FI111647B - New cyclic vasoactive intestinal peptide (VIP) analogues - Google Patents

Abstract

Cyclic peptides of formula (I)-(V) and their salts are new. In the formulae, X = H or a hydrolysable amino protecting gp.; Y = OH or a hydrolysable carboxy protecting gp.; R8 = Asp, Glu or Lys; R12 = Arg, Lys, Orn or Asp; R17 = Met or Nle; R26 = Ile or Val; R28 = Asn or Thr; R'8 = Asp or Asn; R'12 = ARg or Lys; R1 = His, N-CH3-Ala; R2 = Ser or Ala; R6 = (a); Q = lower alkyl-cyclohexyl or lower alkyl-aryl; R28 = Asp, Glu or Ala; R10 = Tyr or R6; R16 = Gln or Ala; R117 = Met, Nle or Ala; R19 = Val or Ala; R22 = Tyr or R6; R24 = Asn or Ala; R126 = Ile, Val or Leu; R27 = Leu or Lys; R128 = Asn, Thr or Lys; Y1 = OH, a hydrolysable carboxy protecting gp. or R29-R30-R31-Z-; R29 = Gly or Ala; R30 = Gly or Ala; R31 = Ala, Met, Cys(Acm) or Thr; Z = OH or a hydrolysable carboxy protecting gp.

Description

111647

This invention relates to a process for the preparation of certain therapeutically useful cyclic peptides as defined in claim 1. These cyclic peptides are analogs of the vasoactive peptide (VIP) and are useful in the treatment of bronchoconstrictive disorders.

The vasoactive intestinal peptide (VIP) was first discovered, isolated and purified from porcine intestine. (U.S. Patent 3,879,371). The peptide has twenty-eight (28) amino acids and has broad homology to secretin and glucagon. [Carlquist et al., Horm. Me-15 tab. Res. 14: 28-29 (1982)]. The VIP amino acid sequence is as follows:

His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser Ile-Leu-Asn-NH2 [(SEQ ID NO: 1) -NH2] VIP is known to have a wide range of biological activities throughout the gastrointestinal tract and the circulatory system. Given its similarity with gastrointestinal hormones, VIP has been found to stimulate pancreatic and bile secretion, hepatic glycogen to glucose, glucagon and insulin secretion, and to activate pancreatic bicarbonate release. [Kerrins, C. and Said, S.I., Proc. Soc. Exp. Biol. Med. 142: 1014-1017 (1972);

Domschke, S. et al., Gastroenterology 73: 478-480 (1977)]. 30 Neurons containing VIP has immunoassay ... lysis localized in the endocrine and avoeri- tysrauhasten, intestinal, and among smooth muscle cells. [Polak, J.M. et al., Gut 15: 720-724 (1974)]. VIP has been found to be a neuro-effector that causes the release of several hor-35s, including prolactin [Frawley, L.S. et al., Neuroendocrinology 33: 79-83 (1981)], thyrokine 111647 2 (Ahren, B. et al., Nature 287: 343- 345 (1980)), and insulin and glucacone (Schebalin, M. et al., Am. J. Physiology E. 232: 197-2007 (1977)]. VIP has also been found to stimulate renin release from the kidney in vivo and in vitro. [Porter, J.P. et al., Neuroendocrinology 36: 404-408 (1983)]. VIP has been found to be present in the nerves and nerve terminals of the airways of various animal species and humans. [Dey, R.D. and Said, S.I., Fed. 39, 1062 (1980); Said, S.I. et al., Ann. N.Y. Acad. Sci. 221: 10 103-1144 (1974)]. The cardiovascular and pulmonary and bronchodilatory effects of VIP are of interest because VIP has been found to be an effective vasodilator and an effective smooth muscle stimulator that affects peripheral, pulmonary and coronary artery blood vessels. [Said, S.I. et al., Clin. Res. 20: 29 (1972). VIP has been shown to have a vasodilating effect on the cerebral blood vessels. [Lee, T.J. and Berszin, I., Science 224 (1989) 898-900]. In vitro studies have shown that the vasoactive intestinal peptide li-20 applied externally to the cerebral arteries caused vasodilatation, suggesting that VIP is a potential transmitter for cerebral vasodilation. (Lee, T. and Saito, A., Science 224: 898-901 (1984)). In the eye, VIP has also been shown to be an effective “25 vasodilator [Nilsson, S.F.E. and Bill, A., Acta

Physiol. Scand. 121: 385-392 (1984)].

VIP can have regulatory effects on the immune system. 0'Dorisio et al. have shown that VIP is able to modulate lymphocyte proliferation and migration. [J. 30 Immunol. 135: 792-796 (1985)].

Since VIP has been found to relax smooth muscle and is normally present in airway tissues, as noted above, it has been suggested that VIP may be an endogenous mediator of bronchial smooth muscle relaxation. 35 [Dey, R.D. and Said, S.I., Fed. Proc. 39 (1962) 1962]. It has been shown in 111647 3 that tissues from asthma patients do not contain detachable reactive VIP compared to tissue from normal patients. This may indicate that a deficiency of VIP or VIP nerve fibers associated with the effect of VIP is associated with asthma. [Ollerenshaw, S. et al., New England J. Med. 320: 1244-1248 (1989)]. In vitro and in vivo testing has shown that VIP relaxes the smooth muscle of the trachea and protects the bronchi from constrictors such as histamine and prostaglan-10-din F2a. [Wasserman, M.A. et al. in Vasoactive Intestinal Peptide, S.I. Said, ed., Raven Press, N.Y., 1982, p. 177-184; Said, S.I. et al., Ann. N.Y. Acad. Sci. 221: 103-114 (1974)]. When administered intravenously, VIP has been found to protect the bronchi from constrictors such as histamine, prostaglandin F2a, leukotrienes, platelet activating factor, as well as antigen-induced bronchoconstriction [Said, S.I. et al., supra, (1982)]. VIP has also been found to inhibit mucus secretion in human airway tissue in vitro. [Coles, 20 S.J. et al., Am. Rev. Respir. Dis. 124: 531-536 (1981)].

In humans, VIP has been found, when administered by intravenous infusion to asthma patients, to cause an increase in peak exhalation rate and to protect against histamine-induced bronchodilation. 25 [Morice, A.H. and Sever, P.S., Peptides 7, 279-280 (1986); Morice, A. et al., The Lancet II (1983) 1225-1227.

However, the pulmonary effects observed with this VIP infusion into the vein were accompanied by cardiovascular side effects, notably low blood pressure and heart rate, and also facial flushing. When VIP was administered intravenously at doses that did not induce cardiovascular effects, VIP did not alter the airway conduction. [Palmer, J.B.

D. et al., Thorax 41, 663-666 (1986)]. The deficiency of activity-35 111647 was explained to be due to the low dose administered and possibly the rapid degradation of the compound.

When the initial VIP was administered to humans by aerosol, it was only very mildly effective in protecting against bronchoconstriction caused by sweating. (Altieri et al., Pharmacologist 25: 123 (1983)). VIP had no significant effect on baseline airway parameters, but had a protective effect against histamine-induced bronchoconstriction when administered by inhalation of drug vapor to humans. [Barnes, P.J. and Dixon, C.M.S., Am. Rev. Res pir. Dis. 130: 162-166 (1984)]. It has been explained that when given with ae rosol, VIP does not produce palpitations! antihypertensive effects in connection with bronchodilation. [Said, S.I. et al. work

Vasoactive Intestinal Peptide, S.I. Said, ed., Raven Press, N.Y., 1928, p. 185-191].

Due to the interesting and potentially clinically useful biological activities of VIP, the substance has been the subject of several reported synthesis programs aimed at improving one or more of the properties of this molecule. Takeyama et al. have reported a VIP analog with aspartic acid substituted at position 8 with glutamic acid. This compound found - ** 25 to be less potent than the original VIP. [Chem.

Pharm. Bull. 28: 2265-2269 (1980)]. Wendlberger et al.

have disclosed the preparation of a VIP analog with norleucine-substituted methionine at position 17 [Peptide, Proc. 16th Eur. Pept. Symp. 290: 295 (1980)]. Peptide 30 din was found to be as effective as the original VIP in its ability to displace radioiodinated VIP from liver membrane preparations. Watts and Wooton have described a series of linear and cyclic VIP fragments containing six to twelve residues of the original sequence. 35 [EP Patents 184,309 and 325,044; U.S. Patent Nos. 4,737,487 and 1,116,447, 4,866,039]. Turner et al. have reported that the fragment VIP (10-28) is an antagonist of VIP. [Peptides 7 (1986)

849-854]. Substituted analog [4-Cl-D-Phe6, Leu17] -VIP

VIP receptor binding and resistance-5 VIP activity have also been described. [Pandol, S. et al., Gastrointest. Liver Physiol. 13: G553-G557 (1986)]. Gozes et al. have reported that the analog [Lys1, Pro2, Arg3, Arg4, -Pro5, Tyr6] -VIP is a competitive inhibitor of VIP binding to its receptor on the surface of nerve cells. 10 (Endocrinology 125: 2945-2949 (1989)). Robberecht et al.

have described a number of VIP analogues having D residues as compensation at the N-terminal of the original VIP. [Peptides 9, 339-345 (1988)]. All of these analogs were less strongly bound to the VIP receptor and showed less activity than the original VIP in c-AMP activation. Tachibana and Ito have described several VIP analogs of the precursor molecule. [In Peptide Chem., T. Shiba and S. Sa-kakibara, editors, Prot. Res. Foundation, 1988, p. ' 481-486, JP-A-1,083,012, US-A-4,822,774]. These compounds were shown to be 1 to 3 times more potent bronchodilators than VIPs, and had 1 to 2 times higher levels of antihypertensive activity. Musso et al. have also described several VIP analogs having substitutions at positions 6-7, 25-9-13, 15-17 and 19-28. [Biochemistry 27 (1988) 8174-8181; European Patent No. 8,271,141; U.S. Patent 4,835,252] These compounds were found to be one or less potent than the original VIP in binding to the VIP receptor and in biological response. Bartfai et al. have reported a series of polysubstituted [Leu17] -VIP analogs * .. genes. [International Patent Application WO 89/05857].

Cyclic vasoactive peptide analogs are obtained according to the present invention. A cyclic peptide is a peptide in which the side chain carboxy terminus of one amino acid in the peptide chain is covalently attached to the side chain amino terminus of another amino acid in the peptide chain to form the amino acid 111647 6. through. This covalent bond between two amino acid residues in the peptide chain produces a ring structure.

The biological activity of the cyclic peptides may be substantially different from that of the starting linear analogs. Cyclic peptides have a more rigid conformation with more restricted structures. These changes are reflected in the biological profiles of cyclic peptides. Converting the peptide analog to the R-10 form may prolong the duration of action of the peptide due to increased rigidity, making it less susceptible to chemical and enzymatic degradation. The bioavailability of cyclic peptides may be increased due to changes in the physical properties of the peptide resulting from stiffening of the structure. In addition, the restricted form of the cyclic peptide may provide greater specificity for the target receptor than does the linear peptide, thereby reducing the tendency for subsequent undesirable biological activities with the desired biological activity.

The present invention provides novel cyclic peptides of formula I

(I) 25

1 I

X-His-Ser-Asp-Aia-Val-Phe-Thr-Re-Asn-Tyr-Thr-R12-30 Leu-Arg-Lys-Gln-R17-Ala-Val-Lys-Lys-Tyr- [X- (SEQ ID NO: 2) -Y] • · • ·

Leu-Asp-Ser-Leu-R26-R28 * Y

Wherein R8 is Asp, Glu or Lys; R 12 is Arg, Lys, Orn or Asp; R 17 is Met or Nle; R26 is Ile or Val; R28 is Asn or Thr; X is hydrogen or a hydrolysable amino protecting group; Y is hydroxyl or a hydrolyzable carboxy protecting group; or pharmaceutically acceptable salts thereof.

Preferred peptides of the formula I are those of the formula:

10 I I

X-His-Ser-Asp-Ala-Val-Phe-Thr-R8-Asn-Tyr-Thr-R12-Leu-Arg-Lys-Gln-Nle-Ala-Val-Lys-Lys-Tyr- [X-. (SEQ ID NO: 3) -Y]

Leu-Asp-Ser-Val-Leu-Thr-Y

Wherein X, Y, R8 and R12 are as defined above for Formula I.

The present invention also provides novel cyclic peptides of formula II

20 di)

1-I

X-His-Ser-Asp-Ala-Val-Phe-Thr-Re-Asp-Tyr-Thr-Lys-25

Leu-Arg-Lys-Gln-R17-Ala-Val-Lys-Lys-Tyr- [X- (SEQ ID NO: 4) -Y]

Leu-Asp-Ser-Leu-2s -Y-R 28

Wherein R5 is Asp or Asn; R 17 is Met or Nle; R26 is Ile or Val; R28 is Asn or Thr; X is hydrogen or a hydrolysable amino protecting group; Y is hydroxyl or a hydrolyzable carboxy protecting group; or pharmaceutically acceptable salts thereof.

111647 8

A preferred peptide of formula II is a peptide of formula:

I I

5-His-Ser-Asp-Ala-Val-Phe-Thr-Asn-Asp-Tyr-Thr-Lys-

Leu-Arg-Lys-Gln-Nle-Ala-Val-Lys-Lys-Tyr- [X- (SEQ ID NO: 5) -Y)

Leu-Asp-Ser-Val-Leu-Thr-Y

Wherein X and Y are as defined above in Formula II.

The present invention further provides novel cyclic peptides of formula III

(III) X-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr * Thr-Lys-i-

Leu-Arg-Lys-Glu-RirAla-Val-Lys-Lys-Tyr- [X- (SEQ ID NO: 6) -Y]

Leu-Asp-Ser-Leu-R26-R28-Y

wherein R 17 is Met or Nle; R26 is Ile or Val; R28 is Asn or Thr; X is hydrogen or an hydrolyzable amino protecting group; Y is hydroxyl or a hydrolyzable carboxy protecting group; or a pharmaceutically acceptable salt thereof.

Preferred peptides of the formula III are peptides of the formula: 30 ::: γ-X-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Lys-I-

Leu-Arg-Lys-Glu-Nle-Ala-Val-Lys-Lys-Tyr- [x- (SEQ ID NO: 7) -y] 35

Leu-Asp-Ser-Val-Leu-Thr-Y

Wherein X and Y are as defined above in Formula III.

According to the parent application, novel cyclic peptides of the formula: 5 (IV) X-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-R 12 - are obtained.

Leu-Arg-Lys-Gln-R 17 -Ala-Val-Lys-Lys-Tyr- [X- (SEQ ID NO: 8) -Y)

10 I

Leu-Asp-Ser-Leu-R 26 -Y-R 2e

wherein R 12 is Arg or Lys; R 17 is Met or Nle; R26 is Ile or Val; R28 is Asn or Thr; X is hydrogen or a hydrolysable amino protecting group; Y is hydroxyl or a hydrolyzable carboxy protecting group; or formula V

(v) X-R1-R2-Asp-Ala-Val-R6-Thr-R5-Asn-R10-Thr-R12-20 1-.

Leu-Arg-Lys-R16-RirAla-R19-Lys-Lys-R22- [X- (SEQ ID NO: 10) -Y] ί6υ-Η24 · Α5ρ-Ρ26 ^ 27 · ^ 28 · Υ ·· 25 where Ri is His, N-CH 3 -Ala; R 2 is Ser or Ala; Rö is

Q

30 H 5 • * • * wherein Q is lower alkylcyclohexyl or lower alkylaryl; R 8 is Asp, Glu or Ala; R 10 is Tyr or R 6; R12 is 35 Arg or Lys; R16 is Gln or Ala; Ri? is Met, Nle or Ala; R 19 is Val or Ala; R 22 is Tyr or R 6; R 24 is Asn or Ala; R26 111647 10 is Ile, Val or Leu; R27 is Leu or Lys; R28 is Asn, Thr or Lys; X is hydrogen or an hydrolyzable amino protecting group; Y is hydroxyl, a hydrolyzable carboxy protecting group, or R 29 -R 30 -R 31 -Z; R 29 is Gly or Ala; R 30 is Gly 5 or Ala; R31 is Ala, Met, Cys (Acm) or Thr; Z is hydroxyl or a hydrolyzable carboxy protecting group; or pharmaceutically acceptable salts thereof.

The peptides obtained according to the invention provide long-term relaxation of the trachea and bronchi-smooth smooth muscle without cardiovascular side effects and are thus useful in the treatment of bronchoconstrictive disorders such as asthma.

They have an increased long-lasting bronchodilator activity with no detectable side-15 effects.

As used herein, the term "lower alkyl" encompasses straight chain and branched chain saturated aliphatic hydrocarbon groups having from 1 to 6 carbon atoms. A preferred lower alkyl group is methyl.

As used herein, the term "aryl" refers to monocyclic aromatic hydrocarbon groups, such as phenyl, which may be unsubstituted or substituted at one or more positions by lower alkyl, lower alkoxy, amino, nitro, mono or dialemyl alkylamino, lower alkoxy or phenylamide.

"Aryl" also means polynuclear aryl groups, such as naphthyl, which may be substituted with one or more of the above groups. Preferred aryl groups are phenyl, unsubstituted or monosubstituted with fluorine, or unsubstituted naphthyl.

As used herein, X is a substituent on the amino nitrogen of an amino terminal amino acid and Y is a substituent on the carbonyl group of a carboxy terminal amino acid. X may be hydrogen or an hydrolyzable amino protecting group. Y may be hydroxyl or a hydrolyzable carboxy protecting group.

111647 11

With respect to the terms "hydrolysable amino protecting group" and "hydrolyzable carboxy protecting group", any conventional protecting groups which may be removed by hydrolysis may be used according to the present invention. Examples of such groups are given below. Preferred amino protecting groups are Λ - -¾ wherein X 3 is lower alkyl or halo lower alkyl. Of these protecting groups, those wherein X 3 is C 1-3 alkyl or halo C 1-3 alkyl are particularly preferred.

Preferred carboxy protecting groups are lower alkyl esters, NH 2 and lower alkyl amides, with C 1-3 alkyl esters, NH 2 and C 1-3 alkyl amides being particularly preferred.

The process for the preparation of the novel peptides and pharmaceutically acceptable salts thereof mentioned above is characterized in that: a) the protected and resin-bound peptide having the corresponding amino acid sequence is selectively deprotected to provide the free side chain amino acid residue and the free residue. ; b) covalently coupling the free side chain amino group and the free side chain carboxyl group with a suitable amide forming reagent; and c) deprotecting and cleaving the peptide that has undergone ring formation by appropriate deprotection and other cleavage, in the presence of additives as cation scavengers, and, if desired, converting the cyclic peptide into a pharmaceutically acceptable salt.

111647 12

The nomenclature used to define peptides is that typically used in the art, with the amino group at the N-terminus to the left and the carboxyl group at the C-terminus to the right. Natural amino acids are any of the naturally occurring amino acids present in proteins, i. Gly, Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met, Phe, Tyr, Pro, Trp and His. When an amino acid has isomeric forms, the L-form of the amino acid is represented unless otherwise specifically indicated.

The following abbreviations or symbols are used to represent amino acids in addition to those described above, protecting groups, solvents, reagents, and the like.

Symbol Meaning 15 Ac acetyl

Orn Ornithine

Nle norleucine

Fmoc 9-fluorenylmethyloxycarbonyl

Fm 9-fluorenylmethyl 20 Boc t-butyloxycarbonyl

Born benzyloxymethyl CH 2 Cl 2 methylene chloride CH 3 CN acetonitrile DMF dimethylformamide · DIPEA N, N-diisopropylethylamine TFA trifluoroacetic acid HOBT N-hydroxybenzotriazole DCC N, N 1 -dicyclohexylcarbodiimide DIC N, N yloxitrile (dimethyl- • «... liamino) phosphonium hexafluorophosphate 35 111647 13 ch3 N-Me-Ala CH3 0

r O

2-Nal I

Η. Λ .OH N

H O p 0 P'FPhe Η. Χ, ΟΗ N ιΓ

15 H O

FAB-MS fast atom bombardment mass spectrometry

Analogs of the original VIP peptide sequence are expressed by indicating substituted amino acids in brackets before the expression "VIP". Derivation of the N-terminal amino group, i. as indicated by X above, is indicated to the left of the brackets in the brackets. Sequence numbers appearing in brackets to the right of "VIP" indicate amino acid deletions and additional ·· 25 units in the original sequence numbering. In other words, for example, Ac- [Lys12, Nle17, Gly29] -VIP (1-29) -NH2 expresses a polypeptide having the amino acid sequence corresponding to the original human VIP, in which the N-terminus is replaced by acetyl, arginine is replaced by lysine at position 30-12 and methionine. has been replaced by norleucine in position 17.

• »

In addition, glycine is linked to the carboxyl site of asparagine 28 and is designated position 29. The suffixes "-OH" and "-NH 2" which follow the term "VIP" or parentheses refer to the free acid and 35 amide forms of the polypeptide, respectively. In the event that neither term is used, the term is intended to include both forms.

As noted above, a cyclic peptide as defined herein is a peptide in which the side chain carboxyl terminus of one amino acid in a peptide is covalently attached to the side chain amino terminus of another amino acid in the peptide chain through amide bond formation. Several nomenclatures and symbols are used to represent the cyclic peptide. The following are examples: 10 i-1

Ac-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Lys-Leu-Arg-Lys-Gln-Nle-Ala-Val-Lys-Lys-Tyr-α Asp-Ser-Val-Leu-Thr-NH2 [Ac- (SEQ ID NO: 20) -NH2l

Ac- [Lys12, Nle17, Val26, Thr28] -VIP-cyclo- (8-> 12) b.

[Ac- (SEQ ID NO: 20) -NH2]

Ac- [Lys12, Nle17, Val26, Thr28] -VIP-cyclo- (Asp8 → Lys12) c. [Ac- (SEQ ID NO: 20) -NH2] .. 25 The above three constructs (a-c) and the following representation, using the expression SEQ ID NO: and the sequence listing information below, all represent and define the same polypeptide having the amino acid sequence. corresponds to the original human VIP, where hydrogen is replaced by the weapon-30 at the N-terminus, arginine is replaced by lysine at position 12, methionine is replaced by norleucine at position 17, isoleucine is replaced by valine at position 26, and asparagine is replaced by threonine at position 28. the amide bond is formed between the side chain carboxyl of the aspartic acid at position 8 and the side chain amine at the position 12 of lysine, thereby forming the cyclic peptide 111647. The above representations of the peptide structure are considered to be equivalent and interchangeable.

As used herein, the terms "Rn" of an amino acid at position n in one of the structures herein and "Xaa" of 5 amino acids at position n of the corresponding sequence in the sequence list below are equivalent and interchangeable in cases where Xaa represents a selection from two or more amino acids .

In the cyclic peptides obtained according to the present invention, the following configurations apply unless otherwise stated.

An amino acid terminal in the chain that is a linked amino acid to provide a cyclic peptide 15 Lys ε-amino (ε = epsilon)

Orn δ-amino (δ = delta)

Asp β-carboxyl (β = beta)

Glu γ-carboxyl (γ = gamma)

Representative of the compounds of the invention obtainable by the present invention are peptides having the following amino acid sequences:

Ac- [Lys12, Nle17, Val26, Thr28) -VIP-cyclo- (Asp8-> Lys12) 25 [Ac- (SEQ ID NO: 20) -NH2]

Ac- [Glu8, Lys12, Nle17, Val26, Thr28) - VIP-Cyclo- (Glu8 → Lys12) [Ac- (SEQ ID NO: 21) -NH2] 30 Ac- [Asn8, Asp9, Lys12, Nle17, Val26 , Thr28] -VIP-cyclo (Asp9- * Lys12) [Ac- (SEQ ID NO: 22) -NH2]

Ac- [Orn12, Nle17, Val26, Thr28) -VIP-cyclo (Asp8 → Orn12) [Ac- (SEQ ID NO: 23) -NH2] 35 111647

Ac- [Lys8, Asp12, Nle17, Val28, Thr28] -VIP-cyclo (Lys8 → Asp12) [Ac- (SEQ ID NO: 24) ~ NH2]

Ac- [Glu8, Orn12, Nle17, Val2,8, Thr28] - VIP-cyclo- (Glu8- »Orn12) [Ac- (SEQ ID NO: 25) -NH2]

Ac- [Lys12, Glul6, Nle17, Val26, Thr28] -V1P-cyclo- (Lys12- »Glu16) [Ac- (SEQ ID NO: 26) -NH2] According to this invention, the compounds of formulas I-HI can be readily synthesized using any known conventional method for forming a peptide bond between amino acids. Such conventional methods include, for example, any solution phase method which permits the condensation of the free alpha amino group of an amino acid or residue protected by a carboxyl group or other reactive groups, and of another amino acid or residue thereof having an amino group or other reactive group. the groups are protected, between the free primary carboxyl group.

A method for synthesizing compounds of Formulas I-III may be performed using the procedure of adding each amino acid in a desired sequence one after the other to another amino acid or residue thereof, or a method of first synthesizing peptide fragments having the desired amino acid sequence and then condensing -30.

Such conventional methods for synthesizing new compounds of formulas I to III include, for example, any solid phase peptide synthesis method. In such a method, the synthesis of novel compounds 35 can be accomplished by sequentially linking the desired amino acid residues to the one-by-one peptide chain according to the general principles of 111647 17 Phase Methods [Merrifield, R.B., J. Amer. Chem. Soc. 85: 2149-2154 (1963);

Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J., editors, Aca-5 demic Press (1980) 1-28.

Common to chemical synthesis of peptides is the protection of reactive side chain groups of different amino acid groups with suitable protecting groups that prevent the chemical reaction from occurring at that point until the protecting group is finally removed. Usually, it is also common to protect an alpha-amino group in an amino acid or fragment while that entity reacts at the carboxyl group, followed by selective removal of the alpha-amino protecting group to allow the subsequent re-15 action at that site. Although specific protecting groups have been introduced for the solid phase synthesis process, it should be noted that each amino acid can be protected by a protecting group conventionally used for that amino acid in solution phase synthesis.

Alpha-amino groups may be protected with a suitable protecting group selected from aromatic urethane-type protecting groups such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-25-bromobenzyl, carbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz); aliphatic urethane-type protecting groups such as t-butyloxycarbonyl (Boc), diisopropylmethyloxycarbonyl, isopropyloxycarbonyl and allyloxycarbonyl.

• · .. Boc is most preferred for alpha amino protection.

Carboxyl groups may be protected with a suitable protecting group selected from aromatic esters, such as benzyl (OBz1) or benzyl substituted with Alem-35 bar alkyl, halogen, nitro, thio or substituted thio, i.e. lower alkyl (1-7 carbon atoms) with 111647 18 thio; aliphatic esters such as lower alkyl, t-butyl (Ot-Bu), cyclopentyl, cyclohexyl (OcHx), cycloheptyl and 9-fluorenylmethyl (OFm). OBz1 and OFm are most preferred for glutamic acid (Glu). OcHx, OBz1 and 5 OFm are most preferred for aspartic acid (Asp).

The hydroxyl groups may be protected with a suitable protecting group selected from ethers such as benzyl (Bzl) or benzyl substituted with lower alkyl, halogen such as 2,6-dichlorobenzyl (DCB), nitro or nitoxy; t-butyl (t-Bu), tetrahydropyranyl and triphenylmethyl (trityl). Bz1 is most preferred for serine (Ser) and threonine (Thr). Bzl and DCB are most preferred for tyrosine (Tyr).

The side chain amino groups may be protected with a suitable protecting group selected from aromatic urethane-type protecting groups such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl such as p-chlorobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, -bromobenzyl-20-oxy-carbonyl, p-diphenyl-isopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz); aliphatic urethane-type protecting groups such as t-butyloxycarbonyl (Boc), diisopropylmethyloxycarbonyl, isopropyloxycarbonyl and allyloxycarbonyl. Z is most preferred for ornithine (Orn). 2-C1-Z and Fmoc are most preferred for lysine (Lys).

The guanidino groups may be protected with a suitable protecting group selected from nitro, p-toluenesulfonyl 30 (Tos), Z, adamantyloxycarbonyl and Boc. Tos is most preferred for arginine (Arg).

The side chain amide groups can be protected with xanthyl (Xan). Unprotected is advantageous in the case of asparagine (Asn) and glutamine (Gin).

Imidazole groups may be protected with a suitable protecting group selected from p-toluenesulfonyl (Tos), 9-111647 19 fluorenylmethyloxycarbonyl (Fmoc), triphenylmethyl (trityl), 2,4-dinitrophenyl (Dnp), Boc and benzoxymethyl ). Tos and Born are most preferred for histidine (His).

Unless otherwise specified, the percentages given below for solids in solid mixtures, liquids in liquids, and solids in liquids are respectively weight / volume, volume / volume and weight / volume. In addition, unless otherwise specified, the suppliers of reagents and instruments mentioned below are not intended to be mandatory. The skilled person will be able to select similar reagents or instruments from other suppliers.

All solvents, isopropanol (iPrOH), methylene chloride (CH 2 Cl 2) and dimethylformamide (DMF) were purchased from Fisher or Burdick & Jackson and used without further distillation. Trifluoroacetic acid was purchased under the trade name Halocarbon and used without further purification. Diisopropylethylamine (DIPEA) was obtained under the trade name 20 Pfaltz and Bauer and distilled from CaO and ninhydrin before use. Dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC) were purchased under the trade name Flu-ka and used without further purification. Hydroxybenzotriazole (HOBT) and 1,2-ethane dithiol (EDT) were purchased from Sigma Chemical Co., Min. and was used without further purification. The protected amino acids were generally of the L configuration and were commercially available from Chemical Dynamics Corp. or Bachem. The purity of these reagents was confirmed by thin layer chromatography, 30 NMR and melting point before use. Boc-O-Me-Tyr, *, Boc-2-Nal and Boc-p-F-Phe were prepared as described.

(Bolin, D.R., U.S. Patent Application 374,503). Boc-Asp (OFm) and Boc-Glu (OFm) were prepared as described. [Bolin, D.R. et al., Org. Prep. Proc. Int. 21: 67-74 (1989)].

Benzhydrylamine Resin (BHA) was a copolymer of styrene and 1% divinylbenzene (100-200 or 111647 20 200-400 mesh) and was purchased from Biomega,

Bachem, Omni or Advanced Chemtech. The total nitrogen content of these resins was generally 0.3 to 1.2 milliequivalents / g.

Thin layer chromatography (TLC) was performed on glass-based pre-coated silica gel 60 F254 plates (Merck) using appropriate solvent mixtures. Detection of the compounds was accomplished by quenching UV fluorescence (absorption at 254 nm), iodine staining, or nin-10 hydrin spray (for primary and secondary amines).

For analysis of the amino acid composition, the peptides were hydrolyzed in 6 N HCl containing 1-4 mg of phenol at 115 ° C for 22-24 hours in sealed, depleted 15 hydrolysis tubes. Analyzes were performed using either a Beckman 121M amino acid analyzer or a Waters HPLC based amino acid analysis system using either a Waters Cat Ex resin or a Pierce AA511 column and detection with ninhydrin.

High pressure liquid chromatography (HPLC) was performed on an LDC instrument comprising Constametric I and III pumps, a Gradient Master solvent programmer and mixer, and a Spectromonitor III adjustable wavelength UV detector. Analytical HPLC was performed by reversed phase using Waters pBondapak C18 columns (0.4 x 30 cm). Preparative HPLC separations were performed on Whatman Magnum 20 partisil 10 ODS-3 columns (2 x 25 cm or 2 x 50 cm) equipped with a Waters Guard-Pak Cis pre-column. Gel chromatography was performed using a Kool-30 2 x 85 cm column, an LKB varioperator pex peristaltic pump, and an IBM adjustable wavelength UV detector.

The peptides were preferably prepared using solid phase synthesis according to the procedure generally described in Merrifield [J. Amer. Chem. Soc. 85, 2149 (1963)], although other similar chemical synthesis known in the art may be used as previously mentioned. Solid phase synthesis is initiated at the C-terminus of the peptide by coupling the protected alpha amino acid to a suitable resin. Such a starting material can be prepared by attaching an alpha-5 amino-protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin or an amide bond to a benzhydrylamine (BHA) or paramethyl benzhydrylamine (MBHA) resin. The preparation of hydroxymethyl resin is known in the art. Chloromethylated resins are commercially available and are well known in the art. BHA and MBHA resin carriers are commercially available and are generally used when the desired peptide to be synthesized contains an unsubstituted amide at the C-terminus.

Generally, the first amino acid to be coupled to the BHA resin was added as the symmetric anhydride of the Boc amino acid using 2 to 10 equivalents of activated amino acid per nitrogen equivalent of the resin. After coupling, the resin was washed and dried in vacuo. Amino acid loading of the resin can be determined by amino acid analysis of a sample of a Boc amino acid resin. The loads were generally between 0.2 and 0.4 mmol / g resin. All unreacted amino groups can be protected by reacting the resin with acetic anhydride and diisopropylethylamine in methylene chloride.

After the addition of 25 Boc amino acids to the resins, several iterations were performed to add the amino acids sequentially. The Boc protection of the alpha amino was removed under acidic conditions. Trifluoroacetic acid (TFA) in methylene chloride, HCl in dioxane or formic acid / acetic acid-30 mixtures can be used for this purpose. Preferably, 50% TFA in methylene chloride (v / v) is used. This may also contain 1-5% by volume of EDT or dimethylsulfide as a t-butylcarbonium ion scavenger. Other general cleavage reagents known in the art can also be used.

111647 22

Following removal of the alpha amino protecting group, the following protected amino acids are gradually coupled in the desired order to provide the intermediate, the protected peptide resin. Activation reagents used for the coupling of amino acids in the solid phase synthesis of peptides are known in the art. For example, benzotriazol-1-yloxitrile (dimethylamino) phosphonium hexafluorophosphate (BOP), dicyclohexylcarbodiimide (DCC), and diisopropyl-10-ylcarbodiimide (DIC) are suitable reagents for such syntheses. DCC and DIC are preferred herein. Other activation reagents described by Barany and Merrifield (in The Peptides, vol. 2, J. Meienhofer, eds., Academic Press, 1979, pp. 1-284) may be used. Various reagents such as 1-hydroxybenzotriazole 15 (HOBT), N-hydroxysuccinimide (HOSu) and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBT) can be added to the coupling mixtures to optimize the synthesis sequences. Here is a preferred HOBT.

Instructions for a typical synthesis cycle were as follows-20:

Instruction 1

Step Reagent Time 1 CH 2 Cl 2 2 x 30 s 2 50% TFA / CH 2 Cl 2 1 min 25 3 50% TFA / CH 2 Cl 2 15 min 4 CH 2 Cl 2 2 x 30 s 5 iPrOH 2 x 30 s 6 CH 2 Cl 2 4 x 30 s 7 6% DIPEA / CH 2 Cl 2 3x2 min 30 8 CH2Cl2 3 x 30 s ♦ · «... 9 switching 10 min - 18 hours 10 CH2Cl2 2 x 30 s 11 iPrOH 1 x 30 s 12 CH2CI2 1 x 30 s 35 13 DM F 2 x 30 s 14 CH2CI2 3 x 30 s 111647 23

Solvents were measured for all washes and couplings in volumes of 10 to 40 ml / g resin. The couplings were performed using either preformed symmetric anhydrides of Boc amino acids or O-acylisourea-5 derivatives. Generally, 2-10 equivalents of activated Boc amino acid were added per equivalent of amine resin using methylene chloride as the solvent. Boc-Arg (Tos), Boc-Gln, Boc-Asn, Boc-His (Tos) and Boc-His (Bom) were coupled in 20-25% DMF / CH 2 Cl 2. Boc-Asn, Boc-Gln and Boc-His (Born) were coupled as their HOBT-active esters to minimize side reactions known.

Peptides were generally ring-shaped using methods known in the art in the following manner. Various protecting groups were used at the amino acid sites of the peptide with the number of side chains to be linked. As amino acids, the ysε and N5-Fmoc derivatives of Lys and Orn were incorporated into the peptide chain. Asp and Glu were attached to the carboxyl site as the Op and 0Y-Fm derivatives. While still attached to the resin, the peptide was treated with 20-20% 40% piperidine in DMF to selectively remove Fmoc and Fm protecting groups. The free side chain amino and carboxyl groups were then covalently linked in the molecule by treatment with a suitable amide forming reagent such as diphenylphosphoryl azide (DPPA), DCC, DIC or BOP. DCC and BOP are preferred herein.

The procedure for a typical tire formation method was as follows:

Instruction 2

Step Reagent Time 30 1 DMF 1 x 30 s 2 20 - 40% Piperine / DMF 1 min 3 DM F 1 x 30 s 4 20 - 40% Piperidine / DMF 20 min 5 iPrOH 1 x 30 s 35 6 · DMF 1 x 30 s 7 iPrOH 2 x 30 s 111647 24 8 DMF 2 x 30 s 9 6% DIPEA / CH 2 Cl 2 2x2 min 10 CH 2 Cl 2 1 x 30 s 11 DMF 1 x 30 s 5 12 coupling 1 x 24 h 13 iPrOH 1 x 30 s 14 CH 2 Cl 2 1 x 30 s 15 DMF 2 x 30 s 16 CH2Cl2 3 x 30 s 10

Throughout the synthesis, coupling reactions were monitored by the Kaiser ninhydrin test to determine the degree of completion [Kaiser et al., Anal. Biochem. 34: 15595-598 (1970)]. Slow reaction kinetics were observed with the amino acids la Boc-Arg (Tos), Boc-Asn and Boc-Gln. For all incomplete coupling reactions, either reconnected using the freshly prepared activated amino acid or protected by treatment of the peptide resin with acetic anhydride as described above. Completely assembled peptide resins were dried under vacuum for several hours.

For each compound, the protecting groups were removed and the peptide cleaved from the resin by the following procedure. Generally, the peptide resins were treated with 25-1 · 25 100 µL of ethanedithiol, 1 mL of anisole and 9 mL of hydrofluoric acid per gram of resin at 0 ° C for 45-60 minutes in a Teflon HF (Peninsula). Alternatively, a modified two-step cleavage procedure [Tam et al., Tetrahedron Letters 23 30: 2939-2940 (1982)], wherein the peptide resin was treated with 3 mL of dimethyl sulfide and 1 mL of hydrogen fluoride for 2 hours at 0 ° C, could be used. evaporated before treatment with 90% HF. The volatile reagents were then removed in vacuo at ice bath temperature. The residue was washed with two or three columns of 20 mL of both Et 2 O and EtOAc and filtered. The peptides were extracted from the resin by washing with three or four volumes of 20 mL of 10% AcOH and filtered. The combined aqueous filtrates were lyophilized to give the crude product.

The crude peptides were initially purified by gel chromatography using Sephadex G-25 fine filler to separate the monomeric substances from the oligomer. The peptides were dissolved in a minimum volume of 10% AcOH and added to a gel column. The column was eluted with 10% AcOH at a flow rate of 0.5-1.5 ml / min. The effluent was monitored at 254 nm and the fractions containing the desired band were pooled and lyophilized to give semi-purified products.

Purification of the semi-purified peptides was generally accomplished by preparative HPLC. Peptides were added to the columns in the smallest volume of either 1% AcOH or 0.1% TFA. Gradient elution was generally started at 10% B buffer, 10-20% 25% B in 10 minutes, and 25-35% B in 3 hours (buffer A: 0.1% TFA / H 2 O, buffer B: 0.1% TFA / CH 3 CN). using a flow rate of 8.0 ml / min. UV detection was performed at 220 nm. Fractions were collected at 1.5-2.5 min intervals and analyzed by analytical HPLC. Fractions estimated to be highly pure were pooled and lyophilized.

The purity of the final products was checked by analytical HPLC using a reverse phase column as mentioned above. Generally, a gradient elution 30 of 20-40% B (Buffer A: 0.022% ... TFA / H2O, Buffer B: 0.022% TFA / CH 3 CN) was used over 15 minutes at a flow rate of 2.0 mL / min. Detection by UV occurred at 210 nm. The purity of all products was estimated to be about 97-99%. Amino acid analyzes of 35 individual peptides were performed and the values obtained were within acceptable limits. In general, all final 111647 26 foam products were also subjected to fast atom bombardment mass spectrometry (FAB-MS). All products produced the expected M + H parent ion within acceptable limits.

The novel y-5 derivatives available in accordance with the present invention have valuable pharmacological properties. They have a tracheal relaxing effect and are effective bronchodilators. The compounds also have cardiovascular side effects. The bronchodilation produced by these new peptides can continue for over two hours. Thus, because the compounds are highly active bronchodilators, they are valuable pharmaceutical agents for the treatment of bronchoconstrictive disorders, e.g., asthma.

The novel compounds of Formulas I-III may be combined with a variety of typical pharmaceutical carriers, preferably non-toxic, inert, therapeutically acceptable carriers, to provide compositions suitable for use in the treatment of bronchoconstrictive disorders such as asthma. The dosage of these compounds in the galenic dosage form will depend on various factors such as the particular compound employed and the particular composition. An effective dosage can be determined by one of skill in the art based on the> ··· 25 effective concentration (EC50) reported herein. Typical dosages for humans would be 0.01-100 pg / kg. For compounds with low EC50 such as 0.1 pg, typical dosages for humans would be about 0.02-20 pg / kg.

The novel compounds of Formulas I-III form pharmaceutically acceptable acid addition salts with a variety of inorganic and organic acids such as sulfuric, phosphoric, hydrochloric, hydrobromic, iodine, nitrogen, sulfamide, citric, such as lactic acid, lactic acid, oxalic acid, maleic acid, succinic acid, tartaric acid, cinnamic acid, acetic acid, trifluoroacetic acid, benzoic acid, salicylic acid, gluconic acid, ascorbic acid and the like.

111647 27

The compounds of the invention may be administered to a patient by parenteral, intravenous, subcutaneous, intramuscular, oral or intranasal routes. A preferred route of administration for parenteral administration is aerosol oral or nasal administration.

The present invention is further illustrated by the following examples.

Example 1

Preparation of Boc-Thr (Bzl) -BHA Resin 10 Benzhydrylamine copolystyrene 1% divinylbenzene crosslinked resin (9.5 g, 3.6 milliequivalents, 200-400 ASTM mesh, Vega Biochem) was swollen using 100 mL of methylene chloride, filtered and washed Steps 7-8 in Guide 1. Boc-Thr (Bzl) (3.35 g, 10.8 mmol) and di-15 cyclohexylcarbodiimide (1.12 g, 5.42 mmol) were reacted in 50 mL of CH 2 Cl 2 for 1 hour, filtered and in 50 mL of CH 2 Cl 2 was added to the swollen resin. This mixture was shaken for 18 hours at room temperature. DIPEA (630 mL, 3.6 mmol) was added and then shaken for an additional 1 hour, filtered, and then steps 10-14 of Step 1 were performed. Kaiser Ninhydrin Analysis was negative. All unreacted amine groups were protected by treating the resin with 1 mL of acetic anhydride and 1 mL of DIPEA in 50 mL of methylene chloride for 30 minutes, filtered and washed according to steps 13-14 of protocol 1.

The resin was dried under vacuum overnight to give 9.8 g of Boc-Thr (Bzl) -BHA resin. A portion of this resin was subjected to amino acid analysis showing a load of 0.17 mmol Thr / g.

Example 2

Preparation of Ac- [Lys12, Nle17, Val26, Thr28] -VIP-cyclo (Asp8 → Lys12) [Ac- (SEQ ID No: 20) -NH2]

The Boc-Thr (Bzl) -BHA resin from Example 1 (9.8 g, 1.7 mmol) was subjected to solid phase synthesis 35 using step 1 above. All couplings were performed using preformed symmetric anhydrides 111647 28 made from Boc amino acid and dicyclohexylcarbodiimide. Boc-Asparagine, Boc-Glutamine and Boc-Histidine (Benzyloxymethyl) were coupled as their respective HOBT-active esters. Reaction times were generally 1 to 5 to 18 hours to complete the coupling step. Five coupling sequences were performed comprising one sequence for each of the amino acids Boc-Leu (1.5 g, 6.0 mmol), Boc-Val (1.3 g, 6.0 mmol), Boc-Ser (Bzl) (1, 8 g, 6.0 mmol), Boc-Asn (773 mg, 3.3 mmol) and Boc-Leu (1.5 g, 6.0 mmol). The resin 10 was dried in vacuo to give 12.2 g of Boc-hexapeptide resin.

A portion of 8.2 g (1.13 mmol) of this resin was coupled via six sequences comprising one sequence of each of the amino acids Boc-Tyr (2,6-DCB) (1.77 g, 4.0 mmol), Boc- 15 Lys (2-Cl-Z) (1.67 g, 4.0 mmol), Boc-Lys (2-Cl-Z) (1.67 g, 4.0 mmol), Boc-Val (876 mg, 4.0 mmol), Boc-Ala (762 mg, 4.0 mmol) and Boc-Nle (932 mg, 4.0 mmol) to give 10.2 g of Boc-dodecapeptide resin.

1.26 g (0.139 mmol) of this resin were subjected to two coupling sequences comprising one sequence of each of the amino acids Boc-Gln (205 mg, 0.93 mmol) and Boc-Lys (2-Cl-Z) ( 627 mg, 1.5 mmol). Half of this resin (0.069 mmol) was subjected to fourteen coupling sequences comprising one cycle of each of the amino acids Boc-25 Arg (Tos) (324 mg, 0.76 mmol), Boc-Leu (188 mg, 0.76 mmol),

Boc-Lys (Fmoc) (354 mg, 0.76 mmol), Boc-Thr (Bzl) (234 mg, 0.76 mmol), Boc-Tyr (2,6-DCB) (333 mg, 0.76 mmol) ), Boc-Asn (97 mg, 0.76 mmol), Boc-Asp (OFm) (311 mg, 0.76 mmol), Boc-Thr (Bzl) (234 mg, 0.76 mmol), Boc-Phe (201 mg, 0.76 mmol), 30 Boc-Val (164 mg, 0.76 mmol), Boc-Ala (143 mg, 0.76 mmol), Boc-Asp (OcHx) (238 mg, 0 , 76 mmol), Boc-Ser (Bzl) (223 mg, 0.76 mmol) and Boc-His (Bom) (156 mg, 0.41 mmol). The peptide resin was subjected to steps 1-8 of protocol 1 and treated with 1.0 mL acetic anhydride and 66 mL 35 DIPEA (0.38 mmol) in 20 mL methylene chloride for 30 minutes. The resin was washed using steps 10-14.

111647 29

The resin was then selectively deprotected by treatment according to Steps 1 to 11 of Step 2 and reacted seven times with DCC (49 mg, 0.24 mmol) and HOBT (32 mg, 0.24 mmol) in 20 mL of distillate-5. DMF for 24 hours. Kaiser ninhydrin analysis was negative. The resin was washed according to steps 13-16 of protocol 2 and dried in vacuo to give 0.72 g.

This resin was treated with 6 mL of dimethyl sulfide and 2 mL of liquid HF for 2 hours at 0 ° C. The reaction mixture was evaporated and the residue was treated with 0.7 ml of anisole and 6 ml of liquid HF for 45 minutes at 0 ° C. The reaction mixture was evaporated and the residue was washed with 1 x 15 mL of Et 2 O and 3 x 15 mL of EtOAc. The resin was extracted with 3 x 20 mL of 10% AcOH. The combined aqueous filtrates were lyophilized to give 227 mg of a white solid.

This crude material was purified by preparative HPLC using a Whatman Magnum-20 ODS-3 column (2 x 25 cm) eluting with a linear gradient of 10-40% B (buffer A: 0.1% TFA / H2O, Buffer solution B: 0.1% TFA / CH 3 CN) over 4 hours. The main peak was separated by analytical HPLC analysis of the pooled fractions, pooled and lyophilized to give 26.7 mg of semi-pure product. This material was re-applied to a Magnum-20 ODS-3 column (2 x 50 cm) and eluted with "25 using the same gradient. The main peak was separated and lyophilized to give 9.5 mg of a white, amorphous powder. This compound was homogeneous by HPLC: FAB-MS: MH calculated 3277.8, found 3276.1.

Example 3 * ♦ .. Preparation of Ac- [Glu8, Lys12, Nle17, Val26, Thr28] -VIP-Cyclo (Glu8-Lys12) [Ac- (SEQ ID No: 21) -NH2]

Benzhydrylamine copolystyrene-1% divinyl-35 benzene cross-linking resin (17.7 g, 2.6 milliequivalents, 200-400 ASTM mesh, Vega Biochem) was swollen in 160 mL of 111647 methylene chloride, filtered and washed according to steps 1 to 8 of step 1. The resin was resuspended in 160 mL of methylene chloride and Boc-Thr (Bzl) (6.25 g, 20.2 mmol) and dicyclohexylcarbodiimide (2.10 g, 10.15 mmol) were added. This mixture was shaken for 8 hours at room temperature, filtered, and then steps 10 to 14 of instruction 1 were performed. Kaiser Ninhydrin Analysis was negative. The unreacted amine groups were protected by treating the resin with 5 ml of acetic anhydride and 5 ml of DIPEA 150 in 10 ml of methylene chloride for 60 minutes, filtered and washed according to steps 13-14. The resin was dried under vacuum overnight to give 18.0 g of Boc-Thr (Bzl) -BHA resin. Part of this resin was subjected to amino acid analysis showing a loading of 0.17 mmol Thr / g. For Boc-Thr (Bzl) -BHA resin (18.0 g, 3.06 mmol), solid phase synthesis was performed using the procedure above 1. All couplings were performed using preformed symmetric anhydrides prepared from Boc amino acid and dicyclohexylcarbodi -imidistä. Boc-asparagine, Boc-glutamine and Boc-histidine (benzyloxymethyl) were coupled as their respective HOBT-active esters. Five coupling sequences were performed comprising one sequence of each of the amino acids Boc-Leu (6.1 g, 24.5 mmol), Boc-Val (5.32 g, 24.5 mmol), Boc-Ser (Bzl) (7.23 g, ··· 24.5 mmol), Boc-Asn (3.13 g, 13.5 mmol) and Boc-Leu (6.1 g, 24.5 mmol). The resin was dried in vacuo to give 22.9 g of Boc hexapeptide resin.

A portion of 7.48 g (1.0 mmol) of this resin was subjected to ten coupling sequences comprising one of 30 cycles of each of the amino acids Boc-Tyr (2,6-DCB) (1.76 g, 4.0 to mmol), Boc-Lys (2-C1-Z) (1.66 g, 4.0 mmol), Boc-Lys (2-C1-Z) (1.66 g, 4.0 mmol), Boc-Val (869 mg) , 4.0 mmol), Boc-Ala (1.5 g, 8.0 mmol), Boc-Nle (925 mg, 4.0 mmol), Boc-Gln (1.08 g, 4.4 mmol), Boc-Lys (2-C1-Z) (1.66 g, 4.0 mmol), 35 Boc-Arg (Tos) (1.71 g, 4.0 mmol), and Boc-Leu (998 mg, 4). 0 111647 31 mmol) to give 9.6 g of Boc-hexadecapeptide resin.

A portion of 7.68 g (0.8 mmol) of this resin was subjected to a single coupling sequence with the amino acid Boc-Lys (Fmoc) (1.5 g, 3.2 mmol) to give 8.32 g of Boc-heptadecapeptide resin.

6.24 g (0.6 mmol) of this resin underwent two coupling divisions consisting of one sequence of each of the amino acids Boc-Thr (Bzl) (742 mg, 2.4 mmol) and 10 Boc-Tyr (2,6- DCB) (1.06 g, 2.4 mmol) to give 6.28 g of Boc-nonadecapeptide resin.

A portion of 2.09 g (0.2 mmol) of this resin underwent nine coupling sequences consisting of one sequence of each of the amino acids Boc-Asn (204 mg, 0.88 mmol), 15 Boc-Asp (OFm) (340 mg, 0 , 8 mmol), Boc-Thr (Bzl) (248 mg, 0.8 mmol), Boc-Phe (212 mg, 0.8 mmol), Boc-Val (174 mg, 0.8 mmol), Boc-Ala (151 mg, 0.8 mmol), Boc-Asp (OcHx) (258 mg, 0.8 mmol), Boc-Ser (Bzl) (236 mg, 0.8 mmol) and Boc-His (Bom) (330 mg, 0.88 mmol). The peptide resin was subjected to steps 1-8 of 20 steps 1 and treated with 0.25 mL of acetic anhydride and 70 mL of DIPEA in 20 mL of methylene chloride for 20 minutes. The resin was washed according to steps 10-14.

The resin was selectively deprotected by hand according to steps 1-11 of step 2. Three quarters of this resin (0.15 mmol) was reacted with DCC (77 mg, 0.37 mmol) and HOBT (51 mg, 0.37 mmol) in 20 mL of distilled DFM for 72 h and 20 min. of toluene for 24 hours. Kaiser ninhydrin analysis was ne-30 gative. The resin was washed according to steps 13-16 of step 2 and dried in vacuo to give 1.72 g.

A portion of 1.14 g (0.099 mmol) of this resin was deprotected by treatment as in Example 2 except that in the second step 1 mL of anisole and 9 mL of 35 HF were used. The reaction mixture was evaporated and the residue was washed with 1 x 15 mL of Et 2 O and 3 x 15 mL of EtOAc. The resin was extracted 3 x 11164732 with 20 mL of 10% AcOH. The combined aqueous filtrates were lyophilized to give 535 mg of a white solid.

This crude material was purified by gel filtration using a Sephadex G-25 fine (2 x 100 cm column) 5 eluting with 10% AcOH. The monomer peak was separated by analytical HPLC analysis of the collected fractions, pooled and lyophilized to give 83.5 mg of semi-purified product. This material was further purified by preparative HPLC as in Example 2, except that a linear gradient of 20-40% over 3 hours was used. The main peak was separated by analytical HPLC analysis of the collected fractions, pooled and lyophilized to give 18.9 mg of a white, amorphous powder. This compound was homogeneous by HPLC and gave correct amino acid analysis. FAB-MS: MH calc. 3292.0, found 3291.8.

Example 4

Preparation of Ac- [Asn8, Asp9, Lys12, Nle17, Val26, Thr28] - VIP-Cyclo (Asp9 Lys12) [Ac- (SEQ ID No: 22) -NH2] 20 1.55 g (0.15 g) a portion of the Boc nonadecapeptide resin obtained from Example 3 was subjected to nine coupling sequences comprising one sequence of each of the amino acids Boc-Asp (OFm) (247 mg, 0.6 mmol), Boc-Asn (153 mg, 0.66 25 mmol). , Boc-Thr (Bzl) (248 mg, 0.8 mmol), Boc-Phe (159 mg, 0.6 mmol), Boc-Val (130 mg, 0.6 mmol), Boc-Ala (113 mg, 0.6 mmol), Boc-Asp (OcHx) (189 mg, 0.6 mmol), Boc-Ser (Bzl) (177 mg, 0.6 mmol) and Boc-His (Born) (248 mg, 0, 66 mmol). The peptide resin was subjected to steps 1-8 of protocol 1 and treated with 0.25 mL acetic anhydride and 70 mL DIPEA in 20 mL methylene chloride for 30 minutes. The resin was washed according to steps 10-14.

The resin was then selectively deprotected by treatment according to Steps 1 to 11 of Step 2 and reacted twice with DCC (77 mg, 0.37 mmol) and HOBT (51 mg, 0.37 mmol) in 20 mL distilled DMF 24 111647 for 33 and 72 hours and twice in 20 ml of toluene for 24 and 48 hours. Kaiser ninhydrin analysis was negative. The resin was washed according to steps 13-15 of step 2 and dried in vacuo to give 1.54 g.

5.26 g (0.122 mmol) of a portion of this resin was deprotected by treatment with HF as in Example 3. The reaction mixture was evaporated and the residue was washed with 1 x 15 mL of Et 2 O and 3 x 15 mL of EtOAc. The resin was extracted with 3 x 20 mL of 10% AcOH. The combined aqueous filtrates were lyophilized to give 485 mg of a white solid.

This crude material was purified by gel filtration using Sephadex G-25 fine as in Example 3 to give 83.5 mg of semi-purified product. This material was further purified by preparative HPLC as in Example 3, except that a linear gradient of 20-45% over 3 hours was used. The main peak was separated by analytical HPLC analysis of the collected fractions, pooled and lyophilized to give 11.5 mg of a white, amorphous powder. This compound 20 was homogeneous by HPLC and gave the correct amino acid analysis. FAB-MS: MH calcd 3277.8, found 3277.6.

Example 5

Preparation of Ac- [Orn12, Nle17, Val26, Thr28] -VIP-cyclo (Asp8 → Orn12) [Ac- (SEQ ID No: 23) -NH2] * 25 L, 92 g (0.2 mmol) for a portion of that obtained in Example 3

The Boc hexadecapeptide resin was subjected to twelve coupling sequences comprising one sequence of each of the amino acids Boc-Or (Fmoc) (364 mg, 0.8 mmol), Boc-Thr (Bzl) (495 mg, 1.6 mmol), Boc-Tyr ( 2,6-DCB) (352 mg, 0.8 mmol), 30 Boc-Asn (102 mg, 0.44 mmol), Boc-Asp (OFm) (329 mg, 0.8 · ... mmol) , Boc-Thr (Bzl) (495 mg, 1.6 mmol), Boc-Phe (212 mg, 0.8 mmol), Boc-Val (174 mg, 0.8 mmol), Boc-Ala (151 mg, 0.8 mmol), Boc-Asp (OcHx) (252 mg, 0.8 mmol), Boc-Ser (Bzl) (236 mg, 0.8 mmol) and Boc-His (Born) (330 mg, 35 , 88 mmol). The peptide resin was subjected to steps 1-8 of protocol 1 and treated with 0.25 mL of acetic acid 111647 34 anhydride and 70 mL of DIPEA in 20 mL of methylene chloride for 30 minutes. The resin was washed in steps 10-14 and dried overnight to give 2.38 g.

1.38 g (0.11 mmol) of a portion of this resin was then selectively deprotected by treatment in steps 2 to 11 of Step 2 and reacted with DCC (55 'mg, 0.27 mmol) and HOBT. (37 mg, 0.27 mmol) in 20 ml of distilled DMF for 24 hours, 20 ml of toluene for 24 hours and five times in 20 ml of distilled DMF for 10 hours. The Kaiser ninhydrin assay was poorly positive. The resin was washed according to steps 13-16 of protocol 2 and dried under vacuum.

0.8 g (0.05 mmol) of this resin was deprotected with treated HF as in Example 3. The reaction mixture was evaporated and the residue was washed with 1 x 15 mL of Et 2 O and 3 x 15 mL of EtOAc. The resin was extracted with 3 x 20 mL of 10% AcOH. The combined aqueous filtrates were lyophilized to give 429 mg of a gummy solid.

This crude material was purified by gel filtration 20 using Sephadex G-25 fine as in Example 3 to give 107 mg of semi-purified product. This material was further purified by preparative HPLC as in Example 3 except that a linear gradient of 10-40% over 3 hours was used. The main 25 peak was separated by analytical HPLC analysis of the collected fractions, pooled and lyophilized to give 17.5 mg of a white, amorphous powder. This compound 011 was homogeneous by HPLC and gave the correct amino acid analysis. FAB-MS: MH calcd 3263.7, found 3264.1.

30 Example 6

Preparation of Ac- [Lys8, Asp12, Nle17, Val26, Thr28] -VIP-cyclo (Lys8 → Asp12) [Ac- (SEQ ID No: 24) -NH2] 1.0 g (0.1 mmol) ) a portion of the Boc-35 hexadecapeptide resin obtained from Example 3 was subjected to twelve coupling sequences which included one sequence of each of the amino acids 111647.

Boc-Asp (OFm) (165 mg, 0.4 mmol), Boc-Thr (Bzl) (124 mg, 0.4 mmol), Boc-Tyr (2,6-DCB) (176 mg, 0.4 mmol) ), Boc-Asn (102 mg, 0.44 mmol), Boc-Lys (Fmoc) (187 mg, 0.4 mmol), Boc-Thr (Bzl) (124 mg, 0.4 mmol), Boc-Phe (106 mg, 0.4 mmol), 5 Boc-Val (87 mg, 0.4 mmol), Boc-Ala (76 mg, 0.4 mmol), Bo-Asp (OcHx) (126 mg, 0.4 mmol) mmol), Boc-Ser (Bzl) (118 mg, 0.4 mmol) and Boc-His (Bom) (150 mg, 0.4 mmol). The peptide resin was subjected to steps 1-8 of the protocol and treated with 0.5 ml acetic anhydride and 35 ml DIPEA 10 in 20 ml methylene chloride for 30 minutes. The resin was washed according to steps 10-14 and dried overnight to give 1.2 g.

0.8 g (0.066 mmol) of a portion of this resin was then selectively deprotected by treatment in steps 15 to 11 of Step 15 2 and reacted with BOP (58 mg, 0.13 mmol) in 20 mL of distilled DMF 24 for an hour. Kaiser ninhydrin analysis was negative. The resin was washed according to steps 13-16 of protocol 2 and dried in vacuo.

This resin was deprotected with treated HF as in Example 3. The reaction mixture was evaporated and the residue was washed with 1 x 15 mL Et 2 O and 3 x 15 mL EtO-Ac. The resin was extracted with 3 x 20 mL of 10% AcOH. The combined aqueous filtrates were lyophilized to give a gummy ··· 25 solid.

This crude material was purified by gel filtration using Sephadex G-25 fine as in Example 3 to give 43.8 mg of semi-purified product. This material was further purified by preparative HPLC as in Example 3 except that a linear gradient of 10-40% over 3 hours was used. The main peak was separated by analytical HPLC analysis of the collected fractions, pooled and lyophilized to give 7.5 mg of a white, amorphous powder. This compound 35 was homogeneous by HPLC and gave correct amino acid analysis. FAB-MS: MH calcd 3277.8, found 3276.4.

111647 36

Example 7

Preparation of Ac- [Glu8, Orn12, Nle17, Val26, Thr28] -VIP-Cyclo (Glu8 → Orn12) [Ac- (SEQ ID No: 25) -NH2] 5 Benzhydrylamine Copolystyrene-1% Divinylbenzene Crosslinked Resin (25, 0 g, 17.5 milliequivalents, 200-400 ASTM mesh, Bachem) was swollen in 160 mL of methylene chloride, filtered and washed according to the steps 7-8 in Table 1. Resin Recurrence-10 was suspended in 160 mL of methylene chloride and Boc-Thr (Bzl) (16.2 g, 52.5 mmol) and dicyclohexylcarbodiimide (5.4 g, 26.2 mmol) were added. This mixture was shaken for 6 hours at room temperature, filtered, and then steps 10 to 14 of instruction 1 were performed. Kaiser Ninhydrin analysis was negative. The unreacted amine groups were protected by treating the resin with 5 ml of acetic anhydride and 5 ml of DIPEA in 150 ml of methylene chloride for 60 minutes, filtered and washed as described in steps 13-14. The resin was dried under vacuum overnight to give 29.6 g of Boc-Thr (Bzl) -BHA resin. A portion of this resin was subjected to amino acid analysis showing a load of 0.21 mmol Thr / g.

A portion of 14.0 g (2.94 mmol) of this resin was subjected to solid phase synthesis using the above oh-25 Jet as in Example 2. Eleven coupling sequences were performed containing one sequence of each of the Boc-Leu amino acids (5.9 g). , 23.5 mmol), Boc-Val (5.1 g, 23.5 mmol), Boc-Ser (Bzl) (6.9 g, 23.5 mmol), Boc-Asn (3.0 g, 13 g). , 0 mmol), Boc-Leu (5.9 g, 23.5 mmol), Boc-Tyr (2,6-DCB) 30 (10.3 g, 23.5 mmol), Boc-Lys (2-Cl) -Z) (9.8 g, 23.5 mmol),

Boc-Lys (2-C1-Z) (9.8 g, 23.5 mmol), Boc-Val (5.1 g, 23.5 mmol), Boc-Ala (4.4 g, 23.5 mmol) ) and Boc-Nle (5.4 g, 23.5 mmol) to give 26 g of Boc decapeptide resin.

A portion of 5.5 g (0.61 mmol) of this resin was coupled via four sequences comprising one sequence of each of the amino acids Boc-Gln (655 mg, 2.7 111647 37 mmol), Boc-Lys (2 -C1-Z) (2.0 g, 4.8 mmol), Boc-Arg (Tos) (2.1 g, 4.8 mmol) and Boc-Leu (1.2 g, 4.8 mmol) . The resin was dried in vacuo to give 6.12 g of Boc hexadecapeptide resin.

A portion of 2.0 g (0.2 mmol) of this resin was subjected to twelve coupling sequences containing one sequence of each of the amino acids Boc-Or (Fmoc) (91 mg, 0.2 mmol), Boc-Thr (Bzl) ( 250 mg, 0.8 mmol), Boc-Tyr (2,6-DCB) (352 mg, 0.8 mmol), Boc-Asn (102 mg, 0.44 mmol), Boc-Glu (OFm) (340 mg, 10 0.8 mmol), Boc-Thr (Bzl) (248 mg, 0.8 mmol), Boc-Phe (212 mg, 0.8 mmol), Boc-Val (174 mg, 0.8 mmol) , Bo-Ala (152 mg, 0.8 mmol), Boc-Asp (OcHx) (252 mg, 0.8 mmol), Boc-Ser (Bzl) (236 mg, 0.8 mmol) and Boc-His ( Born) (150 mg, 0.4 mmol). The peptide resin was subjected to steps 1-8 of step 1 and treated with 0.5 mL acetic anhydride and 38 mL DIPEA in 30 mL methylene chloride for 180 minutes. The resin was washed according to steps 10-14 and dried in vacuo. yielding 2.4 g.

A portion of 1.15 g (0.1 mmol) of this resin was then selectively deprotected by treatment in steps 2 to 11 of Step 2 and reacted twice with BOP (58 mg, 0.13 mmol) and 400 mL: n DIPEA in 20 ml of DMF for 24 and 6 hours. Kaiser ninhydrin analysis was negative. This resin was washed · 25 according to steps 13-16 of step 2 and dried in vacuo to give 1.05 g.

This resin was deprotected by treatment with 9 mL of HF, 1 mL of anisole, and 100 mL of ethane dithiol for 1 hour at 0 ° C. The reaction mixture was evaporated and the ice-30 wash was washed with 2 x 15 mL of Et 2 D and 2 x 15 mL of EtOAc.

• «·,. The resin was extracted with 4 x 20 mL of 10% AcOH. The combined aqueous filtrates were lyophilized to give 385 mg of a light yellow solid.

This crude material was purified by gel filtration 35 using Sephadex G-25 fine as in Example 3 to give 134 mg of semi-purified product.

This material was further purified by preparative HPLC as in Example 3 except that a linear gradient of 26-36% over 3 hours was used. The peak was separated by analytical HPLC-5 analysis of the collected fractions, pooled and lyophilized to give 23.2 mg of a white, amorphous powder. This compound was homogeneous by HPLC and gave correct amino acid analysis. FAB-MS: MH calcd 3277.8, found 3276.3. Example 8 Preparation of Ac- [Lys12, Glu16, Nle17, Val26, Thr28] -VIP-Cyclo (Lys12 → Glu16) [Ac- (SEQ ID No: 26) -1111]

Benzhydrylamine Copolystyrene 1% Divinyl Benzene Crosslinked Resin (30 g, 21.4 milliequivalents, 15,200-400 ASTM Nesh, Bachem) was treated as in Example 22 except 19.9 g Boc-Thr (Bzl) (64.3 mmol) was used. and 6.6 g of dicyclohexylcarbodiimide (32.1 mmol). This mixture was shaken for 18 hours at room temperature, filtered, and then Steps 10-20 of Step 1 of Step 1 were performed. Kaiser Ninhydrin Analysis was negative. The unreacted unreacted amine groups were protected by treating the resin with 8 ml acetic anhydride and 8 ml DIPEA in 200 ml methylene chloride for 60 minutes, filtered and washed according to steps 13-14. The resin was dried under vacuum for 25 hours to give 34.2 g of Boc-Thr (Bzl) -BHA resin. A portion of this resin was subjected to amino acid analysis showing a loading of 0.47 mmol Thr / g.

0.85 g (0.4 mmol) of this Boc-Thr (Bzl) -BHA resin was subjected to solid phase synthesis using an Ap-30 plied Biosystems model 430A peptide synthesizer. All

«· I

couplings were performed using preformed symmetric anhydrides prepared from Boc-amino acid and dicyclohexylcarbodiimide. Boc-Asparagine, Boc-Glutamine and Boc-Arginine (tosyl) were conjugated routinely as the corresponding HOBT-active esters. Eleven coupling sequences were performed containing one sequence of each of the amino acids Boc-Leu (499 mg, 2.0 mmol), Boc-Val (435 mg, 2.0 mmol), Boc-Ser (Bzl) (590 mg, 2.0 mmol), Boc-Asn (464 mg, 2.0 mmol), Boc-Leu (499 mg, 2.0 mmol), Boc-Tyr (2,6-DCB) (880 mg, 2.0 mmol), Boc-Lys (2-C1-Z) 5 (830 mg, 2.0 mmol), Boc-Lys (2-C1-Z) (830 mg, 2.0 mmol),

Boc-Val (435 mg, 2.0 mmol), Boc-Ala (378 mg, 2.0 mmol) and Boc-Nle (462 mg, 2.0 mmol) to give the Boc dodecapeptide resin.

This resin was then subjected to five coupling-10 cycles, as in Example 2, which included one sequence of each of the amino acids Boc-Glu (OFm) (340 mg, 0.8 mmol), Boc-Lys (2-C1-Z) (664 mg, 1.6 mmol), Boc-Arg (Tos) (685 mg, 1.6 mmol), Boc-Leu (398 mg, 1.6 mmol) and Boc-Lys (Fmoc) (375 mg, 0.8 mmol) with. The resin was dried in vacuo to give 2.12 g of Boc-heptadecapeptide resin.

1.06 g (0.2 mmol) of a portion of this resin was then selectively deprotected by treatment in steps 2 to 11 of Step 2 and reacted twice with BOP (177 mg, 0.4 mmol) and 200 mL With DIPEA in 20 ml of distilled DMF for 2 and 8 hours. The Kaiserinhydrin assay was weakly positive. The unreacted amine groups were protected by treating the resin with 0.5 mL acetic anhydride in 20 mL 6% DI-PEA / methylene chloride for 10 minutes, filtered, and ··· 25 washed according to Step 2, Steps 13-16. This resin was then subjected to a single coupling sequence with Boc-Thr (Bzl) (495 mg, 1.6 mmol) and then returned to the Applied Biosystems 430A Peptide Synthesizer as above. Ten coupling sequences were performed which included one sequence of 30 of each of the amino acids Boc-Tyr (2,6-DCB) (880 mg, 2.0 mmol), Boc-Asn (4 64 mg, 2.0 mmol), Boc-Asp (OcHx) (630 mg, 2.0 mmol), Boc-Thr (Bzl) (618 mg, 2.0 mmol), Boc-Phe (531 mg, 2.0 mmol), Boc-Val (435 mg, 2.0) mmol), Boc-Ala (378 mg, 2.0 mmol), Boc-Asp (OcHx) (630 mg, 2.0 mmol), Boc-Ser (Bzl) (590 35 mg, 2.0 mmol) and Boc -His (Tos) (819 mg, 2.0 mmol). The peptide resin was then subjected to Steps 111647 40 1-8 of Step 1 and reacted with 0.5 mL acetic anhydride and 100 mL DIPEA in 20 mL methylene chloride for 30 minutes. The resin was washed according to steps 10-14 and dried in vacuo.

The peptide resin was deprotected as before

7 to give 340 mg of the crude peptide. The peptide was purified by gel filtration as in Example 3 to give 35 mg of semi-pure product. This material was further purified by preparative HPLC as in Example 3 except using a linear gradient of 25-35% to give 15.6 mg of a white, amorphous powder. The compound was homogeneous by HPLC and gave correct amino acid analysis. FAB-MS: MH

calculated 3276.6, found 3278.0.

Experimental Example 1 Tracheal Relaxation Effect of VIP Analogs The relaxant activity of VIP analogs was studied in a model using guinea pig trachea. [Wasserman, M.A. et al., Vasoactive Intestinal Peptide, S.I. 20 Said, Ed., Raven Press, N.Y. 1982, pp. 177-184]. All tissues were taken from male albino guinea pigs weighing 400-600 g and anesthetized with urethane (2 g / kg, ip). After blood was removed from the body, the trachea was removed and divided into 4 · 25 ring segments (length 3 mm). Each ring was suspended with a diameter of 30 stainless steel wires in a 10 mL jacketed tissue bath and secured to a Grass Force Transfer Sensor (Model FT03C, Grass Instruments Co., Quincy, 30 MA) using a 4-0 silk thread for tension recording. Smooth muscle muscle fluid was a modified Krebs solution of the following composition: NaCl 120 mM; KCl 4.7 mM; CaCl2 2.5 mM; MgSO4 .7H2O 1.2 mM; NaHCO3 25 mM; K2HPO4 monobasic 1.2 mM and dextrose 10 mM. Tissue baths were maintained at 37 ° C and continuously pulverized with 95% O 2 and 5% CO 2. Responses were recorded for 8 channels and 4 channels for Hewlett® 111647 41

Packard (models 7702B and 7754A, respectively) using a recording device (Hewlett-Packard, Paramus, NJ). The tracheal rings were placed at a resting tension of 1.5 g, which was determined in the pre-tests to be optimal or near optimal. Numerous tension readjustments were required during the 60 minute stabilization time. The tissues were rinsed at 15 minute intervals.

For each tissue, a cumulative concentration-response curve for the bath was obtained by successive increases of μ1: η of VIP or VIP analogues according to the VanRossum method [Arch. Int. Pharmacodyn. 143: 299-330 (1963)]. Only one cumulative dose-response curve was generated in a single tissue. To minimize inter-tissue variability, relaxation responses II-15 were tasted as a percentage of the maximal response obtained at the end of each concentration-response experiment with added VIP (10-6 M = 100%). Responses from three tissues were pooled and EC 50 values were determined by linear regression.

The results, summarized in Table I, demonstrate the tracheal relaxation effect of the VIP analogs of formulas I to III compared to the original VIP.

Table I

Relaxing effect of VIP analogs on guinea pig tracheal smooth ··· EC50

Compound (nM) VIP [(Seq ID NO: 1) -NH2] 10 30 · · ·

Ac- [Lys12, Nle17, Val26, Thr28] -VIP-cyclo (Asp8 → Lys12) [Ac- (SEQ ID NO: 20) -NH214

Ac- [Glu8 ^ Lys12, Nle17, Val28, Thr28] -VIP-cyclo (Glu8- * Lys12) [Ac- (SEQ ID NO: 21) -NH2] 34 111647 42

Ac- [Asn8, Asp9, Lys12, Nle17, Val26, Thr28] -VIP cyclo- (Asp9-> Lys12) [Ac- (SEQ ID NO: 22) -NH2] 175 Ac- [Orn12, Nle17, Val26, Thr28] -VP-cyclo (Asp8 → Orn12) [AC- (SEQ ID NO: 23) -NH2] 40

Ac- [Lys8, Asp ^ -2, Nle ^ 7, Val26, Thr28] -VIP-cyclo- (Lys8-KAsp12) [Ac- (SEQ ID NO: 24) -NH2]

Ac- [Glu8, Orn12, Nle · ^ · 7, Val26, Thr28) -VIP-cyclo (Glu8 → Orn12) [Ac- (SEQ ID NO: 25) -NH2] 16

Ac- [Lys12, Glu16, Nle17, Val26, Thr28) -V1P-cyclo-15 (Lys12- * Glu16) [Ac- (SEQ ID NO: 26) -NH2) 37

Experimental Example 2 The bronchodilator effect of VIP analogs The in vivo bronchodilator effect of VIP and VIP analogs in guinea pigs was investigated using drip into the trachea. Male guinea pigs (Hartley strain, Charles River) weighing 400 to 600 g were used in this technique. Animals were anesthetized with urethane (2 g / kg) by intraperitoneal administration and a polyethylene cannula was inserted into the jugular vein to administer the drug intravenously.

The animals were subjected to tracheal surgery and dosing solutions containing distilled water or test compound dissolved in distilled water were administered via a pipette to the trachea at approximately three-quarters of the distance between the lower and lower trachea. The concentration of the dosing solution was adjusted to allow a constant volume of 100 ml. Animals were placed on their backs for one minute to facilitate delivery to the lungs. One minute later, spontaneous breathing was stopped by intravenous succinylcholine chloride (1.2 mg / kg) and the animals were assisted in breathing using a Harvard 111647 43 rod Model 680, a small animal respirator set at 40 breaths / min and cylinder stroke volume. 4.0 cm 3. Animals were challenged with a maximal constrictive dose of histamine (50 mg / kg, 5 intravenous) and tracheal pressure (cm of water) was recorded with a Statham pressure transducer (P 32 AA).

The mean change in tracheal pressure was calculated for at least three control and three drug-treated animals, and percent inhibition was calculated.

The relative potency of the compounds administered by drip route was determined by administering various doses of the test compound and calculating the mean effective dose (ED 50). The ED 50 was determined from the dose-response curves obtained with at least three doses producing inhibitory effects of between 10% and 90%. The regression direct correlation coefficient for each compound was always greater than 0.95.

To determine the change in inhibition with time, the time between administration of the compound and irritation with histamine was varied for the different compounds. The change in activity over time was calculated as the time at which inhibition decreased to 40%.

The results, summarized in Table II, show a bronchodilator effect of the VIP analog of formula III in vivo compared to the original VIP.

Table II

The bronchodilator effect of VIP analogues in guinea pigs 30 ED50

Compound (pg) VIP [(Seq ID NO: 1) -NH2J 7.3 35 Ac- [Lys12, Glu ^ Nle17, Val26, Thr28] -VIP-cyclo (Lys12-> Glu16) (Ac- (SEQ ID NO: 1) : 26) -NH 2] 39 111647 44

Claims 1. A process for the preparation of therapeutically useful cyclic peptides of the formula I 5 (I) I-1 X-His-Ser * Asp-Ala-Val-Phe-Thr-R 8 -An-Tyr-Thr-R 12-10.

Leu-Arg-Lys-Gln-R 17 -Ala-Val-Lys-Lys-Tyr- [X- (SEQ ID NO: 2) -Y]

Leu-Asp-Ser-Leu-R26-R28-Y

Wherein R5 is Asp, Glu or Lys; R 12 is Arg, Lys, Orn or Asp; R 17 is Met or Nle; R26 is Ile or Val; R28 is Asn or Thr; X is hydrogen or a hydrolysable amino protecting group; Y is hydroxyl or a hydrolyzable carboxy protecting group; or formula II

20 (II) I f X-His-Ser-Asp-Ala-Val-Phe-Thr-Re-Asp-Tyr-Thr-Lys-25 Leu-Arg-Lys-Gln-R17-Ala-Val-Lys-Lys -Tyr- [x- (SEQ ID NO: 4) -Y]

Leu-Asp-Ser-Leu-p.26 2s -Y

wherein R 5 is Asp or Asn; R 11 is Met or Nle; R 26 is Ile or 30 Val; R2s is Asn or Thr; X is hydrogen or an hydrolyzable amino protecting group; Y is hydroxyl or hydrolysates

a carboxy protecting group in the gum; or formula III

(III) 111647 45 ΓΠ 5 X-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Lys-

Leu-Arg-Lys-Glu-Ala-Ri7-Val'l_ys-Lys-Tyr

Leu-Asp-Ser-R26-Leu-R28-Y [X- (SEQ ID NO: 6) -Y] wherein R17 is Met or Nle; R26 is Ile or Val; R28 is Asn or Thr; X is hydrogen or an hydrolyzable amino protecting group; Y is hydroxyl or a hydrolyzable carboxy protecting group; or for the preparation of pharmaceutically acceptable salts thereof, characterized in that: a) a protected and resin-bound peptide having the corresponding amino acid sequence is selectively deprotected to provide a free side chain amino group and a free side chain carboxyl group; B) covalently coupling the free side chain amino group and the free side chain carboxyl group with a suitable amide forming reagent; in the presence of additives as cation scavengers, and, if desired, converting the cyclic peptide into a pharmaceutically acceptable salt.

Process according to claim 1, characterized in that a cyclic peptide of the formula I is prepared wherein R n is Nle, R 26 is Val and R 28 is Thr.

The method of claim 2,

characterized in that the cyclic peptide is Ac- [Lys12, Nle17, Val26, Thr28] -VIP-cyclo (Asp8 Lys12) [Ac- (SEQ ID NO:

35 NO: 20) -NH 2].

Method according to claim 2, characterized in that the cyclic peptide is Ac- [Glu8, Lys12, Nle17, Val26, Thr28] -VIP-cyclo (Glu8-Lys12) [Ac- (SEQ ID NO: 21). -NH2].

The method of claim 2,

characterized in that the cyclic peptide is Ac- [Orn12, Nle17, Val26, Thr28] -VIP-cyclo (Asp8 Orn12) [Ac- (SEQ ID NO:

NO: 23) -NH2] - The process according to claim 2, characterized in that the cyclic peptide is Ac- [Lys8,

Asp12, Nle17, Val26, Thr28] -VIP-cyclo- (Lys8 → Asp12) [Ac- (SEQ ID NO: 24) -NH2].

A process according to claim 2, characterized in that the cyclic peptide is Ac- [Glu8,15 Orn12, Nle17, Val26, Thr28] -VIP-cyclo (Glu8 → Orn12) [Ac- (SEQ ID NO: 25) - NH2].

A process according to claim 1, characterized in that a cyclic peptide of formula II is prepared which is Ac- [Asn8, Asp9, Lys12, Nle17, 20 Val26, Thr28] -VIP cyclo (Asp9 -> Lys12) [Ac- (SEQ ID NO: 22) -NH2].

9. The method of claim 1, wherein the peptide of formula III which is Ac- [Lys12, G1u1d, Nle17, Val26, Thr28] -25 VIP-cyclo (Lys12-Glu16) [Ac- (SEQ ID NO: 1) is prepared. NO: 26) -NH2].

Claims (9)

A process for the preparation of therapeutically useful cyclic peptides of Formula I (I) II X-His-Ser-Asp-Ala-Val-PheThr-Re-Asn-Tyr-Thr-R12-Leu-Arg-Lys -Gln-Ri7-Ala-Val-Lys-Lys-Tyr- [X- (SEQ ID NO: 2) -Y] Leu-Asp-Ser-R26-Leu-R28 * Y where R8 is Asp, Glu or Lys; R 12 is Arg, Ly, Orn or Asp; R17 is Met or Nle; R26 is Ile or Vai; R 28 is Asn or Thr; X is hydrogen or a hydrolyzable amino protecting group; Y is hydroxyl or a hydrolyzable carboxy protecting group; or Formula II 20 (II) II X-His-Ser-Asp-Ala-Val-Phe-Thr-Rg-Asp-Tyr-Thr-Lys-Leu-Arg-Lys-GIn-RiT-Aia-Val-Lys -Lys-Tyr- [X- (SEQ ID NO: 4) -y] Leu-Asp-Ser-R26-Leu-P.2s-Y where R8 is Asp or Asn; R17 is Met or Nle; R26 is Ile 30 or Vai; R 28 is Asn or Thr; X is hydrogen or a hydrolysable amino protecting group; Y is hydroxyl or a hydrolyzable carboxy protecting group; or Formula III (HI) Γ) X-His-Ser-Asp-Aia-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Lys-Leu-Arg-Lys-GIu-R-17 Ala-Val-Lys-Lys-Tyr-Leu-Asp-Ser-R26-Leu-R28-Y [X- (SEQ ID NO: 6) -Y] wherein R17 is Met or Nle; R26 is Ile or Vai; R 28 is Asn or Thr; X is hydrogen or a hydrolyzable amino protecting group; Y is hydroxyl or a hydrolyzable carboxy protecting group; or pharmaceutically acceptable salts thereof, characterized in that a) a protected and resin-bound peptide with corresponding amino acid sequence is selectively deprotected to give a free side chain amino group and a free side chain carboxyl group; B) the free side chain amino group and the free si chain chain carboxyl group are covalently coupled to a suitable amide-forming reagent; and c) deprotecting and cleavage of the cyclized peptide from the resin by treating with a suitable deprotection and cleavage reagent, if desired in the presence of other suitable additives such as cation eliminator and, if desired, converting the cyclic peptide. a pharmaceutically acceptable site. 2. A process according to claim 1, characterized in that a cyclic pep time of formula I is prepared, wherein R n is Nle, R26 is Vai and R28 is Thr. Method according to claim 2, characterized in that the cyclic peptide is Ac- [Lys12, Nle17, Vai26, Thr28] -VIP-cyclo- (Asp8 - »Lys12) [Ac- (SEQ ID NO: 20) - NH2]. 11164 / The method of claim 2, characterized in that the cyclic peptide is Ac- [Glu8, Lys12, Nle17, Vai26, Thr28] -VIP-cyclo- (Glu8 Lys12) [Ac- (SEQ ID NO: 21 ) -NH 2]. 5. A method according to claim 2, characterized in that the cyclic peptide is Ac- [Orn12, Nle17, Vai26, Thr28] -VIP-cyclo- (Asp8 -> Orn12) [Ac- (SEQ ID NO: 23) -NH 2]. Method according to claim 2, characterized in that the cyclic peptide is Ac- [Lys8, Asp12, Nle17, Vai26, Thr28] -VIP-cyclo- (Lys8 -> Asp12) [Ac- (SEQ ID NO. : 24) -NH 2] - 7. A process according to claim 2, characterized in that the cyclic peptide is Ac- [Glu8, Orn12, Nle17, Vai26, Thr28] -VIP-cyclo- (Glu8 - »Orn12) [Ac- (SEQ ID NO. : 25) -NH 2]. Process according to claim 1, characterized in that a cyclic peptide of formula II is prepared, which peptide is Ac- [Asn8, Asp9, Lys12, Nle17, Vai26, Thr28] -VIP-cyclo- (Asp9 - Lys12) [Ac- (SEQ ID NO: 22) -NH2]. 9. A method according to claim 1, characterized in that a peptide of formula III is prepared, which peptide is Ac- [Lys12, Glu16, Nle17, Vai26,.] Thr28] -VIP-cyclo- (Lys12 -> · Glu16) [Ac- (SEQ ID NO: 26) -NH2]. 30