FI87795B - Method for production of therapeutically useful polypeptides - Google Patents

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 87795

A method for preparing novel therapeutically useful polypeptides.

The invention relates to a process for the preparation of novel therapeutically useful polypeptides of formula (IA):

Cys-Asn-A3-Leu-Ser-Thr-Cys-A8-Leu-Gly-AU ~ A12-A13-Gln * A15-A16_A17_LyS-A19-A20- (IA) 10 Thr-A22-Pro-A24-Thr- A26-A27-Gly-A29-Gly-A31

Pro-NH2 where A3 = Ser, Gly or Ala A8 = Val or Leu 15 An = Lys or Thr A12 = Leu or Tyr A13 = Ser or Thr A15 = Glu or Asp A16 = Leu or Phe 20 A17 = His or Asn A19 = Leu or Phe. A20 = Gln or His, A22 = Tyr or Phe A24 = Arg or Gln -'25 A26 = Asp, Asn or Ala A27 = Val, Thr or Ile A29 = Ala, Ser or Vai A31 = Thr, Vai or Ala 30 or formula (1B): • - (CH2) 5 -, CO-Asn-A'2-Leu-Ser-Thr-NHCHCO-A'7-Leu-Gly-A'io-A, uA, i2-Gln-A ' 14-A'15-A'16-Lys-A'18- (IB) 35 A'19-Thr-A'21-Pro-A'23-Thr-A'25-A'26-Gly-A ' 28-. ··. Gly-A130-Pro-NH2 2 87795 tr, .r> i where A'2 = Ser, Gly or Ala A'7 = Val or Leu A '10 = Lys or Thr A'n = Leu or Tyr 5 A112 = Ser or Thr A '14 = Glu or Asp A' 15 = Leu or Phe A'i6 = His or Asn A '18 = Leu or Phe 10 A' 19 = Gln or His A '21 = Tyr or Phe A' 23 = Arg or Gln A'25 = Asp, Asn or Ala A'26 = Val, Thr or Ile 15 A '2g = Ala, Ser or Val A'30 = Thr, Vai or Ala wherein the compounds of formula (IB) contain α-amino -cork acid (Asu) in which the ω -carboxyl is attached to an amino group of 20 Asn.

The products of formula (IB) are structural analogs of the natural peptides presented by K. Jost and J. Rudinger in the case of the pituitary nerve block (Collect. Czech.

; Chem. Comm., 1967, 32, 1229).

The products of formulas (IA) and (IB) may be regrouped into the following formula: D-A'7-Leu-Gly-A '10-A'n-A' 12-Gln-A '14-A' 15- A '16-Lys-A'18-A' 19-Thr-A '21-Pro-A' 23-Thr-A '25-A' 26-GLY-A '28-Gly-A' 30-Pro NH2 30 where the symbols A'7, A'10, A'u <A'12, A'14, A'15, A'16 ^ 18, A 19 'A 21 / A 23' ^ 25 '^ 26' ^ 28 '3a ^' 30 has the meaning given above and D represents: - either the radical Cys-Asn-A3-Leu-Ser-Thr-Cys-, in which the symbol A3 has the meaning given above, - · f 35 - or the radical: 3 87795 I- (CH2) 5-1 CO-Asn-A '2-Leu-Ser-Thr-NHCHCO- wherein A'2 has the meaning given above.

The invention relates in particular to compounds corresponding to the general formulas (IA) and (IB), as defined above, in which A8 and A'7 represent valine.

The invention more particularly relates to compounds which correspond to the following general formulas (IA) and (IB): - Cys - Asn - Ser - Leu - Ser5 - Thr - Cys - Val - Leu -Gly10 - Lys - Leu - Ser - Gln - Glu15 - Leu - His -

Lys - Leu - Gln20 - Thr - Tyr - Pro - Arg - Thr2b -1 15 Asp - Val - Gly - Ala - Gly30 - Thr - proamide | - (CH2) 5-1 - CO-Asn-Ser-Leu-Ser- Thr5-NH-CH-CO-Val-Leu-Gly-Lys10-Leu-Ser-Gln-Glu-Leu15-His-Lys-Leu-Gln-Thr20-Tyr-Pro-Arg-20 Thr-Asp25-Val-Gly -Ala-Gly-Thr30-proamide. - The polypeptides of the invention reduce calcemia h and phosphatemia; they slow bone fragility, agitation; increase the formation of new bones, increase the absorption of calcium in the gut and reduce calcification.

: · They bind calcium inside the cells. They have an important anti-inflammatory and analgesic effect.

These properties support the use of the polypeptides of formulas (ΙΛ) and (IB) as previously defined as medicaments.

• The main indications for the medicines are Paget's disease, osteoporosis of various causes (common, pore softening, cortisone, post-injury, immobility and idiopathic · - ·· * 35 nen), renal bone growth failure, fractures, hypercalcemia. 37795 mias, bone-joint pain, especially those due to bone metastases, spasmophilia, and disorder of calcification metabolism in normal calcemia.

For pharmaceutical use, pharmaceutical compositions are formed which contain at least one product as previously defined as active ingredient.

These pharmaceutical compositions may be, for example, solid or liquid and are in pharmaceutical forms currently used in human medicine, such as simple or sugar-coated tablets, capsules, granules, liposomes, aerosols, suppositories, injectables; they are prepared according to standard methods. The active ingredient or ingredients may be incorporated into excipients commonly used in these pharmaceutical compositions, such as talc, acacia, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous excipients, fats of animal or vegetable origin, various paraffin derivatives, wetting, dispersing or emulsifying agents, and preservatives.

In particular, pharmaceutical compositions as defined above may be formulated for parenteral administration, oral administration in the form of liposomes, or nasal administration in powder form.

The process of the invention for the preparation of novel polypeptides is characterized in that the necessary Y .: amino acids, peptides or combinations thereof are condensed in the order of the amino acid sequences of formulas (IA) and (IB), and in the preparation of products of formula (IB). The solid phase method for obtaining SEQ ID NOs: 10 to 31 and the classical liquid phase method for obtaining SEQ ID NO: 1 to 9 are combined.

As previously described, the syntheses of the peptides of formula (IA) -35 and the products of formula (IB) 10 5 87795-31 are performed using the solidase method. This makes available the use of a benzylhydrylamino resin (PG Pieta and GR Marshall, Chem. Commun., 1970, 650), the preparation and use of which have been described (P. Ri-5, A. Robinson, M. Kamen and G. Milhaud, Helv ., 1971, 54, 2772).

In a preferred process for the preparation of products of formula (IÄ), the group Ν-α-butyloxycarbonyl (t-Boc) protects the α-amino group of amino acids. The terminal groups of amino acids Ia-10 are protected in the form of: 1) as a benzyl ester to the carboxyl chain of aspartic acid and glutamic acid, 2) as a benzyl ether to the hydroxyl of serine and threonine and as a 2,4-dichlorobenzyl ether to tyrosine, 3) as a methoxybenzyl ether to diphyridine histidine to imidazole, 5) as a benzoxycarbonyl group to the? -amino group of lysine.

The various amino acids are coupled to the peptide chain with dicyclo-20-hexylcarbodiimide (DCCI) and hydroxybenzotriazole (HOBT) to provide excess protection against proline. amino acids first reacted sequentially attached to the resin. The reaction is allowed to proceed for two hours. the t-Boc groups are removed with trifluoroacetic acid in methylene chloride-25 solution; after washing with methylene chloride, the neutralization is carried out with triethylamine or diisopropyl-ethylamine (DIEA) in the same solvent.

Asparagine and glutamine are combined in the form of their paranitrophenyl esters in a solution in dimethylformamide * · _: 30 (DMF). If the control test for coupling ninhydrin according to the method of Kaiser and Colescott is positive, the mixture is treated with pivalic acid (1%); if in this experiment the blue color still remains, the DMF / 1% AcOH mixture is saturated with urea. However, even if this experiment is negative - ‘35 (no blue staining), each coupling can be repeated twice systematically. If the coupling is not total after 48 hours of reaction, as is the case with glutamine at position 20, the unreacted threonine is acetylated with acetylimidazole in methylene chloride or acetic acid and DCCl.

Finally, the imidazole protecting group of histidine is removed while the peptide is still attached to the resin, at pH 8, with mercaptoethanol in DMF solution. The peptide is separated from the resin by treatment with liquid hydrofluoric acid in the presence of anisole and methionine. All protecting groups will be removed during this event.

The peptides are purified by gel filtration. The fractions corresponding to the main peaks are pooled, lyophilized, again transferred to buffer at pH 8 and oxidized for 24 hours with a stream of air to form a disulfide bridge between the two cysteine residues.

The fraction corresponding to the peak with the claimed biological activity is again purified with an ion exchange resin CMC 32 with an acid elution gradient. The resulting peptide is homogeneous in paper electrophoresis and cellulose chromatography.

. - Sequence 10 to 31 of the products of formula (IB) is obtained under the same conditions as those previously presented for the preparation of the products of formula (IA). What was shown for glutamine at position 20 in products of formula (IA) applies, of course, to position 19 of formula. *** (IB) compliant products.

In a preferred method of preparing the products of formula (IB), the synthesis of sequences 1-9 is performed in a liquid medium. Fragment:

(CH L.COOR

H-Asn-Λ ', -Leu-Ser-Thr -NH-CH-OO

S77S5 in which the symbol A'2 has the meaning given above and R = ester is condensed after cyclization with the tripeptide A'7-Leu-Gly. Protecting groups commonly used in peptide syntheses are used.

The peptide of formula (IB) is then obtained as follows:

The above protected nonapeptide is converted to the N-hydroxysuccinimide ester, then coupled with fragment 10-31 attached to the resin in DMF medium at 30 ° C. The peptide is separated from the resin by treatment with liquid hydrofluoric acid in the presence of dimethyl sulfide and p-cresol (J.P. Tam, W.H. Heathand and R.B. Merri-field, J.A.C.S., 1983, 105, 6442). All protecting groups will be removed during this event.

Peptides are purified by gel filtration

The biological activity of the products is determined using male rats weighing 100 to 120 g and fasted for 16 hours. Activity is expressed in MRC units from the reduction in calcemia, measured 20 hours after intravenous injection of the test peptide, suitably diluted in 0.1 N sodium acetate buffer at pH 6 and 0.1% albumin, compared to the activity of appropriately diluted "Research Standard B". Example 1; the biological activity of the peptide is greater than 4000 MRC- 25 units per mg and the activity of the peptide of Example 2 is greater than 4500 MRC units per mg. In view of this activity, the usual dose may be: e.g. 1 to 100 MAC units per day administered intramuscularly or subcutaneously, depending on the product used, depending on the disease already being treated. The activity of the compound of Example 1 has been compared to the calcitonin activity of salmon in an in vitro experiment in which bone loss activity is quantified by measuring the number of wells and the area of the samples. The more calcitonin in the experiment reduces s 87795 the number and area of these wells, the more active it is. To perform the experiment, the long bones of newborn rats were removed, scraped with a scalpel, and samples were collected in buffered Hepes solution. The bone tissue suspension thus obtained is immediately contacted with other bone samples. After incubation at 37 ° C, each bone sample is transferred to a sterile vessel and incubated for 18 h in the presence of calcitonin or solvent. The number and area of the wells are determined with a scanning electro-10 microscope. Results are calculated as bone loss activity as a percentage of control sample value. The results are shown in the following table.

'Number of wells Area of wells in 15 bone samples in bone sample (% of control) (% of control)

Salmon calcitonin 20 (0.1 pg / ml) 35% 40%

Product of Example 1 (0.1 pg / ml) 26% 21%: 25 __,

The results show that the modified chicken calcitonin of Example 1 is more active than salmon calcitonin. The reduction in the number of wells is greater for new calcitonin than for known salmon calcitonin; 26% of control wells and 35%, respectively. The difference is then 35%. The corresponding figures for the reduction in surface area are 21% and 40%, which corresponds to a 90% difference.

35 It is known from the literature (Nieto, Moya and R- ·· _ Candela, Biochim. Biophys. Acta, 322, 1973, 383-391) that chicken calcitonin and salmon calcitonin have very similar properties. The compound obtained according to the present invention is thus also more active against chicken calcitonin.

The following examples illustrate the invention.

Example 1 Preparation of a peptide of the following formula (IA)

Cys - Asn - Ser - Leu - Ser5 - Thr - Cys - Vai - Leu -Gly10 - Lys - Leu - Ser - Gin - Glu15 - Leu - His -10 Lys - Leu - Gln20 - Thr - Tyr - Pro - Arg - Thr25 - (IA) Asp - Val - Gly - Ala - Gly30 - Thr -pramide

T-Boc amino acids containing lateral groups come from BACHEM (Dubendorf, Switzerland) or are prepared in the laboratory by the method of Schnabel (E. Schnabel, Liebigs Ann. Chem. 702, 188, 1967) in an autotitrator. Their purity is determined by polarimetry, melting point and thin layer silica gel chromatography.

Hydrolysis of the purified peptide is performed in a sealed tube under vacuum in 6N HCl medium (110 ° C, 24 hours).

I. Sequence synthesis / a) Preparation of resin (P. Rivaille, A. Robinson, M. Kamen and G. Milhaud, Helv., 1971, 54, 2772).

• 25 Benzyl ketone resin In a 2 liter three-necked flask equipped with a stirrer, 50 g of polystyrene with 1% divinylbenzene Bio-rad SX2 (200-400) is allowed to swell for 2 hours in 350 ml of nitrobenzene. In another flask, 66 g of AlCl 3 are dissolved in 300 ml of nitrobenzene to which 80 ml of benzoyl chloride have been added. The mixture is poured into the first flask with good stirring and kept at ambient temperature for 2 hours. If the resin swells too much, add nitrobenzene (about 100 ml). The mixture is filtered, washed with dioxane (3x), acetic acid (3x), methanol (3x) and finally CH 2 Cl 2 and CH 2 OH alternately to make the resin that it swells and shrinks in each case, leaving sufficient contact time. The mixture is dried in a desiccator under vacuum overnight (yield 75 g).

5 Benzhydrylamine resin In a 1 liter flask fitted with a "Dean Stark" and a stirrer, 500 g of ammonium formate are heated to 120-130 ° C for 1-2 hours. The resin (50 g) is added at once and the temperature is allowed to rise to 160 - 165 ° C - 10 over a period of half an hour with efficient stirring, the temperature was then held for 6 hours. The mixture is washed with water, then treated as above in the case of benzyl ketone resin. The vacuum dried resin is heated to reflux and stirred for 8 hours in 6N HCl. Sit-15 is filtered and washed as above. The attachment capacity varies, depending on the nature of the amino acid, between 0.3 and 0.5 mmol / g.

b) Peptide preparation The device used allows mechanical mixing of a 25 ml pyrex tube 20, the middle part of which contains resin (2 g). Reagents (approximately 15 V ml) are to be added and removed from the porous glass filters at the ends of the tube.

The coupling of amino acids comprises the following steps: '25 Cycle 32

Coupling: 2 g of resin (binding capacity 0.5 mmol / g) are stirred (10 min) in CH 2 Cl 2, (CH 3) 2 NHCH (DMF) medium (1: 1, v / v) in the presence of Boc-L-proline (2 mM, 0 .43 g).

DCCI (4 mmol) and 1-hydroxybenzotriaxol sol (HOBT) (4 mmol) are added and stirring is continued for 2 hours. The resin is washed successively with CH 2 Cl 2 (1x, 2 min), CH 3 OH, abs. (3x, 2 min), and with CH 2 Cl 2 (3x, 2 min). The ninhydrin reaction is performed on a resin sample: it is negative.

Μ 07795

Ainino group exemption:

The resin is treated with CF 3 COOH-CH 2 Cl 2 (1: 1, v / v) for 1 min; the mixture is filtered and the run is repeated, allowing contact to take 15 min. The resin is washed with CH 2 Cl 2 (3 x.

5 1 min). CF3COOH is removed by treating the resin twice

Et3N / CH2Cl2 (12.5: 87.5 v / v) (contact time 1 min, then 5 min). It is washed with CH 2 Cl 2 (8 x, 1 min). The L-proline resin is titrated: it contains 0.5 mmol / g proline.

Cycle 31 Coupling: L-prolyl resin (1 mmol) is mixed (10 min) with 4 mmol of Boc-O-benzyl-L-threonine (1.23 g) in CH 2 -C12-DMF (1: 1, v / v) (10 ml). DCCI (4 mmol) and HOBT (4 mmol) are added and stirring is continued (2 h). The resin is washed successively with CH 2 Cl 2 (1x, 2 min), CH 3 OH abs. (3x, 2 min), CH 2 Cl 2 (3x, 2 min). The ninhydrin reaction is negative.

Amino group release - see Cycle 32

Cycles 30 to 25 The above coupling and release methods are used for the following reactions: '. Cycle 30 4 mmol Boc-glycine (0.71 g). Cycle 29 4 mmol Boc-L-ala (0.76 g). Cycle 28 4 mmol Boc-glycine (0.71 g) ': 25. Cycle 27 4 mmol Boc-L-valine (0.87 g): ... Cycle 26 4 mmol Boc-L-aspartic acid B-benzyl ester (1.29 g)

Cycle 25 4 mmol Boc-O-benzyl-L-threonine: (1.24 g). '/ 30 Cycle 24

Coupling:

The resin peptide of cycle 25, previously washed with -: DMF (2x), is mixed (10 min) with 4 mmol of Boc-N-Y-tosyl-L-arginine (1.41 g) in DMF solution and its ... After 35, the process is the same as described for cycle 31.

i2 3 7795

Cycle 23 Wiring:

The peptide resin from cycle 24 is mixed (10 min) with 4 mmol of Boc-L-proline (0.88 g in CH 2 Cl 2) 5 (10 mL). The DCCI / HOBT mixture is added as described for cycle 32. At the end of the reaction period, the ninhydrin reaction is negative.

Release - see Cycle 32 Cycles 22 to 21 10 The coupling and release processes used in Cycle 23 are used for the following reactions:. Cycle 22 4 mmol Boc-O-benzyl-L-tyrosine (1.49 g). Cycle 21 4 mmol Boc-O-benzyl-L-reonine (1.24 g). Cycle 20 15 Wiring:

The resin peptide from Cycle 21, previously washed with DMF (2x), is mixed with 4 mmol of Boc-L-glutamine-p-nitrophenyl ester (1.5 g) and 10 ml of DMF to which 1 % acetic acid. DCCI (4 mmol) and HOBT 20 (4 mmol) are added and the mixture is stirred for 48 hours. The resin is washed with DMF, CH 2 Cl 2, CH 3 OH, CH 2 Cl 2 (2x). Release - see cycle 32 Cycles 19 to 15 The process for cycle 31 is used for the following reactions: "·. Cycle 19 4 mmol Boc-L-leucine (1.0 g)

Cycle 18 4 mmol Boc-carbobenzyloxy-L-lysine d, 52g). Cycle 17 4 mmol Boc-dinitro-2,4-phenyl (im) L-histidine .. 30 (1.23 g). Cycle 16 4 mmol Boc-L-leucine (1.0 g). Cycle 15 4 mmol Boc-L-glutamic acid benzyl ester (1.3 g)

Cycle 14 35 The process is the same as for Cycle 20 i3 87795

Cycle 13

The process is as for cycle 31, using 4 mmol Boc-O-benzyl-L-serine (1.0 g).

Cycles 12-9 5 The process is the same as for Cycle 31 for the following reactions:. Cycle 12: Derivative used in Cycle 19.

. Cycle 11: Derivative used in Cycle 18.

. Cycle 10: Derivative used in Cycle 30.

10. Cycle 9: Derivative used in Cycle 19.

. Cycle 8: Derivative used in Cycle 27.

Cycle 7 The procedure of Cycle 31 is used to react 4 mmol of S-p-methoxybenzyl ether (1.13 g) of Boc-L-cysteine.

Cycles 6-3. Cycle 6: identical to Cycle 31.

. Cycle 5: identical to Cycle 13.

. Cycle 4: identical to Cycle 19.

20. Cycle 3: identical to Cycle 13.

Cycle 2 '· Coupling::. "The peptide resin from Cycle 3, previously washed with DMF (2x), is mixed (48 h) with 4 mmol of Boc-: · 25 L-asparagine p-nitrophenyl ester ( 1.45 g) in DMF medium in the presence of HOBT (4 mmol) The resin is washed successively with DMF, CH 2 Cl 2, CH 3 OH, CH 2 Cl 2 The ninhydrin reaction is negative.

; Release 30 The process is the same as described for cycle 32.

- Cycle 1 3 mmol of S-p-methoxybenzyl ether of Boc-L-cysteine (1.13 g) are reacted under the conditions of Cycle 31.

: II - Elimination of the dinitrophenyl group of histidine ... 35 2 ml of mercaptoethanol are added to 15 ml of dimethyl4,77795 liformamide (DMF), and the pH is adjusted to 8 with triethylamine. This reagent is introduced into the apparatus and the mixture is stirred for 14 hours. The resin is filtered and washed alternately with methylene chloride (3x) and methanol (3x).

5 After drying in vacuo, the weight of the resin is 2.85 g.

III. Removal of the peptide from the resin and removal of the protecting groups 15 ml of hydrofluoric acid are added dropwise to a mixture of 1.5 g of the resin peptide and 1.5 ml of anisole. The mixture is stirred for 1 h at 30 ° C and for 30 min at ambient temperature. The acid is separated off in vacuo and the residue is taken up again 3 times in 5 ml of acetic acid and mixed with ethyl ether to free the peroxide. The precipitate is centrifuged cold, washed several times with ether, and dried in vacuo. The precipitate is again transferred to water and the insoluble residue is removed by centrifugation. The lyophilized solution gives 800 mg of product.

IV - Peptide Purification 200 mg of crude peptide is placed on the top of a Biogel P6-20 column (diameter 2.5 cm, length 90 cm) (50-100 mesh) and eluted with 0.1 M acetic acid and collected in a 12 '· ml fractions by elution with a 280 nm recorder. The purified peptide is collected from fractions 23-28.

• 25 V- Cyclization

Fractions containing purified peptide are lyophilized and the residue is again transferred to 500 ml of 1M ammonium bicarbonate buffer, pH 8, and subjected to. : air bubbling through a porous plate for 24 hours. The solution is lyophilized and the residue is purified on a CMC 52 Whatman column with an ammonium acetate gradient from 0.6 mho to 7 mho, pH 4, on an LKB Ultrograd 11300. After acid hydrolysis, the amino acid composition is as follows, referring to proline = 2 (theoretical numbers in parentheses): 15 87795

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; Or (2) 2.1.

5

Example 2

Preparation of a peptide of the following formula (IB): (CH2) 5 10 CO-Asn-Ser-Leu-Ser-Thr5-NH-CH-CO-Val-Leu-Gly-Lys10-Leu

Ser-Gln-Glu-Leu15-His-Lys-Leu-Gln-Thr20-Tyr-Pro-Arg- (IB) Thr-Asp25-Val-Gly-Ala-Thr30-proamide t-Boc amino acids with lateral groups , 15 is obtained as shown in Example 1.

In a similar manner, hydrolysis of the purified peptide is performed in a sealed tube under vacuum in 6N HCl medium (110 ° C, 24 hours).

Synthesis of peptide sequence 10-31 20a) Preparation of resin (P. Rivaille, A. Robinson, M. Kamen and G. Milhaud, Helv., 1971, 54, 2772).

Benzyl ketone resin In a 2 l three-necked flask equipped with a stirrer, 50 g of polystyrene containing 1% divinylbenzene Bio-rad SXx (200-400 mesh) are swollen in 350 ml of nitrobenzene. In another flask, 66 g of AlCl3 are dissolved in 300 ml of nitrobenzene to which 80 ml of benzoyl chloride have been added. The mixture is poured; to the first flask with good stirring and keep for two I .. 30 hours at ambient temperature. If the resin swells too much, add nitrobenzene (about 100 ml). The mixture is filtered, washed with dioxane (3x), acetic acid (3x), methanol - *: * (3x) and finally alternately with CH 2 Cl 2 and CH 3 OH to cause the resin to swell and shrink in each case, leaving sufficient contact time. . The mixture is dried overnight. The mixture is dried overnight (yield 75 g).

i6 87795

Benzhydrylamine resin In a 1 liter flask equipped with a "Dean Star" and a stirrer, 500 g of ammonium formate are heated to 120-130 ° C for 1-2 hours. The resin (50 5 g) is added at a time and the temperature is allowed to rise to 160-165 ° C for half an hour with vigorous stirring, and allowed to stand for 6 hours. The mixture is filtered, washed with water, then treated as above in the case of benzyl ketone resin. The vacuum dried resin is heated to reflux for 8 hours in 6N HCl. It is then filtered and washed as before. The attachment capacity varies between 0.3 and 0.5 mmol / g depending on the nature of the amino acid.

Ib) Preparation of Peptide Sequence 10-31 In the apparatus used, it is possible to mechanically mix a 25 ml pyrex tube with a resin (2 g) in the middle. From the 2 porous glass filters at the ends of the tube, the reagents (about 15 ml) are to be added and then removed.

20 Cycle 31

Coupling: 2 g of resin (binding capacity 0.5 mmol / g) are stirred (10 min) in CH 2 Cl 2 '(CH 3) 2 NCHO (DMF) (1: 1, v / v) in the presence of Boc-L-pro] (? mmol, 0.43 g).

.25 DCCl (4 mmol) and 1 hydroxybenzotieto a tso.L ia (HOBT) (4; · .. mmol) are added and stirring is continued for 2 hours. The resin is washed successively with CH 3 OH (1x, 2 min), CH 3 OH, abs. (3x, 2 min), with CH 2 Cl 2 (3x, 2 min). Ninhydrin reaction. : performed on a resin sample: it is negative.

-.30 Amino group exemption:

The resin is treated with CF 3 COOH-CH 2 Cl 2 (1: 1, ... "v / v) for 1 min; the mixture is filtered and the procedure is repeated to allow contact for 15 min. The resin is washed with CH 2 Cl 2 (3 ...: x, 1 min) CF3C00H is removed by treating the resin twice with Et3N / CH2Cl2 (12.5: 87.5 v / v) (contact time i7 87795 1 min, then 5 min) The mixture is washed with CH2Cl2 (8 x, 1 The resin L-proline is titrated: it contains 0.5 mmol / g of proline.

Cycle 30 5 Wiring:

Resin L-prolyl (1 mmol) is mixed with 4 mmol of Boc-O-benzyl-L-threonine (1.23 g) in CH 2 Cl 2 -DFF (1: 1 v / v) (10 mL). DCCI (4 mmol) and HOBT (4 mmol) are added and stirring is continued (2 h). The resin is washed 10 times in succession: CH 2 Cl 2 (1 x, 2 min), CU, 0H abs. (3x, 2 min), CH 2 Cl 2 (3x, 2 min). The ninhydrin reaction is negative.

Amino group release - see Cycle 31

Cycles 29 to 24 Use the above coupling and release processes-15 and to react with the following substances:. Cycle 29 4 mmol Boc-glycine (0.71 g). Cycle 28 4 mmol Boc-L-ala (0.76 g). Cycle 27 4 mmol Boc-glycine (0.71 g). Cycle 26 4 mmol Boc-L-valine (0.87 g) 20. Cycle 25 4 mmol Boc-L-aspartic acid B-benzyl ester-. . (1.29 g) •. Cycle 24 4 mmol Boc-O-benzyl-L-threonine (1.24 g).

Cycle 23

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

· Cycle 22 ... 30 Wiring:

The resin peptide of cycle 23 is mixed (10 min) with 4 ... "mmol of Boc-L-proline (0.88 g) in CH 2 Cl 2 (10 mL). DCCI / HOBT is added as described for cycle 31. At the end of the reaction period, the ninhydrin reaction was negative. 35 Exemption - see Cycle 31 is f 37795.: 4; ' \ .. -

Cycles 21 to 20 The coupling and release processes used in Cycle 22 are used to react the following substances:. Cycle 21 4 mmol Boc-O-benzyl-L-tyrosine (1.49 g) 5. Cycle 20 4 mmol Boc-O-benzyl-L-threonine (1.24 g)

Cycle 19 Wiring:

The resin peptide from cycle 20, previously washed with DMF (2x), is mixed (10 min) with 4 mmol of Boc-10 L-glutamine-p-nitrophenyl ester (1.5 g) and 10 mL of DMF; to which 1% acetic acid has been added. DCCI (4 mmol) and HOBT (4 mmol) are added and the mixture is stirred for 48 hours. The resin is washed with DMF, CH 2 Cl 2, CH 3 OH, CH 2 Cl 2 (2x).

15 Release - see Cycle 31

Cycle 18 _-_ 14

The process used for cycle 30 is used to react the following substances:. Cycle 18 4 mmol Boc-L-leucine (1.0 g) 20. Cycle 17 4 mmol Boc-t-carbobenzyloxy-L-lysine (1.52 g). Cycle 16 4 mmol Boc-dinitro-2,4-phenyl (im) L-histidine (1.23 g). Cycle 15 4 mmol Boc-L-leucine (1.0 g) 25. Cycle 14 4 mmol γ-benzyl ester of Boc-L-glutamic acid (1.3 g)

Cycle 13

The process is the same as for cycle 19.

Cycle 12 30 The process is the same as for Cycle 30 using 4 mmol

Boc-O-benzyl-L-serine (1.0 g).

Cycles 11 and 10

The process is the same as for cycle 30 of the following ai-. · To respond to them:. Cycle 11: Derivative used in Cycle 18.

. Cycle 10: Derivative used in Cycle 17.

19 87795 2. Synthesis of Peptide Sequence 1-9 2a) Partial Sequence Boc-L-Thr (Bzl) -Asu OMe (A) Z-Asu OMe (11.2 g) is taken up in a mixture of MeOH-H 2 O (2: 1, v / v). v) and the mixture is hydrogenated in the presence of palladium on carbon (14 h).

After removal of the catalyst, the mixture is concentrated in vacuo. Dioxane (40 ml) is added, then triethylamine (4.2 ml) is added while cooling, then Boc-L-Thr (Bzl) OSu (16 g) is added. After stirring (72 h), N, N-dimethylamino-1,3-propane-10 didiamine is stirred, and the mixture is stirred (4 h) and then concentrated to one third of its volume. It is extracted with ethyl acetate (AcOEt), then the AcOEt is washed with HCl (IN), then with H 2 O; then distilled in vacuo. The oily residue is taken up in ether, extracted with NaHCO3 (5%), re-extracted with AcOEt and washed successively with H2O, HCl (IN), H2O. The extracts are dried over anhydrous sodium sulfate and the AcOEt is separated. A is obtained in the form of an oily product (10 g).

2b) Partial sequence Boc-L-Ser- (Bzl) -L-Thr- (Bzl) Asu όμθ.οηα (b).

Product A (5 g) is dissolved in TFA (15 ml) under cooling. At ambient temperature, the TFA is removed in vacuo and the residue is dried in vacuo, then dissolved in DMF (10 μl) and the pH is adjusted to 6 with triethylamine (4 ml) under cooling. HOBT (5 g) and Boc-L-Ser (Bzl) OSu are then added with stirring (3 days) at ambient temperature and pH 6. Add N, N-dimethylamino-1,3-propanediamine with stirring (1 h), add H 2 O, and then extracted.

: With AcOEt as described above. Cyclohexylamine 30 (CHA) is added to dry AcOEt and distilled under reduced pressure. Obtained as an oily product B (4 g).

2c) Partial sequence Boc-L-Asn-Ser (Bzl) '-L-Leu-NH-Ni -'! 2 (C).

Dissolve 3 g of Boc - L - Asn - L - Ser - (Bzl) - L - Leu OEt in 20. To 35 ° C-20 h, 10 ml of 80% NH 2 NH 2 H 2 O are added and the mixture is left for 20 37 79 5 14 hours at ambient temperature. It is then mixed with ethyl ether, the precipitate is washed with ethyl ether and recharged with CH3OH-AcOEt-ethyl ether. 2.5 g of the expected product (C) are obtained.

5 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 added to AcOEt in the presence of HCl (IN); the mixture is dried over anhydrous sodium sulfate and concentrated in vacuo. TFA (6 mL) is added cold with stirring at ambient temperature (0.5 h), then the TFA is removed in vacuo and the residue is dried in vacuo. The residue is dissolved in DMF (2 ml) and, after neutralization with triethylamine, is slowly added at -40 ° C to a solution of C Boc-L-Asn-L-Ser (Bzl) -L-Leu-NH-NH 2 (1.7 g) in DMF (6 ml) previously treated with dioxane (2.8 ml), HCl (1N) and isoamyl nitrite (0.6 ml). The pH is adjusted to 7 with triethylamine and the reaction takes place at 5 ° C (72 h).

The reaction mixture is slowly introduced at -5 ° C into 0.5N HCl (60 mL). The precipitate is washed with H 2 O. Extraction is performed. . CHCl 3 (100 mL), and the extracts are washed sequentially with aqueous HCl (1N), NaCl. CHCl3 is removed.

Product D is mixed with CHCl 3 -n-hexane (2 g). Cyclization of (2e) -L-Asn-L-Ser (Bzl) -L-Leu-L-Ser (Bzl) -L-Thr (Bzl) Asu ^ -NH-'25 NH2 (E)

Product D (1.6 g) is dissolved in anhydrous pyridine (15 ml). TFA-ONP (2.5 g) is added, with stirring at 45 ° C (3 h), then concentrated in vacuo, precipitated with ethyl ether, treated with TFA as above, and then. 30 is cyclized in anhydrous pyridine at 50 ° C for 5 hours.

The extraction is performed with CHCl 3 and the extracts are washed as above, concentrated in vacuo, and precipitated with n-hexane.

The precipitate (2 g) is dissolved in DMF (5 ml) and CH 3 OH. 35 hon (25 mL). 15 ml of hydrazine (80%) are added, the mixture 21 87795 is stirred at ambient temperature (14 h) and H 2 O is added. The precipitate is filtered and washed with H 2 O. CH 3 OH (50 mL) is added and the mixture is heated to reflux; product E precipitates (0.8 g).

2 2f) Preparation of SEQ ID NO: 1-9: -L-Asn-L-Ser (Bzl) -L-Leu- L-Ser- (Bzl) -L-Thr (Bzl) -Asul, 7-L-Val-L- Leu-Gly OH (F)

Product E (1 g) is suspended in DMF (4 mL), dioxane and 4N HCl (1.7 mL) are added at -5 ° C. Isoamyl nitrite (0.3 ml) is added at -10 ° C with stirring. L-H-10 Val-L-Leu-Gly (0.9 g) is added at -5 ° C with pH adjusted to 7 with triethylamine. The mixture is stirred in an ice bath (48 h), then product F (1 g) is mixed with 0.5N HCl (150 mL).

3. Peptide preparation

3a) HOBT (111 mg) and DCCL (150 mg) are added to product F

(500 mg) dissolved in DMF (2 ml). Stir for 72 hours in the presence of peptidyl resin 10-31 (300 mg) after removal of Lys10 t-Boc.

3b) Peptide cleavage according to Tam et al., JACS (1983), 20 105, 6442.

The amounts refer to 1 g of peptidyl resin.

1) HF (2.5 ml) is added dropwise to a mixture of resin peptide (1 g), dimethyl sulfide (6.5 ml) and p-cresol (250 mg) with stirring (1 h) at 0 ° C. The acid and dimethyl sulfide are separated in vacuo and the residue is taken up 3 times in 5 ml; AsOEt.

2) The dried peptidyl resin suspended in dimethyl sulfide (1 mL) is treated with liquid HF (10 mL) at 0 ° C (1 h). HF and dimethyl sulfide in vacuo. After removal, the resin is washed with ether (3 times 10 ml); the peptide is taken up in acetic acid, precipitated with ethyl ether, centrifuged cold, washed several times with ether, and dried in vacuo. The precipitate is transferred again to water and the insoluble residue is removed by centrifugation. Lyophilization of the solution gives 500 mg of product.

22 87795 3c) Peptide purification 200 g of crude peptide are applied to the top of a 50-100 mesh Biogel P6 column (diameter 2.5 cm, length 90 cm). It is eluted with 0.1M acetic acid and collected in a 12 ml fraction following elution with a 280 nm recorder. The purified peptide is collected in fractions 23 to 28. These fractions are lyophilized and the residue is purified on a Whatman CMC 32 column with an ammonium acetate gradient from 0.6 mho to 7 mho, pH 4, on an LKB Ultrograd 11300.

10 After acid hydrolysis, the amino acid composition is as follows, referring to proline = 2 (theoretical numbers in parentheses):

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; 15 Pro (2) 2.0; Ser (3) 3.20; Thr (4) 4.10; Tyr (1) 0.80;

Or (2) 2.1; Asu (1) 0.93.

abbreviations:

AcOEt = ethyl acetate

Asu = α-amino-cork acid 20 Bzl = benzyl CHA = cyclohexylamine DCHA = dicyclohexylamine • ONP = p-nitrophenyl ester · - OSu = N-hydroxysuccinimide ester '25 Z = benzyloxycarbonyl

Claims (5)

23 87795 A process for the preparation of novel therapeutically useful polypeptides of formula (IA): Cys-Asn-Aj-Leu-Ser-Thr-Cys-Ag-Leu-Gly-A11-Ai2-A13-Gln-A15-A16- A17-Lys-Aig-A2o- (1A) Thr-A22-Pro-A24-Thr-A26-A27-Gly-A29-Gl'y-A31-Pro-NH2 10 where A3 = Ser, Gly or Ala Αθ = Vai or Leu An = Lys or Thr A12 = Leu or Tyr 15 Aj = Ser or Thr A15 = Glu or Asp A16 = Leu or Phe A17 = His or Asn A19 = Leu or Phe 20 A20 = Gln or His. A22 = Tyr or Phe A24 = Arg or Gln •; A26 = Asp, Asn or Ala A27 = Vai, Thr or Ile • .'25 A29 = Ala, Ser or Vai i '· A31 = Thr, Vai or Ala or formula (IB): .-: -. 30 | - ( CH2) 5 -1; CO-Asn-A '2-Leu-Ser-Thr-NHCHCO-A' 7-Leu-Gly- A '10-A' n - A '12-Gln-A' 14 - A '15-A' 16 - Lys-A '18- (IB) "" A' 19-Thr-A '21 -Pro-A' 23-Thr-A '/ sA' X (i-Gly-A '28- *: ·! Gly- A 130-Pro-NH2 35 87795 where A'2 = Ser, Gly or Ala A'7 = Val or Leu A'10 = Lys or Thr A'n = Leu or Tyr 5 A'12 = Ser or Thr A114 = Glu or Asp A'15 = Leu or Phe A '16 = His or Asn A' 18 = Leu or Phe 10 A11? = Gln or His A1 = Tyr or Phe A'23 = Arg or Gln A ',,, = Asp, Asn or Ala A' 2l, = Val, Thr or Ile A12β = Ala, Ser or Val A '30 = Thr, Val or Ala wherein the compounds of formula (IB) contain an α-amino-capric acid (Asu) in which the ω-carboxyl is attached to the amino group of 20 Asn, characterized in that you need-. . amino acids, peptides or combinations thereof are fused to the amino acid sequences of formulas (IA) and (IB); and in the preparation of products of formula (IB), a solid phase method for obtaining sequences 10 to 31 and a classical liquid phase method for obtaining sequences 1-9 are combined. A method according to claim 1, characterized in that a polypeptide of formula (IA) is prepared, wherein the symbol A8 is intermediate. Process according to Claim 1, characterized in that a polypeptide of the formula (IB) is prepared in which the symbol A'7 is an intermediate. Process according to Claim 1, characterized in that a product of formula (IA) is prepared: 25 87795 Cys-Asn-Ser-Leu-Ser5-Thr-Cys-Val-Leu-Gly10-Lys-Leu-Ser- Gln-Glu15-Leu-His-Lys-Leu-Gln20- (IA) Thr-Tyr-Pro-Arg-Thr2ii-Asp-Val-Gly-Ala-Gly30-5 Thr-proamide Process according to Claim 1, characterized in that a product of formula (IB) is prepared: 10, - (CH 2) 5-1 CO-Asn-Ser-Leu-Ser-Thr 5 -NH-CH-CO-Val -Leu-Gly-Lys10-Leu-Ser-Gln-Glu-Leu15-His-Lys-Leu- (IB) Gln-Thr20-Tyr-Pro-Arg-Thr-Asp2, -Val - Gly-Ala-15 Gly- Thr30-proamide 26 87795