IE863386L

IE863386L - Polypeptides

Info

Publication number: IE863386L
Application number: IE863386A
Authority: IE
Original assignee: Milhaud Gerard
Priority date: 1985-12-24
Filing date: 1986-12-23
Publication date: 1987-06-24
Also published as: FI87795B; FI873632A0; EP0232650A1; DE3667472D1; JPS63502343A; FI873632L; PT84011B; FR2592049A1; CA1273750A; PT84011A; FI87795C; DK436187A; FR2592049B1; GR3000279T3; EP0232650B1; JPH0813838B2; ES2012456B3; DK436187D0; IE59489B1; ATE48616T1

Abstract

Polypeptidic compounds saving the calcium of the organism, preparation thereof, application thereof as medicaments and compositions containing them. Analogues of natural hypocalcemic dotriacontapeptides characterized by the presence of asparagine respectively: a) either in position 2 with a cysteine in position 1,7; b) or in position 1 with an aminosuberic residue in position 1,7. Said polypeptides may be used as active principle for new medicaments which are particularly indicated for osteoporoses of various etiologies.

Description

1 59489 - i - i European Patent Application EP 0146842 describes chicken 10 calcitonin. This calcitonin has an activity similar to that of salmon calcitonin, and inferior to the products of the present application.

The subject of the present invention is new polypeptide 15 compounds, wherein they are chosen from the group formed by the products with the formula Cys-Asn-A-j-Leu-Ser-Thr-Cys-Ag-Leu-Gly-Ajj-A^-Aj-j- G1n"A15"A16"A17"LyS"A19"A20"T'ir"A22~Pro"A24~T'ir"A26 20 dA> -A^-Gly^g-Gly-Agj-Pro nh2 where A3 = Ser, Gly or Ala Ag = Val or Leu Ajj = Lys or Thr Aj2 = Leu or Tyr A13 = ^er or 30 Ajg = Glu or Asp Ajg = Leu or Phe A^ = His or Asn Ajg = Leu or Phe = Gin or His A22 = Tyr or Phe A24 = Ar9 or A2g = Asp, Asn or Ala = Val, Thr or lie k2§ ~ Ala, Ser or Val A31 = Thr, Val or Ala and the products of formula (Ig) which contain^-amino-suberic acid 5 (Asu) of which thed)-carboxyl is fixed to the amino group of Asn: — ^CH2^5 CO-Asn-A'2-Leu-Ser-Thr-NHCHCO-A'7-Leu-Gly-A'1Q- A* n-A' i2-Gln-A' 14~a' 15~A" 16~Lys~A' 18" A' jg-Thr-A'21-Pro-Al23"Thr-A'25-A'2g-Gly-A'gs-Gly-A'sQ-Pro NH2 where: A'2 = Ser, Gly, or Ala Ax-j = Val or Leu A'jq = Lys or Thr A'u = Leu or Tyr 20 A'12 = Ser or Thr A'14 = Glu or Asp A'jjj = Leu or Phe A'jg = His or Asn A^g = Leu or Phe 25 A'jg = Gin or His A'21 = Tyr or Phe A'23 = Arg or Gin A125 = Asp, Asn or Ala A*26 = Val, Thr or lie 30 A*2g = Ala, Ser or Val A'sq = Thr, Val or Ala The products with the formula (Ig) are structural analogues of natural peptides, such as K. Jost and J. Rudinger realized in the 35 case of neurohypophyseal hormones (Collect. Czech. Chem. Comm. 1967, 32, 1229).

The products with the formulae (1^) and (Ig) can be regrouped into the following formula: D-A1y-Leu-Gly-A'iq~A'^-Gln-A114"^'15"^'18 -ig ■Thr_5i -proi3 ■Thris i6 -s,y-$8 -Glyio ■ Pro NH2 in which A'^, A1^q* A'jji ^'12' A 14' A 15' ~A 16' A'i8'A,19* A'21' A23' A*25' A>26' A'28 and A<30 have the previous significance and D represents: - either the residue Cys-Asn-A^-Leu-Ser-Thr-Cys- in which A^ has the previous significance -or the residue [ ^CH2^5 1 CO-Asn-A'o-Leu-Ser-Thr-NHCHC0- in which A'2 has the previous significance.

The subject of the invention is in particular compounds corresponding to the general formulae (1^) and (Ig), as defined previously, in which Ag and A'7 represent a valine.

The subject of the invention is more particularly compounds answering to the following general formulae (IA) and (Ig): -Cyn-Asn-Ser-Leu-Serg-Thr-Cys-Val-Leu-GlyiQ-Lys-Leu-Ser-Gln-GlUjjj -Leu-Hi s-Lys-Leu-Gln^-Thr-Tyr-Pro-Arg-Thr^-Asp-Val-Gly-Ala-Gly^o -Thr-Proamide; -(ch2) 1 -CO-Asn-Ser-Leu-Ser-Thrg-NH-CH-CO-Val-Leu-Gly-LySjQ-Leu-Ser-Gln-Glu-Leujg-His-Lys-Leu-Gln-Thr^-Tyr-Pro-Arg-Thr-Asp^-Val-Gly-Ala-Gly-Thr^Q-Proamide.

The polypeptides according to the invention lower calcemia and phosphatemia; they inhibit bone destruction, accelerate formation of new bones, increase intestinal absorption of calcium and decrease calciurea. They fix calcium inside the cells. They possess an 5 important anti-inflammatory and antalgic effect.

These properties justify the use of polypeptides with the formulae (1^) and (Ig) as defined previously, as medicaments.

The subject of the invention is therefore also polypeptides with the formulae (1^) and (Ig) as defined previously as medicaments.

The subject of the invention is more particularly, as medicaments, the preferred polypeptides mentioned above.

The main indications of the medicaments according to the invention are represented by Paget's disease, osteoporoses of various etiologies (common, poro-malacic, cortisonic, post-traumatic, of immobilization and idiopathic), renal osteodystrophy, fractures, 20 hypercalcemias, osteroarticular pains, in particular those resulting from bone metastases, spasmophilia and normocalcemic tetany.

The subject of the invention is also pharmaceutical compositions containing as active principle at least one of the medicaments as 25 defined previously.

These pharmaceutical compositions can be, for example, solid or liquid and are presented in the pharmaceutical forms currently used in human medicine, like, for example, simple or sugar-coated tablets, 30 capsules, granules, liposomes, aerosols, suppositories, injectable preparations; they are prepared according to the usual methods. The active principle or principles can be incorporated in the excipients usually employed in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, 35 aqueous or non-aqueous vehicles, fatty substances of animal or vegetable origin, paraffin derivatives, glycols, various moistening, dispersing or emulsifying agents, and preservatives.

The subject of the invention is in particular pharmaceutical compositions as defined previously, wherein they are intended for parenteral administration, oral administration in the form of liposomes, or nasal administration in pulverised form.

The subject of the invention is also a preparation process for compounds as defined previously, wherein amino acids, peptides or their combinations, are submitted to condensation reactions in the order of the sequence of amino acids with the formulae (1^) and 10 (Ig)» an£l when the products with the formula (Ig) are prepared, the solid phase technique for obtaining the 10-31 sequence and the classic liquid phase technique for obtaining the 1-9 sequence are combined.

As previously indicated, the synthesis of peptides with the formula (1^) and that of the 10-31 sequence of products with the formula (Ig) are carried out by using the solid phase technique.

This brings into play benzylhydrylamino resin (P.G. Pietta and G.R. Marshall, Chem. Commun., 1970, 650), of which the preparation and use 20 have been described (P. Rivaille, A. Robinson, M. Kamen and G. Milhaud, Helv., 1971, M, 2772).

In a preferred way of preparation of products with the formula (IA), the group N-rt-butyloxycarbonyl (t-Boc) protects the The different amino acids are incorporated in the peptide chain by dicyclohexylcarbodiimide (DCCI) and hydroxybenzotriazol (HOBT) by making protected aminoacids in excess in relation to the proline first fixed on the resin react successively. The reaction is allowed 5 to continue for two hours. The t-Boc groups are eliminated by trifluoroacetic acid in solution in methylene chloride; after washing with methylene chloride, neutralisation is carried out by triethylamine or diisopropylethylamine (DIEA) in the same solvent.

Asparagine and glutamine are introduced in the form of their para-nitrophenyl esters, in solution in dimethylformamide (DMF). If the control test for coupling to ninhydrin according to the method of Kaiser and Colescott is positive, the mixture is treated by pivalic acid (1%); if the blue colouration still persists in this test, the 15 DMF/1% AcOH mixture is saturated with urea. However, even if this test is negative (no blue colouration), each coupling can be repeated twice systematically. If the coupling is not total at the end of 48 hours of reaction, as is the case for glutamine in position 20, the threonine which has not reacted is acetylated by acetyl-imidazol in 20 methylene chloride or by acetic acid and DCCI.

Finally, the protector group of the imidazol of histidine is eliminated, while the peptide is still linked to the resin, at pH 8, with mercapto-ethanol in solution in DMF. The peptide is separated 25 form the resin by treatment with liquid hydrofluoric acid in the presence of anisole and methionine. All the protector groups are eliminated during this operation.

The peptides are purified by filtration on gel. The fractions 30 corresponding to the main peaks are assembled, lyophilized, taken up again in a buffer at pH 8 and oxidized for 24 hours by an air current in order to form the disulphide bridge between the two cysteine residues.

The fraction corresponding to the peak possessing the biological activity sought is purified again on ion exchange resin CMC 32 by an acid elution gradient. The peptide obtained is homogeneous to electrophoresis on paper and to chromatography on cellulose.

The 10-31 sequence of products with the formula (Ig) is realized in the same conditions as those indicated previously for the preparation of products with the formula (IA). What was indicated for glutamine in position 20 for products with the formula (IA) is 5 of course valid for position 19 for products with the formula (Ig).

In a preferred way of carrying out the preparation process for products with the formula (Ig), the synthesis of the 1-9 sequence is carried out in a liquid medium. The fragment (ch2)5coor H-Asn-A12-Leu-Ser-Thr-NH-CH-(j:=0 OH where A'2 has the above significance and R = ester, after cyclization, is condensed with tripeptide A'^-Leu-Gly. Use is made of the protector groups generally used for the synthesis of peptides.

Peptide with the formula (Ig) is then obtained as follows: The above protected nonapeptide is converted into N-hydroxy-succinimide ester, then coupled with the 10-31 fragment fixed on the resin in a DMF medium at 30°C. The peptide is separated from the resin by treatment with liquid hydrofluoric acid in the presence of dimethyl-sulphide and p-cresol (J.P. Tam,- W.H. Heathand and R.B. Merrifield, J.A.C.S., 1983, 105. 6442). All the protector groups are eliminated during this operation.

The peptides are purified by filtration on gel.

The examples which follow illustrate the invention without however limiting it.

Example 1 Obtaining peptide with the following formula (IA): Cys-Asn-Ser-Leu-Serg-Thr-Cys-Val-Leu- GlyiQ-Lys-Leu-Ser-Gln-GlUjg-Leu-His-Lys-Leu-G^g-Thr-Tyr-Pro-Arg-Th^- (IA) Asp-Val-Gly-Ala-Gly^Q-Thr-Proamide The t-Boc aminoacids carrying the lateral functions come from BACHEM (Dubendorf, Switzerland) or are prepared in the laboratory by Schnabel's technique (E. Schnabel, Liebigs Ann. Chem., 702, 188, 1967) in an autotitration apparatus. Their purity is determined by polarimetry, melting point and chromatography on thin layer silica.

The hydrolysis of the purified peptide is carried out in a sealed tube under vacuum in a 6N HC1 medium (110°C, 24 hours).

I - SYNTHESIS OF THE SEQUENCE a) Preparation of the resin (P. Rivaille, A. Robinson, M. Kamen and G. Milhaud, Helv., 1971, 54, 2772).

Benzvl-ketoresin In a 2 1 three-necked flask, fitted with an agitator, 50 g of polystyrene with 1% of divinylbenzene Bio-rad SX^ (200-400) is left to swell for 2 hours, in 350 ml of nitrobenzene. In another flask, 66 g of A1C1^ is dissolved in 300 ml of nitrobenzene to which 80 ml 25 of benzoyl chloride is added. The mixture is poured into the first flask under a good agitation which is maintained for 2 hours at ambient temperature. If the resin swells too much, nitrobenzene is added (about 100 ml). The mixture is filtered, washed with dioxan (3 x), with acetic acid (3 x), with methanol (3 x) and finally, 30 alternately, with CHgClg and CH^OH in order to make the resin swell and contract each time leaving a sufficient contact time. The mixture is dried in a dessicator under vacuum all night (yield 75 g).

Benzylhvdrvlamine resin In a 1 1 flask fitted with "Dean Stark" and an agitator, 500 g of ammonium formiate is heated to 120-130°C, for 1 to 2 hours. The resin (50 g) is added in one go and the temperature is allowed to rise to 160-165°C, over half an hour, under efficient agitation which is maintained for 6 hours. The mixture is washed with water, then treated as above for the benzylketoresin. The resin dried under vacuum is heated to reflux and agitated for 8 hours in 6N HC1. It is 5 then filtered and washed as previously. The fixation capacity varies, according to the nature of the amino acid, between 0.3 and 0.5 mMole/g. b) Preparation of the peptide The apparatus used enables mechanical agitation of a 25 ml pyrex tube the central compartment of which contains the resin (2 g). At the ends of the tube, fritted glass filters are intended for the introduction and then the elimination of the reagents (about 15 ml).

The coupling of the amino acids involves the following times: CYCLE 32 20 Coupling: 2 g of resin (fixation capacity 0.5 mMoles/g) is agitated (10 min) in a C^C^, (CH^NCHO (DMF) medium (1:1, v/v) in the presence of Boc-L-proline (2mM, 0.43 g). DCCI (4 nflloles) and 25 1-hydroxybenzo- triazol (H0BT) (4 mMoles) are added and agitation is resumed for 2 hours. The resin is washed successively with CH2CI2 (1 x, 2 min), CH^OH abs (3 x, 2 min), and C^C^ (3 x, 2 min). The reaction to ninhydrin is carried out on a sample of resin: it is negative.

Liberation of amino group: The resin is treated by a CF^COOH-C^C^ mixture (1:1, v/v) for 1 min; the mixture is filtered and the operation is repeated 35 leaving in contact for 15 min. The resin is washed with CHgC^ (3 x, 1 min). CFjCOOH is eliminated by treating the resin twice with an Et^N/CHgClg mixture (12.5 : 87.5 v/v) (contact time 1 min then 5 min). It is washed with Cl^C^ (8 x, 1 min). The L-proline resin is titrated: it contains 0.5 mMole/g of proline. CYCLE 31 Coupling: The L-prolyl resin (1 mMole) is agitated (10 min) with 4 mMoles of Boc-O-Benzyl-L-threonine (1.23 g) in CH2C12-DMF (1:1 v/v) (10 ml). DCCI (4 mMoles) and HOBT (4 mMoles) are added and the agitation 10 is resumed (2 hrs). The resin is washed successively: CH2C12 (1 x, 2 min), CH^OH abs (3 x, 2 min), CH2C12 (3 x, 2 min). The reaction to ninhydrin is negative.

Liberation of the amino group - See cycle 32.

CYCLES 30 TO 25 The above coupling and liberation processes are used in making react for: - Cycle 30 4 mMoles Boc-glycine (0.71 g) - Cycle 29 4 mMoles Boc-L-ala (0.76 g) - Cycle 28 4 mMoles Boc-glycine (0.71 g) - Cycle 27 4 mMoles Boc-L-valine (0.87 g) - Cycle 26 4 mMoles B-benzyl ester of Boc-L-aspartic acid (1.29 g) - Cycle 25 4 mMoles Boc-O-benzyl-L-threonine (1.24 g).

CYCLE 24 Coupling: The resin-peptide of cycle 25, previously washed with DMF (2 x), is agitated (10 min) with 4 mMoles of Boc-N-y-tosyl-L-arginine (1.41g) in a solution of DMF and then the process is as described for cycle 31.

CYCLE 23 Coupling: The peptide resin of cycle 24 is agitated (10 min) with 4 mMoles of Boc-L-proline (0.88 g in a CH2C12 medium) (10 ml). DCCI/HOBT is added as described for cycle 32. At the end of the reaction period, the reaction to ninhydrin is negative.

Liberation - See cycle 32.

CYCLES 22 TO 21 The coupling and liberation processes used in cycle 23 are used 15 to make react: - Cycle 22 4 mMoles Boc-O-Benzyl-L-tyrosine (1.49 g) - Cycle 21 4 mMoles Boc-O-Benzyl-L-threonine (1.24 g).

CYCLE 20 Coupling: The resin-peptide of cycle 21, previously washed with DMF (2 x) 25 is agitated (10 min) with 4 mMoles of Boc-L-glutamine p-nitrophenylester (1.5 g) and 10 ml DMF with the addition of 1% acetic acid. DCCI (4 mMoles) and H0BT (4 mMoles) are added and the mixture is agitated for 48 hours. The resin is washed with DMF, CH2C12, CH30H, CH2C12 (2 x).

Liberation - See cycle 32.

CYCLES 19 TO 15 35 The process for cycle 31 is used to make react: - Cycle 19 4 mMoles Boc-L-leucine (1.0 g) - Cycle 18 4 nMoles Boc-£-carbobenzyloxy-L-lysine (1.52 g) - Cycle 17 - Cycle 16 - Cycle 15 9)- 4 mMoles Boc-dinitro-2,4-phenyl(im)L-histidine (1.23 g) 4 mMoles Boc-L-leucine (1.0 g) 4 mMoles ^-benzyl ester of Boc-L-glutamic acid (?) (1.3 CYCLE 14 The process is as for cycle 20.

CYCLE 13 The process is as for cycle 31, by using 4 mMoles of Boc-o-benzyl-L-serine (1.0 g).

CYCLES 12 TO 9 The process is as for cycle 31 making react: Cycle 12 Cycle 11 Cycle 10 Cycle 9: Cycle 8: the derivative used in cycle 19. the derivative used in cycle 18. the derivative used in cycle 30. the derivative used in cycle 19. the derivative used in cycle 27.

CYCLE 7 The process for cycle 31 is used making react 4 mMoles of S-p-methoxybenzyl ether of Boc-L-cysteine (1.13 g).

CYCLE 6 TO 3 Cycle 6 Cycle 5 Cycle 4 Cycle 3 identical to cycle 31. identical to cycle 13. identical to cycle 19. identical to cycle 13.

CYCLE 2 Coupling: The peptide resin from cycle 3 previously washed with DMF (2 x) is agitated (48 hrs), with 4 mMoles of p-nitrophenyl ester of Boc-L-asparagine (1.45 g) in a DMF medium, in the presence of HOBT (4 mMoles). The resin is washed successively with DMF, D^C^, CH^OH, CH2CI2. The reaction to ninhydrin is negative.

Liberation - The process is as described for cycle 32.

CYCLE 1 4 mMoles of S-p-methoxybenzyl ether of Boc-L-cysteine (1.13 g) is made to react in the conditions of cycle 31.

II - ELIMINATION OF THE DINITROPHENYL GROUP OF HISTIDINE 2 ml of mercaptoethanol is added to 15 ml of dimethylformamide (DMF), and the pH is adjusted to 8 with triethylamine. This reagent is introduced into the apparatus and the mixture is agitated for 14 hours. The resin is filtered and washed alternately with methylene chloride (3 x), and with methanol (3 x). After drying under vacuum, 25 the weight of the resin is 2.85 g.

III - LIBERATION OF THE PEPTIDE OF THE RESIN AND ELIMINATION OF THE PROTECTOR GROUPS 15 ml of hydrofluoric acid is distilled in a mixture of 1.5 g of resin-peptide and 1.5 ml of anisol. The mixture is agitated for 1 hr at 0°C and for 30 min at ambient temperature. The acid is expelled under vacuum and the residue is taken up again in 3 times 5 ml of acetic acid and precipitated by ethyl ether free from peroxide. The 35 precipitate is centrifuged cold, washed several times with ether, and dried under vacuum. The precipitate is taken up again by water and the insoluble residue is eliminated by centrifuging. The lyophilized solution provides 800 mg of product.

IV - PURIFICATION OF THE PEPTIDE Two hundred milligrams of crude peptide are placed at the top of a column (diameter 2.5 cm, length 90 cm) of Biogel P6, (50-100 mesh), 5 and are eluted with 0.1 M acetic acid and fractions of 12 ml are collected by following elution with a 280 nm recording device. The purified peptide is collected in the 23-28 fractions.

V - CYCLIZATION The fractions containing the purified peptide are lyophilized and the residue is taken up again in 500 ml of 1 M ammonium hydrogenocarbonate buffer, pH 8, and submitted to a bubbling of air through a porous plate for 24 hrs. The solution is lyophilized and 15 the residue purified on a CMC 52 Whatman column by an ammonium acetate gradient of 0.6 mho to 7 mho, pH 4, with an LKB Ultrograd 11300 apparatus. After acid hydrolysis, the composition in amino acids is the following, referred to proline = 2 (theoretical figures in brackets): Ala (1) 1.05; Arg (1) 0.83; Asp (2) 2.10; Cys (2) 1.80; Glu (3) 3.10; Gly (3) 3.30; His (1) 0.80; Leu (5) 5.2; Lys (2) 1.90; Pro (2) 2.0; Ser (3) 3.10; Thr (4) 4.15; Tyr (1) 0.85; Val (2) 2.1.

Example 2 Obtaining the peptide with the following formula (Ig): (CH2)5 O-Asn-Ser-Leu-Ser-Thrg-NH-CH-CO-Val-Leu-Gly-LySjQ-Leu-Ser-Gln-Glu-Leu15-His-Lys-Leu-Gln-Thr20-Tyr-Pro-Arg- (Ig) Thr-Aspgg-Val-Gly-Ala-Gly-Thr^Q-Proamide.

The t-Boc aminoacids carrying the lateral functions are obtained as indicated in example 1.

In the same way, the hydrolysis of the purified peptide is carried out in a sealed tube under vacuum in a 6N HCl medium (110°C, 24 hours). 1. SYNTHESIS OF THE 10-31 SEQUENCE OF THE PEPTIDE la) Preparation of the resin (P. Rivaille, A. Robinson, M. Kamen and G. Milhaud, Helv., 1971, 54, 2772).

Benzyl-ketoresin In a 2 1 three-necked flask, fitted with an agitator, 50 g of polystyrene with 1% of divinylbenzene Bio-rad SXj (200-400 mesh) is left to swell in 350 ml of nitrobenzene. In another flask, 66 g of 15 AICI3 is dissolved in 300 ml of nitrobenzene to which 80 ml of benzoyl chloride is added. The mixture is poured into the first flask under a good agitation which is maintained for two hours at ambient temperature. If the resin swells too much, nitrobenzene is added (about 100 ml). The mixture is filtered, washed with dioxan (3 20 x), with acetic acid (3 x), with methanol (3 x) and finally, alternately, with ^C^ and CH^OH in order to make the resin swell and contract each time leaving a sufficient contact time. The mixture is dried in a dessicator under vacuum all night (yield 75 g).

Benzylhvdrvlamine resin In a 1 1 flask fitted with "Dean Stark" and an agitator, 500 g of ammonium formiate is heated to 120-130°C, for 1 to 2 hours. The resin (50 g) is added in one go and the temperature is allowed to 30 rise to 160-165°C, over half an hour, under efficient agitation which is maintained for 6 hours. The mixture is filtered, washed with water, then treated as above for the benzylketoresin. The resin dried under vacuum is heated to reflux and agitated for 8 hours in 6N HCl. It is then filtered and washed as previously. The fixation 35 capacity varies, according to the nature of amino acid, between 0.3 and 0.5 mMole/g. lb) Preparation of the 10-31 sequence of the peptide.

The apparatus used allows a 25 ml pyrex tube, the central compartment of which contains the resin (2 g), to be agitated mechanically. At the ends of the tube, 2 fritted glass filters are intended for the introduction and then the elimination of the 5 reagents (about 15 ml).

CYCLE 31 Coupling: 2 g of resin (fixation capacity 0.5 mMoles/g) is agitated (10 min) in a medium of CHgClg, (CH^NCHO (DMF) (1:1, v/v) (10 ml) in the presence of Boc-L-proline (2 mM, 0.43 g). DCCI (4 mMoles) and 1-hydroxybenzotriazol (H0BT) (4i#loles) are added and agitation is 15 resumed for 2 hours. The resin is washed successively with CH2CL2 (1 x, 2 min), CH^OH abs (3 x, 2 min), CH CI 2 (3 x, 2 min). The reaction with ninhydrin is carried out on a sample of resin: it is negative.

Liberation of the amino group: The resin is treated by a CF^COOH-Cf^C^ mixture (1:1, v/v) for 1 min; the mixture is filtered and the operation is repeated leaving in contact for 15 min. The resin is washed with C^C^ 25 (3 x, 1 min). CF^COOH is eliminated by treating the resin twice with an ET^N/C^C^ mixture (12.5:87.5 v/v) (contact time 1 min then 5 min). The mixture is washed with CH2C12 (8 x, 1 min). The resin-L-proline is titrated: it contains 0.5 mMole/g of proline.

CYCLE 30 Coupling: The resin-L-prolyl (1 mMoles) is agitated (10 min) with 4 mMoles 35 of Boc-O-Benzyl-L-threonine (1.23 g) in C^C^-DMF (1:1 v/v) (10 ml). DCCI (4 mMoles) and H0BT (4 mMoles) are added and agitation is resumed (2 hrs). The resin is washed successively: C^Clg (1 x, 2 min), CHgOH abs (3 x, 2 min), CH2C12 (3 x, 2 min), CHgOH abs (3x, 2 min), C^C^ (3 x, 2 min). The reaction with ninhydrin is negative.

Liberation of the amino group - See cycle 31.

CYCLE 29 TO 24 The above coupling and liberation processes are used in making react for: - Cycle 29 4 mMoles Boc-glycine (0.71 g) - Cycle 28 4 mMoles Boc-L-ala (0.76 g) - Cycle 27 4 mMoles Boc-glycine (0.71 g) - Cycle 26 4 mMoles Boc-L-valine (0.87 g) - Cycle 25 4 mMoles B-benzyl ester of Boc-L-aspartic acid (1.29 g) - Cycle 24 4 mMoles Boc-O-benzyl-L-threonine (1.24 g).

CYCLE 23 Coupling: The resin-peptide of cycle 24, previously washed with DMF (2 x) is agitated (10 min) with 4 mMoles of Boc-N- -tosyl-L-arginine (1.41 g) in a solution of DMF and the following process is as described for 25 cycle 30.

CYCLE 21 TO 20 Coupling: The resin-peptide of cycle 23 is agitated (10 min) with 4 mMoles of Boc-L-proline (0.88 g) in a medium of CHgC^ (10 ml).

DCCI/H0BT is added as described for cycle 31. At the end of the reaction period, the reaction with ninhydrin was negative.

Liberation - See cycle 31.

CYCLES 21 TO 20 The coupling and liberation processes used in cycle 22 are used in making react: - Cycle 21 4 mMoles Boc-O-Benzyl-L-tyrosine (1.49 g) - Cycle 20 4 mMoles Boc-O-Benzyl-L-threonine (1.24 g).

CYCLE 19 Coupling: The resin-peptide of cycle 20, previously washed with DMF (2 x) is agitated (10 min) with 4 mMoles of Boc-L-glutamine 15 p-nitrophenyl-ester (1.5 g) and 10 ml of DMF with the addition of 1% acetic acid. DCCI (4 mMoles) and H0BT (4 mMoles) are added and the mixture is agitated for 48 hrs. The resin is washed with DMF, CH2C12, CH30H, CH2C12 (2 x).

Liberation - See cycle 31.

CYCLES 18 TO 14 The process for cycle 30 is used in making react: - Cycle 18 - Cycle 17 - Cycle 16 - Cycle 15 30 - Cycle 14 CYCLE 13 The process is as for cycle 19.

CYCLE 12 4 mMoles Boc-L-leucine (1.0 g) 4 mMoles Boc-£-carbobenzyloxy-L-lysine (1.52 g) 4 mMoles Boc-dinitro-2,4 phenyl(im)L-histidine (1.23 g) 4 mMoles Boc-L-leucine (1.0 g) 4 mMoles tf-benzyl ester of Boc-L-glutamic acid (1.3 g).

The process is as for cycle 30 using 4 mMoles of Boc-O-benzyl-L-serine (1.0 g).

CYCLES 11 TO 10 5 The process is as for cycle 30 in making react: - Cycle 11: the derivative used in cycle 18.

- Cycle 10: the derivative used in cycle 17. 2. SYNTHESIS OF THE 1-9 SEQUENCE OF THE PEPTIDE 2a) Partial sequence Boc-L-Thr (Bzl)-Asu OMe (A).

Z-Asu OMe (11.2 g) is introduced into MeOH^O (2:1, v/v) and 15 the mixture is hydrogenated in the presence of palladiated carbon (14 hrs). The catalyst having been eliminated, the mixture is concentrated under vacuum. Dioxan (40 ml) is added, then triethylamine (4.2 ml) is added, while cooling, then Boc-L-Thr (Bzl) OSu (16 g) is added. After agitation (72 hrs), N,N-dimethylamino-20 1,3-propane diamine is introduced, and the mixture is agitated (4 hrs) then concentrated to one third. It is extracted with ethyl acetate (AcOEt), the AcOEt is washed with HCl (IN), then with H2O; then distilled under vacuum. The oily residue is taken up in ether, extracted with NaHCO^ (5%), re-extracted with AcOEt and washed 25 successively with H2O, HCl (IN), H2O. The extracts are dried on anhydrous sodium sulphate and the AcOEt is expelled. A is obtained in the form of an oily product (10 g). 2b) Partial sequence Boc-L-Ser (Bzl)-L-Thr (Bzl) Asu OMe.CHA (B).

Product A (5 g) is dissolved with cooling in TFA (15 ml). At ambient temperature, TFA is expelled under vacuum and the residue is dried under vacuum, then dissolved in DMF (10 ml) and the pH is brought to 6 by triethylamine (4 ml) while cooling. Then H0BT (5 g) 35 and Boc-L-Ser (Bzl) OSu are added with agitation (3 days) at ambient temperature, and at pH 6. N,N-dimethylamino-l,3-propanediamine is added, with agitation, (1 hr), H2O is added, followed by extraction by AcOEt as described above. Cyclohexylamine (CHA) is added to the dried AcOEt, followed by distillation under reduced pressure. The oily product B is obtained (4 g). 2c) Partial sequence Boc-L-Asn-L-Ser(Bzl)-L-Leu-NH-nh2 (C). 3 g of Boc-L-Asn-L-Ser-(Bzl)-L-Leu OEt is dissolved in 20 ml of CH^OH, 10 ml of 80% NH2-NH2-H2O is added, and the mixture is left for 14 hours at ambient temperature. It is then precipitated with ethyl ether, the precipitate is washed with ethyl ether and 10 recrystallized from a mixture of CH^OH-AcOEt-ethyl ether. 2.5 g of expected product is obtained (C). 2d) Partial sequence Boc-L-Asn-L-Ser (Bzl)-L-Leu-L-Ser (Bzl)-L-Thr (Bzl)-Asu OMe (D).

Product B (2 g) is introduced into AcOEt in the presence of HCl (IN); the mixture is dried on anhydrous sodium sulphate and concentrated under vacuum. TFA (6 ml) is added cold, with agitation at ambient temperature (0.5 hr), then the TFA is eliminated under 20 vacuum and the residue is dried under vacuum. The residue is dissolved in DMF (2 ml), and after neutralization with triethylamine, is slowly introduced at -40°C into a solution of C Boc-L-Asn-L-Ser(Bzl)-L-Leu-NH-nh2 (1.7 g) in DMF (6 ml) to which dioxan (2.8 ml) has previously been added, in the presence of HCl 25 (IN) and isoamyl nitrate (0.6 ml). The pH is adjusted to 7 with triethylamine, and reaction takes place at 5°C (72 hrs).

The reactional mixture is slowly introduced at -5°C into 0.5N HCl (60 ml). The precipitate is washed with H2O. Extraction is 30 done by CHC13 (100 ml), the extracts being washed successively with aqueous HCl (IN) NaCl. The CHCl^ is eliminated. Product D is precipitated by a mixture of CHCl^-n-hexane (2 g). 2e) Cyclization -L-Asn-L-Ser (Bzl)-L-Leu-L-Ser (Bzl)-L-Thr (Bzl) 35 Asu1,7-NH-NH2 (E).

Product D (1.6 g) is dissolved in anhydrous pyridine (15 ml). TFA-0NP (2.5 g) is added, with agitation at 45°C (3 hrs), followed by concentration under vacuum, precipitation by ethyl ether, treatment with TFA as above, then cyclization in anhydrous pyridine at 50°C for 5 hours. Extraction is carried out by CHCl^ and the extracts are washed as described above, then concentrated under 5 vacuum, and precipitated by n-hexane.

The precipitate (2 g) is dissolved in DMF (5 ml) and CH^OH (25 ml). 15 ml of hydrazine (80%) is added, the mixture is agitated at ambient temperature (14 hrs) and H20 is added. The precipitate is filtered and washed with H20. CH^OH (50 ml) is added and the mixture is heated to reflux; the product E is precipitated (0.8 g). 2f) Preparation of the 1-9 sequence: -L-Asn-L-Ser (Bzl)-L-Leu-L-Ser (Bzl)-L-Thr (Bzl)-Asu1,7-L-Val-L-Leu-Gly OH (F).

Product E (1 g) is put into suspension in DMF (4 ml), dioxan and 4N HCl (1.7 ml) are added at -5°C. Isoamyl nitrate (0.3 ml) is added at -10°C under agitation. L-H-Val-L-Leu-Gly (0.9 g) is added at -5°, the pH being brought to 7 by triethylamine. The mixture is agitated in an ice bath (48 hrs), then product F (1 g) is precipitated by 0.5N HCl, (150 ml). 3. PREPARATION OF PEPTIDE 3a) H0BT (111 mg) and DCCI (150 mg) are added to product F (500 mg) dissolved in DMF (2 ml). Agitation is carried out for 72 hours in the presence of peptidyl resin 10-31 (300 mg) after elimination of t-Boc of Lys^. 3b) Cleavage of the peptide according to Tam et al. JACS (1983), 105. 6442.

The quantities are referred to 1 g of peptidyl resin. 1) HF (2.5 ml) is distilled in a mixture of resin-peptide (1 g), dimethylsuphide (6.5 ml) and p-cresol (250 mg), with agitation (1 hr) at 0°C. The acid and the dimethylsulphide are expelled under vacuum and the residue is taken up 3 times by 5 ml of AcOEt. > 2) The dried peptidyl resin suspended in dimethylsulphide (1 ml), is treated by liquid HF (10 ml) at 0°C(lh). After elimination of the HF and the dimethylsulphide under vacuum, the resin is washed with ether (3 times, 10 ml); the peptide is taken up with acetic acid, precipitated by ethyl ether, centrifuged cold, washed several times with ether, and dried under vacuum- The precipitate is taken up again by water and the insoluble residue is eliminated by centrifuging. The lyophilized solution produces 500 mg of product. 3c) Purification of the peptide. 200 mg of crude peptide is placed at the top of a column (diameter 2.5 cm, length 90 cm) of Biogel P6, 50-100 mesh. It is eluted by 0.1M acetic acid and fractions of 12 ml are collected by following the elution with a 280 nm recording device. The purified peptide is collected in the fractions 23-28. These fractions are lyophilized and the residue is purified on a Whatman CMC 32 column by ammonium acetate gradient of 0.6 mho to 7 mho, pH 4, with an LKB Ultrograd 11300 apparatus.

After acid hydrolysis, the composition in amino acids is the following, referred to proline = 2 (theoretical figures in brackets): Ala (1) 1.15; Arg (1) 0.85; Asp (2) 2.15; Glu(3) 3.10; Gly (3) 2.9; His (1) 0.83; Leu (5) 5.3; Lys (2) 1.95; Pro (2).0; Ser (3) 3.20; Thr (4) 4.10; Tyr (1) 0.80; Val (2) 2.1; Asu (1) 0.93.

Abbreviations AcOEt = ethyl acetate Asu = o<-aminosuberic acid Bzl = Benzyl CHA = Cyclohexylamine DCHA = Dicyclohexylamine 0NP = p-nitrophenyl ester OSu = N-hydroxysuccinimide ester Z = Benzyloxycarbonyl BIOLOGICAL ACTIVITY OF THE PRODUCTS OF THE EXAMPLES This is determined using male rats weighing from 100 to 120 g and having been without food for 16 hours. The activity is expressed 5 in MRC units starting with the decrease in calcemia, measured one hour after intravenous injection of the peptide under test suitably diluted in a buffer of 0.1N sodium acetate pH 6 and 0.1% albumin, in comparison with the activity of the "Research Standard B" suitably diluted. The biological activity of the peptide of example 1 is 10 higher than 4000 MRC units per mg and that of the peptide of example 2 is higher than 4500 MRC units per mg.

In the light of this activity, the usual dose, which is variable depending on the product used, the subject treated and the cause of 15 infection, could be for example from 1 to 100 MRC units per day intramuscularly or subcutaneously.

Claims

- 24 -CLAIMS

1. New polypeptides, wherein they are chosen from the group formed 5 by the products of formula UA): Cys-Asn-A^-Leu-Ser-Thr-Cys-Ag-Leu-Gly- AirA12"A13*Gln"A15"A16*A17"Lys" A19"A20"Tlir"A22"Pro"A24"T^r"A26~A27" 10 Gly^g-Gly-Ajj-Pro NH2 where: 15 A 20 25 A 30 8 *11 *12 *13 *15 *16 *17 *19 *20 *22 24 *26 *27 *29 31 and = Ser, Gly or Ala = Val or Leu = Lys or Thr = Leu or Tyr = Ser or Thr = Glu or Asp = Leu or Phe = His or Asn = Leu or Phe = Gin or His = Tyr or Phe = Arg or Gin = Asp, Asn or Ala = Val, Thr or lie = Ala, Ser or Val = Thr, Val or Ala the products of formula (Ig): (ch2)5- 35 uO-Asn-A'2-Leu-Ser-Thr-NHtHC0-A'?-Leu-Gly-A'1Q- A'11"A'12*G1n"A'14"A'15"A'16"Lys"A'18" A'~i9"Thr"A'21~Pro~A'23"Thr-A12g-A12g-Gly-A'gg-Gly-A'^-Pro NH2 (In) where: - 25 - A12 = Ser, Gly, or Ala A'^ = Val or Leu 5 A'jq = Lys or Thr A1^ = Leu or Tyr A'12 = ^er or Thr A'14 = Glu or Asp A'^ = Leu or Phe 10 A1^ = His or Asn A'jg = Leu or Phe A'jg = Gin or His A'21 = Tyr or Phe A123 = Arg or Gin 15 A125 = Asp, Asn or Ala A'26 = Val, Thr or lie A'2q = Ala, Ser or Val A130 = Thr, Val or Ala 20

2. New polypeptides corresponding to the formula (IA) as defined in claim 1 in which the symbol Ag represents a valine.

3. New polypeptides corresponding to the formula (Ig) as defined in claim 1 in which the symbol A1^ represents a valine. 25 35

4. The following product with the formula (1^) according to claim 1: Cys-Asn-Ser-Leu-Serg-Thr-Cys-Val-Leu-Glyio-Lys-Leu-Ser-Gln-Gluig-Leu-His-30 Lys-Leu-G^Q-Thr-Tyr-Pro-Arg-Th^jj-Asp-Val-Gly-Ala-G^g-Thr-Proamide.

5. The following product with the formula (Ig) according to Claim 1: ■(ch2)5 CO-Asn-Ser-Leu-Ser-Thrg-NH-CH-CO-Val-Leu-Gly-LySjQ-l-eu- Ser-Gln-Glu-Leuig-His-Lys-Leu-Gln-Th^g-Tyr-Pro-Arg- (Ig) - 26 - Thr-Asp25-Val-G1y-Ala-G1y-Thr2g-Pn3cmri

6. Process for the preparation of the new polypeptides specified in claims 1 to 5, wherein amino acids, peptides or their combinations are 5 submitted to condensation reactions in the order of sequence of the amino acids with the formulae (IA) and (Ig) and during the preparation of the products of the formula (Ig) the solid phase technique for obtaining the 10-31 sequence and the standard liquid phase technique for obtaining the 1-9 sequence are combined. 10

7. As medicaments, the polypeptides mentioned in claims 1 to 5.

8. Pharmaceutical compositions containing one medicament according to claim 7 as active principle. 15

9. The pharmaceutical preparations containing as active principle one or more polypeptides mentioned in claims 1 to 5, as well as appropriate vehicles intended for parenteral administration, oral administration in the form of liposome and nasal administration in the form of a spray. 20

10. A process for the preparation of polypeptides as claimed in claims 1 to 5, substantially as hereinbefore described by way of Example.

11. Polypeptides according to Claim 1 whenever prepared by a process 25 as claimed in Claim 6 or Claim 10. T0MKINS & CO. 30 35