DERIVATIVES OF RETRO-ENANTIO-SOMATOSTATIN, INTERMEDIATES THEREFOR AND PROCESS THEREFOR

DERIVES DE RETROENANTIOSOMATOSTATINE

English Abstract

DERIVATIVES OF RETRO-ENANTIO-SOMATOSTATlN.
INTERMEDIATES THEREFOR,AND PROCESS THEREFOR
Abstract of the Disclosure
Compounds of the formula I or Ia
<IMG>
(I.)
<IMG>
(Ia.)
in which R is hydrogen or CONHCH2CONHCH2CH3 are disclosed. The
compounds are obtained by a process which comprises the following
step: preparing peptide fragments II, III, V and VII see below,
by a series of condensations involving the reaction of an appropriately
protected peptide having an activated ester group with an appropriately
protected peptide having a free amino group; condensing
<IMG> (II)
by means of the azide method with <IMG> (III)
followed by hydrogenolysis of the reaction product to obtain
<IMG> (IV),
condensing the latter by means of the azide method with
<IMG> (V) followed by treating the
resulting compound with hydrazine hydrate to obtain
<IMG>
(VI), condensing the latter by means of the azide method with
-1-

HNH?HCH2STrt (VII) in which R is hydrogen or CONHCH2CONHCH2CH3 to
obtain the linear protected peptide <IMG>
(VIII) in which R is
as defined herein; thereafter said linear peptide is transformed into the
desired cyclic peptide of formula I by deprotecting and oxidizing
processes. In addition, the linear, reduced form of the peptide of
formula Ia is obtained by deprotection of the aforementioned linear
peptide or by reduction of the cyclic peptide. The peptides of
formulae I and Ia are useful for the management of diabetes and the
treatment of acromegaly. Methods for their use are also disclosed.
-2-

Claims

The embodiments of this invention in which an exclusive
property or privilege is claimed are defined as follows:
1. A process for preparing a peptide of formula I or Ia
<IMG>
(1)
<IMG>
(Ia)
in which R is hydrogen or CONHCH2CONHCH2CH3, or a pharmaceutically
acceptable salt thereof, which comprises selecting a process from
the group of processes consisting of:
(a) when a compound of formula I is required,
reacting a decapeptide of formula VI
<IMG>
with a reagent that furnishes nitrous acid in situ in the presence of
a strong acid to convert said decapeptide to the corresponding azide
and reacting said azide with a compound of formula VII, HNH?HCH2STrt,
in which R is as defined herein to obtain the corresponding linear pro-
tected peptide of formula VIII
<IMG>
in which R is as defined herein followed by oxidizing said
linear protected peptide with iodine or thiocyanogen to obtain the
corresponding cyclic disulfide derivative of formula IX
66

<IMG>
in which R is as defined herein and subsequently removing all remaining
protecting groups under moderately acidic condition to obtain the
corresponding peptide of formula I; or
subjecting said linear peptide to treatment with either
mercuric acetate, mercuric chloride, silver acetate or silver nitrate to
remove selectively the sulfhydryl protecting groups to obtain the
mercuric or disilver salt, respectively, of the corresponding di-
sulfhydryl derivative; converting the latter salt to its corresponding
free disulfhydryl derivative of formula X by treatment with hydrogen
sulfide, oxidizing said last-named derivative by treatment with
oxygen, 1,2-dilodoethane, sodium or potassium ferricyanide or iodine
to obtain the corresponding cyclic disulfide and removing the
remaining protecting groups under moderately acidic conditions to
obtain the desired peptide of formula I;
(b) when a compound of formula Ia is required,
removing the protecting groups under moderately acidic conditions
from said linear protected peptide of formula VIII to obtain said
corresponding peptide of formula Ia; and
(c) when a pharmaceutically acceptable salt of said compound
of formula I or Ia is required,
reacting said compound of formula I or Ia with a pharmaceutically
acceptable acid to obtain the corresponding pharmaceutically acceptable
salt of the corresponding compound of formula I or Ia.
67

2. A process as claimed In Claim I in which the decapeptide
of formula VI is prepared by reacting a tripeptide of formula V,
<IMG> , with a reagent that furnishes
nitrous acid in situ in the presence of a strong acid to convert said
tripeptide to the corresponding azide and reacting said azide with a hepta-
peptide of formula IV, <IMG> , to
obtain the decapeptide of formula
<IMG>
, followed by reacting said last-named compound with hydrazine
hydrate and isolating said decapeptide of formula VI.
3. A process as claimed in Claim I in which the compound of for-
mula VII, HNH?HCH2STrt, in which R is CONHCH2CONHCH2CH3 (VIIb) is prepared by
reacting the dipeptide of formula <IMG> with ethylamine to
obtain the corresponding dipeptide of formula <IMG> and
removing the terminal amino protecting group (Trt) of said last-
named compound under mildly acidic conditions to obtain said compound of
formula HNH?HCH2STrt in which R-is CONHCH2CONHCH2CH3.
68

4. A process as claimed in Claim 2 in which the tripeptide
of formula V, <IMG> , is prepared by
reacting a tripeptide of formula <IMG> witn an
activated ester of Trt-S-CH2CH2CO2H to obtain <IMG>
, followed by reacting the last-named compound with hydrazine
hydrate and isolating said tripeptide of formula V.
5. A process as claimed in Claim 4 in which the tripeptide
of formula <IMG> is prepared by reacting a dipeptide
of formula <IMG> with an activated ester of <IMG> to
obtain <IMG> and removing the terminal amino protecting
group (Z) of said last-named compound by hydrogenation, in the presence of
a noble metal catalyst to obtain said tripeptide.
6. A process as claimed in Claim 5 in which the dipeptide of
formula <IMG> is prepared by reacting H-D-Phe-OMe witn an
activated ester of <IMG> to obtain <IMG> and removing the
terminal amino protecting group (Z) of said last-named compound by
hydrogenation in the presence of a noble metal catalyst to obtain said
dipeptide.
7. A process as claimed in Claim 2 in which the heptapeptide
of formula IV, <IMG> , is prepared
by reacting the tetrapeptide of formula II, <IMG> ,
with a reagent that furnishes nitrous acid in situ in the presence of a
strong acid to convert said tetrapeptide to the corresponding azide and
reacting said azide with a tripeptide of formula III, <IMG>,
69

to obtain a heptapeptide of formula <IMG>
and removing the terminal amino protecting group (Z) of
said last-named compound by hydrogenation in the presence of a noble
metal catalyst and isolating said heptapeptide.
8. A process as claimed in Claim 7 in which the tetrapeptide of
formula II, <IMG> , is prepared by reacting a
tripeptide of formula <IMG> with an activated ester of
<IMG> to obtain a tetrapeptide of formula <IMG> ,
followed by reacting said last-named compound with hydrazine hydrate and
isolating said tetrapeptide.
9. A process as claimed in Claim 8 in which the tripeptide
of formula <IMG> , is prepared by reacting a dipeptide of
formula H-D-Trp-D-Phe-OMe with an activated ester or <IMG> to obtain
a tripeptide of formula <IMG> and removing the terminal
protecting group (Z) of said last-named compound by hydrogenation in the
presence of a noble metal catalyst to obtain said tripeptide.
10. A process as claimed in Claim 9 in which the dipeptide
of formula H-D-Trp-D-Phe-OMe is prepared by reacting H-D-Phe-OMe with an
activated ester of Z-D-Trp-OH to obtain a dipeptide of formula
Z-D-Trp-D-Phe-OMe and removing the terminal amino protecting group (Z)
of said last-named compound by hydrogenation in the presence of a noble
metal catalyst to obtain said dipeptide.

11. A process as claimed in Claim 7 in which the tripeptide
of formula III, <IMG> , is prepared by reacting a dipeptide
of formula <IMG> with an activated ester of Z-D-Phs-OH to obtain
a tripeptide of formula <IMG> and removing the terminal
amino protecting group (Z) of said last-named compound by hydrogenation
in the presence of a noble metal catalyst to obtain said tripeptide.
12. A process as claimed in Claim 11 in which the dipeptide
of formula <IMG> is prepared by reacting <IMG> with an
activated ester of Z-D-Asn-OH to obtain a dipeptide of formula
<IMG> and removing the terminal amino protecting group (Z) of
said last-named compound by hydrogenation in tne presence of a noble metal
catalyst to obtain said dipepttde.
13. A process as claimed in Claim 1 in which the linear protected
peptide of formula VIII as defined therein is subjected to moderately acidic
conditions to obtain the corresponding compound of formula Ia
HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-
NH?HCH2SH in which R is as defined therein.
14. A process as claimed in Claim 1 in which the corresponding
disulfhydryl derivative as defined therein is subjected to moderately
acidic conditions to obtain the corresponding compound of formula Ia
HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-Asn-D-Lys-
NH?HCH2SH in which R is as defined therein.
71

15. The process as claimed in Claim 1 wherein said linear pro-
tected peptide of formula VIII is subjected to treatment with iodine in the
presence of a lower alkanol or acetic acid to obtain the corresponding
cyclic disulfide derivative of formula IX.
16. The process as claimed in Claim 1 wherein said linear
protected peptide of formula VIII is subjected to treatment with iodine
at from about 0° to 30°C for about 30 to 180 minutes in a lower alkanol
or acetic acid to obtain the corresponding cyclic disulfide derivative of
formula IX.
17. The process according to step (a) of Claim 1 for preparing
the compound of formula I, as defined in Claim 1, in which R is hydrogen.
18. The process according to step (a) of Claim 1 for preparing
the compound of formula I, as defined in Claim 1, in which R is CONHCH2CONHCH2CH3.
19. The process according to step (b) of Claim 1 for preparing
the compound of formula Ia, as defined in Claim 1, in which R is hydrogen.
20. The process according to step (b) of Claim 1 for preparing
the compound of formula Ia, as defined in Claim 1, in which R is
CONHCH2CONHCH2CH3.
21. The process according to step (c) of Claim 1 wherein a
compound of formula I or Ia is reacted with acetic acid to obtain the
acetic acid addition salt of the corresponding compound of formula I or Ia.
22. The process according to step (c) of Claim 1 wherein a
compound of formula I or Ia is reacted with hydrochloric acid to obtain
the hydrochloric acid addition salt of the corresponding compound of
formula I or Ia.
23. A compound of formula I or Ia
<IMG>
(I)
72

HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NH?HCH2SH
(Ia)
in which R is hydrogen or CONHCH2CONHCH2CH3 or a pharmaceutically acceptable
salt thereof, when prepared by the process of Claim 1, or an obvious chemical
equivalent thereof.
24. The compound of formula I
<IMG>
in which R is hydrogen, when prepared by the process of Claim 17, or an obvious
chemical equivalent thereof.
25. The compound of formula I
<IMG>
in which R is CONHCH2CONHCH2CH3, when prepared by the process of Claim 18 or an
obvious chemical equivalent thereof.
26. The compound of formula Ia
HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NH?HCH2SH
in which R is hydrogen, when prepared by the process of Claim 19, or an
obvious chemical equivalent thereof.
27. The compound of formula Ia
HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NH?HCH2SH
in which R is CONHCH2CONHCH2CH3, when prepared by the process of Claim 20, or an
obvious chemical equivalent thereof.
73

28. The acetic acid addition salt of a compound of
formula I or Ia of Claim 23, when prepared by the process of
Claim 21, or an obvious chemical equivalent thereof.
29. The hydrochloric acid addition salt of a compound
of formula I or Ia of Claim 23, when prepared by the process of
Claim 22, or an obvious chemical equivalent thereof.
74

Description

AHP-6550
104~623
- Backqround of this Invention
a. Field of Invention
This invention relates to deriva~ives of the te1radecapeptide
- somatostatin. More particularly, this invention concerns peptide
derivatives of retro-enantio-somatostatin and salts thereof, a process
for preparing the peptide derivatives and salts, intermediates used in
the process and methods for using the peptide derivatives and their ~ ,
salts.
; b. Prior Art
The name "somatos+atin" has been proposed for the factor
found in hypothalamic extracts which inhibits the secretion of growth
~ ,. . .
hormone (somatotropin). The structure~of this factor has been -~
elucidated by P. Brazeau, et al., Science, 179, 77 (1973) and reported
to be the following tetradecapeptide structure: -
H-Ala-Gly-Cys-Lys-Asn-phe-phe-Trp-Lys-Thr-phe-Thr-ser-cys-oH.
:1 . I I '':
The abbreviations used herein for the various amino acids
are Ala, alanine; Asn, asparagine; Cys, cysteine; Gly, glycine; Lys,
Iysine; Phe, phenylalanine; Ser, serine; Thr, threon7ne; and Trp,
tryptophane.
' The constitution of the tetradecapeptide somatostatin has
been confirmed by synthesis; for example, see D. Sarantakis and W. A.
McKinley, Biochem. Biophys. Res. Comm., 54, 234 (1973), J. Rivier,
et al., Compt. Rend. Ser. D, 276, 2737 (1973) and H.U. Immer et al.,
Helv. Chim. Acta, 57, 730 (1974).
The important physiological activity of this tetradecapeptide
established it as a compound of significance for clinical pharmacology
relating to the treatment of acromegaly and the management of diabetes;
for example, see K. Lundbaek, et al., Lancet, 2, 131 (1970) and
R. Guillemin in "Chemistry and Biology of Peptides" J. Meienhofer, Ed.,
3rd American Peptide Symposium Boston 1972, Ann Arbor Science
Publications, Ann Arbor, Mich., 1972, pp 585 - 600.
3 ~i ~

AHP-6550
1046)6Z3
The linear form of somatostatin, having two sulfhydryl
groups instead of a disulfide bridge, has been prepared recently
by J.W.F. Rivier, J. Amer. Chem. Soc., 96, 2986 (1974). He reports
that the linear form is equipotent to somatostatin based on the
ability of 1he two compounds 1o inhibit the rate of secretion of
growth hormone by rat pituitary cells in monolayer tissue cultures.
Only recently have there been reported polypeptides, other
than the natural hormone and its linear form, having somatostatin-
like activity. D. Sarantakis, et al., Biochem. Biophys. Res. Comm.,
55, 538 t1973) reportèd~`the synthesis of the somatostatin analog,
~AIa ]-somatostatin, by solid phase methods. This analog ¦ : -
exhibited a very small amount of activity~ about 0.01% of somato-
statin s potency. P. Brazeau, et al., Biochem. Biophys. Res. Comm.,
60, 1202 (1974) recently reported the synthesis of a number of
acylated des[Ala -Gly ]-somatostatin compounds, by solid phase methods.
The prcsent invention discloses new analogs of somatostatin
based on the principle of the retro-enantio system. This system
is achieved by construction of a reversed sequence of amino acids
havtng opposlle configuration, i.e., D Tnstead of L, to give the ¦
retro-enantio tsomer of the natural peptide. It is surprising
that the retro-enantio derivatives of somatostatin of formulae I or
la have been found to retain the activity of the natural hormone
somatostatin notwithstanding the fact that other hormones of the
retro-enantio system have shown a range of retffntil)n of full activity
to complete loss-Df activity, as reported in the review by
J. Rudinger, The Design of Peptide Hormone Analogs, pp 368 - 369 in I
Drug Design, Vol. II, Ed. E.J. Ariëns, Academic Press, New York and :
London, 1971.
. ' ':':
4 . ~:
, ::
. ,
~,~ , .. .. . ,, . : ..... . . .
. . .
.

AHP-6550
''
104~623
The present inven~ion discloses retro-enantio peptlde ~ ¦
derivatives which retain the activity of the natural hormone
somatostatin. The derivatives are prepared readily by a convenient
process, which includes the following advantages: the process starts .,
from readily available materials, avoids noxious reagents, is executed .
facilely and utilizes easily removable protecting groups.
The foregoing advantages and attributes render the peptides
of this invention useful for the management of diabetes and ¦'
the treatmenl of acromegaly.
SummarY of the Invention'.
,, . ~
The pept,id,es ot this invention are represented by formulae I
and la; formula I representing the cyclic peptides of this invention
and formula la representing the linear reduced form S-CH CH C0-D-Ser-
; 2 3 4 5 6 7 7 9 1O ~ 2 2 1, ~
'. D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NHICHCH2~ !1, .'
R 1:
'' ' (I)
,~ H-S-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn- .:
.I D-Lys-NHlCHCH2SH
.I R
', 20 . - . (la) .
'I In-which R is hydrogen or CONHCH2ÇONHCH2CH3.
, When R is CONHCH2CONHCH2CH3,the terminal group NHCIHCH25
~ R
1 may be written alternatively as D-Cys-Gly-NHEt, and formulae I and la
1 25 may be written as
;D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn- .
D-Lys-D-Cys-Gly-NHEt
(I) ~
or
HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-
Lys-D-Cys-Gly-NHEt
(la) , ~N
. I~ .
~ . ' :~
.,
. ~, ' ' ., . "
' ' . , '
. ' . . . ' . .
~ .'

AHP-o550
104~6Z3
The pharmaceutically acceptable salts of the compounds of
formulae I and la are also included within the scope of this ir,vention.
The peptides of this invention are prepared by a process
comprising:
Preparing peptide fragments 11, III,IV, V, Vl and Vll, see
below, by a series of condensations involving the reactlon of an
appropriately protected peptide having an activated ester group with
an appropriately protected peptide having a free amino group.
The tetrapeptide of formula Z-D-TIhr-D Llys-D-Trp-D-Phe-NHNH2
Bu 8 c
~, ~
tll) containing the amino!and residues 4 - 7 is condensed by means ~ -
of the azide me,t"hod with a tripeptide (8 - 10) of formula
,' H-'D-Phe-D-Asn-D-Lys-OMe (111) to yield the heptapeptide (4 ~ 10) of ''
,,, - Boc
formula'Z-D-TLr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lrs-OMe. The latter is . ;;
hydrogenoltzed by means of hydrogen and a noble metal catalyst to
yield the heptapeptide (4 - 10) of formula
H-D-TIlt-D-Lrs-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-OMe (IV). .
Bu Boc Boc
Sald last-named compound (IV? is condensed by means of the
azide method with the tripeptide (I - 3) of formula
2CH2CO D Sltr D Thltr D-P~e-NHNH2 (V) to yield the decapeptide ~
~, ~ of formula n
Trt-s-cH2cH2co-D-ser-D-T7r-D-phe-D-T~r-D-Lys-D-Trp-D-phe-D Phe D As
' Blt Bu Bu Boc
D-Lys-NHNH2 (Vl) containing the amino acid residues I - 10. ;~
, BbC ''~, ~
Said last-named compound (Vl) is condensed by means of the ;
azide method with HNH~HCH25 Trt (Vll) in which R is hydrogen ~VIIa) or
11
:
11'
.: ---`-- _ :'
, ~ . , ~...... .. .. . ..

A~IP-G550
104~1623
CONHCH2CONHCH2CH3 (Vllb) to yield the linear protected peptide of
formula tVIII) ''
' r S CH2CH2C-D sTt, D T~tr-D-phe-D-Tsr-D-LJs-D-Trp-D-phe-D-phe
Lys-N ~ HCH2S Trt tV111);'1n which R is as defined herein followed by
B c R
oxidizing said linear protected peptide tVIII) with iodine or thio-
cyanogen to obtain the corresponding cyclic disulfide of formula tlX)
2 H2C0 D S~t D Thl~tr-D-Phe-D-T~r-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-
I I B Bu B~ Boc
Lys-NH~HCH2S 7n whTch R is as defined herein and subsequently
B c ,; ~
removing all remaining protecting groups under moderately acidic
conditions to obtain the corresponding peptide of formula l; or
followed by subjecting saTd ITnear peptTde of formula tVlll)to
; 15 treatment with eTther mercuric.acetate, mercuric chloride, silver
, acetate or sl'lver n1trate to remove selectively the sulfhydryl pro-
~ tecting groups to obtaTn the mercuric or dTsTIver salt, respectTvely,
of the correcpondlng dlsulfhydryl derTvatlve; converttng the latter
salt to its correspondTng free dTsulfhydryl derivatTve by treatment
wlth`hydrogen sulfide, oxTdizTng said last-named derivative by
treatment wTth oxygen, 1,2-diiodoethane, sodium or potassium ferrTcy- '
anide or TodTne to obtain the correspondTng cyclic d'isulfide der-
. .
7vative, and removing the remaining protectTng groups under moderately
acidic conditions to obtain the desired peptide of formula 1.
'~ 25 Alternatively, saTd cyclTc disulfTde derivatlve Ts reduced to saTd
~ correspondTng free dTsulfhydryl derivative by agents known to be
:~ effective for reducTng known cyclTc disulfTdes to their corresponding
dTsulfhydryl derTvatTves.
:
.
~ ~,

_ AHP-6550
~104V623
A further aspect of this invention comprises the removal
of all the protec1ing groups from the aforementioned linear protected
peptide of formula (Vlll) or the aforementioned disulfhydryl der- j
ivatives under moderately acidic conditions to obtain the linear
reduced form of the peptide of this invention of formula la,
H-S-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-
D-Lys-NH~HCH~SH in which R is as defined herein.
R
! The latter compound is also obtained by direct reduction of
the cyclic peptide of .formula I by agents known to be effective for
reducing known cyclic disulfides to their corresponding disulfhydryl
i derivatives. If désired said reduced form of the cyclic peptide
y is converted to the corresponding derivative of formula I by one of
;1 the above oxidizing agents.
Details of the Invention
In general the abbreviations used herein for designating the ¦;
j amino acids ana the protective groups are based on recommendations
¦ of the IUPAC-IUB Commission on Biochemicàl Nomenclature, see
¦ Btochernlstry, Il, 1726-1732 (1972). For instance, Gly, Cys~ Lys, Asn,
Phe, Trp, Thr, and Ser represent the "residues"of glycine, cysteine,
~ ;
Iyslne, asparagine, phenylalanine, tryptophane, threonine and serine,
respectivsly. By the residue is meant a radical derived from the ¦
corresponding D-amino acid by eliminating the OH portion of the ¦
carboxyl group and the H portion of the amino group. All the amino
acids have the unnatural D-configuration.
~i A number of procedures or techniques for the preparation of l ~ I
peptid~s have hitherto been well established. For instance, the I
I functional groups which are not involved in the peptide bond ~ -
; ; formation reaction are optionally protected by a protecting group
or groups prior to the condensation reaction. For example, pro- -
tecting groups which may be chGsen for an amino function of a
'. .'
~: : : 8 - :-` , ~. ..
.,~.................................. . .
~ ' .
~ .. . :
:'~ .~ I : .~,
~ ~ ., ' , . , ' , ~''

AHP-6550
1~40623 :
peptide or amino acid not involved in the peptide bond formation
are the alkoxycarbonyls which include benzyloxycarbonyl (represented
- by Z), t-butyloxycarbonyl (represented by Boc), ~Ja-dimethyl-3~5
j dimethoxybenzyloxycarbonyl (represented by Ddz), 2-(P-biphenyl)-
isopropyloxycarbonyl (represented by Bpoc), i~-chlorobenzyloxy-
carbonyl, D~methoxybenzyloxycarbonyl, isopropyloxycarbonyl, or
ethoxycarbonyl; the acyl type protecting groups which include
~ormyl, trifluoroacetyl, phthalyl, acetyl, or toluenesulfonyl; the
alkyl type protecting groups which include triphenylmethyl or trityl
(represented by Trt) or benzyl, The preferred protecting groups
are benzyloxyca~bonyl, t-butyloxycarbo~yl, triphenyImethyl and
dimethyl-3~5-dimethoxybenzyloxycarbonyl~ The protecting groups
~ ~ .
for the hydroxyl of serine and tyrosine are represented by acetyl,
tosyl, benzoyl, tert-butyl trepresented by But), trityl, and benzyl.
The preferred protecting group is tert-butyl. The protecting group
on the sulfur of cysteine or modified cysteine is illustrated by
~enzyl, triphenylmethyl or trltyl trepresented by Trt), benzyloxy-
carbonyl, or acetamldomethyl trepresented by Acm), the preferred
protecting groups are trltyl and acetamidomethyl. The carboxylic
acid function of a peptide or amino acid can be considered protected
by lower alkyl or lower aralkyl esters which include methyl
(represented by OME), ethyl ~represented by OEt), or benzyl
, (represented by OBzl), and also by substituted hydrazides which -
include t-butyloxycarbonyl hydrazide (represented bV NHNH Boc),
~; 25 benzyloxycarbonyl hydrazide (represented by NHNH Z),or a,~-dimethyl-
3,5-dimethoxybenzyloxycarbonyl hydrazide (represented by NHNH Ddz).
'1 '
1~ ~ 9 1
r ~ ~i
.,~ .. i1~ -
~1~

AHP-6550
.. :~
104~623 -
To promote facile condensation of the peptide carboxyl
group with a free amino group of another peptide to form a new
peptide bond, the terminal carboxyl group mus~ be activa1ed.
Descriptions of such carboxyl-activating groups are found in general
textbooks of peptide chemistry; for example K.D. Kopple, "Peptides
and Amino Acids", W.A. Benjamin, Inc., New York, 1966, pp. 45 - 51
and E. Schroder and K. LUbke, "The Peptides"; Vol. 1, Academic Press,
New York, 1965, pp. 77 - 128. Examples of the activated form of the
; terminal carboxyl are acid chloride, anhydride, azide, activated
ester, or _-acyl urea of a dialkylcarbodiimide. The following activated
esters have proved to be particularly su~table in the process of
this invention: 2,4,5-trichlorophenyl (represented by OTcp), ~ -
pentachlorophenyl trepresented by OPcp), D-nitrophenyl (represented
by ONp), or l-benzotriazolyl. The succlnim7do group is also useful
for activating a carboxyl. -
The term "azide method" as used herein refers to th~ method
of coupling two pepttde fragmenTs wh7ch comprlses the reactlon of a
pept7de hydrazide wtth a reagent which furnishes nitrous acid In ~ ;
sltu. Suitable reagents for this purpose include organic n7trites
(e.g. _-butyl nitrite, isoamyl n7tr7te) or an alkal7 metal n7trite salt
~e.g. sodium nitrite, potassium nitrite) in the presence of a strong
acid such as hydrogen chloride or sulfur7c or phosphoric ac7d. The
;; corresponding peptide azide thus obtained is then reacted with a
peptide having a free amino group to obtain the desired peptide.
Preferred condit70ns for the az7de method of coupl7ng cornprises reacting
:!: . . -.
~ , . '. ' '
, ,:
~ '
:~ . .. .
~--
. . . : : ' : ,; , . : '

AHP~6~50
1~ 623
. ' . .
the peptide hydrazide with nitrous acid, generated In situ frorn an
organic nitrite in the presence of a mineral acid, preferably hydrogen
chloride, (pH ranging usually from 0.1 to 2), in an anhydrous inert
organic solvent, for example, dimethylformamide, dimethyl sulfoxide,
ethyl acetate, methylene dichlorids, tetrahydrofuran, dioxane, and the
like at -30 to 20C, preferably at about -15C, for 10 to 30 minutes
to obta1n the corresponding azide. The peptide azide can be isolated
and crystallized or is prèferably allowed to remain in the reaction
mixture, and thereafJer reacting the azid~e in the said mixture with
the peptide unit having the free amino group at temperatures rangtng ;
from -30 to 20C for about one to two hours and then at 0 to 30C for
10 to 30 hours. An acid acceptor, preferably an organic base, for ~¦
Zl example N-ethyldiisopropylamine, N-ethylmorpholine or triethyl-
amine, is present in the reaction medium in order to make the reaction
medium sllghtly alkaline, preferably pH 7.0 to 7.5. See also the
above clted textbooks of Kopple or Schr~der and LUbke for add7tional
descriptions of thls method.
The terms "peptide, polypeptide, tripeptide, hexapeptide,
and the like" as used herein are not limited to refer to the respective
parent peptides but also are used in reference to modified peptides
; havlng functionalized or protecting groups. The term "peptide" as ;~
iZ~ used herein is used in reference to a peptide with two to twelve
amTno ac7d residues. In addition the residue "NH~HCH2S" as
- R
defTned herein is written as H-D-Cys-Gly-NHEt when R is CONHCH2CONHCH2CH3,
~ and is written as a modified residue of cysteine when R 7s H, viz., 2-
"'t~ ~ thioethylamine.
~: : ' ,,~,, '
:
.~ ,
~: ''' ~ lZ
. .. ,~
:- . ~ .. ... . :. :
:: - ~ . : : . . .. .
:: . .

AHP-6550
1~4~)623
The abbreviation Me represents a methyl group and NHN~12
represents a hydrazide group. In addition, the following abbreviations
are used: dimethylformamide (DMF), tetrahydrofuran (THF), dimethyl
sulfoxide (DMSO), methanol (MeOH), ethyl acetate (EtOAc), methylene
dichlortde (CH2C12), N,N'-dicyclohexylcarbodiimide (DCC), sodium
chloride (NaCI), sodium bicarbonate (NaHC03), sodium sulfate (Na2S04)
magnesium sulfate (MgS04), and 5~ palladium on charcoal (5% Pd/C).
The term l'lower alkyll' as used herein contemplates hydro- ~-
carbon radicals having one to three carbon atoms and includes methyl,
slO ethyl and propyl.
The term 'Imineral acidll as used herein contemplates the
strong inorganic acids and includes hydrochloric, hydrobromic,
suJfuric, phosphoric and the like. When the term is used in con-
1~ junction wtth an anhydrous system, hydrogen chloride is the preferred
jl5 mlneral acid.
! The term llmiIdly acidtc conditionsll as used herein contem- ~ '
plates conditlons in which a dilute aqueous solutlon of an organic ~ i
aclc, for example 30 - 80% or mixi~ures thereof, is a prlncipal çr-
component of the reaction medlum. ~ -
~o The term "moderately acidlc conditions" as used herein
contemplates conditions in which concentrated organic acids or ~ ~ -
f aqueous solutions of the mineral acids are used as a principal -~
component of the reaction medium,at temperatures ranging from
' ~ about -30 to 30C. Examples of preferred conditions in this j
case include the use of 50 to 100% trifluoroacetic acid at 0
... . . . .
~i~ to 30C or 0.1 - 12N hydrochloric acid in aqueous or anhydrous organic ' - ~ -
solvents at -20 to 10C.
:1 ' .. ,
he term 'lorganic nitrite'l includes the commercially
available alkyl nitrites~ for instance, t-butyl nitrite or isoamyl
nitrite.
:
f
~'
., ' .
-.f

Al-IP-6550
104~623
:'
The term organic base as used herein includes triethyl-
amine, N-ethylmorpholine, or N-ethyldiisopropylamine.
~ The term strong base as used herein contemplates both
- organic bases, as described above, and strong inorganic bases
including the hydroxldes and carbonates of sodium and potassium.
The peptldes of thls invention, includlng the cycllc and
the llnear reduced forms, are obtained in the form of the free base
or a~ an acid additlon salt thereof elther directly from the process
of thls inventlon or by reacting the peptide with one or more
equivalenTs of the appropriate acTd. Examples of preferred salts are
those wlth pharmaceuttcally acceptable organic acids, e.g. acetic, -~
lactlc, succlnic, benzolc, salicyllc, methanesulfonlc or toluene- ;
sulfonlc acid, as well as polymeric acids such as tannic acld or
carboxymethyl cellulose, and salts wlth inorganic acids such as -
` 15 hydrohallc aclds, e.g. hydrochlorlc acid, or sulfurlc acld, or phos-
, phorlc acld. It should be noted that the peptldes have two baslc
! nltrogens givlng rlse to addltlon salts wlth one to posslbly two
i equlvalenTs of acid. If deslred a partlcular acid addition salt
J, is converted into another acid addition salt, e.g., a salt with a ~ -
non-toxic, pharmaceutically acceptable acld, by treatment with the
,~ - appropriate ion exchange resln In the manner descrlbed by R. A.
-~ ; Boissonas, et al., Helv. Chlm. Acta, 43, 134g (1960). Suitable
ion exchange reslns are cellulose based cation exchangers, for
example carboxymethylcellulose or chemically modlfled, cross-llnked
dextran cation exchangers, for example, those of the Sephadex*C type,
and strongly basic anion exchange resins, for example those listed in
J.P. Greenste7n and M. Wtnitz Chemistry of the Amlno Acids , John
J Wiley and Sons, Inc., New York and London, 1961, Vol. 2, p. 1456.
t * Sephadex is a trade mark
~, 13
., ,
., .
~ .. }
~ - ,

AHP-6550
1041)623
The peptides of this invention of formulae I and la give
complex sal-l-s wi-rh heavy metal ions. An example of a pharmaceutically
acceptable heavy metal complex is a complex formed with zinc or
with zinc protamine.
The peptides of formulae I or la, as well as their ;
corresponding pharmaceutically acceptable salts, are useful
because they possess the pharmacological activity of the
natural hormone somatostatin. Their activity is demonstrated ~;
readily in pharmacological tests such as a modification
[A.V. Schally, et al., B~i`ochem. Biophys. Res. Commun., 52, 1314
(Ig73~; J. RivierJ et al., C.R. Acad. Sci. Paris, Ser. D, 276, ~ ~
2737 (1973)] of the n vitro method of M. Saffran and A.V. Schally, ~-
Can. J. Biochem. Physiol., 33, 405 (1955).
The activity of the peptides of formulae I or la is
, 15 demonstrated also In vivo 7n a modification of the pentobarbital- ;
! 7nduced increase in plasma growth hromone level in the rat as
described by Brazeau, et al., cited above. In this test the
peptldes of this Inventlon show a level of actlvity which is of the
same order as that of somatostatin.
The peptides of formulae I or la and their salts are useful
for the treatmert of acromegaly and other hypersecretory endocrine
states and in the management of diabetes in mammals; see for example, -
P. Brazeau, et al., cited above. When a peptide of formula t or la
or a salt thereof is employed for such treatment or management, it
1~ 25 is administered systemically, preferably parenterally " n combination
¦~ - with a pharmaceutically acceptable liquid or solid carrier. The
proportion of the peptide or salt thereof is determined by its
,~ solubility in the given carrier, by the given carrier, or by the
`~ chosen route of administration, and by standard biological practice.
For parenteral administration to animals the peptide or a salt
: - ~
thereof is used in a sterile aqueous solution which may also contain
: : 14
i
... :.. , .. ... .. . ,.. , . ,.. , ,. ....... . , ,. :.. .. .. . .... .. . . ...
: . ,: . .... . . . . . . . . .. . ,. , ~ . .

Al-IP-6550
~ .
1()40~Z3
other solutes such as buffers or preservatives, as well as sufficient
pharmaceutically acceptable salts or glucose to make the solutlon
isotonic. The dosage will vary with the form of administration
and with the particular species of animal to be treated and Ts
preferably kept at alevel of from 5 mcg to 300 mcg per kilogram -
body weight. However, a dosage level in the range of from about
10 mcg to about 50 mcg per kilogram body weight is most desirably
employed in order to achieve effecttve results.
The peptides or salts thereof may also be administered
in one of the long acting,~slow-release or depot dosage for~s
descrtbed below~ preferably by intramuscular injection or by
Implantation. Such dosage forms are designed to release from
about 0.5 mcg to about 50 mcg per kilogram body weight per day.
It is often desirable to administer a peptide of formula I
or ~ continuously over prolonged periods of time in long-acting,
slow-release, or depot dosage forms. Such dosage forms may elther
contatn a pharmaceutically acceptable salt of the peptide having
¦ a low degree of solubillty In body flulds, for example one of those
salts descrlbed below, or they may contain the peptlde in the form of
a w3ter-soluble salt together with a protective carrier whtch preve~ts
¦~ rapld release. In the latter case, for example, the peptide may beformulated wtth a non-antigenic par~ially hydrolyzed gelatin
~,~ in the form of a viscous liquld; or the peptide may be absorbed on~1 a pharmaceutically acceptable solid carrier, for example ztnc
hydroxide, and may be administered in suspension in a pharmaceutlcally
acceptable liquid vehTcle; or the peptide may be formulated in gels or
I: :
suspensions with a protective non-antigenic hydrocolloid, for example
,~ sodium carboxymethylcellulose, polyvtnylpyrrolidone, sodium alg7nate,
gelatine, polygalacturonic acids, for example, pectin, or certaln
mucopolysaccharTdes, together with aqueous or non-aqueous pharmaceuti-
cally acceptable liquid vehtcles, preservatives, or surfactants.
. .

AHP-6550
1 :
104~623 : ~
Examples of such formulations are found in standard pharmaceutical
- texts, e.g. in Remington's Pharmaceutical Sciences, 14th Ed., Mack
y;~ ~ :
Publishing Co., Easton; Pennsylvania, 1970. Long-acting, slow-
release preparations of the peptide of formulae I or la may also
be obtained by microencapsulation in a pharmaceutically acceptable
coating, for exampie gelatine, polyvinyl alcohol or ethyl cellulose.
Further examples of coat7ng mater7als and of the processes used
for m7croencapsulat70n are descr7bed by J.A. Herb7g 7n "Encycloped7a
of Chemical Technology", Vol. 13, 2nd Ed., Wiley, New York 1967,
pp 436 - 456. Such formulat70ns, as well as suspens70ns of salts ¦;
of the agent wh7,ch are only sparingly soluble in body fluidsJ for
' example salts with pamoic acid or tannic acid, are designed to j-
release from about 5.0 mcg to about 100 mcg of the active compound
per k7logram body weight per day, and are preferably administered by ~! : I
i 15 7ntramuscular 7nject70n. Alternat7vely, some of the solid dosage forms
17sted above, for example certa7n spar7ngly water-soluble salts or
~' dlspers70ns 7n or adsorbates on solld carr7ers of salts of the agent,
. :.., ,.,, . ,-: ... .
~ for example d7spers70ns 7n a neutral hydrogel of a polymer of ethylene -
¦ glycol methacrylate or s7m71ar monomers cross-17nked as descr7bed inU.S. Patent 3,551,556 may also be formulated 7n the form of pellets 1 1
releas7ng about the same amounts as shown above and may be 7mplanted
subcutaneously or 7ntramuscularly. i
Process i
For conven;ence and clar7ty 7n the following dtscussion the
indlv7dual pept7de un7t (i.e., amino acid) is designated sometlmes by
a number which refers to the pos7tion in which the part7cular am7no ac7d
appears 7n the sequence of the amino acids as illustrated in the formula 1.
.:~, . ... .:
, : , .,
:' ' Ij,; :'
I ...~
:

AHP-6550
~:
1046)623 t:
Thef process of this invention is carrier out in the
following manner. .'
: With reference to the tripeptide fragment 1-3, the ff~ `
tripeptide is prepared by reacting a protected activated ester of ~ -
D-threonine, preferably Z-D-T3r-OH with a lower alkyl ester of
'~ ,,
D-phenylalanine, preferably H-D-Phe-OMe, to obtain the corresponding ~ ;:
lower alkyl ester of the dipeptide Z~D-Thr-D-Phe-OH, which after removal
B~t : .
the terminal protecting grou~ (Z) using hydrogen in the presence of ~ -
a noble metal catf~kys~t yields the corresponding lower alkyl ester of
:. H-D-T~r-D-Phe-OH, preferably H-D-T~r-D-Phe-OMe. In turn the latter ~ .
Blt BLt' , , ., :,- ,-,,
compound is reacted with a protected activated ester of D-serine, :: -
preferably the benzotriozolyl ester to obtain the corresponding lower ¦ r,~
alkyl ester of Z-D-Selr-D-T3r-D-Phe-OH, preferably Z-D-Sftr-D-T~r-D-Plle-OMe.
:,~ ' . .
j Subsequent removal of the terminal amino protecting group of the latter .
f compound usTng hydrogen in the presence of a noble met31 catalyst yields
20 the corresponding lower alkyl ester of H-D-Ser-D-Thr-D-Phe-OH, pre-
~; ferably H-D-SIer-D-Thlr-D-Phe OMe. Condensation of said last-named
compound with a protected activated esler of thiopropionic acid,
: preferably the benzotriazolyl ester, gives the corresponding protected ;
f 25 lower :Ikyl ester of Trt-S-CH~CH2CO-D-Ser-D-T~r-D-Phe-OH, preferably : ;
Trt S H2CH2CO D Sltr D r3tr-D-Phe-OMe. The latter compound is treated ~
with hydrazine hydrate to obtain the hydrazide of the tripeptide ~ :
ragment 1 3 of formula Trt S CH2CH2CO-D-SIt-D-T~r-D-Phe-NHNH2 (V). ;
~ 7
.. ~`'
': . ~ :
``` ~.

Al-IP-6550
104~623 ~
In a preferred embodiment of the preparation of the above
tripeptide fragment 1-3, a mixture of substantially equimolar
amounts of Z-D-TIhr-OH, prepared from D-threonine in the same manner
B t
1 5 as described for the corresponding L-isomer by E. Schr~der, Justus
Liebigs Ann. Chem.,- 670, 127 (1963) and H-D-Phe-OMe HCI, prepared from
D-phenylalanine in the same manner as described for the L-isomer by
F. Bergel, J.M. Jchnson, and R. Wade, J. Chem. Soc., 3802 (1962), in
an Inert organic solvent, preferably DMF or THF, at -20 to 10C, ¦~
preferably at 0C, is tre~ated with a molar excess, preferably with 1.1
~i to 1.3 molar equivalents of a strong organic base, preferably N-ethyl-
morpholine, to p~ 7-8. A molar excess~preferably 1.1 to 1.3 molar
equivalents, of l-hydroxybenzotriazole is added followed by the
dropwise aodition of a substantTally molar equivalent of DCC (1.0 to I
1.3 molar equivalents) in an inert organic solvent, preferably DMF or
THF. The mixture is kept at -20 to 10C, preferably at 0C from
~ 30 rninutes to 2 hours and then at 20 to 30C for ar, additional hour,
;7 filtered, and the filtrate evaporated. The residue is taken up in a ,
] substantlally water-lmmtsclble organic solven~, preferably dlethyl ~
etller, washed, drled, and evaporated. The res1due is taken up in a ¦ ;
mixture of a lower alkyl ester of a lower alkanoic acid, preferably ¦
ethyl acetate, and a hydrocarbon, preferably hexane, and is purified
by chromatography on silica gel to yield the ~ipeptide of formula
Z-D-T~r-D-Phe-OMe. Said last-named compound is then subjected to : -
i~ 25
hydrogenation in the presence of a noble metal catalyst, preferably 5%
palladium on charcoal (5% Pd/C), and of an e~iimolar amount of pyridine ;~
hydrochloride or of an excess of acetic acid. Methanol, ethanol,
acetic acid, or mixtures thereof are convenie~t solvents for this
hydrogenation. In this manner the terminal a~ano protecting group (Z)
, 18 I :
,~,:
... .
: -- --` . '
!
. . .` - , . . .

AHP-6550
1040623
of the above dipeptide is removed to give the corresponding dipeptide
of formula H-D-T~r-D-Phe-OMe as its acetic acid or hydrochloric acid
,~ , Blt
,
addition salt. Said last-named compound and a substantially equimolar
amount of Z-D-S~r-OH, prepared as described for the L-isomer by
, Bbt
E. Schr~der, Justus Liebigs Ann. Chem., 670, 127 (1963), in an inert
organtc solvent, preferably DMF or THF, at -20 to 10C, preferably at
0C, is treated with a molar excess, preferably 1.1 to 1.3 molar
equivalents, of a strong organic base, preferably N-ethylmorpholine, to ?r
pH 7 - 8. A substantially-equimolar amount of l-hydroxybenzotriazole is
added followed by the addition of a substantially molar equivalent -
of DCC (1.0 to 1.3 molar equivalents) in an inert organic solvent,
preferably DMF or THF, and the mixture is cooled to -20 to 10C, pre-
~I ferably to 0C. The mixture is kept at -20 to 10C, preferably at 0C~ from 30 mTnutes to two hours and then at 20 to 30C for an additionalj Hour, fiItered, and the fiItrate is evaporated. The residue is taken
up tn a substantially water-immiscible organic solvent, preferably
dlethyl ether, washed, dried, and evaporated. The resldue is taken
up In a mixture of a lower alkyl ester of a lower alkanoic acid, pre-
ferably ethyl acetate, and a hydrocarbon, preferably hexane, and is
puriiied by chromatography on silica gel to yield Z-D-Selr-D-T~r-D-Phe-OMe.
~1~j Said last-named compound is dissolved in a lower alkanol or a lower
alkanoic acid or a mixture thereof, preferably in acetic, a noble metal
catalyst, preferably 5% Pd/C, is added and ~he mixture is agitated in
an atmosphere of hydrogen at room temperature for 10-30 hours, preferably
for about 20 hour, until substantially one molar equivalent of hydrogen :
has been taken up. FiItration of the catalyst and evaporation of
the filtrate yields the tripeptide of formula H-D-Ser-D-Thr-D-Phe-OMe
.,~ , .,
~9
. ':
., ' ,:':
::
` ~ .

Al iP-6550
1~623
as the acetic acid addition salt. Said last-named compound is
dissolved in an inert organic solvent, preferably DMF or THF, ~-
at -20~ to 10C, preferably at 0C, and 1-reated with a molar
- excess, preferably 1.1 to 1.3 molar equivalents, of a strong
organic base, preferably N-ethylmorpholine ,to pH 7 - 8. A
substantially equimolar amount of 3-tritylthiopropionic acid, pre-
pared as described by E. Biliman and N.V. Due, Bull. Soc. Chim.
Fr., 35, 384 (1924), in an inert organic solvent, preferably DMF
or THF, is added followed by the addition of a substantially
molar equivalent of l-hydro,xybenzotriazole. A molar excess of
DCC (1.1 to 1.3~mp~ar equivalents) in an inert organic solvent,
1 prefeably DMF or THF, is added and the mixture is kept at -20
:11 to .10C, preferably at 0C from 30 minutes to 2 hours and then
-I at 20 to 30C for an additional hour, fiItered, and the fiItrate
eva?orated. The residue Ts taken up in a substantially water-
Immisctble organic solvent, preferably diethyl ether, the pre-
.
, cTpttate is removed by fiItration, washed, dried, and evarporated.
J The residue 7s taken up In a mlxture of a lower alkyl ester of a
j lower alkanolc acid, preferably ethyl acetate, an aromatic hydro-
¦ 20 carbon, preferably benzene, and a strong organic base, preferably ~ :
, trlethylamine, and is purified by chromatography on silica gel.
:~ Crystallization of the purified matRrial yields
H2C0 D S~tr D T~tr-D-Phe-OMe. Said last-named compound is
dtssolved in an inert organic solvent, for example methanol, ethanol,
1; DMF, preferably methanol, and the solutlon i5 treated with an excess
`~ of hydrazine hydrate, for example with 20 to 50 molar equivalents. ;
m The reaction mixture is kept at -20 to 10C, preferably at 0C, from :
1 30 minutes to 2 hours and then at 20 to 30C for 15 to 30 hours,
1 ~ ~
3~ ::
. f ~ O
, ' .
'
.~', ',

AHP--6550
- :
104~)6Z3
; preferably for 24 hours. Water is added, the resulting precipitate is
collected by fil1ration and dried to yield the tripeptide fragment 1-3
of formula Trt-S~OH2CH2CO-D-S~r-D-T~r-D-Phe-NHNH2 (V).
~ B Bu r~-~With reference to the tetrapeptide fragment 4-7, the
tetrapeptide is prepared by reacting an amino protected D-tryptophane,
preferably Z-D-Trp-OH, with a lower alkyl ester of D-phenylalanine,
preferably H-D-Phe-OMe, to obtain an amino protected lower alkyl
ester of the dipeptide H-D-Trp-D-Phe-OH, preferably Z-D-Trp-D~Phe-OMe,
I which after removal of thè terminal protecting group (Z) using hyarogen
in the presence,o~ a noble metal catalyst yields the corresponding
! lower alkyl ester of H-D-Trp-D-Phe-OH, preferably H-D-Trp-D-Phe-OMe.
In turn, the latter compound is reacted with an activated ester of
Z-D-Lys-OH to give the corresponding lower alkyl ester of
Boc
Z-D-Lys-D-Trp-D-Phe-OH, preferably Z-D-Lys-D-Trp-D-Phe-CMe. Subsequent
Boc Boc
removal of the terminal amlno protecting group of the latter compound ¦
(Z) using hydrogen in the presence of a noble metal catalyst gtves
¦ the corresponding lower alkyl ester of H-D-Lys-D-Trp-D-Phe-OH, pre-
¦~ 20 Boc
ferably H-D-Lrs-D-Trp-D-Phe-OMe. Condensatlon of the last-named
ompound with an activated ester of Z-D-TIr-OH yields the corresponding 1¦
~ Z5 ~ But Bo
! ~ Z-D-T~r~D-Lrs-D-Trp-D-Phe-OMe. The latter compound Is treated w7th
hydrazine hydrate to obtain the tetrapeptida fragment 4-7 of formula
Z~D-TIlr-D-L~s-D Trp-D-Phe NHNH2 --
~ 30 ~ Bu Boc 21 ~ ¦
.':: :, .: ~ .
~ ~. . `1.
.: : ` - .,.. , .
"".. ~ , ,, ;,...... ., . : ~ : ; : . ~ , ' : . ,

AHP-6550
10406Z3
In a preferred embodiment of the preparation of'the above
tetrapeptide fragment 4-7, substantially equimolar amounts of ''
Z-D-Trp-OH, prepared as described for the L-isomer hy E. Klieger, -~
E. Schr~der, and H. Gibian, Justus Liebigs' Ann. Chem., 640, 157
(1961), and ~ Phe-OMe-HCI (see F. Bergel et al, cited above) with
an excess, preferably 1.5 to 2.5 molar equivalents, of l-hydroxybenzo-
triazole, in an inert organic solvent, prefeably DMF, at -20 to 10C,
; preferably 0C, is treated with an excess, preferably 1.1 to 1.3 molar
; equivalents, of an organic base, preferably N-ethylmorpholine, to pH
7 - 8. A substantially equ~imolar amount of DCC in an inert organic
solvent, preferab,ly~DMF, at -10 to 10C, is added dropwise. The
.. .. . .
-1 mixture is kept at -20 to 10C for an additional hour, cooled to -10
.i..................................... . .
'~; to 10C, filtered, and the filtrate evaporated. The residue is taken '
up in a subslantially water-imm7scible solvent, preferably ethyl ';~-1
! ,: .. .
acetate, washed, dried and evaporated. The residue is taken up in "' -
' a mixture of a halogenated hydrocarbon solvent, preferably chloroform, -
and a lower alkanol, preferably methanol. The solution is passed
through a column of sil7ca gel. Evaporatton of the eluate and
crystalllzation of the residue ylelds the dipeptlde fragment 6-7 of
formula Z-D-Trp-D-Phe-OMe. Said last-named compound is then subjected ~' '
to hydrogenation in the presence of a noble metal catalyst, preferably
' 5~ Pd/C. Methanol, ethanol, acetic acid, or mixtures thereof are
conven7ent solvents for this hydrogenation. When acetic acid is used ¦
, the product will be isolated as the acetic acid add7tion salt. ¦
~' 25 Filtration of the catalyst, and evaporation of the filtrate yields
the dipeptide fragment 6-7 of formula H-D-Trp-D-Phe-OMe.
i ''
:i
: ~ :. -..... . ... : . , . : , .

~\HP-6~C~O
1041)623
Said last-named compound in an inert organic solv~nt, preferably DMF,
at -20 to 10C, preferably 0C, is treated with an excess, preferably
1.1 to 1.3 molar equivalents, of a strong organic base, preferably
N-ethylmorpholine, to pH 7 - 8,the mixture is then treated with a
su~stantially molar equivalent of a protected activated ester of
D-lysine, preferably Z-D-Lys-ONp prepared in the same manner from
Boc
D-lysin~ as described for the L-isomer by E. Sandrin and R. A.
Boissonnas, Helv. Chim. Acta., 46, 1637 (1963). The solution is
stlrred at about 0C for 30`minutes to two hours, at 20 - 30C for
two to four days, and e,vaporated. The residue is taken up in a ~ ~
substantially water-immiscible solvent, preferably ethyl acetate, ~ -
washed, dried, and evaporated. The residue is taken up in a
halcgenated hydrocarbon solvent, preferably chloroform, a lower
alkanol, preferably methanol, and a strong organic base, pre-
ferably pyr;dine. The solution is passed through silica gel. , -
After evaporation of the eluate the residue is crystallized to ` ¦
yield the tripeptide fragment 5-7 of formula Z-D-L~s-D-Trp-D-Phe-OMe.
Boc
Said last-named compound is then subjected to hydrogenation in the
presence of a noble metal catalyst, preferably 5~ Pd/C. Methanol,
ethanol, acetic acid, or mixtures thereof are convenient solvents ~1
for thts hydrogenation,when acetic acid is used the product will be ~`
isolated as the acetic acid addition salt. FiItration of the catalyst,
, .
and evaporation of the fiItrate yields the tripeptide fragment 5-7 of
formula H-D-Lys-D-Trp-D-phe-oMe. Said last-named compound, sub-
Bocstantially equimolar amount of a protected D-threonine, preferably
.
- :
: :~ 23 ~
: - .
.
: . . .` .... . . ... .
~ . .. . , . , . , . :

AHP-6550
104~)6Z3
Z-D-TIhr-OH (see E. Schr~der cited above), and about one to two ~ -
Bu
molar equivalents of l-hydroxybenzotriazole in an inert organic
solvent, preferably DMF, at -20 to 10C, preferably 0C, is
treated with an excess, preferably 1.1 to 1.3 moiar equivalents, of a
- 5
strong organic base, preferably N-ethylmorpholine,to pH 7 - 8.
A substantially equimolar amount of DCC in an inert solvent, pre-
ferably DMF, at about 0C is slowly added dropwlse. The mixture
is stirred at about 0C for 30 minutes to two nours, at 20 - 30C
for one to two hours, fiItered, and evaporated. The residue is
.~ 1O J"" ~
taken up in a halogen3ted hydrocarbon sJolvent, preferably chloroform,
and a lower alkanol, preferably methanol. The solution is passed
.~ . - -:
~` through a column of silica gel. Evaporation of the eluate and
crystallization of the residue yields the tetrapeptide fragment 4-7
~ of formula Z-D-T~r-D-Lyls-D-Trp-D-Phe-OMe. Said last-named compound
,~ ,,
is dissolved in an inert organic solvent, for example methanol,
ethanol, or DMF, preferably DMF. The solution is treated with an
excess of hydrazine hydrate, for example 20 to 50 molar equivalents,
. .
and is kept at -20 to 10C, preferably at 0C, for one and a half
to three hours, preferably two hours. Water is added; the pre-
cipitate is collected by f71tration, dried, and crystallized to
1~ :
, yield the tetrapeptide fragment 4-7 of formula Z-D-T~r-D-Lys-D-Trp-
D-Phe-NHNH2 ( I I ,.
~ ,
With reference to the tripeptTde fragment 8-10, the
~¦ tripeptide is prepared by reacting a protected activated ester of
1~ D-asparagine, preferably Z-D-Asn-OTcp, with a lower alkyl ester of
- a protected D-lysine, preferably H-D-Lys-OMe, to obtain the corresponding
1~ Boc
2 ~ .
:~ ................................. .
.. :
..
.. . . .. . . ~. . : ;,
.. = .: ` , . ., . . , ~ ~ ..
.. .,, . . ~ . . ., .. ., :
; . ` ; . . . .. . .
` . . .. . .. . . . . . . . .

AHP-6550
104V623
protected lower alkyl ester of the dipeptide D-asparaginyl-D-lysine,
preferably Z-D-Asn-D-Lyls-OMe, which after removal of the terminal amino
Boc
protecting group (Z) using hydrogen in the presence of a noble metal
catalyst gives the corresponding lower alkyl ester of H-D-Asn-D-L~s-OH,
~ Boc
preferably Z-D-Phe-D-Asn~D-Lys-OMe. Subsequent removal of the terminal
Boc
amlno protecting group of the latter compound (Z) using hydrogen in
the presence of a noble metal catalyst gives the corresponding lower
alkyl ester of H-D-Phe-D-Asn-D-Lyls-OH, preferably the tripeptide fragment -
, B c - -
8-10 of formuia~H,D~Phe-D-Asn-D-Lrs-OMe (111).
Boc
', ' :
In a preferred embodiment of the preparat70n of the tri-
peptide fragment 8-10, substantially equtmolar ambunts of Z-D-Asn-OTcp, ,;
¦ prepared from D-asparagine in the same manner as described for the
~` corresponding L-isomer by J. Beacham, G. Dupuis, F.M. Finn, H.T. Storey,
C. Yanaihara, N. Yanaihara, and K. Hofmann, J. Amer. Chem., Soc., 93,
5526 ~1971), an~ H-D-Lys-OMe, prepared from D-lysine tn the same
B c
monner as described for the corresponding L-isomer by R. Schwyzer and
W. Rittel, Helv. Chim. Acta, 44, 159 (1961), in an inert organic
I solvent, preferably DMF, at -20 to 10C, preferably 0C, is treated
with~a substantiDlly molar equivalent of an organic base,-preferably
i : . ~ 1
3~ 25 N-ethylmorpholine and stirred at -20 to 10C, preferably 0C, for
; two to four hours, and then at 20 to 30C for 15 to 30 hours. The
solutlon is evaporated, the residue is triturated with an alkyl ether,
.1 ,- .
preferably diethyl ether and dried to yield the dTpeptide fragment 9-10 ~`
of formula Z-D-Asn-D-L,ys-OMe. Said last-named compound is then subjected
oc
2 ~
r : .
,
,
- ,, , . , .. , ., .. , ., ,, , . ~ .

AHP-6550
1~)4~6Z3
to hydrogenation in the presence of a noble metal catalyst, pre-
ferably 5% Pd/C. Methanol, ethanol, acetic acid or mixtures
~ thereof are convenient solvents for this hydrogenation. The
catalyst is removed by fiItration, the fiItrate is treated with
~ . .
a substantially equimolar amount of a mineral acid, prefera~ly
hydrochloric acid, an a evaporated to yield the dipeptide fragment
9-10 of formula H-D-Asn-D-Lys-OMe as the hydrochloric acid addition --
60c
salt. Said last-named compound and a substantially equimolar
; amount of Z-D-Phe-OTcp, prepared from D-phenylalanine in the
same manner described for the corresponding L-isomer by J. Pless -~-
and R. A. Boissonnas, Helv. Chim. Acta.~ 46 IGO9 (1963), in
an inert organic solvent, preferably DMF or THF, at -20 to 10C,
preferably 0C, is treated with an excess, prefer~bly 1.1 to 1.3
^1 15 molar equivalents, of a organic base, preferably N-ethylmorpholine. ;~
j The solutlon is stirred at about 0C for 20 to 30 hours. The
precipitate is collected by f71tratTon and crystallized to obtain
the trtpepttde fragment 8-10 of formula Z-D-Phe-D-Asn-D-Lys-OMe.
80c
The latter compound is subjected to hydrogenatton in the presence
of a noble metal catalyst, preferably 5% Pd/C. Methanol, ethanol,
acet!c actd, or mixtures thereof are convenient solvents for this
hydrogenation, when acettc actd ts ùsed the product wtll be tsolated
as the acettc acid addttion salt. The catalyst is removed by
~tltrat70n, the filtrate evaporated, the residue taken up in an aromatic
hydrocarbon, preferably benzene, and evaporated to obtain the tri-
pepttde fragment 8-10 of formula H-D-Pho-D-Asn-D-L~s-OMe ~111). ~
j~ Boc ,~ -
1 , .
2 6
i. ~ ' .
;
. . , .. ,~ . . ..
.- , . . . . .. . . .
,. . . i . , ~ . . ..
..

AHP-6550
` 104~623 j
With reference to the fragment HNHCIHCH25 Trt (Vll) in which f:: :
R is CONHCH2CONHCH2CH3, i.e. 1-he fragmant VlIb alternatively written as
I H-D-C~s-Gly-NHEt, said fragment is prepared by reacting a protected
Trt
lower alkyl ester of the dipeptide D-cysteinyl-glycine, preferably !~ -
Trt-D-Cys-Gly-oMe~ with ethylamine, to obtain the correspondingly 1
Trt
protected ethylamide of the dipeptide D-cysteinyl-glycine, preferably ,
Trt-D-Cys-Gly-NHEt. Removal of the termTnal amino protecting group
~Trt) using miIdly acidic conditions, preferably a mixture of water
and acetic acid or formic acid yields the corresponding addition salt
of the fragment H NHC~HCH2S Trt ~VII~ in which R is CONHCH2CONHCH2CH3,
~' . . . .
i.e. H-D-C~s-Gly-NHEt (Vllb).
Trt
In a preferred embodiment of the preparation ot the
fragment (Vll) in which R Is CONHCH2CONHCH2CH3,the dipeptide
Trt-D-Crs-Gly-OMe, prepared from D-cysteine in the same manner
I as described for the corresponding L-isomer by G. Amlard, Bull.
;1 Soc. Chim. ~Fr.), 1956, 698, Is treated with a molar excess,
preferably 50 - 200 molar equivalents, of ethylamine at -15 to
15C, preferably 5C, for 20 to 30 ~hours. The solution is
evaporated, and the residue is dissolved in a solution of a
- lower alkyl ester of a lower alkanoic acid, preferably ethyl
acetate, and an aromatic hydrocarbon, preferably ben7ene, and
purified by chromatography on silica gel to obtain the dipeptide of
'~ ~ formul a Trt- D-C~s-Gly-NHEt. Said last-named compound, is subjected ~j
' to miIdly actdic conditions, preferably 70 to 90% acetic acid at ¦~
30 to 50C, preferably 45C, for ten to 20 minutes, preferably ~-
- 30 15 minutes. Water is added, the mixture is filtered, the filtrate ,
;` 2 7 1 ~
: ~ ,~.
,;., ` ii
, - - - - - `= _ ~.
~ ...... .. , . . . , . . . . . . . ......... .... .... .

- AHP-6550
',: '. '''
- 104~6Z3
is treated with a substantially molar equivalent of a mineral acid,
;~ preferably hydrochloric acid, to obtain the fragment HNHIHCH2STrt (Vll)
in which R is CONHCHzCONHCH2CH3 alternatively written as H-D-Cys-Gly-
NHEt (Vllb).
The heptapeptide fragment 4-10 is conveniently prepared by
coupling the fragment 4-7 and th~ fragment 8-10 according to the
azide coupl7ng method in the following manner. A solution of the -
tetrapeptide fragment 4 7~of formula Z-D-T~r-D-L~s-D-Trp-D-Phe-NHNH2 (Il),
obtatned as desc~SIbéd above, in an inert anhydrous organic solvent,
preferably DMF, is cooled to a temperature of from about -30C to ~
about -10C and mixed with a solution of about two to five molar -~ -
jJ equivalents, preferably three molar equivalents, of a mineral acid, I
J 15 preferably hydrogen chloride, in an inert anhydrous organic solvent,
preferably ethyl acetate. An organic nitrite, preferably t-butyl
nitrltTe or isoamyl nitrite in a substantially equimolar amount is
added with stirring. The solutTon Is stlrred for 10-30 minUTes,
preferably for about 15 mlnutes, at a temperature of from about
-20C to about -10C. Keeplng the solution at a temperature of
from about -30C to about -10C, a solution of a substant7ally ¦
equimolar amount of the tripept7de fragment 8-10 of formula ¦
H-D-Phe-D-Asn-D-Lyls-OMe (lil), preferably as the acetic acid add7t70n
salt obtalned as descr7bed above, and of about three to f7ve molar
t~ ~ 25 equivalents, preferably abo_T 3.5 molar equ7valents, of an organ7c ,
base, preferably N-ethyldi7sopropylam7ne, 7n an 7nert anhydrous ;
organ7c solvent, prefe mbly DMF, 7s added slowly with stirr7ng. -~
,:
'
:', - 2~ ~1
.,~ ~ .... j~ .
,~,
~.. ,......... . ~ ~ - .- .. . . ... . .

AHP-6550
104V623
~he mixture is stirred for 30 - 60 rninules at about -20C to about
-10C, then at about 2~ to 30C for 20 to ~0 hours. The solution
is evaporated and the resid~Je is triturated with cold aqueous citric
acid (0.5 to 2N), water and dried. The residue is taken up in a
mixture of a halogenated hydrocarbon, preferably ch!oroform, and
a lower alkanol, preferably methanol, and purifie~ by chromatography
on silica gel. The solvent is evaporated and the residue crystallized
to yleld the heptapeptide fragment 4-10 of formula
Z-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-oMe.
Bl Boc Boc
Said last-named ~ompound is subjected to hydrogenation in
the presence of~a;n~ble metal catalyst, preferably 5% Pd/C.
Methanol, ethanol, acetic acid, or mixtures thereof are suitable
solvents for this hydrogenation, and when acetic acid is used the
'3 15 product is isolated as the acetic acid addition sait. The catalyst
, 1s removed by filtration; the filtrate evaporated, the residue taken
up In an aromatic hydrocarbon, preferably benzene, evaporated, and
1 drled over strong alkali, preferably potassium hydroxide or sodium
hydroxlde, to obtain the heptapeptide frag~ent 4 10 of formula
H-D-T~rD-L~s-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-OMe (IV).
The decapeptide fragment~~l-10 is conveniently prepared
by coupling the fragment 1-3 and th~ fragment 4-10 according to -
- the azide coupling method in the following manner. A solution of
~j 25 the trTpeptide fragment 1-3 of formula ~- -
S CH2CH2CO D Sltr D Tltr-D-Phe-NHNH2 (V), obtained as described
. . .
~~ above, in an inert anhydrous solvent, preferably DMF, is cooled tc
~, -.:: - ~. :a temperature of from about -30C to about -10C and mixed with a `~
solution of about two to five molar equivalents, preferably three
molar equivalents, of a strong mineral acid, preferably hydrogen
hloride, in an anhydrous organic solvent, preferably ethyl acetate.
2~ ~:
. 1 ' .
- . i
.- -. . , ,. .. . .. .. ., ., ,. ., : .
. .~; ~. ~ . , .. . ` ,, . :. . .
-: . : .. . , . , : ` - . . .

AHP-~550
1040623
An organic nitrite, preferably t-butyl nitri1e or isoamyl n7trite,
in a substantially equimolar amount, is added with stirring. The
solution is stirred for lO - 30 minutes~ preferably for about
15 minu~es, at a temperature of from about -20C to about -10C.
Keeping the solution at a temperature of from about -30C to about
-10C, a solution of a substantially equimolar amount of the
heptapeptide fragment 4-10 of formula H-D-Thr-D-Lys-D-Trp-D-Phe-D-
Bl Boc
Phe-D-Asn-G-Lys-OMe ~IV) obtained as described above, and of about
Boc
:; 10 ~`
about three to five molar e!quivalents, preferably about 3.5 molar
equivalents, of ~a~ organic base, preferably N-ethyldiisopropylamine,
in an inert anhydrous organic solvent, preferably DMF, is slowly added
with stirring. Stirring of the resulting mixture is continued for
30 - 60 minutes at about -20C to about -10C, then at about 2~ to
30C for 20 to 30 hours, The solution is evaporated, the residue is
trlturated with cold aqueous citric acid (0.5 to 2N), water and
dried. The residue is taken up in a mixture of a halogenated
' hydrocarbon, preferably chloroform, and a lower alkanol, preferably
methanol, purified by~chromatography on silica gel and crystallized
i20
to yieid the decapeptide fragment 1-lO of formula Trt-S-CH2CH2CO-D-Sir-
D T~tr D Phe D-Thit-D-L~5-D-Trp-D-phe-D Phe_D_A5n-D-Lys-oMe.
Bu Bu B c Boc
Said last-named compound is taken up in an inert organic
solvent, for example methanol, ethanol, or DMF, preferably DMF, and
` treated with an excess of hydrazine hydrate, for example 20 to 50
molar equivalents. The mixture is kept at -20 to 10C, preferably
.`~ . ' . ' ,
.
3 0
, .
.
.
: :

AHP-6550
'~' ' "
1040623
; 0C, for 15 to 45 minutes and at 20 to 30C, for 20 to 30 hours. ,
Water is added, the precipitate is collected by fiItration, washed
- with water, and dried to yield the decapeptide fragment 1-10 of
formula Trt-S-CH2CH2CO-D-Ser-D-Th,r-D-Phe D-T~r-D-Lys-D-Trp-D-Phe~-Phe- ''
Bl B~t Blt Boc ~'
D-Asn-D-Ly,s-NHNH2 (V I ) . . .
Boc `-
The linear peptide Vlll is convenient;y prepared by coupling
the fragment 1- ! (Vl) and the fragment ~11) according to the azide
', coupling method in the fo~'lowing manner. A solùtion of the decapeptide
fragment 1-10 (Vl)~ obtained as described above, is taken up in an
inert anhydrous organ?c solvent, preferably a mixture of DMF and DMSO,
cooled to a temperature of from about -30C to about -10C and mixed
i with a solution of about two to five molar equivalents, preferably
three molar equivalents of a mineral acid, preferably hydrogen
chloride, in an anhydrous organic solvent, preferably efhyl acetate. -
A sùbstantially equimolar amount of an organTc nitrite, preferably
t-butyl nitrite or tsoamyl nitrite,is added, and the solution is
stlrred for 10-30 mlnutes, preferably for about 15 minutes, at a
; temperature of from about -20C to about -10C. Keeping the stirred '-
!; solution at a temperature of from'ai30ut -30C to about -10C, a
soiution of a substantially equimolar amount of the fragmen~ ; ~'
HNHCHeH2STrt (Vll) in which R is CONHCH2CONHCH2CH3(Vllb) obtained as des- ~i~
cribed above or the fragment HNH~HCH2STrt (Vll) in which R is hydrogen (Vlla)
- :~ 25 ~ ~ R
described by F. 1. Carroll et al., J. Org. Chem. 30, 36 (1965)], and
of about three to five molar equivalents, preferably about 3.5 molar
equivalents, of an organic base,~preferably N-ethyldiisopropylamine,
' ~' ~
. , : ': :.
: ~ : :~ :.-, :.
,~
3 1 ::
~-'
-,
` . ,., : .::
' :.
,

AHP-6550
.
104~)623
in an inert anhydrous organic solvent, preferably DMF, is added
slowly. Stirring of the resulting mixture is continued for 45 - 75
minutes at about -20C to aboul -10C, then at about 2Q to 30C
. for 20 to 30 hours. Evaporation of the solution, trituration of
, 5 the residue with cold aqueous citric acid (0.5 to 2N), water,
methanol, and drying yields the corresponding linear protected
peptlde of formula (Vlll) Trt-S-CH CH C0-D-S~r-D-T~r-D-Phe-D-Thr-D-
Ly,s-D-Trp-D-Phe~D-Phe~Asn~-L~s-NH~HCH25 Trt (Vlll) 7n which R is as
"4; Boc Boc R
o " ~
defined herein. J
The conversion of the above linear protected peptide,
1 obtained as described above, to the compound of formula I is ~ ;~
3' accomplished conveniently and efficiently by first subjecting the
linear protected peptide to the action of iodine, preterably in the
presence of a lower alkanol or a lower alkanoic acid whereby
slmultaneous removal of the sulfhydryl protecting groups, i,e. Trt,
~ and formatlon of tne disulflde brldge occurs to glve the corresponding
I cycli~c disulfide of formula IX
S-CH2CH2C0-D-Sfr-D-TIr-D-Phe-D-T~r~-D-Llys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-
Bu Bu Bu Boc B c
~; N ~HCH2~ (IX) in which R is as defined hercin, subsequent treafment of
j~ ~ of the latter compound under moderately acidic conditions removes the
3- ~ ~ remaining protecting groups (i.e. Boc and Bu ) to gTve the correspondlng
compound of formula 1.
- J :: ~
,~, .
., - . :
, . i
. ~ , .
,.' : ~ ' :
,~ -32-
j,,, : . ~ . .

AHP-6550
: 104~623
In a preferred embodiment of the above transformation, the
Iinear protected peptide (Vll!) is dissolved in a lower alkanol or
a lower alkanoic acid, preferably acetic acid, and added to an 1;
excess of iodine (5 to 25 molar equivalents, preferably 10 mclar
equivalents) dissolved in a lower-alkanol or a lower alkanoic acid,
preferably methanol, at a concentration of about 2 - 5~ iodine.
The time and temperature of this reaction is not critical; however,
it is desirable to keep the reaction between 0 and 30C by
regulat7ng the addition to the iodine solution or by cooling of
the reactton mixture, or by a combination of both. Under these ¦-
~ conditions the addition usually takes 30 to 60 minutes. After the
,J' addit7On the mixturé is stirred at 20 to 30C for 30 to 120 minutesJ
1 preferably 60 minutes. Thereafter the mixture is cooled to about 0C -
i and an excess of a miId reducing agent, preferably sodium thiosulfate in ~
aqueous solution is added. The mixture is concentrated and the residue
Is suspended in water. Collection of the solid material affords the
deslred corresponding cyclic disulfide of formula IX.
7 Alternatively, the linear protected peptide (Vll) is converted
to the aforementioned corresponding cyclic disulfide by the method of
¦ 20 R.G. Hlskey and R. L. Smith, J. Amer. Chem. Soc., 90, 2677 (1968) using
thlocyanogen. ;
Again alternatively, the cyclic disulfide (1X) is also obtained
by selectively removing the sulfhydryl protecting groups of t,le above - ¦
linear protected peptide (Vlll)by the action of a mercuric or silver salt, !
for example, mercuric acetate, mercuric chloride, silver acetate or silver
nitrate, in an inert organ7c solvent, for example DMF or acetic acid,
according to known methods; for example, see B. Kamber, and N. Rlttel,
Heiv. Chem. Acta, 52, 1074 tl964), L. Zervas, et al., J. Amer. Chem.
- Soc.,87, 4922 (1965) and R. G. Denkewalter et al., ~. Amer. Chem. ~
.: ~ - . . : :'
Soc., 91, 502 (1969). -The mercuric or disilver salt thus obtained is
~ . :
., , - :.
-33-

AHP-6550
1040623
then converted to the corresponding free disulfhydryl derivative (X)
2 2 Itr D T~tr D Phe D-TIhr-D-Lyls-D-Trp-D-Phe-D-Phe-D-Asn-D-
Lys-NHCHCH2SH (X)in which R is as defined herein
B c
by treatment with hydrogen sulfide, see L. Zervas, et al., cited above.
The latter derivative is then converted to the aforementioned cyclic
disulfide ~IX) by a mild oxidizing agent selected from the group
consisting of iodine according to the method described hereinbefore,
oxygen according to the~me~thod of J. Rivier, et al., C.R. Acad. Sci.
Ser. D, 276, 2737 (1973), 1,2-diiodoethane according to the method of
, , ~,:
F. Weygand and G. Zumach, Z. Naturforsch~ 17b, 807 (1962), and sodium
or potassium ferricyanide according to the method of D. Jarvis, et al.,
J. Amer. Chem.-Soc., 83, 4780 (1961).
,, Finally, the aforementioned cyclic disulfide of formula IX is
transformed into the corresponding cyclic peptide of formula 1, by
subJecting the former to moderately acidTc conditions whereby the
re~aining protecting groups of tne cyclic disulfide (IX) are removed.
~, Generally this step is carried out by treating the cyclic disulflde (IX)
wlth 50 - 100~ trifluoroacetic acid or with an aqueous solution con-
I tain'ng a mineral acid at 0 to 20C for 10 to about 60 minutes~
.j . . ~ . ~:Examples of such mineral acids are 10 to 20% aqueous sulfuric ac7d, -
~ 10% phosphoric acid, 10 - 30% hydrobromic acid and 10 to 36% hydrochloric
; acid. An extremely useful acid is concentrated hydrochloric acid.
i Preferred conditions for the present step include dissolving ~he
cyclic di 5U Ifide in a minimum of concentrated hydrochloric acid
cooled to O~C and stirring the mixture at 0C for five to ten minutes
under a nitrogen atmosphere. Thereafter glacial acetic acid
,..
,, - , ,~
,,, , .
.
.
..
-3~-
, .
. . . . ~ .
;: . . .
; ' ' ' '. , ' ' ,'~; : ~

AHP-6550
._
104~623
(10 X vols.) is added, the solution is cooled to about -70C and
Iyophilized to give the corresponding cyclic peptide of formula
The latter product is purified further by ion exchange chromatography
using a carboxymethylcellulose cation exchanger and ammontum acetate
as the eluant. In the latter case the product Ts obtained in the form
of its acid addition salt with acetic acid. Alternatively, the
product is purified by partltlon chromatography on a chemically
modified cross-llnked dextran, for example Sephadex*LH 20 or
Sephadex G-25, using methanol or acetlc acld, respectively, as
the eluting solvent. In the case where Sephadex*LH-20 and methanol
as the eluting solvent is employed, the product Ts obtalned In the
form of its hydrochloric acid addition salt. In the case where - -
Sephadex G-25~,and acet7c acid is employed, the product is obtained
in the form of its acetic ac1d addltion salt. Repeated IyophllizatTon
.. .
from water of the product In the form of its acetic acid additlon salt
! ylelds the substantlally pure cycllc peptlde of formula I in which
R Is as deflned hereln, In the form of the free base.
The llnear reduced form of the peptlde of formula la -
is obtained by removal of the protectlng groups from the aforem,entioned
llnear protected peptide of formula (Vl~
~, Trt-S-CH2CH2CO-D-Selr-D-T~r-D-Phe-D-T~,r-D-Lys-D-Trp-D-phe-D-phe-D-Asn
81 BL Bl Boc
D-Lys-NHCHCH2S Trt (Vll~)
!~ 80c R ~ ~
in whlch R is as defined hereln from the aforementloned dlsulfhydryl
~;j derivatlve of formula (X)
t 8~t D T~t~ D-L~s-D-Trp-D-Phe-D-Phe-D-Asn
D-Lys-NH~HCH2SH (X) -' -
Boc R
-
, * Sephadex is a trade mark
~35~

AI~P-6550
` 1041)623
in which R is as defined herein under moderately acidic conditions.
Preferred conditions for this deprotection step comprises dissolving
the linear protected peptide (Vlll) or the disulfhydryl derivative (X)
in concentrated hydrochloric acid at 0 to 5C in an inert atmosphere,
for example, nitrogen or argon. The mixture is kept at this
temperature for five to ten minutes. Subsequent isolation of the -
linear reduced form is accomplished in the same manner as described
previously for the isolation of the cyclic peptide of formula 1.
Also, the linear reduced form (la) is obtained directly
lo by reduction of the cycltc peptide of formula 1. Reduction with
dithiothreitol according to the method~of W.W. Cleland, Biochem. _,
, 480 (1964) is preferred, although other agents known to be effective
j for the reduction of cyclic disulfides to the correspondTng disulf-
hydryl derivative are applicable, for example, sodium bisulfite ~ -
followed by hydrolysis of the intermediate dithiosulfate derivat.ive.
j Finally it will be apparent to those skilled in the art
J fhat: equ7valent amino, hydroxy or thtol protecting groups,
¦ ~ equlvalent methods of coupling peptide fragments, and equlvalent
¦ metnods for removal of amlno, hydroxy or thiol protecting groups,
j 20 other than those disclosed herein could be used in the embodimentsof this tnvention without departing from the scope and spirit of
the tnventTon. Such apparent alterations are intended to be included
withtn the scope of this invention. ~ -
The following flow diagrams and examples illustrate further
this invention.
.
::~
.
. . .
1-
-36-
!
. ' .. , . ... ,- . .. ... ... . . .
, . . .. .. . .. . . .. .

AHP-6550
:
.: .
4 5 6 7104~623 8 9 10
Z-D-T~r-D-L~s-D-Trp-D-Phe-NHNH + H-D-Phe-D-Asn-D Lys-OMe
Bu Boc Boc
.
4 5 6 7 ~ 9 1 o : ~ -
~: H-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-OMe
Blt Boc . Boc ; ~ :
'. ( l V ) ~
::
.1 1 2 3
. ~ Trt-S-CH2CH2CO-D-Ser-D-T~r-D-Phe-NHNH :
~ ~S'" ~) , J ~---- ~
< ,~
i 1 2 3 4 S 6 7 8 9 IO
r-D-Tlr-D-phe-D-T~r-D-Lyls-D-Trp-D-phe-D-phe-D-Asn-D-Lys-oMe
Bu B Bu Boc Boc
Corresponding hydrazide (Vl)
1 ~ + HNHCH2CH2STrt or + HNH-IH-CH2S-Tr~
¦ ~ ~Vllaj ONHCH2CON~H2CH3 -
alternatively written as
\ H D-C~s-Gly-NHEt
b
Linea~r protected peptid~e (V~ L~ :
(R = ~ \ (R = CONHCH2CONHCH2CH3'
idation \ I) Ag or Hg
/ \ 2) H2S
dation ~ ~ . .
. Cycl7c disul ide derivative (IX) Disulfhydryl derivative (X)
: Reduction /
Depr tection Depl otection / . ~ :
Reduction~
- Cyclic peptide (I) Linear reduced for~ of
peptide (la)
~ ~ ~ Oxidation
i:~, . : : . :
' . : .. -
~: : ' ,"' :'' -.
'I ~ . , . ' . , ,, ' , , . , ' , ~ ' " " " ~ "

AHP-6550
~04~)623 ,,~ ~.
t -t
Z ~ / ~ _ ~
~0 ~m~m ;i~ t~ i~m ~,m ~ m ~m~ ~
_ _ . _ _ _
O ~ ~ ~ s I ~¦ --I ~¦
=1 ~ ~ lo~
~ I ¦ ¦~ ¦ 1~ ¦~
L ~ ~m ~ ~ ~m
'~ ~ O ~ O O Z , ~ C~ .
T/m 1~ l~o /m /m ~ /a~ ~ ~ _ I ,
N
-:
3û-

AHP-6550
1040623 ~..
- EXAMPLE-I - -
.
BenzYloxvcarbonYI~ t-butYl)-D-threonYl-l3-phenylalan7ne MethYI Ester
A' . (Z-D-Tt)r-D-Phe-OMe j
Blt . . - I
A mixture of Z-D-Ttlr-OH (3.1 9, 10 mmoles) and h-D-phe-oMe-H
Bu ;;~
(2.16 9, 10 mmoles) in THF (15 ml) is cooled to 0C and N-ethylmorpholine
(1.8 ml) added to attain a pH of 7 - 8. I-Hydroxybenzotriazole (1.4 g,
10.3 mmoles) is added followed by dropwise addition of DCC t2.1 9, 10
1O mmoles) in THF (20 ml). The mixture is stirred at 0C for 45 min and at
T room temperature for I hr. After filtration, the filtrate is con-
centrated under reduced pressure and the~residue dissolved in ether~
1 After fiItration of the precipitate, the fiItrate is washed with
t saturated NaHC03 solution,saturated NaCI solution, 5~ aqueous citric, 15 acid solution, and saturated NaCI solution. The ether solution is dried .
over Na2S04 and evaporated under reduced pressure. The residue is
subjected to chromatography on silica gel (200 9) using 25% EtOAc in !~ -
hexane. Evaporation of tne solvent under reduced pressure g7ves the
tl~le compound as an oil, EOl]D = -25.1 tc = 1, DMF~, nmr
20 tCDC13) ~ 1.08 (d,J = 6.5Hz, 3H), 1.15 (S, 9H), 3.70 (s, 3H), 5.18
;~ (s~ 2H), 7.35 tm, IOH).
;, - . .. `~.` "
.~ . :
~ ~ ''''
~1 : '
: ''
t ~
1 . :
'. .: '-
-39-
. . l . ~
.,,
. ,
- .. ~ : . . : . . .. . .

AHP-6550
Benzyloxvcarbonvl-(O-t-butYI)-D-serYl-(O-t-butYl)-D-threon~l-D~phenvl-
- alanine MethYI Ester (Z-D-Se,r-D-Thr-D-Phe-OMe)
., Blt B¦t , ..
Z-D-T~r-D-Phe-OMe (7.5 9, 15.9 mmoles, described in Example 1)
Bu
- dissolved in MeOH (90 ml) containing pyridine hydrochloride (1.83 9, 15.9 '
mmoles) is hydrogenated with 5% Pd/C as a catalyst. The mixture is
flItered and the fiItrate taken to dryness under reduced pressure to
givè H-D-Thr-O-Phe-OMe-HCI. The above product and Z-D-Ser-OH
~ 10 t4.67 9, 15.9 mmoles) are d7ssolved in dry THF (45 ml), cooled to OqC
'' and 2.7 ml N-ethylmorphol7ne'is added. I-Hydroxybenzotriazole (2.16 9,
. . .
15.9 mmoles) is addéd followed by a cold ~0C) solution of DCC (3.27 9,
15.9 mmoles) in THF (30 ml). The mixture is stirred for 45 min at 0C
and then I hr at room temperature. After filtratlon, the THF is
removed under reduced pressure, the residue taken up in ether and filtered.
I The ftItrate Is washed with saturated NaHC03 solution, saturated NaCI
¦ solution, ice-cold 5% citric acid solution, saturated NaCI solution,satu'rated NaHC03 solution and saturated NaCI solutlon. The resldue
(9.2 9) obtained after drylng the ether layer wlth Na2S04 and evaporating
under reduced pressure Is subjected to chromatography on a column of
slllca gel (200 9) using 30% EtOAc !n hexane. The solvent is evaporated
`~ under reduced pressure to glve the title compound as an oil, nmr (CDC13)j 1.05 ~d, J = 6.5 Hz, 3H), 1.18 (s, 18H), 3.75 (s, 3H), 5.18 (s, 2H),'3~ '25 7.4 (m, IOH).
~. -
~40- ` '
,

AHP-6550
104~)623
EXAMPLE-3
(O-t-butyl)-D-seryl-tO-t-butYI)-D-threonyl-D-phenYlalanine MethYI Ester
Acetate (H D-S~t D Tlht 3 2 ;
Z-D-Ser-D-Thr-D-Phe-OMe (8.4 9, 13.6 mmoles, described in
Blt B¦t . ,
Example 2), dissoived in acetic acid (84 ml), is hydrogenated with 5%
Pd/C as a catalyst for 20 hr. The mixture is filtered and the fiitrate
- taken to dryness under reduced pressure to gtve the title compound as
an oil: nmr (CDC13) ~ 1.05 (d, J = 6.5 Hz, 3H), 1.19 (s, 18H), 2.06
(s, 3H), 3.76 (s, 3H).
., ~
1 . '
'' ''~'~ '
.~
.
':
.' .,
. ~
,- . ~
:
'
'

AHP-6550
:
104~623
EXAMPLE-4
N-rl-oxo-3-(tritvl)thiolpropvl-(O-t-h~ltYl)-D-seryl-(o-t-buty!---D-threon
D-phenylalanine Mel-hYl Ester (Trt-S-CH2 H ~ Tr-D-T~r-D-Phe-~Me)
A solutior, of H-D-S~r-D-TIhr-D-Phe-OMe-CH3CO2H (1'.6 mmoles
described in Example 3) in dry THF (25 ml) is cooled to 0C
and N~ethylmorphollne (1.7 ml) added until pH 7 is attained. A
solution of 3-tritylthiopropionic acid (4.7 g, 13.6 mmoles) in THF
(25 ml) is added followed ~by l-hydroxybenzotriazole (1.~ g, 13.6 mmoles)
,
and DCC (2.8 9, 13.6 mmoles~ in THF (25 ml). The mixture is stirred
at 0C for 45 min "and at room temperatur0 for 2 hr. The precipitate
is removed by filtration, and the filtrate is concentrated under
reduced pressure. The residue is dissolved in ether and the precipitate
` 15 removed by fiItra~ion. The fiItrate is washed with saturated NaHC03
¦ solution, saturated NaCI solution, cold 5% citric acid solution, saturated
¦ NaCI solution, saturated NaHC03 solution and saturated NaC! solution.
The ether solutton Is dried over Na2S04 and evaporated under reduced
pressure. The residue is subjected to chromatography or a column of
silica gel (220 g) using 30% EtOAc-in benzene containing 0.2% tri~thyl-
amine. After evaporation of the eluates, under reduced pressure the
residue j~5 crystallized from ether to give the title compound, mp 117 -
125C [~]D = 18.6 ~c = 1, DMF)~
, ~ ''' ,~- '
- ,
j - .. : .
. .
''':
~ : . .
.'~ .
,, . . - 1. ~-
1 .:
;~
-~2- Y
, . . .
~: . : .' . ~ '

AHP-6550
10406Z3
EXAMPLE 5 -
N-rl-oxo-3-(trityl)thiolpropyl-lo-t-bu1-yl)-D-seryl-(o-t-butyl)-D--hreon
D-phenvlalanine Hydra~ide (V, Trt-~-CH2__ ~ elr-D-TIr~D~phe--NHNH2)
H2 2 D Sltr D T~r-D-Phe-OMe (2.025 g, described in
Example 4) in MeOH (70 ml) and hydrazine hydrate (2 ml) is stirred
at 0C for I hr and at room temperature for 24 hr. The precipitate
which is obtained upon addttion of water is collected on a sTntered glass
flIterand dried over phosphorus pentoxide to give the title compound;
nmr (CDC13) ~ 0.98 (s, 3H),I1.16 (s, 18H), 7.4 (m, 20H). ~ -
. ' " J ,'~ ~ -
, ' ~ ' ' '. ' ' ''
4, 1 '. ':
i j, 1'.'"
., .
, . . :
. 4~ : ..
1 ;~ `~ 1' ' '
,:
.1 " ''
'~ . ...
.,
-43-
' ~ : . ~ .
.;
.,
~, -- - . ..
, `;:
: . `, : - :, . , ` , . . . .. :.:i

AHP-6550
104~623
EXAMPLF=_
Benzyloxycarbonvl-D-tryptophyl-D-phenylalanine Methvl Ester
(Z-D-lrp-D-Phe-OMe)
A solution of Z-D-Trp-OH (3.62 9, 10.7 mmoles), H-D-Phe-OMe-HCI
(2.3 9, 10.7 mmoles) and l-hydroxybenzotriazole (2.89 9, 21.4 mmoles)
in dry DMF (25 ml) is cooled to 0C and N-ethylmorpholine (1.37 ml,
10.7 mmoles) is added. A cold (0C) solution of DCC (2.27 9, 11 mmoles)
In DMF (6 ml) is added dropwise and the reaction mixture is stirred
for I hr at 0C and I hr at room temperature. The reaction mixture is
then cooled to 0C, fiItered, the fiItrate evaporated under reduced
pressure and taken up in EtOA~. The EtOAc solution is washed with
saturated NaHC03 soii~tion, water, cold citric acid (2N), water, saturated
NaHC03 solution and saturated NaCI solution, dried with MgS04, and
evaporated under reduced pressure to afford the crude title product I
whtch is subjected to chromatography on a co;lumn of silica gel
'1 . : .
~500 9) with CHC13 containing MeOH (1%) as eluent. Tha eluates are
evaporaled under reduced pressure and the residue is crystallized
;~ from EtOAc-petroleum ether to give the t;tle compound; mp 130 - 131C,
[~X]D5 = +34.1 (c = I, DMF).
: .
11 . ` ~' .
:. ..
,~ l
,, . ': '
1 ~ ~ '':.
:.
."~ .
1~
~` --44-- ,,
: . . . . ., . . . , , , ,.,.,, ,.,.. ; ", .... .. . . ..
::.,.-: : ~ -. . :: :,.. : .

~ 550
.
104()~
EXAMPLE 7
BenzyloxYcarbon~l-N~-t-butvloxycal-bonyl-D- Iysvl-D-trY~tor)hy!-D-
phenylalanine Methyl Ester (Z-D-Lys-D-Trp-D-Phe-O~le)
Boc
A mixture of Z-D-Trp-D-Phe-OMe (2.00 g, 4.0 mmoles, described -
I in Example 6) and 5% Pd/C (0.25 g) in acetic acid is stirred
rapidly under an atmosphere of hydrogen. After completion of
hydrogen uptake a stream of nitrogen is passed tllrough the mixture
for 15 m7n, the catalyst is removed by filtration, and the acetic
IO acid is rcmoved under reduced pressure. The residue is taken
; 7n~o benzene, evaporate~d under reduced pressure (twice), and
dried under reduced pressure over KOH pellets to give
H-D-Trp-D-Phe-~Mé CH3C02H. The above product is dissolved in
! dry DMF (20 ml) and cooled to 0C. N-ethylmorpholine (0.512 ml,
4 mmoles) is added, followed by Z-D-L~s-ONp (2.00 g) 4 mmoles). -`~
Boc
.i, : .
The solutTon is stirred at 0C for 30 min and for 3 days at room
temperature. The solvent is evaporated under reduced pressure,
the res7due i~ taken Tnto EtOAc and washed with cold sa1urated
NaHC03 solutlon, water, cold citric actd (2N), water, saturated
NaHC03 solution and saturated NaCI solution. The EtOAc solution
is dried with MgS04 and evaporated under reduced pressure to ~;~
afford the crude title product. After chromatography on a column
of silica gel ~300 9) with CHCI3 containing MeOH (5%) and pyridine
2 (0-1%) as eluent, the chromatographically pure product is triturated ~ I
with ether, filtered, dried under reduced pressure, and crystallized
from MeOH-CH2CI2-isopropyl ether to give the title compound; mp 172 -
174C, [~]D = +18.9 (c = I, DMF).
.i i .
1~. ' ,.
.
:.
. - ,:
:~
. .
,
-45-
:: . , : . : ~ :
' :' ~ .: ` :.' ' .' ., ' '

B~IP-6550
104~6Z3
EXAMPLE 8
N~-t-ButyloxYcarbonv!-D-lYsYI-D-tryptophyl-D--p-enylalanine
Methyl-Ester Acetate (H-D-L~vs-D-Trp-D-Phe-OMe CH3CO
` Boc - --
A mixture of Z-D-L~s-D-Trp-D-Phe-OMe (2.218 9, 3.04 mmoles, :~
B c
~: described in Example 7) and 5% Pd/C (0.25 g) in acetic acid is
~ rapidly stirred under an atmosphere of hydrogen for 20 hr. The
'~, catalyst is removed by filtration and the filtrate evaporated
under reduced pressure to give the title compound as an oil~
nmr (CDC13) ~ 1.4 (s, 9H), 2.07 (s, 3H), 3.6 (s, 3H). -
", , .
, . '
., - ~''-'.
j ' ' . . '~, ''
` 3 ~
~ '
.' ~ ,.
- , i
. '
:~ ``-- 46- - ; :.
~ 1
: ~ . . . . :, :.- . , ~ -

AHP-6550
~ 1040623
EXAMPLE 9
BenzvloxYcarbonY!-(O-t-butYI)-D-threonyl-N~-t-butYlox~arb
D-lYs~l-D-t~y~tophvl-D-phenvlalanine Methvl Ester (Z-D-Thr-D-Lys-D-
Trp-D-phe--oMe) ~ BLt B~c
5- A solut;on of H-D-Lys-D-Trp-D-Phe-OMe-CH3C02H (3.04 mmoles,
- - B c - ,
,~ . -. . ' : .
,,1, described in Example 8), Z-T~$-OH (0.940 9, 3.0A mmoles) and 1- ;'
hydroxybenzotriazole (0.820 9, 6.08 mmoles) in dry DMF 15 ml), is
cooled to 0C and N-ethylmorpholine (0.39 ml, 3.04 mmoles) is ,,
I added. A cold ~0C')'~solution of DCC (0.626 9, 3.04 mmoles)
in DMF ~4 ml) is added dropwise and the reaction mixture is stirred '-
I hr at 0C and I hr at room temperature. After filtration the DMF
is removed under reduced pressure. The oily residue obtained after
drying is subjected to chromatography on a column of silica gel (270 9) ,'
wtth 2% MeOH in CHC13 as eluent. The chromatographically pure product ,
Is'crystalltzed from MeOH/isopropyl ether to give the
' tltle compound;,mp 124 - 126C, [a]D25 = +3.9o (c = 1, DMF).
.
I: ' ~,!
.
'I -~
.: .
-':: -
:
-47- ~

AHP-6550
~04~)6Z3
EXAMPLE 10
Ben~yloxycarbonyl-~0-t-b~y1~__threonv!-N~-t-bulyloxycarbonyl-D-
IYsYl-D-l-rvptop-hyl-D-~henvl~laninc HYdrazide ( ! ! . Z-D-T~r-D-Lys-D-
Trp-D-Phe-NHNH2) But Boc
A solution of Z-D-T~$-D-Lys-D-Trp-D-phe-oMe (1.307 9, 1.475 : :
Bb Bdc
mmoles, described in Example 9) and hydrazine hydrate (2.74 ml) in : :
:~ DMF (8 ml) is stirred at 0C for 2 hr. Water is added, and the
precipitate is collected by filtration and dried (1.267 g) over
phosphorus ; pentoxide. The dried residue is crystallized from
: 10 MeOH-CH2C12-isopropyt~.ther to give the l-itle compound; mp 203 -204C,
[a]D5 - +11.6~ ~c=l, DMF).
-' .. , "' ''
., ". :
:.
: . :
'i` ' i : -
: .. ' 1
., l -
! --
-4a- :
~: i : ~
: ~ -- -, ~:
I :
, . . ~. ~ . . . - , .:
: . :.: . ~ . . - : - . .. .
. - :: . . ~ , :,
..... : . . , . : , . . . .
:. . . . . : . . ... ~ .

AHP-6550
,\
1040623 -
` EXAMPLE II
BenzyloxYcarbonyl-~-asparaqinyl-N ~ butyloxvcarbonyl-D-lysine
Methyl Ester ~Z-D-Asn-D-L~s-OMe) ~
Boc - ;
A solution of Z-D-Asn-OTcp (1.48 9, 6.0 mmole) and H-D-Lys-nMe I
Boc
(2.67 g, 6.0 mmole) in DMF (40 ml) and .'~-ethylmorpholine (0.5 ml)
is stirred at 0C for 3 hr and at 25C for 20 hr. The soivent is
removed under reduced pressure and the residue triturated with
~,' 1Oether. The residu~ i~s dried under reduced pressure to give the
title compound; mp 147 - 149C, nmr (MeOH-d4): ~ 1.4
~, 9H~, 3,7 Is, 3H), 5.15 (s, 2H),~7.37 (s, 3H).
,1' ' ' . ~'' .''
1~ ' .. . '
I' .
~
~ .
~
I
~ ,~
1 1.
~ ~ .
. ~ .
-49- ,
I .
, ~ ~ ..' ' ' ' , ' . : . .. '

AHP-6 5 50
10406Z3
EXAMPLE_12
Benzyloxycarbonyl-D-phenylalanine-D-asparaginyl-N~-t-butyloxy-
carbonyl-D-lysine Methyl-Ester ~Z-D-Phe-D-Asn-D-Lys~t3Me~
130c
- 5 A mixture of Z-D-Asn-D-Lys-OMe (2.32 9, 4.67 mmoles, ~' Boc ~
described in Example 11) and 5% Pd/C tO.30 9) in acetic acid
(30 ml) Is rapidly stirred under an atmosphere of hydrogen for
1.5 hours. The mixture is ftItered and IN hydrochloric acid
; 1O (4.67 ml, 4.67 mmoles) is added to the filtrate. The filtrate
¦ is concentrated under'reduced pressure, MeOH (50 ml) is added,
and the-solve~t i,s removed under reduced pressure to give
H-D-Asn-D-Lyls-OMe-HCI.
Boc
1, A solution of the above compound (0.88 9, 2.14 mmoles)
'I ' Z-D-Phe-~Tcp (1.023 9, 2.14 mmoles), and N-ethylmorpholine (I ml)
1 - 1n dry THF (30 ml) is stirred at 0C for 24 hr. The precipitate
Is collected by filtration and crystallized from MeOH-isopropyl
ether to gTve the t7tle compound; mp 173 - 175C, ~D5 = ~13-4
(c - 1, DMF~. -
., . : ,
~ ~ ~ .
~ ~ . .:
:'~
~ '' ' . .,, ~'":'
: :: _ ~
2`" ' ~
'~ ' ',
! ~
....... l
-50-

Al-IP-6~50 ¦ .
. ~ . ,' '
: 104U623 ~
.EXAMPLE 13 ..
D-Pheny!alanYI-D-asp ~ uty!oxycarbonyl~D-l~L~ne Me~hY!
Ester hcetate~ H-D-Phe~D-Asn=D~L~s~~Me C!-I ~ ~ ;~
~ ^ B c - .. ~
A mixture of Z~D-Phe-D-Asn-D-L~s-OMe (0.641 9, 0.994 mmole, . ~ :
. Boc ~ . .
described in Example 12) and 5%.Pd/C (0.065 9) in acetic acid
(9 ml) is rapidly stirred under an atmosphere of hydrogen for 20 hr.
: The mixture is fiItered and the fiItrate is evaporated under :.
.' 1O reduced pressure. The residue is dissolved in benzen~ and : :
, evaporated under reduced pressure (twice~ and dried -
~ under reduced;préssure over KOH pell,ets to give the title compound .
.1 as an oil: nmr (CDC13) 6 1.38 (s, 9H), 2.02 (s, 3H), 3.7 (s, 3H), ;-: ;
1~ 7.4 (m, 5H).
~ ' ', . : .,
,1 . .
,~' '' ' ' , ,~
~
.
:
~ -
.,. .
.1 ,. , :'~
.
3~ . ~:
"
''~,'`' ~ ~: ' ' ,
.:
,
51-
i ~ ,:
1: '
, . . . . . .. . . .
~ .' : . :.. : ~ :'. ::: , .

AHP-6550
_,
1040623
EXAMPLE 14
Benzyloxycarbonyl-~O-t-butyl)-D-threonyl-N~-t-butvloxycarbonyl-D-l~y !-
D-tryptop~ l-D-phenylalanyl-D-phenvlalanyl-D-asparaainyl-N~=t-but ~ -
carbonyl-D-lysine Methvl Ester-(Z-D-T;r-D-L~s-D-Trp-D-Phe-D-Phe-D-Asn-D-
- 5 l~s-OMe) - But Boc
B c - -
Z-D-T3r-D-Lys-D-Trp-D-Phe-NHNH2 (0.88 g, 0.994 mmole, described
B B c
In Example 10) is dissolved in dry DMF ~20 ml) with slight heating and
the clear solution is cooled to -20C. Hydrochloric acid in EtOAc
~2N: 1.25 ml) is added followed by t-butyl nitrite (0.137 ml, 1.2 mmole).
The mixture is stirr~d for 15 min at -15Cv The mixture is stirred
for 15 mln at -15C. A soluflon ot H-D-Phe-D-Asn-D-Lys-OMe CH3CO~H -
(0.994 mmole, described in Example 13) in DMF (6 ml) containing N-ethyl-
d7isopropylamine (0.60 ml, 3.5 mmoles) is cooled to -15C and added
dropwise to the above mixture. Stirring is continued at -15C for I hr
1 and at room temperature overnight. The reaction mixture is evaporated ¦~
¦ under reduced pressure at 35C, the resTdue triturated with ice cold
citrlc acld (IN), the m1xture is filtered and the precipitate is washed
, with wate~ and dried over phosphorus pentoxide (1.27 g). The solid
¦ residue is subjected to chromatography on a column of silica gel (127 9)
with 5~ MeOH in CHC13 as eluent. The chromatographically pure product
~ is evaporated under reduced pressure and the residue is crystallized
1~ from MeOH-CH2C12~isopropyl ether to give the title compound; mp 213 - ~ ~-
25 215C, [a]D = ~13.4 (c = 1, DMF). `
' .'
. . .-
! . .
- 3 2 ~
~'
, `:
,'.,' . .

~ AHP-6550
~04~)623
'- EXAMPLE-15 ' ''
(O-t-Butyl)-D-threonyl-N -t-butYloxYcarbonYI-D-lYsyl-D-tryptophyl-D-
phenylalanyl-D-pheny!a anYl-D-asParaqinY!-N~-t-butyloxycarbonyl-D-lysine ~ ' ',
Methyl Ester Acetate-(IV~ H-D-T~r-D-Lys-D-l'rP-D-Phe-D-Phe-D-Asn-D-
~- 5 L s-OMe-CH CO H) Bl Bo~
-r 3- ~
, Boc
A mixture of Z-D-T3r-D-Lyls-D-Trp-D-Phe-D-Phe-D-Asn-D-LIys-OMe
B Boc Boc
(0.71 9, 0.515 mmole, described in Example 14) and 5~ Pd/C (0.065 9) in
acetic acid is rapidly stiired under an atmosphere of hydrogen for 20 hr.
The mixture is filtered and the filtrate evaporated under reduced pressure.
,~ The residue is dissolved in benzene, evaporated under reduced pressure
. , ,
(~twice),and dried under reduced pressure over KOH pellets
to give the title compound as an oil: nmr (CDC13) ~ 0.95 (s, 9H), 1.50
~, 15 ~5/ 9H), 2.04 (s, 3H), 3.81 (:, 3H).
1 ' .' ~'
~'
.' ~
. . ''' :
$ ,, . :~
I !
3~
1~ .
. , .
, ~ .
j '~
;:1
~: :
, -53-

AHP-6550
10406Z3
EXAMPLE 16
N-rl-Oxo-3-(tritv!)thiolpr~a~!L ~ butyl)-D-seryl-(O-t-butyl)-D-~threonyl-
D-phenYlalanYI-(O-t-but~l)-D-threony__N -t-butyloxycarbonvl-D-lvsYI-D-
try~Dto~l-D--æhen-ylalanyl-D-~henylalanyl-D-asDaraqin~-Ne-t-but~
carbonyl-D-lvsine MethYI Ester (Trt-S-CH2 H~CO-D-S~rr-D-T~r-D-Phe-D-T~r-D-
L~s-D-~u~ _ Phe-D-Phe-D-A n-D-L ~ Bbt Bbt Bbt
B c Boc
; The tripeptlde hydrazide (Y) Trt-S-CH2CH2CO-D-Ser-D-Thr-D-Phe-
NHNH2 (0.418 9, 0.515 mmole, described In Example 5) is dissolved in dry
DMF (6 ml) and cooled to -20C. Hydrogen chloride in EtOAc (2N,
0.645 ml) is added;followed by t-butyl n7trite (0.0706 ml, 0.619
mmole). The mixture is stirred for 15 min at -15C. A solution of
H-D-T~r-D-Ly,s-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-OMe-CH3C02H (0.515 mmole,
Bu Boc Boc : ,
described in Example 15) in DMF (10 ml) containing N-ethyldiisopropylamine
(0.310 mi, 1.8 mmole) is cooled to -15C and added dropwise to the ~ ~ -
;1 above mixture. Stirrlng Is continued at -15C for I hr and at room
temperature overn19ht. The reactlon mtxture Ts evaporated under reduced
pressure at 35C, the resldue Is trlturated wlth Ice cold cltrlc acld
;1~ iltered, washed With Water and dried over phosphorus pentoxlde.
~ The ~sorld resldue is subJected to chromatography on a column of silica
-I gel~(100 g) wlth 5% MeOH in CHC13 as eluent. The chromatographically
purë product is crystallized from MeOH/CH2C12/isopropyl ether to glve
the title compound mp 205 - 207C [a]D5 = ~4.3 (c = 1, DMF).
,
'
.. ~ -,
'' , ' ' ' ' ' ' ' ' `

AHP-6550
1040623 ~
EXAMPL~. 17
l~'-rl-Oxo-3-(trityl)thiol?ropvl-(0-t-butyl)-D-se_~-(0-t-butYI)-D-threonyl-
D-phenylalanyl-(0-t-butyl)-D-threonyl-N~-;-butyloxycarbonyl-D- IYSVI_D ~ .
~: trvptophy!-D~phenYlalanyl-D-phenylalanyl-D-asparaqlnyl-N~-t-butyl_xy-
carbonyl-D-Iysine Hydra~ide (VI, Trt-S-CH2 II~CO-D-Selr-D-Thlr-D-Phe-D-TIhr-D-
lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NHNH2) But But Bu
Boc Boc .
A solution of Trt-S-CH2CH2C0-D-SIr-D-T~r-D-Phe-D-T~r-D-Llys D-
B B B Boc
:, Trp-D-Phe-D-Phe-D-Asn-D-Lyls-OMe (0.504 9, 0.249 mmole, described in
Boc .l .
Example 16) in pMl~t~ ml) and hydrazine fhydrate (0.464 ml) is stirred
,. at 0C for 30 min and at room temperature for 23 hr. The product is :
precipitated by addition of cold water and filtered. The precipitate
is washed several times with water and dried under reduced pressure
over phosphorus pentoxide to give the title compound:.amino acid ~
analysis: Lys, 1.91, Thr, 1.62,Ser, 0.76, Asp, 1.00, Phe, 2.90. ~ :
.i ~ ,
-_.
.
! : ;
i~', ''
. j 1i'"
.. . .
`. : - 1'
!-~
~
.~ ................................ . 1,
-55- 1
'''` ' .
,
'' ' :'. ' ~' ' '' ' ': :' `
". ,`, ': ` - :~ :
~' , ', . . ` : .

AHP- 6550
~ 104~6Z3
EXAMPLE 18
N;S-Ditrityl-D-cysteinylqlvcine-ethylarr.?ide tTrt-D-C~s-Gly-NHEt)
Trt
A solution of Trt-D-C~Is-Gly-OEt (10 9, prepared as described
Trt
by G. Amiard, Bull. Soc. Chim. (Fr.), 1956, 698 for the L-isomer of
cysteine) and ethylamine (15 mlj is allowed to stand at 5C for 24 hr.
The solution is evaporated under reduced pressure and the residue is
subjected to chromatography on a column ofstlicagelt200 9) uslng 15%to
30% EtOAc In benzene as eluent. The eluants are evaporated under reduced
pressure to give the titlë c,ompound; nmr (CDC13) a o.s
(t, J = 7 Hz, 3H)"~7.35 (m, 30H). --
.~, ~ ,.
., . : .
; ' ' . : :
~1 ~
., . , ;,.,;.. '
,. , ~ ~ .
. 1 ~ . : ~ .
'
,
:i:
,? ~
' ': ` ` .' , .' :
J,
f
-~ ~ ` ? . .
;,'~ '' .
:j: : :
. ~ , .
1 -56-
'. .
.j~ . .

AHP-6550
1040623
EXAMPLE 19
S-Tritvl-D-cysteinvlqIycine-ethylamide ~Yd-roc-h!or-ide
~ _S Trt, R = C0NHCH~CONHCH2CH3LVIIb)
A solution of Tr1--D-Clys-Gly-NHEt (1.03 9, 1.52 mmoles,
Trt
~ described in Example 18) in acetic acid t8 ml) and water (1.9 ml) -~
-~ is stirred at 45C for 15 min. Water (8 ml) is added an~ the
precipitate is removed by fiItration. Hydrochloric acid (IN, 1.5 ml)
is added to the flItrate and the soluTTon Iyophilized. The dry
product is washed with ether to give the title compound; nmr (DMS0-d6)
,1 ~ 1.02 (t J=7 Hz, 3H), 3.20 (m, 2H), 7.40 (s, 15H).
, ~ , J ,. ~ .~
'
. ' _
.
.A .,
~. " . .
-'1 , ' '
'"J~ . :
.i ' ' ' ' '
: .
. .
!` - ,. .
~q~ ~ - ~ ',,. -
,~ ~
"~
,`, ~ , . .
i
; ~ ,
, ..
I
... . . .. . . . .
- - . ; ~ .. .
. . ~ . . , . .` :
.

AHP-6550
` 10406Z3
cXAMPLE 20
N-rl-Oxo-3-(tritYl)thiolpropyl-(o-t-butyl)-D-seryl-(o-t-butyl)-D-threonyl- . .:
D-phenYlalanyl-(o-~-butyl)-D-threonYl-N -t-butYloxycarbonYl-D-lysYl-D
tryptophyl-D~phenylalanyl-~-phenylalanyl-D-asparaginyl-N~-t-butvloxy
- 5 carbonyl-D-lysvl-(S-trityl)-~-cvsteinylqlycine-ethylamide.
t Bl't ~y s-D-Trp-D-Phe-D- Phe-D-Asn-D-
Lys-Nl~CH~S Trt, ~ ~ -~
Boc R
Trt-S-CH2CH2CO-p-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-phe-D-phe
¦t 1 Blt Boc
D-Asn-D-L~s-NHNH2 (~.467 9, 0.231 mmole, ~escribed in Example 17) is
Boc
dissolved in a mixture of dry distilled DMF (8 ml) and DMSO (2 ml) and
cooled to -20C. Hydrogen chloride in EtOAc (1.4 N, 0.412 ml, 0.577
mmole) is added followed by t-butyl nitrite (0.0313 ml, 0~276 mmole?.
The mixture is stirred for 15 min at -15C. A solution of H-D-Cys-Gly-
Trt
j NHEt-HCI (0.112 9, 0.231 mmole, described in Example 19) in DMF
(2.5 ml) containlng N-ethyldiisopropylamine (0.138 ml), 0.807 mmole) is
cooled to -15C and added dropwise to the above reaction mixture.
Stirring is continued at -15C for l~hr and at room temperature over- j~
night. The reaction mixture is evaporated under reduced pressure, the
residue is triturated with ice cold citric acid (IN), filtered and
washed with water. The solid residue is washed three times with MeOH
and dried under reduced pressure to give the title compound; amino
~ .., . , ..: , .
~ acid analysis: ratio Asp/Gly = 1.08:1.
i; ; "'8 : :
': .
.. . . .
., .,, .. , - . ... . ..... . ... . ... .. ,. ",. .........
.. .. .. . . .... , . ,".. " ... .:. . , ~ . ' ... ~ .. . .

AHP-6550
4~6Z3
EXAMPL E 2 !,
Cvclic Disulfide of-N~ oxo-3-thio)propyl-(0-t-butyl)-D-servl-(O-t-butyl)-
D-threonYI-D-phenYtalanYl-~O-t-butvl)-D-threonYl-N3-t-butyloxycarbonyl-D
lYsYl-D-trvPtophYl-D-phenyialanyl-D-~henvlalanyl-D-asparaqinyl-N -t-butvl-
oxvcarbonyi-~-lysYI-D-cvsteinYlt~lYcine-ethvlamids (S-CH~CH~CO-D-SIer-D-T
Phe-D-T ~ ys-D-Trp-D-Phe-D-Phe-D-Asn-D-L~s-NH~HCH~S,R~ CONHCH2CONHCHC~,IX)
B Boc B c -
. . _ _ _ _ _ _ _D- .
A solution of Trt-S-CH2CH2CO-D-S~tr-D-TIh~r D Phe D T~tr D Lyjs
10 Trp-D-Phe-D-Phe-D-Asn-D-L~-D-Cys-Gly-NHEt (0.32 9, 0.132 mmole,
B Tr~ -
,~ I
described in Exam,p;le,20) in acetic acid is slowly added to a stirred
solution of iodine (0.336 9, 1.32 mmole) in MeOH (66 ml) at room
; temperature. After completion of addition the solution is stirred at :
¦ 15 room temperature for I hr. The sctiution is coo~ed to 0C and a
solution of sodium thiosulfate in water (IN) iS slowly added to destroy
the excess of iodine until a colorless solution is obtained. The solvent
Ts evaporated under reduced pressure almost to dryness; the residue
is dissolved in MeOH and added to 7ce cold water. The precipitate
is collected by filtration, washed with water and dried under reduced
pressure over phosphorus pentoxide to give the title compound; amino 1~ 1
acid analysis: Lys, 1.92; Ser, 0.75; Asp, !.05; Gly, 1.00; Thr, 1.78; ¦
Phe, 3.02; Cysteic acid, 0.93.
.
g
1.
.
,~
I
::
.' ~ ' .
. ~ .:
~ l!
. . - .. . , . . . ~. . . .
. : . ~.. .. . . .. ~ .. . . ~

Afl-lp-r~j5o
1040623 ~
EXAMj~LE 22
CYCI;C Disulfide of N-(l-oxo-3-thio)DroDvl-D-serYl-D-thrffonyl-D-Dllcnyl-
alanYl-D-threonYI-D-lvsYl-D-tryDtoDnyl-D-phenylalanyl-D-Dhenylalanyl-D
asDaraqinYl-D-IYsvl-D-cvsteinYlalvcine-ethylamide
; 5 ~ ~ ~C0-D-Ser-D-Thr-D-Phe-D-Thr-D-LYs-D-TrD-D-Phe-D-Phe-D-Asn-D-
LYs-Nllt~H2Ch2 . 1. R = CONHCH2CONHCH2fCH
A solution of the cyclic disulfide of ~CH2CH2C0-D-Ser-D-TIhr-
D-Phe-D-TIhr-D-Livs-D-Trp-D-Phe-D-Phe-D-Asn-D-Lyls-D-C s-Gly-NHEt (0.250 9,
But Bdc Boc
1 0
j 0.132 mmole, described in Example 21) is vigorously stirred at 0C under
an atmosphere of nTtrogen for 10 min in conc. hydrochloriG acid (11 ml).
Acetic acid (150 ml) is added and the solution is Iyophilized. The
residue is taken in water, filtered and again Iyophilized. The residue
(0.20 9) is dissolved in 0.01 M ammonium acetate (10 ml), the solution
obtained is app~ied to a column (2.1 cm x 30 cm) of carboxymethyl ~ ~-
ii cellulose (Whatman CM-23) and eluted first with O.OIM ammonlum acetate
(200 ml), to remove the impurlties. The pure compound is eluted with 0.05 M
I and 0.06 M ammonium acetate sfnd Iyophllized to give the tltle
i compound as a whlte solld in the form of its acetic acid
,~ 20 addition salt; vMmeaXoH 289 (6310), 283 nm (6 6310~. Repeated Iyophilization
i of the latter product from water gives the title compound
as the free base;amino acid analysis: Lys, 1.88; Asp, 1.00;
Thr, 1.89; Ser, 0.92; Gly, 0.71; Cys, 0.43; Phe, 2.93. Lyophilization
¦ 25 of the latter product from IN hydrochlor!c acid gives the title compound
in the form of its hydrochloric acid additlon salt; amino acid
~ analysls: Lys, 1.92; Asp, 1.00; Thr, 1.90; Ser, 0.90; Gly, 0.78; Cys,
1 0.52; Phe, 2.90.
i . .
~ * Whatman is a trade mark
1 . .`,
.,. '
.:
,
~: - 60- :

1040623 AHP-6550
EXAMPLE 23
N-(!-Oxo-3-thio)propyl-D-seryl--D-threonyl-D-~-henyla!a-nyl-D-th~l--D-
lysYI-D-trYptophyl-D-phenyla_lanyl-D-phenylalanYl-D-asparaainvl-D-lysyl-D-
cysteinvlqlycine-ethylamide tH-S-CH2__C0-D-Ser-D-Thr-D-Phe-D-Thr-D-LYs-D- ~ -
he-D-phe-D-Asn-D-Lvs-NHci-2 _~SH. Ia. R = CONHCH ~ ~C_ ~
By following the procedure of Example 22 but replacing the
title cyclic disulfide of Exa~nple 21 with an equivalent amount of the
tTtle compound of Example 20 Trt-S-CH2CH2C0-D-Selr-D-Thr-D-Phe-D-Thr-D-
But Bbt B~t
Lyls-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-D-cys-Gly-NHEt the title compound
B c ~ ~ Boc T~t
of this example is obtained; amino acid analysis: Lys, 2.02; Asp, 1.00;
Thr, 1.95; Ser, 0.~8; Gly, 0.82; Cys, 0.49; Phe, 2.97.
In the same manner, by replacing the title compound of Example
' 20 with the corresponding disulfhydryl derivative of formula X in which
! R is CONHCH2CONHEt HS-CH2CH2CO-D-Se,r-D-T~t-D-Phe-D-TIht D LYis D Trp D
Bu B Bu B c
Phe-D-Phe-D-Asn-D-Lys-D-C~s-Gly-NHEt and proceding as above the title
J B~c Trt
compound Is also obtained.
, .
. '' .
. .
.
:J . I
.. i~ '''
.,~ :
', '~
:. '
61
..
. . ,, . . ' ~ . :

104~623 AHP-6550
EXAMPLE ?~4
N-rl-Oxo-3-ttritYl)thiolpropYI-~O-t-butyl)-D-serYl-(o-t-butyl)-D-threon
D-PhenYlalanYI-(O-t-butyl)-D-threonyl-N~-t-butvloxycarbonvl-D-lYsyl-D
tryptophyl-D-phenYlalanYl-D-Dhenylalanyl-D-asparaqinYI-N -t-butvloxy-
carbonyl-D-lysine 2-(tritYlthio)ethYlamide tTrt-S-CH2CH_CO-D-Sle~
Phe-D-Thr-D-Lyjs-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NHCH2_~S Trt. Vlll. R - H)
B~ Boc B~c
Trt-S-CH2CH CO-D-Sçr-D-T~r-D-Phe-D-T~r-D-Lys-D-Trp-D-Phe-D-Phe-
2 B~t B~t B1t Boc
D-Asn-D-Lys-NHNH2 ~0.458 g, 0.227 mmole, described in Example 17) is
B c
' dissolved in a mixture of dry distilled DMF (8 ml) and DMSO (2 ml) and't . cooled to -20C. Hydrogen chloride in EtOAc (2.0 N; 0.286 ml, 0.567
~ mmole) is added f~oliowed by t-butyl nitrite (0.0314 ml, 0.276 mmole). '-
.. , :'
The mixture i5 stirred for 15 min at -15C. A solution of 2-tritylthio-
ethylamine [0.080 9, 0.250 mmole, prepared as described by F.l Carroll
,j et al., J. Ora. Chem~, 30, 36 (1965)] in DMF (3 ml) containing N-ethyl-diisopropylamine (0.055 mlj 0.348 mmole)'is cooled to 15C and added '
dropwise to the above reaction mixture. Stirring is continued at -15C
for l hr and at room temperature for 3 days. The reaction mixture is
~ evaporated under reduced pressure, the resldue Is trlturated wlth Ice
'i 20
"¦ cold cltric acid (IN) and filtered. The precipitate is washed with
! -
water, drled and crystallized from chloroform-methanol to give the
title compound,mp 234 - 237C. anal: calcd for C130 H165 N15 019
S2 : C, 67.71; H, 7.21; N, 9.11. Found: C, 67.64; H, 7.28; N, 9.24. ¦
I' . .
: ~ l . . ',
.~ . ' . '
~:1 . ... ..
3 ~ :
.. ; ~ ,
- ~ .:
.
-62-
:, .
-
.,

AHP-6550
1~)40623
EXAMPLE 25
Cyclic DisulfidQ of N-(l-Oxo-3-thio)propvl-(0-t-butYI)-D-serYl-(O-t-butYi)-,
D-threonvi-D-phenvialanYI-(O-t-butYl)-D-threonyl-N~-t-but~loxycarbonvl-D-
lysvl-D-trYDtophyl-D-phenYIalanyl-D-phenylalanyl-D-asparaqinyl-N -t-butvl-
oxycarbonyl-D-Ivsine_2-thioethylamide (5 ~ 2C l--e~3t -~- -
D-L~s D-Tr~-D-Phe-D-Phe-D-Asn-D-L ~ 2__2S. IX, R = H
B c B c
B~t B~t B~t B~c
B~" 2 2 9, 0.096 mmole,
described in Example ,24) in acetic acid (40 ml) is slowly added to a
stirred solution of iodine (0.956 mmole) in acetic acid (50 ml) at room ~-
temperature. After completion of addition, the solution is stirred at
. , ,
room temperature for I hr. The solution is cooled to 0C and a
solution of sodium thiosulfate in water (IN) is slowly added to destroy
, the excess of iodine until a colorless solution is obtained. The
solvent Is evaporated under reduced pressure almost to dryness and the
~ restdue Is triturated wi ih water. The preclp1tate is collected by
3~ 20 filtration and dried under reduced pressure over phosphorus pentoxlde ;
~t ~o give the title compound; amino acid analysis: Lys, q.89; Ser, 0.71;
" ~
Y Asp, ~.06; Thr, 1.75; Phe 3.12. -~-
'' ' ' ., :
'~
,., ~: '
': ',
~ '
ij :
~ 1 ~
-63~
'~ !

- 1~4~6Z3 AHP-6550
:: EXAMPLE 26
CYCIjC Disulfide of N-(!-Oxo-3-thio)propyl-D--ser~l-D-threony!--D-phen
alanYI-D-threonvl-D-lysy!-D-tryptophyl-D-phenylalanyl-D-pheny!a!anyl-D-
asparaainyl-D-lysine 2-thioethylamide (Sl-CH2___C0-D-Ser-D-Thr-D-Phe-D-Thr-
D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-LYs-NHCH2CH_~ 1. R = H)
A solution of the cyclic disulfide of S ~ C0 D Sltr-D-T~r-
D Phe D T~r D LYIS-D-TrP-D-Phe-D-Phe-D-ASn-D-~YIS-NHC~2CH2 (0.096 mmole,
BU BOC B C 1.
described in Example 25) is vigorously stirred at 0C under an
:'
atmosphere of nitrogen for~10 min in conc. hydrochloric acid (10 ml). -
Acetic acid (100 ml) is added and the solution is Iyophilized. The
, " ~ ~ .
residue is taken up in 5% acetic acid in water and again Iyophilized
The resldue is dissolved in the upper phase of n-butanol-acetic
acid-water (4:1:5) and fiItered. The fiItrate is subjected to
partition chromatography on a column of a chemically modified cross- ~;
linked dextran ("Sephadex G-25 M", 3 x 50 cm, equilibrated in the
lower phase of n-butanol-acetic acid-water (4:1:5) and then equilibrated
In the upper phase) using the upper phase to desorb the cyclic peptide .
The fractions containing the pure cycllc peptide are combined and
;yophilTzed to give the title compound in the form of its acetic acid
addition salt; ~MmaxH 290 (~ 5,415)-, 282 (~ 6,000) and 274 nm (~ 5,660). ,1
Repeated Iyophilization of the latter compound from water gives the '¦
tTtle compound in the form of its free base; amino acid analysis: ~
Lys, 2.10; Asp, 0.93; Thr, 1.83; Ser, 0.93; Phe, 3.00. !
. `' ' . l :'
': . ; ,
~ ~ .
, : : .
,: :
:~:
-
, ` . :
:
i- '~
: : :
'- . : :.
.,, ~,
-` . . . . . . . : .
.. . : , , , . ~ . . .

- A~IP-G550
11;)4~623
EXAMPLE 27
N~ Oxo-3-thio)propyl-D-seryl-D-threonVI-D-phenYlalanYI-D-threonYl-D-
lysYl-D-tryptophvl-D-phenylalanvl-D-phenylalanyl-D-asr)araqinyl-D-lyslne
2-thioetnv!amide (H-S-CH2CH~CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lvs-D-
TrD-D-Phe-D-Phe-D-Asn-D-LYs-NHCH2CH~SH. Ia, R = H)
By following the procedure of Example 26 but replacing the
title cyclic disulfide of Exampl e 25 with an equivalent amount of the
title compound of Example 24 Trt-S CH2cH2co-D-ser-D-Thlr-D-phe-D-Ter-D-L
D-Trp-D-Phe-D-Phe-D-Asn-D-L~s-NHCH2CH2S Trt the title compound of thls
B c
example is obtain~ amino acid analysis Lys, 1.95; Asp, 1.13; Thr, 1.91;
Ser, 0186; Phe, 3.00.
; In the same manner, but replacing the title compound of
Example 24 with the correspondingdisulfhydryl derivative of formula X in
which R is hydrogen HS-CH2CH2CO-D-S~r-D-T3r-D-Phe-D-Tt~r-D-LYjs-D-TrP-D-
. B B But Boc
j Phe-D-Phe-D-Asn-D-Lys-NHCH2CH25H and proceeding as above the title
compound Is also obtained. ;
~ ~ -
.
, ::
.
,
.
-65-
.~
.. . ~ .
.. . .
- ,: . .