Novel and biologically useful peptides of the formula: A.sub.1 -A.sub.2 -L-Phe-X-L-Thr-L-Ser-R.sub.1 -Y-L-Ser-R.sub.2 -L-Thr-L-Pro-L-Leu-L-Val-L-Thr-B are disclosed (together with pharmaceutical compositions comprising a pharmaceutically effective amount of same) wherein: PA1 (a) A.sub.1 and A.sub.2 each represent an aminoacid residue of the formula H.sub.2 N-ALK-CO-, where ALK is an alkylidene group with 1 to 6 carbon atoms inclusively; PA1 (b) X is an amino-acid residue selected from the group consisting of L-Met, L-Met(O), L-Met(O.sub.2) and L-Leu; PA1 (c) R.sub.1 and R.sub.2 each represent one of the amino-acid residues selected from the group consisting of L-Glu and L-Gln; PA1 (d) Y represents an amino acid residue selected from the group consisting of L-Lys and D-Lys; and PA1 (e) B represents one of the amino-acid or peptide moieties selected from L-Leu-OH, D-Leu-OH, L-Leu-L-Phe-OH, L-Leu-L-Phe-L-Lys-OH, L-Leu-L-Phe-D-Lys-OH, L-leucinol and L-MeLeu-OH or a functional derivative thereof; these peptides have psychopharmacological properties capable of accelerating the inhibition of the conditioned flight response, so that they are eminently suitable for the treatment of certain mental disorders in which reduction of the brain function is desired; more particularly the peptides have neuroleptic activity. Peptides of formula (I) wherein one of R.sub.1 and R.sub.2 is L-Glu and the other L-Gln are preferred, and compositions containing compositions of formula (I) wherein R.sub.1 is L-Glu and R.sub.2 is L-Gln are especially suitable.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention described and claimed herein relates to 
psychopharmacologically-active peptides, methods of preparing such 
compounds, and pharmaceutical compositions in a form suitable for 
therapeutic administration containing these peptides. In particular, the 
present invention relates to novel psychopharmacologically active peptides 
and peptide derivatives which have been derived from a certain fragment of 
the hormone .beta.-lipotropin (.beta.-LPH). .beta.-Lipotropin is a 
polypeptide, consisting of 91 amino-acids, which is formed in the 
posterior lobe of the hypophysis and shows fat-mobilising activity. 
2. Description of the Prior Art and Other Information 
Some .beta.-LPH fragments are already known and have been described in the 
literature. See, for example, the articles in Chem. & Eng. News of Aug. 
16, 1976, page 18 and Nov. 15, 1976, page 26. 
Thus it is known that the fragment .gamma.-lipotropin, .beta.-LPH-(1-58), 
possesses fat-mobilising properties just as .beta.-LPH itself. The 
fragment .beta.-LPH-(41-58), called .beta.-melanotropin, is capable of 
influencing the pigmentation of the skin by stimulating the melanocytes. 
The sequence .beta.-LPH-(61-91), called .beta.-endorphin, is known to 
possess analgesic activity, which just as that of morphine, can be 
antagonised by naloxone so that the assumption that both morphine and 
.beta.-endorphin act at the same receptor is logical and obvious. 
In the meantime, it has also been found that a certain affinity for the 
opiate receptor is also shown by smaller peptide fragments of 
.beta.-endorphin, for example .beta.-LPH-(61-76) (.alpha.-endorphin), the 
fragment .beta.-LPH-(61-69), and the fragment .beta.-LPH-(61-65) 
(Met-enkephalin). See Nature 258, 577 (1975). 
Affinity for the opiate receptor has also been described for the endogenous 
peptide Leu-enkephalin, [Leu.sup.65 ]-.beta.-LPH-(61-65), and for the 
synthetic D-Ala-Met-enkephalin,[D-Ala.sup.62 ]-.beta.-LPH-(61-65). See 
e.g. Science 194, 330 (1976). 
It has furthermore already been ascertained that .beta.-endorphin, 
.beta.-LPH-(61-91), possesses certain psychopharmacological properties. 
For example, this peptide inhibits the extinction of the (active) flight 
response in the well-known pole-climbing test (pole jumping avoidance 
behavior). This property of .beta.-endorphin cannot be diminished by known 
morphine antagonists, such as naloxone or naltrexone, so that a conclusion 
to one skilled in the art that the psychopharmacological activity of 
.beta.-endorphin is completely independent of the opiate receptor sites in 
the brains, is certainly justifiable. 
Apart from .beta.-endorphin, the smaller peptide fragments derived from 
this polypeptide, namely .alpha.-endorphin, the fragment 
.beta.-LPH-(61-69) and Met-enkephalin have been shown to inhibit the 
extinction of the flight response in a similar way. 
The peptide .gamma.-endorphin, .beta.-LPH-(61-77), which only differs from 
.alpha.-endorphin through the presence of one extra amino-acid at the 
C-terminal end, has also proved to possess psychopharmacological activity 
albeit of a completely different nature than that of .alpha.- and 
.beta.-endorphin. While .alpha.-endorphin retarded this extinction of the 
flight response, .gamma.-endorphin was shown, in contrast, to accelerate 
the extinction of the flight response. It is remarkable that the mere 
addition of one amino-acid residue to the C-terminal part of 
.alpha.-endorphin should bring about such a dramatic reversal of the 
behavioural activity. 
Surprisingly, it has now been found that peptides with an amino-acid 
sequence .beta.-LPH-(62-77) or closely related analogues derived from such 
a peptide, accelerate the extinction of the flight response to a greater 
extent than is the case with .gamma.-endorphin. Furthermore, these 
peptides according to the invention, in contrast to .gamma.-endorphin, do 
not possess affinity to the opiate receptors. 
In U.S. Pat. No. 4,097,471 (Sarantakis) there is disclosed a peptide of the 
formula 
H-Tyr-Gly-Gly-Phe-Leu-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-OH or a 
salt thereof. U.S. Pat. No. 4,127,517 (Coy) discloses a peptide of the 
formula 
H-Tyr-X-[Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr]-Leu-Phe- 
Lys-Asn-Ala-Ile. U.S. Pat. No. 4,127,518 (Coy) discloses a peptide 
H-Tyr-X-[Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr]-Leu-Y, 
wherein Y is OH, alkoxy, amine, and salts thereof. U.S. Pat. No. 4,127,519 
shows a peptide of a formula analogous to 4,127,518 wherein the Leu moiety 
outside the bracketed portion is replaced with Leu-Phe moiety. U.S. Pat. 
No. 4,127,520 also described a peptide analogous to 4,127,518, but 
replaces 4,127,518's Leu-Y with a Leu-Phe-Lys-Y moiety. 
SUMMARY OF THE INVENTION 
Surprisingly, novel peptides have been found of the formula: 
EQU A.sub.1 -A.sub.2 -L-Phe-X-L-Thr-L-Ser-R.sub.1 -Y-L-Ser-R.sub.2 
-L-Thr-L-Pro-L-Leu-L-Val-L-Thr-B I 
wherein: 
(a) A.sub.1 and A.sub.2 represent each an L-amino-acid residue of the 
formula H.sub.2 N-ALK-CO-, wherein ALK is an alkylidene group with 1 to 6 
carbon atoms; 
(b) X is an amino-acid residue selected from the group consisting of L-Met, 
L-Met(O), L-Met(O.sub.2) and L-Leu; 
(c) R.sub.1 and R.sub.2 each represent one of the amino-acid residues 
selected from the group consisting of L-Glu and L-Gln; 
(d) Y represents an amino-acid residue selected from the group consisting 
of L-Lys and D-Lys; and 
(e) B represents one of the amino-acid- or peptide moieties selected from 
L-Leu-OH, D-Leu-OH, L-Leu-L-Phe-OH, L-Leu-L-Phe-L-Lys-OH, 
L-Leu-L-Phe-D-Lys-OH, L-Leucinol and L-MeLeu-OH 
or a suitable functional derivative thereof. 
Especially preferred are the peptides (and compositions containing an 
effective amount of same) in which one of R.sub.1 and R.sub.2 L-Glu and 
the other L-Gln, in particular R.sub.1 is Glu and R.sub.2 is Gln. By 
"alkylidene" of one to six carbons it is meant an alkylidene hydrocarbon 
of one to six carbons unsubstituted by other moieties; the "alkylidene" 
species may comprise straight-chain or branched-chain species. 
The amino-acid residues covered by A.sub.1 and/or A.sub.2 are for example 
glycyl, alanyl, valyl, leucyl or isoleucyl, preferably Gly and L-Ala. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The peptides and peptide derivatives according to the general formula I are 
prepared in steps each of which are known to those in the art. The methods 
which are most frequently used for the preparation of the compounds herein 
referred to may be summarized as follows in three alternative processes: 
(a) condensation in the presence of a condensing agent of (1) an amino-acid 
or peptide containing a free carboxyl group (and in which other reactive 
groups have been protected) with (2) a compound (amino-acid, peptide or 
amine) containing a free amino group (and in which other reactive groups 
have likewise been protected); or 
(b) condensation of (1) an amino-acid or peptide containing an activated 
carboxyl group, and in which other reactive groups have optionally been 
protected, with (2) a compound (amino-acid, peptide or amine) containing a 
free NH.sub.2 group and in which other reactive groups have been 
protected, or 
(c) condensation of (1) an amino-acid or peptide containing a free carboxyl 
group (and in which other reactive groups have been protected) with (2) a 
compound (amino-acid, peptide or amine) containing an activated amino 
group (and in which other reactive groups have optionally been protected); 
after which the protecting groups, if desired, are removed. 
Methods of activating the carboxyl group are known to those skilled in the 
art, and include conversion of same into an acid halide, an azide, 
anhydride, imidazolide, or an activated ester such as the 
N-hydroxysuccinimide ester or the p-nitrophenyl ester. 
The amino group may be activated by known methods to those in the art, 
including converting the amino group into a phosphite amide, or by using 
the "phosphor-azo" method. See for both methods of activating: 
Houben-Weyl, Methoden der Organischen Chemie, 4th ed., Volume XV/2 (Georg 
Thieme Verlag), incorporated herein by reference. 
The most usual methods for the above-noted condensation reactions are: the 
carbodi-imide method, the azide method, the mixed anhydride method, and 
the activated ester method, as described in E. Schroder and K. Lubke, "The 
Peptides", volume I, 1965 (Academic Press), incorporated herein by 
reference. The so-called "solid phase" method of Merrifield, described in 
85 J. Amer. Chem. Soc. 2149 (1963), also incorporated herein by reference, 
may furthermore also be used for the preparation of the peptides and 
peptide derivatives herein described. 
The reactive groups which are not to participate in the condensation 
reaction are effectively protected by suitable so-called "protecting" or 
"protective" groups which in turn are later readily removed by hydrolysis 
or reduction. Thus a carboxyl group may be effectively protected by known 
methods, for example, esterification with at least a stochiometrically 
effective amount of methanol, ethanol, tertiary butanol, benzyl alcohol or 
p-nitrobenzyl alcohol, or in the alternative, by conversion by known means 
into an amide, as for example, described in Houben-Weyl, Methoden der 
Organischen Chemie, 4th ed., Volume XV/1, page 315 seq. This last 
protecting group is however very difficult to remove, so that it is 
recommendable that this group only be used to protect the carboxyl group 
of the C-terminal amino-acid in the final peptide or the .gamma.-carboxyl 
group of the glutamic acid. In this case, the the peptides synthesis leads 
directly to the amide of the peptide according to the general formula (I). 
Groups which may effectively protect an amino group are generally suitable 
acid groups, for example, an acid group derived from suitable aliphatic, 
aromatic, araliphatic or heterocyclic carboxylic acids (such as acetic 
acid, benzoic acid, pyridine-carboxylic acid), or an acid group derived 
from carbonic acid (such as ethoxy-carbonyl, benzyloxy-carboyl, 
t-butyloxycarbonyl or p-methoxybenzyloxy-carbonyl), or an acid group 
derived from a sulphonic acid (such as benzene-sulphonyl or 
p-toluene-sulphonyl). Other groups may also be used, such as substituted 
or unsubstituted aryl- or aralkyl-groups, for example benzyl and 
triphenyl-methyl, or groups such as o-nitrophenylsulphenyl or 
2-benzoyl-1-methylvinyl. (See Houben-Weyl, Vol. XV/1, page 46 seq.). 
It is preferably to protect also the .epsilon.-amino group of lysine, and 
optionally the hydroxyl groups of serine and threonine. This latter 
protection is however not invariably necessary. The usual protective 
groups in this connection are a tertiary-butyloxy-carbonyl or a tosyl 
moiety for the .epsilon.-amino group of lysine, and a t-butyl or benzyl 
moiety for the hydroxyl group of serine and threonine. 
The protecting groups may be cleaved by various conventional methods, 
depending on the nature of the group concerned, for example with the aid 
of trifluoroacetic acid, or by mild reduction, for example with hydrogen 
and a catalyst, such as palladium, or with HBr in glacial acetic acid. 
Peptides according to the present invention with the amino-acid residue 
L-Met(O) may be prepared from the corresponding Met-containing peptide by 
mild oxidation using methods known in the art, for example with dilute 
hydrogen peroxide or a peracid. Such an oxidation results in a mixture of 
the S- and R-sulphoxide, which can be resolved into the separate 
diastereo-isomers by known methods, for example by selective 
crystallization. The separate diastereoisomers may also be obtained 
directly by use of L-methionine-S (or R)-sulphoxide in the peptide 
synthesis. 
The sulphone-peptides according to the present invention with the 
amino-acid residue Met(O.sub.2) may be obtained by oxidation of the 
corresponding Met-peptide I or by use of methionine-sulphone in the 
peptide synthesis. 
Under the term suitable functional derivatives of the peptides according to 
the general formula (I) are understood: 
(a) salts of the peptides according to the invention, in particular the 
acid addition salts and metal salts; 
(b) esters preferably derived from aliphatic alcohols with one to about 
eighteen carbon atoms, in particular from alkanols with one to about six 
atoms, such as methanol, ethanol, propanol, isopropanol, butanol, sec. 
butyl alcohol, amyl alcohol, iso-amyl alcohol and hexyl alcohol; 
(c) amides or mono or di-alkyl-substituted amides, where the alkyl group(s) 
possess(es) 1 to about 6 carbon atoms; preferably methyl or ethyl; 
(d) N-acyl derivatives, derived from an aliphatic carboxylic acid with one 
to about six carbons; and 
(e) metal complexes, formed by bringing the peptides herein referred to 
into contact with a sparingly soluble salt, hydroxide or oxide of a metal, 
preferably zinc. 
Salts may be obtained directly from the reaction milieu in which the 
peptides are prepared or they may be prepared later by the reaction of the 
peptide with a base. 
The acid addition salts mentioned in (a) above may be obtained directly by 
isolating the peptide from the desired acid milieu, or the peptide 
obtained may be converted later into an acid addition salt by reaction of 
the peptide with an acid such as HCl, HBr, phosphoric acid, sulphuric 
acid, acetic acid, maleic acid, tartaric acid, citric acid, polyglutamic 
acid, or carboxymethylcellulose etc. 
The metal salts mentioned in (a) above, in particular the alkali metal 
salts, are obtained by reaction of the peptide with the desired metal 
base, such as NaOH, Na.sub.2 CO.sub.3, NaHCO.sub.3, etc. 
N-acyl derivatives mentioned in (d) above, by which is understood 
specifically the N-terminal acyl derivatives, are preferably prepared by 
the use in the peptide synthesis of an amino-acid which already bears the 
acyl group concerned. This acyl group then also functions as a protective 
group in the peptide synthesis. In this way, the desired acyl derivatives 
is prepared directly. It is however also possible to introduce the desired 
acyl group later, by acylating the peptide in the usual way known to those 
in the art. 
The N-acyl group which is most preferred is the acetyl group. 
Esters and amides in (b) and (c) are also preferably prepared by using in 
the peptide synthesis an amino-acid which already bears the desired ester 
or amide group; they may however also be prepared later by esterifying the 
peptide obtained in the usual way known to those in the art, or by 
converting the same into an amide. 
The amides in (c) which are most preferred are the unsubstituted amide; 
e.g., the mono-methyl- or dimethyl-amide, or the mono-ethyl- or 
diethyl-amide. 
The metal complexes in (e) above may be obtained by bringing the peptide 
into contact with sparingly soluble metal salts, metal hydroxides or metal 
oxides. The metal phosphates, metal pyrophosphates and metal 
polyphosphates are generally used as sparingly soluble metal salts. The 
metals which may be used in this process are the metals which belong to 
the "b" subgroups of the Periodic Table, for example cobalt, nickel, 
copper, iron and preferably zinc, as well as metals from the main groups 
of the Periodic Table which are capable of forming complexes, such as 
magnesium and aluminium. The preparation of these metal complexes takes 
place in the usual way. A metal complex may for example be obtained by 
addition of the peptide and a sparingly soluble metal salt, metal 
hydroxide, or metal oxide to an aqueous medium. The metal complex may also 
be obtained by addition of an alkaline medium to an aqueous solution of 
the peptide and a soluble metal salt, such that the insoluble 
peptide-metal hydroxide complex is formed. The metal complex may 
furthermore be obtained by addition of the peptide, a soluble metal salt 
and a further soluble salt to an aqueous, preferably alkaline, medium, 
such that an insoluble peptide-metal salt complex is formed "in situ". 
The metal complexes may be used directly without further processing as 
suspensions, or they may for example be freeze-dried and later 
resuspended. 
The peptides according to general formula I, and the functional derivatives 
as defined above, accelerate (as already noted above) the extinction of 
the (active) flight response in rats in the so-called "pole climbing" test 
to a very considerably extent. 
The passive flight behaviour of rats is also considerably reduced on use of 
the peptides according to the invention. 
In addition to the above-noted effects on behaviour, in higher dosages the 
peptides herein referred to cause a pronounced reduction in ambulation of 
rats, a surprising effect that could not be observed with 
.alpha.-endorphin or even with .gamma.-endorphin. 
The present peptides are furthermore surprisingly active in the so-called 
"grip test". The rats treated with the peptides according to the invention 
hang suspended above the floor of the cage with their front paws grasping 
a pencil for a significantly longer time than rats treated with saline 
(placebo) or .alpha.-endorphin. 
This pharmacological profile renders the peptides and peptide derivatives, 
herein referred to, particularly suitable for use in the treatment of 
certain mental disorders in which a reduction of the cerebral functions is 
desired. In particular the present peptides have neuroleptic activity and 
are thus suitable in the treatment of for example schizophrenic syndromes. 
The peptides are used in effective amounts with known carriers, and 
preferably used in a dosage of 1 .mu.g to 1 mg per kg body weight per day, 
depending on the form in which they are administered. Humans are 
preferably treated with a daily dosage of 0,1 mg to about 10 mg, more 
particularly between 0,5 and 2 mg. 
The peptides according to the invention may be administered by either the 
oral, rectal or the parenteral routes, by means of a pharmaceutically 
effective carrier known to those in the art. The peptides are preferably 
used as injectable preparations. For the purposes of injection they are 
dissolved, suspended or emulsified in a suitable fluid. Mixed with 
suitable excipients and fillers, the peptides herein referred to may 
further be provided in a form suitable for oral administration, such as 
pills, tablets, dragees or capsules. The peptides herein described may 
furthermore be administered in the form of a suppository or spray. 
Particularly valuable preparations are obtained when the peptides herein 
referred to are provided in a form conferring prolongation of activity. 
Preferably, the metal complexes are used. These metal complexes may be 
obtained by bringing the peptides into contact with sparingly soluble 
metal salts, metal hydroxides or oxides known to those in the art. The 
metal phosphates, metal pyrophosphates and metal polyphosphates are 
generally used as sparingly soluble metal salts. 
Peptides according to the general formula I which are especially preferred 
are those peptides in which, 
A.sub.1 and A.sub.2 are Gly or L-Ala, 
X represents L-Met, L-Met(O) or L-Met(O.sub.2), 
R.sub.1 represents L-Glu, 
Y represents L-Lys, 
R.sub.2 represents L-Gln, and 
B represents L-Leu-OH, D-L-Leu-OH or L-Leu-L-Phe-D-Lys-OH, 
as well as the acid addition salts, lower aliphatic esters and amides 
thereof. 
With respect to the examples below, the following observations and rules 
are made: 
I. If no optical configuration is given, the L-form is implied. 
II. The following abbreviations have been used for the protecting or 
activating groups employed: 
Boc=tertiary butyloxycarbonyl 
tBu=tertiary butyl 
Me=methyl 
ONp=p-nitrophenyloxy 
Z=benzyloxycarbonyl 
III. The following abbreviations have been assigned to the solvents or 
reagents used: 
To=toluene 
EtOH=ethanol 
Bu=butanol 
Py=pyridine 
Ac=acetic acid 
EtOAc=ethyl acetate 
Am=amyl alcohol 
I.A.N. or IAN=iso-amyl nitrite 
DMF=dimethylformamide 
THF=tetrahydrofuran 
DCCI=dicyclohexylcarbodi-imide 
DCHU=dicyclohexylurea 
TEA=triethylamine 
TFA=trifluoro-acetic acid 
Wa=water 
N.E.M.=N-ethylmorpholine 
HOBt=N-hydroxybenztriazole 
IV. The following abbreviations have been used for the amino-acid groups: 
Met=methionyl 
Met(O)=sulphoxide of methionyl 
Met(O.sub.2)=sulphone of methionyl 
Phe=phenylalanyl 
Pro=prolyl 
Ser=seryl 
Lys=lysyl 
Thr=threonyl 
Glu=glutamyl 
Gln=glutaminyl 
Gly=glycyl 
Val=valyl 
Leu=leucyl 
Ala=alanyl 
MeLeu=N.sup..alpha. -methylleucyl 
Although the invention has been described with respect to the specific 
embodiments above, numerous variations and modifications will become 
evident to those skilled in the art, without departing from the scope and 
spirit of the invention as described above, defined in the appended 
claims, and as shown in the following examples: 
EXAMPLES--STARTING MATERIALS 
A. Synthesis of Boc-Gly-Gly-Phe-Met-OH and analogues 
(1) H-Phe-Met-OMe.HCl 
11.83 g of Boc-Phe-Met-OMe (see Biochemistry, 8, 4183 (1969), incorporated 
herein) is dissolved in 100 ml methylene chloride after which HCl is 
passed into the solution for about 40 minutes. After evaporation of the 
solution to dryness, 75 ml ethyl acetate is added, resulting in a 
precipitate. The solid substance is separated by filtration, washed with 
petroleum ether, and dried. Melting point 123.degree.-124.degree. C. Rf in 
To:EtOH (8:2)=0.43 on SiO.sub.2. 
(2) Boc-Gly-Gly-OH 
The procedure followed was that of Tetrahedron 25, 2119 (1976), 
incorporated herein. Rf in Bu:Py:Ac:Wa (4:0.75:0.25:1)=0.42 on SiO.sub.2 ; 
decomposition at 125.degree. C.-127.degree. C. 
(3) Boc-Gly-Gly-Phe-Met-OMe 
4.43 g of Boc-Gly-Gly-OH, A (2) is dissolved in 30 ml DMF and cooled to 
0.degree. C., after which 1 equivalent TEA (triethylamine) is added. The 
mixture is cooled further to about -10.degree. C., after which 1 
equivalent (eq) ethyl chloroformate is added and the whole is stirred for 
about 10 minutes. 6.6 g H-Phe-Met-OMe.HCl (A.1) in 30 ml DMF and 1.1 eq 
TEA is then added to the mixture, which is stirred for about 30 minutes at 
about -10.degree. C. and for a further 20 hours at room temperature. After 
cooling to about -10.degree. C., TEA.HCl is separated by filtration and 
the filtrate is evaporated to dryness. The residue is dissolved in 235 ml 
EtOAc and 55 ml water and is subsequently washed with 30% NaCl solution, 
0.1 N HCl, 30% NaCl solution, 5% NaHCO.sub.3 solution and 30% NaCl 
solution. The solution is then dried over Na.sub.2 SO.sub.4 filtered and 
evaporated to dryness. Melting point 100.degree.-101.degree. C.; Rf in 
CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.94 on SiO.sub.2. 
(4) Boc-Gly-Gly-Phe-Met-OH 
2.62 g of the peptide obtained in A(3) is dissolved in 30 ml dioxan/H.sub.2 
O-(9:1). After addition of 1.2 eq 2.17 N NaOH, the whole is stirred at 
room temperature for about 1 hour, after which the pH of the mixture is 
adjusted to about 6 and it is evaporated to dryness. The residue is 
subsequently dissolved in 50 ml EtOAc, after which the pH is adjusted to 2 
with 1 N HCl. After washing with 30% NaCl (3.times.), drying over Na.sub.2 
SO.sub.4 and filtering, the solution is evaporated to dryness. Rf=0.65. 
Melting point 97.degree.-98.degree. C. 
(5) The following peptides are prepared in an analogous manner: 
Boc-Gly-Ala-Phe-Met-OH; Rf=0.72; 
Boc-Ala-Gly-Phe-Met-OH; Rf=0.73 
Boc-Leu-Gly-Phe-Met-OH; Rf=0.77 
The system used in A(4) and A(5) is Bu:Py:Ac:Wa (4:0.75:0.25:1). 
B. Synthesis H-Thr-Ser-Glu(OtBu)-Lys(Boc)-OMe and analogues 
(1) H-Glu(OtBu)-Lys(Boc)-OMe 
(a) 35.3 g Z-Glu(OtBu)-OH and 27.0 g HOBt are dissolved in 150 ml DMF, 
after which the mixture is cooled to about -22.degree. C. 29.7 g 
H-Lys(Boc)-OMe.HCl in 100 ml DMF and 1 eq NEM is then added to the cooled 
mixture. The pH of the mixture is adjusted to 6.4 with NEM and 23 g DCCI 
is then added. After stirring for about 15 minutes at about -22.degree. C. 
and about 12 hours at room temperature, DCHU is separated by filtration 
and the filtrate is evaporated to dryness. 
The residue is dissolved in 400 ml EtOAc and washed consequently with 15% 
NaCl solution, 5% KHSO.sub.4 solution, 5% NaHCO.sub.3 solution and 15% 
NaCl solution. After drying and filtering, the filtrate is evaporated to 
dryness. The residue is crystallized from ether/petroleum ether (1:2). 
Yield 86.6%; melting point 54.degree.-56.degree. C. 
(b) The peptide obtained in B(1) is dissolved in DMF, after which Pd/C 
(10%) is added and H.sub.2 is passed through until the evolution of 
CO.sub.2 ceases. After filtering, the filtrate is evaporated to dryness. 
Rf in To:EtOH (8:2)=0.24 on SiO.sub.2. 
(2) Z-Thr-Ser-N.sub.2 H.sub.3 
38.05 g Z-Thr-Ser-OMe (See Recueil 83, 255, (1964), incorporated herein) is 
dissolved in 12 ml ethanol, after which 43 ml hydrazine hydrate is added. 
After stirring for about 2 hours, the solid substance is separated by 
filtration, washed with ethanol/ether (1:1) and dried. 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.58 on SiO.sub.2 ; 
decomposition 215.degree.-216.degree. C. 
(3) H-Thr-Ser-Glu(OtBu)-Lys(Boc)-OMe 
(a) 1.22 g of the hydrazide obtained in B(2) is suspended in 15 ml DMF 
after which 4.28 ml 2.42 N HCl/DMF is added. The clear solution is cooled 
to about -20.degree. C. 0.7 ml IAN is then added and the mixture is 
stirred for about 30 minutes at about -20.degree. C. 
1.5 g of the peptide obtained in B(1) in 10 ml DMF is then added. The pH of 
the reaction mixture is then adjusted to 7.2, and the whole is placed in a 
refrigerator for about 6 days. 
The solvent is then removed by evaporation, the residue is dissolved in 
EtOAc and the resultant solution is washed. Evaporation to dryness gives a 
solid substance. Yield 61.9%; melting point 130.degree.-132.degree. C. 
(b) In a way similar to that described in B(1)(b) the Z-protected peptide 
is hydrogenated in methanol with palladium on charcoal as catalyst. Yield 
99%; Rf in Bu:Py:Ac:Wa (38:24:8:30)=0.73 on SiO.sub.2. 
(4) The following protected peptides are obtained in analogous ways: 
EQU H-Thr-Ser-Glu(OtBu)-D-Lys(Boc)-OMe 
Rf in Bu:Py:Ac:Wa (38:24:8:30)=0.77 on SiO.sub.2 ; 
EQU H-Thr-Ser-Gln-Lys(Boc)-OMe 
Rf in Bu:Py:Ac:Wa (38:24:8:30)=0.65 on SiO.sub.2. 
C. Synthesis Z-Ser-R.sub.2 -Thr-Pro-OH (R.sub.2 =Glu or Gln) 
(1) H-Thr-Pro-OtBu 
In the way described in B(1)., 0.33 mol Z-Thr-OH and 0.35 mol H-Pro-OtBu 
are coupled with the aid of HOBt and DCCI in DMF. Yield 64%; melting point 
65.degree.-67.degree. C. 
The Z-Thr-Pro-OtBu obtained in this way is hydrogenated in the way 
described above (see B.3.b.). 
Rf in To:EtOH (8:2)=0.10 on SiO.sub.2. 
(2) H-Gln-Thr-Pro-OtBu 
1.36 g H-Thr-Pro-OtBu is dissolved in 10 ml DMF, after which 1.93 g 
Z-Gln-ONp is added and the reaction mixture is stirred for about 20 hours 
at room temperature. 
After evaporation of the mixture to dryness, the residue is dissolved in 
EtOAc and washed consecutively with 5% KHSO.sub.4 solution, 5% NaHCO.sub.3 
solution and a saturated NaCl solution. The solution is then dried over 
Na.sub.2 SO.sub.4 and filtered, and the filtrate is evaporated to dryness. 
Melting point 89.degree.-90.degree. C.; yield 59%. 
The Z-protected peptide obtained is hydrogenated in DMF in the way 
described above. 
Rf in CHCl.sub.3 :CH.sub.3 OH (8:2)=0.08 on SiO.sub.2. 
(3) Z-Ser-Gln-Thr-Pro-OtBu 
In a way analogous to that described in C(1), 20.5 g Z-Ser-OH is coupled 
with the peptide obtained in C(2) with the aid of DCCI and BOBt. 
Yield 70%. Melting point 104.degree.-106.degree. C. 
(4) Z-Ser-Gln-Thr-Pro-OH 
1.43 g of the peptide obtained in C(3) is stirred in 15 ml 90% TFA at room 
temperature for about 30 minutes. The mixture is then poured into ether. 
The solid material is separated by filtration, washed with ether and 
dried. Yield 90%; melting point 111.degree.-113.degree. C. 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.23 on SiO.sub.2. 
(5) Z-Ser-Glu(OtBu)-Thr-Pro-OH 
H-Thr-Pro-OMe, obtained by coupling Z-Thr-OH and H-Pro-OMe with the aid of 
the HOBt/DCCI method followed by hydrogenation, is consecutively coupled 
with Z-Glu(OtBu)-OH and Z-Ser-OH. Both coupling reactions are performed by 
the HOBt/DCCI method, and after the first coupling of Z-protected peptide 
obtained is hydrogenated. 
The resultant peptide, Z-Ser-Glu(OtBu)-Thr-Pro-OMe, is subsequently 
saponified by dissolving in dioxan/water (9:1) and addition of 0.2 N NaOH 
(see A(4)). 
Rf in CHCl.sub.3 :CH.sub.3 :CH.sub.3 :OH:Wa (70:30:5)=0.29 on SiO.sub.2. 
D. Synthesis of H-Leu-Val-Thr-(L or D)-Leu-OtBu 
(1) H-Thr-Leu-OtBu 
In a way corresponding to that described in J.A.C.S. 95, 877 (1973), 
incorporated herein, Z-Thr-Leu-OtBu is prepared via the HOBt/DCCI coupling 
method. 
Melting point 81.5.degree.-83.degree. C. 
The Z-protected dipeptide is then hydrogenated in the way described above. 
Yield 100%; Rf in To:EtOH (8:2)=0.20 on SiO.sub.2. 
(2) H-Thr-D-Leu-OtBu 
Obtained by hydrogenation of Z-Thr-D-Leu-OtBu with melting point 
89.degree.-92.degree. C. 
Rf in To:EtOH (8:2)=0.18 on SiO.sub.2. 
(3) H-Val-Thr-Leu-OtBu 
Coupling of 7.85 g Z-Val-ONp with 5.53 p H-Thr-Leu-OtBu in 160 ml DMF in 
the way described in C(2) provides Z-Val-Thr-Leu-OtBu in 72% yield. 
Melting point 127.degree.-129.degree. C. 
Hydrogenation of this Z-protected peptide in methanol gives 9.1 g of an 
oily product. 
Rf in CHCl.sub.3 :CH.sub.3 OH (8:2)=0.60 on SiO.sub.2. 
(4) H-Val-Thr-D-Leu-OtBu 
Obtained in a way analogous to that described in D(3). 
Rf in CHCl.sub.3 :CH.sub.3 OH (8:2)=0.55 on SiO.sub.2. 
(5) H-Leu-Val-Thr-Leu-OtBu 
Coupling of 6.7 g Z-Leu-ONp and 6.1 g of the peptide obtained in D(3), in 
160 ml DMF, in the way described in C(2), gives the Z-protected peptide in 
a yield of 7.7 g (77%). Melting point 153.degree.-155.degree. C. This 
Z-protected peptide is hydrogenated in methanol. Rf=0.75 l on SiO.sub.2. 
(6) H-Leu-Val-Thr-D-Leu-OtBu 
Rf=0.80 
(7) H-Leu-Val-Thr-leucinol 
Rf=0.50 
(8) H-Leu-Val-Thr-MeLeu-OtBu 
R.sub.f =0.68, prepared in a way analogous to D(5). 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5) on SiO.sub.2. cl E. Synthesis of 
Boc-Gly-Gly-Phe-Met-Thr-Ser-Glu(OtBu)-Lys(Boc)-OMe and analogues 
(1) Boc-Gly-Gly-Phe-Met-Thr-Ser-Glu(OtBu)-Lys(Boc)-OMe 
2.4 g Boc-Gly-Gly-Phe-Met-OH, A(4), and 3.15 g 
H-Thr-Ser-Glu(OtBu)-Lys(Boc)-OMe, B(3), are coupled with the aid of 2 eq 
HOBt and 1 eq DCCI in the way described in B(1). 
After removal of the DCHU by filtration, the filtrate is evaporated to 
dryness and the residue is crystallized from methanol. Melting point 
207.degree.-209.degree. C. (decomposition); yield 61%. 
(2) Boc-Gly-Gly-Phe-Met-Thr-Ser-Glu(OtBu)-Lys(Boc)-OH 
2.92 g of the peptide obtained in E(1) is dissolved in 30 ml dioxan/water 
(9:1) after which 14.4 ml 0.217 g NaOH is added to the solution. 
The reaction mixture is stirred for 18 minutes at room temperature. 
The pH of the mixture is then adjusted to 2 with N HCl. After the addition 
of about 10 ml water, a solid crystallizes and this is filtered off and 
dried. 
Yield 78%; melting point 215.degree.-216.degree. C. (dec.). 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.49 on SiO.sub.2. 
The following are prepared in a corresponding way: 
(3) Boc-Ala-Gly-Phe-Met-Thr-Ser-Glu(OtBu)-Lys(Boc)-OH 
Rf in CHCl.sub.3 :CH.sub.3 :OH:Wa (70:30:5)=0.52 on SiO.sub.2. 
(4) Boc-Gly-Ala-Phe-Met-Thr-Ser-Glu(OtBu)-Lys(Boc)-OH 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.51 on SiO.sub.2. 
(5) Boc-Leu-Gly-Phe-Met-Thr-Ser-Glu(OtBu)-Lys(Boc)-OH Rf in CHCl.sub.3 
:CH.sub.3 OH:Wa (70:30:5)=0.54 on SiO.sub.2. 
(6) Boc-Gly-Gly-Phe-Met-Thr-Ser-Glu(OtBu)-D-Lys(Boc)-OH 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.50 on SiO.sub.2. 
(7) Boc-Gly-Gly-Phe-Met-Thr-Ser-Gln-Lys(Boc)-OH 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.40 on SiO.sub.2. 
F. Synthesis of H-Ser-R.sub.2 -Thr-Pro-Leu-Val-Thr-(L or D)-Leu-OtBu 
(R.sub.2 =Glu(OtBu) or Gln) and analogues 
(1) H-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OtBu 
1.17 g Z-Ser-Gln-Thr-Pro-OH of C(4) is coupled to 930 mg 
H-Leu-Val-Thr-Leu-OtBu D(5) with the aid of 2 eq HOBt and 1 eq DCCI, 
according to the method described in B(1). After removal by filtration of 
the DCHU formed, the filtrate is evaporated to dryness and dissolved in a 
mixture of sec. butanol and CHCl.sub.3 (2:3), after which the solution is 
washed and evaporated to dryness. The residue is crystallized from 
DMF/EtOAc (1:20); melting point 210.degree.-212.degree. C. 
Yield 72%. 
The Z-protected peptide obtained is hydrogenated in methanol in the way 
described above. Yield 86%. 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.25 on SiO.sub.2. 
The following peptides are prepared in a corresponding way: 
(2) H-Ser-Glu(OtBu)-Thr-Pro-Leu-Val-Thr-Leu-OtBu, C(5)+D(5). 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.35 on SiO.sub.2. 
(3) H-Ser-Gln-Thr-Pro-Leu-Val-Thr-D-Leu-OtBu, (C(4)+D(6)) 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.30 on SiO.sub.2. 
(4) H-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-NHCH.sub.3 
100 mg of the peptide Z-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH (see F.5) is 
dissolved in 2 ml DMF, after which the solution is cooled to about 
-10.degree. C. 1 eq TEA and 1 eq ethylchloroformate are then added, after 
which the mixture is stirred for 10 minutes. After addition of an excess 
of monomethylamine, the mixture is stirred for about 30 minutes at about 
-10.degree. C. and 2 hours at 0.degree. C., after which the whole is 
evaporated to dryness. The residue is dissolved in a mixture of sec. 
butanol and chloroform (2:3), after which the solution is washed, dried, 
and evaporated to dryness. Yield 65 mg, melting point 
223.degree.-225.degree. C. 
The Z-protected peptide-monomethylamide obtained is hydrogenated in DMF in 
the usual way. 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.26 on SiO.sub.2. 
(5) H-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OMe 
100 mg of the peptide Z-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OtBu (see F(1)) in 
2 ml 90% TFA is stirred for 20 minutes at room temperature. The mixture is 
then evaporated in dryness and the solid material is filtered off and 
dried. 
The solid (80 mg) is dissolved in DMF and esterified with caesium carbonate 
and methyl iodide by the method described in J.O.C. 42, 1286 (1977), 
incorporated herein. The Z-protected peptide methyl-ester is then 
hydrogenated in DMF in the usual way; yield 45 mg. 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.34 on SiO.sub.2. 
(6) H-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys(Boc)-OtBu 
0.992 g Z-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH of F(5) is coupled to 540 mg 
H-Phe-Lys(Boc)-OtBu in DMF with the aid of DCCI (1 eq) and HOBt (2 eq). 
After removal of the DCHU by filtration, the filtrate is evaporated to 
dryness. The residue is subsequently dissolved in 75 ml sec. 
butanol/chloroform (2:3) and the solution washed with water, 0.1 N HCl, 5% 
NaCl solution and water, after which it is dried over Na.sub.2 SO.sub.4, 
filtered and the filtrate evaporated to dryness. 
Yield of Z-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys(Boc)-OtBu: 1 g; melting 
point 221.degree.-222.degree. C. (decomposition). 
This peptide is hydrogenated in DMF with Pd/C as catalyst in the way 
described above. 
Rf In CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.27 on SiO.sub.2. 
(7) H-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-OtBu 
Rf=0.34 on SiO.sub.2. 
(8) H-Ser-Gln-Thr-Pro-Leu-Val-Thr-MeLeu-OtBu 
Rf=0.30 on SiO.sub.2 
(9) H-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leucinol 
Rf=0.16 on SiO.sub.2. 
(10) H-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-D-Lys(Boc)-OtBu 
Rf=0.48; (7), (8), (9) and (10) being prepared in a way corresponding to 
that described in F(6). Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5).

EXAMPLE I 
Synthesis of 
H-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH 
1.28 g of Boc-Gly-Gly-Phe-Met-Thr-Ser-Glu(Otbu)-Lys(Boc)-OH of E(2) above 
and 308 mg HOBt were dissolved in 10 ml DMF and the mixture was cooled to 
about -22.degree. C. 1.05 g H-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OtBu (F(1)) 
in 5 ml DMF and 1 eq NEM was then added to the cooled mixture. The pH of 
the mixture was adjusted to 6.5 with NEM and 247 mg DCCI was added. After 
stirring for 15 minutes at about -22.degree. C., 8 hours at room 
temperature and finally for 12 hours at 35.degree. C., under N.sub.2, the 
DCHU formed was separated by filtration and the filtrate was washed and 
dried. 
The precipitate formed was washed and dried. 
Yield 77%; decomposition at 212.degree.-214.degree. C. 
Rf in CHCl.sub.3 :CH.sub.3 OH:Wa (70:30:5)=0.79 on SiO.sub.2. 1.65 g of the 
protected peptide thus obtained was placed in 30 ml 90% TFA, and a few 
drops of tert. butyl sulphide were added. The mixture was stirred for 1 
hour at room temperature, after which it was poured into ether. The solid 
thus obtained was separated by filtration and dried. The substance was 
then dissolved in 30 ml tert. butanol/water (1:1), an ion exchange resin 
in acetate form (LEWATIT) is added, and the mixture is stirred for about 
30 minutes. The ion exchange resin is subsequently removed by filtration, 
and the filtrate is evaporated to dryness. Yield: 1.2 g. 
Rf in Bu:Py:Ac:Wa (2:0.75:0.25:1)=0.26 on SiO.sub.2. 
The substance is purified by counter-current distribution in the solvent 
system Bu:Ac:Wa (4:1:5). Yield 680 mg. 
EXAMPLE II 
Synthesis of 
H-Gly-Gly-Phe-Met(O)-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH 
200 mg of the peptide obtained in Example I is dissolved in 20 ml glacial 
acetic acid, after which 0.08 ml 30% hydrogen peroxide is added. The 
mixture is stirred for about 1 hour at room temperature, after which 300 
mg platinum Black in glacial acetic acid is added to the mixture and the 
whole is stirred for about a further 15 minutes. 
The solid material is separated by filtration and the filtrate is 
evaporated to dryness. 
Yield 190 mg. 
The peptide thus obtained is further purified by counter-current 
distribution chromatography in the solvent system Bu:Ac:Wa (4:1:5). 
Yield 150 mg (as acetate). 
Rf in Bu:Py:Ac:Wa (2:0.75:0.25:1)=0.20 on SiO.sub.2. 
EXAMPLE III 
Synthesis of 
H-Gly-Gly-Phe-Met(O.sub.2)-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu 
-OH 
200 mg of the peptide obtained in Example I is introduced into 5 ml water 
after which 0.025 ml 0.5 M ammonium molybdate, 0.125 ml HClO.sub.4 and 
0.075 ml 30% hydrogen peroxide are added. The mixture is stirred for about 
4 hours at room temperature, after which 5 ml tert. butanol/water (1:1) 
and an ion exchange resin in acetate form are added. After stirring for 
about 30 minutes, the ion exchange resin is separated by filtration and 
the filtrate is evaporated to dryness. 
Yield 180 g peptide (in acetate form). 
Rf in Bu:Py:Ac:Wa (2:0.75:0.25:1)=0.23 on SiO.sub.2. 
EXAMPLE IV 
The following are prepared in a way corresponding to that described in 
Example I: 
1. H-Ala-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH 
(E.3+F.1) Rf=0.28 
2. H-Gly-Ala-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH 
(E.4+F.1) Rf=0.27 
3. H-Leu-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH 
(E.5+F.1) Rf=0.30 
4. H-Gly-Gly-Phe-Met-Thr-Ser-Glu-D-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH 
(E.6+F.1) Rf=0.27 
5. H-Gly-Gly-Phe-Met-Thr-Ser-Gln-Lys-Ser-Glu-Thr-Pro-Leu-Val-Thr-Leu-OH 
(E.7+F.2) Rf=0.29 
6. H-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-D-Leu-OH 
(E.2+F.3) Rf=0.33 
7. 
H-Gly-Gly-Phe-Met-thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-NHCH.sub 
.3 
(E.2+F.4) Rf=0.31 
8. 
H-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OCH.sub. 
3 
(E.2+F.5) Rf=0.37 
9. 
H-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys- 
OH 
(E.2+F.6) Rf=0.24 
10. 
H-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-OH 
(E.2+F.7) Rf=0.30 
11. 
H-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-D-Ly 
s-OH 
(E.2+F.10) Rf=0.29. 
12. H-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leucinol 
(E.2+F.9) Rf=0.28 
13. H-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-MeLeu-OH 
(E.2+F.8) Rf=0.32 
All Rf values in Bu:Py:Ac:Wa (2:0.75:0.25:1) on SiO.sub.2. 
EXAMPLE V 
Pole-jumping avoidance behavior 
Rats were trained to jump onto a pole within 5 sec. following presentation 
of the conditioned stimulus (CS) which was a light on top of the cage. 
Rats which did not jump within 5 sec. received scrambled footshocks (0.2 
mA) as the unconditioned stimulus (UCS) until the response was made, or 
for 30 sec. maximally. 10 trials a day were given in one session with an 
average intertrial interval of 60 sec. Intervals between trials were 40, 
60 and 80 sec. which were presented in a random fashion. Rats were trained 
for 4 days. Extinction was studied the day following acquisition. During 
this first post-acquisition session failure to respond within 5 sec. to 
the CS were not followed by the UCS. All rats were given a 10 trial 
extinction session. Those animals which made 8 or more avoidances were 
used for further experimentation. These rats received peptide or placebo 
(saline) in a volume of 0.5 ml per rat s.c. immediately after completion 
of the first extinction session. Extinction was studied again 2 and 4 h. 
later. 
______________________________________ 
Results 
Extinction (10 trials) 
Treatment 0 2 4 
______________________________________ 
.gamma.-Endorphin (reference) 
0.03 .mu.g.sup.3 
8.8 .+-. 0.3.sup.2 
6.7 .+-. 0.8 
4.0 .+-. 0.7 
0.1 .mu.g 9.3 .+-. 0.3 
5.5 .+-. 0.6 
2.3 .+-. 0.4 
0.3 .mu.g 9.0 .+-. 0.4 
4.6 .+-. 0.3 
0.8 .+-. 0.4 
Saline 0.5 ml 9.4 .+-. 0.4 
8.0 .+-. 0.5 
7.8 .+-. 0.7 
.alpha.-Endorphin (reference) 
0.3 .mu.g 9.0 .+-. 0.4 
8.3 .+-. 0.3 
7.8 .+-. 0.3 
Haloperidol (reference) 
0.03 .mu.g 9.3 .+-. 0.3 
6.5 .+-. 0.7 
4.0 .+-. 0.6 
0.1 .mu.g 9.5 .+-. 0.4 
3.0 .+-. 0.0 
0.5 .+-. 0.4 
Saline 0.5 ml 9.8 .+-. 0.3 
9.0 .+-. 0.4 
7.8 .+-. 0.5 
[Des-Tyr.sup.1 ].gamma.-endorphin 
0.01 .mu.g 9.0 .+-. 0.3 
6.2 .+-. 0.4 
4.2 .+-. 0.7 
0.1 .mu.g 8.8 .+-. 0.4 
2.8 .+-. 1.0 
1.2 .+-. 0.5 
0.3 .mu.g 9.3 .+-. 0.5 
3.0 .+-. 0.7 
1.3 .+-. 0.5 
Saline 0.5 ml 9.7 .+-. 0.4 
9.7 .+-. 0.4 
8.7 .+-. 0.6 
[Des-Tyr.sup.1, Met(0).sup.5 - 
.gamma.-endorphin 
0.003 .mu.g 9.0 .+-. 0.3 
7.8 .+-. 0.8 
4.4 .+-. 0.9 
0.01 .mu.g 9.4 .+-. 0.3 
4.8 .+-. 0.7 
1.4 .+-. 0.7 
______________________________________ 
.sup.1 h after injection 
.sup.2 Mean .+-. S.E. 
.sup.3 Dose per rat s.c.