Note: Descriptions are shown in the official language in which they were submitted.
20050~6
O. Z . 0050/40390
NOVEL TNF PEPTIDES
The present invention relates to novel peptide~ derived
from tumor necro~is factor tTNF), the preparation thereof
and the use thereof as dru~s.
Carswell et al. (Proc. Natl. Acad. Sci. USA 72 (1975~
3666) reported that the serum of endotoxin-treated
animals which had previously been infected with the
Calmette-Guerin ~train of Mycobacteria (BCG) brought
about hemorrhagic necrosi~ in variou~ mouse tumors. ~his
activity was ascribed to tumor necrosis factor. TNF also
has a cytostatic or cytotoxic effect on a large number of
transformed cell line~ in vitro, whereas normal human and
animal cell lines are unaffected (Lymphokine Report~ Vol.
2, pp 235-275, Academic Press, New York, 1981). Recently,
the biochemical characterization and the g~ne for human
TNF have been described (Nature 312 (1984) 724, J. Biol.
Chem. 260 (1985) 2345, Nucl. Acids Re~. 13 (1935) 6361).
It i~ possible to deduce from thi~ data the following
protein structure for mature human TNFs
V~l~n~eD~r~ te~noD ~u~ysProVal~ sValVaLU3AY~Ro
G~ uGl
V~lGl. ~ ~ ~ D~IYalV~lP ~ Ll~lyl ~ l ~yrS
GlnV~l~Eh~nklyG~L~yCy~B~6~rrlio~V~lT~T~rUi~Ile
S ~ gIl~l~lValSe~ n ~ ysV ~ lalleLy~SerPro
CysG~dh~Glurhrbxf~uGlyAk~uAla~y~hl~kp~yCluProIld~deu
GlyGly ValPhPr~l nT ~1 uLysGayAnpknyels L~f1UI1e~59Pn~O
~ ValTyrPh0GlyIleIleA~u
The TNF gen~ of cattle, rabbits and mice have also been
described (Cold Spring Harbor Symp. Quant. Biol. 51
(1986) 597).
BeYide~ its cytotoxic properties, ~NF i~ one of the main
~OIU50~
- 2 - o.Z. 0~g~J40390
sub~tances involved in inflammatory reaction~ (Pharmac.
Re~. 5 ~1988~ 1293. Animal models have shown that TNF is
involved in septic shock (Science 229 ~1985) 869) and
graft-vers~-ho~t disea~e (J. ~xp. Med. 166 (1987) 1280).
We have now found that peptides with a considerably lower
molecular weight ha~e beneficial properties.
The present invention relàteq to peptide~ of the
formula I
X-Al~-Hi~-A-Y
where
A is Val, Leu, Ile or -NH-(CH2)m-CO- (with m being an
integzr from 1 to 12),
X is G-NH-CHM-CO-, G-NH-CHN-CO-W-, G-R-NH-CHM-CO- or
G-R-~H-CH~-CO-W- and5 Y is -3, -NH-CHQ-CO-Z, -V-NH-CHQ-CO-Z, -NH-CHQ-CO-U-Z
or -V-NH-CHQ-CO-U-Z,
where, in X and Y,
G is hydrogen or an amino-protective group,
Z is OH or ~H2 or a carboxyl-prot~ctive group, or
R is -Leu-Arg-Ser-Ser-Ser-Gln-Asn-Ser-Ser-Asp-Ly~-Pro-,
-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-~y~-Pro-,
-Leu-Arg-Ser-Ser-Ser-Gln-Ala-Ser-Ser-Asn-Lys-Pro-,
-Leu-Arg-Ser-Ala-Ser-Arg-Ala-LQu-Ser-Asp-Lys-Pro-
or a ~equence of 5-11 amino acid re~idue4 from ona
o~ the~e peptide chains or a-chain composed of 1-4
naturally occurring ~-amino acids,
U, V, and W are chain~ compos~d of 1-4 naturally occur-
ring ~-amino acid~, and
M and Q are hydrogens or on~ of the following
-CH(CH3)2, -CH(CH3)-C2H5, -C6H5, -CH(OH)-CH3
~H 2~ ~H Z~ or --t CH 2 ) b--T
H
(with b b0ing from 1 to 6 and ~ ~eing hydrogen or
'~C~5056
- 3 - O. Z . 0050/40390
OH, CH30, CH3S ~ ( CH3 ) zCH ~ C6X5 ~ p-HO--CbH4, HS, H2~,
HO-CO, H2N-CO or H2N-C ( =N~ ) -NH ) or
M and Q together are a - ( CH2 ) c-S-S- ( CH2 ) d- ~ - ( CH2 ) ,-CO-~3H-
( CH2 ) ~- or - ( CH2 ) "-NH-CO- ( CH2 ) ~-NH-CO- ( CH2 ) ~- bridge
(with c and d being from 1 to 4, e and f being from
1 to 6 and g being from 1 to 12),
a~ well a~ the ~alt~ thereof with physiologically
tolerated acids.
~he peptide of the formuls I are constructed of ~-amino
acid~, but they can contain 1 or 2 D-a~ino acids. The
side-chains of the trifunctional amino acids can carry
protective group3 or be unprotected.
Particularly preferred phy~iologically tolerated acid~
ares hydrochloric acid, citric acid, tartaric acid,
lactic acid, phosphoric acid, methanesulfonic acid,
acetic acid, formic acid, male~c acid, fumaric acid,
malic acid, succinic acid, malonic acid, ~ulfuric acid,
L-glutamic acid, L-aspartic àcid, pyruvic acid, mucic
acid, benzoic acid, glucuronic ac~d, oxalic acid, ascor-
bic acid and acetylglycine.
The novel peptide~ can be open-chain (G 3 H, 2mino-
protective group; Z = OH, NH2, car~oxyl-protective sroup
M and Q not connected together) and, in particular, ha~e
a disulfide bridgQ (G z H, amino-protective group;
z D 0~ NH2~ carboxyl-protective group; ~ + Q = -(CH2)C-
S-S-(CH2)d-) or a side chain bridge (G = H, amino-protec-
tive group, Z - OH, NH2, car~oxyl-protectiYe group, ~ ~ Q
= -(CH2),-NH-CO-(CH2)s~ or -(CH2).-NH-CO-(CH2)~-N~-CO-
( CH2 ) ~
The novel compound~ can be prepared by conventional
methods of peptide chemi~try.
~hu~, the pept$des can be con~tructed sequentially from
Z005056
_ 4 _ o z. 0050/40390
amino acids or by linking together suitable smaller
peptide fragments. In the sequential construction, the
peptide chain is extended stepwi~e, by one amino acid
each time, starting at the C terminus. In the case of
coupling of fragments it is possible to link together
fragments of different length~, these in turn being
obtainable by sequential construction from amino acids or
coupling of other fragments. The cyclic peptides are
obtained, after ~ynthesi~ of the open-chain peptide~, by
a cycliza~ion reaction carried out in high dilution.
In the ca~e both of sequential construction and of
fragment coupling it i~ nece~sary for the building block~
to be linked by formation of an amide linkage.
Enzymatic and chemical method3 are suitable for thi~.
Chemical method~ for forming amide linkages are dealt
with in detail by M~ller, Methoden der Organi~chen Chemie
(Method~ of Organic Chemistry) Vol. XV/2, pp 1-364,
Thieme Verlag, Stuttgart, 1974; Stewart, Young, Solid
Phase Peptide Synthe~i~, pp 31-34, 71-82, Pierce Chemical
Company, ~ockford, 1984; Bodan~zky, ~lausner, Ondetti,
Peptide Synthesis, pp 85-128, John Wiley & Sons, New
York, 1976 and other ~tandard works of peptide chemistry.
Particularly preferred are the azide method, the ~ymmetr-
ical and mixed anhydride method, active e8t8r8 generated
in situ or preformed and the formation of amide linkageA
u8ing coupling reagent~ (activator~), in particular
dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide
(DIC), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
(EEDQ), l-~thyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDCI), n-propanephosphonic anhydride
(PPA), N,N-bi~(2-oxo-3-oxazolidinyl)amidopho~phoryl
chloride (BOP-Cl), diphenylpho~phosyl azide (DPPA),
Castro's reagent (BOP), O-benzotriazolyl-~,N,N~,N~-tetra-
methyluron~um 8~1t8 (HBTU), 2,5-diphenyl-2,3-dihydro-3-
3~ oxo-4-hydroxythiophone dioxide (Steglich'3 reagent;
5 0 5 6
_ 5 _ o.z. 0050/40390
HOTDO) and l,l~-carbonyldiLmidazole (CDI). The coupling
reagents can be employed alone or in combination with
additives such as N,N'-dLmethyl-4-aminopyridine (DMAP),
N-hydroxybenzotriazole (HOBt), N-hydroxybenzotriazine
(HOOBt), N-hydroxysuccinLmide (HOSu) or 2-hydroxy-
pyridine.
Where~s it i~ normally po~sible to di~pense with protec-
tive group~ in enzymatic peptide ~ynthesis, for chemical
synthe~i~ it is neceqsary for there to be reversible
protection of the reactive functional groups which are
not involvèd in the formation of the amide linkage on the
two reactant~. Three conventional protective group
technique~ are preferred for chemical peptide synthe~es:
the benzyloxycarbonyl (Z), the t-butyloxycarbonyl (Boc)
and the 9-fluorenylmethyloxycarbonyl (~moc~ techniques.
In each ca~e the protective group on the -amino group of
the chain-extending building block is identified. The
~ide-chain protective groups on the trifunctional amino
acids are cho~en 80 that they are not necessarily elimin-
ated together with the ~-amino protective group. A
detailed review of amino acid protective group~ i~ gi~en
by ~ller, Nethoden der Organischen Che~ie Vol XV/l, pp
20-906, Thiem~ Verlag, Stuttgart, 1974.
The building blocks used to construct the peptide chain
can be reacted in solution, in suspension or by a method
simllar to that de~cribed by Merrifield in J.Amer. Chem.
Soc. 8~ (1963) 2149. Particularly preferred methods are
those in which peptide~ are constructed ~equentially or
by fragment coupling by use of the Z, Boc or Fmoc protec-
tive group technique, in which case the reaction takesplace in ~olution, a~ well as tho~e in which, similar to
the Nerrifield technique, one reactant i9 bound to an
in~oluble polymeric Rupport ~al~o called re~in herein-
after). ~hi~ typically entails thQ peptide being con-
~tructed ~equentially on the polymeric ~upport, by use of
0~5 ~
- 6 - O.Z. 0050/40390
the Boc or Fmoc protective group technique, with the
growing peptide chain being covalently bonded at the
C terminus to the insoluble resin particle~ (cf. Figures
1 and 2). This procedure allow~ reagent~ and byproducts
to be removed by filtration, and thus recrystallizatio~
of intermediates is superfluou~.
The protected amino acids can bs bonded to ~ny ~uitable
polymers which merely need to be in~oluble in the ol-
vent~ used and to have a table physical form which
allows easy filtration. The polymer mu~t contain a
functional group to which the fir~t protected amino acid
can be firmly linked by a covalent bond. A wide variety
of polymers i8 suitable for thi~ purpose, for example
cellulose, polyvinyl alcohol, polymethacrylate, sulfon-
ated polystyrene, chloromethylated copolymer of ~tyreneand divinylbenzen~ lMerrifield resin), 4-methylbenz-
hydrylamine-resin (MBHA-resin), phenylac~tamidomathyl-
re~in (Pam-resin), p-benzyloxybenzyl alcohol-resin,
benzhydrylamine-re~in (BHA-resin), 4-hydroxymethyl-
benzoyloxymethyl-re~in, the resin u3ed by Breipohl et al.
(Te~rah~dron Lett. 28 (1987) 565; from ~ACHEM), HYCR~M
re~in (from ORPEG~N) or SASRIN resin (from BACHEM~.
Solv6nts suitable for peptide synth2~is in solution are
all those which are inert under the resction condition~,
in particular water, N,N-di~ethylformamide (DMF),
dimethyl sulfoxida (DMS0), acetonitrile, dichlorome~hane
(DCM), 1,4-dioxane, tetrahydrofuran (THF), N-methyl-2-
pyrrolidone (N~P) and mixturea of the said solvents.
Peptide synthesis on polymeric suppor~ can be carried
out in all inert organic solvents which dis~olve ~he
amino acid derivatives u~ed; however, solvents which have
re3in-~welling propsrtie~ are preferred, ~uch a~ DMF,
DCM, NMP, acetonitrLlo and DMS0, as well a~ mixtures of
the~o solvent~.
;~005056
- 7 - O.Z. 00~0/40390
After the peptide has been synthesized it is cleaved off
the polymeric support. The cleavage condition~ for the
various type~ of resins are di~closed in the literature.
The cleavage reactions most commonly use acid and
pa71adium catalysis, in particular cleavage in anhydrous
liquid hydrogen fluoride, in anhydrous trifluoromethane-
sulfonic acid, in dilute or concentrated trifluoroacetic
acid or palladium-catalyzed cleavage in THF or T~F-DCM
mixtures in the presence of a weak ba~e such as morpho-
line. The protective groups may, depending on the choicethereof, be retained or likewi~e cleaved off under the
cleavage conditions. Partial deprotection of the peptide
may also be worthwhile if the intention i8 to carry out
certain derivatization reaction~ or a cyclization.
Some of the novel peptide~ have good cytotoxic proper-
tie~. Some others of the peptide~ ha~e high affinity for
the cellular TNF receptor without, however, having
cytotoxic activity. ~hey are therefore TNF antagonist~.
They compete with natural TNF for binding to the cellular
TNF rereptor and thus ~uppres~ the TNF effect. The novel
peptide~ are valuable drugs which can be employed for
treating neopla tic di~ease~ and autoi~mune diseases a~
well a~ for controlling and preventing infection~
inflammations and transplant re~ection reactions. Simple
experiment~ can be used to elucidate the mode of action
of ths indi~idual peptide~. Th~ cytotoxicity of the
peptid~ is determined by incubating a TNF-~ensitive cell
line in the presence of the peptide. In a ~econd experi-
mental approach, the cell line i8 incubated with the
relevant peptid~ in the presence of a lethal amount of
~NF. It is possible in this way to detect the
$NF-antagon~tic effect. In addition, the affinity of the
peptid~ for the cellular TNF receptor i~ deter~ined in an
in vitro binding experiment.
The follo~ing te~t ~y~tems ware used to ch~r~cterize the
200505~
- 8 - O.Z. 0050/40390
agonistic and antagonistic effects of the novel peptide~:
I. Cytotoxicity test on TNF-sensitive indicator cells,
II. Cytotoxicity antagonism te~t on TNF-~ensitive
indicator cell~,
III. Competitive receptor-binding te~t on indicator cell~
expressing TNF receptor.
I. Cytotoxicity te~t
The agoni~tic effect~ of the novel peptides are
a~sessed on the ba3i~ of their cytotoxic effect on
TNF-~ensitive cells (e.g. L929, MCF-7, A204, U937).
The te3t with L929 and NCF-7 wa~ carried out a~
followss
1. 100 ~1 of culture medium containing 3 to 5 x 103
fra~hly trypsiniz~d, exponentially growing, L929
cell~ (mou~e) or MCF-7 cells (human) were
pipetted into the wells of a 96-well flat-bottom
culture plate. The plate was incubated at 37C
overnight. ThQ air in the incubator was saturated
with water vapor and contained 5% CO2 by volume.
The L929 culture medium contained 500 ml of lx
~arle's MEM (Boehringer ~annheim), 50 ml of heat-
nactivated (5SC, 30 min) fetal calf 8erUm
(FCS), 50 ml of L-gluta~ins (200 mM), 5 ml of
lOOx non-e~sential amino acid~, 3 ml of LN HEPES
buffer pH 7.2,and 50 ml of gentamicin (50 mg/ml).
The MCF-7 culture medium contained 500 ml of lx
Dulbecco's MBM (~oehringer Mannheim), 100 ml of
heat-inactivated t56-C, 30 min) FCS, 5 ~1 of
~-glutamine and 5 ml of lOOx non-e3sential amino
acids.
2. Th~ next day 100 ~1 of the peptide ~olution to be
;~05056
_ g _ o.z. 0050/40390
tested were added to the cell cultures and
sub~ected to serial 2-fold dilution. In addition,
some cell control~ ti.e. cell culture3 not
treated with peptide dilution) and some rhu-~NF
controls (i.e. cell cultures treated with recom-
binant human ~NF~ were also made up. The culture
plate wa~ incubated at 37~C in an atmosphere of
air saturated with water vapor and containing 5%
C02 by volume for 48 h.
3. The percentage of ~urviving cell~ in the cultures
treated with peptLde dilution was determined by
staining with cry~tal viole~. For this purpose,
the liquid wa~ removed from the well~ of the test
plate by tapping it. 50 ~1 of crystal violet
solution were pipetted into each well.
The composition of the crystal violet ~olution
wa~ as follows:
3.75 g of cry~tal violet
1.75 g of NaCl
161.5 ml of ethanol
43.2 ml of 37% formaldehyde
water ad 500 ml
The crystal violet solution wa~ left in the wells
for 20 min and then likewi~e removed by tapping.
The plates were then washed 5 times by immersion
in wat~r in order to remove dy~ not bound to the
cells. Tho dye bound to tha cells was extracted
by adding 100 ~1 of reagent solution (SOS etha-
nol, 0.1~ glacial acetic acid, 49.9~ water) to
each well.
4. The plata~ were ~haken for 5 min to obtain a
~olution of uniform color in each well. The
5 ~ S 6
- 10 - O.Z. 0050/40390
~urviving cells ware determined by mea~uring the
extinction at 540 nm of the colored solution in
the individual wells.
5. 5ubsequently, by relating to the cell control,
the 50% cytotoxicity value was definet, and the
reciprocal of the sample dilution which resulted
in 50~ cytotoxicity was calculated a~ the cyto-
toxic activity of the te~t sa~ple.
II. Cytotoxicity antagonism test
The antagonistic effect of the peptide~ wa3 a~ses~ed
on the basis of their property of antagonizing the
cytotoxic effect of rhu-TNF on TNF ~en~itive cell~
(e.g. L929, ~C~-7, A204, U937). The cytotoxicity
antagonism te~t with L9~9 and MCF-7 cells wa3
carried out as follow~s
1. 100 ~1 of culture medium containing 3 to 5 x 103
fre~hly tryp~inized, exponsn~ially growing, Lg29
cells (mouse! or MCF-7 cell~ (human) were
pipetted into the wells of a 96-well flat-bottom
culture plate. The plate wa~ incubated at 37C
overnight. The air in the incubator wa~ ~aturated
with water vapor and contained 5% CO2 by volume.
The L929 culture medium contained 500 ml of lx
Earle's MEM (Boehringer Ma~nheim), 50 ml of heat-
inactivated (56-C, 30 min) FCS, 5 ml of ~-gluta-
min~ (200 mM), S ml of lOOx non-essential amino
acid~, 3 ml of lM ~EP8S ~uffer pN 7.2, and 500 ~1
of ~entami~in (50 m~/ml).
ThQ N~F-7 culture mediu~ contained 500 ml of lx
DulbQcco's MEM (Boehringer Nannheim), 100 ml of
heat-i~activated (56-C, 30 min) FCS, 5 ml of
L-glutamine (200 mM) and 5 ml of lOOx non-~sen-
tial amino acid~.
~305056
~ O.Z. 0050/40390
2. The next day 10~ ~1 of the peptide ~olution to be
te~ted were added to the cell culture~ and
sub~ected to ~erial 2-fold dilution. Then, 100 ~1
of a rhu-TNF dilution in culture medium, which
dilution had an 80-100% cytotoxic effect in the
. final concentration in the cell culture, were
added to these cell cultures. In additlon, ~ome
cell controls (i.e. cell cultures not treated
with peptide solution or with rhu-TNF solution)
and ~ome rhu-TNF controls (= cell cultures
treated only with rhu-TNF solution) were also
made up. The culture plate wa3 then incubated at
37~C in an atmosphere of air ~aturated with water
vapor and containing 5% CO2 by volume for 48 h.
3. ~he percenta~e of surviving cell3 in the cultures
treated with substance solution wa~ determined by
~taining with cry~tal violet. For this purpose,
the liquid wa~ removed from the wells of the test
plate by tapping Lt. 50 ~1 of cry~tal violet
~olution were pipetted into each well.
The crystal violet ~olution had the composition
specif~ed in I.3
The crystal violet solution was left in the wells
for 20 min and then likewisQ removed by tapping.
The plates were then washed 5 time~ by immer~ion
in water in order to remove dye not bound to the
cell~. ~he dye bound to the cells wa~ extracted
by adding 100 ~1 of reagent ~olution (S0~ etha-
nol, 0.1~ glaciAl acetic acid, 49.9% water) to
each well.
4. The plate~ were shaken for 5 min to obtain a
solution of uniform color in each well. The
ZIQ0~0~6
- 12 - O.Z. 0050/40390
surviving cells were determined by mea~uring the
extinction at 540 nm of the colored solution in
the individual well~.
5. Subsequently, by relating to the cell control and
the rhu-TNF control, the 50% antagoni~m value wa~
defined, and the ~ample concentration which
resulted in 50% antagonism of rhu-TNF cytotox-
icity at the rhu-TWF concentration u~ed was
calculated a~ antagonistic activity of the ~ample
te~ted.
III. Competitive receptor-binding te~t
Both the agoni~tic and antagoni~tic effect~ of
peptide~ are conditional on th~ latter binding to
the TMF receptor. Thi~ mean~ that peptides with an
agonistic or antagoni~tic effect compete with
rhu-TNF for binding to the TNF receptor on TNF-
sensitive indicator cells (e.g. U937). ~he competi-
tive receptor-binding te~t wa~ carriad out a~
follow~ 5
1. 100 ~l of medium containing various concentra-
tions of the peptide to b~ te~ted and of rhu-T~F
(= control) were pipetted into the reaction
vessel~. The medium comprised 500 ~1 of PBS
(Boehringer Mannheim), lO ml of heat-inactivatsd
(56C, 30 min) FCS and 100 mg of sodium azide.
2. Sub~equently, lO0 ~l of medium containing 1 ng of
~Z~I-labeled rhu-~NF (Bol~on lactoperoxida~e
method) were placed in the reaction ve~8els and
mixed. The non-specific bindinq (N5B) was deter-
~ined by ~ixing in the reaction ve~8el~ the
l2~I-labeled rhu-TNF (1 ng of l2~I-rhu-TNF in lO0 ~l
of medium) with a 200-fold exce~8 of unl~beled
rh~-TNP (200 ng of rhu-TWF in 100 ~l of medium).
21~(~50S~
- 13 - O.Z. 0050/40390
3. Then 100 ~1 of medium containing 2 x 10~ U937
cell~ (human) were pipetted into the reaction
ve~sel~ and mixed. The reaction vessel3 (test
volume 300 ~1) w~re incubated at 0C for 90 min.
The reaction mixtures were remixed after 45 min.
4. After the incubation the cells were centrifuged
at 1800 rp~ and 4C for 5 min, washed 3 times
with medium and transferred quantitatively into
counting vial~, and the cell-bound radioactivity
wa~ determined in a Clini gamma counter 1272
(LXB Wallac).
5. After the mea~uremant~ had ~ee~ corrected for the
non-specific binding, thè 50% competition value
w~ defined by relat~on to th~ overall binding,
and tha ~ample concentration which led to 50%
competition of l25I-rhu-TNF binding at the125I-rhu-
TNF concentratio~ used wa~ calculated as the
competitive activity of the ~ample tested.
The Example~ which follow are intended to ~xplain the
invention in more detail. The proteinogenous a~ino acids
are abbreviated in th~ ~xample8 using the con~entional
three-l~tter code. Other meaning~ ares
Ab~ = 4-aminobutyric acid, ~c - acetic acid,
Ade - 10-amlnodecanoic acid, Ahx = 6-aminohexanoic acid,
A~o ~ g_~minononanoic acid, Aoc = 8-aminooctanoic acid,
Ape - 5-aminopentanoic acid, ~cy - homocyYteine,
Hly ~ homolysine, Orn - ornith~ne,
Dap - 2,3-diaminopropionic acid.
A. General procedure
I. The peptides claim2d in clai~ 1 were ~ynthesized
using ~tandard mothod~- of solid-pha~e p~ptide
ynthe~is in a compl~tely au~o~tic mod~l 430A
Z~3~505~
- 14 - O.Z. 0050/4039~
synthe~i~ in a completely automatic model 430A
peptide synthesizer from APPLIED BIOSYSTEMS. The
apparatus uses different synthesis cycle~ for the
Boc and Fmoc protective group techniques.
a) Synthesis cycle for the Boc protective group
technique
1. 30% trifluonwLetic acid in DCM 1 x 3 mun
2. 50% triflu=nYY~oic acid in ~CM 1 x 17 mLn
3. DCMwashing 5 x 1 min
4. 5% diL~QylethyL~ne in DCM 1 x 1 min
5. 5% dii~rpylethyla~Ie in NMP 1 x 1 min
6. NMP was~ing 5 x 1 min
7. Addition of pn~tivated prJls~Id a~
acid (activation ky 1 eqlivalent of DOC
and 1 equivalent of HOBt in NMP/DCM);
pepkide coupling (lst part) 1 x 30 min
8. A~ition of nMSO to the n~ n mi~h~e
u~l it c ~ 20% nMSO by volume
9. Pq~i~b ooup~ng ~2n~ ~ t) 1 x 16 min
l0.Ad~iticn of 3.8 ~YZ~ of d~
11. p~ e coupling (3rd Eart) 1 x 7 min
l2.DCMwad~ng 3 x ln~n
13.1f r3x~cn i~ ~xxoplete, rq~ition
of coup~ (neturn bo 5.)
14. l0~ ~o~c a~ride, 5% dilx}D~Qyl-
ethyL~ne in DCM 1 x 2 min
15. 10% ao~i~ a~ride Ln DC~ 1 x 4 min
16.DoMwa~n~ 4 x l min
17 R~lIn to l.
b) Sy~is cy~le for ths Fmoc p~æ~ive ~x~ toc~l4Fe
1. N~P w~*~ng 1 x l min
2. 20% p~ ne in NMP 1 x 4 min
3. 20% plp3i~lne in NMP 1 x 16 m~n
4. NMP wa~hing 5 x 1 min
~;~G0505;~
- 15 - O.Z. 0050/40390
5. A~dition of pn~tivabed pr~lr~ed
acid (a~tivation by 1 eqyivalent of DOC
and 1 ~ alent of HOBt in NMP/DCM);
p~lde coupling 1 x 61 min
6. NMP wzshin~ 3 x 1 min
7. ~f r3~0n i ~Yx~plete, ~ition of
coupling (~ n to 5.)
8. 10% ~ a~ride in NMP 1 ~ 8 min
9. NMP w2shing 3 x 1 min
10. R~n bo 2.
II. Working up of peptide-re~ins obtained a~ in Ia
The peptide-resin obtained a~ in Ia was drisd under
reduced pressure and tran~ferred into a reaction
ves~el of a Teflon HF apparatus (from P~NINSULA).
Addition of a scavenger, preferably ani~ole (1 ml/g
of re~in), and of a thiol, in the ca~e of tryptophan-
containing peptide~, to remove the indole formyl
group, preferably ethanedithiol (0.5 ml/g of re~in)~
was followed by condensation i~ of hydrogen fluoride
(10 ml/~ o resin) while cooling with liquid N2. The
mixture waa allowed to wPrm to O-C, and waQ ~tirred
at thi~ temperature for 45 min. The hydrogen fluor-
id~ wa~ then stripped off under reduced pre~aure a~d
the residue wa~ wa~hed with e~hyl acetate in order
to rem~ve remaining 3cavenger. The peptid~ w~
extracted with 30% stren~th acetic acid and
filtered, and the filtrate was freezQ-dried.
To prepare peptide hydr~zides, the peptlde-resin
(Pam- or Merrifield resin) was ~u~pended in DMF
(15 ml/g of re~in), hydrazine hydrate (20 equiva-
lenta) was added, and the mixtur~ w~s stirred at
room t~mpsra~ure for 2 day~. To work up, the reain
was filtered off and the filtrate wa~ evaporated to
dry~ess. The reaidue was cry~tallized from DM~/~t2O
or ~eOH/Et2O.
:,
.
;?dO05056
- 16 - O.Z. 0050/40390
III. Working up of the peptide-resin~ obtained as in Ib
~he peptide-resin obtained as in Ib was dried under
reduced pressure and subYequently sub~ected to one
of the following cleavage procedures, depending on
S the amino acid compo~ition (Wade, Tregear, ~oward
Florey Fmoc-Workshop Manua1, ~el~ourne 1985).
2~3~SOS~j
- 17 - O.Z- 0050/4039
Peptide containing Clea~age conditions
Arg(Mtr) Met Trp TFA Scavenger Reaction
Time
S _
no no no 95% 5% H20 1.5 h
yes no no 95% 5% thioanicole > 3 h
no yes no 95~ 5~ ethyl methyl 1.5 h
sulfide
no no yes 95% 5% ethanedithiol~ 1.5 h
anisole (1:3)
no yes yes 95% 5% ethanedithiol/ 1.5 h
ani~ole/ethyl methyl
sulfide (1:3:11
ye~ yes ye~ 93% 7% ethanedithiol/2 3 h
anisole/ethyl methyl
~ulfids (1:3:3)
The ~uspension of the peptide-resin in the suitable
TFA mixture was stirred at room temperature for the
stated time and then the resin was filtered off and
wa~h~d with TFA and with DC~. The filtrate and the
wa~hings were exten~ively conce~trated, and the
peptide was precipitated by addition of diethyl
ether. The mixture wa~ cooled in an ice bath, and
the precipitat~ was filtered off, taken up in 30%
acetic acid and freeze-dried.
IV. Purification and characterization of the peptides
Purification wa~ by gel chromatography (SEPHAD~X~
G-10, G-15/10% HOAc; SEPHAD~X~ LH20/MeOH) and 8ub-
sequent medium prs~sure chromato~raphy (~tationary
pha~e: HD-SIL C-18, 20-45 ~, 100 ~; mobile phase:
gradient with A = 0.1% TFA/MeOH, B = 0.1% TFA/H20).
056
- 18 - O.Z. 0050/40390
The purity of the final products wa3 detelmined by
analytical HPLC (~tationary pha~e: 100 x 2.1 mm
VYDAC C-18, 5 ~, 300 ~; mobile phase = CH3CN/H20
gradient buffered with 0.1% TFA, 40C). Charac-
S terization was by means of amino acid analy~i~ and
fast atom bombardment ma3s spec~rometry.
B. Specific procedures
EXAMPLE 1
Ac-Pn~br-Asp-~ys-kx~Val-Ala-Hi~-Ap~y-Ile-Ile-Ala-Leu-CH
1.11 g o~ Boc-~eu-Pam-resin ~ub~titution 0.45 mmol/g),
corresponding to a batch size of 0.5 mmol, were reacted
in as in AIa with 2 mmol each of
Boc-Ala-OH Boc-Hi8(Z)-OH ~oc-Lys(Cl-Z)-OH
Boc-Ile-OH Boc-Ala-OH Boc-Asp(OChx)-OH
Boc-Ile-OH Boc-Val-OH Boc-Ser(Bzl)-OH
Boc-Gly-OH Boc-Pro-OH Boc-Pro-OH
Boc-Ape-OH
(~tep~ 14-16 were di~pensed with in all the couplings
follo~ing His).
After th~ synthesi~ was co~pletec the N terminus was
acetylated (8tep~ 1-6 ~nd 14-16 a~ in AIa) and the
peptide-resin w~ dried under reduced pressure; the yield
was 1.91 g.
0.95 g of the re~in obtained in this way was ~ub~ected to
HF cleavage as in AII. The crude product (245 mg) was
purified ~y gel filtration (SEPHAD~ G-10) and medium
presQure chro~atography (cf. AIV; 60-80% A; 0.25~ min~l).
127 mg of final prod~ct were obtained.
'~C~ 5 U ~i
- 19 - O.Z. 0050/40390
EXANPLE 2
~-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-LYS-PrO-Val-
Ala-Hi~-Aoc-Gly-Ile-Ile-Ala-Leu-OH
0.48 g of Fmoc-~eu-p-alkoxybenzyl alcohol-resin (sub~tit-
S ution 0.52 mmol/g~, corresponding to a batch size of
0.2S mmol, was reacted a~ in AIb with 1 mmol each of
Fmoc-Ala-OH Fmoc-Val-OH Fmoc-Arg(Mtr)-OH
Fmoc-Ile-OH Fmoc-Pro-OH Fmoc-Ser(tBu)-OH
Fmoc-Ile-OH Fmoc-Ly~(Boc)-OH Fmoc-Ser(tBu)-OH
Fmoc-Gly-OH Fmoc-A~p(OtBu)-OH Fmoc-Ser(t~u)-OH
Fmoc-Aoc-OH Fmoc-Ser(tBU)-OH Fmoc-Arg~Mtr)-OH
Fmoc-His~Trt)-OH Fmoc-Pro-OH Fmoc-~al-ON
Fmoc-Ala-OH Fmoc-Thr(tBU3-OH
After the synthesis wa~ complete, th0 N terminu~ wa~
deprotected (step~ 2-4 a~ in AI~). The resulting
peptide-resin was dried under reduced pressure; thQ yield
was 1.24 g.
The crude peptide (475 mg) obtain~d after TFA cleavage as
in AIII wa~ purified by gel filtr~tion (SFPHADEX~ G-10)
and medium pressur~ chromatography (cf. AIV; 50-70~ A;
0.25% min~';). 197 mg of pure product were obtained.
The following can be prepared in a 8~m~ lar manner to
Examples 1 and 2:
3. H-Vla-A~br~r~r-Arg-lhr-P~br-AsF-Ly~-Pro-Val-Ala-Hi~-
~p~y-Ile-Ile-Ala-Ieu-oH
4. Ac-Val-A ~ -Ser-S3r-An~hr-Pn~br-A~p-Ly~-Pro-Val-Ala-His-
Ap~y-Ile-Ile-Ala-Lsu-N~
5. Ac-Pn~bo~Aop-Ly~-P~o-Val-Ala~ p~y-~Ile,Ala-leu-N~
6. Ac-Pn~br-A~p-Ly~-Pro-V~l-Ala-~R-Aoc~Gly-Ile-Ile-AL~-T~-oH
7. H~Ser-A~2-Ly~-Prc-V~l-Ala~ p~y-Ile_~Ala-Ieu-Cff
S0S~;
- 20 - O.Z. 00~0/40390
8. Ac-SQr-A~p-Lys-~Val-Ala-Hi~-Ap~y-Ile-I~e-Ala-Leu-oH
9. Ac-Ser-Asp-Lys~x~Val-Ala-His-Ap~ly-Ile-Ile-Ala-Leu-
~
EXA~LE 1 0
H-Asp-Lys-Pro-Cys-Ala-His-Ape-Gly-Cys Ile Ala-Leu-OH
1.11 g of Boc-Leu-Pam-resin (substitution 0.45 mmol/g),
corresponding to a batch size of 0.5 mmol were reacted a~
in AIa with 2 mmol each of
Boc-Ala-OH Boc-Ape-O~ Boc-Pro-O~
Boc-Ile-OH Boc-Hi8(Z)-ON Boc-Ly~(Cl-Z)-OH
Boc-Cy~(pMB)-OH Boc-Ala-OH Boc-A~p(OChx)-OH
Boc-Gly-OH Boc-Cys(p~B)-OH
(steps 14-16 were dispensed with in all the couplings
following Hi3).
The re~ulting peptide-resin was dried under reduced
pres~urQ; the yield wa~ 1.76 g.
0.88 g o the re~in obtained in this way wa~ sub~ected to
HF cleavage a3 in AII. The ~reeze-dried crude product was
tsken up in 2 1 of 0.1% strength ace~ic acid, and the pH
wa~ then ad~u~ted to 8.4 with aqueous ammonia. Under an
argon atmo~phere, 0.01 N g3[Fe(CN)a~ ~olut~on wa~ ~lowly
added dropwise until the yellowiah-green color parsi~ted
for at l~a~t 15 min. Tha mix~ure was then stirred for ~ h
~nd then acidified to pK 4.5 with glacial ace~ic acid,
and 15 ml of ~n aqueous suspension of ~n an$on exchanger
(BIORAD- 3 x 4A, chloride form) were added. After 30 min,
the ion exchAnger re~in wa~ filtered off, and the filt-
rate wa~ concentrated to 100 ml in ~ rot~ry evaporator
and ~ub~equently freeze-dried.
;~G~35l)5t;
- 21 - O.Z. 0050/40390
All the sol~ent~ u~ed had previously been ~aturated with
nitrogen in order to prevent any oxidation of the free
cyqteine residues.
The crude product wa~ purified by gel chromatography
S (SEPHADEX~ G-15) and medium pressure chromatography (cf.
AIV; 60-80% A; 0.25~ minl). 72 mg of pure product were
obtained.
The following can be prepared in a ~imilar manner to
Example lO (p-~BHA-re~in wa~ used for the prepara~ion of
the peptide amides):
Il. Ac-C~ls-Ala-HiS-Val-C~5-NH2
12. Ac-Cys^Ala-H1s-Leu-C~s-NHf
13. Ae-Cys-Ala-H1S-~t--CYs~NH2
14. Ac-C~s-Al~-H1s-Aoc-Cys-NH2
15. Ac-C~s-Al~-H1s-Ano-Cys-NH2
16. ~c-Cys-Al~-H1s-Ade-Cys-Nh2
17. H-C~S-AI~-H1S-A~S-Gly-Cys-OH
18. Ae-Cys-~ H1s-Abs-Gty-Cys-~H2
I9. ~-CyS-~la-~ls-~po-61y-CyS-OH
20. Ac-Cys-At~-H1s-~p--6ty-Cys-NH2
21. H-C~s-Al~-H1s-~hx-GI~-C~s-OH
22. Ac-Pro-C~s-Ala-Hls-Ap~-6l~-Cys-ll--N~2
" 2~05056
- 22 - 0~ Z . 0050/40390
23. Ac-Lys-Pro-Cys-Ala-His-Ape-Gly-Cys-lle-AI~-NH2
24. H-Asp-Lys-Pro-Cys-Ala-His-Abs-Gly-Cys-lle-Ala-Leu-OH
25. Ac-Asp-Lys-Pro-Cys-Ala-His-Abs-Gly-Cys-lle-Ala-Leu-O~
26. H-Asp-Lys-Pro-Cys-Ala-His-Abs-Gly-Cys-lle-Ala-Leu-NH2
27. Ac-Asp-Lys-Pro-Cys-Ala-His-Abs-Gly-Cys-Ile-~la-Leu-NH2
28. H-Asp-Lys-Pro-Hcy-Ala-His-Abs-Gly-Cys-lle-Ala-Leu-OH
29. Ac-Asp-Lys-Pro-Hcy-Ala-His-Abs-Gly-Cys-lle-Ala-Leu-NH2
30. H-Asp-Lys-Pro-Cys-Ala-His-Abs-Gly-Hcy-lle-Ala-Leu-OH
31. Ac-Asp-Lys-Pro-Cys-Ala-His-Abs-Gly-Hcy-lle-Ala-Leu-NH2
32. H-Asp-Lys-Pro-Hcy-Ala-His-Abs-Gly-Hcy-lle-Ala-~eu-OH
33. Ac-Asp-Lys-Pro-Hcy-Ala-H3s-Abs-Gly-Hcy-lle-Ala-Leu-NH2
34. Ac-Asp-Lys-Pro-Cys-Ala-His-Ape-Gly-Cys-~le-Ala-Leu-OH
, _ .
35. H-Asp-Lys-Pro-Cys-Ala-H3S-Ape-Gly-Cys-lle-Ala-Leu-NH2
36. Ac-Asp-Lys-Pro-Cys-Ala-tl3s-Apo-Gly-Cys-lle-Ala-Leu-NH2
37. H-Asp-Lys-Pro-Hcy-Ala-H3s-Ap~-Gly-CyS-lle-Ala-Leu-OH
i
38. Ac-Asp-Ly5-Pro-Hcy-Ala-His-Ape-Gly-Cys-llc-Ala-Leu-NH2
39. H-Asp-Lys-Pro-Cys-~la-His-Apa-Gly-Hcy-Ilo-Ala-Leu-OH
40. Ac-Asp-Lys-Pro-Cys-Ala-H~s-Ape-Gly-Hcy~ -Ala-Leu-NH2
r
41. H-Asj-Lys-Pro-Hcy-~la-H~s-Apo-Gly-Hcy-lle-Ala-Leu-OH
I_ ,
42. Ac-Asp-Lys-Pro-Hcy-Ala-His-Ape-Gly-Hcy-lle-Ala-Leu-NH2
43. H-Asp-Lys-Pro-Cys-~la-H3s-Ahx-GI~-Cys-Ito-~la-Leu-OH
44. Ac-Asp-Lys-Pro-Cys-Ala-H3s-Ahx-Gly-Cys-llo-~la-Lou-OH
45. H-Asp-~ys-Pro-Cys-Ala-H1s-Ahx-Gly-Cys-llo-~la-Leu-NH2
46. Ac-Asp-Lys-Pro-Cy~-~la-His-~hx-Gly-Cys-~ls-Ala-Lau-NH2
~ ;)5056
- 23 - O Z 0050/40390
47 H-Asp-Lys-~ro-Hcy-Ala-His-Ahx-Gly-Cys-lle-Ala-Leu-OH
4~ Ac-Asp-Lys-Pro-Hcy-Ala-His-Ahx-Gly-Hcy-Ile-Ala-Leu-NH2
49 H-Asp-Lys-Pro-Cys-Ala-His-Ahx-Gly-Hcy-~le-Ata-Leu-OH
Ac-Asp-Lys-Pro-Cys-Ala-His-Ahx-Gly-Hcy-Ile-Ala-Leu-NH2
Sl H-Asp-Lys-Pro-Hcy-Ata-~s-Ahx-Gly-Hcy-Ile-Ala-Leu-OH
S2 Ac-Asp-Lys-Pro-Hcy-Ala-His-Ahx-Gly-Hcy-Ile-Ala-Leu-NH2
53 ~-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys-
Pro-Cys-Ala-H1s-Ape-Gly-Cys-Ile-Ala-Leu-OH
54 H-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-LyS-
Pro-Cys-~la-His-Abs-Gly-Cys-Ile-Ala-~eu-OH
~-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys-
Pro-Cys-~la-Hts-Ahx-Gty-CyS-Ite-Ata-Leu-OH
56 H-Asp-~ys-Cys-Val-Ala-H~s-Ape-Gly-Cjs-lle-~la-~eu-OH
I
57 Ac-Asp-Lys-Cys-Val-Ala-Hls-Apo-Gly-Cys-Ile-Ala-Leu-NH2
58 H-Asp-~ys-Pro-Cys-Ala-His-Ape-Gly-II~-Cys-Ata-Leu-OH
59 Ac-~sp-Lys-Pro-Cys-Ala-His-Ap--61y-Ile-Cys-~la-Leu-NH2
~-~sp-Ly5-Cys-Val-~l--Ht5-~p~-Gly-Ilo-Cys-Ala-Lau-O~
61 Ac-Asp-~ys-Cys-~al-~la-His-~p~-Gly-lle-Cjs-~la-~eu-NH2
I
62 H-~sp-Ly~-pro-c~s-~la-Hts-ApJ-~la-c~s-lls-~la-L~u-oH
63 Ac-A~p-L~s-Pro-Cys-~ta-His-~p--~la-Cys-Ila-~la-~eu-HH2
64 H-Lou-~rg-Sor-Ser-S~r-Gln-Asn-S~r-Ser-~sp-Lys-Pro-
CyS-~ta-Hts-Ap~-Gly-Cys-Ilo-~la-Leu-O*
H-Leu-Arg-S~r-S-r-S~r-Gln-Asn-Sor-Ser-ASp-LyS-Pro-
Cys-Ala-His-~bs-Gly-Cys-lle-~la-Leu-OH
66 H-Leu-~rg-S-r-Ser-S~r-Gln-Asn-5er-5er-Asp-Lys-Pro-
Cys-Ala-H1s-~hx 61y-Cys-lt~-Ala-Lsu-OH
67 H-Lys-Pro-Cys-~la-Hts-~hx-Gly-Cys-Ilo-~la-Leu-OH
68 H-Lys-Pro-Cys-Ata-~ts-~oc-Gly-C~s~ Al~-L~u-OH
05056
- 24 - O.Z. 0050/40390
ExAMæLE 69
Ac-Pro-Oap-Ata-~is-Aoc-Gly-ASp-lle-Ala-Leu-~H2
1.02 g of Boc-Leu-MBHA-resin (~ubstitution 0.49 mmol/g),
corresponding to a batch size of 0.5 mmol, were reacted
a~ in AIa with 2 mmol each of
Boc-Ala-OH Boc-Gly-OH Boc-Ala-OH
~oc-Ile-OH Boc-Aoc-OH Boc-Dap(Z)-OH
Boc-Asp-(OChx)-OH Boc-Hi8(Z)-OH ~oc-Pro-OH
(step~ 14-16 were dispen~ed with in ~11 the couplings
following Hi~. After the ~ynthesi~ was complete, the N
terminu~ was acetylated (~tep~ 1-6 and 14-16 a~ in AI~.
The resulting peptide-re~in was dried under reduced
pres~ure; tha yield was l.S8 g.
The crude product (385 mg) obtained after HF cleavage a~
in AII wa~ di~olved in 500 ml of degas~ed DMP, and
0.43 ml of triethylamine and, at -25-C, 0.43 ml of
diphenylphosphoryl azide were added. The mixture was
stirred at -25-C for 2 h, stored at -20-C for 2 day~, at
4C for 2 day~ and at roo~ temperature for 2 days, and
3ubsequently evapor~ted to dryne~s. The crude peptide was
purified by gel chromatography (SEPHAD~X~-LH 20) and ~ub-
~equ~nt medlum pre~ure c~romatography (cf. A IV,
50-70% A, 0.254 min1). 122 mg of pure product were
obtained.
EXANP~E 70
Ac-~ys-Prcl-Oap-~la-~~1S-~hx-Gty-~sp-Ile-~la-Lou-NH2
1 g of resin dew ribed by Breipohl ~t al. (fram BASHE~),
correspondinq to a batch size of 0.5 mmol, wa~ reacted
a~ in AIb with 2 ~ol e~ch of
200505t~
- 25 - o.z. 0~50/4~39
Fmoc-Leu-OH Fmoc-Gly-OH Fmoc-DaptZ)-OH
Fmoc-Ala-OH Fmoc-Ahx-OH Fmoc-Pro-OH
Fmoc-Ile-OH Fmoc-His(Trt)-OH Fmoc-Ly~(Z)-OH
Fmoc-Asp(OtBu)-OH Fmoc-Ala-OH
After the synthesic wa~ completed, the M terminu~ was
acetylated (steps 2-4 and 8-9 as in AIb). The peptide-
resin was dried under reduced pressure; yield 1.75 g.
The crude product (518 mg) obtained after TFA cleavage as
in AIII wa~ di~olved in 500 ml of degas~ed DMF. After
addition of 0.43 ml of triethylamine and (at -25C~
0.43 ml of diphenylphosphoryl azide, the mixture was
~tirred at -25C for 2 h, and stored at -20C for 2 days,
at 4C for 2 days and at room temperature for 2 days. It
was then evaporated to dryness, and the crude peptide wa~
purified by gel chromatography (SEPHADEX~ LH 20). The
i olated monomer (182 mg) wa~ deprotected with HF as in
AII and purified by medium pressure chromatography
(cf. AIV; 55-75% A; 0.25~ min~l). 144 mg of pure product
were obtained
EXAMPLF 71
Ac-Dap-Ala-His-Aoc-Glu-OH
6.45 g of F~oc-Glu(OtBu)-Merrifield resin (substitution
O.31 mmol/g), corre~ponding to a batch size of 2 mmol,
were reacted as in AIb with 8 mmol each o~
Fmoc-Aoc-O~ Fmoc-Ala-OH
Fmoc-Hi~(Tos)-OH Fmoc-Dap(Boc) OH
Subsequently, N-termin~l deprotection and acetylation
were carried out (steps 2-4 and 8-9 as in AIb) and the
t-butyl and Boc protective group~ were eliminated (~tep~
1-6 a~ in AIa). The cyclization on the reain took place
Z11,30505~;
- 26 - O.Z. 0050/40390
in NMP with the addition of 3.54 g of BOP and 3.5 ml of
dii~opropylethylamine (16 h). The peptide-resin was dried
under reduced pressure. The yield was 7.0 g. The crude
product obtained after HF cleavage a~ in AII was purified
by gel filtration (SEPHADEX~ G-15) and medium pre~ure
chromatography (cf. AIV; 5-20% A; 0.25% min~1). 21 mg of
pure product were obtained.
The following can be prepared in a ~imilar manner to
Examples 69, 70 and 71:
72. Ac-oap-Ala-H1s-Aoc-~sp-NH2
73. Ac-Oap-~la-Hi5-AOC-GlU~~Ha
74. Ac-Lys-~1a-His-Ano-Glu-NH2
75. H-L~s-~la-~is-~h~-G~u-OH
76. Ac-Lys-Ala-His-Ahx-61u-OH
77. H-Dap-~la-H1s-Aoe-Gtu-OH
78. Ac-oap-Ala-His-~d~-~sp-NH2
79. Ac-Asp-Ala-H~s-Aoc-Oap-NH2
ao . Ac-Glu-Ala-His-Ano-oap-NH 2
81 . AC-ASp-Al a-H~s-Ap~-Gly-Ljs-NH2
82. Ac-Lys-Al~-His-Ahx-Gly-Asp-NH2
,_ ~
83 . AC-ASp-A 1 a-His-Leu-Gly-Oap-NH2
84. Ac-Pro-Asp-Ala-H1s-Apo-61y-Oap~ -NH2
85. Ac-Lys-Pro-Asp-Ala-His-Ape-Gly-Lys-Ile-Ala-NH2
86. Ac-Asp-Lys-Pro-Asp-Ala-His-Ape-Gly-Lys-lte-Ala-Leu-NH2
2'~30505~i
- 27 - O.Z. 0050/40390
87 Ac-Asp-Lys-Pro-Asp-Ala-His-Ape-Gly-Lys-lle-Ala-Leu-OH
88 Ac-Asp-Lys-Pro-Glu-Ald-His-Ape-Gly-Lys-lle-Ala-Leu-NH2
-
9 Ac-Asp-Lys-Pro-Glu-Ala-His-Ape-Gly-Lys-lle-Ala-Leu-OH
90. Ac-Asp-Lys-Pro-Lys-Ala-His-Ape-Gly-Asp-lle-Ala-Leu-NH2
91. Ac-Asp-Lys-pro-Lys-Ala-His-~pd-Gly-Asp-Ile-p~la-Leu-oH
92 H-Asp-Lys-Pro-Lys-Ala-His-Ahx-61y-Asp-ll~-Ala-L2u-NH2
93 Ac-Asp-~ys-Pro-Oap-Ala-His-~hx-Gly-Asp-lle-Ala-Leu-OH
94. Ac-Pro-~sp-Lys-Asp-~/al-~la-His-Ape-Gly-orn-lle-~ta-Leu-NH2
Ac-Pro-~sp-Lys-~sp-Yal-Ala-His-~pe-Gly-Orn-ll~-~la-Leu-OH
96 Ac-Asp-L~s-Pro-~sp-~la-His-~p~-Gly-ll~-Orn-~la-Leu-NH2
97 H-Asp-Lys-Pro-Asp-~la-His-Ap~-Gly-ll~-Orn-Ala-Leu-O~
_
98 Ac-Pro-Asp-~ys-~sp-Val-Ala-His-Ape-Gly-ll~-Dap-Ala-~eu-NH2
99 Ac-Pro-Asp-Lys-Asp-Yal-Ala-His-Ap~-Gly~ -Dap-Ala-Leu-OH
100. H-Asp-~ys-Pro-Asp-~la-His-Ap~-~la-Oap-llo-Ala-~eu-NH2
101 Ac-Asp-~s-Pro-Asp-Ala-H~s-Ap--Ala-Oap-lle-Ala-L~u-OH ,
