Acylpeptides derived from somatostatin or from a derivative thereof having analogous action, in which the amino acids sequence is modified by omitting individual amino acids and/or by exchanging them for other amino acids, and in which the .epsilon.-amino group of the lysine residue in the 9-position, and optionally also the .epsilon.-amino group of the lysine residue in the 4-position and/or the N-terminal .alpha.-amino group carries the acyl radical of an optionally substituted alkanecarboxylic acid, and salts and complexes thereof can be used as antidiabetics and/or for combating gastrointestinal bleeding. They are manufactured by conventional methods of peptide chemistry.

The invention relates to novel acylpeptides derived from somatostatin and 
its analogues, processes for the manufacture of these acylpeptides, 
pharmaceutical preparations containing the same and the use of these 
compounds and preparations for therapeutic purposes. The acylpeptides 
according to the invention are derivatives of somatostatin and of 
analogues derived therefrom, in which the .epsilon.-amino group of the 
lysine residue in the 9-position, and optionally also the .epsilon.-amino 
group of the lysine residue in the 4-position and/or the N-terminal amino 
group is substituted by the radical of a carboxylic acid, it being 
possible to modify the amino acids sequence of somatostatin by omitting 
individual amino acids or by exchanging them for other amino acids. 
As is known, somatostatin, a cyclic tetradecapeptide of the formula 
##STR1## 
[Science 179, 77 (1973)], inhibits the pituitary-controlled secretion of 
the somatotrophic hormone (somatotrophin). It also inhibits the secretory 
activity of the endocrine pancreas, such as the secretion of insulin and 
glucagon. In the case of somatostatin itself, these valuable properties 
cannot be used fully in practice since this compound has too short a 
duration of action. In addition, it is often preferable for the active 
substance to exercise its inhibitory effect predominantly on one of the 
hormones mentioned. For this reason, attempts are being made to achieve a 
dissociation of the inhibitory effects and a duration of action which is 
as long as possible by modifying the basic sequence, especially by 
omitting individual original amino acids and/or exchanging them for other, 
often "unnatural", amino acids. 
Surprisingly, it has now been found that, by a very unusual modification of 
the basic structure, which comprises acylating the .epsilon.-amino group 
of the lysine residue in the 9-position of somatostatin or a structural 
analogue thereof having a similar action with an optionally substituted 
alkanecarboxylic acid, an acylpeptide is produced in which the original 
activity of the basic structure is not only maintained but often increased 
and heightened still further in the sense discussed above, especially in 
respect of the duration of action. Such a result is all the more 
surprising in view of the fact that the basic character of the terminal 
amino group of the Lys.sup.9 residue, which is considered indispensable 
for the biological action of somatostatin and analogous active substances, 
is thereby eliminated. 
The compounds according to the invention are often advantageous also from 
the technical standpoint since, in their synthesis from smaller building 
blocks, the selective protection of the amino groups concerned, 
specifically the .epsilon.-amino groups of lysine residues, is no longer 
necessary and hence the synthesis is simplified. 
From this point of view, of the acylpeptides of the invention in general, 
and also of their representatives which are given special emphasis 
hereinbelow, there are preferred those in which all the amino groups 
concerned carry the same acyl radical. 
The present invention relates especially to acylpeptides derived from 
somatostatin and analogues thereof, having the general formula 
##STR2## 
in which 
Ac represents an acyl radical of an optionally substituted alkanecarboxylic 
acid, present at the free amino group, 
A represents the radical of the partial formula 
##STR3## 
(wherein cys represents L-Cys or D-Cys and Bmp represents the 
desaminocysteine residue), or the residue of an .omega.-amino-lower 
alkanecarboxylic acid of the partial formula --NH--CH(R)--(CH.sub.2).sub.n 
--CO-- (wherein n represents 0 or an integer from 1 to 6 and R represents 
hydrogen or carboxyl) which, if n=2 and R is hydrogen, can also be 
substituted by a cyclic hydrocarbyl radical and, in that case, is 
designated hereinafter as Gaba(Ar), 
B represents Lys, Lys(Ac) or Lys(X) (wherein X is an 
.epsilon.-amino-protecting group), 
C represents Asn, Ala or His, 
D represents Phe or, if no sulphur-containing amino acid residues are 
present in the radical A, together with E may represent the radical 
##STR4## 
trp represents L-Trp, D-Trp or an analogous radical, which carries in the 
indole nucleus, for example in the 5-position, a halogen atom, especially 
fluorine, 
E represents Phe or Tyr or, together with D, has the meaning given above. 
G represents L-Ser, D-Ser or the residue of a secondary .alpha.-amino acid 
having a maximum of 8 carbon atoms, and 
a, b, c, f and g each represents, independently of one another, 0 or 1, and 
non-toxic salts and pharmacologically acceptable complexes thereof. 
The alkanecarboxylic acid forming the basis of the acyl radical Ac has 
preferably not more than 18 carbon atoms if it is unsubstituted and 
preferably not more than 8 carbon atoms if it is substituted. The 
substituents are, on the one hand, hydroxyl, mercapto, lower alkylthio, 
such as methylthio, guanidino, carboxyl, carboxamido and especially 
primary amino groups, or an imino group bonded at two different carbon 
atoms, and, on the other hand, mono- or bicyclic hydrocarbyl or 
heterocyclyl radicals, such as especially phenyl, p-hydroxyphenyl, 1- or 
2-naphthyl, 2-, 3- or 4-pyridyl, 2- or 3-indolyl, 2- or 4-imidazolyl, 2-, 
4- or 5-thiazolyl, 2-thienyl or 2-furyl. The acid may carry one or more 
substituents of the same kind or different kinds, the total number of 
carbon atoms, including the carbon-containing substituents, being 
preferably not more than 18. Especially preferred are acyl radicals that 
are derived from singly branched or especially straight-chained 
unsubstituted alkane-(mono or di)-carboxylic acids, the former having a 
maximum of 18, and the latter a maximum of 9, carbon atoms, such as those 
derived from acetic, propionic, butyric, isobutyric, valeric, isovaleric, 
caproic, oenanthic, undecanoic, lauric, myristic, palmitic and stearic 
acid on the one hand, and malonic, succinic, glutaric, adipic, pimelic and 
suberic acid on the other hand. 
Especially preferred are also acyl radicals that are derived from 
.alpha.-amino acids of the L-series which are naturally occurring, 
especially as peptide building blocks, and their closely related 
analogues, such as, especially, the enantiomers of the "unnatural" 
D-series. Of the preferred .alpha.-amino acids, for example the following 
are most especially suitable: glycine, alanine, valine, leucine, 
isoleucine, phenylalanine, aspartic acid, glutamic acid, arginine, lysine 
and histidine, also .beta.-alanine, .alpha.-aminobutyric acid, 
.gamma.-aminobutyric acid, norvaline, isovaline, norleucine and ornithine, 
and also asparagine, glutamine, tyrosine, tryptophan, methionine, 
threonine, serine, and, most especially, proline and hydroxyproline in 
which the .alpha.-amino group is cyclised with the alkyl radical to form a 
ring. 
The .epsilon.-amino-protecting group of the Lys.sup.4 residue denoted by 
the symbol X has the meanings given hereinbelow. It differs fundamentally 
from the above-characterised acyl group Ac in that it can be split off 
selectively with the amino group being liberated, whereas the acyl group 
Ac of the .epsilon.-amino group cannot be detached without at the same 
time impairing the peptidic amide bonds. 
The radical denoted Gaba(Ar) is more precisely defined by the formula 
##STR5## 
in which one of the symbols R.sub..alpha., R.sub..beta. and R.sub..gamma. 
is an unsubstituted or substituted cyclic hydrocarbyl radical Ar and the 
other two represent hydrogen. The substituted .gamma.-aminobutyric acid 
corresponding to the radical Gaba(Ar) has the short form H--Gaba(Ar)--OH. 
the cyclic hydrocarbyl radical Ar is a mono-, di- or polycyclic cycloalkyl 
radical or a corresponding aryl radical containing at least one aromatic 
ring and having a maximum of 18, preferably a maximum of 12, ring carbon 
atoms. Of the cycloalkyl radicals, those that are preferred have 3- to 
8-membered, and especially 5- and/or 6-membered, rings, such as, for 
example, cyclopropyl, cyclobutyl, cycloheptyl, cyclooctyl and more 
especially cyclopentyl and cyclohexyl, also 1-bicyclo[2,2,2]-octyl, 
2-bicyclo[2,2,2]octyl, 2-bicyclo[2,2,1]heptyl, 1- or 2-adamantyl, and 1- 
or 2-perhydronaphthyl, i.e. bicyclo[4,4,0]decyl. An aryl radical is 
especially a naphthyl radical, such as 1- or 2-naphthyl, a corresponding 
partially hydrogenated naphthyl radical, such as, especially, 1-, 2-, 5- 
or 6-(1,2,3,4-tetrahydronaphthyl), phenyl, anthryl, fluorenyl or azulenyl. 
All of these cyclic hydrocarbyl radicals may carry one or more lower 
aliphatic hydrocarbyl radicals, especially alkyl radicals having a maximum 
of 4 carbon atoms, for example methyl, ethyl, propyl, isopropyl or butyl, 
and/or further cyclic, especially monocyclic, hydrocarbyl radicals, such 
as those defined above, the total number of carbon atoms being a maximum 
of 18. Examples of such cyclic hydrocarbyl radicals are 
4,4-dimethylcyclohexyl, tolyl, such as 2-, 3- or 4-tolyl, and biphenylyl, 
for example 4-biphenylyl. 
The aromatic moiety of the cyclic hydrocarbyl radicals may be substituted 
by one, two or more identical or different substituents, such as halogen, 
for example chlorine, bromine, iodine and especially fluorine, phenoxy, 
lower alkoxy, for example one derived from one of the above-mentioned 
lower alkyl radicals having a maximum of 4 carbon atoms, including, 
especially, methoxy, also nitro and amino, especially primary amino, 
di-lower alkylamino and acylamino, such as lower alkanoylamino, for 
example acetamino. Especially preferred are phenyl radicals substituted by 
the mentioned substituents. 
The radical Ar is found in the .alpha.-, .gamma.- or preferably the 
.beta.-position of the chain of .gamma.-aminobutyric acid; accordingly, 
especially preferred radicals of the formula Gaba(Ar) are derived from the 
following butyric acids: 4-amino-3-phenyl-, 4-amino-3-cyclohexyl-, 
4-amino-3-(2-naphthyl)- and especially 4-amino-3-(1-naphthyl)- butyric 
acid and 4-amino-3-(3-phenoxyphenyl)-butyric acid. 
The secondary .alpha.-amino acid having a maximum of 8 carbon atoms 
mentioned in the symbol G is an .alpha.-lower alkylamino-lower 
alkylcarboxylic acid in which the two lower alkyl radicals may be 
connected to each other by a C-C bond, an oxygen atom, a sulphur(II) atom 
or an optionally lower alkylated nitrogen atom, each individual lower 
alkyl radical containing a maximum of 6 carbon atoms and both together 
containing a maximum of 7 carbon atoms. The lower alkyl radical forming 
the basis of the carbon skeleton of the carboxylic acid has preferably 
more than one carbon atom and is especially one that occurs in natural 
amino acids, such as butyl, isobutyl, pentyl and especially ethyl or 
isopentyl. The lower alkyl radical that occurs as a substituent of the 
amino group or the nitrogen bridge is preferably methyl. The C-C bond 
which optionally connects the two lower alkyl radicals is preferably a 
single bond. The .alpha.-amino group is preferably in a steric 
configuration that corresponds to the natural amino acids, i.e. the 
L-amino acids. Preferred radicals of such secondary .alpha.-amino acids 
are especially those that are known as naturally occurring amino acids, 
such as, especially, L-proline, or those that are directly analogous to 
these in structure, such as, on the one hand, 4-oxaproline and especially 
4 -thiaproline of the formula 
##STR6## 
in which Z is oxygen or sulphur, and, on the other hand, an N-lower 
alkylated, especially N-methylated, aliphatic amino acid, especially 
N-methyl-L-leucine. 
Of the acylpeptides according to the invention, there should be emphasised 
those which are derived from especially valuable somatostatin-like basic 
peptides known per se and are characterised by specific formulae, such as 
those given below: 
Thus somatostatin, D-Trp.sup.8 -somatostatin, [D-Trp.sup.8 -D-Cys.sup.14 
-]-somatostatin, [(5-F)-D-Trp.sup.8 ]-somatostatin and des-[Ala.sup.1 
-Gly.sup.2 ]-somatostatin form the basis of acylpeptides of the following 
formula IA: 
##STR7## 
in which A' represents Ala-Gly-, Ac-Ala-Gly-, H- or Ac- and Ac, B, trp and 
cys have the meanings given at the beginning; derived from des-[Ala.sup.1 
-Gly.sup.2 -]-desamino-Cys.sup.3 -somatostatin and its structural 
analogues are the acylpeptides of the formula IB: 
##STR8## 
in which Ac, Bmp, B, trp and G have the meanings mentioned at the 
beginning and C' represents Asn or His; derived from oligopeptides in 
which one or more of the amino acids in the positions 1, 2, 4, 5, 12 and 
13 of [D-Trp.sup.8 ]-somatostatin or [D-Trp.sup.8 -D-Cys.sup.14 
]-somastatin are omitted, are acylpeptides of the formula IC: 
##STR9## 
in which Ac, B and cys have the meanings mentioned at the beginning and a, 
b, c, f and g each represents, independently of one another, 0 or 1; 
derived from analogues having the (6-11)-cystine bridge are the bicyclic 
acylpeptides of the formula I in which A represents the residue of 
.omega.-aminoheptanoic acid, D and E together represent the radical 
##STR10## 
a represents 1 and b, c, f and g represent 0, and which correspond to the 
formula ID: 
##STR11## 
in which Ac and trp have the meanings mentioned at the beginning; and 
finally sulphur-free cyclopeptides having partial sequences of at least 6 
and not more than 11 of the amino acids of the somatostatin ring form the 
basis of the acylpeptides of the formula IE: 
##STR12## 
in which a, b, c, f and g each represents, independently of one another, 0 
or 1, Ac, trp and B have the meanings mentioned at the beginning. A" 
denotes the radical --NH--CH(R)--(CH.sub.2).sub.n --CO-- or Gaba(Ar) 
characterised at the beginning and C" represents Asn or Ala. 
Especially preferred acylpeptides of the formula IE are those in which Ac 
has the general and especially emphasised meanings defined at the 
beginning, trp represents D-Trp, A" denotes an .omega.-amino-lower 
alkanecarboxylic acid residue, in which R represents hydrogen and n 
represents 0 or an integer from 1 to 3, B represents Lys, Lys(Ac) or 
Lys(INOC) (wherein INOC denotes the isonicotinyloxycarbonyl present at the 
.epsilon.-amino group), f and at least one of the symbols a, b, c and g 
are equal to 1, whilst the others each represent, independently of one 
another, 0 or 1, such as especially those compounds in which a=0 and b, c, 
f, g=1 or a, b=0 and c, f, g=1 or a, b, c=0 and f, g=1 or alternatively a, 
b, g=0 and c, f=1. 
Most especially preferred acylpeptides of the formula IE are those in which 
Ac has the general and especially emphasised meanings defined at the 
beginning, trp represents D-Trp, A" represents the radical Gaba(Ar) 
defined at the beginning, especially one having the hydrocarbyl Ar in the 
.beta.-position, or an .omega.-amino-lower alkylcarboxylic acid residue in 
which n represents 0 or an integer from 1 to 6, especially from 1 to 3, 
and more especially 2, and R represents carboxyl and especially hydrogen, 
C" represents Ala and especially Asn, a equals 1, c equals 0 or especially 
1 and b, f and g equal 0. Of these, there should be especially emphasised 
compounds in which, in the radical A", n=5 and R represents hydrogen or 
carboxyl, a=1 and b, c, f and g all equal 0, and especially compounds of 
the formula 
##STR13## 
in which trp.sub.o represents D-Trp which may also carry fluorine in the 
5-position, Ac has the general and especially emphasised meanings defined 
at the beginning and U represents hydrogen or the radical Ar defined at 
the beginning, especially the phenyl, cyclohexyl, 2-naphthyl and 
especially 1-naphthyl or m-phenoxyphenyl radical. 
As a result of substitution with the radical Ar a centre of asymmetry is 
produced at the .beta.-carbon atom of the .gamma.-aminobutyric acid which 
results in the presence of in each case two diastereoisomeric forms of the 
cyclopeptide according to the invention which may, if desired, be used 
separately or, alternatively, together, as a mixture of diastereoisomers, 
for the same purposes. 
Most especially preferred are acylpeptides of the above formulae I to IF, 
in which Ac represents an acyl radical according to the examples of 
carrying out the process, or which have the basic structure of one of the 
acylpeptides shown in the Examples. The acylpeptides shown in the Examples 
are then most of all preferred. 
Those acylpeptides of the formula I containing a free carboxyl group which 
are characterised above either in general terms or as being preferred may 
alternatively be in the form of salts, for example sodium, potassium, 
calcium or magnesium salts, or alternatively in the form of ammonium salts 
derived from ammonia or a physiologically tolerable organic 
nitrogen-containing base. Those acylpeptides of the formula I containing a 
free amino group which are characterised above either in general terms or 
as being preferred may alternatively be in the form of their salts, that 
is their acid addition salts. Suitable acid addition salts are especially 
physiologically tolerable salts with conventional therapeutically 
acceptable acids; of the inorganic acids, mention should be made of 
hydrohalic acids, such as hydrochloric acid, and also of sulphuric acid 
and phosphoric or pyrophosphoric acid; of the organic acids, mention 
should be made especially of sulphonic acids, for example benzenesulphonic 
acid and p-toluenesulphonic acid, or of lower alkanesulphonic acids, such 
as methanesulphonic acid, also of carboxylic acids, such as acetic acid, 
lactic acid, palmitic and stearic acid, malic acid, tartaric acid, 
ascorbic acid and citric acid. 
The acylpeptides of the formula I according to the invention may 
alternatively be in the form of complexes. Complexes should be understood 
as being compounds the structures of which have not yet been fully 
clarified and that are formed when certain inorganic or organic substances 
are added to peptides and that impart to these a prolonged action. Such 
substances are described, for example, for ACTH and other 
adrenocorticotropically active peptides. Those that should be mentioned 
are, for example, inorganic compounds that are derived from metals, such 
as calcium, magnesium, aluminium, cobalt and especially zinc, especially 
sparingly soluble salts, such as phosphates, pyrophosphates and 
polyphosphates, as well as hydroxides of these metals, also alkali metal 
polyphosphates, for example Calgon.RTM.N, Calgon.RTM.322, Calgon.RTM.188 
or Polyron.RTM.B 12. Organic substances that prolong action are, for 
example, non-antigenic types of gelatin, for example polyoxygelatin, 
polyvinylpyrrolidone and carboxymethylcellulose, also sulphonic or 
phosphoric acid esters of alginic acid, dextran, polyphenols and 
polyalcohols, especially polyphloretin phosphate and phytic acid, andalso 
polymers and copolymers of basic or, especially, acidic, amino acids, for 
example protamine or polyglutamic acid. 
Unless otherwise indicated, the short forms of the amino acid residues 
refer to residues of the .alpha.-amino acids of the L-series that occur 
naturally. 
Unless otherwise indicated, the term "lower" wherever it occurs in 
connection with an organic radical or a compound, indicates such a radical 
or compound having a maximum of 7 carbon atoms and preferably a maximum of 
4 carbon atoms. 
The novel acylpeptides according to the invention have a physiological 
action that is fundamentally similar to the action of somatostatin. They 
can therefore be used advantageously in therapeutic indications similar to 
those of somatostatin, for example especially for the treatment of 
functional disorders in which the secretion of the somatotrophic hormone 
or glucagon is abnormally high, such as in the case of acromegaly or 
diabetes. Since they also inhibit blood losses in the gastrointestinal 
tract they can also be used successfully in this area of indication. They 
can also be used as valuable intermediates for the manufacture of other 
therapeutically valuable compounds, for example those having a further 
modified acyl radical Ac. 
The acylpeptides according to the invention are obtained by using 
conventional manufacturing processes of peptide chemistry which are known 
per se. 
Thus, they are manufactured, for example, by acylating a corresponding 
peptide having a free .epsilon.-amino group of the lysine residue in the 
9-position, optionally while temporarily protecting any free hydroxyl 
groups and/or other free amino groups. The acylation is effected 
especially by treating a peptide of the formula 
##STR14## 
in which A.sub.o represents a radical corresponding to the radical A 
defined at the beginning in which the .alpha.-amino group of an N-terminal 
amino acid residue may carry an .alpha.-amino-protecting group X' having 
the meaning defined in detail below, Y.sub.o represents a hydrogen atom 
present at the oxygen atom, or a hydroxyl-protecting group Y having the 
meaning defined in detail below, and in which the other symbols have the 
meanings given at the beginning, with an alkanecarboxylic acid Ac.sub.o 
OH, in which Ac.sub.o represents a radical corresponding to the acyl 
radical Ac defined at the beginning in which any amino and hydroxyl groups 
present may carry protecting groups X and X', and Y respectively, or with 
a reactive derivative of such an acid and, if desired or necessary, 
liberating the amino groups and hydroxyl groups in the resulting product 
by splitting off the protecting groups X and X', and Y respectively. 
The above-mentioned residue of an N-terminal amino acid does not occur in 
all meanings of A or A.sub.o but only in those that are indicated by the 
symbol A', and is represented by the radical 
##STR15## 
The meaning of the symbol X' corresponds fairly extensively to that of the 
.alpha.-amino-protecting groups that are used in the synthesis of the 
peptide chain and described in detail hereinbelow. Preferably, similar or, 
more preferably, identical, protecting groups are used both in the radical 
A.sub.o and in the radical Ac.sub.o and are split off simultaneously 
following the acylation reaction. 
A reactive derivative of an acid Ac.sub.o OH is, for example, an anhydride, 
especially a symmetric anhydride of the formula Ac.sub.o --O--Ac.sub.o or 
a cyclic anhydride of a dicarboxylic acid, such as succinyl anhydride or 
glutaryl anhydride, or alternatively a mixed anhydride with a different 
organic acid, for example with trifluoroacetic acid, or especially with an 
inorganic acid, for example an acid azide or acid halide, especially an 
acid chloride. A reactive acid derivative is preferably an activated 
ester, for example one in which the acid Ac.sub.o OH is esterified with 
2,4,5-trichlorophenol, pentachlorophenol, pentafluorophenol, 2-nitrophenol 
or especially 4-nitrophenol, or with an N-hydroxy compound, such as 
N-hydroxysuccinimide, 1-hydroxybenzotriazole or N-hydroxypiperidine, or 
alternatively with an N,N'-disubstituted isourea, such as especially 
N,N'-dicyclohexylisourea, or a similar activating component known from 
peptide chemistry, cf. Houben-Weyl: Methoden der organischen Chemie; 4th 
edition, vol. 15/I and II, E. Wunsch (editor): Synthese von Peptiden 
(Georg Thieme Verlag, Stuttgart; 1974). 
The acylation is effected in a manner known per se, preferably in customary 
solvents, for example dioxan, tetrahydrofuran, acetonitrile, pyridine, 
dimethylformamide, dimethylacetamide, dimethyl sulphoxide, 
N-methylpyrrolidone, hexamethylphosphoric acid triamide, chloroform and 
methylene chloride, and in advantageous mixtures thereof. Alternatively, 
an organic base, for example a quaternary or especially a tertiary amine, 
such as triethylamine, N-ethylmorpholine or N-methylpiperidine, can be 
added to obtain the amino group which is to be acylated in deprotonated 
form. The reaction temperature is usually from -20.degree. to +70.degree. 
C., preferably from approximately 0.degree. C. to room temperature. 
Active esters are generally advantageous as acylating agents because they 
preferentially acylate amino groups before hydroxyl groups and thus render 
protection of hydroxyl groups virtually superfluous. In the case of 
dicarboxylic acids, however, cyclic anhydrides are preferred, if they are 
available. In order to avoid undesired O-acylation, only one equivalent of 
the acylating agent is usually used for each free amino group of the 
starting material of the formula II. 
If, however, it is more advantageous for some reason to dispense with 
selective acylation, as may be the case especially in the reaction with 
acid chlorides, the acylating agent is used in excess and the co-acylated 
hydroxyl groups are liberated subsequently in the same conventional manner 
as the protected hydroxyl groups, especially by basic hydrolysis, for 
example with sodium or potassium hydroxide in the presence of water. 
The subsequent splitting off of any protecting groups present depends on 
their type and is carried out in each case in a conventional manner known 
per se, as described in detail hereinbelow. The splitting off of any 
hydroxyl-protecting groups Y and .alpha.-amino-protecting groups X' 
present in the radicals A.sub.o and Ac.sub.o is an obligatory measure; on 
the other hand, an .epsilon.-amino-protecting group X in the lysine 
residue in the 4-position is split off only if desired. 
The acylpeptides according to the invention can also be manufactured by 
cyclising a linear peptide corresponding to the acylpeptide, optionally 
while temporarily protecting any free hydroxyl, carboxyl and/or amino 
groups. A corresponding linear peptide is one which has the same amino 
acids in a sequence identical to that of the cyclic peptides according to 
the invention, a bond between any two adjacent ring-forming amino acids 
being, however, interrupted and replaced by corresponding terminal 
functional groups which may be present also in an activated form. If the 
ring is interrupted at the amide bond between any two successive amino 
acids, the terminal groups of the linear peptide are a carboxyl group and 
an amino group; if, however, the ring is interrupted between two cysteine 
residues, such as especially between those in the 3- and 14-positions, 
then a disulphide bond is broken, and the corresponding linear peptide has 
as terminal groups two free or functionally modified mercapto groups. 
Depending on the specific type of terminal groups, appropriate cyclisation 
processes are used. 
The compounds according to the invention can thus be manufactured by 
cyclisation by cyclising a corresponding linear peptide of the formula 
EQU H--[I.sub.a ]--V (III), 
in which 
I.sub.a represents a radical corresponding to the formula I in which the 
amide bond between any two adjacent amino acid residues of the peptide 
ring is interrupted, and 
V represents a free hydroxyl group, a hydroxyl group modified by an 
activating group or represents the hydrazino group --NH--NH.sub.2, 
any amino, carboxyl and hydroxyl groups present that do not participate in 
the cyclisation reaction being, as required, in protected form and 
liberated subsequently. 
Of the linear peptides of the formula III, those in which the radical A is 
present as a terminal amino acid in the radical [Ia] are preferred. These 
preferred starting materials are characterised by the formulae 
EQU H--(B).sub.b --(C).sub.c --D--Phe--trp--Lys(Ac)--Thr--E--(Thr).sub.f 
--(G).sub.g --(A).sub.a --V (IIIa) 
and especially 
EQU H--(A).sub.a --(B).sub.b --(C).sub.c 
--D--Phe--trp--Lys(Ac)--Thr--E--(Thr).sub.f --(G).sub.g --V (IIIb) 
in which Ac, A, B, C, D, trp, E and G, and also a, b, c, f and g have the 
meanings given at the beginning and V has the meanings given immediately 
above. Most especially preferred are compounds of the formulae IIIa and 
IIIb in which there are no sulphur-containing amino acids in the radical 
A. 
A functional group represented by the symbol V supplements the carbonyl 
group of the C-terminal amino acid residue and forms together with that 
group a free carboxyl group, an activated ester group or the carbazolyl 
group, as the case may be. 
The activating group by which the hydroxyl group is modified is especially 
one that forms the activated ester of N-hydroxysuccinimide, 
1-hydroxybenzotriazole, N,N'-dicyclohexylisourea, 2,4,5-trichlorophenol, 
2-nitrophenol, 4-nitrophenol, pentachlorophenol or pentafluorophenol but 
may also be a different activating group of this type known from peptide 
chemistry, cf. Houben-Weyl, volume 15/II. 
The cyclisation according to the invention of the linear peptides of the 
formula III is carried out in a manner known per se by means of 
conventional coupling methods customarily used for the formation of the 
amide bond, the peptide starting materials, however, being used in a very 
low concentration in order to influence the course of the coupling 
operation in favour of intramolecular cyclisation at the expense of 
intermolecular polycondensation. 
The linear peptides are advantageously used in an approximately 1.10.sup.-4 
molar to approximately 1.10.sup.-2 molar concentration, preferably an 
approximately 1.10.sup.-3 molar concentration, which corresponds to a 
weight/volume concentration of approximately 0.01 to 1.0%, preferably 
0.1%. The reaction mixture can be correspondingly diluted from the start 
or this dilution can be produced continuously by the slow dropwise 
addition of the starting material, and optionally the other reagents, to 
the reaction mixture. 
Cyclisation is preferably carried out, at a starting concentration 
indicated above, by (a) treating a starting material of the formula III, 
in which V represents a free hydroxyl group, while temporarily protecting 
other amino, carboxyl and hydroxyl groups, with a carbodiimide, optionally 
in the presence of an active ester-forming component, or (b) reacting with 
an organic base a starting material of the formula III, in which V 
represents a hydroxyl group modified to form the activated ester and the 
terminal amino group is present in protonated form, at least the amino 
groups and carboxyl groups not participating in the cyclisation reaction 
being protected, or (c) first treating a starting material of the formula 
III, in which V represents the group --NHNH.sub.2 and at least the amino 
groups not participating in the cyclisation reaction are protected, with 
nitrous acid or a lower alkyl ester thereof under acidic conditions and 
then cyclising with excess organic base at an above-mentioned low 
concentration. 
A carboxyl group is protected by a protecting group W in the manner 
described hereinbelow. For the protection of the amino and hydroxyl 
groups, the groups X and X', and Y respectively, are advantageously used. 
The cyclisation is carried out in suitable solvents, for example dioxan, 
tetrahydrofuran, acetonitrile, pyridine, dimethylformamide, 
dimethylacetamide, dimethyl sulphoxide, N-methylpyrrolidone, 
hexamethylphosphoric acid triamide, chloroform, methylene chloride or 
ethyl acetate, and mixtures thereof. 
In process variant (a) the cyclisation is brought about by a carbodiimide, 
preferably N,N'-dicyclohexyl carbodiimide, which is advantageously used in 
excess; it is to be assumed that the starting material of the formula III 
having a free carboxyl group is first converted into an activated ester of 
dicyclohexylisourea (or an analogous isourea) and this active ester formed 
in situ immediately reacts further. The intermediate formation of an 
active ester can doubtless be attributed to the addition of an active 
ester-forming component as an auxiliary reagent; for this purpose, active 
ester-forming components customary in peptide chemistry may be used, such 
as, especially, 2,4,5-trichlorophenol, 2- or 4-nitrophenol, 
pentachlorophenol and pentafluorophenol, but, more especially, N-hydroxy 
compounds, among which N-hydroxysuccinimide, N-hydroxypiperidine and above 
all 1-hydroxybenzotriazole are especially advantageous. In the case of 
this variant, the operating temperature is generally 0.degree.-70.degree., 
preferably 35.degree.-55.degree.. 
In the case of variant (b) which is carried out with ready-prepared active 
esters, especially those already pointed out, cyclisation takes place 
spontaneously as soon as the terminal amino group is deprotonated by the 
organic base. The bases used are preferably quaternary or especially 
tertiary amines, for example triethylamine or N-ethylmorpholine. The 
operation is preferably carried out at 10.degree.-30.degree., especially 
at room temperature. 
In the case of variant (c), the first phase, i.e. the formation of the acid 
azide by treating with nitrous acid or an ester thereof, may 
advantageously be carried out at a considerably higher concentration of 
the starting materials than in the case of the subsequent cyclisation. The 
operation is advantageously carried out with approximately one equivalent 
of a lower alkyl nitrite, such as ethyl nitrite, isoamyl nitrite and 
especially tert.-butyl nitrite, in a hydrochloric acid medium at 
temperatures of from approximately -30.degree. to approximately 
-5.degree., preferably approximately -20.degree.; a slight excess of 
nitrite is permissible. The solution of the azide formed is then, after 
the necessary dilution, rendered basic at a temperature of from 
approximately 0.degree. to approximately 35.degree. by means of excess 
organic base, for example one of those mentioned above, and thereby made 
to cyclise spontaneously as in the case of process variant (b). 
In special cases, the compounds according to the invention, if they contain 
a pair of sulphur-containing amino acid residues, such as those of D- or 
L-cysteine or .beta.-mercaptopropionic acid, can be manufactured by 
oxidising a corresponding linear peptide of the formula 
EQU T.sub.o --[I.sub.s ]--T.sub.o (IV), 
in which I.sub.s represents a radical corresponding to the formula I in 
which the disulphide bond between the sulphur-containing amino acid 
residues is interrupted, and T.sub.o represents hydrogen or a 
mercapto-protecting group T, to form the disulphide bridge, optionally 
having split off the mercapto-protecting groups T beforehand or splitting 
them off at the same time, and by liberating amino, carboxyl and/or 
hydroxyl groups present optionally in protected form. 
The linear peptide IV is especially one of the formula 
EQU T.sub.o --A.sub.1 --(B).sub.b --(C).sub.c 
--Phe--Phe--trp--Lys(Ac)--Thr--E--(Thr).sub.f --(G).sub.g --A.sub.2 
--T.sub.o (IVa), 
in which 
A.sub.1 represents 
##STR16## 
T.sub.o has the meaning given immediately above and the other symbols have 
the meanings given at the beginning. 
The linear peptide IV is, however, alternatively one of the formula 
##STR17## 
in which A" denotes a sulphur-free amino acid residue mentioned under the 
meanings of A, T.sub.o has the meaning given immediately above and the 
other symbols have the meanings given at the beginning. 
Cyclisation by oxidation is carried out in the conventional manner 
generally known per se. It is possible to oxidise a linear peptide of the 
formula IV in which all the protecting groups have previously been split 
off. If, however, the linear peptide of the formula IV is in a form having 
protected amino, hydroxyl and/or carboxyl groups, as is obtained in most 
instances in the case of prior synthesis, it is advantageous to carry out 
the cyclisation first and only then to split off the protecting groups 
(i.e. the groups X, X', Y and W). In that case, preferably any carboxyl 
groups present are protected as tert.-butyl esters, .epsilon.-amino groups 
are protected by the tert.-butoxycarbonyl group, the hydroxyl groups of 
the serine and threonine residues are protected, if at all, as tert.-butyl 
ethers, and the mercapto groups are protected by trityl, acetaminomethyl, 
p-methoxybenzyl, PCH or MPCH, or by tetrahydropyranyl groups (Thp). Apart 
from acetaminomethyl, all these functional groups can be split off in one 
step by the action of acids (acidolysis). Mercapto-protecting groups of 
the trityl, acetaminomethyl or tetrahydropyranyl type may, however, if 
desired, be split off selectively with heavy metal salts, for example 
mercuric acetate, and hydrogen sulphide, while protecting groups of the 
tert.-butyl type are retained. In this manner, there is obtained a linear 
peptide having free mercapto groups which can be cyclised by oxidation in 
a manner known per se, for example with iodine, with diiodoethane in 
organic solvents, or with oxygen, especially atmospheric oxygen, such as 
with atmospheric oxygen in liquid ammonia. It is even more advantageous to 
remove the trityl, tetrahydropyranyl or acylaminomethyl groups protecting 
the mercapto groups while simultaneously forming the disulphide bridge 
with iodine, for example in methanol, acetic acid or especially 
dimethylformamide, the other protecting groups of the type mentioned being 
retained and split off subsequently. 
The narrower choice of protecting groups is determined by the specific 
purpose, it being necessary especially in the case where several 
functional groups are to be protected to select advantageous combinations. 
As .epsilon.-amino-protecting groups X it is possible to use any of the 
amino-protecting groups that are customary in peptide chemistry, as are 
described synoptically in the appropriate reference works, for example in 
Houben-Weyl: Methoden der oganischen Chemie; 4th edition, vol. 15/I, E. 
Wunsch (editor): Synthese von Peptiden; (Georg Thieme Verlag, Stuttgart; 
1974). 
Thus, for example, amino-protecting groups that can be split off by 
reduction or by bases can be used, for example especially the 
benzyloxycarbonyl group and benzyloxycarbonyl groups that are substituted 
in the aromatic moiety by halogen atoms, nitro groups, lower alkoxy groups 
and/or lower alkyl radicals, such as p-chloro- and 
p-bromobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 
p-methoxybenzyloxycarbonyl or p-tolyloxycarbonyl groups, or alternatively 
by the isonicotinyloxycarbonyl group, and also by acyl groups, such as 
p-toluenesulphonyl, benzenesulphenyl, o-nitrobenzenesulphenyl groups or 
formyl, trifluoroacetyl or phthaloyl. 
An advantageous .epsilon.-amino-protecting group X is an ethoxycarbonyl 
group which carries, in the .beta.-position, a silyl group substituted by 
three hydrocarbon radicals, such as triphenylsilyl, dimethylbutyl-silyl or 
especially a trimethylsilyl group. A 
.beta.-(trihydrocarbylsilyl)-ethoxycarbonyl group of this type, such as a 
.beta.-(tri-lower alkylsilyl)-ethoxycarbonyl group, for example especially 
the .beta.-(trimethylsilyl)-ethoxycarbonyl group, forms, together with the 
.epsilon.-amino group that is to be protected, a corresponding 
.beta.-trihydrocarbylsilylethoxycarbonylamino group (for example, the 
.beta.-trimethylsilylethoxycarbonylamino group), which is stable under the 
conditions of acid hydrolysis and hydrogenolysis, but can be split off by 
the action of fluoride ions under quite specific, very mild conditions. In 
this respect, it behaves analogously to the .beta.-silylethyl ester group 
described hereinbelow as a carboxyl-protecting group. (This similarity 
must be given particular consideration during the synthesis: but for 
isolated cases, the use of one of these protecting groups excludes the 
simultaneous use of the other protecting group.) Further details are given 
below in connection with the protection of the carboxyl group as 
.beta.-silylethyl ester. 
Groups that can be split off by acidolysis are most especially preferred, 
such as especially the tert.-butoxycarbonyl group and analogous groups, 
for example the tert.-amyloxycarbonyl, isopropoxycarbonyl, 
diisopropylmethoxycarbonyl, allyloxycarbonyl, cyclopentyloxycarbonyl, 
cyclohexyloxycarbonyl, d-isobornyloxycarbonyl and adamantyloxycarbonyl 
groups, and groups of the aralkyl type, such as benzhydryl and 
triphenylmethyl (trityl), or certain aralkoxycarbonyl groups of the 
2-(p-biphenylyl)-2-propoxycarbonyl type, which are described in Swiss 
Patent Specification No. 509 266. 
As the hydroxyl-protecting group Y there can be used any of the groups 
customarily used for this purpose in peptide chemistry, cf. the work cited 
above (Houben-Weyl). Groups that can be split off by acidolysis, such as 
2-tetrahydropyranyl and more especially tert.-butyl, or 
tert.-butoxycarbonyl, are preferred. Alternatively, however, 
hydroxyl-protecting groups that can be split off by reduction or by means 
of bases can be used, for example benzyl and benzyloxycarbonyl groups that 
may be substituted in the aromatic moiety by halogen, nitro and/or lower 
alkoxy, or lower alkanoyl radicals, such as acetyl, or aroyl radicals, 
such as benzoyl. It is also possible to proceed without protecting 
hydroxyl groups if certain limiting measures are observed. 
As carboxyl-protecting groups W there can be used any group customarily 
used for this purpose, cf. the work cited above (Houben-Weyl). Thus, 
carboxyl groups are protected, for example, by the formation of hydrazides 
or by esterification. Suitable for esterification are, for example, lower 
optionally substituted alkanols, such as methanol, ethanol, cyanomethyl 
alcohol, 2,2,2-trichloroethanol, benzoylmethyl alcohol or especially 
tert.-butyl alcohol, or alternatively an optionally substituted benzyl 
alcohol. An especially advantageous category of substituted alkanols is 
ethyl alcohols that contain in the .beta.-position a tri-substituted silyl 
group, such as a triphenylsilyl, a dimethyl-butylsilyl or especially a 
trimethylsilyl group. As described, for example, in Belgian Patent 
Specification No. 851,576, these alcohols are especially suitable for 
protecting carboxyl groups because, although the corresponding 
.beta.-silylethyl esters, for example .beta.-(trimethylsilyl)-ethyl ester, 
have the stability of conventional alkyl esters, they can be split off 
selectively under mild conditions by the action of fluoride ions while all 
the other protecting groups are retained. 
As a mercapto-protecting group T there can be used any of the groups 
customarily used for this purpose in peptide chemistry, the mercapto 
groups being protected especially by suitable acylation or alkylation. 
Suitable for acylation is, for example, the acetyl or benzoyl radical, a 
lower alkylcarbamoyl group, (for example ethylcarbamoyl), or a 
benzyloxycarbonyl group optionally substituted, for example, as stated 
above. Suitable for alkylation are, for example, tert.-butyl, 
isobutoxymethyl, benzylthiomethyl or tetrahydropyranyl radicals or 
arylmethyl radicals optionally substituted by halogen, lower alkoxy or 
nitro, such as benzyl, p-methoxybenzyl, diphenylmethyl, 
dimethoxybenzhydryl or more especially trityl, and also phenylcyclohexyl 
(PCH), p-methoxyphenylcyclohexyl (MPCH), thien-2-ylcyclohexyl, et.al., cf. 
Ber. 101, 681 (1968). An acylaminomethyl radical of the general formula 
R.sub.t --CO--NH--CH.sub.2 --, in which R.sub.t --CO-- denotes the radical 
of a carboxylic acid is also very advantageous, cf. Tetrahedron Letters 
1968 (26), 3057 and German Offenlegungsschrift No. 2 060 969. The acyl 
radical R.sub.t --CO-- can be derived from an aliphatic, cycloaliphatic, 
aromatic, araliphatic or heterocyclic carboxylic acid or a carbonic acid 
mono-derivative (such as a carbonic acid monoester or a carbamic acid). 
The symbol R.sub.t represents especially an optionally substituted lower 
alkyl radical, for example a methyl, ethyl, propyl, isopropyl, n-butyl or 
tert.-butyl radical, which may contain as substituents, for example 
chlorine, trifluoromethyl or the nitro group. R.sub.t also represents, for 
example, an optionally substituted cycloalkyl radical having 3-8, 
preferably 5 or 6, ring atoms, such as the cyclopentyl or cyclohexyl 
radical, or an optionally substituted aromatic or araliphatic, preferably 
monocyclic, radical, especially an optionally substituted phenyl or benzyl 
radical, for example unsubstituted phenyl or benzyl, or phenyl or benzyl 
substituted in the phenyl moiety by lower alkyl, lower alkoxy, halogen or 
nitro, or a monocyclic heterocyclyl radical, for example thienyl or furyl. 
Of the acylaminomethyl groups, the acetylaminomethyl group is especially 
preferred. 
The protecting groups Y and W, and also the .alpha.-amino-protecting group 
X' characterised in more detail below, are preferably so chosen that they 
can be split off under similar conditions; especially preferred in this 
connection are the groups already pointed out above that can be split off 
by acidolysis. All these protecting groups can thus be split off 
advantageously in a single operation; it is also possible, however, to use 
different kinds of protecting group and to split off each one 
individually. 
If, however, a protecting group X, i.e. an .epsilon.-amino-protecting group 
present in the Lys.sup.4 residue, is to be retained in the end product of 
the formula I, the radicals X', Y and W should be so chosen that they can 
be split off while group X is retained. 
The protecting groups are split off in the generally known manner; acid 
hydrolysis (acidolysis) is carried out, for example, by means of 
trifluoroacetic acid, hydrochloric acid or hydrofluoric acid, or, in the 
case of protecting groups that are sensitive to acids, by means of a lower 
aliphatic carboxylic acid, such as formic acid and/or acetic acid, in the 
presence of water and optionally a poly-halogenated lower alkanol or lower 
alkanone, such as 1,1,1,3,3,3-hexafluoropropan-2-ol or hexafluoroacetone. 
The groups that can be split off by reduction, especially those which 
contain benzyl radicals, are preferably removed by hydrogenolysis, for 
example by hydrogenation under palladium catalysis. The 
isonicotinyloxycarbonyl group is split off preferably by zinc reduction. 
Those end products according to the invention which contain basic groups 
are obtained as bases or as acid addition salts depending on the method of 
isolation; these can subsequently be inter-converted in a manner known per 
se. Similarly, end products having acidic groups may also be in the form 
of salts, it being possible to convert each form into the other in known 
manner. 
The formation of the above-mentioned complexes also is carried out 
according to known methods; complexes with sparingly soluble metal 
compounds, for example aluminium or zinc compounds, are produced 
preferably in a manner analogous to that known for ACTH, for example by 
reaction with a soluble salt of the metal concerned, for example zinc 
chloride or zinc sulphate, and precipitation with an alkali metal 
phosphate and/or hydroxide. Complexes with organic compounds of the type 
polyoxygelatine, carboxymethylcellulose, polyvinylpyrrolidone, 
polyphloretin phosphate, polyglutamic acid, etc. are obtained by mixing 
these substances with the peptide in aqueous solution. In the same manner, 
insoluble compounds may also be manufactured with alkali metal 
polyphosphates. 
The starting materials of the above-characterised formulae III and IV and, 
unless stated otherwise, the intermediates used for the synthesis thereof, 
are new and, in some cases, can be used advantageously also for the 
synthesis of other somatostatin analogues, for example those having 
analogous amino acid partial sequences. The present invention relates to 
these starting materials and the processes for the manufacture thereof. 
They are obtained by methods known per se, by condensing with one another, 
in any time sequence, the amino acids and smaller peptide units necessary 
for their synthesis with the formation of CO--NH bonds, it being possible 
to protect temporarily any functional groups not participating in the 
reaction. 
In the manufacture of these starting materials, and also of all the 
necessary intermediates, suitable protecting groups for the terminal 
.alpha.-amino and carboxyl groups are especially the protecting groups 
that are customarily used in the synthesis of long-chain peptides and that 
can be split off readily and selectively, for example by solvolysis or 
reduction. They have already been mentioned above several times under the 
symbols X' and W, respectively. 
Examples of .alpha.-amino-protecting groups X' are: di- or triaryl-lower 
alkyl groups optionally substituted, for example, by halogen, nitro, lower 
alkyl or lower alkoxy, such as diphenylmethyl or triphenylmethyl groups, 
for example benzhydryl, trityl, di-(p-methoxy)-benzhydryl, or especially 
groups derived from carbonic acid that can be split off by hydrogenolysis, 
such as benzyloxycarbonyl groups optionally substituted in the aromatic 
moiety by halogen atoms, nitro groups, lower alkyl or lower alkoxy groups, 
for example benzyloxycarbonyl, p-bromo- or p-chlorobenzyloxycarbonyl, 
p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl; also 
2-(p-biphenylyl)-2-propoxycarbonyl and similar aryloxycarbonyl groups 
described in Swiss Patent Specification No. 509 266. It must be ensured 
that the .alpha.-amino-protecting group X' can be split off selectively 
while the optionally present .epsilon.-amino-protecting group X of the 
lysine residue in the 4-position is retained. Furthermore, it is often 
advantageous if, during the splitting off of the .alpha.-amino-protecting 
group, an optionally present carboxyl- or hydroxyl-protecting group W or Y 
also remains undamaged. 
The carboxyl-protecting groups for this purpose are the same as those 
discussed above in connection with the corresponding meaning of the symbol 
W. 
These protecting groups can be split off in known manner. Thus, the 
benzyloxycarbonyl group can be split off by hydrogenolysis, the N-trityl 
group by mineral acids, such as hydrohalic acids, for example hydrofluoric 
acid or preferably hydrochloric acid, or an organic acid, such as formic 
acid, acetic acid, chloroacetic acid or trifluoroacetic acid, in aqueous 
or absolute trifluoroethanol as the solvent (cf. German 
Offenlegungsschrift DT No. 2 346 147) or with aqueous acetic acid; the 
tert.-butoxycarbonyl group can be split off with trifluoroacetic acid or 
hydrochloric acid, the 2-(p-biphenylyl)-isopropoxycarbonyl group with 
aqueous acetic acid or, for example, a mixture of glacial acetic acid, 
formic acid (82.8% strength) and water (7:1:2) or in accordance with the 
process described in DT-OS No. 2 346 147. 
The .beta.-silylethyl ester groups are split off preferably with reagents 
that yield fluoride ions, for example fluorides of quaternary organic 
bases, such as tetraethylammonium fluoride. Alternatively, however, they 
may be split off, in the same manner as conventional alkyl esters, by 
alkaline hydrolysis, for example by means of alkali metal hydroxides, 
carbonates or bicarbonates, or may be converted into the corresponding 
carbazoyl groups by hydrazinolysis, for example by means of hydrazine 
hydrate. Acidolysis is preferably used for splitting off tert.-butyl 
esters and hydrogenolysis for benzyl esters. 
The condensation of the amino acids and/or peptide units which must be 
effected for the manufacture of the starting materials of the formula III 
or IV is carried out in a manner known per se, preferably by linking an 
amino acid or a peptide having a protected .alpha.-amino group and an 
optionally activated terminal carboxyl group (=active component) to an 
amino acid or a peptide having a free .alpha.-amino group and a free or 
protected, for example esterified, terminal carboxyl group (=passive 
component), liberating the terminal amino group in the product so formed 
and reacting this peptide, which contains a free .alpha.-amino group and 
an optionally protected terminal carboxyl group, with a further active 
component, i.e. an amino acid or a peptide having an activated carboxyl 
group and a protected .alpha.-amino group, and so on. The carboxyl group 
can be activated, for example, by converting it into an acid azide, 
anhydride, imidazolide, isoxazolide or an activated ester, such as one of 
those mentioned hereinbelow, or by reacting it with a carbodiimide, such 
as N,N'-dicyclohexyl carbodiimide, optionally with the addition of 
N-hydroxysuccinimide or an unsubstituted or, for example, a halogen-, 
methyl- or methoxy-substituted 1-hydroxybenzotriazole or 
4-hydroxybenzo-1,2,3-triazine-3-oxide (inter alia cf. DT No. 1 917 690, DT 
No. 1 937 656, DT No. 2 202 613), or especially 
N-hydroxy-5-norbornene-2,3-dicarboximide, or with 
N,N'-carbonyldiimidazole. The most usual coupling method is the 
carbodiimide method, also the azide method, the activated esters method 
and the anhydride method, the Merrifield method and the method using 
N-carboxyanhydrides or N-thiocarboxyanhydrides. 
Suitable for the formation of activated esters, such as those mentioned 
above, are, for example, phenols and thiophenols optionally substituted by 
electron-attracting substituents, such as phenol, thiophenol, thiocresol, 
p-nitrothiophenol, 2,4,5- and 2,4,6-trichlorophenol, pentachlorophenol, o- 
and p-nitrophenol, 2,4-dinitrophenol, and p-cyanophenol, and also, for 
example, N-hydroxysuccinimide, N-hydroxyphthalimide and 
N-hydroxypiperidine. 
In an especially preferred method of manufacture of the peptides of the 
formulae III and IV, the coupling method used is the carbodiimide method 
with N,N'-dicyclohexyl carbodiimide in the presence of 
1-hydroxybenzotriazole. The terminal carboxyl group is protected in the 
form of the .beta.-(trimethylsilyl)-ethyl ester, and the .alpha.-amino 
group of the active component is protected by the benzyloxycarbonyl group 
which is split off by hydrogenolysis after each coupling step. In order to 
protect the .epsilon.-amino group of the lysine residue in the 4-position, 
acylation with the tert.-butoxycarbonyl group is used and, for the 
hydroxyl group of the serine and threonine residues, etherification with 
the tert.-butyl group. These two protecting groups may, if desired, be 
split off finally in one step by acid hydrolysis, for example by means of 
trifluoroacetic acid, hydrochloric acid or hydrofluoric acid. The 
.epsilon.-amino group of the lysine residue in the 9-position is present 
in acylated form and therefore requires no protection. 
The sulphur-containing amino acid residues (Bmp and cys) are introduced 
preferably only in the last stages of the synthesis since, as is known, 
the presence of sulphur can impair the activity of the hydrogenation 
catalysts and hence plate in doubt the use of the otherwise very 
advantageous groups that can be split off by hydrogenolysis. The mercapto 
groups in the said acids are advantageously protected by the trityl groups 
which are especially suitable for carrying out preferred variants of the 
process. 
Depending on the procedure used, the compounds of the formulae III and IV 
are obtained, depending on their character, in the form of bases or acid 
addition salts or, alternatively, in the form of acids or their salts. The 
bases can be obtained from the acid addition salts in a manner known per 
se. Therapeutically acceptable acid addition salts can, in their turn, be 
obtained from the bases by reacting with acids, for example with those 
that form the above-mentioned salts. Acids and their salts stand in a 
similar relationship to each other. Compounds that have both a free 
carboxyl group and a basic group may be in the form of internal salts. 
Owing to the close relationship between the new compounds in free form and 
in the form of their salts, hereinbefore and hereinafter the term "free 
compounds" shall, if desired, also include the salts thereof and the term 
"salts" shall, if desired, also include the free compounds, here 
appropriate according to meaning and purpose. 
The invention relates also to those embodiments of the process in which a 
compound obtained as an intermediate at any stage of the process is used 
as starting material and the remaining process steps are carried out, or 
in which a starting material is formed under the reaction conditions or is 
used in the form of a derivative, for example a salt. 
In the process of the present invention, the starting materials used are 
preferably those that result in the compounds described at the beginning 
as especially valuable. 
The present invention relates also to pharmaceutical preparations that 
contain compounds of the formula I or pharmaceutically acceptable salts or 
complexes thereof. These preparations may be used especially in the 
abovementioned indications if they are administered intraperitoneally, 
such as intravenously, intramuscularly or subcutaneously, or also 
intranasally. The necessary dose depends on the particular disorder to be 
treated, its severity and the duration of therapy. The number and quantity 
of the individual doses and also the administration scheme can best be 
determined on the basis of an individual examination of the patient 
concerned. The method of determining these factors is known to the man 
skilled in the art. As a rule, however, in the case of injection, a 
therapeutically active quantity of a compound of this type lies in the 
dosage range of approximately 0.001 to approximately 0.2 mg/kg body 
weight. Preferably, the range is approximately 0.0015 to approximately 
0.15 mg/kg body weight and administration is by intravenous infusion or 
subcutaneous injection. Accordingly, pharmaceutical preparations for 
parenteral administration in unit dose form contain per dose, depending on 
the type of medication, approximately 0.08 to approximately 15 mg of one 
of the compounds according to the invention. Apart from the active 
substance, they usually also contain a buffer, for example a phosphate 
buffer, that is to maintain the pH between approximately 3.5 and 7, and 
also sodium chloride, mannitol or sorbitol for adjusting the isotonicity. 
They may be in freeze-dried or dissolved form and solutions may 
advantageously contain an antibacterially active preservative, for example 
0.2-0.3% of 4-hydroxybenzoic acid methyl ester or ethyl ester. If the 
active substance in such preparations is to be in the form of a complex 
having a prolonged duration of action then it may be formed directly by 
adding the complex-forming components to an injection solution that is 
prepared, for example, according to the above-mentioned methods. A 
suitable additive is, for example, 0.1-1.0% by weight of a zinc(II) salt 
(for example sulphate) in conjunction with 0.5-5.0% by weight of protamine 
(for example as a sulphate), calculated on the total volume of the 
injection solution; this preparation is in the form of a solution having a 
pH of 3.5 to approximately 6.5 or in the form of a suspension having a pH 
of approximately 7.5 to 8.0. 
A preparation for intranasal administration may be an aqueous solution or 
gel, an oily solution or suspension, or alternatively a fat-containing 
salve. A preparation in the form of an aqueous solution is obtained, for 
example, by dissolving the active substance of the formula I, or a 
therapeutically acceptable acid addition salt thereof, in an aqueous 
buffer solution having a pH of up to 7.2 and adding a substance producing 
isotonicity. A polymeric adhesive, for example polyvinylpyrrolidone, 
and/or a preservative are advantageously added to the aqueous solution. 
The individual dose is approximately 0.08 to approximately 15 mg, 
preferably 0.25 to 10 mg, which are contained in approximately 0.05 ml of 
a solution or 0.05 g of a gel. 
An oily form of medication for intranasal administration is obtained, for 
example, by suspending a peptide of the formula I, or a therapeutically 
acceptable acid addition salt thereof, in an oil, optionally with the 
addition of swelling agents, such as aluminium stearate, and/or 
interfacially active agents (surfactants), the HLB value 
("hydrophilic-lipophilic balance") of which is less than 10, such as fatty 
acid monoesters of polyhydric alcohols, for example glycerine 
monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan 
monoleate. A fat-containing salve is obtained, for example, by suspending 
the active substance according to the invention in a spreadable fat base, 
optionally with the addition of a surfactant having a HLB value of less 
than 10. An emulsion salve is obtained by triturating an aqueous solution 
of the peptide active substance in a soft, spreadable fat base with the 
addition of a surfactant the HLB value of which is less than 10. All these 
intranasal forms of medication may also contain preservatives. The 
individual doses are approximately 0.08 to approximately 15 mg, preferably 
0.25 to 10 mg, contained in approximately 0.05 to approximately 0.1 g of 
the base substance. 
Also suitable for intranasal administration are inhalation or insufflation 
preparations, such as insufflation capsules that permit the active 
substance to be insufflated in the form of a powder with respiratory air, 
or aerosols or sprays that can disperse the pharmacological active 
substance in the form of a powder or in the form of drops of a solution or 
suspension. Preparations having powder-dispersing properties generally 
contain adjuncts in addition to the active substance: insufflation 
capsules contain, for example, solid carriers, such as lactose; aerosol or 
spray preparations contain, for example, a liquid propellant having a 
boiling point of below room temperature and, if desired, other carriers, 
such as liquid or solid non-ionic or anionic surfactants and/or solid 
diluents. Preparations in which the pharmacological active substance is in 
solution contain, in addition to this, a suitable propellant and also, if 
necessary, an additional solvent and/or a stabiliser. Instead of the 
propellant, it is also possible to use compressed air which is produced 
when required by means of a suitable compressing and releasing device. 
The invention relates also to the use of the novel compounds of the formula 
I and therapeutically acceptable acid addition salts thereof as 
pharmacologically active compounds, especially in the indications 
mentioned at the beginning, preferably in the form of pharmaceutical 
preparations. The daily dose administered to a warm-blooded animal 
weighing approximately 70 kg is from approximately 0.1 to approximately 
120 mg.

The invention is illustrated in the following Examples but is not limited 
by these. Temperatures are given in degrees Centigrade; the conventional 
short forms, for example those compiled in "Synthese von Peptiden" 
(editor: E. Wunsch), volume XV of "Methoden der org. Chemie" (Houben-Weyl) 
(1974; G. Thieme, Stuttgart) are used as abbreviations, for example for 
denoting amino acids, peptides, protecting groups, etc. The following 
abbreviations, in particular, are used: 
______________________________________ 
Boc tert.-butoxycarbonyl 
But tert.-butyl (as ether-forming group) 
OTmse 2-(trimethylsilyl)-ethoxy (as ester- 
forming group) 
Z benzyloxycarbonyl 
TLC thin layer chromatography. 
______________________________________ 
In TLC, unless otherwise indicated, silica gel is used as the adsorbent and 
the following systems are used as eluants: 
______________________________________ 
System 
52: n-butanol/acetic acid/water 
(71.5:7.5:21) 
101: n-butanol/pyridine/acetic acid/water 
(38:24:8:30) 
104: chloroform/methanol/17% aqueous ammonia 
(41:41:18) 
111B: n-butanol/pyridine/25% aqueous ammonia/ 
water (40:24:6:30) 
112A: n-butanol/pyridine/formic acid/water 
(42:24:4:20) 
151: n-butanol/pyridine/acetic acid/water 
(38:20:6:24) 
157: chloroform/methanol/water/acetic acid 
(70:42:10:0.5) 
157B: chloroform/methanol/water/acetic acid 
(85:13:1.5:0.5) 
157C: chloroform/methanol/water/acetic acid 
(75:27:5:0.5) 
157E: chloroform/methanol/water/acetic acid 
(55:47:13:5) 
______________________________________ 
EXAMPLE 1 
##STR18## 
50 mg of somatostatin (peptide content 82%, remainder water and acetic 
acid) in 175 .mu.l of dimethylformamide are stirred with 16 mg of 
4-nitrophenyl acetate and 12 .mu.l of triethylamine for 2 hours at room 
temperature. The product, precipitated with ether/hexane, is filtered in 
chloroform/methanol (1:1) over a Sephadex LH-20.RTM. column and the 
fractions that are pure according to TLC are isolated. Yield: 44 mg. 
TLC: [chloroform/methanol/water/glacial acetic acid (55:47:13:0.5)] 
R.sub.f : 0.46. 
EXAMPLE 2 
##STR19## 
100 mg of 
##STR20## 
(in the form of the acetate) together with 15 .mu.l of N-methylmorpholine 
and 20 mg of p-nitrophenyl acetate are dissolved in 1.0 ml of 
dimethylformamide and kept at room temperature for 20 hours. For working 
up, the reaction mixture is taken up in 20 ml of ethyl acetate and washed 
3 times using 5 ml of water each time. After drying over sodium sulphate, 
the organic phase is concentrated by evaporation in vacuo. The residue is 
triturated with 5 ml of ether, filtered off and washed with ether. The 
crude product is chromatographed on a silica gel column (70 g) by means of 
a chloroform/methanol mixture (with the proportion of methanol gradually 
increasing from 5 to 15%). Suitable fractions that are uniform according 
to thin-layer chromatography are combined, concentrated by evaporation in 
vacuo, dissolved in 20 ml of tert.-butanol and lyophilised. 
______________________________________ 
TLC: [CHCl.sub.3 /CH.sub.3 OH/H.sub.2 O (14:6:1)] 
R.sub.f : 0.65 
[CHCl.sub.3 /CH.sub.3 OH (85:15)] 
R.sub.f : 0.20 
[n-butanol/acetic acid/H.sub.2 O (3:1:1) 
R.sub.f : 0.77 
______________________________________ 
The following N.sup..epsilon. -acyl-Lys.sup.9 derivatives are obtained from 
the same peptide (in the form of the free base) in an analogous manner 
while observing the other conditions, and serve as intemermediates for the 
end products of the following Example 3: 
(a) The N.sup..epsilon. -(N-benzyloxycarbonylglycyl)-Lys.sup.9 derivative 
(Ac=Z-Gly) is obtained with 33 mg of Z-Gly-p-nitrophenyl ester 
______________________________________ 
TLC: [CHCl.sub.3 /CH.sub.3 OH/H.sub.2 O (14:6:1)] 
R.sub.f : 0.74 
[CHCl.sub.3 /CH.sub.3 OH (85:15)] 
R.sub.f : 0.29 
______________________________________ 
(b) The N.sup..epsilon. -(N-benzyloxycarbonylleucyl)-Lys.sup.9 derivative 
(Ac=Z-Leu) is obtained with 38 mg of Z-Leu-p-nitrophenyl ester 
______________________________________ 
TLC: [CHCl.sub.3 /CH.sub.3 OH/H.sub.2 O (14:6:1)] 
R.sub.f : 0.80 
[CHCl.sub.3 /CH.sub.3 OH (85:15)] 
R.sub.f : 0.35 
______________________________________ 
(c) The N.sup..epsilon. -(N-benzyloxycarbonylphenylalanyl)-Lys.sup.9 
derivative (Ac=Z-Phe) is obtained with 42 mg of Z-Phe-p-nitrophenyl ester. 
______________________________________ 
TLC: [CHCl.sub.3 /CH.sub.3 OH/H.sub.2 O (14:6:1)] 
R.sub.f : 0.84 
[CHCl.sub.3 /CH.sub.3 OH (85:15)] 
R.sub.f : 0.39 
______________________________________ 
(d) The N.sup..epsilon. -(N-benzyloxycarbonylprolyl)-Lys.sup.9 derivative 
(Ac=Z-Pro) is obtained with 39 mg of Z-Pro-p-nitrophenyl ester. 
______________________________________ 
TLC: System 157B R.sub.f : 0.35 
______________________________________ 
(e) The N.sup..epsilon. -(N.sup..alpha. 
-tert.-butoxycarbonyl-N.sup..epsilon. -benzyloxycarbonyllysyl)-Lys.sup.9 
derivative [(Ac=Boc-Lys(Z)] is obtained with 49 mg of 
Boc-Lys(Z)-p-nitrophenyl ester. 
______________________________________ 
TLC: [CHCl.sub.3 /CH.sub.3 OH (8:2)] 
R.sub.f : 0.55 
______________________________________ 
EXAMPLE 3 
##STR21## 
After the addition of 10 mg of palladium-on-carbon (10%) a solution 78 mg 
of 
##STR22## 
(see Example 2a) in 10 ml of methanol and 0.5 ml of 1 N aqueous acetic 
acid is hydrogenated for 10 hours at room temperature and normal pressure. 
For working up, the catalyst is filtered off, the filtrate is concentrated 
by evaporation in vacuo and the residue is dissolved in 20 ml of 
tert.-butanol and lyophilised. 
The corresponding N.sup..epsilon. -leucyl-(Ac=Leu), N.sup..epsilon. 
-phenylalanyl- (Ac=Phe), N.sup..epsilon. -prolyl- (Ac=Pro) and 
N.sup..epsilon. -(N.sup..alpha. -tert.-butoxycarbonyl)lysyl derivative 
(Ac=Boc-Lys) is manufactured in analogous manner: 
__________________________________________________________________________ 
TLC: (silica gel): 
System 
R.sub.f [Ac.dbd.Gly] 
R.sub.f [Ac--Leu] 
R.sub.f [Ac.dbd.Phe] 
R.sub.f [Ac.dbd.Pro] 
R.sub.f [Ac.dbd.BocLys] 
__________________________________________________________________________ 
101 0.60 0.69 0.71 -- -- 
111B 
0.62 0.80 0.80 -- -- 
112A 
0.58 0.70 0.82 -- -- 
151 0.59 0.70 0.70 -- -- 
157 -- -- -- -- 0.65 
157C 
-- -- -- 0.73 -- 
__________________________________________________________________________ 
The last-named N.sup..epsilon. -(N.sup..alpha. -tert.-butoxycarbonyl)lysyl 
derivative (Ac=Boc-Lys) is converted further to form the corresponding 
peptide having the lysyl radical free, that is to say, to form the 
[D-Trp.sup.8 -N.sup..epsilon. -lysyl-Lys.sup.9 -Gaba.sup.12 
]cyclosomatostatin(5-12)octapeptide of the formula 
##STR23## 
in the following manner: 
100 mg of 
##STR24## 
are dissolved at 5.degree., under nitrogen, in 1.9 ml of a mixture of 89% 
by volume of trifluoroacetic acid, 10% by volume of water and 1% by volume 
of thioglycolic acid, the solution is immediately heated to 25.degree. and 
left to stand under nitrogen for 25 minutes at room temperature. The 
product is precipitated with 20 ml of ether and lyophilised from 
tert.-butanol. 
______________________________________ 
TLC: System 101 R.sub.f : 0.55 
______________________________________ 
EXAMPLE 4 
[D-Trp.sup.8 -N.sup..epsilon. -octanoyl-Lys.sup.9 -Gaba.sup.12 
]cyclosomatostatin(5-12)-octapeptide 
##STR25## 
10 .mu.l of triethylamine and 23 mg of caprylic acid anhydride (octanoic 
acid anhydride) are added to 69 mg of 
##STR26## 
(in the form of the acetate) in 0.25 ml of dimethylformamide and the 
mixture is left for 15 hours at room temperature. The product, 
precipitated with water, is subsequently filtered in chloroform/methanol 
(1:1) over a column of Sephadex LH-20.RTM. for purification. The fractions 
that are pure according to TLC are isolated. 
______________________________________ 
TLC: [chloroform/methanol/water/glacial acetic acid 
(90:10:1:0.5)] R.sub.f : 0.23 
______________________________________ 
EXAMPLE 5 
[D-Trp.sup.8 -N.sup..epsilon. -stearoyl-Lys.sup.9 -Gaba.sup.12 
]cyclosomatostatin(5-12)-octapeptide 
##STR27## 
A solution of 66 mg of 
##STR28## 
(in the form of the acetate), 26 mg of stearic acid p-nitrophenyl ester 
and 9 .mu.l of triethylamine in 0.2 ml of dimethylformamide is left 
overnight at room temperature. The material, precipitated with water, is 
subsequently filtered in chloroform/methanol (1:1) over a column of 
Sephadex LH-20.RTM. and the fractions that are pure according to TLC are 
isolated. 
______________________________________ 
TLC: [chloroform/methanol/water/glacial acetic acid 
(75:26:5:0.5)] R.sub.f : 0.18 
______________________________________ 
EXAMPLE 6 
[D-Trp.sup.8 -n.sup..epsilon. -(.beta.-carboxypropionyl)-Lys.sup.9 
-Gaba.sup.12 ]cyclosomatostatin(5-12)octapeptide 
##STR29## 
100 g of 
##STR30## 
(in the form of the acetate) are dissolved in 0.8 ml of dimethylformamide, 
and 9 .mu.l of N-methylmorpholine and 8.2 mg of succinic acid anhydride 
are added thereto. After a reaction period of 1 hour at room temperature, 
5 ml of (peroxide-free) ether are added. For purification, the oily crude 
product that precipitates out is triturated with 5 ml of ether, dissolved 
in 5 ml of tert.-butanol and lyophilised. 
______________________________________ 
TLC: [n-butanol/acetic acid/water (3:1:1)] 
R.sub.f : 0.65 
System 52 R.sub.f : 0.63 
System 104 R.sub.f : 0.60 
System 111B R.sub.f : 0.50 
System 157E R.sub.f : 0.58 
______________________________________ 
EXAMPLE 7 
##STR31## 
A solution of 283 mg of crude 
H--Asn--Phe--Phe--(D--Trp)--Lys(Ac)--Thr(But)--Phe--Gaba--OH (stage 7.7), 
324 mg of N-hydroxybenzotriazole and 495 mg of DCCI in 240 ml of 
dimethylformamide is kept at 50.degree. for 20 hours. For working up, the 
solvent is evaporated off in a high vacuum at approximately 30.degree. and 
the residue is triturated with 10 ml of ethyl acetate. The 
dicyclohexylurea that precipitates out is filtered off, the filtrate is 
diluted with ethyl acetate to 50 ml, washed three times using 20 ml of 1 N 
aqueous oxalic acid each time and then washed with water until neutral, 
dried over sodium sulphate and concentrated by evaporation in vacuo. For 
purification, the crude product is subjected to countercurrent 
distribution in the system methanol/water/chloroform/carbon tetrachloride 
(2700:675:900:1575 parts by volume) over 430 stages. The phases (K=1.16) 
that are contained in elements 210 to 254 are combined and concentrated by 
evaporation in vacuo. The residue is dissolved in 20 ml of tert.-butanol 
and lyophilised, resulting in a material that is uniform according to 
thin-layer chromatography and has the formula 
##STR32## 
This protected cyclopeptide (186 mg) is dissolved at 5.degree., under 
N.sub.2, in 1.5 ml of a mixture of 89% by volume of trifluoroacetic acid, 
10% by volume of water and 1% by volume of thioglycolic acid, the solution 
is immediately heated to 25.degree. and, after 90 minutes at room 
temperature under N.sub.2, is precipitated with 15 ml of ether. The 
resulting crude product is lyophilised from tert.-butanol. 
The title compound obtained is identical, according to TLC using three 
systems, to the product of Example 2. 
The linear octapeptide used as starting material is obtained as follows: 
STAGE 7.1 
Z--(D--Trp)--Lys(Ac)--OH 
A solution of 21.87 g of DCCI in 100 ml of acetonitrile is added dropwise 
at 0.degree. to 5.degree., over a period of 45 minutes, to a solution of 
33.84 g of Z--(D--Trp)--OH and 17.42 g of 8-hydroxyquinoline in 50 ml of 
acetonitrile. After a further 30 minutes at 5.degree., the 
dicyclohexylurea that precipitates out is removed by filtration and 
washing out with 50 ml of acetonitrile. A solution of 20.70 g of 
H--Lys(Ac)--OH in 25.9 ml of 4.25 N potassium hydroxide solution and 80 ml 
of acetonitrile is added to the filtrate and the mixture is left to stand 
for 15 hours at room temperature. For working up, the reaction mixture is 
concentrated by evaporation in vacuo and the residue is taken up in 1 
liter of ethyl acetate, washed three times using 200 ml of 1 N 
hydrochloric acid each time at 0.degree. and three times using 200 ml of 
water each time, the organic phase is dried over sodium sulphate and 
concentrated by evaporation in vacuo. The resulting brown oil is dissolved 
in 150 ml of chloroform and, while stirring vigorously, is introduced 
dropwise into 1.5 liter of hexane. The flocculent, glutinous precipitate 
is filtered off, washed with 500 ml of hexane and dried in vacuo. For 
further purification, the material is dissolved in 150 ml of carbon 
tetrachloride/ethyl acetate (6:4 parts by volume) and is chromatographed 
with this solvent mixture over a silica gel column. Suitable fractions 
that are uniform according to thin-layer chromatography are concentrated 
by evaporation in vacuo, yielding the pure product in the form of a 
foamlike substance. 
______________________________________ 
TLC: [chloroform/methanol/water (14:6:1)] 
R.sub.f : 0.45 
______________________________________ 
STAGE 7.2 
Z--(D--Trp)--Lys(Ac)--Thr(But)--Phe--OTmse 
At 5.degree., 2.28 g of N-hydroxybenzotriazole and 3.38 g of DCCI are added 
to a solution of 7.24 g of crude (87% according to titration) 
H--Thr(But)--Phe--OTmse and 7.58 g of Z--(D--Trp)--Lys(Ac)--OH (stage 7.1) 
in 100 ml of dimethylformamide and the reaction mixture is kept at 
5.degree. for 1 hour and at room temperature for a further 15 hours. For 
working up, the dicyclohexylurea that precipitates out is filtered off and 
the filtrate is concentrated by evaporation in a high vacuum. The residue 
is reprecipitated twice from ethyl acetate/petroleum ether and dried in 
vacuo. 
______________________________________ 
TLC: [chloroform/ethyl acetate (1:1)] 
R.sub.f : 0.18 
[toluene/acetone (1:1)] 
R.sub.f : 0.50 
______________________________________ 
STAGE 7.3 
H--(D--Trp)--Lys(Ac)--Thr(But)--Phe--OTmse 
After the addition of 0.50 g of palladium-on-carbon (10%) a solution of 
5.00 g of Z--(D--Trp)--Lys(Ac)--Thr(But)--Phe--OTmse (stage 7.2) in 300 ml 
of methanol is hydrogenated for 5 hours at room temperature and normal 
pressure. For working up, the catalyst is filtered off and, after the 
filtrate has been concentrated by evaporation, the residue that remains is 
used immediately in stage 7.4. 
STAGE 7.4 
Z--Asn--Phe--Phe--(D--Trp)--Lys(Ac)--Thr(But)--Phe--OTmse 
210 mg of N-hydroxybenzotriazole and 276 mg of DCCI are added to a solution 
of 632 mg of Z--Asn--Phe--Phe--OH and 803 mg of 
H--(D--Trp)--Lys(Ac)--Thr(But)--Phe--OTmse (stage 7.3) in 5 ml of 
dimethylformamide and the mixture is left for 15 hours at room 
temperature. For working up, the dicyclohexylurea that precipitates out is 
filtered off and the filtrate is concentrated by evaporation in a high 
vacuum. The oily residue is triturated with 5 ml of methanol and filtered 
by suction. For purification, the undissolved material is again triturated 
with 5 ml of methanol at 50.degree., filtered with suction, washed with 
methanol and dried in vacuo. The product is uniform according to 
thin-layer chromatography. 
______________________________________ 
TLC: [chloroform/methanol (85:15)] 
R.sub.f : 0.78 
[chloroform/methanol/water (14:6:1)] 
R.sub.f : 0.82 
______________________________________ 
STAGE 7.5 
Z--Asn--Phe--Phe--(D--Trp)--Lys(Ac)--Thr(But)--Phe--OH 
900 mg of Z--Asn--Phe--Phe (D--Trp)--Lys(Ac)--Thr(But)--Phe--OTmse (stage 
7.4) are dissolved in 23 ml of a freshly prepared anhydrous 0.15 N 
tetraethylammonium fluoride solution in dimethylformamide and the mixture 
is kept at 25.degree. for 30 minutes. After cooling to 5.degree., 0.68 ml 
of 1 N aqueous hydrochloric acid are added to the reaction mixture, while 
stirring well, and the product is precipitated by the addition of 70 ml of 
water. The material filtered off is washed with 5 ml of water, dried in 
vacuo over phosphorus pentoxide and used directly in stage 7.6. 
STAGE 7.6 
Z--Asn--Phe--Phe--(D--Trp)--Lys(Ac)--Thr(But)--Phe--Gaba--OBzl 
61 ml of 1-hydroxybenzotriazole and 93 mg of DCCI are added to a mixture of 
411 mg of Z--Asn--Phe--Phe--(D--Trp)--Lys(Ac)--Thr(But)--Phe--OH (stage 
7.5) and 82 mg of Gababenzyl ester p-toluenesulphonate in 2 ml of 
dimethylformamide and the mixture is left for 20 hours at room 
temperature. For working up, 15 ml of ice-cold methanol are added to the 
mixture and the mixture is filtered. For further purification the solid 
obtained is stirred for 10 minutes with 5 ml of warm methanol, the 
suspension is cooled to 0.degree., and the pure product is filtered off 
and dried in vacuo. 
______________________________________ 
TLC: [chloroform/methanol (85:15)] 
R.sub.f : 0.75 
______________________________________ 
STAGE 7.7 
H--Asn--Phe--Phe--(D--Trp)--Lys(Ac)--Thr(But)--Phe--Gaba--OH 
After the addition of 50 mg of palladium-on-carbon (10%), a solution of 400 
mg of Z--Asn--Phe--Phe--(D--Trp)--Lys(Ac)--Thr(But)--Phe--Gaba--OBzl 
(stage 7.6) in 25 ml of dimethylformamide is hydrogenated for 6 hours at 
room temperature and normal pressure. For working up, after filtering off 
the catalyst, the solution is concentrated to 2 ml in a high vacuum and 
the product is precipitated with 25 ml of peroxide-free ether, filtered 
off and dried in vacuo. The crude product is subjected to the final stage 
(cyclisation) without further purification.