FI109541B - A process for the preparation of bradykinin antagonists - Google Patents

Abstract

Peptides of the formula I A-B-C-E-F-K-(D)-Tic-G-M-F'-I (I> in which A is hydrogen, alkyl, alkanoyl, alkoxycarbonyl, alkylsulphonyl, cycloalkyl, aryl, arylsulphonyl, heteroaryl or an amino acid, each of which can optionally be substituted, B is a basic amino acid, C is a di- or tripeptide, E is the residue of an aliphatic or alicyclic-aliphatic amino acid, F is, independently of one another, an amino acid which is optionally substituted in the side chain or is a direct bond, G is an amino acid, F' is defined as F, can be -NH-(CH2)2-8 or a direct bond, I is -OH, -NH2 or -NHC2H5, and K is a radical -NH-(CH2)1-4-CO-, or is a direct bond, have a bradykinin-antagonistic action. Their therapeutic uses comprise all pathological states which are mediated, induced or assisted by bradykinin and bradykinin-related peptides. The peptides of the formula I are prepared by known methods of peptide synthesis.

Description

109541

A process for the preparation of bradykinin antagonists

The invention relates to a process for the preparation of novel peptides having bradykinin antagonistic activity.

WO 86/07263 describes bradykinin antagonist peptides in which e.g. the 7-position L-Pro of the peptide hormone bradykinin or other bradykinin analogs has been replaced by a D-amino acid such as D-Phe, D-Thia, 10 D-Pal, CDF, D-Nal, MDY, D-Phg, D-His, D-Trp, D-Tyr, D-hPhe, D-Val, D-Ala, D-His, D-Ile, D-Leu or DOMT.

It is an object of the invention to provide novel effective peptides having a bradykinin antagonist activity.

15 This task is solved by preparing peptides,

of formula I

ABCEFK- (D) -Tic-GM-F'-I (I) wherein A is (D) -H-Arg, B is Arg C is Hyp-Pro-Gly or Pro-Hyp-Gly, · · ·. .. E is Leu, Tbg or Cha, •, 25 F is Ser, K is a direct bond, • 'M is a direct bond, • · ·

ί ί: (D) -Tic is a group of formula V

H O

(V) • G is cis-endo-octahydroindole-2-carboxylic acid; '... * F' is Arg and 2 109541 I is -OH.

Examples of particularly preferred peptides of formula I are: H- (D) -Arg-Arg-Pro-Hyp-Gly-Leu-Ser- (D) -Tic-Oic-Arg-OH 5 H- (D) -Arg- Arg-Pro-Hyp-Gly-Cha-Ser- (D) -Tic-Oic-Arg-OH H- (D) -Arg-Arg-Pro-Hyp-Gly-Tbg-Ser- (D) -Tic-Oic Arg-OH.

The process of the invention for the preparation of bradykinin antagonists of the formula I

ABCEFK- (D) -Tic-GM-F'-I (I) wherein A is H- (D) -Arg, B is Arg, 15 C is Pro-Hyp-Gly or Hyp-Pro-Gly, E. is Leu, Tbg or Cha, F is Ser, K and M represent a direct bond,

(D) -Tic is a group of formula V

20 HO

m ocr <v »25 G is Oic, *» 1 »1 F 'is Arg, and I is -OH, and their physiologically acceptable salts are characterized in that. ; A) a fragment having a C-terminal free carboxyl group or an activated derivative thereof · · is reacted with a corresponding fragment having an N-terminal group; is a free amino group, or • 1 · · · · · 3 109541 b) the peptide is formed stepwise, optionally removing one or more protecting groups temporarily attached to protect the other groups from the compound obtained in (a) or (b); The compounds of formula I are optionally converted into their physiologically acceptable salts.

The peptides were prepared according to the present invention by well known peptide chemistry methods, see e.g. Houben-Weyl, Method 15, Band 15/2, preferably by solid phase synthesis as described e.g. in B. Merrifield, J. Am. Chem. Soc. 8: 5, 2149 (1963) or R.C. Sheppard, Int. J. Peptide Protein Res. 21, 118 (1983), or similar methods known in the art, 15 urethane protecting groups such as, for example, tert-butyloxycarbonyl (Boc) or fluorenylmethyloxycarbonyl (Fmoc) protecting group are used. If necessary to prevent side reactions or in the synthesis of specific peptides, the side chain functional groups of the amino acids are further protected with suitable protecting groups (see, e.g., TW Greene, "Protective Groups in Organic Synthesis"), with Arg (Tos) being preferred. ), Arg (Mts), Arg (Mtr), Arg (PMC),! *. Asp (OBzl), Asp (OBut), Cys (4-MeBzl), Cys (Acm), Cys (SBut),

Glu (OBzl), Glu (OBut), His (Tos), His (Fmoc), His (Dnp), ', 25 His (Trt), Lys (Cl-Z), Lys (Boc), Met (0), Ser (Bzl), Ser (But),

Thr (Bzl), Thr (But), Trp (Mts), Trp (CHO), Tyr (Br-Z), '· Tyr * (Bzl) or Tyr (But).

The solid phase synthesis begins by attaching a protected amino acid at the C-terminus of the peptide to the corresponding resin. Such starting materials may be obtained by attaching a protected amino acid by an ester or amide bond to a chloro-methyl, hydroxymethyl, benzhydrylamino (BHA) or methyl-benzhydrylamino (MBHA)-modified polystyrene or polyacrylamide resin. Resins for use as carrier material 35 are commercially available. Typically, BHA and MBHA resins are used when synthesizing the t-* 4 109541 ring peptide must have a free carbamoyl group. In the event that the C-terminus of the peptide must have a secondary amide group, a chloromethyl or hydroxymethyl resin is used and cleavage is performed with the corresponding amines. If it is desired to provide, for example, ethylamide, the ethylamine-containing peptide may be cleaved from the resin, followed by removal of the side chain protecting groups with other suitable reagents. If the amino acid side-chain tert-butyl protecting groups on the peptide are to be retained, then the synthesis is carried out using an Fmoc protecting group for the temporary protection of the α-amino group of the amino acid and using, for example, the procedure described in R.C. Sheppard, J. Chem. Soc., Chem. Comm. 1982, 587, whereby the guanidino group of arginine is protected by protonation with pyridinium perchlorate, and the protection of other amino acids having side-chain functionalities is achieved by catalytic transfer hydrogenation (A. Felix et al., J. Org. Chem. L3 (1978) 4194) or benzyl protecting groups. sodium in liquid ammonia (W. Roberts, J. Am. Chem. Soc., 6, 6203 (1954)).

A: When the amino protecting group of an amino acid attached to a resin is removed with a suitable reagent such as e.g.

• trifluoroacetic acid in methylene chloride for Boc protecting group, or 20% piperidine in dimethylformamide for Fmoc protecting group, the following protected amino acids are added sequentially! I go in the order I want. Intermediate N-terminal protected peptide resins are deprotected. With the reagents described above before combining with the following amino acid derivative.

*; · All possible reagents can be used as coupling reagent.

• I

I: For activating reagents for peptide synthesis, see, e.g., Houben-Weyl, Methods 35, Chem 15, Band 15/2, but especially carbodiimides such as N, N '-dicyclohexylcarbodiimide, N, N '-diisopropyl · 10 · 109541 -cylcarbodiimide or N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide. The coupling can then be carried out directly by the addition of an amino acid derivative with an activating reagent and optionally with the addition of a racemization inhibitor such as 1-hydroxybenzotriazole (HOBt) (W. König, R. Geiger, Chem. Ber. 103 (1970) 708). or 3-hydroxy-4-oxo-3,4-dihydrobenzotriazine (HOObt) (W. König, R. Geiger, Chem. Ber. 103, 2054 (1970)), as a resin, or also as a symmetric anhydride or HOBt or 10 HOObt. the preactivation of the esteric amino acid derivative can be carried out separately, and a solution of the activated agents in a suitable solvent is added to the peptide resin to which the coupling may occur.

The coupling or corresponding activation of the amino acid derivatives with one of the above activation reagents can be carried out in dimethylformamide, N-methylpyrrolidone or methylene chloride or in a mixture of said solvents. The activated amino acid derivative is usually added in excess of 1.5 to 4 times. In cases where incomplete coupling occurs, the coupling reaction is repeated without first removing the α-β amino protecting groups of the peptide resins necessary for the insertion of the following V: amino acid.

* · ... # The successful course of the coupling reaction can be • 1 1 ', 25 studied by the ninhydrin reaction as described e.g.

in E. Kaiser et al. Anal. Biochem. 59, 595 (1970).

* · Synthesis can also be performed automatically using, for example, the Model 430A peptide synthesizer from Applied Biosystems from the device manufacturer, ·,; 30 synthesis programs obtained or available to the user.

* · 1 ... "The latter are particularly used when using * | 'amino acid derivatives protected by Fmoc group.

»» <1 »» f 1 t »t» i i »•« I · »tl • I I» t »·» • »fi 109541

Once the peptide synthesis is carried out as described above, the peptide can be cleaved from the resin by reagents such as liquid hydrogen chloride (preferred for Boc-prepared peptides) or trifluoroacetic acid (preferred for Fmoc-synthesized peptides). These reagents not only cleave the peptide from the resin but also other side chain protecting groups of the amino acid derivatives. In this way, the peptide is obtained as free hap-10 pona, except in the case of BHA and MBHA resins. BHA or MBHA resins provide the peptide as the acid amide when cleaved with hydrogen fluoride or trifluoromethanesulfonic acid. Other methods for the preparation of peptide amides are described in EP-A 287 882 and EP-A 322 348. The cleavage of peptide amides from the resin is then carried out by treatment with medium acids (e.g. trifluoroacetic acid) commonly used in peptide synthesis to add cation , anisole, thioanisole, ethanedithiol, dimethylsulfide, ethylmethylsulfide or the like in solid phase synthesis:! ordinary cationic binders, alone or in admixture with two or more such adjuvants. Tri- *: *. Fluoroacetic acid can also be used as a suitable solvent, such as methylene chloride, diluted.

If the tert-butyl or benzyl side chain protecting groups of the peptides are to be retained, then the peptide synthesized on the specially modified carrier resin is cleaved with 1% trifluoroacetic acid, as described in e.g. RC Sheppard, J. Chem. Soc., Chem. Comm. 1982, 587. If individual tert-butyl or benzyl side chain protecting groups are to be retained, a suitable combination of synthesis and cleavage methods is used.

A modified carrier resin as described by Sheppard is also used to synthesize peptides having a carbamoyl group or an ω-amino or a) -guanidinoalkyl group at the 7-109541 C terminus. After synthesis, the fully protected peptide from its side chain is cleaved from hart-5 and then reacted with the conventional one; by solution synthesis with the corresponding amine or co-aminoalkylamine or co-guanidinoalkylamine, respectively, whereby any other functional groups present may be temporarily protected in a known manner.

A further method for preparing peptides containing the? -Aminoalkyl group is described in EP-A 264 802.

In accordance with the present invention, the peptides were preferably synthesized using solid phase technique according to two general protecting group procedures:

Synthesis was performed on Applied Biosystems Automated Peptide Synthesizer Model 430 A using Boc or Fmoc protecting groups for the temporary protection of the α-amino group.

When using the Boc protecting group, pre-programmed synthesis cycles obtained for synthesis: from the device manufacturer were used.

Peptides having C-terminal free carboxyl; group, was synthesized with the corresponding Boc amino acid functionalized 4- (hydroxymethyl) phenylacetamidomethylpolystyrene resin of the Applied Biosystems (R.B.

'' Merrifield, J. Org. Chem. 4: 3, 2845 (1978). MBHA resin from the same company was used to prepare the peptide amides V · '. The activating reagents used were N, N'-dicyclohexylcarbodiimide or N, N'-diisopropylcarbodiimide. Activation, ···, was performed as a symmetric anhydride, HOBt ester or t HOObt ester in CH 2 Cl 2, a mixture of ClbCl 2 and DMF or • · • t · NMP. 2-4 equivalents were used for the coupling reaction: ***: activated amino acid derivative. In cases where the · · · .h 35 attachment was incomplete, the reaction was repeated * ··· *.

"109541

When the Fmoc protecting group was used for the temporary protection of the α-amino group, its own synthesis programs were used for synthesis using the Modell 430A automatic peptide synthesizer from Applied Biosystems.

The synthesis was carried out on Bachem p-benzyloxybenzyl alcohol resin (S. Wang, J. Am. Chem. Soc. 95, 1328 (1973)) which had been esterified according to known methods (E. Atherton et al., JCS Chem. Comm. 1981, 336) with the corresponding amino acid. Activation of amino acid derivatives 10 as an HOBt or HOObt ester was performed directly on the amino acid cartridges provided by the device manufacturer by adding a solution of diisopropylcarbodiimide in DMF to a pre-weighed mixture of the amino acid derivative and HOBt or HOObt. Likewise, Fmoc amino acid OObt esters prepared in the material as described in EP-A 247 573 may be used. The Fmoc protecting group cleavage was performed with a 20% solution of piperidine in DMF in a reaction vessel. The excess reactive amino acid derivative used was 1.5 to 2.5 equivalents. If the 20 attachment was incomplete, it was repeated as in the Boc method.

• ·

The peptides prepared according to the invention, alone or in combination, have a bradykinin antagonist *: ·. effect that can be studied with different models (see.

> Tti. 25 Handbook of Exp. Pharmacol. Butter. 25, Springer Verlag, 1970, pp. 53-55) such as, for example, isolated rat uterus, guinea pig ileum or isolated guinea pig lung: Timo.

In order to study the peptides <'· [30] of the invention in isolated pulmonary artery, guinea pigs (Dunkin. ···. Hartley) weighing 400-500 g are killed by a blow to the neck.

• »• · •« »« 4 1 1 9 109541

The thorax is opened and the pulmonary artery is carefully prepared. The surrounding tissue is carefully removed and the pulmonary artery is incised spirally at an angle of 45 °.

5 A 2.5 cm long and 3-4 mm wide vein strip is attached to a 10 ml volume bath filled with Ringer's solution.

The composition of the solution in mmol / l is as follows: i 10 NaCl 154 KCl 5.6

CaCl2 1.9

NaHCO 3 2.4 glucose 5.0 15

95% O 2 and 5% CO 2 are blown through the solution and warmed to 37 ° C. pH 7.4, preload 1.0 g.

Isometric contraction changes are measured using a lever-20 apparatus and an HF modem (distance meter), manufactured by Hugo Sachs, and recorded with a compensation plot :: (BEC, Goerz Metrawatt SE 460).

· '·. After the one hour equilibration period, the test is started.

·; ·. After the vein strips have reached their maximum sensitivity for bradykinin at 2 x 10'7 mol / L - the bradykinin causes vasoconstriction - the peptides are exposed to the veins for 10 minutes at 5 x 10 ~ 5 mol / l, and when the bradykinin the increase is repeated comparing the reduction in the effect of bradykinin with the control.

· *. To detect a partial agonist effect, the peptides are used in doses of 1 x 10 ~ 5 to 1 x 10 3 mol / l.

; The IC 50 values calculated from the dose-response curve of the peptides of the invention are shown in Table 1.

.;. 35 10 109541

Table 1:

Compound IC50 [M] H- (D) -Arg-Arg-Pro-Hyp-Gly-Leu-Ser- (D) -Tic-Oic-Arg-OH 5, 9.10-9 5 H- (D) -Arg- Arg-Pro-Hyp-Gly-Cha-Ser- (D) -Tic-Oic-Arg-OH 3,7.1CT8 H- (D) -Arg-Arg-Pro-Hyp-Gly-Tbg-Ser- (D) -Tic-Oic-Arg-OH 6.0.1CT6

The therapeutic use of the peptides prepared according to the invention encompasses all pathological conditions mediated, triggered or promoted by bradykinin and bradykinin-related peptides. These include e.g. lesions such as wounds, burns, rashes, erythema, edema, angina, arthritis, asthma, allergies, allergic rhinitis, shock, inflammation, hypertension, pain, itching and altered sperm motility.

The invention therefore also relates to pharmaceutical preparations containing peptides of the formula I.

The pharmaceutical compounds contain an effective amount of the active ingredient of the formula I - alone or in combination - with an inorganic or organic pharmaceutically acceptable carrier.

Use may be enteral, parenteral - such as subcutaneous, i.m. it i.v. - under the tongue, epi- ·; ·. cutaneous, nasal, rectal, intravaginal, intrabuccal or inhalation. Active substance. the dosage depends on the species, weight, age and route of administration of the warm-blooded animal.

The pharmaceutical preparations described herein are prepared by known methods of dissolving, mixing, granulating or granulating.

. **. For oral administration or use on mucous membranes, the active compounds are admixed herein as required. · In admixture with conventional additives such as carriers, stabilizing agents or inert diluents, and by conventional means, suitable for administration as tablets, granules, inhalation capsules, aqueous, aqueous or oily suspensions, or aqueous, oily solutions. Inert carriers include, for example, gum arabic, magnesium oxide, magnesium carbonate, potassium phosphate, milk sugar, glucose, magnesium stearyl lifumarate, or starch, in particular corn starch. The preparation can then be carried out by both dry and wet granulation. Suitable oil carriers or solvents are, for example, vegetable or animal oils such as sunflower oil and cod liver oil.

The topical preparation may be in the form of an aqueous or oily solution, lotion, emulsion or gel, ointment or ointment, or if possible a spray, whereby adherence may be enhanced by the addition of a polymer.

For intranasal use, the compounds are admixed for this purpose with conventional additives such as stabilizers or inert diluents and conventionally formulated into suitable dosage forms such as aqueous, alcoholic or oily suspensions or aqueous, alcoholic or oily solutions. The aqueous intra-nasal formulations may be supplemented with chelating agents, ethylenediamine-N, N, N ', N'-tetraacetic acid, citric acid, tartaric acid or salts thereof. Nasaaliliuos- ·; ·. administration may be by means of a metered dose nebulizer or nasal drops containing a viscosity enhancer or a nasal gel or nasal ointment, respectively.

For inhalation use, nebulizers or compressed gas packs containing inert carrier gas may be used.

j 30 Intravenous, subcutaneous, epicutaneous or intra-. ··. for dermal use, the active compounds, or their physiologically acceptable salts, are reconstituted; suspension or emulsion, if desired together with pharmaceutically acceptable excipients, for example isotonic or pH-adjusting agents, and solubilizing agents, emulsifiers or other excipients.

12 109541

Because some of the drugs described have a short half-life in body fluids, the use of slow-release injectables is appropriate. The pharmaceutical forms can be, for example, oily crystal suspensions, microcapsules, rods or grafts, the latter being formed from tissue-compatible polymers, in particular biodegradable polymers such as poly-lactic acid-polyglycolic acid copolymers or human albumin.

Solutions of 0.01 to 5 mg / ml for topical and inhalation administration form, for systemic applications, 0.01 to 10 mg / kg are suitable.

List of Abbreviations: The abbreviations used for amino acids correspond to abbreviations for peptide chemistry, which are usually three-letter codes as described in Europ. J. Biochem. 138 (1984) 9. Other abbreviations used are listed below.

20 Acm acetamidomethyl ε-Ahx ε-aminohexanoyl. N Aoc cis, endo-2-azabicyclo [3.3.0] octane-3-S-carbonyl

Boc tert-butyloxycarbonyl 25 But tert-butyl

Bzl Benzyl • »CDF Chloro- (D) -phenylalanyl * · '' Cha Cyclohexylalanyl

Chg cyclohexylglycyl: *,: 30 Cl-Z 4-chlorobenzyloxycarbonyl DM F dimethylformamide DOMT O-methyl- (D) -threonyl · »; ί Dnp 2,4-Dinitrophenyl

Fmoc 9-Fluorenylmethyloxycarbonyl ··· 35 MDY O-methyl- (D) -Tyrosyl

Me methyl 13 109541 4-Mebzl 4-methylbenzyl

Mtr 4-Methoxy-2,3,6-trimethylphenylsulfonyl

Mts mesitylene-2-sulfonyl 5 Nal 2-naphthylalanyl NMP N-methylpyrrolidine

Npg neopentylglycyl

Oic cis-endo-octahydroindole-2-carbonyl

Opr Isoxazolidin-3-ylcarbonyl 10 Pal pyridylalanyl

Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl

Tbg tert-butylglycyl TFA trifluoroacetic acid 15 Thia 2-thienylalanyl

Tcs 4-methylphenylsulfonyl

Tic 1,2,3,4-tetrahydroisoquinolin-3-ylcarbonyl

Trt trityl 20

The following examples are intended to illustrate preferred methods for synthesizing the peptides of the invention by solid phase synthesis, without limiting the invention thereto.

The following amino acid derivatives were used: _. Fmoc-Arg (Mtr) -OH, Boc- (D) -Arg-OH, Fmoc-Arg (Pmc) -OH, Fmoc-

Hyp-OH, Fmoc-Pro-OObt, Fmoc-Gly-OObt, Fmoc-Phe-OObt, Fmoc- * · · '· * * Ser (tBu) -OObt, Fmoc- (D) -Tic-OH, Fmoc- Gln-OH, Fmoc-Aoc-OH,

Fmoc-Thia-OH, Fmoc-Opr-OH, Fmoc- (D) -Asn-OH, Fmoc-5-Ala-OH ,; 30 Fmoc-Oic-OH.

Example 1: H- (D) -Arg-Arg-Hyp-Pro-Gly-Leu-Ser- (D) -Tic-Oic-Arg-OH hi: stepwise generated with the peptide synthesizer Model; Applied Biosystems, using 35 Fmoc process for p-benzyloxybenzyl alcohol esterified with Fmoc-Arg (Mtr) -OH, provided by 14 109541

Novabiochem (filling about 0.5 mmol / g resin). 1 g of resin was added and synthesis was performed using a modified synthesis program for the Fmoc process.

Each synthesizer cartridge was charged with 5 l mmol of an amino acid derivative having a free carboxyl group, along with 0.95 mmol HOObt. These amino acids were directly activated in the cartridges by dissolving them in 4 ml of DMF and adding 2 ml of a 0.55 M solution of diisopropylcarbodiimide in DMF. The HOObt esters of the other 10 amino acids were dissolved in 6 ml of NMP, and then, as in situ pre-activated amino acids, were coupled to the deprotected 20% piperidine in DMF. When the synthesis was complete, the peptide was cleaved from the resin by simultaneous removal of the side chain protecting groups with trifluoroacetic acid using thioanisole and ethane dithiol as cationic binders. The residue obtained after removal of the trifluoroacetic acid was extracted with acetic acid and centrifuged several times. The remaining residue was chromatographed on Sephadex LH 20 with 10-20% acetic acid. The pure peptide contains | The fractions were combined and freeze-dried.

MS (FAB): 1264 (M + H) * The following examples were prepared analogously to pre-I *: *. with character 1.

• * * 2 5 Example 2: • ·

. H- (D) -Arg-Arg-Hyp-Pro-Gly-Cha-Ser- (D) -Tic-Oic-Arg-OH

MS (FAB): 1304 (M + H) @ 1 · J · * ·. * · Example 3: H- (D) -Arg-Arg-Hyp-Pro-Gly-Tbg-Ser- (D) - Tic-Oic-Arg-OH 30 MS (FAB): 1264 (M + H)

«I I

* · · * · T t · 11 »·» t>

Claims (3)

A process for making bradykinin antagonists of the formula I A-B-C-E-F-K- (D) -Tic-G-M-F'-I (I) | wherein A is H- (D) -Arg, 10. is Arg, C is Pro-Hyp-Gly or Hyp-Pro-Gly, E is Leu, Tbg or Cha, F is Ser, K and M are a direct bond, (D) -Tic is a group of the formula VHO OCT 20 G is Oic, F 'is Arg, and I is -OH, ·; ·. and their physiologically acceptable salts. Characterized in that a) a fragment having a free carboxyl group or an activated derivative thereof as a C-terminus is reacted with a corresponding fragment having an N-terminus as a free amino group, or; B) the peptide is formed stepwise, optionally removing one or more protecting groups temporarily attached to protect the other functional groups from the compound obtained in accordance with (a) or (b); the compounds of formula I thus obtained are optionally converted into their physiologically acceptable salts. 16 109541 Förfarande för framställning av bradykininanta-gonister med formeln I 5 A-B-C-E-F-K- (D) -Tic-G-M-F'-I (I) Color A and H- (D) -Arg, 10 B Ar Arg, C Pro Pro-Hyp-Gly eller Hyp-Pro-Gly, E Le Leu, Tbg eller Cha, F Ser Ser, K och M betecknar en direct bindning, 15 (D) -Tic är en group med formeln VI? (V) CCT 20 G är Oic:: F 'är Arg och • · I är -OH, ·; ·, samt för f ramställning av deras physiologiskt godtagbara! · · T salter, kännetecknat av att i ·: ··: 2. a) that fragment, stem C-band group en en fri! 1 * carboxyl group eller dess Aktiverade derivat, fäs att · · · · 'a reagent med moiety, stem N-fri amino group, eller jb) peptide bildas stegvis, ···, 30 frän föreningen som erhällits enligt punkt ( a) eller (b) avlägsnas eventuellt en eller flera skyddsgrupper som • · ·, · | infogats temporärt som paneld för andra functionella, * grupper, och de sälunda erhällna föreningarna enligt formeln I förvandlas eventuellt account you physiologist t · '* ·· * 35 godtagbara salter. • ·