VIP type peptides

Peptides comprising, in sequence, units selected from the amino acid residues 11 to 23 of vasoactive intestinal peptide (VIP) and consisting at least of the amino acid residues 15 to 20, or an analogue thereof wherein one or more of the amino acid residues is replaced by an equivalent other amino acid, or a pharmaceutically acceptable salt thereof; having pharmacological activity, a process for their preparation and their use as pharmaceuticals.

The invention relates to VIP fragments and analogues, processes for their 
preparation, pharmaceutical preparations containing them and their use in 
medicine. 
Vasoactive intestinal peptide (VIP) was originally isolated from the small 
intestines of the hog, but it has since been isolated from other species, 
such as the chicken, and has been shown to have a wide distribution 
throughout body tissues. 
It has systemic vasodilator activity. It includes systemic hypotension and 
increases cardiac output on intravenous infusion. It increases hepatic 
arterial blood flow, increases blood sugar levels, and has the ability to 
bring about tracheal relaxation, and relaxation of gut smooth muscle, as 
well as stimulation of the output of bicarbonate from intestinal 
secretions. It therefore appears to be useful in treatment of hypertension 
and peripheral vascular disease on parenteral administration, and as a 
bronchodilator on aerosol or parenteral administration. 
Vasoactive intestinal peptide comprises a peptide having a sequence of 28 
amino acids in a single chain. The sequence of VIP (pig) is shown in table 
1. 
TABLE 1 
__________________________________________________________________________ 
VIP (pig) 
__________________________________________________________________________ 
NTerminus 
##STR1## 
##STR2## 
##STR3## 
##STR4## 
CTerminus 
__________________________________________________________________________ 
Abbreviations used are as follows: 
______________________________________ 
Amino Acid Residue Abbreviations 
______________________________________ 
alanine Ala 
arginine Arg 
asparagine Asn 
aspartic acid Asp 
glutamine Gln 
histidine His 
isoleucine Ile 
leucine Leu 
lysine Lys 
methionine Met 
norleucine Nle 
phenylalanine Phe 
serine Ser 
threonine Thr 
tyrosine Tyr 
valine Val 
______________________________________ 
The amino acid components are in the L-form. 
VIP (chicken) is closely related, differing in the 11, 13, 26 and 28 
positions; the peptide has: 
______________________________________ 
a serine residue in position 11, 
a phenylalanine residue in position 13, 
a valine residue in position 26 and 
a threonine residue in position 28. 
______________________________________ 
A number of C-terminal fragments have been produced, mostly in the 
synthetic programme required to prove the structure of VIP. Few structures 
have been obtained from the N-terminus, and very little work has been 
carried out on fragments from the centre of the molecule. 
It has, however, been concluded (Robberecht, Gut Hormones (1978) edited by 
Bloom, p 97 to 103) that the C-terminus of VIP holds the receptor 
recognition site, and that the N-terminus holds the activation site, 
together with a minimal capacity for binding. 
Counter to the commonly held views regarding the activity of VIP, we have 
found that there is pharmacological activity even in the absence of the 
amino acid units at the C- and N-termini of the molecule. 
The present invention provides a peptide comprising, in sequence, units 
selected from the amino acid residues 11 to 23 of VIP and consisting at 
least of the amino acid residues 15 to 20, or an analogue thereof wherein 
one or more of the amino acid residues is replaced by an equivalent other 
amino acid. 
The present invention also provides a peptide consisting, in sequence, of 
the VIP units selected from the amino acid residues 11 to 23, and 
comprising at least the amino acid residues 15 to 20, or an analogue 
thereof having pharmacological activity. 
Preferably in a peptide of the present invention the amino acid units are 
selected from residues 13 to 23 or 11 to 21, more especially from residues 
13 to 21, of VIP. In an analogue thereof, one or more than one amino acid 
unit may be replaced by an equivalent amino acid unit. 
Amino acids can be considered as members of different classes; such 
groupings are well known. Replacement of an amino acid of the peptide by 
an equivalent amino acid may be by another amino acid of the same class, 
and where an amino acid can be grouped into two or more classes, 
replacement may be made from one or more of these classes. 
All amino acids in an analogue of the present invention may, for example, 
be naturally occurring amino acids, i.e. L-amino acids, or amino acids in 
the D- or DL-form. 
It seems reasonable to suppose that the activity of a peptide bears some 
relationship to its secondary structure (which could be inherent, or 
adopted at the receptor site). Thus the expressed activity could be 
related to a potential for formation of a highly ordered arrangement of 
some of the amino acids. 
Where there is replacement of one or more amino acids, the replacement may, 
for example, be such that the essential structure of the fragment is 
maintained. 
Without intending to be limited to the following hypothesis, we presently 
believe it is possible that, for peptides of the present invention, a 
helical structure may be a contributory factor in the pharmacological 
activity. The replacing amino acid or acids in an analogue thereof may 
therefore, if desired, be selected so as to have at least as good a 
helical-forming character as the replaced amino acid(s). However, lack of 
a helical structure may not impair the activity of a peptide or analogue 
of the present invention; for example, it may be preferred, for 
pharmacological reasons or otherwise, to incorporate D-amino acid(s) as 
the replacing amino acid(s) and it will, of course, be understood that 
unless all amino acids in the resulting analogue are in the D-form, the 
structure will not be of a helical nature. 
Thus, for example: 
the threonine at position 11 of VIP (pig) may, if desired, be replaced by 
another hydroxy amino acid, e.g. serine (Ser); and the serine at position 
11 of VIP (chicken) may, if desired, be replaced by another hydroxy amino 
acid, e.g. threonine (Thr); 
the arginine at position 12 and/or at position 14 may, if desired, be 
replaced by another basic amino acid, e.g. lysine (Lys) or ornithine 
(Orn); 
the leucine at position 13 of VIP (pig) and/or at position 23, and the 
phenylalanine at position 13 of VIP (chicken) may, if desired, be replaced 
by another hydrophobic amino acid, in the case of leucine, by, for 
example, valine (Val), and, in the case of phenylalanine, by, for example, 
tyrosine; 
the lysine at any one or more of positions 15, 20 and 21 may, if desired, 
be replaced by another basic amino acid, e.g. ornithine (Orn) or arginine 
(Arg); 
the glutamine at position 16 may, if desired, be replaced by another 
carboxamido amino acid, e.g. asparagine (Asn); 
the methionine at position 17 may, if desired, be replaced by another 
neutral amino acid, e.g. the isosteric norleucine (Nle) or leucine (Leu); 
the alanine at position 18 may, if desired, be replaced by another 
hydrophobic amino acid, e.g. glycine (Gly) or norvaline (Nva); 
the valine at position 19 may, if desired, be replaced by another 
hydrophobic amino acid, e.g. leucine (Leu); 
the tyrosine at position 22 may, if desired, be replaced by another 
hydrophobic amino acid, especially an aromatic amino acid, e.g. 
phenylalanine (Phe). 
Especially, there should be mentioned analogues in which one or more of the 
amino acid residues 15 to 20 is replaced by an equivalent other amino acid 
and any additional amino acid residues present correspond to those in VIP. 
Especially, the present invention provides a hexapeptide amide with the 
amino acid sequences of the residue 15 to 20 of VIP, or an analogue 
thereof in which one or more of the amino acids is replaced as indicated 
above. 
Very especially, the present invention provides the hexapeptide 
EQU Lys Gln Y Ala Val Lys 
where Y represents Met or Nle; and also the hexapeptide 
EQU Lys Gln Y Ala Leu Lys 
where Y represents Met or Nle. 
Fragments and analogues of VIP (pig) should especially be mentioned, but 
the basic structure may correspond to VIP from any source. 
The following fragments and analogues should especially be mentioned: 
______________________________________ 
Arg [A]; 
Leu Arg [A]; 
Arg Leu Arg [A]; 
Thr Arg Leu Arg [A]; 
[A] Lys; 
[A] Lys Tyr; 
[A] Lys Tyr Leu; 
Arg [A] Lys; 
Arg [A] Lys Tyr; 
Arg [A] Lys Tyr Leu; 
Leu Arg [A] Lys; 
Leu Arg [A] Lys Tyr; 
Leu Arg [A] Lys Tyr Leu; 
Arg Leu Arg [A] Lys; 
Arg Leu Arg [A] Lys Tyr; 
Arg Leu Arg [A] Lys Tyr Leu; 
Thr Arg Leu Arg [A] Lys; 
Thr Arg Leu Arg [A] Lys Tyr; 
Thr Arg Leu Arg [A] Lys Tyr Leu. 
______________________________________ 
where 
[A] denotes Lys Gln Y Ala Val Lys 
in which Y represents Met or Nle. 
The amino acids may, for example, be in the L-form; although one or more 
D-amino acids may, if desired, be present in the structure. 
The carboxy-terminus of the peptides or analogues of the present invention 
may be in the form of the acid (--OH); an ester, for example an alkyl 
ester, especially a (C.sub.1 -C.sub.4)-alkyl ester, e.g. the methyl ester, 
(--OCH.sub.3), the hydrazide (--NH--NH.sub.2), or an amide, usually the 
unsubstituted amide (--NH.sub.2). Preferably the carboxy-terminus is in 
the form of the unsubstituted amide. 
The amino-terminus of the peptides or analogues of the present invention 
may be in the form of the unsubstituted amine (--NH.sub.2) or protected 
amine (--NHR) where R represents, for example, acetyl or 
tert.-butyloxycarbonyl, or benzyloxycarbonyl, or in the form of an acid 
addition salt, preferably a physiologically tolerable, pharmaceutically 
acceptable acid addition salt, of the amine. 
Acid addition salts may be, for example, salts with inorganic acids such, 
for example, as hydrochloric acid, hydrobromic acid, orthophosphoric acid 
or sulphuric acid, or organic acids such, for example, as methanesulphonic 
acid, toluenesulphonic acid, acetic acid, trifluoroacetic acid, propionic 
acid, lactic acid, citric acid, tartaric acid, fumaric acid, malic acid, 
succinic acid, salicylic acid or acetylsalicylic acid. 
Thus, more particularly, the present invention provides a polypeptide of 
the general formula 
EQU X--(Y').sub.n --Y.sub.15 Y.sub.16 Y.sub.17 Y.sub.18 Y.sub.19 Y.sub.20 
--(Y").sub.m --Z I 
in which 
X represents a hydrogen atom or an amine-protecting group, preferably a 
hydrogen atom; 
(Y').sub.n represents a direct bond or 
EQU Y.sub.11 Y.sub.12 Y.sub.13 Y.sub.14, Y.sub.12 Y.sub.13 Y.sub.14, Y.sub.13 
Y.sub.14 or Y.sub.14 
in which 
Y.sub.11 represents Thr, Ser or the residue of another hydroxy amino acid, 
Y.sub.12 represents Arg or the residue of another basic amino acid, 
Y.sub.13 represents Leu, Phe or the residue of another hydrophobic amino 
acid, 
Y.sub.14 represents Arg or the residue of another basic amino acid; 
Y.sub.15 represents Lys or the residue of another basic amino acid, e.g. 
Orn, 
Y.sub.16 represents Gln or the residue of another carboxamido amino acid, 
Y.sub.17 represents Met or the residue of another neutral amino acid, e.g. 
Nle, 
Y.sub.18 represents Ala or the residue of another hydrophobic amino acid, 
Y.sub.19 represents Val or the residue of another hydrophobic amino acid, 
Y.sub.20 represents Lys or the residue of another basic amino acid, e.g. 
Orn, 
(Y").sub.m represents a direct bond or 
EQU Y.sub.21, Y.sub.21 Y.sub.22 or Y.sub.21 Y.sub.22 Y.sub.23 
in which 
Y.sub.21 represents Lys or the residue of another basic amino acid, 
Y.sub.22 represents Tyr or the residue of another hydrophobic amino acid, 
Y.sub.23 represents Leu or the residue of another hydrophic amino acid; and 
Z represents a hydroxyl group, or a group of the formula OR such that COZ 
represents an ester, or a hydrazino group such that COZ represents a 
hydrazide, or NH.sub.2 such that COZ represents an amide, preferably 
NH.sub.2 ; and salts thereof, preferably physiologically tolerable salts 
thereof, especially physiologically tolerable acid addition salts thereof. 
The compounds of formula I are preferably in pharmaceutically acceptable 
form. By pharmaceutically acceptable form is meant, inter alia, of a 
pharmaceutically accpetable level of purity excluding normal 
pharmaceutical additives such as diluents carriers, and including no 
material considered toxic at normal dosage levels. A pharmaceutically 
acceptable level of purity will generally be at least 50% excluding normal 
pharmaceutical additives, preferably 75%, more preferably 90% and still 
more preferably 95%. 
A peptide or analogue of the invention may be prepared by those methods 
known in the art for the synthesis of compounds of analogous structure and 
in this regard reference is made, by way of illustration only, to the 
following literature: 
(a) Y. S. Klausner and M. Bodanszky, Bioorg. Chem. (1973), 2, p 354-362. 
(b) M. Bodanszky, C. Yang Lin and S. I. Said, Bioorg. Chem. (1974), 3, p 
320-323. 
(c) S. R. Pettit, "Synthetic Peptides", (Elsevier Scientific Publishing Co. 
1976). 
(d) Stewart and Young, "Solid Phase Peptide Synthesis" (W. H. Freeman and 
Co. 1969). 
(e) E. Atherton, C. J. Logan and R. C. Sheppard, J. C. S. Perkin I, (1981) 
p 583-546. 
(f) E. Brown, R. C. Sheppard and B. J. Williams, J. C. S. Perkin I, (1983) 
p 1161-1167. 
The present invention also provides a peptide or analogue of the present 
invention which has been prepared synthetically. 
A peptide or analogue of the present invention may, for example, be formed 
by the sequential coupling of appropriate amino acids or by the initial 
preparation and subsequent coupling of peptide subunits, themselves 
prepared in stepwise manner; in either case either classical solution 
chemistry methods of peptide synthesis or solid phase procedures may be 
used. 
The coupling reactions may be effected by, for example, activating the 
reacting carboxyl group of the ingoing amino acid, and reacting this with 
the amino group of the substrate unit. Details of suitable, optional 
activating and protecting (masking) groups and of suitable reaction 
conditions (for the coupling reactions and for the introduction and 
removal of protecting groups) giving, preferably, the minimum of 
racemisation, may be found in the above-referenced literature. 
Accordingly, the present invention further provides a process for the 
preparation of a peptide or analogue of the present invention, which 
comprises coupling a suitable amino acid or amino acid sequence in which 
the carboxyl group is activated with an appropriate amino acid or amino 
acid sequence and repeating, if necessary, the coupling procedure until 
there is obtained a peptide comprising, in sequence, units selected from 
the amino acid residues 11 to 23 of VIP consisting at least of the amino 
acid residues 15 to 20, or an analogue thereof in which one or more of the 
amino acid residues is replaced by an equivalent other amino acid, 
wherein, if desired or required, non-reacting functional groups are 
protected during the coupling procedure and, if desired, subsequently 
deprotected. 
A polypeptide of the general formula I may thus be prepared by reacting a 
reagent of the general formula 
EQU H--Y.sup.1 --OH (II) 
wherein 
Y.sup.1 represents an amino acid unit or a partial radical sequence 
identical with the corresponding N-terminal amino acid unit or partial 
radical sequence in formula I, 
with a reagent of the general formula 
EQU H--Y.sup.2 --OH (III) 
wherein 
Y.sup.2 represents an amino acid unit or a partial radical sequence 
identical with that in the balance of the above-defined product peptide, 
the reagents (II) and (III) being optionally protected and/or activated 
where and as appropriate, followed if desired or required by one or more 
of the following: 
deprotection of the products, 
conversion of one carboxy terminus into another carboxy terminus, 
conversion of a free peptide into a salt thereof. 
For example, an appropriate peptide ester of the general formula 
EQU X--Y.sup.1 --Y.sup.2 --OR (IV) 
wherein X, Y.sup.1 and Y.sup.2 have the meanings given above and R 
represents, for example, an alkyl group and preferably an alkyl group 
having 1 to 4 carbon atoms, may be converted into an amide by reaction 
with ammonia. 
Compounds of the general formulae II, III and IV may themselves be prepared 
by standard techniques analogous to those described above. 
It will be appreciated that a protected forms of a peptide or analogue of 
the present invention are useful novel intermediates and form an aspect of 
the invention. 
A peptide or analogue of the present invention may also be prepared on a 
solid phase support, for example a polyamide or a polystyrene resin, using 
amino acids protected at the N-terminus, for example with the 
fluorenylmethyloxycarbonyl group or the t-butyloxycarbonyl group and with 
appropriate protection of any side-chain functional groups. 
One such reaction scheme for solid-phase peptide synthesis is, for example, 
illustrated below. 
##STR5## 
This technique involves the addition of the first protected amino acid to a 
solid resin support. After removal of the protecting group (deprotection) 
the amino acid-resin is coupled with the next protected amino acid in its 
activated form. The deprotection/coupling procedures are repeated until 
the required peptide is obtained. The peptide is then cleaved from the 
resin before final removal of the protecting groups Alternatively, when 
desired or necessary, the protecting groups may be removed before cleavage 
of the peptide from the resin. 
Advantageously the Fmoc group is the form of protection used for the 
.alpha.-amino function of the amino acids involved (but not for side chain 
protection). 
However, the last amino acid in each synthesis is generally protected as 
its t-BOC or FMOC derivative. This allows the peptide to remain fully 
protected on cleavage from the resin. 
The use of alternative resins may also require the need for removal of 
protecting groups prior to resin cleavage. In this case it is likely that 
the Fmoc-protecting group would be used for N-alpha protection throughout 
the syntheses. 
The peptides and analogues of the present invention have smooth muscle 
relaxant activity such as gastro-intestinal, bronchodilator and 
vasodilator actions, and in addition, anti-ulcer activity. They may be 
useful in preventing the pain and constipation frequently encountered in 
some irritable bowel syndrome (IBS) patients and may be a useful new 
approach to duodenal ulcer therapy. 
The present invention further provides a peptide or analogue of the present 
invention, for use in a method of treatment of the human or animal body. 
Where the fragment or analogue is in the form of a salt thereof, it should 
of course be understood that this is a physiologically tolerable salt, 
which is pharmaceutically acceptable. 
The peptide or analogue of the invention may be administered per se or, 
preferably, as a pharmaceutical composition also including a 
pharmaceutically suitable carrier. 
Accordingly, the present invention provides a pharmaceutical composition, 
which comprises a peptide or analogue of the present invention, in 
admixture or conjunction with a pharmaceutically acceptable carrier. 
The preparation may, if desired, be in the form of a pack accompanied by 
written or printed instructions for use. 
In accordance with conventional pharmaceutical practice the carrier may 
comprise a diluent, filler, disintegrant, wetting agent, lubricant, 
colourant, flavourant or other conventional additive. 
Preferably, a pharmaceutical composition of the invention is in unit dosage 
form. 
The suitable dosage range for compounds of the invention may vary from 
compound to compound and may depend on the condition to be treated. It 
will also depend, inter alia, on the relation of potency to absorbability 
and on the mode of administration chosen. 
Suitable formulations are, for example, intravenous infusions, aerosols and 
enteric coated capsules. 
The present invention further provides a method of treatment of a human or 
non-human animal, which comprises administering an effective, non-toxic, 
amount of a peptide or analogue of the present invention to a human or 
non-human animal; and a peptide or analogue of the present invention for 
use as a pharmaceutical, in particular for the treatment of disorders and 
complaints described below. 
A peptide or analogue of the present invention may be used to treat the 
following disorders and complaints; abnormalities of gut motility, e.g. 
hypermotility as in IBS or oesophageal spasm; peptic ulceration; bronchial 
spasm; vascular conditions such as hypertension and ischaemia; mental 
disorders. 
Conveniently, the active ingredient may be administered as a pharmaceutical 
composition hereinbefore defined, and this forms a particular aspect of 
the present invention. 
A suitable dose is, for example, in the range of from 1 .mu.g to 2.5 mg/kg 
i.v. in the rat. A possible daily dose for humans is, for example, 0.01 to 
50 mg by intravenous infusion, 0.01 to 250 mg by aerosol or 0.1 to 500 mg 
by enteric coated capsule. 
No adverse toxicological effects are indicated at the aforementioned dosage 
ranges. 
In the following, the various derivatives protecting groups, reagents and 
solvents are referred to by abreviations for convenience. 
______________________________________ 
Derivatives, Protecting 
Abbreviated 
Groups, Reagents, Solvents 
Designation 
______________________________________ 
Tertiary-butyl Bu.sup.t 
Tertiary-butyloxycarbonyl 
t-Boc 
N--hydroxysuccinimide ester 
OSu 
Methyl ester OMe 
Trifluoroacetic acid TFA 
Dicyclohexylcarbodiimide 
DCC 
Benxyloxycarbonyl CBZ 
Dimethylformamide DMF 
Tetrahydrofuran THF 
p-Nitrophenyl ester ONP 
Hydrochloride salt .HCl 
Ethyl acetate EtOAc 
Methanol MeOH 
Ammonium Acetate NH.sub.4 OAc 
1-Hydroxybenzotriazole HOBT 
Chloroform CHCl.sub.3 
Pyridine Pyr 
n-Butanol BuOH 
Ammonium hydroxide NH.sub.4 OH 
Sodium hydrogen carbonate 
NaHCO.sub.3 
Sodium chloride NaCl 
Ether Et.sub.2 O 
Sodium sulphate Na.sub.2 SO.sub.4 
Potassium hydroxide KOH 
Acetic acid AcOH 
______________________________________ 
T.L.C. (Merck) silica gel plates) with solvent systems 
E.sub.4 MeOH--CHCl.sub.3 (1:9) 
H n-BuOH:AcOH:Pyr:H.sub.2 O (15:3:10:12) 
A.sub.3 n-BuOH:AcOH:H.sub.2 O (4:1:1)

EXAMPLE 1 
H-Lys-Gln-Nle-Ala-Val-Lys-NH.sub.2 (V) 
The hexapeptide amide (V) was prepared as illustrated in Scheme I and the 
experimental details are given below. 
##STR6## 
t-Boc-Val-Lys(CBZ)-OMe (VI) 
A mixture of t-Boc-Val-OSu (3.14 g, 10 mmol) and H-Lys(CBZ)-OCH.sub.3.HCl 
(3.31 g, 10 mmol) in THF (300 ml) was treated at room temperature with 
triethylamine (1.38 ml) and left stirring for 17 hr. The resulting 
solution was evaporated in vacuo and the residue was dissolved in EtOAc 
(500 ml). The organic solution was washed successively with water 
(2.times.200 ml), 5% citric acid (2.times.200 ml), water (2.times.200 ml) 
and dried over Na.sub.2 SO.sub.4. The dried solution was filtered and 
evaporated in vacuo to give (VI) (4.9 g; 99%) as a foam. T.l.c. R.sub.f 
E.sub.4 =0.64. 
H-Val-Lys(CBZ)-OMe trifluoroacetate (VII) 
The protected dipeptide (VI) (4.9 g, 10 mmol) was dissolved in TFA (20 ml) 
and stirred for 10 minutes at room temperature. The solution was 
evaporated in vacuo, azeotroped with toluene (2.times.20 ml) and 
triturated with Et.sub.2 O (2.times.50 ml). The mother liquors were 
decanted to leave the partially deprotected dipeptide as the 
trifluoroacetate salt VII (2.41 g; 51% R.sub.f E.sub.4 =0.22. 
t-Boc-Ala-Val-Lys(CBZ)-OMe (VIII) 
(VII) 2.4 g, 6.1 mmol) was added to t-Boc-Ala-ONP (1.9 g, 6.1 mmol) in THF 
(50 ml) containing triethylamine (0.85 ml). The mixture was stirred at 
room temperature for 4 days, evaporated in vacuo and partitioned between 
EtOAc (250 ml) and water (100 ml). The organic layer was washed 
successively with 0.45M NH.sub.4 OH (4.times.50 ml), 2% citric acid 
(4.times.50 ml) and water (4.times.50 ml). The organic layer was dried 
over Na.sub.2 SO.sub.4, filtered and evaporated in vacuo. The residue was 
recrystallised from EtOAc-hexane to give (VIII) (1.62 g; 47%) as 
colourless microcrystals, mp 104.degree. C. [.alpha.].sub.D.sup.26 
=-48.degree. C. (C=1, MeOH). 
t-Boc-Ala-Val-Lys(CBZ)-NH.sub.2 (IX) 
(VII) (2.0 g, 35 mmol) was added to a solution of ammonia (ca. 50 ml) in 
methanol (50 ml). The mixture was kept in a sealed pressure vessel for 24 
hrs, then evaporated to dryness. The residue was taken up in EtOAc, 
evaporated to dryness and triturated with Et.sub.2 O give (IX) (1.75 g; 
90%) as colourless microcrystals mp 195.degree.-196.degree. C. 
[.alpha.].sub.D.sup.26 =-48.8.degree. (C=1, MeOH). 
H-Ala-Val-Lys(CBZ)-NH.sub.2.trifluoroacetate (X) 
The protected tripeptide amide (IX) (1.0 g, 1.8 mmol) was dissolved in cold 
TFA (10 ml). After 10 minutes, the mixture was evaporated in vacuo and the 
residue was triturated with ether (2.times.50 ml). The mother liquors were 
decanted and the residue was dried under vacuum to give (X) as a foam 
(0.85 g; 83%). 
t-Boc-Gln-Nle-OMe (XI) 
t-Boc-Gln-ONP (12.3 g, 32 mmol) and HOBT (5.0 g, 37 mmol) were added to a 
solution of H-Nle-OMe.HCl (5.99 g, 33 mmol) and triethylamine (4.9 ml) in 
DMF (55 ml). 
The mixture was stirred at room temperature overnight, EtOAc (100 ml) was 
added and the organic phase was washed with 2% citric acid, 0.45M NH.sub.4 
OH until free of nitrophenol, 5% NaHCO.sub.3, 2% citric acid, water until 
neutral, and a saturated solution of NaCl. The solution was dried over 
Na.sub.2 SO.sub.4, filtered and concentrated in vacuo. Petroleum ether 
(bpt 40.degree.-60.degree. C.) was added, the precipitate was filtered, 
washed with the same solvent and dried in vacuo over silica gel to give 
(XI) (10.2 g, 85%) mpt 108.degree.-109.degree. C., [.alpha..sub.D.sup.26 
=-14.89.degree. (C-1, DMF), T.l.c. R.sub.f A.sub.3 =0.72. 
H-Gln-Nle-OMe.trifluoroacetate (XII) 
The protected dipeptide ester (XI) (4.8 g, 13 mmol) was dissolved in cold 
TFA (40 ml). After 10 minutes, the TFA was removed in vacuo and dry ether 
(200 ml) was added. The ether was decanted and the residue was washed with 
more ether (100 ml). The oily material was dried over KOH to give (XII) as 
a white foam that was used immediately, T.l.c. R.sub.f A.sub.3 =0.42. 
BOC-Lys(CBZ)-Gln-Nle-OMe (XIII) 
H-Gln-Nle-OMe.TFA salt (XII) (13 mmol) triethylamine (1.76 ml, 13 mmol), 
HOBT (2.16 g, 16 mmol) and BOC-Lys(CBZ)-ONP (7.6 g, 15 mmol) were 
dissolved in DMF (30 ml). The reaction mixture was kept basic with small 
amounts of triethylamine. The mixture was stirred overnight at room 
temperature, concentrated in vacuo and treated with unsymmetrical 
dimethylethylenediamine (2 equivs). After 2 hours, EtOAc was added and the 
product was isolated as described for compound (XI). The solid was washed 
with petroleum ether (bpt 40.degree.-60.degree. C.) and dried in vacuo 
over silica gel to give (XIII) (5.5 g; 65%) [.alpha..sub.D.sup.26 
=-16.49.degree. (C=1, DMF) T.l.c. R.sub.f A.sub.3 0.8. 
BOC-Lys(CBZ)-Gln-Nle-NHNH.sub.2 (XIV) 
The tripeptide methyl ester (XIII) (1 g, 1.6 mmol) was suspended in DMF (5 
ml), hydrazine (0.8 g, 0.78 ml, 16 mmol) was added and the mixture was 
stirred overnight. The solvent was removed in vacuo and the residue 
solidified using MeOH/EtOAc to give (XIV) (0.9 g, 90%). T.l.c. R.sub.f 
A.sub.3 =0.70 This was used immediately in the next step. 
t-Boc-Lys(CBZ)-Gln-Nle-Ala-Val-Lys(CBZ)-NH.sub.2 (XV) 
(XIV) 0.58 g, 0.9 mmol) was dissolved in anhydrous DMF (18 ml) and cooled 
to -30.degree. C. 4.56M HCl in dioxane (0.90 ml) was added followed by 
t-butyl nitrite (0.12 ml). The reaction was left for 30 to 40 min at 
-30.degree. C. then cooled to -60.degree. C. Triethylamine (0.60 ml) was 
added followed by the deprotected amide (X) (0.384 g, 0.68 mmol) and a 
further addition of triethylamine (0.1 ml). The mixture (M) was left to 
stand, reaching ambient temperature over 2 days. A further amount of (XIV) 
(0.29 g, 0.45 mmol) in DMF (10 ml) was treated at -30.degree. C. with 4.5M 
HCl in dioxane (0.45 ml), t-butyl nitrite (0.06 ml) and triethylamine (0.3 
ml) as described above and added to the reaction mixture (M) at 
-30.degree. C. The whole was left to stand, reaching ambient temperature 
over a further 4 days. 
The mixture was evaporated in vacuo and the residue was triturated with 
EtOAc:MeOH (1:1) (50 ml) to give (XV) (0.62 g; 85%) as a greyish solid. 
H-Lys-Gln-Nle-Ala-Val-Lys-NH.sub.2 (V) 
The protected hexapeptide (XV) (0.2 g, 0.2 mmol) was dissolved in TFA (5 
ml) and treated with HBr gas over 1 hr. The mixture was evaporated in 
vacuo and triturated with Et.sub.2 O (2.times.50 ml) to give (V) as a 
hydrobromide salt (0.13 g) R.sub.f H=0.14. This and a subsequent batch of 
product were purified by adsorption on to an ion exchange column (CM 25 
Sephadex, Pharmacia) which was washed with 10-100 mmol NH.sub.4 OAc at pH 
7. The product was eluted with 100 mmol NH.sub.4 OAc at pH 8.5. 
Lyophilisation and subsequent preparative HPLC [.mu.Bondapak ODS.; 
CH.sub.3 CN: 50 mmol NH.sub.4 OAc(aq) (15:85) gave (V) as an acetate salt 
(0.15 g) mp 253.degree.-255.degree. C. T.l.c. R.sub.f H=0.14, MH.sup.+ 
(FAB)=685. 
EXAMPLE 2 
H-Arg-Lys-Gln-Nle-Ale-Val-Lys-Lys-NH.sub.2.Acetate (XVI) 
The octapeptide amide (XVI) was prepared as illustrated in Scheme II and in 
the experimental details given below. 
t-Boc-Lys(CBZ)-Lys(CBZ)-OCH.sub.3 (XVII) 
A mixture of t-Boc-Lys-(CBZ)-OH (1.14 g, 3 mmol), Lys-(CBZ)-OCH.sub.3.HCl 
(0.99 g, 3 mmol), DCC (0.62 g, 3 mmol), HOBT (0.41 g, 3 mmol) and 
triethylamine (0.42 ml) in dry amine-free DMF (20 ml) was stirred for 17 
h. Work up as described for VI gave XVII (1.2 g; 61%) as colourless 
microcrystals, mp 109.degree.-110.degree. (ex acetone-light petroleum 
ether 40.degree.-60.degree.) [.alpha.].sub.D.sup.26 =-11.9.degree. (C=1 
MeOH) R.sub.f E.sub.4 =0.71. 
H-Lys-(CBZ)-Lys-(CBZ)-OCH.sub.3.trifluoroacetate (XVIII) 
The protected dipeptide (XVII) (1.8 g, 2.7 mmol) was partially deprotected 
as for VII to give XVIII as a foam (1.8 g; 99%) R.sub.f E.sub.4 =0.22. 
t-Boc-Val-Lys-(CBZ)-Lys-(CBZ)-OCH.sub.3 (XIX) 
A mixture of t-Boc-Val-OSu (0.86 g 2.7 mmol), XVIII (1.8 g, 2.7 mmol) and 
triethylamine (0.4 ml) in THF (50 ml) was stirred, under N.sub.2, for 2 
days. Work up as described for VI gave XIX (1.25 g; 60%) as colourless 
microcrystals, mp 145.degree.-147.degree. (ex EtoAc) R.sub.f E.sub.4 =0.40 
[.alpha.].sub.D.sup.26 =-24.59.degree. (C=1 MeOH). 
H-Val-Lys-(CBZ)-Lys-(CBZ)-OCH.sub.3.trifluoroacetate (XX) 
The protected tripeptide XIX (2.16 g, 2.9 mmol) was partially deprotected 
as for VII to give XX as a flaky solid (2.02 g; 92%). 
Boc-Ala-Val-Lys-(CBZ)-Lys-(CBZ)-OCH.sub.3 (XXI) 
A mixture of XX (2.0 g), Boc-Ala-ONP (1.0 g, 3.2 mmol), HOBT (0.80 g) and 
triethylamine (0.50 ml) was stirred at room temperature in DMF (5 ml) for 
24 h. The mixture was evaporated to 1/4 volume and taken up into 
CHCl.sub.3 (100 ml). The organic solution was washed successively with 
0.45M NH.sub.4 OH (4.times.50 ml), 2% citric acid (4.times.50 ml) and 
water (4.times.50 ml). The organic layer was dried over Na.sub.2 SO.sub.4, 
filtered and evaporated in vacuo to 1/4 volume. The solution was 
chromatographed on Kieselgel 60 PF.sub.254 on a `Chromatotron` and the 
product was eluted with an increasing concentration of MeOH (0-5%) in 
CHCl.sub.3 to give XXI (1.98 g; 84%) as colourless microcrystals, mp 
175.degree.-177.degree. [.alpha.].sub.D.sup.26 =-36.89 (C=1 MeOH). 
MH.sup.+ =827 (FAB). 
Boc-Ala-Val-Lys-(CBZ)-Lys-(CBZ)-NH.sub.2 (XXII) 
XXI (1.78 g, 2.2 mmol) was added to a solution of ammonia (ca. 50 ml) in 
methanol (50 ml). The mixture was kept in a sealed vessel for 48 h. The 
resulting precipitate was filtered, washed with dry Et.sub.2 O to give 
XXII (1.78 g; 98%) as colourless microcrystals mp 243.degree.-244.degree.. 
H-Ala-Val-Lys-(CBZ)-Lys-(CBZ)-NH.sub.2.trifluoroacetate (XXIII) 
The protected tetrapeptide XXII (1.75 g, 2.2 mmol) was suspended in acetic 
acid (3 ml), cooled to 10.degree. and treated with TFA (9 ml). The 
solution was stirred for 20-25 min. Work-up as described for VII gave 
XXIII (1.71 g) R.sub.f E.sub.1 =0.7. 
H-Lys-(CBZ)-Gln-Nle-OMe (XXIV) 
The protected tripeptide ester (XIII) (2.0 g, 3 mmol) was dissolved in cold 
TFA (12.6 ml) and glacial acetic acid (5.4 ml). After 25 minutes, the 
solvents were removed in vacuo and dry ether (100 ml) was added. The ether 
was decanted and the residue was washed with more ether (100 ml). The oily 
material was dried in vacuo over KOH to give the trifluoroacetate salt of 
(XXIV) as a white foam. The foam was dissolved in water (40 ml) and a cold 
solution of sodium carbonate (0.15 g) in water (10 ml) added. The free 
base was extracted into ethyl acetate (100 ml, then 4.times.30 ml) and 
this organic phase was washed with water (2.times.20 ml), saturated NaCl 
(20 ml), dried over Na.sub.2 SO.sub.4, filtered and evaporated in vacuo to 
give XXIV (1.5 g; 89%). T.l.c. R.sub.f =0.3 in 30% MeOH/CHCl.sub.3. 
BOC-Arg(H.sup.+)-Lys-(CBZ)-Gln-Nle-OMe (XXV) 
The free amine (XXIV) (1.5 g, 2.8 mmol) was dissolved in DMF (9 ml). The 
solution was cooled, then BOC-Arg(H.sup.+)OH (1.23 g, 4.5 mmol), DCC (0.82 
g, 4 mmol) and HOBT (0.57 g, 4 mmol) were added. After 2 hours, additional 
portions of BOC-Arg(H.sup.+)OH (0.45 g, 1.6 mmol), DCC (0.3 g, 1.5 mmol) 
and HOBT (0.2 g, 1.6 mmol) were added and the reaction was allowed to 
proceed for 3 days. 
The dicyclohexylurea was removed by filtration and washed with DMF 
(3.times.5 ml). The solvent was removed in vacuo and the residue was 
applied in methanol to a Sephadex LH-20 column (2.5.times.100 cm) 
pre-equilibrated with the same solvent. Fractions of 5 ml were collected 
at a flow rate of 1 ml/3 mins. The fractions containing the desired 
product were pooled, evaporated and re-chromatographed under the same 
conditions. 
The product was further purified on Kieselgel 60 Pf.sub.254 using a 
`Chromatotron` (20% MeOH/CHCl.sub.3 as eluant) to give (XXV) (1.3 g, 56%) 
[.alpha.].sub.D.sup.26 =-24.9.degree. (C=1, MeOH). 
BOC-Arg(H.sup.+)-Lys-(CBZ)-Gln-Nle-NHNH.sub.2 (XXVI) 
The tetrapeptide methyl ester (XXV) (1.3 g, 1.6 mmol) was suspended in 
methanol (6 ml), hydrazine hydrate (0.8 g, 0.78 ml, 1.6 mmol) was added, 
and the stirring was continued for 6 hours. The product was filtered, 
washed with cold methanol (3.times.10 ml), water (8.times.5 ml), and dried 
in vacuo to give (XXVI) (1.2 g; 92%). The product was used immediately. 
BOC-Arg(H.sup.+)-Lys-(CBZ)-Gln-Nle-Ala-Val-Lys-(CBZ)-Lys-(CBZ)-NH.sub.2.Chl 
oride (XXVII) 
The protected tetrapeptide (XXVI) (0.37 g, 0.45 mmol) was dissolved in 
anhydrous DMF (5 ml) and cooled to -30.degree.. 4.56M HCl in dioxane (0.45 
ml) was added followed by t-butylnitrite (0.06 ml). The reaction was left 
for 30-40 min at -30.degree. then cooled to -60.degree.. Triethylamine 
(0.30 ml) was added followed by the deprotected amide XXIII (0.28 g, 0.3 
mmol) and a further addition of triethylamine (0.05 ml). The mixture 
M.sub.2 was left to stand, reaching ambiant temperature over 2 days. A 
further amount of XXVI (0.21 g, 0.26 mmol) in DMF (5 ml) was treated at 
-30.degree. C. with 4.56M HCl in dioxane (0.25 ml), t-butyl nitrite (0.04 
ml) and triethylamine (0.17 ml) as described above and added to the 
reaction mixture M.sub.2 at -30.degree. C. The whole was left to stand, 
reaching ambiant temperature over a further 4 days. 
The mixture was treated with methanol and the whole centrifuged. The 
resulting solid and mother liquors were both shown to contain the desired 
product XXVII (1.25 g) MH.sup.+ =1471 (FAB). 
H-Arg-Lys-Gln-Nle-Ala-Val-Lys-Lys-NH.sub.2.Acetate (XVI) 
The protected octapeptide (XXVII) (1.25 g, 0.85 mmol) was dissolved in TFA 
at 10.degree. and treated with hydrogen bromide gas for 2 h. The whole 
mixture was evaporated in vacuo, triturated with ether (4.times.15 ml) and 
filtered to give the free peptide as its hydrobromide salt (1.0 g). The 
peptide was purified with concomitant conversion to an acetate salt, XVI 
(0.22 g) (MH.sup.+ (FAB)=969) in the same manner as that described for 
(V). 
##STR7## 
Solid Phase Synthesised Peptides 
(a) The following examples were synthesised by solid phase methods using 
the 4-hydroxymethylbenzoylnorleucyl derivatised polydimethylacrylamide gel 
resin Pepsyn B (1.0 mequiv/g or 0.3 mequiv/g) as supplied by Cambridge 
Research Biochemicals Ltd. 
DMF was fractionally distilled in vacuo from ninhydrin before use and 
stored over pre-activated molecular sieves (4 A). Piperidine was freshly 
distilled from a suitable drying agent. Dichloromethane (A.R.) was dried 
over pre-activated molecular sieves (4 A). 
The amino acids were chosen as their Fmoc-derivatives with BOC- to t-Bu- 
side chain protection where necessary. 
The symmetrical anhydride of the first amino acid (2.5 equiv), (prepared as 
described by E. Brown et al in J.C.S. Perkin I, 1983, 80) was added to the 
resin (1 equiv) in DMF (10-15 ml) in the presence of a catalytic quantity 
of dimethylaminopyridine. The mixture was agitated with N.sub.2 and the 
reaction was allowed to proceed for 1 h. The resin was drained and the 
addition procedure was repeated. The drained resin was then washed with 
DMF (10-15 ml.times.1 min.times.10). The removal of the Fmoc protecting 
groups was achieved by agitation of the peptide-resin with piperidine (10 
ml; 20% DMF) for 3 min then 7 min. 
Subsequent addition of each amino acid was carried out using the Fmoc 
symmetrical amino acid anhydrides (2.5 equiv) or the preformed 
hydroxybenzotriazole ester (3.0 equiv) (from Fmoc-amino acid, DCC and 
HOBT). 
Amino acids containing amidic side chains (e.g. Gln or Asn) were coupled as 
their p-nitrophenyl activated esters (3.0 equiv) in the presence of 
hydroxybenzotriazole (6.0 equiv). 
Fmoc-Arginine was coupled to the peptide resin via its hydroxybenzotriazole 
ester. This was prepared by suspending Fmoc-Arginine (10 equiv) in DMF (10 
ml) and adding HOBT (30 equiv). The clear solution was added to the resin 
and agitated for 1 minute. DCC (10 equiv) was then added and the reaction 
was allowed to proceed to completion. 
The final amino acid in the chosen sequence was added as its N.alpha. Boc 
derivative either as the symmetrical anhydride or as the preformed 
hydroxybenzotriazole ester. 
Boc-Arginine was coupled as its hydrochloride and activated by addition of 
DCC (5 equiv) to the protected hydrochloride salt (10 equiv) in DMF (10-15 
ml) 5 minutes prior to addition of the whole reaction mixture to the 
peptide-resin (1 equiv). 
In some cases, Fmoc-amino acid anhydrides (eg Phe, Ala, Gly) coprecipated 
with DCU during their formation. In these cases, the anhydrides were 
prepared in the presence of 10% DMF in dichloromethane. Dichloromethane 
was removed in vacuo before addition of the whole mixture to the peptide 
resin. Couplings in general were carried out for 1-2 h and repeated if 
necessary. Completeness of acylation was verified by a qualitative Kaiser 
ninhydrin test as described by E. Kaiser et al in Anal. Biochem. (1970) p. 
34. 
Peptide cleavage from the resin was accomplished via ammonolysis to provide 
the protected peptide amide. To this end, when the final coupling was 
complete, the peptide-resin was washed with DMF (10-15 ml.times.1 
min.times.10), anhydrous dichloromethane (10-15 ml.times.1 min.times.10) 
and dry ether (10 ml.times.1 min.times.10). The collapsed resin was dried 
over silica gel for 1 hour in a vacuum desiccator: The resin was 
re-swollen as previously described, drained and treated with a saturated 
solution of ammonia in methanol at -10.degree.. The vessel was sealed and 
allowed to reach ambiant temperatures for 2 days. The apparatus was 
cooled, opened and the contents were allowed to warm to room temperature. 
The suspension was filtered under suction and the resulting residue was 
washed with methanol (5.times.5 ml) and DMF (5.times.5 ml). The combined 
washings and filtrate were evaporated in vacuo. The resulting residue was 
triturated with dry ether and filtered to give the protected peptide. 
The final acidolytic deprotection procedure removed all protecting groups 
(e.g. BOC, t-Bu) from the peptide amide. Thus the protected peptide was 
dissolved in trifluoroacetic acid (4 ml/100 mg of peptide) and stirred at 
room temperature for 3 h. In some cases, hydrogen bromide gas was bubbled 
though the mixture during this time. The mixture was evaporated in vacuo 
and the resulting solid was triturated with dry ether (7.times.5 ml) to 
give the required peptide either as its trifluoroacetate or its 
hydrobromide salt. The peptides were purified by one or a combination of 
methods listed below. 
(a) Conversion to acetate salt 
The peptide salt was dissolved in a minimum amount of water and passed down 
a strong anion exchange resin which was in its acetate form (e.g. Sephadex 
QAE-A-25). Eluant was fractionated and the fractions containing desired 
materials were lyophilised. 
(b) Selective adsorbtion chromatography 
The peptide salt was dissolved in a minimum amount of water and adsorbed 
onto a weak cation exchange resin (e.g. Sephadex CM-25). The peptide 
acetate was recovered during elution with an increasing concentration of 
NH.sub.4 OAc (0.05M-0.5M) at pH 7, an increasing pH gradient (pH 7-pH 9) 
or a combination of both. 
(c) High Performance Liquid Chromatography.HPLC 
The peptide was purified by preparative HPLC on reverse phase C.sub.18 
silica columns (e.g. .mu.bondapak, Hypersil ODS). 
The peptides were characterised by 24 h acidolytic cleavage and PITC 
derivatised amino acid analysis (Waters Picotag system) and fast atom 
bombardment (FAB) mass spectrometry (Jeol DX 303). 
EXAMPLE 3 
H-Leu-Arg-Lys-Gln-Nle-Ala-Val-Lys-Lys-NH.sub.2.Acetate (XXVIII) 
XXVIII was prepared using the 0.3 mequiv/g Pepsyn B resin. 
[MH].sup.+ =1081 (FAB). 
Amino acid analysis. Glu (1.0) Arg (1.0) Ala (1.0) Val (0.88) Nle (1.0) Leu 
(1.0) Lys (2.88). 
EXAMPLE 4 
H-Lys-Gln-Nle-Ala-Leu-Lys-NH.sub.2.Acetate (XXIX) 
XXIX was prepared using the 1.0 mequiv/g Pepsyn B resin. [MH].sup.+ 
(FAB)=699. Amino acid analysis; Glu (0.9), Ala (0.8), Leu (0.8), Nle 
(0.8), Lys (1.8). 
EXAMPLE 5 
H-Lys-Gln-Nle-Ale-Val-Orn-NH.sub.2.Acetate (XXX) 
XXX was prepared using the 1.0 mequiv/g Pepsyn B resin [MH].sup.+ 
(FAB)=671. Amino acid analysis; Glu (1.01), Ala (0.9), Val (0.8), Nle 
(1.1), Lys (1.0), Orn (1.1). 
EXAMPLE 6 
H-Orn-Gln-Nle-Ala-Val-Orn-NH.sub.2.Acetate (XXXI) 
XXXI was prepared using the 1.0 mequiv/g Pepsyn B resin [MH].sup.+ 
(FAB)=657. 
EXAMPLE 7 
H-Lys-Gln-Leu-Ala-Val-Lys-NH.sub.2.Acetate (XXXII) 
XXXII was prepared using the 1.0 mequiv/g Pepsyn B resin [MH].sup.+ 
(FAB)=685. 
EXAMPLE 8 
H-Arg-Lys-Gln-Nle-Ala-Val-Lys-Lys-Tyr-Leu-NH.sub.2.Acetate (XXXIII) 
XXXIII was prepared using the 0.3 mequiv/g Pepsyn B resin. [MH].sup.+ 
(FAB)=1245. 
EXAMPLE 9 
H-Lys-Gln-Nle-Ala-Val-Lys-Lys-Tyr-Leu-NH.sub.2.Acetate (XXXIV) 
XXXIV was prepared using the 0.3 mequiv/g Pepsyn B resin. [MH].sup.+ 
(FAB)=1089. 
(b) Use of the Beckmann model 990B Peptide Synthesiser 
The following example was synthesised using leucine resin ester. This was 
prepared by reacting chloromethylated resin (3.5 g, 0.7 mequiv Clg.sup.-1 
; 1% cross-linked styrene/divinylbenzene as supplied by Merseyside 
Laboratories) at 50.degree. C., for 17 h, in DMF (40 ml) with the 
anhydrous cesium salt obtained from Boc-L-leucine monohydrate (0.5 g, 2 
mmol). The resulting Boc-L-leucine resin ester was exhaustively washed 
with DMF, 50% aqueous DMF, H.sub.2 O, DMF and finally CH.sub.2 Cl.sub.2, 
then dried (3.68 g; 0.22 mmol leucine/g). 
Removal of the BOC group (from 3.6 g resin) was achieved by reaction with 
50% TFA in CH.sub.2 Cl.sub.2 (50 ml) for 5 min then 25 min. The leucine 
resin ester salt was washed with CH.sub.2 Cl.sub.2 (7.times.50 ml), 
neutralised with 10% di-isopropylamine in CH.sub.2 Cl.sub.2 for 5 min 
(3.times.50 ml) and washed with CH.sub.2 Cl.sub.2 (8.times.50 ml). 
The first amino acid of the required sequence was coupled to the leucine 
resin ester by the following procedure. Fmoc-L-tyrosine-t-butyl ether (6 
mmol) and di-isopropylcarbodiimole (6 mmol) were reacted with the leucine 
resin ester in CH.sub.2 Cl.sub.2 /DMF (35 ml) for 12 h then the 
Fmoc-Tyr(Bu.sup.t)-Leu-resin ester was washed with CH.sub.2 Cl.sub.2 
(5.times.50 ml). 
To remove the Fmoc protecting group, the peptide resin was washed with DMF 
(5.times.50 ml), reacted with 55% piperidine in DMF (50 ml) for 5 min then 
20 min, then washed with DMF (5.times.50 ml). 
Subsequent Fmoc amino acids were coupled using the procedure described 
above except for Fmoc-glutamine which was incorporated using the HOBT/DCC 
pre-activation procedure of Konig and Geiger (Chem. Ber., 103, 788-98, 
(1970)). 
Couplings, in general, were carried out for 1-2 h and repeated if 
necessary. Completeness of acylation was verified by a qualitative 
ninhydrin test as described by E. Kaiser et al, in Anal. Biochem., (1970), 
p34. 
Deprotection was carried out by reaction with 55% piperidine in DMF, as 
described above, followed by reaction with a mixture of TFA (45 ml), 
CH.sub.2 Cl.sub.2 (45 ml), anisole (10 ml) and methionine (1 g) for 84 
min. The peptide resin was then washed with CH.sub.2 Cl.sub.2 (5.times.50 
ml) and dried. 
Peptide cleavage from the resin was accomplished via ammonolysis to provide 
the peptide amide. To this end, the peptide resin was stirred with 
ammonia-saturated methanol (120 ml) for 44 h, filtered and washed with 
methanol. Evaporation of the combined washings followed by lyophilisation 
from aqueous acetic acid gave the crude peptide amide. The ammonolysis was 
repeated if FAB mass spec showed the presence of peptide ester. 
Purification was carried out via HPLC on a Lichoprep RP8 column 
(25.times.1.6 cm) with 0.1% aqueous TFA (A) and 90% acetonitrile/10% A (B) 
as a gradient from 0% B to 100% B over 60 min at 12 ml/min. 
EXAMPLE 10 
H-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-NH.sub.2.Acetate (XXXV) 
[MH].sup.+ (FAB)=1007. 
Amino acid analysis; Glu (0.92), Ala (0.92), Tyr (0.98), Val (0.99), Met 
(0.85), Leu (1.08), Lys (3.24). 
EXAMPLE 11 AND 12 
The following examples are prepared in accordance with the methods 
described for examples 3 to 9. 
##STR8## 
PHARMACOLOGICAL DATA 
I. Colonic Motility 
(a) In vivo 
Male albino rats, Wistar strain (Charles River UK) 300-500 g were 
anaesthetised with urethane. A segment of proximal colon was prepared for 
intraluminal pressure recording after the method of Maggi and Meli (Maggi, 
C. A. and Meli, A. (1982), J. Pharmacol. Methods 8, 39-46). The activity 
of the compound (V) was assessed from its action on the spontaneous 
motility of the preparation after intravenous administration. The compound 
was found to be active at 3 .mu.mol/kg. 
(b) In vitro 
Segments of circular muscle cut from the proximal colon of rats were 
mounted in Krebs solution in isolated tissue baths after the methods of 
Couture et al. (Couture R. et al. (1981), Can. J. Physiol. Pharmacol., 59, 
957-964; and Couture R. et al., (1982), Pharmacol., 24, 230-242). The 
activity of the compound V was assessed from the effect on the spontaneous 
contractile responses generated by this tissue. The ED.sub.50 was found to 
be 10.sup.-5 M. 
II. Anti-ulcer Activity 
Anti-ulcer activity may be related to the enhanced capacity to dispose of 
gastric acid. Acid disposal capacity may be enhanced by increased 
intestinal secretions and enhanced acid disposal capacity is believed to 
be useful in the treatment of peptic ulcer disease. 
Method for estimating the acid disposal capacity of the rat proximal 
duodenum 
Male Wistar rats, 180-250 g bodyweight, fasted overnight, are anaesthetized 
with urethane (150 mg/100 g bodyweight i.m.). The trachea is cannulated, 
and a gastric cannula, 0.5 cm i.d., 3 cm long, is inserted into the 
non-glandular forestomach via a mid-line abdominal incision. The 
intragastric cannula is exteriorized via a stab wound in the body wall. A 
triple lumen catheter, 0.3 cm o.d., is passed via the gastric cannula 
through the pylorus. The duodenum is ligated 1 cm below the pylorus, and 
the pylorus ligated around the cannula, thus creating a 1 cm proximal 
duodenal pouch excluding pancreatic and biliary secretions. The two 
ligatures enclosing the duodenal pouch are placed so as to avoid occluding 
the blood supply to the duodenal segment. Gastric secretions are allowed 
to drain freely from the gastric cannula. Compounds are administered 
dissolved in 0.9% sodium chloride (saline) as a 1.2 ml/h infusion via a 
catheter inserted in a jugular vein. 
The triple lumen catheter is connected as follows. Lumen 1 delivers 
perfusing medium at 0.1 ml/min via a peristaltic pump. 
Lumen 2 collects the perfusate and delivers it to a flow cell containing a 
pH microelectrode. Outflow pH is recorded throughout the experiment. Lumen 
3 is connected to a pressure transducer to monitor intraluminal pouch 
pressure. Body temperature is maintained at 34.degree. C. throughout the 
experiments. 
After preparation, the duodenal segment is perfused with saline, adjusted 
to pH 6.5 with hydrochloric acid, for 30 minutes. The perfusing medium is 
then changed successively to saline adjusted with hydrochloric acid to pH 
4, 3.5, 3 and 2.5 in increasing order of acidity. Each solution is 
perfused for 40 minutes. At the end of the pH 2.5 infusion period, saline 
pH 6.5 is perfused for 30 minutes, and the descending pH series repeated. 
This procedure produces two series of input pH/output pH values, 
designated 1st and 2nd passes. 
A group size of 6 animals or larger is used and the effect of compounds on 
the output pH compared to control data determined. For comparisons between 
groups, Student's `t` test is used. Significance is taken at P&lt;0.05. 
The compound of example 4 caused a significant increase in acid disposal at 
input pH 3 and 2.5 on the first pass and input pH 2.5 on the second pass 
at a dose of 150 nmol/kg/h, and at input pH 2.5 on the first pass at a 
dose of 30 nmol/kg/h.