q₁ is a mercapto-protecting group and/or when the side-chain mercapto group of Cys is protected, removing the or each said protecting group; (iii) oxidising the free mercapto groups to form a disulfide bridge; (iv) removing any side-chain functional group-protecting groups optionally present and, when Q₂ is not -NH₂, converting the terminal carboxy group of Phe into an amide group; and (v) if desired, converting the resulting compound of formula (1) into a physiologically acceptable salt thereof. The present invention further provides a pharmaceutical composition comprising a compound of formula (I) or a physiologically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent. A process by liquid phase synthesis: When R is H, a peptide of formula (1) synthesized by converting a carboxyl in phenylalanyl at the C-terminal to an amide, condensing successively a protected amino acid and/or a protected lower peptide in the order of amino acid sequence as shown in formula (1), when R is H, removing the protective group for L-cysteinyl and mercapto in the β-mercaptopropionyl group and the protective group for the functional group in the other side chain by acid hydrolysis, and when R is H-Ala-NH-, removing the protective group for mercapto in the L-cysteinyl and the protective group for the functional group in the other side chain by acid hydrolysis, and oxidizing L-cysteinyl ( or β-mercaptopropionylgroup ) and mercapto in L-cysteinyl to form a disulfide bridge at a final stage of the condensation reaction. The condensation reaction can be effected by repeating the removal and addition of a protective group and the condensation reaction according to the conventional process for peptide synthesis. Protective groups for a starting material and intermediates used in the process for the production of a peptide of formula (1) of the present invention are known in peptide chemistry, for example protective groups which can be easily removed by a known procedure such as hydrolysis, acid decomposition, reduction, aminolysis or hydrazinolysis. The protective groups are well known in the literature of peptide chemistry. Examples of preferred protective groups are butyloxy carbonyl, benzyloxy carbonyl or p-methoxybenzyloxy carbonyl for an α-amino group, benzyloxy carbonyl or p-chlorobenzyloxycarbonyl for a side chain amino group such as the ε-amino group in lysine, a methyl ester or benzyl ester for an α-carboxyl group, benzyl ester for a side chain carboxyl group such as the side chain carboxyl in aspartic acid or glutamic acid, benzyl for a hydroxyl group in serine or threonine, mesitylene-2-sulfonyl or tosyl for the amino group of guanidino in arginine and p-methoxylbenzyl or acetamidomethyl for the mercapto group of cysteine or β-mercaptopropionic acid. In the synthesis of a peptide of formula (1) of the present invention, the condensation of each amino acid and/or lower peptide can be effected by reacting an amino acid or a lower peptide having a protected α-amino and activated terminal α-carboxyl, with an amino acid or a lower peptide having free α-amino and a protected terminal carboxyl group, or alternatively by reacting an amino acid or a lower peptide having an activated α-amino and protected terminal carboxyl with an amino acid or a lower peptide having a protected α-amino and free terminal carboxyl group. A carboxyl group can be activated by conversion, for example, to acid azide, acid anhydride, acid imidazolide or an activated ester such as cyanomethyl ester, p-nitrophenyl ester or N-hydroxysuccinimide ester. Furthermore, it can be activated by using a condensation reagent, for example a carbodiimide such as N,N'-dicyclohexyl-carbodiimide (DCC), N-ethyl-N'-3-dimethylaminopropyl-carbodiimide or N,N'-carbonyl -diimidazole. Examples of preferred condensation reactions used in the present invention are the azide method, the activated ester method, the mixed anhydride method and the carbodiimide method. In the condensation reaction, it is preferred to avoid or at least to minimize the racemization reaction; preferred processes are the azide method, the activated ester method, the Wunsch method [Z. Naturforsch., 21b, 426 (1966) ] or the Geiger method [Chem. Ber., 103, 788 (1970) ] . A peptide of formula (1) can be synthesized by any procedure : however, it is preferable to construct an amino acid sequence by connecting an amino acid and/or lower peptide in order from the C-terminal. A peptide of formula (1) wherein R is H can be obtained by removing the protective groups in the protected peptide chain, namely β-mercaptopropionyl pentatriaconta-peptideamide having protected ε-amino, side chain carboxyl, hydroxyl, guanidino and mercapto groups. These protective groups are preferably removed by one step removal with acid hydrolysis using, for example, trifluoro-methane sulfonic acid or anhydrous hydrogen fluoride to obtain β-mercaptopropionyl pentatriaconta-peptideamide having free mercapto group. A peptide of formula (1) wherein R is H-Ala-NH- can be obtained by removing the protective groups in the protected peptide chain, namely heptatriaconta-peptideamide having protected ε-amino, side chain carboxyl, hydroxyl, guanidino and mercapto groups. These protective groups are preferably removed by the same way as above to obtain heptatriaconta-peptideamide having a free mercapto group. In the said peptide-amide, an inner molecular disulfide linkage is formed by oxidation to obtain the peptide of formula (1). Disulfide linkage can be effected generally by oxidation with oxygen in water, diiodo-ethane in an organic solvent, iodine in glacial acetic acid or potassium ferricyanide in water. A process by solid phase synthesis: In the present invention, a solid phase peptide synthesis method can be used in part or in total as a synthesis of a peptide of formula (1). For example, a peptide fragment (3-37) [hereinafter a peptide of an amino acid sequence constituted of amino acids Nos. 3 to 37 is referred to as peptide fragment (3-37) or peptide (3-37 ) ] is synthesized by a solid phase method, and the α-amino group in the said peptide is acylated with β-mercaptopropionic acid to obtain a protected pentatriaconta-peptide-bound resin or a protected heptatriaconta-peptide-bound resin. These protective groups and the resin are removed by a known method such as using trifluoromethane sulfonic acid or anhydrous hydrogen fluoride in a one-step removal to obtain a β-mercaptopropionyl pentatriaconta-peptideamide having a free mercapto group or a heptatriaconta-peptideamide having a free mercapto group. A peptide of formula (1) can be obtained by constructing an inner molecule disulfide bond as set forth in the process by liquid phase synthesis hereinabove. Examples of a resin used in the solid phase method are conventional resins such as benzhydrylamine resin or p-methyl-benzhydrylamine resin. A resin with the desired functional equivalent or cross-linkage can be synthesized, but is also commercially available. In the solid phase method, an amino acid is condensed in the resin, in the order of the amino acid sequence of formula (1), from the C-terminal amino acid to the third amino acid (amino acid No. 3) or from the C-terminal amino acid to the 1st amino acid (amino acid No.1), and in the case of a peptide wherein R is H, it is acylated with β-mercaptopropionic acid at the condensation to the third amino acid. A functional group in the amino acid is protected by a known method. Examples of protective groups are set out hereinbefore. In the solid phase reaction, a resin in a reaction vessel is swelled by adding dichloromethane, chloroform, dimethylformamide, benzene or a solvent for swelling the resin, in a ratio of 2-20 ml solvent per 1 g resin. In another reaction vessel, 1-6 equivalents of t-butyloxycarbonyl (hereinafter designated Boc-) amino acid per 1 equivalent of the amino group in the resin are first reacted with DCC, and the obtained acid anhydride, which is separated from a by-product dicyclohexylurea (hereinafter designatd DCU), is added to the resin hereinabove. The amount of condensation agent (DCC) is 0.5-3 equivalents per 1 equivalent of Boc-amino acid. The reaction generally proceeds for 5-60 minutes. The coupling amount of amino acid or peptide can be determined according to a conventional method [T. Fairwell et al., Biochemistry, 22 : 2691 (1983) ] by checking the amount of Boc-amino acid upon sampling the Boc-amino acid-resin or Boc-peptide-resin obtained in each process. The protective group for the α-amino group, Boc, is removed by an acid such as trifluoroacetic acid and the condensation reaction is performed. An automatic solid phase synthesizer can be used; however, a manual procedure can also be used. The entire operation is preferably performed under a nitrogen gas atmosphere. In this way, a peptide fragment (3-37) or (1-37) bound to a resin can be obtained. A peptide fragment (3-37) bound to a resin is acylated with β-mercaptopropionic acid in the final step to obtain a β-mercaptopropionyl-protected pentatriaconta peptide-amide-bound resin. The thus-obtained protected pentatriaconta peptideamide-bound resin or protected heptatriaconta peptide amide-bound resin is, as previously described, treated with anhydrous hydrogen fluoride to remove the protective group and resin in one-step, whereby β-mercaptopropionyl pentatriaconta peptide-amide having a free mercapto group or heptatriaconta peptide-amide having a free mercapto group can be obtained. A peptide of formula (1) can be obtained by forming intra molecular disulfide bonds in the above peptide-amide. Isolation and purification: The thus-obtained peptide of formula (1) can be purified by purification methods which are well known in peptide or protein chemistry. For example, a gel-filtration method can be carried out using Sephadex G-25, Sephadex G-50 or Sephadex LH-20 (trade names), or ion-exchange chromatography using carboxymethyl cellulose, or another resin, or HPLC can be used. Examples of a peptide of formula (1) of the present invention are: A peptide of formula (1) of the present invention can be obtained according to the process in the form of a free base or a salt. For example a salt with a known organic acid such as acetic acid, citric acid, malic acid or tartaric acid can be prepared. The abbreviations used in this specification are as follows: Ala: L-alanine Asp: L-aspartic acid Glu: L-glutamic acid Asn: L-asparagine Thr: L-threonine Cys: L-cysteine Val: L-valine His: L-histidine Arg: L-arginine Leu: L-leucine Gly: glycine Phe: L-phenylalanine Ser: L-serine Lys: L-lysine Pro: L-proline Met: L-methionine Boc: t-butyloxycarbonyl Cl-Z: p-chlorobenzyloxycarbonyl Bzl: benzyl MBzl: p-methoxybenzyl Tos: tosyl OBzl: benzyl ester TFA: trifluoroacetic acid DMF: N,N'-dimetnylformamide DCM: dichloromethane DCC: N,N'-dicyclohexylcarbodiimide DIEA: diisopropylethylamine HOBt: 1-hydlroxybenzotriazole MBHA-resin: p-methylbenzhydrylamine resin Serum calcium and serum phosphate reducing activities: Assay method: The peptide of formula (1) of the present invention and known h-CGRP (each 80 µg) dissolved in citrate buffer, pH 6.5, containing 0.1% bovine serum albumin (hereinafter designated as dissolving medium) (1 ml) were administered intravenously into the tail vein of Wistar rats, body weight 80-90 g, 5-6 rats in one group, at 80 µg /kg. After 30 and 60 mins. of administration, blood samples were collected from the abdominal descending aorta. The serum calcium concentration was measusred by atomic adsorption spectrophotometry. Serum phosphate was measured by a method according to Goldenberg et al. [Clin. Chem., 12: 872-882 (1966) ] . Figure 1 is a graph showing the rat serum calcium reducing effect of h-CGRP and peptides of formula (1) of the present invention; and Figure 2 is a graph showing the rat serum inorganic phosphate reducing effect of h-CGRP and peptides of formula (1) of the present invention. As shown in Figs. 1 and 2, the concentrations of serum calcium and phosphate were reduced by more than 20-30% in desalanyl-deamino- [Asp¹⁴ ] h-CGRP(2-37), 80 µg /kg, administered group [ -  - ] , [Asp¹⁴ ] h-CGRP(1-37) [ - □ - ] , [Asn³,Phe¹⁵,Gly²³] h-CGRP(1-37) [ - ▪ - ] , [Asn³,Asp¹⁴, Gly²³ ] h-CGRP(1-37) [ - ▵ - ] , and [Asn³,Asp¹⁴,Phe¹⁵ ] h-CGRP(1-37) [ - ▴ - ] as compared with the control group (dissolving medium administered group) [ - × - ] and these activities were observed to continue after 2 hours. These activities were stronger than those of h-CGRP [ - ○ - ] and the effects were prolonged significantly. As illustrated hereinabove, the peptide of formula (1) of the present invention has stronger serum calcium reducing activity and serum phosphate reducing activity as compared with known h-CGRP. Furthermore, it has a longer-lasting activity than h-CGRP. It is useful for the treatment of calcium metabolic disorders, cardiac disease and ulcers, or for the improvement of cerebral circulation. The following Examples further illustrate the present invention. Example 1 Production of [Asn³,Phe¹⁵,Gly²³] h-CGRP(1-37): Anisole (2 ml), dimethylsulfide (2 ml) and ethanedithiol (0.4 ml) were added to [Asn³,Phe¹⁵,Gly²³] h-CGRP(1-37)-MBHA-resin of the formula (1.09 g). Anhydrous hydrogen fluoride (20 ml) was added thereto and the mixture was stirred at 0°C for 1 hour. After the anhydrous hydrogen fluoride was distilled off in vacuo, the residue was washed with ether and 20% acetic acid (30 ml) was added thereto to extract the peptide. The extract was passed through a Dowex WGR (trademark) column (2.5× 15 cm) and eluted with 1 M acetic acid (150 ml). The eluate was freeze dried to obtain a white powder (400 mg). This powder (200 mg) was dissolved in 50 mM Na₂HPO₄ buffer (pH 7.5, 15 ml) containining 8 M urea and 5mM dithiothreitol, and stirred for 1 hour, then diluted with 50 mM Na₂HPO₄ buffer (pH 7.5, 1300 ml), and thereafter 20 mM K₃Fe(CN)₆ aqueous solution (9 ml) was added thereto. The solution was charged on a column of CHP-20P (Mitsubishi Kasei Kogyo, tradename) (2.5 × 10 cm) and was subjected to linear gradient elution with 0.1 N aqueous formic acid (600 ml) containing 5% acetonitrile ∼ 0.1 N aqueous formic acid (600 ml) containing 45% acetonitrile. Samples (100 µl) from the fractions (each 10 ml) were measured colorimetrically by the Folin-Lolly method at 750 nm. Fractions Nos. 44-65 were collected and lyophilized to obtain a white powder (60 mg). Residual powder (200 mg) was treated by the same way to obtain a further white powder (60 mg). The combined powder was charged on a column (2.5 × 15 cm) of CM-Toyopearl 650 M (trademark), and eluted by linear gradient elution with 0.1 N aq. acetic acid (700 ml) ∼ 0.4 M aq. ammonium acetate (pH 5, 700 ml). Fractions (each 10 ml) Nos. 105-123 were collected and freeze dried to obtain a white powder. The thus-obtained white powder dissolved in 0.1 M acetic acid was charged on a column of Sephadex G-25 Fine (trade name) (2.6×90 cm) and eluted with 0.1 M acetic acid. Fractions Nos. 23-36 (each 10 ml) were collected and lyophilized to obtain a white powder (42.8 mg). This powder was purified by reverse phase HPLC to obtain purified [Asn³, Phe¹⁵,Gly²³ ] h-CGRP(1-37)(20.3 mg). Column: Nucleosil 5C₁₈ (8 mm ID × 250 mm) Buffer: 0.1% TFA-acetonitrile (a gradient elution with acetonitrile concentration from 28 to 32% for 20 minutes) Flow: 2.5 ml/min. Fraction: collected at a peak at retention time 16.3 min. Physical properties: [α] D 26.5 : -56. 63° (c = 0.113, 0.1 M acetic acid) Amino acid analysis (6N-HCl hydrolysis): Amino acid analysis (enzymatic hydrolysis by trypsin-aminopeptidase M): Arg is analysed by citrulline. The above protected [Asn³,Phe¹⁵,Gly²³] h-CGRP(1-37)-MBHA-resin was obtained by the following procedure: Solid phase peptide synthesizer: 430-A peptide synthesizer Applied Biosystems Inc. MBHA-resin (Applied Biosystems Inc., amino group: 0.48mM/g)(1.04 g) in a reaction vessel for solid phase peptide synthesis was treated with DCM (8 ml)(4 times, each 1 min.), DCM solution (8 ml) containing 60% TFA (20 min.), DCM (4 ml) (3 times, each 15 sec.), DMF solution (3 ml) containing DIEA (1 ml)(2 times, each 1 min.) and DMF (8 ml)(6 times, each 40 sec.),in this order, under a nitrogen gas atmosphere with stirring. The material was filtered after each treatment. DCC (0.5 M-DCM solution)(2 ml) was added to Boc-Phe (2 mM, amino acid sequence No. 37) dissolved in DCM (5 ml) in a vessel for amino acid activation and reacted for 5 mins. The filtered reaction mixture was transferred to a vessel for concentration, and DMF (3 ml) was added therein. DCM was then distilled off under a nitrogen gas atmosphere. Further DMF (3ml) was added, and the mixture was transferred to the above reaction vessel, then reacted for 25 mins. The reaction mixture was washed 6 times with DCM (8 ml), 20 sec. each time, and the mixture was filtered to obtain a Boc-Phe-MBHA-resin. The above Boc-Phe-MBHA-resin was washed 4 times with DCM (8 ml, each 1 min.) in the reaction vessel and filtered. 40% DCM solution (8 ml) containing 60% TFA was added thereto and stirred for 20 min. to remove Boc. The thus-obtained resin was washed 3 times with DCM (4 ml, each 15 sec.), 2 times with DMF solution (3 ml) containing DIEA (1 ml)(each 1 min.) and 6 times with DMF (8 ml, each 40 sec.), in this order, and filtered. DCC (0.5 M-DCM solution)(2 ml) was added to Boc-Ala (2 mM, amino acid sequence No. 36) dissolved in DCM (5 ml) in a vessel for amino acid activation and reacted for 5 mins., then treated the same as Boc-Phe hereinabove. The reaction mixture was concentrated after adding DMF under a nitrogen gas atomosphere, transferred into a reaction vessel and reacted for 20 mins. washed 6 times with DCM (8 ml, each 20 sec.) and filtered to obtain a Boc-Ala-Phe-MBHA-resin. Subsequently, amino acids (sequence from Nos. 1 to 35) were subjected to a coupling reaction and at a final step of the reaction Boc was removed by DCM solution containing 60% TFA, then washed with DCM, DIEA and DMF to obtain protected [Asn³, Phe¹⁵,Gly²³ ] h-CGRP(1-37)-MBHA-resin. The protected amino acids used in the process are as follows: In the above solid phase synthesis, when Asn and Arg were used, DCC solution (3 ml) and HOBt solution (0.5 M-DMF solution) (4 ml) were added to the amino acids (2 mM) the mixture was reacted for 35 min., then treated the same as the other amino acids. The mixture was transferred into a reaction vessel for the coupling reaction, washed with DCM and filtered. Further DCC solution (3 ml) and HOBt solution (0.5 M-DMF solution)(4 ml) were added again to the amino acids (2 mM), the mixture was reacted for 35 mins., and then the mixture was transferred into a reaction vessel for the coupling reaction according to the so-called double coupling method. Example 2 Production of [Asn³,Asp¹⁴,Gly²³] h-CGRP(1-37): Anisole (2 ml), dimethylsulfide (2 ml) and ethanedithiol (0.4 ml) were added to [Asn³,Asp¹⁴,Gly²³] h-CGRP(1-37)-MBHA-resin of the formula (1.173 g). Anhydrous hydrogen fluoride (20 ml) was added thereto and the mixture was stirred at 0°C for 1 hour. After the anhydrous hydrogen fluoride was distilled off in vacuo, the residue was washed with ether and 20% acetic acid (30 ml) was added thereto to extract the peptide. The extract was passed through a Dowex WGR (trademark) column (2.5 × 15 cm) and eluted with 0.1 M acetic acid (150 ml). The eluate was freeze dried to obtain a white powder (460 mg). The powder dissolved in 0.1 M acetic acid was charged on a column (3.0 × 90 cm) of Sephadex G-25 Fine and eluted with 0.1 M acetic acid. The eluate was separated at each 10 ml fraction. Fractions Nos. 28-40 were collected and lyophilized to obtain a white powder (306.6 mg). The powder (152 mg) was dissolved in 50 mM Na₂HPO₄ buffer (pH 7.5, 10 ml) containing 8 M urea and 5 mM dithiothreitol, and stirred for 1 hour, then diluted with 50 mM Na₂HPO₄ buffer (pH) 7.5, 1125 ml), thereafter 20 mM K₃Fe(CN)₆ aqueous solution (8 ml) was added thereto. The solution was charged on a column of CHP-20P (Mitsubishi Kasei Kogyo, tradename) (2.5 × 14 cm) and was subjected to linear gradient elution with 0.1 N aqueous formate (500 ml) containing 5% acetonitrile ∼ 0.1 N aqueous formate (500 ml) containing 45% acetonitrile. Samples (100 µl) from the fractions (each 10 ml) were measured colorimetrically by the Folin-Lolly method at 750 nm. Fractions Nos. 41-55 were collected and lyophilized to obtain a white powder (60 mg). The residual powder (154 mg) was treated by the same way to obtain a further white powder (40 mg). The combined powder was charged on a column (2.5 × 20 cm) of CM-Toyopearl 650 M (trademark), and eluted by linear gradient elution with distilled water (700 ml) ∼ 0.4 M aq. ammonium acetate (pH 5,700 ml). Fractions (each 10 ml) Nos. 108-116 were collected and freeze dried to obtain a white powder. The thus obtained white powder dissolved in 0.1 M acetic acid was charged on a column of Sephadex G-25 Fine (trade name) (2.6× 90 cm) and eluted with 0.1 M acetic acid. Fractions Nos. 8-14 (each 10 ml) were collected and lyophilized to obtain a white powder (13.0 mg). This powder was purified by reverse phase HPLC to obtain purified [Asn³,Asp¹⁴,Gly²³] h-CGRP(1-37)(4.7 mg). Column: Nucleosil 5C₁₈ (8 mm ID x 250 mm) Buffer: 0.1% TFA-acetonitrile (a gradient elution with acetonitrile concentration from 28 to 32% for 25 minutes) Flow: 2.5 ml/min. Fraction: collected at a peak at retention time 17.3 min. Physical properties: [α] D ²⁵ : -62.29° (c = 0.122, 0.1 M acetic acid) Amino acid analysis (6N-HCl hydrolysis): Amino acid analysis (enzymatic hydrolysis by trypsinamino-peptidase): Arg is analysed by citrulline. The above protected [Asn³,Asp¹⁴,Gly²³] h-CGRP(1-37)-MBHA-resin was obtained by the same way as in Example 1. The protected amino acids used in the process are as follows: Example 3 Production of [Asn³,Asp¹⁴,Phe¹⁵] h-CGRP(1-37): Anisole (2 ml), dimethylsulfide (2 ml) and ethanedithiol (0.4 ml) were added to [Asn³,Asp¹⁴,Phe¹⁵] h-CGRP(1-37)-MBHA- resin of the formula (1.173 g). Anhydrous hydrogen fluoride (20 ml) was added thereto and the mixture was stirred at 0°C for 1 hour. After the anhydrous hydrogen fluoride was distilled off in vacuo, the residue was washed with ether and 20% acetic acid (30 ml) was added thereto to extract the peptide. The extract was passed through a Dowex WGR (trademark) column (2.5× 15 cm) and eluted with 0.1 M acetic acid (150 ml). The eluate was freeze dried to obtain a white powder (410 mg). This powder (150 mg) was dissolved in 50 mM Na₂HPO₄ buffer (pH 7.5, 10 ml) containing 8 M urea and 5mM dithiothreitol, and stirred for 1 hour at room temperature, then diluted with 50 mM Na₂HPO₄ buffer (pH 7.5, 1125 ml), there-after 20 mM K₃Fe(CN)₆ aqueous solution (8 ml) was added thereto.The solution was charged on a column of CHP-20P (Mitsubishi Kasei Kogyo, trade name)(2.5 × 12 cm) and was subjected to linear gradient elution with 0.1 N aqueous formate (500 ml) containing 5% acetonitrile ∼ 0.1 N aqueous formate (500 ml) containing 45% acetonitrile. Samples (100 µl) from the fractions (each 10 ml) were measured colorimetrically by the Folin-Lolly method at 750 nm. Fractions Nos. 48-58 were collected and lyophilized to obtain a white powder (43 mg). Residual powder (260 mg) was treated by the same way to obtain a further white powder (73 mg). The combined powder was charged on a column (2.5 × 10 cm) of CM-Toyopearl 650 M (trademark), and eluted by linear gradient elution with distilled water (500 ml) ∼ 0.4 M aq. ammonium acetate (pH 5, 500 ml). Fractions (each 10 ml) Nos. 65-75 were collected and freeze dried to obtain white powder. The thus-obtained white powder dissolved in 0.1 M acetic acid was charged on a column of Sephadex G-25 Fine (trade name) (3 × 30 cm) and eluted with 0.1 M acetic acid. Fractions Nos. 22-24 (each 10 ml) were collected and lyophilized to obtain white powder (19.0 mg). This powder was purified by reverse phase HPLC to obtain purified [Asn³,Asp¹⁴,Phe¹⁵] h-CGRP(1-37) (11 mg). Column: Nucleosil 5C₁₈ (8 mm ID × 250 mm) Buffer: 0.1% TFA-acetonitrile (a gradient elution with acetonitrile concentration from 30 to 33% for 20 minutes) Flow: 2.5 ml/min. Fraction: collected at a peak at retention time 12.9 min. Physical properties: [α] D 25 : -63.68° (c = 0.179, 0.1 M acetic acid) Amino acid analysis (6N-HCl hydrolysis): Amino acid analysis (enzymatic hydrolysis by trypsin-aminopeptidase M): Arg is analysed by citrulline. The above protected [Asn³,Asp¹⁴,Phe¹⁵] h-CGRP(1-37)-MBHA-resin was obtained by the same way as in Example 1. The protected amino acids used in the process are as follows: Example 4 Production of [Asp¹⁴] h-CGRP(1-37): Anisole (2 ml), dimethylsulfide (2 ml) and ethanedithiol (0.4 ml) were added to [Asp¹⁴] h-CGRP(1-37)-MBHA-resin of the formula (1.10 g). Anhydrous hydrogen fluoride (20 ml) was added thereto and the mixture was stirred at 0°C for 1 hour. After the anhydrous hydrogen fluoride was distilled off in vacuo, the residue was washed with ether and 1M acetic acid (20 ml) was added thereto to extract the peptide. The extract was passed through a Dowex WGR (trademark) column (2.5× 15 cm) and eluted with 1 M acetic acid (150 ml). The eluate was freeze dried to obtain a white powder (390 mg). This powder was dissolved in 50 mM Na₂HPO₄ buffer (pH 7.5, 20 ml) containing 8 M urea and 5mM dithiothreitol, and stirred for 1 hour, then diluted with 50 mM Na₂HPO₄ buffer (pH 7.5, 2000 ml), thereafter 20 mM K₃Fe(CN)₆ aqueous solution (10 ml) was added thereto. The solution was charged on a column of CHP-20P (Mitsubishi Kasei Kogyo, trade name)(2.5 × 24 cm) and was subjected to linear gradient elution with 0.1 N aqueous formate (800 ml) containing 5% acetonitrile 0.1 N aqueous formate (800 ml) containing 45% acetonitrile. Samples (100 µl) from the fractions (each 10 ml) were measured colorimetrically by the Folin-Lolly method at 750 nm. Fractions Nos. 72-90 were collected and lyophilized to obtain a white powder (160 mg). The powder was charged on a column (2.5 × 10 cm) of CM-Toyopearl 650 M (trademark), and eluted by linear gradient elution with distilled water (600 ml) ∼ 0.4 M aq. ammonium acetate (pH 5, 600 ml). Fractions (each 10 ml) Nos. 48-52 were collected and freeze dried to obtain a white powder. The thus obtained white powder dissolved in 0.1 M acetic acid was charged on a column of Sephadex G-25 Fine (trade name) (2.6× 90 cm) and eluted with 1 M acetic acid. Fractions Nos. 16-30 were collected and lyophilized to obtain a white powder (30 mg). This powder was purified by reverse phase HPLC to obtain purified [Asp¹⁴ ] h-CGRP(1-37)(21 mg). Column: YMC-GEL ODS S-5 (20 mm ID x 250 mm) Buffer: 0.1% TFA-acetonitrile (a gradient elution with acetonitrile concentration from 29 to 34% for 30 minutes). Flow: 7 ml/min. Fraction: collected at a peak at retention time 18.4 min. Physical properties: [α] D ²⁵: -74.74° (c = 0.099, 0.1 M acetic acid) Amino acid analysis (6N-HCl hydrolysis): Amino acid analysis (enzymatic hydrolysis by trypsin-amino-peptidase M): Arg is analysed by citrulline. The protected amino acids used in the process are as follows: Example 5 Production of desalanyl-deamino- [Asp¹⁴ ] h-CGRP(2-37): Anisole (2 ml), dimethylsulfide (2 ml) and ethanedithiol (0.4 ml) were added to protected-desalanyl-deamino- [Asp¹⁴ ] h-CGRP(2-37)-MBHA-resin of the formula (1.26 g). Anhydrous hydrogen fluoride (20 ml) was added thereto and the mixture was stirred at 0°C for 1 hour. After the anhydrous hydrogen fluoride was distilled off in vacuo, the residue was washed with ether and 1M acetic acid (20 ml) was added thereto to extract the peptide. The extract was passed through a Dowex WGR (trademark) column (2.5 × 15 cm) and eluted with 1 M acetic acid (150 ml). The eluate was freeze dried to obtain a white powder (428 mg). This powder was dissolved in 50 mM Na₂HPO₄ buffer (pH 7.5, 20 ml) containing 8 M urea and 5mM dithiothreitol, and stirred at room temperature for 1 hour, then diluted with 50 mM Na₂HPO₄ buffer (pH 7.5, 2000 ml), thereafter 20 mM K₃Fe(CN)₆ aqueous solution (12 ml) was added thereto. The solution was charged on a column of CHP-20P (Mitsubishi Kasei Kogyo, trade name)(2.5 × 24 cm) and was subjected to linear gradient elution with 0.1 N aqueous formate (800 ml) containing 5% acetonitrile ∼ 0.1 N aqueous formate (800 ml) containing 45% acetonitrile. Samples (100 µl) from the fractions (each 10 ml) were measured colorimetrically by the Folin-Lolly method at 750 nm. Fractions Nos. 89-102 were collected and freeze dried to obtain a white powder (116 mg). The powder dissolved in 1 M acetic acid was charged on a column (2.6× 85 cm) of Sephadex G-25 Fine (trademark), and eluted with 1 M acetic acid. Fractions (each 10 ml) Nos. 17-30 were collected and lyophilized to obtain a white powder (54 mg). This powder was purified by reverse phase HPLC as hereinbelow to obtain purified desalanyl-deamino- [Asp¹⁴ ] h-CGRP(2-37)(13 mg). Column: YMC-GEL ODS S-5 AM type (20 mm ID × 250 mm) Buffer: 0.1% TFA-acetonitrile (a gradient elution with acetonitrile concentration from 31.5 to 32.5% for 30 minutes) Flow: 7 ml/min. Fraction: collected at a peak at retention time 19.5 min. Physical properties: [α] D 25 : -81.38° (c = 0.091, 0.1 M acetic acid) Amino acid analysis (6N-HCl hydrolysis): The above protected-desalanyl-deamino [Asp¹⁴ ] h-CGRP (2-37)-MBHA-resin was obtained by the following procedure: Amino acids (sequence from Nos. 3 to 37) were subjected to a coupling reaction by the solid phase synthesis as in Example 1 and the peptide was acylated with MBzl- β - mercaptopropionic acid at the final stage of reaction to obtain protected-desalanyl-deamino- [Asp¹⁴ ] h-CGRP (2-37)-MBHA-resin. The protected amino acids used in the process are as follows:

Claims: A compound of the formula (I) wherein R is H or H-Ala-NH- and: A is Asp, B is Asp or Glu, C is Leu and D is Val; A is Asn, B is Gly, C is Phe and D is Gly; A is Asn, B is Asp or Glu, C is Leu and D is Gly; or A is Asn, B is Asp or Glu, C is Phe and D is Val; or a pharmaceutically acceptable salt thereof. A process for the preparation of a compound of formula (1) as defined in claim 1 or a physiologically acceptable salt thereof, which process comprises (i) synthesising a peptide which has a chain structure of the formula wherein R,A,B,C and D are as defined in claim 1, Q₁ is hydrogen or a mercapto-protecting group and Q₂ is optional but, if present, is -NH₂ or together with the carboxyl group from the Phe represents a protected amide group, and in which side-chain functional groups are optionally protected; (ii) when Q₁ is a mercapto-protecting group and/or when the side-chain mercapto group of Cys is protected, removing the or each said protecting group; (iii) oxidixing the free mercapto groups to form a disulfide bridge; (iv) removing any side-chain functional group-protecting groups optionally present and, when Q₂ is not -NH₂, converting the terminal carboxy group of Phe into an amide group; and (v) if desired, converting the resulting compound of formula (1) into a physiologically acceptable salt thereof. A process according to claim 2 wherein the peptide employed in step (i) has been prepared by solid phase synthesis. A process according to claim 2, wherein the peptide employed in step (i) has been prepared by solution phase synthesis. A pharmaceutical composition comprising a compound of formula (1) as defined in claim 1 or a physiologically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

Inventors: HORI, MASAYUKI, MORITA, KAORU, NODA, TOSHIHARU, UZAWA, TOYONOBU
Applicants: ASAHI KASEI KOGYO KABUSHIKI KAISHA
Application year: 1988
Main classification: C07K7
IPC codes: A61K38, A61P3, C07K14