EP0000252A1

EP0000252A1 - Peptides, pharmaceutical compositions containing the peptides and a process for the preparation of the peptides

Info

Publication number: EP0000252A1
Application number: EP78300046A
Authority: EP
Inventors: Peter Roy; Brian George Overell; Denis Raymond Stanworth
Original assignee: Beecham Group PLC
Current assignee: Beecham Group PLC
Priority date: 1977-06-29
Filing date: 1978-06-15
Publication date: 1979-01-10
Also published as: CA1105006A; DK292778A; EP0000252B1; IL54967A; ES471243A1; ZA783699B; US4223016A; IE781290L; DE2861593D1; IL54967A0; IE47105B1; JPS5416402A; AU522641B2; AU3762878A; NZ187638A; IT7850095A0

Abstract

Peptides (6-12 amino acids) and their salts, useful in' allergy desensitisation composition, particularly vaccines, have the formula

where R is optional and, if present, is a group resistant to enzyme hydrolysis; R_i is a basic amino acid residue optionally linked to basic and/or neutral nonhydrophobic amino acid residues; R₂ is one or more neutral nonhydrophobic . amino acid residues; R₃ is one or more hydrophobic amino acid residues optionally linked to neutral nonhydrophobic amino acid residues; X is hydrogen or an N-protecting group; and Y is hydroxyl or a C-terminal protecting group. Examples are:

Description

This invention relates to certain peptides useful for desensitisation therapy, and to desensitisation compositions containing them.
It is well known that many substances are capable of releasing histamine from mast cells. Such release is often due to disruption of the cell membrane, and reagents behaving in this way are said to be non-selective. One of the physiological systems causing release of histamine involves attachment of an antibody to the cell surface. When the antibody reacts with its appropriate antigen, a process is initiated leading to release of histamine. It has been postulated that such antigen-antibody reaction causes distortion of the antibody molecule, so that a portion of it is brought into contact with the cell surface,' thereby initiating the response. Release of histamine brought about in this fashion does not involve rupture of the cell membrane, and is said to be selective (see D. R. Stanworth, "Immediate Hypersensitivity", Chapter 8 North Holland Publishing Company London, 1973).
Experimentally, non-selective and selective release of histamine can be distinguished respectively by the presence or absence either of simultaneously released intracellular enzymes, or of radioactive chromium previously absorbed by

that substances capable of causing selective release of histamine may be used in lasensitisation therapy of allergic humans.
An object of this invention is to provide a novel class of peptides having this property, and desensitisation compositions, particularly vaccines containing such peptides.
Accordingly the present invention provides a peptide of formula (I):
and salts thereof, consisting of 6 to 12 naturally occurring amino acid residues, wherein R is an optionally present group, capable of confering on a peptide resistance to enzyme breakdown; R_l represents a residue of a basic amino acid, optionally linked to one or more residues of neutral non-hydrophobic amino acids and/or basic amino acids; R₂ represents a residue of a neutral non- hydrophobic amino acid, optionally linked to one or more durtner residues of neutral non-hydrophobic amino acids; R₃ represents a residue of a hydrophobic amino acid, tionally linked to one or more residues of neutral non- drophobic amino acids and/or hydrophobic amino acids; is hydrogen, or a N^. - protecting group; and Y is hydroxyl,
C - terminal protecting group.
Unless otherwise stated, the amino acids referred to --eafter are in the L- configuration.
When R is present, it is a group capable of confering a peptide resistance to enzyme breakdown. Examples of
groups R are given in J-. Rudinger,"The Design of reptide Hormone Analogues",Chapter 9 in Drug Design, volume (II) edited by E. J. Ariens, Academic Press, New ark and London, 1971.
Thus suitable examples of R, when present, include
hydroxyprolyl, the D- form of a common amino acid
or an amino acid residue with omission of the
amino group.
Suitable examples of R_i
lysyl and ornithyl and combinations of such
optionally with residue(s) of neutral non-hydrophobic amino acids such as threonyl and seryl. Particularly suitable examples of R₁ include Lys-Thr-Lys, Arg-Lys-Thr-Lys and the like. Normally R₁ will consist of 1 to 5 amino acid residues, suitably 3 to 5 residues. R₁ will often contain at least two basic amino acid residues and at least one neutral non- hydrophobic amino acid residue.
Suitable examples. of neutral non-hydrophobic amino acids R₂ include glycyl, alanyl, seryl and threonyl and combinations of such residues. A particularly suitable example of R₂ is Gly-Ser-Gly. Preferably R₂ consists of 1 to 5 amino acid residues, for example 3 amino acid residues.
Suitable examples of hydrophobic amino acids R₃ include residues of amino acids notionally derived from alanine β-substituted by an aromatic or aliphatic hydrophobic group, such as phenylalanyl, valyl and leucyl; and combinations of such residues. Particularly suitable examples of R₃ include Phe-Phe and Phe-Phe-Val-Phe. Preferably R₃ consists-of 1 to 4 amino acid residues, for example 2 or 4 residues.
X.is hydrogen or a N-protecting group. Suitable examples of N-protecting groups X include those conventionally known for this use in peptide chemistry. Examples of such groups include carboxylic acid groups such as acetyl, chloroacetyl, trifluoroacetyl, butyryl, benzoyl, phenylacetyl, pyridine-carbonyl; or an acid group derived from carbonic acid such as ethoxycarbonyl, benzyloxycarbonyl, t-butyloxycarbonyl, biphenylisopropoxycarbonyl, p-methoxy-benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-phenylazobenzyloxycarbonyl, p-(p'-methoxyphenylazo)-benzyloxycarbonyl, t-amyloxycarbonyl; or an acid group derived from a sulphonic or p-toluenesulphonic acid; or other.groups such as benzyl, trityl, formyl., phthaloyl, o-nitrophenylsulphenyl,.benzylidene or nitro. Preferred N-protecting groups X include t-butyloxycarbonyl or benzyloxycarbonyl.
protecting groups Y include ester
residues of C_1-6 alkyl esters such as methoxy, ethoxy and t-butoxy; benzyloxy, p-nitrobenzyloxy,
The peptides of the invention have 6 to 12 amino acid esidues. Preferably they have 8 to 10 amino acid residues ...
One particularly suitable.group of peptides is of formula (II):
wherein X, Y and R are as defined; c and e are lysyl, arginyl or ornithyl; d is threonyl or seryl; b is an optionally present arginyl, lysyl or ornithyl; f and h are glycyl or alanyl; g is seryl or threonyl; i and j are phenylalanyl, valyl or leucyl; and k and 1 are optionally present phenylalanyl, valyl or leucyl; and salts thereof.
Preferably in formula (II) X is hydrogen and Y is hydroxyl,-NH₂ or C₁_₄ alkoxy such as methoxy, and,when R is present, it is prolyl or hydroxyprolyl.
Examples of peptides within the scope of the invention ^.e:- .
herein Y is hydroxyl, NH₂ or methoxy.
The peptides of this invention may be prepared by methods known in the art of peptide synthesis comprising dupling the amino acids from which the peptide is derived
ally to build up the desired peptide.
Methods of sequential
no acids to form peptides by forming amide links
in the art. In general the amino acids, provided with protecting'groups where necessary, are coupled in the correct order, or smaller peptides are combined into larger units. The amide linkage is usually prepared by condensing an amino acid, or peptide, having a protected a-amino group and a free or activated terminal carboxyl group, with an amino acid or peptide with a protected carboxyl group and a free a-amino group.
Activation of the carboxyl group can be effected, for example, by converting the carboxyl group into an acid halide, an azide, anhydride or imidazolide, or into an activated ester such as the cyanomethyl ester, p-nitrophenyl ester, 2,4,5-trichlorophenyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester, benztriazole ester.
The most widely used methods of condensation of amino acids or peptides include the carbodiimide method, the azide method, the anhydride method, and the activated esters method, as described, for example, by Schroder and Lubke in "The Peptides", Volume 1 (1969), (Academic Press). An alternative method is the solid phase method of Merrifield (J. Am. Chem. Soc., 85, 2149 (1963)).
Any reactive groups in the amino acid or peptide which are not to take part in the condensation ,reaction should be protected by any:of the N-protecting groups or carboxyl protecting groups described above which can be readily removed after the condensation.
The removal of the protecting group(s) present in the resultant peptide may be effected by an appropriate procedure depending upon the kind(s) of the protective group(s). Some typical procedures are as follows: hydrogenation in the presence of palladium catalyst (e.g. palladium carbon, palladium black) for benzyloxycarbonyl, p-hitrobenzyloxycarbonyl, p-bromo-benzyloxycarbonyl, p-phenylazobenzyloxycarbonyl, p-(p'-methoxyphenylazo)-benzyloxycarbonyl and trityl groups protecting the amino end; treatment.with hydrogen bromide in glacial acetic acid
mucosa as a liquid spray or as
snuff.
Yet another possible route of administration would be by application to the buccal mucosa, again as a liquid or dry composition.
Accordingly, the present invention includes a pharmaceutical composition adapted for use in desensitisation therapy, comprising a peptide or pharmaceutically acceptable salt of formula (I) together with a pharmaceutically acceptable carrier suitable for parenteral, intra-nasal or buccal administration.
A preferred composition of the invention is a desensitisation vaccine.
. The compositions of the invention may be administered in conventional manner for desensitisation therapy.
The preparation and properties of some of the peptides of this invention are illustrated by the following examples.
Peptides were synthesised by classical methods of peptide synthesis described in the literature of peptide chemistry, for example by means of classical solution synthesis or solid phase peptide synthesis (SPPS), or by use of a combination of these methods.
Where appropriate amino acids refer to the L-configuration unless otherwise stated, and the following abbreviations are used:

(ii) Phe.Phe.Ome.HCl:

The intermediate (i) (4.65g) was BOC-deprotected using a solution of 2N HCL in EtAc (30ml) over 2 hours at room temperature. The product precipitated from solution in 78% yield (3.10g) and had m.p. 205°C;[&]²⁵ D= 43.3° (C = 1, AcOH).

(iii)BOC.Gly.Phe.Phe.OMe:

BOC.Gly.OSu (2.l8g,0.008M) was coupled to (ii)(2.90g, 0.008M) in DMF in the presence of 1 equivalent of Et₃N . (1.12ml) at room temperature over 3 days. Similar work-up described for isolation of (i), gave the product in 67% yield (2.60g); m.p. 159-161^oC, after crystallisation from EtAc (40ml) [&]D⁵= -9.5⁰ (C = 1, DMF); amino acid analysis:

required: 1.00 Gly: 2.00 Phe.
found: 1.00 Gly: 2.02 Phe.

(iv) GlyPhePheOMe.HCl:

The intermediate (iii) (2.60g) was BOC-deprotected in a similar manner to that described for (ii). The product deposited as an oil which was triturated with ether to give a white crystalline solid in almost quantitative yield. The material was purified further on Sephadex LH20 column eluting with water and had m.p. 196-199°C; TLC in 9:1 CHC1₃: MeOH showed one spot with I₂ stain at Rf. 0.22. Amino acid analysis:

required: 1.00 Gly: 2.00 Phe.
found: 1.00 Gly: 1.94 Phe.

(v) BOC.Ser(Bzl)GlyPhePheOMe:

BOC.Ser(Bzl)OH (1.66g, 0.0056M) was coupled to (iv) (2.36g, 0.0056M) -in MDC (20ml) at 0°C using DCCI (1.16g, 0.0056M) and Et₃N (0.79 ml: 1 equivalent). The reaction mixture was stirred at O°C for ½ hour, room temperature for 2 hours, filtered and filtrate evaporated in vacuo. Crystallisation of the residue from EtAc/petrol (80-100°C) afforded a 67% yield (2.50g) of product, m.p. 163-167°C. TLC in 9:1 CHC1₃: MeOH (I₂ stain) showed product at Rf 0.68;

The.NMR spectrum was consistent with structure amino acid analysis:

required: 1.00 Ser: 1.00 Gly: 2.00 Me.
found: 0.94 Ser: 1.12 Gly: 2.00 Phe.

(vi) Ser(Bzl)GlyPhePheOMe.HCl (II):

Intermediate (v) (1.75g) above was BOC-deprotected in a similar manner to that described for (ii). Addition of ether to the reaction mixture gave product as a solid in 96% yield (1.52g), [&]²⁵ D= 21.0° (C = 1, AcOH). It was 25 purified on Sephadex LH20 eluting with 1M AcOH, [&]²⁵ D=21.0° (C = 1, AcOH) TLC examination in 9:1 CHC1₃: MeOH (I₂ stain) showed product (acetate salt) as^.one spot at Rf 0.27. The NMR spectrum was consistent with structure. Amino acid analysis:

required: 1.00 Ser: 1.00 Gly: 2.00 Phe.
found: 1.00 Ser: 1.11 Gly: 1.92 Phe.

(III)BOC.Lys(Z)Thr(Bzl)Lys(Z)GlySer(Bzl)GlyPhePheOMe

Tertiary-butyl nitrite (0.32ml,0.00266M) was added with vigorous stirring to a solution of (I) (1.60g, 0.00177M) in DMF (30ml) containing 60 equivalents 2N HCL in THF (5.5ml, O.OO11M) at -20°C. After 30 minutes, (II) (1.05g, 0.00177M) in DMF (5ml) with sufficient Et₃N (2.11ml) resent to neutralise all HCl present, was added, and the reaction mixture stirred for 18 hours at 4°C, filtered and filtrate concentrated in vacuo. Addition of cold water gave product which was obtained in 50% yield (1.28g) after crystallisation from EtOH. TLC in 9:1 CHCI₃:MeOH (I₂ stain) was homogeneous - 25 and showed product at.Rf 0.6. M.p. 202-203°C; [&]²⁵ D= -5.9° (C = 1, DMF). The NMR spectrum was consistent with structure. Amino acid analysis:

required: 2.00 Lys: 2.00 Phe: 2.00 Gly: 1.00 Thr: 1.00 Ser.
found: 2.00 Lys: 1.94 Phe: 2.06 Gly: 0.99 Ser: 0.96 Thr,

(IV) Lys (Z)Thr (Bzl)Lys (Z)GlySer(Bzl)GlyPhePheOMe.HCl

Fully protected octapeptide (II) (1.20g) was BOC-deprotected in 2N HCl solution in a 6:14 DMF/EtAc solvent
of 4 hours was used .
and addition of ether deposited product..
from MeOH/ether gave a 52% yield (0.60g) of product. TLC in 9:1 CHC1₃: MeOH (I₂stain) showed one Ajor spot at Rf 0.4; [&]²⁵ D= l0.l° (C = 1, AcOH). Amino acid analysis :

required: 1.00 Thr: 1.00 Ser: 2.00 Gly: 2.00 Phe: 2.00 Lys.
found: 1.00 Thr: 1.00 Ser: 2.17 Gly: 2.19 Phe: 2.32 Lys.

(V) LysThrLysGlySerGlyPhePheOMe

Partially protected octapeptide (IV) (O.lOg) was hydrogenated in 85% AcOH (70ml) with 10% Pd/C catalyst (0.20g) over a steady stream of hydrogen for 20 hours. The mixture was filtered, evaporated in vacuo and residue filtered on Sephadex LH20 eluting with water to give the desired octapeptide methyl ester (V) (0.03g, 46% yield). TLC examination showed 1 spot at Rf 0.2 in 5:2:2 BAW (t-BuOCl/KI-starch stain) and Rf 0.5 in 5:2:3 BAW (Ninhydrin stain). Amino acid analysis: required: 1.00 Ser: 1.00 Thr: 2.00 Gly: 2.00 Phe: 2.00 Lys. found: 1.00 Ser: 1.03 Thr: 2.02 Gly: 2.06 Phe: 1.98 Lys. Isotachophoretic examination showed one band in >95% amount (leading electrolyte lOmM KOH + MES pH 6.0 and terminating electrolyte lOmM β-alanine and HC1 pH 4.23). The NMR 80 .
spectrum was consistent with structure.

EXAMPLE 2: The preparation of ArgLysThrLysGlySerGlyPhePheOM

This nonapeptide was prepared by coupling of (IV) above with Z .Arg (Z )₂ ,, .OSu, followed by hydrogenolysis of the resultant fully protected nonapeptide.

(i) Z.Arg(Z)₂Lys(Z)Thr(Bzl)Lys(Z)GlySer(Bzl)GlyPhePhe0Me:

To octapeptide (IV) (0.344g, 0.30025M) above in DMF (3ml) at O°C was added (1 equivalent) Et₃N (0.025g in lml OMF) and Z-Arg ( Z ) ₂0Su (0.17g, 0.00025M in 2ml DMF). The olution was left at 4°C for 65 hours, diluted with water (8ml) and the deposited product filtered off and dried
yield). Crystallisation from DMF/EtOH gave product with m.p. 204-210^°C (decomposition). TLC examination in 9:1 CHCl₃: 'MeOh (I₂ stain) showed on U.V. visualisation 1 spot at Rf 0.69. The NMR spectrum was consistent with structure. Amino acid analysis:

required: 1.00 Thr: 1.00 Ser: 2.00 Gly: 2.00 Phe: 2.00 Lys: 1.00 Arg.
found: 1.00 Thr: 1.00 Ser: 2.10 Gly: 2.01 Phe: 2.04 Lys: 1.04 Arg.

(ii) ArgLysThrLysGlySerGlyPhePheOMe

Fully protected nonapeptide (i) (0.07g) above was dissolved in a minimum amount of DMF and 5 times the volume of AcOH added. The mixture was hydrogenated in the presence of 10% Pd/C catalyst (2.5 times weight of compound) for 19 hours at room temperature using a steady stream of hydrogen. Water was added to give a 15% aqueous solution and the mixture hydrogenated for a further 3 hours. Filtration and evaporation in vacuo at 45^oC gave product as a glassy solid. Purification was performed on a Sephadex LH20 column eluting with 1M AcOH and product isolated in 26% yield (0.018g). TLC in 5:3:5 BAW (ninhyrin stain) showed product at Rf 0.34. Amino acid analysis:

required:l.00 Thr:1.00 Ser:2.00 Gly:2.00 Phe:2.00 Lys:1.00 Arg.
found: 1.00 Thr:1.01 Ser:2.07 Gly:2.00 Phe:2.07 Lys:1.00 Arg.

EXAMPLE 3: The preparation of LysThrLysGlySerGlyPhePheVal

- PheOMe The decapeptide, methylester was synthesised by a 4+2+4 fragment condensation strategy as follows:-

Prepared in three stages:-.

(d) BOC . Ser (Bzl) GlyOMe

BOC.Ser(Bzl)OH (5.Og, 0.017M) was coupled to Gly .OMe.HCl (2.13g, 0.017M) in M.D.C. (100ml) at R.T. for
hours in the presence of 1 equivalent of Et₃N^.and using ACCI (3.5g, 0.017M) as the condensing agent. The preecipitate was filtered off and the solution washed X 2 with water, aqueous NaHC0₃, water, dried and evaporated in vacuo to leave an oil (7.lg).
T.L.C. 9:1 CHCo₃: MeOH (I₂ stain) showed one spot at ^{AF 0.64}; [&]²⁵ D = 4.7⁰(C = 1,MeOH) .

(ii) BOC.Ser(Bzl)GlyOH

Compound (i) above (7.Og) was dissolved in dioxan (25ml) and treated with an equal volume of IN NaOH (25ml) and the solution stirred for1 2 hour at R.T. N HCl (25ml) was added to a slight excess and the oil that formed extracted into EtAc. The organic layer was back-extracted into NaHC0₃and acidified to pH 3.8 with 20% citric acid, extracted with EtAc, the organic layer washed with water, brine, dried and evaporated to leave the product as a grup (4.Og) .

.L.C. 1:1 CHC1_3/EtOH .(I₂ stain) showed product at Rf 0.59. the NMR spectrum was consistent with structure.

ii)BOC . Ser (Bz l) GlyOSu

Compound (ii) above (4g 0.01135M) was treated with .Su (1.3g O.O11M) and DCCI (2.34g O.O11M) in dioxan (50ml) - R.T. overnight. The precipitate that formed was
off, solvent removed and the product crystallised from I.P.A. (lOOml) in 59% yield (3.00g).
.L.C. 9:1 CHC1₃/MeOH (I₂ stain) showed one major spot 16 0.57; _M._P. 132-134°C; [&]^{25 D=} -1.8° (C = l, MeOH).

II) PhePheValPheOMe Prepared in six steps:-

Was coupled to PheOMe . HC1 (6.85g 0.0318M) in
overnight and in the presence of Et.N 1 equivalent) . The mixture was filtered and filtrate washed with IN HC1, saturated NaCl solution; dried and evaporated in vacuo to give product (11.31g) as a white crystalline compound in 94% yield.
T.L.C. in 9:1 (CHC1₃:Me0H) (I₂ stain) shows one spot at . Rf 0.77 [&]²⁵ D=-27.4° (C=l, _MeOH).

(ii) ValPheOMe.HCl

Compound (i) (9.25g) was BOC-deprotected in 2N HCL in EtAc (100m1) for 24 hours at room temperature when the product precipitated. The mix was diluted with dry EtAc and product filtered off in 78% yield (6.Og). The product was finally purified on Sephadex LH20. M.P. 193-193.5°; T.L.C. 9:1 CHCL3/MeOH (I₂ stain) shows one spot at Rf 0.60. [&]²⁵ D= [&]²⁵ D= 63.3° (C=1, _AcOH)

(iii)BOC.PheValPheOMe

Compound (ii) (5.34g, O.OI7M) was coupled to BOC.PheOSu (6.15g, 0.017M) .in 25%.D.M.F. in Toluene (250ml) at room temperature for 65 hours in the presence of Et3_N (1equiv.) The mixture was then filtered, solvent removed in vacuo and the syrup quenched with water. The white precipitated (8.5g) was filtered off and recrystallised from-EtAc/8O-100° petrol; yield 80%.
T.L.C. 9:1 CHC1₃/MeOH (I₂ stain) shows one spot at Rf 0.69 25 [&]²⁵ D = -31.O° (C = 1 MeOH). NMR consistent with structure.

(iv) PheValPheOMe.HCl

Compound (iii) (6.87g) was BOC-deprotected in 2N HCl in EtAc (100m1) for 2 hours at room temperature when a white solid precipitated. (5.84g) representing 97% yield of product. M.P. 243-245° (decomposition) T.L..C. in 9:1 CHCl₃:MeOH.(I₂ stain) shows one spot at Rf 0.59.
8.8°. (C = 1. AcOH)

(v) BOC.PhePheValPheOMe ...

BOC.Phe0Su-(4.30g 0.0119M) was coupled to compound . (iv) (5.5g 0.0119M) in toluene (1O0m1) at room temperature for 65 hours in the presence of sufficient D.M.F. to produce solution, ant Et₃N (1 equivalent). The solvent was evaporated in vacuo and the syrup quenched with water and product filtered off. The product was then triturated with hot ethanol, cooled and collected (6.38g, 80% yield). M.P. 218-219^oC
T.L.C. in 9:1 CHCl₃:MeOH (I₂ stain) shows one spot at 25 Rf 0.62. [&.]²⁵ D= -15.9⁰ (C = 1, D.M.F.) The NMR. spectrum was consistent with structure.

(vi) PhePheValPheOMe.Tfa

Compound (v) (5g) was BOC-deprotected in T.F.A (25ml) at O°C for1 2 hour, and at room temperature for 1 2 hour. The solution was then quenched with ether (75ml) and product filtered off (4.48g, 88% yield). M.P. 224-226° (decomposition) T.L.C. 9:1 CHCI₃:MeOH (I₂ stain) shows one spot at Rf 0.49. [&]²⁵ D= 10.6 (C = 1, AcOH). The NMR spectrum was consistent with structure.

(VIII)Ser(Bzl)GlyPhePheValPhe.OMe Prepated in two stages:-

(i) BOC.Ser(Bzl)GlyPhePheValPheOMe

Intermediate VI (2.37g, 0.00528M) was coupled to intermediate VII (3.62g, 0.00528M) in toluene (500ml) overnight at room temperature in the presence of Et₃N (0.74ml, 0.00528M). The mixture was washed with water, and solvent evaporated in vacuo. The solid obtained was triturated with water, dried and recrystallized from EtOH (4.42g, yield 93%).
T.L.C. 9:1 CHCI₃:MeOH (I₂ stain) shows one spot at Rf 0.61. 25° 0 [&]²⁵D --13.5 (C = 1 D.M.F.). The NRM spectrum was consistent with structure.

(ii) Ser (Bzl) GlyYhePheValPheOMe

Compound (i) (2.8g) was BOC-deprotected in T.F.A. (30ml) for 40 minutes at O°C. The solution was quenched with ether (200ml) and the precipitated product obtained : in quantitative yield.
T.L.C. 9:1 CHCI₃:Me0H (I₂ stain) shows one spot at Rf 0.2. M.P. 214-216°C (decomposition). [&]^25°D = 3.7° (C = l,_AcOH) The NMR spectrum was consistent with structure.

BOC.Lys ( Z) Thr (Bzl) Lys (Z ) GlySer (Bzl) GlyPhePheValPheOMe _.

Peptide I (2.25g) (see Example 1) was coupled to VIII (2.19g) by the Henzyl-Rudinger modification of the azide method, as previously described for the octapeptide. The product was recrystallized from EtOH and obtained in (2.5g, 61% yield). M.P. 243-244°.
Amino acid analysis: calculated: lThr: lSer: 2GLy: lVal:

3Phe: 2Lys
found: 1.13Thr: 1.11Ser: 2.15Gly:
0 l.OOVal: 3.04Phe: 1.98Lys.

²⁵

⁰

_R

LysThrLysGlySerGlyPhePheValPheOMe

The intermediate above (0.18g) was BOC, Z and Bzl- deprotected by treatment with 33% HBr in dioxan (5ml) at room temperature for 1 hour when a precipitate.formed. Additional HBr/dioxan (5ml) and water (lml) was then added which effected solution and reaction.continued.for a further 1 2 hour. Acetone (50m1) was then added and the solution quenched with ether (100ml). The supernatent was decanted and solid dissolved in water (7ml) and freeze- dried to give.0.145g product as:the tri-hydrobromide salt. Amino acid analysis: required: lThr: lSer: 2Gly: lVal: 3Phe:

2Lys
found: 1.05Thr: 1.00Ser: 2.14Gly:
1.02Val: 3.13Phe: 2.OOLys

The decapeptide free acid was synthesised by a 4+2+4 fragment condensaiton strategy as follows:-

(IX) PhePheValPheOBz Prepared in six stages:-

(i) BOC.ValPheOBz

BOC.ValOSu (15.7g, 0.050M) was coupled to PheOBZ.pTsa (21.35g, 0.050M) in dioxan (200ml) at R.T. for 41 2 hours in the presence of 1 equivalent of Et₃N. The reaction mixture was evaporated at reduced pressure and the resulting residue dissolved in EtAc and the solution washed with water, dried and evaporated in vacuo to leave a crystalline solid (21.3g).
L.C. 9:1 CHCI₃:Me0H (I₂ stain) showed one spot at Rf 0.93; &]25° D = -31.8° (C = l, MeOH) &]^25° D = -31.8° (C = 1, MeOH).

ii) ValPheOBz . HC1

Compound (i) (21.3g) was BOC-deprotected in 2N HCl in -
Ac (240ml) for 41 2 hours at R.T. when the product
. The mix was diluted with dry ether and product
. off in 78% yield (15.25g). M.P. 180-182°;
" 9:1 CHC₂: MeOh (I₂ stain) . shows one spot at Rf O. 44.
= 24,4° (C = 1, A_COH).

iii. ) BOC . Ph eValPheOBz

from EtAc/40-60° petrol; yield 85%. M.P. 160-162 T.L.C. 9:1 CHCI₃:MeOH (I₂stain) shows one spot at Rf 0.72. 25⁰ _[&]25_{D = -36}.₀° (_C = 1, _MeOH).

(iv) PheValPheOBz.HC1

Compound (iii) (20.Og, 0.033M) was BOC-deprotected in 2N HC1 in EtAc (240ml) for 2 hours at R.T. when a white solid precipitated (15.23g) representing 85% yield of product. M.P. 228-229° (decomposition)
T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) shows one spot at Rf 0.63. [α]^25° _D= -6.9° (C = 1, AcOH).

(v) BOCPhePheValPheOBz

BOC.PheOSu (10.26g, 0.0283M) was coupled to compound (iv) (15.23g, 0.0283M) in 50%dioxan/DMF (250ml) at R.T. for 4 hours in the presence of Et₃N (1 equivalent). The mixture was poured into iced water and the resulting white precipitate filtered off and recrystallised from EtAc/40-60° petrol in quantitative yield (21.41g). M.P. 191-193°.
T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) shows one spot at Rf 0.72. [α]^25° _D= -12.8° (C = 1, DMF).

(vi) PhePheValPheOBz.HCl

Compound (v) (21.15g, 0.028M) was BOC-deprotected in 2N HC1 in EtAc (500ML) for 2 hours at R.T. - The product (17.9g) was precipitated in 92% yield upon addition of dry ether. M.P. 242° (decomposition).
T.L.C. 9:1 CHCl₃:MeOH (I₂stain) shows one spot at Rf 0.74. [α]^25° _D = -5.9 (C = 1, AcOH). The NMR spectrum was consistent with structure.

(X) Ser(Bzl)GlyPhePheValPheOBz Prepared in two stages:-

(i) BOC.Ser(Bzl)GlyPhePheValPheOBz

Intermediate (VI) (4.49g, O.OlOM) was coupled to intermediate (IX) (6.85g, 0.0010M) in 35% DMF/dioxan (75ml) at R.T. for 4 hours in the presence of Et₃N (1 equivalent). The mixture was poured into iced water and the precipitated product (9.39g) recrystallised from methanol in 91% yield.

(ii) Ser(Bzl)GlyPhePheValPheOBz.HCl

Compound (i) (5.0g, 0.0051M) was BOC-deprotected in N HCl in EtAc(150ml). for 2 hours at R.T. The product '4.42g) was precipitated in 94% yield upon addition of dry ether. M.P. 232 -234° (decomposition)
T.L_.C. 9:1 CHCl₃:Me0H (I₂ stain) shows one spot at Rf 0.44. [α^25° _D = -4.3 (C = l, AcOH). The NMR was consistent with structure.

(XI) ZLys(Z)Thr(Bzl)Lys(Z)GlyOTcp Prepared in seven stages:-

(i) BOC.Lys(Z)GlyOMe

BOC.Lys(Z)OSu (23.85g, 0.050M) was coupled to GlyOMe. HCl (6.25g, 0.050M) in 50% dioxan/DMF at R.T. for 4½ hours ..n the presence of 1 equivalent of Et₃N. The reaction mixture was evaporated in vacuo and the residue dissolved in EtAc. The solution was washed, dried, filtered and aporated to a colourless oil which solidified on
in 89% yield.
E.C. 9:1 CHCl₃:MeOH (I₂ stain) showed one spot at Rf 0.54.

L) Lys(Z)GlyOme.HCl

Ccmpound (i) (20.00g, 0.0443M) was BOC-deprotected in HCl in EtAc (250ml) for 2 hours at R.T. when the product
. The mixture was diluted with dry ether and product filtered off in 96% yield (15.67g).
P. 158-159°.

.C. EtAc (I₂stain) showed one spot at Rf 0.52.

) BOC. Thr (Bzl) Lys (Z ) GlyOMe

BOC.Thr(Bzl)OH (6.18g, 0.02oM) was coupled to compound (ii) (7.76g, 0.020M) in 30% DMF/dioxan (75ml) in iced water
then at R.T. for a further 2 hours in the presence of DCCI (1 equivalent)
The reaction mixture was filtered and evaporated in vacuo and the residue purified by silica column chromatography eluting with chloroform. The product was isolated as a colourless solid in 49% yield. M.P. 135-136°.
T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) showed one spot at Rf 0.57. The F.T. ¹³C NMR was consistent with structure.

(iv) Thr(Bzl)Lys(Z)GlyOMe.HCl

Compound (iii) (3.48g, 0.0054M) was BOC-deprotected in 2N HCl in EtAc(lOOml) for 2 hours at R.T. The product (2.88g) was precipitated in 91% yield upon addition of dry ether. M.P. 100-101°.
T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) showed one spot at Rf 0.52. 0 [α]^25° _D = -13.5° (C = 1, AcOH).

(v) ZLys(Z)Thr(Bzl)Lys(Z)GlyOMe

ZLys(Z)OTcp (1.80g, 0.003M) was coupled to compound (iv) (1.72g, 0.003M) in dioxan (45ml) at R.T. for 4 hours in the presence of Et₃N (1 equivalent). The product was filtered off, washed with water and dried in vacuo (1.36g, 50% yield). M.P. 185-188°.
T.L.C. 9:1 CHCl₃: MeOH (1₂ stain showed one spot at Rf 0.76.

(vi) ZLys (Z) Thr (Bzl) Lys (Z) GlyOH

A solution of compound (v) (0.92g, O.OO1M) in 50% DMF/methanol was treated with IN NaOH solution (2.5ml) and stirred at R.T. for 1 hour. Upon acidification the precipitated product (0.45g) was recrystallised from methanol in 49% yield. M.P. 171-173°.
T.L.C. 2:1 CHCl₃:MeOH (t.butyl chloroformate/NaI - starch spray) showed one spot at _Rf 0.50. [α]^25° _D = -5.4° (_C = 1, AcOH). The F.T.¹³C NMR was consistent with structure.

(vii) ZLys (Z) Thr (Bzl) Lys (Z) GlyOTcp

A solution of TcpOH (0.10g, 0.0005M) and compound (vi) (0.46g, 0.0005M) in DMF was treated with DCCI (0 11g. 0.0005M) and stirred at 5° for 1 hour then at R.F. overnight. The reaction mixture was filtered product (0.60g) isolated as a crispy solid upon evaporation in vacuo. M.P. 176-178°.
T.L.C. 9:1 CHCl₃:Me0H (I₂ stain) showed one spot at Rf 0.71. [α]^25° _D = -6.0° (_C= ₁, _AcOH).

ZLys (Z) Thr (Bzl) Lys (Z) GlySer (Bzl) GlyPhePheValPheOBz

Peptide (XI) (0.55g, 0.0005M) was coupled to compound (X) (0.46g, 0.0005M) in DMF at R.T. for 4 hours in the presence of Et₃N (1 equivalent). The reaction mixture was poured into iced water and the resulting precipitate filtered off and dried in vacuo. Purification by silica column chromatography, eluting with CHCl₃, gave the product (0.92g) in 85% yield.
T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) showed one spot at Rf 0.34. The F.T.¹³C NMR was consistent with structure.

LysThrLysGlySerGlyPhePheValPheOH

The intermediate above (0.76g, 0.0003M) was deprotected by continuous hydrogenation in 85% acetic acid with IN HC1 (lmM) for 18 hours in the presence of 10% Pd/charcoal (0.80g). The product was purified on a Biogel P2 column eluting with 1M ammonium acetate and subsequently on a CM32 cellulose column eluting with O.lM ammonium acetate pH5. Final isolation of the product in 23% yield was by lyopholisation.
T.L.C. butanol/acetic acid/water (5:2:2) (ninhydrin spray) showed one spot at Rf 0.22. Amino acid analysis:

calculated:2Lys: lThr: 2Gly: lSer: 3Phe: 1Val
found: 1.80Lys: 0.92Thr: 2.OOGly: 1.07Ser: 3.24Phe: 0.91Val

EXAMPLE 5: The preparation of ProArgLysThrLysGlySerGlyPhe PheOMe

This decapeptide was synthesised by a 1+1+4+4 fragment condensation strategy as follows:-

(XII) Lys (Z) Thr (Bzl) Lys (Z) GlyOMe Prepared in two steps from Thr(Bzl)Lys(Z)GlyOMe described in example 4.

(i) BOC.Lys (Z) Thr (Bzl) Lys (Z) GlyOMe

BOC.Lys(Z)OSu (2.38g, 0.005M) was coupled to Thr(Bzl) Lys(Z)GlyOMe.HCl (2.87g, 0.005M) in dioxan (60ml) at R.T. for 4 hours in the presence of Et₃N (1 equivalent). The reaction mixture was poured into iced water to give the required product (3.80g) as a crystalline white solid in 84% yield. M.P. 103-105°.
T.L.C. 9:1 CHCl₃:Me0H (I₂ stain) showed one spot at Rf 0.55. [α]^25° _D = -10.4° (C = 1, AcOH).

(ii) Lys (Z) Thr (Bzl) Lys (Z) GlyOMe.HCl

Compound (i) (3.80g, 0.0042M) was BOC-deprotected in 2N HCl in EtAc (100ml) for 2 hours at R.T. The product (3.30g) was precipitated in 93% yield upon addition of dry ether. M.P. 184-186°.
T.L.C. 9:1 CHCl₃:MeOH (I₂stain) showed one spot at Rf 0.80. [α]^25° _D = 5.6° (C = 1, AcOH).

(XIII)Arg (N0₂) Lys (Z) Thr (Bzl) Lys (Z) GlyOMePrepared in two stages:-

(i) BOc.Arg (N0₂) Lys (Z) Thr (Bzl) LYS (Z) GlyOMe

BOC.Arg(NO₂)OSu (1.40g, 0.0033M) was coupled to compound (XII) (3.30g, 0.004M) in 10% DMF/
R.T. for 3 hours in the presence of Et₃N (1 equivalent). Unreacted (XII) was filtered off and the reaction mixture poured into iced water, extracted with EtAc to give the product (2.60g) which was recrystallised from IPA in 71% yield. M.P. 133-135°.
T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) showed one spot at Rf 0.42. [α]^25° _D= -6.7° (C = 1, AcOH).

(ii) Arg (N0₂) Lys (Z) Thr (Bzl) Lys (Z) GlyoMe.HCl

Compound (i) (2.Og, 0.0018M) was BOC-deprotected in 2N HC1 in EtAc(50ml) for 2 hours at R.T. The product (1.75g) was precipitated in 92% yield upon addition of dry ether. M.P.. 157° (decomposition)
T.L.C. 2:1 CHCl₃:MeOH (I₂ stain) showed one spot at Rf 0.53. 250 ° [α]^25° _D = -5.6 (C = 1, MeOH).

(XIV) ZProArg (NO₂) Lys (Z) Thr (Bzl) Lys (Z) GlyOH Prepared in two stages:-

(i). ZProArg (NO₂) Lys (Z) Thr (Bzl) Lys (Z) GlyOMe

ZProOsu (0.57g, 0.0016M) was coupled to compound (XIII) (1.71g, 0.0016M) in 20% DMF/dioxan (30ml) at R.T. for 2 hours in the presence of Et3N (1 equivalent). The reaction mixture was poured into iced water and extracted with EtAc to give the product (l.llg) in 54% yields.
T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) showed one spot at Rf 0.36.

(ii) ZProArg (NO₂) Lys (Z) Thr (Bzl) Lys (Z) GlyOH

A solution of compound (i) (0.87g, 0.0007M) in 50% DMF/ methanol (30ml) was treated with 1N NaOH solution (1.7ml) and stirred at R.T. for 2 hours. Acidification precipitated the product (0.43g) in 50% yield.
T.L.C. 2:1 CHCl₃:MeOH (I₂stain) showed one spot at Rf 0.77.
= -4.1° (C = 1, AcOH). The NMR was consistent with structure.

ZProArg (N0₂) Lys (Z) Thr (Bzl) Lys (Z) GlySer (Bzl) GlyPhePheOMe

Peptide (XIV) 0.40 g, 0.0033M) was coupled to compound (VIII) (0.20g, 0.0034M) in DMF (5ml) in the presence of Et₃N (1 equivalent), DCCI (0.07g, 0.0035M) and hydroxybenzotriazole (0.044g, 0.0035M) at 5° for 1 hour then at R.T. for 1 hour. The precipitated urea was filtered off and the required product (0.50g) isolated by pouring the reaction mixture into iced water and isolating by .filtration in 88% yield.
T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) showed one spot at Rf 0.52. The NMR was consistent with structure.

ProArgLysThrLysGlySerGlyPhePheOMe

The intermediate above (0.40g, 0.0022M) was deprotected by continuous hydrogenation in 85% acetic acid for 18 hours' in the presence of 10% Pd/charcoal catalyst (0.40g). The product was purified on a Biogel P2 column eluting with water and subsequently on an LH20 Sephadex column again with aqueous elution. Final isolation of the product in 34% yield was by lyopholisation.
T.L.C. butanol/acetic acid/water (5:2:2) (ninhydrin spray) showed one spot at Rf 0.34.
Amino acid analysis calculated: 1Pro: 1Arg: 2Lys: 1Thr:

2Gly: 1Ser: 2Phe
found: 0.95Pro: 0.99Arg: 1.88Lys: l.OOThr: 2.00Gly: 1.OlSer : 1.96Phe.

EXAMPLE 6: The preparation of LysThrLysGlySerGlyPhePheOH

The octapeptide free acid was synthesised by a 4+4 fragment condensation strategy as follows:-

(XV) Ser(Bzl)GlyPhePheOBz Prepared in six stages:-

(i) BOCPhePheOBz

BOCPheOH (11.88g, 0.045M) was coupled to PheOBz.pTsa-19.4g, O.o45M) in MDC (200 ml) at 0° for 1 hour then at R.T. overnight in the presence of Et₃N (1 equivalent) and DCCI (1 equivalent). The reaction mixture was filtered and the product (14.92g) isolated in 64% yield upon evaporation in vacuo and recrystallisation from EtOAc/80-100° petrol (14.92g). _M._P. 123.5-124.5⁰.
T.L.C. 1:1 EtAC: 80-100° petrol (I2 stain) showed one spot at Rf 0.68. [α]^25° _D = -16.7° (C = 1, MeOH).

ii) PhePheOBz.Tfa

Compound (i) (14.0g, 0.028M) was BOC-deprotected in 50%Tfa in MDC (1OOml) for ½ hour at 0°. The solution was quenched with dry ether and the product (14.23g) filtered off in quantitative yield. M.P. 180° (decomposition) T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) showed one spot at Rf 0.73.
= 17.5° (C = 1, AcOH).

(iii) BOCGlyPhePhe OBz

BOC.GlyOSu (9.6g, 0.0353M) was coupled to compound
R.T. overnight in the
The reaction mixture was evaporated at
and the resulting residue dissolved in EtAc, washed, dried and evaporated in vacuo to leave a crystalline solid _.(19.71g) in quantitative yield. M.P. 127-130°.
^T.L.C. 9:1 CHCl₃:Me0H (I₂ stain) showed one spot at Rf 0.0 [α]^25° _D = -17.4° (C = 1, MeOH).

(iv) GlyPhePheOBz.Tfa

Compound (iii) (19.3g, 0.0346M) was BOC-deprotected in 50% Tfa in MDC (130ml) for 1½ hours at 0°. The solution was quenched with ether and the product (17.79g) filtered off in 90% yield.
T.L.C. 9:1 CHCl₃:Me0H. (I₂ stain) showed single spot at _Rf 0.35. [α]^25° _D = 6.0° (C = 1 , AcOH).

(v) BOC.Ser(Bzl)GlyPhePheOBz

BOC.Ser(Bzl)OSu (11.03g, 0.0282M) was coupled to compound (iv) (16.13g, 0.0282M) in 15% DMF/toluene (350ml) at R.T. overnight in the presence of Et₃N (1 equivalent). The reaction mixture was evaporated at reduced pressure and the resulting residue dissolved in EtAc, washed, dried and evaporated in vacuo to give the product (14.88g) 72% yield upon recrystallisation from EtAc/petrol. M.P. 149-151°.
^T.L.C. ^9:1 CHCl₃:MeOH (I₂ stain) showed a single spot: at Rf 0.65. [α]^25° _D = -11.2 (C = 1, MeOH).

(vi) Ser (Bzl) GlyPhePheOBz

Compound (v) (14.45g, 0.0196M) was BOC-deprotected in 50% Tfa in MDC (140ml) for 1 hour at 0°. The solution was quenched with ether and the product (13.1)g filtered off in 89% yield. M.P. 185-187° (decomposition). T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) showed one spot at

= 1O.1° (C = 1, AcOH).

(XVI) BOC.Lys (Z) Thr (Bzl) Lys (Z) GlyOSu Prepared in four es from BOC.Thr (Bzl) Lys (Z) GlyOMe described in

(i) Thr(Bzl)Lys(Z)GlyOMe.Tfa

BOC.Thr(Bzl)Lys(Z)GlyOMe (2.87g, 0.0045M) was BOC-deprotected in 50% Tfa in MDC (50ml) for 1 hour at 0°. The solution was quenched with ether and the product (2.1Og) filtered off in 72% yield.
T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) showed one spot at Rf 0.26.

(ii) BOC.Lys (Z) Thr (Bzl) Lys (Z) GlyOMe

BOC.Lys(Z)OSu (2.00g, 0.003M) was coupled to compound (i) (1.45g, 0.003M) in 10% DMF/toluene at R.T. overnight. The reaction mixture was evaporated at reduced pressure and the resulting residue dissolved in EtAc, washed, dried and evaporated in vacuo to give the product (2.04g) in 74% yield upon recrystallisation from EtAc/petrol. M.P. 117-119°.
T.L.C. 9:1 CHCl₃:Me0H (I₂ stain) showed one spot at Rf 0.6₀.

(iii) BOC.Lys (Z) Thr (Bzl) Lys (Z) GlyOH

A solution of compound (ii) (1.98g, 0.0022M) in DMSO (30ml) was treated with IN NaOH solution (1½ equivalents) and stirred at R.T. for 1 hour. Acidification gave the product (1.82g) in quantitative yield.
T.L.C. 5:1 CHCl₃:MeOH (I₂ stain) showed compound just above baseline.

(iv) BOC.Lys(Z)Thr(Bzl)Lys(Z)GlyOSu

HOSu (0.24g, 0.002M) was coupled to compound (iii) (1.82g, 0.002M) in dioxan (25ml) at R.T. for 4 hours in the presence of DCCI (1 equivalent). The reaction mixture was filtered and the filtrate evaporated at reduced pressure. Recrystallisation of the residue from EtOH gave the product (0.30g) in 15% yield. M.P. 118-122⁰.
T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) showed one spot at Rf 0.50. [α ]^25° _D = -4.0° (C = 1, DMF). The NMR was consistent with structure.

BOC.Lys (Z) Thr (Bzl) Lys (Z) GlySer (Bzl) GlyPhePheOBz

Peptide (XVI) (0.22g, 0.0023M) was coupled to compound (XV)
overnight in the presence of
reaction mixture was evaporated at reduced
residue recrystallised from EtOH to give the
in 85% yield. M.P. 195-199°.
T.L.C. 9:1 CHCl₃:MeOH (I₂ stain) showed one spot at Rf The NMR was consistent with structure.

LysThrLysGlySerGlyPhePheOH

The intermediate above (0.25g, 0.0017M) was dissolved in Tfa (lOml) and deprotected by bubbling through HBr at R.T. for 1 hour. The solution was quenched with dry ether and the product dried in vacuo over P₂0₅ and KOH. The product was purified on a Biogel P2 column eluting with water and subsequently on a CM32 cellulose column eluting with a linear ionic strength gradient of ammonium acetate pH5 which gave separation of the free acid and some benzyl ester contaminant. Final isolation of the product was by lyopholisation.
T.L.C. butanol/acetic acid/water (5:2:2) (ninhydrin spray) showed one spot at Rf 0.20. Amino acid analysis:

calculated: 2 Lys: lThr: 2Gly: lSer: 2Phe
found: 1.77Lys: 0.96Thr: 2.00Gly: 1.06Ser:

Biological Activity

Methods

(1) Histamine, (2) Cr⁵¹ and (3) Lactic Dehydrogenase Release from Rat Peritoneal Mast Cells (Rat Mast Cell in vitro test)

cells, derived from the peritoneal washings of
male, outbred Wistar rates (250-300g), were purified
the procedure according to Cooper and Stanworth reparative Biochem. 4(2), 105, 1975).
The purified cells were washed twice in Dulbecco's complete (i.e. free from mineral salts) buffer and then resuspended in Dulbecco's medium to the required volume.
a typical experiment, sufficient cells were available
duplicate challenges, i.e. 60 samples and in this
resuspension volume employed was 6.1 mls. 0.1 ml
were taken for estimating the cell

(1) Histamine release:

One third of the cell suspension was employed.
0.9 ml duplicate aliquots of challenge solution,
in complete Dulbecco's medium and prewarmed to 37°C, was added 0.1 ml of cell suspension. The solutions were then shaken gently, and allowed to incubate for 5 minutes at 37°C. The reaction tubes were then quickly removed from the incubator and placed in an ice bath. Supernatants were then separated from the cell population following centrifugation for 3 minutes at 1000 r.p.m. The cell residues were then treated with 2 mls of 0.4N perchloric acid and allowed to stand for approximately 30 minutes at ambient temperature. The precipitated protein was removed by centrifugation and the supernatant solutions set aside for histamine analysis. The original supernatant solutions were treated with 1.0 ml of 0.8N perchlorate and then treated in a similar manner to the cell residues. Histamine was measured by the method according to Evans, Lewis and Thompson (Life Sciences, 12, 327, 1973) using a Technicon Auto-analyser. Histamine release was calculat as a percentage of total histamine available in each challenge solution.

(2) Cr⁵¹ release:

One third of the cell.suspension was employed. To approximately 2.0 ml of cell suspension in Dulbecco's medium was added 0.1 ml of a solution of Cr⁵¹ labelled sodium chromate. Approximately 50-1OOµCi Cr⁵¹ was employed (specific activity: 300-500 µCi/mg Cr). The cells were allowed to stand for 30 minutes at ambient temperature and then excess chromium was removed by washing the cells thee times in Dulbecco's buffer. The cell pellet was finally resuspended in the same buffer and 0.1 ml of cell suspension was then added to 0.9 ml of each challenge
Tracer Laboratory Spectromatic γ counter. The percentage of Cr⁵¹ released was assessed in relation to the values obtained for the positive and negative control solutions.

(3) LDH measurement: ^-

One third of the cell suspension was employed. The incubation procedure was identical to that described above and carried out simultaneously until the challenge solution supernatants were separated from the cell residues. Lactic dehydrogenase activity was then estimated directly in the supernatant solutions by the method according to Johnson and Erdos (Proc. Soc. Exp. Biol. _Med. 142. 1252. 1973). To 0.5 ml of supernatant was added 0.5 ml of NAD (lmM in. o.2M Tris buffer, pH 8.5). 0.5 ml of this solution was then taken and treated with 50pl of lactic acid (50mM in O.2M Tris buffer, pH 8.5); as control, 50pl of O.2M Tris buffer (pH 8.5) was added to a second aliquot (0.5 ml) of the NAD solution. The solutions were incubated at ambient temperature for 20 minutes and the fluorescence emission was then measured. The excitation and emission wave lengths used were 340 and 460nm respectively. All measurements were carried out using a Baird Atomic automatic spectrofluorimeter (Fluoripoint). The LDH activity was assessed in terms of the increase of fluorescence over control due to NADH formation following lactate addition. The percentage of LDH released was assessed in relation to the fluorescence intensity obtained in the positive control challenge solution supernatants (i.e. Triton X 100 challenge).

(b) Skin Test Method

Skin tests were carried out in the shaved backs of animals (rats and baboons) immediately after intravenous injection of pontamine sky blue (5%) in aqueous sodium chloride solution (0.9%) at a dose of 0.1 ml per kilogram of body weight in the case of rats and 5 ml per animal in the case of baboons.
Peptide in aqueous sodium
or saline control, were injected
or 0.10 ml volumes. Skin reactions were read 20 minutes after intradernal challenge.

(c) Cross Desensitisation in the in vitro rat mast cell system between antigen and peptide

Brown Norway rats were immunised intraperitoneally with lOOµg of ovalbumen (XOA) in 1mg 'alum'. On day 27, peritoneal mast cells were removed, bulked and washed. Aliquots of cells were desensitised by the addition of 4 x 5 minute incubations with various XOA or peptide concentrations or buffer alone. The cells were then submitted to an optimal histamine releasing challenge of peptide, XOA, or challenged with buffer alone.

Claims

1. A peptide of formula (I):

and salts thereof, consisting of 6 to 12 naturally occurring amino acid residues, wherein R is an optionally present group, capable of confering on a peptide resistance to enzyme breakdown; R1 represents a residue of a basic amino acid, optionally linked to one or more residues of neutral non-hydrophobic amino acids and/or basic amino acids; R₂ represents a residue of a neutral non- hydrophobic amino acid, optionally linked to one or more further residues of neutral non-hydrophobic amino acids; R₃ represents a residue of a hydrophobic amino acid, optionally linked to one or more residues of neutral non- hydrophobic amino acids and/or hydrophobic amino acids; X is hydrogen, or a N-protecting group; and Y is hydroxyl, or a C-terminal protecting group.

2. A peptide as claimed in claim 1 wherein the basic amino acid residues are selected from arginyl, lysyl and ornithyl; the neutral, non-hydrophobic amino acid residues are selected from glycyl, alanyl, seryl and threonyl; and the hydrophobic amino acid residues are selected from phenylalanine, valine and leucine.

3. A peptide as claimed in claim 1 or claim 2 wherein R is present and is selected from prolyl, hydroxyprolyl, a D-amino acid residue and an amino acid residue with the omission of the terminal amino group.

4. A peptide as claimed in any one of the preceding claims wherein X is hydrogen and Y is hydroxyl, amino or methoxy.

5. A peptide as claimed in any one of the preceding claims having from 8 to 10 amino acid residues.

6. A peptide as

X-R-[b-c-d-e]-[f-g-h]-[i-j-k-l]-1 wherein X, Y and R are as defined in claim 1; and

lysyl, arginyl or ornithyl; d is threonyl or seryl;

optionally present arginyl, lysyl or ornithyl; f and h are glycyl or alanyl; g is seryl or threonyl; i and j are phenylalanyl, valyl or leucyl; and k and 1 are optionally present phenylalanyl, valyl or leucyl; and salts thereof.

7. A peptide as claimed in claim 6 wherein X is hydrogen, Y is hydroxyl, amino, or methoxy, and R when present is prolyl or hydroxyprolyl.

8. A peptide as claimed in claim 1 selected from the group consisting of:

wherein _Y ¹ is hydroxyl, -NH₂ or methoxy.

9. A pharmaceutical composition, adapted for use in desensitisation therapy, comprising a peptide as claimed in any one of the preceding claims or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier, suitable for parenteral, intra-nasal buccal administration.

10. A composition as claimed in claim 9 in the form

desensitisation vaccine.