Aminoacyl derivatives

Peptide and pseudopeptide derivatives of the formula: ##STR1## wherein R.sup.1 and R.sup.2 stand for defined hydrocarbyl or halo-hydrocarbyl substituents, for example alk-2-enyl radicals; .dbd.N--A-- stands for a defined amino acid residue; B, D and E, which may be the same or different, stand for a valency bond or a defined amino acid residue; F stands for a defined amino acid residue; X stands for a carboxy, ester or amide group; and the linkages between the amino acid residues are peptide linkages or at least one of said linkages is a defined pseudo linkage; and pharmaceutically-acceptable salts thereof. Processes for the manufacture of the compounds. Pharmaceutical compositions comprising one of the compounds and a pharmaceutical diluent or carrier. The compounds are antagonists at the opiate receptors, and most of them are selective .delta.-receptor antagonists.

This invention relates to aminoacyl derivatives, and more particularly it 
relates to peptide and pseudopeptide derivatives which are active as 
antagonists at the so-called opiate receptors in warm-blooded animals. 
It is generally recognised that in warm-blooded animals there are at least 
two distinct types of opiate receptor, i.e. the .mu.-receptor and the 
.delta.-receptor (see Robson & Kosterlitz, Proc. R. Soc. London (B), 1979, 
205, 425-432, Goodman et al., Proc. Natl. Acad. Sci. U.S.A., 1980, 77, 
6239-6243, and Simon, Trends in Pharmacol. Sci., 1981, 2, 155). Compounds 
are known, for example naloxone, which are antagonists of natural 
endogenous agonist substances for example enkephalins, at opiate 
receptors. However, all of these known antagonists are more potent at the 
.mu.-receptors than at the .delta.-receptors. That is, all of the known 
opiate antagonists are selective .mu.-receptor antagonists. 
The preparation of N-mono-allyl-[Leu]enkephalin and 
N-mono-allyl-[Met]enkephalin has been described by Hahn et al., Res. 
Commun. Chem. Pathol. Pharmacol., 1977, 18, 1, who stated that those 
compounds have both agonist and antagonist properties. Up to the present 
time there has been no description in the literature, including the patent 
literature, of an enkephalin or chemically similar compound in which the 
N-terminus bears two lower alkenyl or like substituents. We have found 
that such compounds are opiate antagonists which are virtually devoid of 
agonist properties, and, in contrast to the above-mentioned known 
antagonists, they are at least as potent at the .delta.-receptors as they 
are the .mu.-receptors, and most of them are more potent at the 
.delta.-receptors than they are at the .mu.-receptors. That is, most of 
them are selective .delta.-receptor antagonists. 
According to the invention there are provided compounds of the formula: 
##STR2## 
wherein: R.sup.1 stands for an alk-2-enyl, haloalk-2-enyl or alk-2-ynyl 
radical of not more than 5 carbon atoms, or a furylmethyl or 
tetrahydrofurylmethyl radical; 
R.sup.2 stands for an alk-2-enyl, haloalk-2-enyl, alk-2-ynyl or alkyl 
radical of not more than 5 carbon atoms, a phenylalkyl radical of not more 
than 10 carbon atoms, or a furylmethyl or tetrahydrofurylmethyl radical; 
or R.sup.1 and R.sup.2 are joined to form, together with the adjacent 
nitrogen atom, a morpholino, piperidino, methylpiperidino or 
1-aza-3,6-methancycloheptan-1-yl radical; 
&gt;N-A stands for the residue of D-, L-, D,L- or aza-tyrosine, phenylalanine 
or p-aminophenylalanine; 
B stands for a single valency bond or for the residue of D-, L- or D,L-, 
where the amino acid contains a chiral centre, or aza-, glycine, 
methionine, alanine, serine or sarcosine; 
D stands for a single valency bond or for the residue of D-, L- or D,L-, 
where the amino acid contains a chiral centre, or aza-, glycine, alanine, 
phenylalanine, sarcosine, serine, O-benzylserine, cysteine or 
S-benzylcysteine; 
E stands for a single valency bond or for the residue of D-,L- or D,L-, 
where the amino acid contains a chiral centre, or aza-, glycine, 
phenylalanine, N-methylphenylalanine, p-nitrophenylalanine, 
p-chlorophenylalanine or tryptophan; 
F stands for the residue of D-,L- or D,L-, where the amino acid contains a 
chiral centre, or aza-, glycine, leucine, methionine, alanine, 
phenylalanine, proline, serine, O-benzylserine or norleucine, or a 
dipeptide residue which is D-,L-, D,L- or aza-, leucine-arginine, 
leucine-glutamic acid, leucine-leucine, leucine-phenylalanine or 
leucine-threonine; and 
X stands for a group of the formula --CO.sub.2 R.sup.3 or --CONHR.sup.4, 
wherein R.sup.3 stands for hydrogen or an alkyl or alkenyl radical of not 
more than 4 carbon atoms, and R.sup.4 stands for hydrogen, an alkyl, 
hydroxyalkyl, cycloalkyl or alkoxycarbonylalkyl radical of not more than 6 
carbon atoms, a phenylalkyl or phenyl(hydroxy)alkyl radical of not more 
than 9 carbon atoms, or a phenyl, phenylcyclopropyl, 2-benzylthioethyl, 
2-(2-phenylethylthio)ethyl or indanyl radical; 
and wherein the linkages between the amino acid residues are peptide 
linkages or at least one of said linkages is a pseudo linkage selected 
from --CH.sub.2 S--, --NHCO--, --CO.NH.O--, trans--CH.dbd.CH-- and 
--CH.sub.2 CH.sub.2 --; 
and pharmaceutically-acceptable salts thereof. 
It is to be understood that throughout this specification the standard 
abbreviations for amino acids are used (see Pure and Applied Chemistry, 
1974, 40, 317-331, and Neuropeptides, 1981, 1, 231-235). An 
.alpha.-aza-amino-acid is one in which the .alpha.-CH part of the amino 
acid has been replaced by a nitrogen atom. The abbreviation for an 
.alpha.-aza-amino-acid is derived from that for the corresponding amino 
acid by adding the prefix "Az". Thus, for example, Azala stands for 
.alpha.-aza-alanine, Azgly stands for .alpha.-aza-glycine, and so on. When 
the configuration of an amino acid is not designated herein, it is to be 
understood that it has the natural L configuration (except for acids 
having no chiral centre). In this specification the word "pseudo" has the 
meaning that, in the compound in question, at least one of the 
conventional peptide linkages (--CO.NH--) has been replaced by a linkage 
selected from --CH.sub.2 S--, --NHCO--, --CO.NH.O--, trans--CH.dbd.CH-- 
and --CH.sub.2 CH.sub.2 --. The change from the peptide linkage is 
indicated by the use of the Greek symbol psi (.psi.); see Neuropeptides, 
1981, 1, 231-235. It is to be understood that in the compounds of this 
invention any particular chiral centre may be in the D-, L- or 
D,L-configuration. 
R.sup.1 may, for example, stand for an allyl, crotonyl, 2-chloroallyl, 
3-chloroallyl, propargyl, 2-furylmethyl, 3-furylmethyl or 
2-tetrahydrofurylmethyl radical. 
R.sup.2 may, for example, stand for an allyl, crotonyl, 2-chloroallyl, 
3-chloroallyl, propargyl, methyl, ethyl, n-propyl, n-butyl, benzyl, 
2-phenylethyl, 2-furylmethyl, 3-furylmethyl or 2-tetrahydrofurylmethyl 
radical. 
Alternatively, R.sup.1 and R.sup.2 may be joined to form, together with the 
adjacent nitrogen atom, a morpholino, piperidino, 4-methylpiperidino, 
3-methylpiperidino or 1-aza-3,6-methancycloheptan-1-yl radical. 
&gt;N-A may, for example, stand for Tyr or Phe(p-NH.sub.2). 
B may, for example, stand for a single valency bond or Gly, Azgly, D-Met, 
D-Ala, D-Ser or Sar. 
D may, for example, stand fo a single valency bond or Gly, Azgly, Ala, 
D-Ala, Azala, Phe, D-Phe, Sar, D,L-Ser, Ser, Ser(Bzl), D-Ser(Bzl) or 
D,L-Cys(Bzl). 
E may, for example, stand for a single valency bond or Gly, Phe, D-Phe, 
Azphe, MePhe, Phe(p-NO.sub.2), D,L-Phe(p-Cl) or Trp. 
F may, for example, stand for Gly, Azgly, Leu, D-Leu, D,L-Leu, Met, D-Ala, 
Phe, Pro, Ser, Ser(Bzl), Nle, Leu-Arg, Leu-Glu, Leu-D-Glu, Leu-Leu, 
Leu-Phe or Leu-Thr. 
R.sup.3 may, for example, stand for hydrogen or a methyl, ethyl or allyl 
radical. 
R.sup.4 may, for example, stand for hydrogen or an ethyl, 
4-methyl-2-pentyl, 1-hydroxy-4-methyl-2-pentyl, cyclohexyl, 
3-(ethoxycarbonyl)propyl, 2-methoxycarbonyl-2-butyl, benzyl, 
2-phenylethyl, 3-phenylpropyl, 1-phenyl-2-propyl, R-1-phenyl-2-propyl, 
2-phenylpropyl, 1-hydroxy-3-phenyl-2-propyl, phenyl, phenylcyclopropyl, 
2-benzylthioethyl, 2-(2-phenylethylthio)ethyl or 1-indanyl radical. 
One preferred embodiment of the invention consists of compounds of the 
formula I wherein R.sup.1 and R.sup.2 stand for allyl radicals, &gt;N-A 
stands for Tyr, and B, D, E, F and X have the meanings stated above, all 
of the linkages between the amino acid residues or .alpha.-aza-amino-acid 
residues are peptide linkages or one of said linkages is a pseudo linkage 
selected from --CH.sub.2 S--, --NHCO--, --CO.NH.O--, trans--CH.dbd.CH-- 
and --CH.sub.2 CH.sub.2 --, and pharmaceutically-acceptable salts thereof. 
A group of preferred compounds of the invention consists of 
diallyl-Tyr-Gly-Gly-.psi.(CH.sub.2 S)-Phe-Leu-OH, 
diallyl-Tyr-Gly-Azgly-Phe-Leu-OH, 
diallyl-Tyr-Gly-Gly-Phe-.psi.(NHCO)-D,L-Leu-OEt, 
diallyl-Tyr-Gly-Azgly-Phe-Leu-D-Glu-OH, and 
##STR3## 
and pharmaceutically-acceptable salts thereof. 
The salts of the invention may, in the case where the compound of the 
formula I is sufficiently basic, be pharmaceutically-acceptable 
acid-addition salts or, in the case where the said compound is 
sufficiently acidic, pharmaceutically-acceptable base-addition salts. The 
acid-addition salts are derived from an inorganic or organic acid which 
affords a pharmaceutically-acceptable anion, for example hydrochloric, 
phosphoric, acetic, citric or trifluoroacetic acid. The base-addition 
salts are derived from a base which affords a pharmaceutically-acceptable 
cation, for example ammonia, N-methyl-D-glucosamine or arginine, or they 
may, for example, be alkali metal salts. 
The compounds of the invention can be prepared by procedures which are 
conventional in peptide chemistry. 
According to a further feature of the invention there is provided a process 
for the manufacture of the compounds of the formula I, wherein R.sup.1, 
R.sup.2, A, B, D, E, F and X have the meanings stated above, and 
pharmaceutically-acceptable salts thereof, which comprises removing one or 
more conventional protecting groups from a corresponding protected 
compound by conventional means. 
In the case where &gt;N-A stands for Tyr, D-Tyr and D,L-Tyr, the protecting 
group may be a t-butyl radical which may be removed by treatment of the 
protected derivative with hydrogen chloride or trifluoroacetic acid. 
Hydrogen chloride may be used in the form of an aqueous solution, at a 
concentration between 1 M and that of a saturated solution, or it may be 
used as a solution in an organic solvent, for example ethyl acetate, at a 
concentration in the range 2 M to 6 M. The process is preferably carried 
out at a temperature between 0.degree. C. and ambient temperature, and 
optionally in the presence of a scavenger compound, for example anisole, 
thioanisole, methionine or dimethyl sulphide. Trifluoroacetic acid may be 
used as a de-protecting agent by itself or it may be diluted with 5-10% by 
volume of water. The process involving trifluoroacetic acid is preferably 
carried out at ambient temperature, and optionally in the presence of a 
scavenger compound, for example 2-mercaptoethanol or anisole. 
According to a further feature of the invention there is provided a process 
for the manufacture of those of the compounds of the formula I wherein X 
stands for a carboxy radical, and pharmaceutically-acceptable salts 
thereof, which comprises hydrolysing a corresponding (1-6C)alkyl or benzyl 
ester under alkaline conditions. 
A suitable ester is a methyl, ethyl or benzyl ester. A suitable hydrolytic 
agent is an alkali metal hydroxide, for example sodium hydroxide. The 
hydrolysis is carried out in the presence of water, and preferably in the 
presence of methanol or ethanol. The hydrolysis is conveniently carried 
out at ambient temperature. 
According to a further feature of the invention there is provided a process 
for the manufacture of those of the compounds of the formula I which are 
amides containing the group --CONHR.sup.4, wherein R.sup.4 has the meaning 
stated above, and pharmaceutically-acceptable salts thereof, which 
comprises reacting a corresponding carboxylic acid or (1-4C)alkyl ester 
thereof with a compound of the formula R.sup.4 NH.sub.2. 
The process is conveniently carried out in methanol or ethanol. 
According to a further feature of the invention there is provided a process 
for the manufacture of those of the compounds of the formula I which 
contain the group: 
##STR4## 
wherein R.sup.5 and R.sup.6, which may be the same or different, stand for 
an alk-2-enyl, haloalk-2-enyl or alk-2-ynyl radical of not more than 5 
carbon atoms, and pharmaceutically-acceptable salts thereof, which 
comprises reacting the corresponding compound containing the group: 
##STR5## 
with a compound of the formula R.sup.5 Hal or R.sup.6 Hal, wherein R.sup.5 
and R.sup.6 have the meanings stated above and Hal stands for a halogen 
atom, in the presence of an acid-binding agent, and wherein &gt;N-A- has the 
meaning stated above. 
Hal may, for example, stand for a chlorine or bromine atom. A suitable 
acid-binding agent is, for example, an alkali metal carbonate or 
bicarbonate, for example potassium carbonate or sodium bicarbonate. The 
process is conveniently carried out in aqueous ethanol, at an elevated 
temperature. It is to be understood that, by varying the conditions of the 
reaction and/or the amounts of the reactants, it is possible to obtain 
products containing two identical N-substituents or containing two 
different N-substituents (in the latter case by first making the R.sup.5 
-NH-A- compound and then converting it into the R.sup.5 -NR.sup.6 -A- 
compound). 
According to a further feature of the invention there is provided a process 
for the manufacture of those of the compounds of the formula I which 
contain the group: 
##STR6## 
wherein R.sup.7 stands for a furylmethyl or tetrahydrofurylmethyl radical 
and R.sup.8 stands for an alkyl radical of not more than 5 carbon atoms, a 
phenylalkyl radical of not more than 10 carbon atoms, or a furylmethyl or 
tetrahydrofurylmethyl radical, and pharmaceutically-acceptable salts 
thereof, which comprises reacting the corresponding compound containing 
the group: 
##STR7## 
with the appropriate aldehyde or ketone and an alkali metal hydride 
reducing agent, and wherein &gt;N-A- has the meaning stated above. 
The aldehyde or ketone may, for example, be propionaldehyde, 2-furaldehyde, 
benzaldehyde or acetone. The alkali metal hydride may, for example, be an 
alkali metal borohydride, for example sodium cyanoborohydride. The process 
is conveniently carried out in ethanol, optionally together with acetic 
acid, at ambient temperature. It is to be understood that, by varying the 
conditions of the reaction and/or the amounts of the reactants, it is 
possible to obtain products containing two identical N-substituents or 
containing two different N-substituents (in the latter case by first 
making the R.sup.7 -NH-A-compound and then converting it into the R.sup.7 
-NR.sup.8 -A-compound). 
Compounds of the formula I containing the group: 
##STR8## 
wherein R.sup.5 and R.sup.8 have the meanings stated above, and 
pharmaceutically-acceptable salts thereof, can be obtained by reacting the 
corresponding compound containing the group of the formula III with 
R.sup.5 Hal in the presence of an acid-binding agent, as described above, 
so as to obtain a compound containing the group of the formula IV, and 
then reacting the latter compound with the appropriate aldehyde or ketone 
and an alkali metal hydride reducing agent so as to obtain the desired 
product. Alternatively, the order of these two reactions can be reversed. 
The peptide and pseudopeptides used as starting materials in the processes 
of the invention can be prepared by procedures which are conventional in 
peptide chemistry, as is illustrated hereinafter by the descriptions of 
the preparation of various starting materials and by the reaction 
diagrams. Similarly, the pharmaceutically-acceptable salts of the 
invention are obtainable by conventional procedures. 
The activity of the compounds of the invention as antagonists at opiate 
receptors has been demonstrated in the guinea pig ileum test ("ileum 
test") and the mouse vas deferens test ("vas test"); see the article by 
Shaw et al. in "Characteristics and Functions of Opioids", edited by Van 
Ree and Terenius, Elsevier/North-Holland Biomedical Press, 1978, 185-195. 
It is generally recognised that in the guinea pig ileum the .mu.-receptor 
predominates, and that in the mouse vas deferens the .delta.-receptor 
predominates. The potency of a compound in the above-mentioned tests is 
expressed as a Ke value, i.e. the concentration of the compound 
(antagonist) in the presence of which the agonist concentration has to be 
doubled in order to maintain a constant response. [Leu]-enkephalin is used 
as the agonist in both tests. The potency of any particular compound in 
the tests depends upon its precise chemical structure, but the compounds 
of the invention are active in the ileum test at a concentration in the 
range 1 .mu.M to 30 .mu.M, and in the vas test at a concentration in the 
range 10 nM to 10 .mu.M (.mu.M stands for micromole, i.e. 10.sup.-6 mole, 
and nM stands for nanomole, i.e. 10.sup.-9 mole). 
Data illustrating the lack of toxicity of a compound of the invention, 
namely: (allyl).sub.2 &gt;Tyr-Gly-Gly-.psi.(CH.sub.2 S)-Phe-Leu-OH is as 
follows: 
(1) When administered subcutaneously, the compound exhibits no toxic 
effects at doses up to 100 mg./kg. in the rat. 
(2) In male and female mice, subcutaneous doses of up to 100 mg./kg. have 
been administered without significant toxic effect. 
(3) An intravenous dose of 100 mg./kg. (which is a very high dose) in the 
mouse produced no toxic effects. 
(4) The compound has been administered orally to rats at 150 mg./kg. 
without producing any toxic effects. 
Because of their activity as opiate receptor antagonists, the compounds of 
the invention may be used for the treatment of the following conditions 
and/or diseases in man: schizophrenia and other mental illnesses, stress, 
shock, anorexia nervosa, epilepsy, disorders of the endocrine function 
including post-menopausal flushing, and gastro-intestinal disorders. The 
compounds may also be used as sedatives. When a compound of the invention 
is used for the treatment of man, it may be administered orally, or 
parenterally, for example by intraveous, subcutaneous or intramuscular 
injection or by infusion, or sub-lingually or rectally. A recommended 
daily oral dose for man is in the range 1 mg. to 1.0 g. Such a dose may be 
administered as a single daily dose or it may be divided into, for 
example, three doses per day. A recommended parenteral dose for man is 1 
mg. to 250 mg., a recommended sub-lingual dose is 1 mg. to 250 mg., and a 
recommended rectal dose is 2 mg. to 1.0 g. 
The compounds of the invention may also be used as research tools or 
diagnostic agents in pharmacological or related studies. 
According to a further feature of the invention there are provided 
pharmaceutical compositions comprising a compound of the formula I, 
wherein R.sup.1, R.sup.2, A, B, D, E, F and X have the meanings stated 
above, or a pharmaceutically-acceptable salt thereof, and a 
pharmaceutically-acceptable diluent or carrier. 
The pharmaceutical compositions of the invention may be in a form suitable 
for oral, parenteral, sub-lingual or rectal administration. Thus, for 
example, they may be in an orally-administrable unit dosage form, for 
example tablets or capsules, which may optionally be adapted for sustained 
or controlled release, or they may be in an injectable form, for example a 
sterile injectable solution or suspension, or in the form of a 
suppository. The pharmaceutical compositions of the invention are 
obtainable in conventional manner using conventional diluents or carriers. 
The pharmaceutical compositions of the invention may optionally contain, in 
addition to a peptide or pseudopeptide derivative of the invention: 
(1) a known opiate antagonist, for example naloxone; 
(2) a known psychotropic agent, for example an antipsychotic agent, for 
example chlorpromazine, or a antidepressant agent, for example imipramine, 
or an anxiolytic agent, for example chlordiazepoxide; 
(3) a known analgesic agent, for example morphine; or 
(4) a known anticonvulsant agent, for example primidone. 
The invention is illustrated but not limited by the following Examples, in 
which the R.sub.f values refer to ascending thin layer chromatography on 
silica gel plates (Kieselgel G). The solvent systems used, unless 
otherwise stated, were as follows (the ratios are by volume): 
______________________________________ 
R.sub.f A 
n-butanol/acetic acid/water (4:1:5) 
R.sub.f B 
n-butanol/acetic acid/water/pyridine (15:3:12:10) 
R.sub.f C 
s-butanol/3% w/v aqueous ammonium hydroxide (3:1) 
R.sub.f D 
acetonitrile/water (3:1) 
R.sub.f H 
cyclohexane/ethyl acetate/methanol (1:1:1) 
R.sub.f K 
chloroform/methanol/water (55:40:10) 
R.sub.f K' 
chloroform/methanol/water (55:40:1) 
R.sub.f K.sub.HOAc 
chloroform/methanol/acetic acid (60:30:5) 
R.sub.f K.sub.NH.sbsb.3 
chloroform/methanol/ammonium hydroxide 
(S.G. 0.880) (60:30:5) 
R.sub.f L 
methanol/ethyl acetate (5:95) 
R.sub.f M 
methanol/ethyl acetate (7:93) 
R.sub.f P 
chloroform/methanol (19:1) 
R.sub.f P.sub.NH.sbsb.3 
chloroform/methanol (19:1) containing 1% v/v 
ammonium hydroxide (S.G. 0.880) 
R.sub.f Q 
chloroform/methanol (9:1) 
R.sub.f Q.sub.HOAc 
chloroform/methanol (9:1) containing 1% v/v 
acetic acid 
R.sub.f Q.sub.NH.sbsb.3 
chloroform/methanol (9:1) containing 1% v/v 
ammonium hydroxide (S.G. 0.880) 
R.sub.f R 
chloroform/methanol (4:1) 
R.sub.f R.sub.HOAc 
chloroform/methanol (4:1) containing 1% v/v 
acetic acid 
R.sub.f S 
chloroform/methanol (7:3) 
R.sub.f T 
chloroform/methanol (6:4) 
______________________________________ 
The symbol D,L-indicates that the product in question was obtained as a 
mixture of diastereoisomers containing not less than 30% of any one 
diasteroisomer, the remainder of the mixture consisting of the other 
diasteroisomer. 
In the footnotes column in the Tables hereinafter, there is a reference if 
the preparation of the starting material is described in another Example, 
or described in detail in the "Preparation of Starting Materials" section 
(SM1 to SM9), or outlined in a diagram (indicated by the letter D followed 
by the number of the appropriate Example). 
The following abbreviations are used in this specification: 
______________________________________ 
Me methyl OCP 2,4,5-trichlorophenoxy 
Et ethyl DCCI dicyclohexylcarbodiimide 
Pr.sup.n 
n-propyl DCHA dicyclohexylamine 
Pr.sup.i 
isopropyl DMF dimethylformamide 
Bu.sup.n 
n-butyl mM millimole 
Bu.sup.i 
isobutyl NCDI N--carbonyldiimidazole 
Bu.sup.t 
t-butyl NMR nuclear magnetic resonance 
Ph phenyl TFA trifluoroacetic acid 
Bzl benzyl THF tetrahydrofuran 
OSu 1-succinimidolyloxy 
t.l.c. thin layer chromatography 
Boc t-butoxycarbonyl 
Z benzyloxycarbonyl 
______________________________________

EXAMPLES 1-72 
The protecting group(s) in the protected polypeptide or pseudopeptide was 
or were removed by cleavage with TFA using one of the procedures A1 to A5 
described below. The products thus obtained are listed in Table I. 
A1. The starting material (ca. 0.2 g.) was dissolved in sufficient TFA 
containing 5 to 10% v/v of water to give an approximately 10% w/v 
solution. The mixture was stirred at 20.degree. to 25.degree. C. for 0.5 
to 2 hours, and the product was isolated by evaporation of the solvent in 
vacuo. 
A2. As A1, but using a 2:1 v/v mixture of TFA and anisole. 
A3. As A1, but in the presence of 5 molecular equivalents of methionine per 
molecular equivalent of starting material. 
A4. As A1, but in the presence of 10 molecular equivalents of dimethyl 
sulphide per molecular equivalent of starting material. 
A5. As A1, but using a 2:1 v/v mixture of TFA and thioanisole. 
TABLE I 
__________________________________________________________________________ 
Example R.sub.f .times. 10.sup.2 
Foot- 
No. Product A B C D H P Q Other 
Process 
notes 
__________________________________________________________________________ 
1 
##STR9## 34 
66 
21 K80 A1 2,6 freeze- 
dried D1 
2 
##STR10## 46 
50 
17 
60 A1 1,8 D2 
3 
##STR11## 53 
60 
17 K85 A1 1 
4 
##STR12## 67 
32 K33 A1 1 
5 
##STR13## 66 
66 
40 
65 K73 A1 1 
6 
##STR14## 40 
63 
33 
73 K68 A1 4 D6 
7 
##STR15## 57 
66 K'69 A1 1 
8 
##STR16## 8 16 
K95 A1 3,6 
9 
##STR17## Q.sub.HOAc 45 
A1 3,14 
10 
##STR18## R.sub.HOAc 33 
A1 3,15 
11 
##STR19## R17 A1 1 
12 
##STR20## 75 
40 
80 
85 A1 23 
13 
##STR21## 25 
49 
K'78 A3 3,15 D13 
14 
##STR22## 42 A3 17 
15 
##STR23## 0 K'59 A1 3,7 
16 
##STR24## 25 
K'78 A1 3,10,17 D16 
17 
##STR25## 42 
62 
36 
75 A1 1,16 
18 
##STR26## 57 
60 
34 
53 A1 1 D18 
19 
##STR27## S35 A1 1 
20 
##STR28## T47 A1 1 
21 
##STR29## T46 A1 1 
22 
##STR30## 36 
K89 K'85 
A1 3,15 
23 
##STR31## 66 
76 
77 Al 3 
24 
##STR32## 60 
70 
40 
77 A1 3,11 SM5 
25 
##STR33## 56 
76 
71 A1 3 D25 
26 
##STR34## R29 A2 3,15 
27 
##STR35## R27 A2 3,10 SM9 
28 
##STR36## 33 K'65 Al 1 D25 
29 
##STR37## T52 Al 1 
30 
##STR38## P50 A4 3,14 D30 
31 
##STR39## 84 
76 
20 
58 A1 1 
32 
##STR40## 58 
68 
43 
70 K90 A1 1,16 D32 
33 
##STR41## 44 
64 
36 
68 K68 A1 3 D33 
34 
##STR42## 70 20 
K'80 Al 3 
35 
##STR43## 83 
80 
0 20 A1 1 
36 
##STR44## 80 
80 13 A1 2 
37 
##STR45## 43 
66 
40 K80 A1 1 
38 
##STR46## 71 
70 
33 K80 A1 3,8 
39 
##STR47## 66 
70 
36 
78 6 K'84 A1 1 
40 
##STR48## 33 
K84 A1 1 
streak K'80 
41 
##STR49## 38 A1 1 
42 
##STR50## K77 K'67 
A1 3 
43 
##STR51## 5 39 A1 1 
44 
##STR52## 32 
R53 A2 2,14 D44 
45 
##STR53## 26 
R43 A2 2,14 
46 
##STR54## K91 K'71 
A2 3,9 SM7 
47 
##STR55## 66 
70 
45 
70 K78 A1 3,10 D47 
48 
##STR56## 60 
66 
38 
68 K'68 A1 3,12 D48 
49 
##STR57## 79 
87 
74 K93 A1 1 SM6 
50 
##STR58## 12 
38 
K83 K'74 
A1 3 
51 
##STR59## 68 
29 K66 K'66 
A5 1 D51 
52 
##STR60## 78 79 K93 A2 3,14 D52 
53 
##STR61## 61 
70 
63 A2 3,10 
54 
##STR62## 47 
R78 A1 1,13 
55 
##STR63## K55 A1 1 SM2 
56 
##STR64## T36 A1 1 SM3 
57 
##STR65## A35 A1 1 SM3 
58 
##STR66## T38 A1 1 SM3 
59 
##STR67## T38 A1 1 SM3 
60 
##STR68## T41 A1 1 SM3 
61 
##STR69## T37 A1 1 SM3 
62 
##STR70## T33 A1 1 SM4 
63 
##STR71## T32 A1 1 SM4 
64 
##STR72## M41 A1 1 SM4 
65 
##STR73## T36 A1 1 SM4 
66 
##STR74## T40 A1 1 SM4 
67 
##STR75## T48 A1 1 SM4 
68 
##STR76## K'50 A1 1 SM4 
69 
##STR77## K59 A1 1 SM4 
70 
##STR78## K'59 A1 1 SM4 
71 
##STR79## K46 A1 1 SM4 
72 
##STR80## R30 A1 1 SM4 
__________________________________________________________________________ 
Footnotes 
1. Product triturated with diethyl ether and collected by filtration. 
2. As 1 but triturated with 50% v/v diethyl ether/petroleum ether (b.p. 
60-80.degree. C.). 
3. Product obtained as a powder by freezedrying from water or tbutanol 
containing 5% v/v of water. 
4. Product crystallised from methanol/diethyl ether. 
5. Product purified by gel permeation chromatography using Sephadex C15 i 
5% v/v aqueous acetic acid. 
6. As 5 but using Sephadex G10. 
7. As 5 but using 0.1M ammonium acetate. 
8. As 5 but using 0.05M ammonium hydroxide. 
9. As 5 but using Sephadex G10 in 10% v/v aqueous acetic acid. 
10. Product purified by column chromatography on silica gel (Merck 
Kieselgel 7754), eluting with chloroform containing 5% v/v of methanol. 
11. As 10 but eluting with chloroform containing 12% v/v of methanol. 
12. As 10 but eluting with chloroform containing 15% v/v of methanol. 
13. As 10 but eluting with chloroform containing 0 to 2% v/v of methanol, 
the methanol concentration being increased in a stepwise manner. 
14. As 10 and 13 but eluting with chloroform containing 0 to 5% v/v of 
methanol. 
15. As 10 and 13 but eluting with chloroform containing 0 to 10% v/v of 
methanol. 
16. As 10 but eluting with a mixture of chloroform, methanol and water 
(55:40:1 v/v). 
17. As 5 but using Sephadex 1H 20 in methanol. 
18. As 5 but using Sephadex G10 in 0.1M ammonium acetate. 
19. As 5 but using Sephadex G10 in 10% w/v ammonium hydroxide. 
20. As 5 but using Sephadex G10 in 0.5M ammonium hydroxide. 
21. As 5 but using Sephadex LH 20 in DMF. 
Unless otherwise stated below, in those of Examples 1 to 72 in which the 
products were of the general chemical type: 
##STR81## 
the starting materials had the corresponding general structure: 
##STR82## 
Similarly, unless otherwise stated below, in those of Examples 1 to 72 in 
which the products were esters or amides of the general chemical type: 
##STR83## 
the starting materials has the corresponding general structure: 
##STR84## 
(a) In the case of Example 1, the arginine residue in the starting material 
was in the form of the N.sup..omega., N.sup..epsilon. 
-bis-adamantyloxycarbonyl derivative. 
(b) In the case of Examples 10, 15, 44, 45 and 48, the starting material 
was of the general chemical type: 
##STR85## 
EXAMPLES 73-90 
The protecting group(s) in the protected peptide or pseudopeptide was or 
were removed by cleavage with hydrogen chloride using one of the 
procedures B1 and B2 described below. The products thus obtained are 
listed in Table II. 
B1. The starting material (0.1 to 10 g.) was dissolved in sufficient 2 M- 
to 5 M-hydrogen chloride in ethyl acetate to give an approximately 10% w/v 
solution. The mixture was stirred at 20.degree. to 25.degree. C. for 0.5 
to 1 hour, and the product was isolated either by evaporation of the 
solvent in vacuo or, where appropriate, by the collection of the solid 
product by filtration. 
B2. As B1 but using M-hydrogen chloride in acetic acid. 
TABLE II 
__________________________________________________________________________ 
Example R.sub.f .times. 10.sup.2 Foot- 
No. Product A B C D H P Q Others 
Process 
notes 
__________________________________________________________________________ 
73 
##STR86## K.sub.NH.sbsb.3 85 
B1 1 
74 
##STR87## R60 B1 3 D74 
75 
##STR88## 61 
71 
62 R60 B1 3,15 
76 
##STR89## K.sub.NH.sbsb.3 86 
B1 1,14 
77 
##STR90## 28 
K86 B1 1,14 SM8 
78 
##STR91## 11 
R54 B1 1 
79 
##STR92## 61 
73 
65 20 
R42 B1 3,15 
80 
##STR93## 14 
R47 B1 3,15 
81 
##STR94## 66 P.sub.NH.sbsb.3 31 
B1 3,14 
82 
##STR95## 68 
79 
32 K83 B2 3 D82 
83 
##STR96## 35 B1 1 D83 
84 
##STR97## 35 B1 1 
85 
##STR98## 37 B1 3 
86 
##STR99## 19 
R56 B1 3 D86 
87 
##STR100## 25 
R71 B1 3 
88 
##STR101## 22 
R62 B1 3 
89 
##STR102## 35 
R72 B1 3,14 
90 
##STR103## Q.sub.NH.sbsb.3 50 
B1 3 
__________________________________________________________________________ 
The general comments after Examples 1 to 72 concerning the starting 
materials also apply to Examples 73 to 90. However, in the case of Example 
82 the starting material was of the type: 
##STR104## 
EXAMPLES 91-101 
The appropriate ester (see below for information on esters used as starting 
materials) was hydrolysed by one of the following procedures: 
C1. A sufficient quantity of the ester was dissolved in methanol to give a 
5 to 20% w/v solution. Aqueous sodium hydroxide (M to 4 M; 2 to 15 molar 
equivalents) was added, and the mixture was stirred for 0.5 to 2 hours at 
room temperature. The reaction mixture was neutralised by the addition of 
an appropriate amount of M-hydrochloric acid, and the solvent was then 
evaporated in vacuo. 
C2. As C1 but using ethanol in place of methanol. 
The products thus obtained are listed in Table III. The footnotes in the 
Table (see the end of Table I for information on the footnotes) indicate 
how the products were purified. 
TABLE III 
__________________________________________________________________________ 
Example R.sub.f .times. 10.sup.2 Foot- 
No. Product A B C D H P Q Other Process 
notes 
__________________________________________________________________________ 
91 
##STR105## K'58 K.sub.HOAc 74 
C1 3,9 Ex. 8 
92 
##STR106## K74 K'57 
C1 3,19 D92 
93 
##STR107## K69 K'58 
C1 3,9,18 Ex. 13 
94 
##STR108## K82 K'53 
C1 3 Ex. 16 
95 
##STR109## K76 K'51 
C1 3 Ex. 22 
96 
##STR110## 69 
76 
33 K85 C1 3,20 
97 
##STR111## K67 K'51 
C1 3,9 Ex. 40 
98 
##STR112## K73 K'50 
C1 3,17 
99 
##STR113## K75 K'59 
C1 3,17 
100 
##STR114## 57 
75 
50 K'73 C2 3 Ex. 49 
101 
##STR115## 72 
45 C1 1 D101 
__________________________________________________________________________ 
The esters used as starting materials in Examples 91 to 101 were as 
follows: 
Examples 91 to 95, 97 to 99, and 101: the corresponding methyl ester 
Example 96: the corresponding benzyl ester 
Example 100: the corresponding ethyl ester 
EXAMPLES 102 AND 103 
Example 102 
H-Tyr-Gly-Gly-Phe-Leu-OMe.HCl (0.20 g., 0.33 mM) was dissolved in ethanol 
(7 ml.). To the solution were added furfuraldehyde (0.031 ml., 0.4 mM), 
acetic acid (0.016 ml., 0.20 mM) and sodium cyanoborohydride (0.025 g., 
0.4 mM). The solution was stirred for 20 hours at room temperature and the 
solvent was then evaporated in vacuo. The residue was dissolved in a 
mixture of ethyl acetate (25 ml.) and saturated aqueous sodium bicarbonate 
solution (20 ml.). The two layers were separated and the organic layer was 
washed with saturated brine (20 ml.) and dried (Na.sub.2 SO.sub.4). The 
solvent was evaporated in vacuo and the gum-like residue was 
chromatographed using the dry column chromatography technique with ethyl 
acetate/methanol (92.5:7.5 v/v). The product band was eluted from the 
column using ethyl acetate/methanol (85:15 v/v). Evaporation of the 
solvent in vacuo gave 2-furylmethyl-Tyr-Gly-Gly-Phe-Leu-OMe having R.sub.f 
0.3 (t.l.c. on silica gel; chloroform/methanol 92.5:7.5 v/v). 
The above-mentioned furylmethyl derivative (0.10 g., 0.15 mM) was dissolved 
in ethanol (3.5 ml.). To the solution were added acetic acid (0.037 ml., 
0.62 mM), propionaldehyde (0.033 ml., 0.46 mM) and sodium cyanoborohydride 
(0.029 g., 0.46 mM). The solution was stirred for 20 hours at room 
temperature and the solvent was then evaporated in vacuo. The residue was 
dissolved in a mixture of ethyl acetate (25 ml.) and saturated aqueous 
sodium bicarbonate (20 ml.). The two layers were separated and the organic 
layer was washed with saturated brine (20 ml.) and dried (Na.sub.2 
SO.sub.4). The solvent was evaporated in vacuo to give a gum-like residue. 
This residue was chromatographed on two 20.times.20 cm silica preparative 
t.l.c. plates using ethyl acetate/methanol 93:7 v/v as eluent. The product 
band was eluted with ethyl acetate/methanol 80:20 v/v. Evaporation of the 
solvent in vacuo gave 
##STR116## 
having R.sub.f 0.48 (t.l.c. on silica gel; ethyl acetate/methanol 93:7 
v/v). 
Example 103 
In analogous manner to that described in Example 102 there was obtained 
bis-(2-furylmethyl)-Tyr-Gly-Gly-Phe-Leu-OMe having R.sub.f L 0.44. 
EXAMPLE 104 
Diallyl-Tyr-Gly-N.sub.2 H.sub.3 (462 mg., B 1.01 mM; see D 104) was 
dissolved in DMF (5 ml.) and the solution was cooled to -20.degree. C. A 
solution of hydrogen chloride in dioxan (8.0 M, 0.25 ml., 2.0 mM) was 
added, followed by t-butyl nitrite (138 .mu.l., 1.2 mM), and the mixture 
was stirred for 3 minutes at -20.degree. C. Triethylamine (0.56 ml., 4.0 
mM) was added, followed by a suspension of 
H-Gly-.psi.(-transCH.dbd.CH)-D,L-Phe-Leu-OH hydrochloride (300 mg., 0.84 
mM; see D104) and triethylamine (238 .mu.l., 1.7 mM) in DMF/H.sub.2 O (50% 
v/v, 10 ml.). The temperature was increased to 0.degree. C. and the pH of 
the resulting suspension (monitored with moist pH paper) was adjusted to 
pH 8 by the addition of triethylamine. Stirring was continued for a 
further 0.5 hour while maintaining the temperature at 0.degree. C. The pH 
was again adjusted to 8 by the addition of triethylamine, and the reaction 
mixture was stirred for a further 24 hours at 0.degree. C. The solvent was 
evaporated and the residue was partitioned between ethyl acetate (20 ml.) 
and water (20 ml.). The two phases were separated and the aqueous phase 
was extracted with more ethyl acetate (20 ml.). The combined ethyl acetate 
phases were washed successively with water (2.times.20 ml.) and saturated 
brine (20 ml.), and then dried (Na.sub.2 SO.sub.4). The solvent was 
evaporated in vacuo, and the residue was applied to a column of silica 
(Merck Kieselgel 7754, 24.times.2.0 cm.) and eluted with chloroform 
containing 2-5% v/v methanol. The appropriate fractions were combined and 
the solvent was evaporated in vacuo. The residue was dissolved in 
t-butanol containing 5% v/v water and the solvent was removed by 
freeze-drying. There was thus obtained 
diallyl-Tyr-Gly-Gly-.psi.(transCH.dbd.CH)-D,L-Phe-Leu-OH, R.sub.f R 0.30. 
EXAMPLE 105 
H-Tyr-D-Met-Gly-Phe-OMe (1.6 g., 4 mM) was dissolved in ethanol/water (9:1 
v/v, 25 ml.). To the solution were added sodium bicarbonate (1.008 g., 12 
mM) and allyl bromide (1.44 g., 12 mM). The mixture was refluxed for 2 
hours. The solvent was evaporated in vacuo and the residue was partitioned 
between ethyl acetate (50 ml.) and water (50 ml.). The mixture was 
separated and the organic phase was washed successively with water (50 
ml.), M-citric acid (50 ml.), water (50 ml.), M-sodium bicarbonate (50 
ml.), water (50 ml.) and saturated brine (50 ml.). The organic phase was 
then dried (Na.sub.2 SO.sub.4) and the solvent evaporated to give a clear 
yellow oil. The product was applied to a column of silica (Merck Kieselgel 
7754, 24.times.2.0 cm.) and eluted with chloroform containing 0-2% v/v 
methanol. The appropriate fractions were combined and the solvent 
evaporated. The residue was freeze-dried from t-butanol containing 5% v/v 
water to give dially-Tyr-D-Met-Gly-Phe-OMe, having R.sub.f R 0.70. 
EXAMPLE 106 
DCCI (0.150 g., 0.73 mM) was added to a stirred, cooled (0.degree. C.) 
solution of diallyl-Tyr-OH (0.181 g., 0.69 mM), 
H-Gly-D,L-Cys(Bzl)-Phe-Leu-OMe hydrochloride (0.400 g., 0.69 mM), 
1-hydroxybenzotriazole (0.187 g., 1.38 mM) and triethylamine (92 .mu.l., 
0.69 mM) in DMF (12 ml.). The cooling bath was removed and the reaction 
mixture was stirred for 16 hours at 20.degree. C. Dicyclohexylurea was 
removed by filtration and the filtrate was evaporated in vacuo. The 
residue was partitioned between ethyl acetate (30 ml.) and water (20 ml.). 
The mixture was separated and the organic phase was washed successively 
with M-sodium bicarbonate (20 ml.), water (20 ml.), M-citric acid (20 
ml.), water (20 ml.) and saturated brine (20 ml.). The organic solution 
was then dried (MgSO.sub.4) and evaporated. The residue was applied to a 
column of silica (Merck Kieselgel 7754, 30 g.) and eluted with 50% v/v 
ethyl acetate/petroleum ether (b.p. 60.degree.-80.degree. C.). The 
appropriate fractions were combined and the solvent was evaporated. The 
oily residue was freeze-dried from t-butanol containing 5% v/v water to 
give diallyl-Tyr-Gly-D,L-Cys(Bzl)-PHe-Leu-OMe as a white powder having 
R.sub.f Q 0.45. 
EXAMPLE 107 
Diallyl-Tyr-Gly-Azgly-OPh (0.200 mg., 0.442 mM) and 2-phenylpropylamine 
(70.7 .mu.l., 0.486 mM) in methylene dichloride (1 ml.) were stirred for 3 
days at 20.degree. C. The solvent was evaporated in vacuo, and the residue 
was applied to a column of silica (Kieselgel 7754, 1.8.times.15 cm.) and 
eluted with chloroform containing 0.5% v/v methanol. The appropriate 
fractions were combined and the solvent removed by evaporation. The 
residual glassy solid was freeze-dried from t-butanol containing 5% v/v 
water to give dially-Tyr-Gly-Azgly-R,S-NHCH.sub.2 CHMePh as a white powder 
having R.sub.f Q 0.24. 
EXAMPLE 108 
A solution of diallyl-Tyr-Glyz-Azgly-OPh (0.200 mg., 0.442 mM) and 
(2S)-2-amino-3-phenyl-1-propanol (73.5 mg., 0.42 mM) in methylene 
dichloride (1 ml.) was stirred for 2 days at 20.degree. C. The solvent was 
evaporated in vacuo and the residue was partitioned between ethyl acetate 
(10 ml.) and water (10 ml.). The mixture was separated and the organic 
phase was washed successively with 1 M-sodium dihydrogen phosphate 
(2.times.5 ml.), water (5 ml.), and saturated brine (5 ml.). The solution 
was dried (MgSO.sub.4) and the solvent removed by evaporation. The residue 
was applied to a column of silica (Merck Kieselgel 7754, 19.times.1.5 cm.) 
and eluted with chloroform containing methanol (0.5% v/v). The appropriate 
fractions were combined and the solvent was evaporated in vacuo. The 
residue was freeze-dried from t-butanol containing water (5% v/v) to give 
diallyl-Tyr-Gly-Azgly-(2S)-NHCH(Bzl)CH.sub.2 OH as a white powder having 
R.sub.f Q 0.15. 
EXAMPLE 109 
Diallyl-Tyr-GLy-Gly-Phe-Leu-OMe (55 mg., see Ex. 8) was dissolved in 
anhydrous ethylamine (5 ml.). The solution was kept at room temperature 
for 48 hours, and the solvent was then evaporated in vacuo. The residue 
was diallyl-Tyr-Gly-Gly-Phe-Leu-NHEt having R.sub.f D 0.68 and R.sub.f Q 
0.39. 
EXAMPLE 110 
A mixture of H-Tyr-Gly-Gly-Phe-Leu-OH (50 mg., 0.09 mM), allyl bromide (1 
ml., ca. 100 equivalents), sodium bicarbonate (1 g., ca. 100 equivalents), 
ethanol (20 ml.) and water (1 ml.) was refluxed for 5 hours. The mixture 
was cooled to room temperature and filtered, and the solvent was 
evaporated in vacuo from the filtrate. The residual oily solid was 
partitioned between ethyl acetate (5 ml.) and 5% v/v aqueous acetic acid 
(5 ml.). The mixture was separated and the ethyl acetate phase was washed 
with 5% v/v aqueous acetic acid (2.times.2 ml.). The combined ethyl 
acetate phases were dried (MgSO.sub.4) and the solvent evaporated in 
vacuo. The residue was freeze-dried from t-butanol containing 5% v/v water 
to give diallyl-Tyr-Gly-Gly-Phe-Leu-Oallyl having R.sub.f A 0.70, R.sub.f 
C 0.79, R.sub.f K 0.94 and R.sub.f K' 0.88. 
PREATION OF STARTING MATERIALS 
The peptides and pseudopeptides used as starting materials in the above 
Examples were obtained by procedures which are conventional in peptide 
chemistry. By way of illustration, typical preparative procedures are 
described below (SM1 to SM9). Following that, the preparation of other 
starting materials is outlined in diagrams (each diagram has a number 
preceded by the letter D; the number indicates the corresponding Example). 
SM1 
H-Tyr(Bu.sup.t)-Gly-Gly-Phe-Leu-OBu.sup.t 
This compound, which was used in the preparation of various starting 
materials (see SM2, SM3 and SM4), was obtained as follows: 
Z-Gly-OH (25.2 g., 132 mM) was dissolved in DMF (300 ml.) and the solution 
was cooled to -10.degree. C. N-methylmorpholine (14.8 ml., 132 mM) was 
added, followed by ethyl chloroformate (12.1 ml., 126 mM). After 3 minutes 
at -10.degree. C., a solution of H-Phe-Leu-OBu.sup.t (40.1 g., 120 mM) in 
DMF (100 ml.) was added, and the mixture was stirred at 0.degree. C. 
overnight. The solvent was evaporated and the residue was dissolved in 
ethyl acetate (600 ml.) and washed successively with water (2.times.60 
ml.), 2 N-aqueous potassium bicarbonate (3.times.60 ml.), and water 
(3.times.60 ml.). The solution was dried (MgSO.sub.4) and filtered. The 
solvent was evaporated to give Z-Gly-Phe-Leu-OBu.sup.t. 
The last-named compound (55.7 g., 105 mM) was dissolved in methanol (270 
ml.), and 5% palladium-on-carbon catalyst (5 g.) in water (30 ml.) was 
added. The mixture was vigorously stirred and a slow stream of hydrogen 
gas was bubbled through it at ambient temperature for 5 hours. The 
catalyst was filtered off (Kieselguhr) and washed with methanol. The 
filtrate was evaporated to give H-Gly-Phe-Leu-OBu.sup.t as a white solid. 
The last-named compound (29.4 g., 75 mM) was dissolved in DMF (100 ml.), 
the solution was cooled to 0.degree. C., and Z-Gly-OCP (32.4 g., 83 mM) 
was added. The mixture was stirred at 0.degree. C. overnight. The solvent 
was evaporated and the residue was dissolved in ethyl acetate (1 l.), 
washed with water (5.times.250 ml.) and then dried (MgSO.sub.4). The 
mixture was filtered and the solvent evaporated. The residue was collected 
and thoroughly washed with diethyl ether to give 
Z-Gly-Gly-Phe-Leu-OBu.sup.t. 
The last-named compound (42 g., 72 mM) was dissolved in methanol (270 ml.) 
and 5% palladium-on-carbon catalyst (4 g.) in water (30 ml.) was added. 
The mixture was vigorously stirred and a slow stream of hydrogen gas was 
bubbled through it at ambient temperature for 5 hours. The catalyst was 
filtered off (Kieselguhr) and washed with methanol. The filtrate was 
evaporated to give H-Gly-Gly-Phe-Leu-OBu.sup.t. 
Z-Tyr(Bu.sup.t)-OH dicyclohexylammonium salt (44.2 g., 80 mM) was suspended 
in ethyl acetate (500 ml.) and the suspension was shaken with 1 N-aqueous 
citric acid (4.times.50 ml.) and water (4.times.50 ml.). The resulting 
solution in ethyl acetate of Z-Tyr(But)-OH was dried (MgSO.sub.4) and 
filtered, and the solvent was evaporated to give an oil, which was 
dissolved in DMF (300 ml.). N-Methylmorpholine (9 ml., 80 mM) was added 
and the solution was cooled to -10.degree. C. Ethyl choroformate (7.2 ml., 
76 mM) was added and, after 3 minutes at -10.degree. C., a solution of 
H-Gly-Gly-Phe-Leu-OBu.sup.t (72 mM, see above) in DMF (100 ml.) was added. 
The mixture was stirred at -10.degree. C. for 1 hour and then at 0.degree. 
C. overnight. The solvent was evaporated, the residue was dissolved in 
ethyl acetate (1 l.) and washed successively with water (2.times.250 ml.), 
1 N-aqueous citric acid (3.times.150 ml.), water (150 ml.), 2 N-aqueous 
potassium bicarbonate (3.times.100 ml.) and water (2.times.100 ml.). The 
solution was dried (MgSO.sub.4) and filtered and the solvent was 
evaporated to give Z-Tyr(Bu.sup.t)-Gly-Gly-Phe-Leu-OBu.sup.t. 
The last-named compound (40 g., 50 mM) was hydrogenolyzed as described 
above to give the required starting material 
H-Tyr(Bu.sup.t)-Gly-Gly-Phe-Leu-OBu.sup.t. 
SM2 
##STR117## 
This compound, which was used as the starting material in Example 55, was 
obtained as follows: 
H-Tyr(Bu.sup.t)-Gly-Gly-Phe-Leu-OBu.sup.t (0.222 g., 0.33 mM.) was 
dissolved in ethanol (10 ml.). To the solution were added sodium 
bicarbonate (0.153 g., 1.65 mM.) and propargyl bromide (0.163 ml., 1.65 
mM). The mixture was stirred and refluxed for 20 hours. The solvent was 
evaporated in vacuo and the residue dissolved in a mixture of ethyl 
acetate (30 ml.) and water (20 ml.). The two phases were separated and the 
organic phase was washed with saturated brine (20 ml.) and dried (Na.sub.2 
SO.sub.4). The solvent was evaporated in vacuo to give a gum-like residue 
which was chromatographed using the dry column chromatography technique 
with ethyl acetate/methanol 97:3 v/v as eluent. The product band was 
eluted from the column using ethyl acetate/methanol 90:10 v/v. Evaporation 
of the solvent in vacuo gave 
dipropargyl-Tyr(Bu.sup.t)-Gly-Gly-Phe-Leu-OBu.sup.t as a glassy solid 
having R.sub.f 0.49 (t.l.c. on silica gel; ethyl acetate/methanol 97:3 
v/v). 
SM3 
##STR118## 
This compound, which was used as the starting material in Example 56, was 
obtained as follows: 
H-Tyr(Bu.sup.t)-Gly-Gly-Phe-Leu-OBu.sup.t (2.0 g., 3 mM) was dissolved in 
ethanol (90 ml.). To the solution were added sodium bicarbonate (0.378 g., 
4.5 mM) and allyl bromide (0.387 ml., 4.5 mM). The solution was stirred 
and refluxed for 20 hours. The solvent was then evaporated in vacuo and 
the residue dissolved in a mixture of ethyl acetate (120 ml.) and water 
(80 ml.). The two phases were separated and the organic phase was washed 
with saturated brine (80 ml.) and dried (Na.sub.2 SO.sub.4). The solvent 
was evaporated in vacuo to give a gum-like residue which was 
chromatographed using the dry column chromatography technique with ethyl 
acetate/methanol 90:10 v/v as eluent. The product band was eluted from the 
column with ethyl acetate/methanol 85:15 v/v. The solvent was evaporated 
in vacuo from the eluate to give 
allyl-Tyr(Bu.sup.t)-Gly-Gly-Phe-Leu-OBu.sup.t having R.sub.f 0.48 (t.l.c. 
on silica gel; ethyl acetate/methanol 90:10 v/v). 
The above-mentioned allyl derivative (0.20 g., 0.28 mM) was dissolved in 
ethanol (10 ml.), and to the solution were added acetic acid (0.050 ml., 
0.84 mM), propionaldehyde (0.060 ml., 0.84 mM) and sodium cyanoborohydride 
(0.035 g., 0.56 mM). The mixture was stirred for 20 hours at room 
temperature, the solvent was then evaporated in vacuo, and the residue 
dissolved in a mixture of ethyl acetate (25 ml.) and aqueous sodium 
bicarbonate (1 M; 20 ml.). The two phases were separated and the organic 
phase was washed with saturated brine (20 ml.) and dried (Na.sub.2 
SO.sub.4). The solvent was evaporated in vacuo and the residue was 
chromatographed using the dry column chromatography technique with ethyl 
acetate as the eluent. The product band was eluted from the column using 
ethyl acetate/methanol 90:10 v/v as the eluent, and evaporation of the 
solvent in vacuo gave 
allyl(n-propyl)-Tyr(Bu.sup.t)-Gly-Gly-Phe-Leu-OBu.sup.t as a glassy solid 
having R.sub.f 0.40 (t.l.c. on silica gel; ethyl acetate). 
The following compounds were prepared in analogous manner: 
______________________________________ 
##STR119## 
Used as starting 
R material in Example No. 
______________________________________ 
Me 57 
Pr.sup.i 58 
Bu.sup.n 59 
phenylethyl 60 
2-tetrahydrofurylmethyl 
61 
______________________________________ 
SM4 
The following compounds were prepared in analogous manner to that described 
in Example 102: 
______________________________________ 
##STR120## 
Used as starting material 
R.sup.1 R.sup.2 in Example No. 
______________________________________ 
2-furylmethyl 
Et 62 
2-furylmethyl 
Bu.sup.n 63 
3-furylmethyl 
Pr.sup.n 64 
3-furylmethyl 
3-furylmethyl 
65 
2-tetrahydro- 
Pr.sup.n 66 
furylmethyl 
2-tetrahydro- 
2-tetrahydro- 
67 
furylmethyl furylmethyl 
(CH.sub.2).sub.5 68 
(CH.sub.2).sub.2CHMe(CH.sub.2).sub.2 
69 
CH.sub.2 CHMe(CH.sub.2).sub.3 
70 
(CH.sub.2).sub.2O(CH.sub.2).sub.2 
71 
##STR121## 72 
______________________________________ 
SM5 
(allyl).sub.2 -Tyr(Bu.sup.t)-Gly--Azgly-Phe-Leu-OBu.sup.t 
This compound, which was used as the starting material in Example 24, was 
obtained as follows: 
H-Phe-Leu-OBu.sup.t (0.669 g., 2 mM) was dissolved in DMF (5 ml.) and the 
solution was cooled to 0.degree. C. N-carbonyldimidazole (0.33 g., 2 mM) 
was added and the mixture was stirred for 45 minutes at 0.degree. C. 
Benzyloxycarbonylglycine hydrazide (0.45 g. 2 mM) was added and the 
mixture was stirred at 0.degree. C. overnight. The solvent was evaporated 
and the residue was dissolved in ethyl acetate (100 ml.) and washed with 
water (3.times.15 ml.), dried (MgSO.sub.4) and filtered. The filtrate was 
evaporated and the residue was chromatographed on a column of silica gel 
equilibrated and eluted with methanol/chloroform 1:19 v/v. The product was 
located by t.l.c. of the fractions from the column. Those which contained 
pure product were combined and evaporated to give a foam. This was 
triturated with petroleum ether (b.p. 60.degree.-80.degree. C.) to give 
Z-Gly-Azgly-Phe-Leu-OBu.sup.t. 
The last-named compound (0.5 g., 1 mM) was dissolved in methanol (45 ml.), 
and 5% palladium-on-carbon catalyst (100 mg.) in water (5 ml.) was added. 
The mixture was vigorously stirred and a slow stream of hydrogen gas was 
bubbled through it at ambient temperature for 4 hours. The catalyst was 
filtered off (Kieselguhr) and washed with methanol. The solvent was 
evaporated from the filtrate and washings and the residue was dried in 
vacuo over phosphorus pentoxide to give H-Gly-Azgly-Phe-Leu-OBu.sup.t as a 
foam. 
(Allyl).sub.2 -Tyr(Bu.sup.t)-OH (0.7 g., 2.2 mM) was dissolved in DMF (5 
ml.) and the solution was cooled to -10.degree. C. N-methylmorpholine 
(0.25 ml., 1 equivalent) was added, followed by ethyl chloroformate (0.2 
ml., 0.95 equivalent). After 3 minutes at -10.degree. C., a solution of 
H-Gly-Azgly-Phe-Leu-OBu.sup.t (see above; 0.5 g., 1 mM) in DMF (5 ml.) was 
added and the mixture was stirred at 0.degree. C. overnight. The solvent 
was evaporated and the residue was dissolved in ethyl acetate (100 ml.), 
and washed with water (2.times.10 ml.), 2 N-aqueous potassium bicarbonate 
(3.times.10 ml.) and water (3.times.10 ml.). The solution was dried 
(MgSO.sub.4) and filtered. The solvent was evaporated to give the crude 
product which was chromatographed on a column of silica gel equilibrated 
and eluted with methanol/chloroform 1:19 v/v. The product was located by 
t.l.c. of the fractions from the column. Those which contained the pure 
product were combined and evaporated and there was thus obtained 
(allyl).sub.2 -Tyr(Bu.sup.t)-Gly-Azgly-Phe-Leu-OBu.sup.t. 
SM6 
(allyl).sub.2 -Tyr(Bu.sup.t)-Gly-Gly-Phe-.psi.(NHCO)-D,L-Leu-OEt 
This compound, which was used as the starting material in Example 49, was 
obtaned as follows: 
A. Boc-Gly-Gly-NH-CH(Bzl)-NH.sub.2 
Boc-Gly-GLy-Phe-OMe (8.0 g., 20 mM) was dissolved in methanol (25 ml.), and 
95% v/v hydrazine hydrate (3.2 ml.) was added. The mixture was kept at 
room temperature for 24 hours and then evaporated. The residue was 
chromatographed on a column of silica gel equibrated and eluted with 
methanol/chloroform 1:9 v/v. The fractions which contained the required 
hydrazide were combined and evaporated to give Boc-Gly-Gly-Phe-NHNH.sub.2, 
R.sub.f P 0.6, R.sub.f Q 0.3. 
The last-named compound (7.9 g., 20 mM) was suspended in 2 N-aqueous 
hydrochloric acid (30 ml., 3 equivalents) at 0.degree. C. A solution of 
sodium nitrite (1.52 g., 1.1 equivalents) in water (10 ml.) was then 
added. The precipitated azide was extracted immediately into ethyl acetate 
(200 ml.), and the organic phase was washed successively with ice-cold 
water (2.times.20 ml.) and 2 N-aqueous potassium bicarbonate (3.times.15 
ml.), and then dried (MgSO.sub.4). The solution was filtered from drying 
agent and warmed gently to 50.degree. C. Benzyl alcohol (17 ml., 10 
equivalents) was then added and the mixture was kept at ambient 
temperature for 24 hours. The solvent was evaporated and the residue was 
triturated with cyclohexane. The resulting crystalline solid was 
recrystallised from ethyl acetate/cyclohexane to give 
Boc-Gly-Gly-NHCH(Bzl)NHCOOBzl, the structure of which was confirmed by 
NMR. 
The last-named compound (1.45 g., 3 mM) was dissolved in DMF (15 ml.) and 
5% palladium-on-carbon catalyst (0.2 g.) was added. The mixture was 
vigorously stirred and a slow stream of hydrogen gas was bubbled through 
it at ambient temperature for 6 hours. The catalyst was filtered off 
(Kieselguhr) and washed with DMF (10 ml.). There was thus obtained a 
solution of Boc-Gly-Gly-NHCH(Bzl)NH.sub.2 in DMF ("Solution A"). 
B. OCP-CO-CH(Bu.sup.i)-CO.sub.2 Et 
Ethyl 2-(ethoxycarbonyl)-4-(methyl)pentanoate (6 g., 28 mM) was dissolved 
in ethanol (9 ml.) and 4 N-aqueous sodium hydroxide (7 ml.) was added. The 
mixture was stirred at ambient temperature for 1 hour and then evaporated. 
The residue was diluted with water (30 ml.) and the aqueous solution was 
washed with diethyl ether (3.times.10 ml.). The combined ethereal washings 
were backextracted with water (5 ml.). The combined aqueous phases were 
acidified to pH 3 at 0.degree. C. with citric acid, and the resulting oil 
was extracted into ethyl acetate (4.times.20 ml.). The combined extracts 
were washed with water (2.times.20 ml.) and dried (MgSO.sub.4). The 
mixture was filtered and the solvent was evaporated to give 
2-ethoxycarbonyl-4-methylpentanoic acid as an oil. The structure was 
confirmed by NMR. 
The said acid (4.7 g., 25 mM) and 2,4,5-trichlorophenol (5.9 g., 30 mM) 
were dissolved in diethyl ether (100 ml.), and the solution was cooled to 
0.degree. C. DCCI (5.2 g., 25 mM) was added and the mixture was stirred at 
0.degree. C. for 16 hours. The dicyclohexylurea which was formed was 
filtered off and washed with diethyl ether. The filtrate and washings were 
combined and the solvent was evaporated to give an oil. This was purified 
on a column of silica gel equilibrated and eluted with petroleum ether 
(60.degree.-80.degree. C.) to give 2-ethoxycarbonyl-4-methyl-pentanoic 
acid 2,4,5-trichlorophenyl ester as an oil. The structure was confirmed by 
NMR. 
C. Solution A (see section A above) was cooled to 0.degree. C. and the 
ester named immediately above (2.2 g., 6 mM) was added. The mixture was 
kept at 0.degree. C. for 16 hours, and the solvent was then evaporated. 
The residue was dissolved in ethyl acetate (50 ml.), and the crude product 
was precipitated by the addition of petroleum ether (b.p. 
60.degree.-80.degree. C.). The crude product was separated by filtration 
and purified on a column of silica gel equilibrated and eluted with 
methanol/chloroform 1:19 v/v. The fractions which contained pure product 
were combined and evaporated to give 
Boc-Gly-Gly-Phe-.psi.(NHCO)-D,L-Leu-OEt. 
The last-named compound (1.2 g., 2 mM) was dissolved in ethyl acetate (50 
ml.), and a solution of hydrogen chloride in ethyl acetate (6 molar, 15 
ml.) was added. The mixture was kept at ambient temperature for one hour. 
The solvent was then evaporated and the residue ("compound B") was dried 
in vacuo over phosphorus pentoxide and sodium hydroxide. 
(Allyl).sub.2 -Tyr(Bu.sup.t)-OH (0.93 g., 2.5 mM) and 
1-hydroxybenzotriazole (0.4 g., 3 mM) were dissolved in DMF (3 ml.), and 
DCCI (0.52 g., 2.5 mM) was added at 0.degree. C. After 15 minutes at 
0.degree. C., compound B (2 mM) was added as a suspension in DMF (5 ml.), 
followed by triethylamine (0.42 ml., 3 mM). The mixture was stirred at 
0.degree. C. for 16 hours. The precipitated dicyclohexylurea was filtered 
off and washed with DMF. The combined filtrate and washings were 
evaporated and the residue was dissolved in ethyl acetate (75 ml.), washed 
successively with water (2.times.15 ml.) and 2N-aqueous potassium 
bicarbonate (3.times.15 ml.), and dried (MgSO.sub.4). The mixture was 
filtered and the solvent was evaporated to give an oil. This was purified 
on a column of silica gel equilibrated and eluted with methanol/chloroform 
1:19 v/v. The product was located by t.l.c. of the fractions from the 
column and those which contained pure product were combined and evaporated 
to give (allyl).sub.2 -Tyr(Bu.sup.t)-Gly-Gly-Phe-.psi.(NHCO)-D,L-Leu-OEt 
having R.sub.f H 0.88, R.sub.f P 0.16 and R.sub.f Q 0.34. 
SM7 
(allyl).sub.2 -Tyr(Bu.sup.t)-Gly-Gly-.psi.(CH.sub.2 S)-Phe-Leu-OMe 
This compound, which was used as the starting material in Example 46, was 
obtained as follows: 
R-2-Bromo-3-phenylpropionic acid (11.46 g., 50 mM), followed by cysteamine 
hydrochloride (17.04 g., 150 mM), were added to a stirred solution of 0.5 
M-aqueous sodium bicarbonate (300 ml.) which was continuously purged with 
a slow steam of nitrogen. After one hour the reaction flask was sealed and 
left to stand at room temperature for three days, by which time a white 
precipitate had separated. The mixture was cooled in an ice-bath and then 
filtered, and the filtrate was evaporated in vacuo to a small volume. At 
that stage a solid precipitated. The mixture was filtered and the solid 
was dissolved in warm water (500 ml.). The solution was applied to a 
column of cation-exchange resin (BIO-REX 70; H.sup.+ form; 20.times.3.0 
cms.). The product was washed from the resin with water (500 ml.), and the 
solution obtained was evaporated in vacuo until a crystalline solid began 
to separate. The mixture was set on one side for 16 hours and then 
filtered. The solid residue was washed with ethanol and then crystallised 
from ethanol. There was thus obtained H-Gly-.psi.(CH.sub.2 S)-Phe-OH, 
[.alpha.].sub.D.sup.21 -6.8.degree. (c 1, in water). 
The last-named compound (2.3 g., 10.2 mM) and triethylamine (1.42 ml., 10.2 
mM) were dissolved in DMF/water (1:2 v/v; 45 ml.). The solution was cooled 
to 4.degree. C. and to it was added Boc-Gly-OSu (2.8 g.; 10.2 mM). The 
mixture was stirred for 16 hours at 4.degree. C. More Boc-Gly-OSu (250 
mg.) was added, and stirring was continued for another 16 hours. The 
solvent was evaporated in vacuo and the oil residue was partitioned 
between ethyl acetate (100 ml.) and M-aqueous citric acid (100 ml.). The 
two phases were separated, and the organic phase was washed successively 
with M-aqueous citric acid (100 ml.) and saturated brine (100 ml.), and 
then dried (MgSO.sub.4). The solution was evaporated in vacuo. The residue 
was dissolved in 50% v/v aqueous methanol (200 ml.), the solution applied 
to a column of ion-exchange resin (AG 1-X2; OAc.sup.- form; 16.times.30 
cms.) and eluted with a gradient of 0 to 4% v/v acetic acid in 50% v/v 
aqueous methanol. The appropriate fractions were combined, the solvent 
evaporated in vacuo, and the residue was dried in vacuo over sodium 
hydroxide pellets. There was thus obtained Boc-Gly-Gly-.psi.(CH.sub.2 
S)-Phe-OH, the structure of which was confirmed by NMR. 
A solution of the last-named compound (2.88 g., 7.5 mM), 
1-hydroxybenzotriazole (1.5 g., 11.25 mM) and methyl leucinate 
hydrochloride (1.5 g. 8.25 mM) in DMF (15 ml.) was stirred at 0.degree. C. 
To the solution was added a solution of triethylamine (1.15 ml., 8.25 mM) 
and DCCI (1.75 g., 8.25 mM) in DMF (4 ml.). The mixture was stirred at 
4.degree. C. for 48 hours and then filtered. The filtrate was evaporated 
in vacuo and the residue was partitioned between ethyl acetate (50 ml.) 
and water (50 ml.). The two phases were separated and the organic phase 
was washed successively with M-aqueous citric acid (50 ml.), saturated 
brine (50 ml.), 10% w/v aqueous sodium bicarbonate (50 ml.) and saturated 
brine (50 ml.), and then dried (MgSO.sub.4). The solution was evaporated 
in vacuo and the residue was purified by column chromatography on silica 
(Merck Kieselgel 7754; 250 g.) using chloroform containing 0-2.5% v/v of 
methanol as the eluent. The appropriate fractions were combined and the 
solvent evaporated in vacuo, and there was thus obtained 
Boc-Gly-Gly-.psi.(CH.sub.2 S)-Phe-Leu-OMe, R.sub.f P 0.46 and R.sub.f Q 
0.58. 
The last-named compound (1.58 g.) was dissolved in 2 M hydrogen chloride in 
ethyl acetate (70 ml.), and the solution kept at 23.degree. C. for 1.5 
hours. The solvent was evaporated in vacuo, the residue was dissolved in 
methanol (50 ml.), and the solvent was again removed in vacuo. The residue 
was mixed with diethyl ether, the solvent was decanted away, and the 
residual gum was dissolved in 50% v/v aqueous methanol (200 ml.). The 
solution was passed through a column of an anion-exchange resin (AG 1-X2, 
OAc.sup.- form). The resulting eluate was applied to a cation-exchange 
resin (BIO-REX 70; H.sup.+ form), and the product was eluted with a 
gradient of 0-4% v/v acetic acid in 50% v/v aqueous methanol. The 
product-containing fractions were combined and concentrated by evaporation 
to yield H-Gly-Gly-.psi.(CH.sub.2 S)-Phe-Leu-OMe, R.sub.f K 0.55, R.sub.f 
K' 0.34. 
Isobutyl chloroformate (0.13 ml., 1 mM) was added to a stirred, cooled 
(-20.degree. C.) solution of (allyl).sub.2 -Tyr(Bu.sup.t)-OH (320 mg., 1 
mM) and N-methylmorpholine (0.11 ml., 1 mM) in THF (10 ml.). The reaction 
mixture was stirred for 4 minutes at -18.degree. C. A solution of 
H-Gly-GLy-.psi.-(CH.sub.2 S)-Phe-Leu-OMe (470 mg.) and triethylamine (0.14 
ml., 1 mM) in THF (10 ml.) and DMF (2 ml.) was added. The reaction mixture 
was kept at -18.degree. C. for 30 minutes and then at 4.degree. C. for 16 
hours. The solvent was evaporated in vacuo and the residue was partitioned 
between ethyl acetate (25 ml.) and water (25 ml.). The two phases were 
separated and the organic phase was washed successively with saturated 
aqueous potassium bicarbonate (25 ml.) and saturated brine (25 ml.), and 
then dried (MgSO.sub.4). The solvent was evaporated and the residue was 
applied to a column of silica (Merck Kieselgel 7754; 120 g.) and eluted 
with chloroform containing 0-2.5% v/v methanol. The appropriate fractions 
were combined and the solvent was evaporated. The residue was dissolved in 
t-butanol containing 5% v/v water and the solution was freeze-dried. There 
was thus obtained (allyl).sub.2 -Tyr(Bu.sup.t)-Gly-Gly-.psi.(CH.sub.2 
S)-Phe-Leu-OMe having R.sub.f P 0.3 and R.sub.f Q 0.53. 
SM8 
(Allyl).sub.2 -Tyr(Bu.sup.t)-Gly-Gly-Phe-NHBzl 
This compound, which was used as the starting material in Example 77, was 
obtained as follows: 
Ethyl chloroformate (80 .mu.l., 0.86 mM.) was added to a stirred, cooled 
(-20.degree. C.) solution of (allyl).sub.2 -Tyr(Bu.sup.t)-Gly-Gly-Phe-OH 
(500 mg., 0.86 mM) and N-methylmorpholine (95 .mu.l. 0.86 mM) in DMF (5 
ml.). After 1 minute, benzylamine hydrochloride (286 mg., 2.0 mM) was 
added, followed by N-methylmorpholine (220 .mu.l., 2.0 mM). The reaction 
mixture was stirred for 1 hour with no further cooling, and the solvent 
was then evaporated in vacuo. The residue was partitioned between diethyl 
ether (25 ml.) and saturated aqueous sodium bicarbonate (25 ml.). The two 
phases were separated and the organic phase was washed successively with 
water (25 ml.) and saturated brine (25 ml.), and then dried (Na.sub.2 
SO.sub.4). The solvent was evaporated in vacuo and the residue was 
purified on a column of silica (Merck Kieselgel 7754) using 0-2% v/v 
methanol in chloroform as the eluent. The appropriate fractions were 
combined and the solvent evaporated in vacuo to give (allyl).sub.2 
-Tyr(Bu.sup.t)-Gly-Gly-Phe-NHBzl. 
SM9 
(allyl).sub.2 -Tyr(Bu.sup.t)-Gly-D-Ala-Phe-Leu-OBu.sup.t 
This compound, which was used as the starting material in Example 27, was 
obtained as follows: 
A. (allyl).sub.2 -Tyr(Bu.sup.t)-OH 
This intermediate was obtained by either of the following alternative 
procedures: 
(1) A mixture of O-t-butyltyrosine (22.41 g., 94.5 mM), sodium bicarbonate 
(39.64 g., 470 mM) and allyl bromide (40.3 ml., 470 mM) in 10% v/v aqueous 
methanol (315 ml.) was refluxed for 4 hours. The solvent was removed by 
evaporation and the residue partitioned between water (150 ml.) and ethyl 
acetate (300 ml.). The two phases were separated and the organic phase was 
washed with saturated brine (100 ml.), dried (MgSO.sub.4), and 
concentrated by evaporation to give an oily residue. The residue was 
purified by column chromatography (silica, eluted with chloroform), and 
there was thus obtained (allyl).sub.2 -Tyr(Bu.sup.t)-Oallyl. 
Aqueous 2 M-sodium hydroxide (88 ml.) was added to a solution of the 
last-named compound (57 g., 159 mM) in methanol (400 ml.), and the mixture 
was heated under reflux. Water was added to the reaction mixture in 20 ml. 
portions at intervals until no more turbidity was observed (the total 
volume of water added was approximately 200 ml. over 2 hours). The 
reaction mixture was then cooled and most of the methanol was removed by 
evaporation in vacuo. The resulting solution was diluted to 500 ml. with 
water and washed with diethyl ether (2.times.100 ml.). The aqueous phase 
was acidified to pH 4 with solid citric acid and the product was extracted 
with diethyl ether (2.times.200 ml.). The combined ethereal extracts were 
washed with saturated brine (100 ml.), dried (MgSO.sub.4), and 
concentrated by evaporation to give an oily residue. Petroleum ether (b.p. 
40.degree.-60.degree. C.; 300 ml.) was added and the mixture was stirred a 
few minutes until a thick white precipitate had formed. More petroleum 
ether (b.p. 40.degree.-60.degree. C.; 500 ml.) was added and after 
trituration the solid was collected by filtration, washed with petroleum 
ether and dried. There was thus obtained (allyl).sub.2 -Tyr(Bu.sup.t)-OH, 
having m.p. 76.degree.-8.degree. C., R.sub.f 0.5 in methanol/chloroform 
4:1 by volume and containg 1% v/v acetic acid. 
(2) Z-Tyr(Bu.sup.t)-OMe (23.7 g.; 61.5 mM) was dissolved in isopropanol 
(190 ml.). A suspension of 10% palladium-on-charcoal (2.4 g.) was added 
and a slow stream of hydrogen gas was bubbled through the vigorously 
stirred reaction mixture which was maintained at room temperature. 
Throughout the hydrogenolysis a solution of 10% oxalic acid in water (w/v) 
was added by means of an acetotitrator to maintain a pH of 3. When the 
theoretical amount of oxalic acid had been consumed the reaction mixture 
was diluted with water (100 ml.) to dissolve a small amount of solid which 
had precipitated, and the catalyst was removed by filtration. The filtrate 
was concentrated by evaporation, the residual white solid was 
recrystallised from ethanol (100 ml.), and there was thus obtained 
H-Tyr(Bu.sup.t)-OMe monooxalate hemihdyrate, m.p. 155.degree.-6.degree. C. 
The last-named compound (13 g.) was partitioned between ethyl acetate (200 
ml.) and saturated aqueous sodium bicarbonate (100 ml.). The two phases 
were separated, the organic phase was dried (MgSO.sub.4), and the solvent 
was evaporated to leave a yellow oil. The oil was dissolved in methanol 
containing 10% v/v water. Sodium bicarbonate (14.86 g., 177 mM) and allyl 
bromide (15 ml., 174 mM) were added and the mixture was heated under 
reflux for 2 hours. The solvent was evaporated in vacuo and the residue 
was partitioned between water (100 ml.) and diethyl ether (100 ml.). The 
two phases were separated, both being retained. The aqueous phase was 
extracted with more diethyl ether (50 ml.). The combined ethereal extracts 
were washed successively with 20% w/v aqueous citric acid (50 ml.) and 
saturated brine (50 ml.), and then dried (MgSO.sub.4) and concentrated by 
evaporation to give a yellow oil. The oil was purified by column 
chromatography (silica; Merck Kieselgel 7734; eluted with chloroform). The 
appropriate fractions were combined and the solvent removed by evaporation 
to give (allyl).sub.2 -Tyr(Bu.sup.t)-OMe. 
The last-named compound (7.90 g., 23.86 mM) was dissolved in methanol (35 
ml.). Aqueous 2 M-sodium hydroxide (13.20 ml.) was added and the mixture 
was refluxed for 3 hours. The solvent was removed by evaporation in vacuo. 
The residual oil was dissolved in water (200 ml.), washed with diethyl 
ether (3.times.25 ml.), and acidified with citric acid (6.0 g.). The 
aqueous phase was saturated with solid sodium chloride and the insoluble 
oil which separated was extracted into ethyl acetate (3.times.50 ml.). The 
combined ethyl acetate extracts were washed successively with water 
(2.times.20 ml.), M-aqueous potassium bicarbonate (20 ml.), water 
(2.times.20 ml.) and saturated brine (20 ml.). The solution was dried 
(MgSO.sub.4), and the solvent was removed by evaporation in vacuo. The 
residual oil was triturated with petroleum ether (b.p. 
60.degree.-80.degree. C.), whereupon a white solid was formed. The solid 
was collected by filtration, washed with petroleum ether (b.p. 
60.degree.-80.degree. C.), and dried. There was thus obtained 
(allyl).sub.2 -Tyr(Bu.sup.t)-OH, having m.p. 70.degree.-78.degree., 
R.sub.f 0.5 in methanol/chloroform 4:1 v/v and containing 1% v/v acetic 
acid. 
B. (allyl).sub.2 -Tyr(Bu.sup.t)-Gly-OMe 
This intermediate was obtained by either of the following alternative 
procedures: 
(1) Ethyl chloroformate (4.4 ml., 47.3 mM) was added to a stirred, cooled 
(-20.degree. C.) solution of (allyl).sub.2 -Tyr(Bu.sup.t)-OH (14.84 g., 
46.67 mM; see section A immediately above) and N-methylmorpholine (5.2 
ml., 47.3 mM) in DMF (150 ml.). After 45 seconds methyl glycinate 
hydrochloride (5.86 g., 46.67 mM) and N-methylmorpholine (5.2 ml. 47.3 mM) 
were added, and the mixture was stirred for 1.5 hours with no further 
cooling. Sufficient water was then added to give a clear solution and the 
solvent was removed by evaporation. The residue was partitioned between 
ethyl acetate (100 ml.) and water (100 ml.), the two phases were 
separated, and the ethyl acetate phase was washed successively with 
aqueous 20% w/v citric acid (50 ml.), saturated aqueous sodium bicarbonate 
(50 ml.), and saturated brine (50 ml.). The solution was dried (Na.sub.2 
SO.sub.4), the solvent was evaporated, and there was thus obtained 
(allyl).sub.2 -Tyr(Bu.sup.t)-Gly-OMe. 
(2) To a solution of Z-Tyr(Bu.sup.t)-Gly-OMe (1.47 g., 3.32 mM) in methanol 
(20 ml.) was added a suspension of 10% palladium-on-charcoal (180 mg.) in 
water (5 ml.). The mixture was stirred vigorously and a slow stream of 
hydrogen was bubbled through it for 3 hours at room temperature. The 
catalyst was removed by filtration. Allyl bromide (1.42 ml., 16.6 mM) and 
sodium bicarbonate (1.4 g., 16.6 mM) were added to the filtrate, and the 
mixture was refluxed for 4.5 hours. The mixture was then cooled to 
20.degree. C., and the supernatant liquid was decanted from the 
precipitated solid. The liquid was concentrated by evaporation. The 
residue was partitioned between ethyl acetate (30 ml.) and water (20 ml.). 
The two phases were washed successively with water (2.times.20 ml.) and 
saturated brine (20 ml.), dried (Na.sub.2 SO.sub.4), and concentrated by 
evaporation to give a partially solid residue. This was purified by column 
chromatography (silica; Merck Kieselgel 7754; eluted with chloroform 
containing 0 to 2% v/v methanol) and the appropriate fractions were 
evaporated in vacuo to give (allyl).sub.2 -Tyr(Bu.sup.t)-Gly-OMe, the 
structure of which was confirmed by NMR. 
C. (allyl).sub.2 -Tyr(Bu.sup.t)-Gly-OH 
Aqueous 2 M sodium hydroxide (22 ml.) was added to a stirred solution of 
(allyl).sub.2 -Tyr(Bu.sup.t)-Gly-OMe (15.6 g., 40.3 mM; see section B 
above) in methanol (150 ml.). After 45 minutes the solvent was removed by 
evaporation to give a partially solid residue. The residue was partitioned 
between water (50 ml.) and ethyl acetate (50 ml.). The two phases were 
separated, and the aqueous phase was acidified with aqueous citric acid 
(20% w/v, 30 ml.) and extracted with ethyl acetate (4.times.50 ml.). The 
combined ethyl acetate extracts were dried (MgSO.sub.4) and concentrated 
by evaporation to leave a clear yellow oil. The oil was dissolved in 50% 
v/v aqueous methanol and applied to a column of anion exchange resin (AG 
1-X2; OAc.sup.- form). The column was eluted with increasing 
concentrations of acetic acid (0 to 0.5 M increased in stepwise manner) in 
50% v/v aqueous methanol, and the fractions containing pure product were 
combined. The solvent was removed by evaporation to yield a clear yellow 
oil. The oil was freeze-dried from t-butanol containing 5% v/v water. The 
freeze-dried solid was dissolved in ethyl acetate (65 ml.), and 
dicyclohexylamine (7.5 ml.) was added, whereupon a white precipitate 
slowly formed. This was collected by filtration and washed with ethyl 
acetate to give the dicyclohexylammonium salt of (allyl).sub.2 
-Tyr(Bu.sup.t)-Gly-OH, m.p. 152.degree.-152.5.degree. C. 
D. (allyl).sub.2 -Tyr(Bu.sup.t)-Gly-D-Ala-Phe-Leu-OBu.sup.t 
Pivaloyl chloride (0.63 ml., 5.12 mM) was added to a stirred, cooled 
(-10.degree. C.) suspension of the above-mentioned dicyclohexylammonium 
salt (2.85 g., 5.14 mM) in DMF (20 ml.). The mixture was stirred for 15 
minutes at -10.degree. C. A solution of H-D-Ala-Phe-Leu-OBu.sup.t (2.07 
g., 5.10 mM) in DMF (20 ml.) was added and the mixture was stirred for 1 
hour at 0.degree. C. and then for 2.5 hrs. at 25.degree. C. Water (100 
ml.) was added, the mixture was extracted with ethyl acetate (3.times.100 
ml.), and the combined ethyl acetate extracts were washed successively 
with 20% w/v aqueous citric acid (50 ml.), saturated aqueous sodium 
bicarbonate (50 ml.) and saturated brine (100 ml.). The organic solution 
was dried (MgSO.sub.4), and the solvent was evaporated in vacuo. The 
residue was purified by column chromatography on silica (Merck Kieselgel 
7754) and elution with 0-1 % v/v methanol in chloroform. The appropriate 
fractions were combined and the solvent evaporated in vacuo. There was 
thus obtained (allyl).sub.2 -Tyr(Bu.sup.t)-Gly-D-Ala-Phe-Leu-OBu.sup.t, 
the structure of which was confirmed by NMR. 
##STR122##