Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
Z0050~
O.Z. ~050/40385
Novel TNF PeDtide3
The pre~ent invention relate~ to novel peptide~
derived from tumor necro~i~ factor (TNF), the preparation
thereof and the u~e thereof a~ drug~.
Car~well et al. ~Proc. Natl. Acad. Sci. USA 72
(1975) 3666) reported that the serum of endotoxin-treated
animal~ which had previously been infected with the
Calmette-Guerin strain of Mycobacteria (BCG) brought
about hemorrhagic necro~is in variou~ mou~e tumors. This
activity was ascribed to tumor necrosis factorO TNF al~o
ha~ a cyto~tatic or cytotoxic effect on a large number of
transformed cell lines in vitro, whereas normal human and
animal cell line~ are unaffected (Lymphokine Report~ Vol.
2, pp 235-275, Academic Press, New York, 1981). Recently,
the biochemical characterization and the gene for human
TNF have been de~cribed (Nature 312 (1984) 724, J. Biol.
Chem. 260 (1985) 2345, Nucl. Acids Re~. 13 (1985) 6361).
It i8 pos~ible to deduce from the~a data the
following pro~ein structure for mature human ~NP:
V ~ VAl~l A~
G~aGluGl~
~alGl ~ 1 ~ r
GlnVaLLaU~eLyEGayG~yCy~nebr~D~o~Va~ e~ le
SOE~gIl~lPVa]SeCI~l~Va~ ~le~
cyDGiu~klurhoRs~auGly~lp~-luAlaLy~?n~r~5luFrolleq~leu
GayGayVel~G1nl~Lluty~G~y
Tyr~agUçPh3L ~lu~ ~ nValIy~xæ~yIleIl o~l
The TNF gene~ of cattle, rabbits and mice have
also been de~cribed (Cold Spring Harbor Symp. Quan~.
Biol. 51 (1986) 597).
Be~ide~ it~ cytotoxic propertie~, T~F i8 ons of
the m~in ~ub~tance~ involved in inflamm~tory reactions
(Pharmac. Re~. 5 (1988) 129). Animal modelff have shown
that TNF i8 involved ~n septic shock (Science 229 (1985)
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869) and graft-ver~us-host di~ease (J. Exp. Med. 166
(1987) 1280).
We have now found that peptide~ with a consider-
ably lower molecular weight have beneficial properties.
The pre ent invention relates to peptide~ of th~
formula I
X-A-B-Pro-E-Y I,
where
A is Gly, Ala or Ser,
B -i~ Cy~, ~yr or Thr,
E i4 Ser or Pro,
X i~ G-NN-CHM-CO-, G-NH-CXM-CO-W-, G-R-NH-CHM-CO- or
G-R-~H-CHM-CO-W- and
Y is -Z, -NH-CHQ-CO-Z, -V-NH-CHQ-CO-Z, -NN-CXQ-CO-U-Z
or -V-N~-CHQ-CO-U-Z,
where, in X and Y,
G is hydrogen or an amino-protective group,
Z i~ 0~ or NH2 or a carboxyl-protective group, or
G and Z together are al~o a covalent bond or the group
-CO-(CN2).-NH- where a i~ fro~ 1 to 12,
R, U, V and W are peptide chains composed of 1-4 natur-
ally occurring u-amino acids and
M and Q sre h~drogens or one of the groups -CH(CH3)2,
-CH(CH3)-C2H~, -C5H5, -CH(OH)-CN3
--CH~ --CH~ or -(CH2lb-T
H H
(~ith b being from 1 to 6 and T being hydrogen or
0~, CH30, CH3S, (CH3)2CH, C~H" p-HO-C6H~, HS, H2N,
HO-CO, H8N-CO or HpN-C(=NH)-NH) or
M and Q together are a -(CH2)~-S-S-~CH2)~ CH2).-CO-NH-
-(CH2~s- 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 be~ng from 1 to 12), ns well a~ the
~alts thereof with phy~iologically tolerated acid3.
The peptides of the formula I are con~tructed of
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L-amino acids, but they can contain 1 or 2 D-amino acids.
The side-chain~ of the trifunctional amino acids can
carry protective groups or be unprotected.
Particularly preferred phy3iologically tolerated
acid~ are: hydrochloric acid, citric acid, tartaric acid,
lactic acid, phosphoric acid, methane~ulfonic acid,
acetic acid, formic acid, maleic acid, fumaric acid,
malic acid, succinic acid, malonic acid, sulfuric acid,
~-glutamic acid, L-aspartic acid, pyruvic acid, mucic
acid, benzoic acid, glucuronic acid, oxalic acid, ascor-
bic acid and acetylglycins.
The novel peptide~ can be open-chain tG = H,
amino-protective group; Z = OH, ~H2t carboxyl-protective
group, M and Q not connected together) and, in particu-
lar, have a di~ulfide bridge (G = ~, 2mino-protective
group; Z = OH, NH2~ carboxyl-protective group M + Q = -
(CH2)c~S~S~(CH2)d~) or a side chain bridg0 (G = H, amino-
protective group, Z = OH, NH2~ carboxyl-protective group,
M + Q = -(CH2).-NH-CO-(CH2)~- or -(CH2).-NH-CO-(CH2)6-NH-CO-
(CH2)~-) or be linked head-to-tail (G + Z = covalent bond
or -CO-(CH2),-NH-).
~he novel compound~ can be prepared by conven-
tional method~ of peptide chemi~try.
Thus, the peptides can be con~tructed sequenti-
ally from a~ino acids or by linking together suitable
s~aller peptide fragment~. In the sequential con~truc-
tion, the peptide chain i~ extended stepwise, by one
amino acid each time, starting at the C terminus. In the
ca~e of fraqment coupling it i8 pos~ible to link together
fragment~ of different lengths, the~e in turn being
obtain~ble by seque~tial con~truction frQm amino acids or
coupling of other fra~ments. The cyclic peptides are
obtained, after synthesis of the open-chain peptides, by
a cyclization reaction carried out in high dilution.
In the case both of ~equential construction and
of fragment coupling it i8 neceE~ary for the building
block~ to be linked by formation of an amide linkage.
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Enzymatic and chemical methodq are quitable for thi~.
Chemical methods for forming amide linkage~ are
dealt with in detail by M~llsr, Methoden der Organischen
Chemie Vol. XV/2, pp 1-364, Thieme Verlag, Stuttgart,
lg74; Stewart, Young, Solid Phase Peptide Synthesi~, pp
31-34, 71-82, Pierce Chemical Company, Rockford, 1984;
Bodan~zky, Rlausner, Ondetti, Peptide Synthesi~, pp
85-128, John Wiley & Sons, New York, 1976 and other
standard works of peptide chemistry. Particularly prefer-
red are the azide method, the symmetrical and mixedanhydride method, active e~ter~ generated in situ or
preformed and the formation of amide linkage~ u~ing
coupling reagent~ (activators), in particular dicyclo-
hexylcarbodiimide (DCC), diisopropylc~rbodiimide (DIC),
1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (~DQ),
l-ethyl-3-(3-dimethylaminopropyl)carbodiimlde hydrochlor-
ide (EDCI), n-propanepho~phonic anhydride (PPA), N,N-bis-
(2-oxo-3-oxazolidinyl)amidophosphoryl chloride (BOP-Cl),
diphenylphosphsryl azide (DPPA), Ca~tro's reagent (BOP),
O-benzotriazolyl-N,N,N',N~-tetra-methyluronium salt~
(HBTU), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene
dioxide (Steglich'c reagent; HOTDO) and l,1'-carbonyldi-
imidazole (CDI~. The coupling reagent~ can be employed
alone or in co~bination with additives such a3 N,N'-dime-
thyl-4-aminopyridine (DMAP), N-hydroxybenzotr~azole
(HOBt), N-hydroxybenzotriazine (HOOBt), N-hydroxy~uccin-
imide (XOSu) or 2-hydroxypyridine.
Wherea~ it i~ normally po~ible to di~pen~e with
protectiv~ group~ in enzymatic peptide ~ynthe~i~, for
ch d c~l ~ynthe~i~ it ~ necessary for there to be
reversible protection of the reactive functional ~roups
wh~ch are not involved in the formation of the amite
linkage on the two reactant~. Three conventional pro-
tective group technique~ are preferred for chemical
peptide 3ynthe~ess the benzyloxycarbonyl (Z), the
t-butyloxycarbonyl (Boc) and tha 9-fluorenylmethyloxy-
carbonyl (Fmoc) technique~. In each case the protective
Z0~5(36~
-- 5
group on the ~-amino group of the chain-extending building
block is identified. The side-chain protective groups on
the trifunctional amino acids are chosen so that they are
not necessarily eliminated together with the a-amino
protective group. A detailed review of amino acid protec-
tive groups is given by Muller, Methoden der Organischen
Chemie Vo. XV/1, pp 20-906, Thieme Verlag, Stuttgart, 1974.
The building blocks used to construct the peptide
chain can be reacted in solution, in suspension or by a
method similar to that described by Merrifield in J.Amer.
Chem. Soc. 85 (1963) 2149. Particularly preferred methods
are those in which peptides are constructed sequentially or
by fragment coupling by use of the Z, Box or Fmoc protec-
tive group technique, in which case the reaction takes place
in solution, as well as those in which, similar to the
Merrifield technique, one reactant is bound to an insoluble
polymeric support (also called resin hereinafter). This
typically entrails the peptide being constructed
sequentially on the polymeric support, by use of the 8OC or
Fmoc protective group technique, with the growing peptide
chain being covalently bonded at the C terminus to the
insoluble resin particles (cf. Figures 1 and 2). This
procedure allows reagents and by ~ oved by
filtration, and thus recrystallization of intermediates is
superfluous.
The protected amino acids can be bonded to any
suitable polymers which merely need to be insoluble in the
solvents used and to have a stable physical form which
allows easy filtration. The polymer must contain a
functional group to which the first protected amino acid can
be firmly linked by a covalent bond. A wide variety of
polymers is suitable for this purpose, for example
cellulose, polyvinyl alcohol, polymethacrylate, sulfonated
polystyrene, chloromethylated copolymer of styrene and
divinylbenzene (Merrifield resin), 4-methylbenzhy-
drylamine-resin (MB~A-resin), phenylacetamidomethyl-
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resin (Pam-re~in), p-benzyloxybenzyl alcohol-resin,
benzhydrylamine-resin (BHA-resin), 4 hydroxymethyl-
benzoyloxymethyl-re~in, the resin used by Breipohl et al.
(Tetrahedron Lett. 28 (1987) 565; from 2ACHEM~, HYCRAM
resin (from ORPEGEN) or SASRIN resin (from BACHEM).
Solvents suitable for peptide synthe~i~ in
solution are all those which are inert under the reaction
conditions, in particular water, N,N-dimethylformamide
(DMF), dimethyl sulfoxide (DMS0), acetonitrila, dichloro-
m~thane (DCM), 1,4-dioxane, tetrahydrofuran (THF),
N-methyl-2-pyrrolidone lN~P) and mixturss of the said
solvents. Peptida synthesi~ on polymeric support~ can be
carried out in all inert organic solvents which dis~olve
the amino acid derivatives used; however, solvents which
15 al80 have r~in-~w~lling propertie~ are preferred, such
as DMF, DCM, NMP, acetonitrile and DNS0, as well a~
mixtures of the~e ~olvents.
After the peptide ha~ been synthe~ized it i8
cleaved off the polymeric support. The cleavage condi-
tion~ for the various types of re~ins are disclo~ed inthe literature. The cleavage reactions most commonly u~e
acid and palladium catalysis, in particular cleavage in
anhydrou~ liquid hydrogen fluoride, in anhydrou~ tri-
fluoromethanesulfonic acid, in dilute or concentrated
trifluoroacetic acid or palladium-catalyzed cleavage in
THF or THF-DC~ mixture~ in the pre~ence of a weak base
such as morpholino. The protective groups may, depending
on the choice thereof, be retained or likewi~e cleaved
off under th~ cl~avage condition~. Parti~l deprotection
of the peptide may also be worthwhile if the intantion i8
to carry out certain derivatization reactions or a
cyclization.
Some of the novel peptides have good cytotoxic
proparti~s. So~e others of the peptide3 have high affin-
3S ity for th~ cellular TNF receptor without, however,having cytotoxic activity. They ~re tharefore TNF antago-
nists. They compete with natural TNF for bindinq to the
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cellular TN~ receptor and thu~ ~uppres~ the TNF effect.
~he novel peptideY are valuable drug~ which can be
employed for treating neoplastic di~ea~e~ and autoimmune
di~eases as well a~ for controlling and preventing
infections, inflammations and tran~plant re~ection
reaction~. Simple experiments can be used to elucidate
the mod~ of action of the individual peptides. The
cytotoxicity of the peptide i~ determined by incubating
a TNF-sen~itive cell line in the pre~ence of the peptide.
In a second experimental approach, th~ cell line is
incubated with the relevant peptide in the presence of a
lethal amount of TNF. It is pos~ible in this way to
detect the TNF-antagoni~tic effect. In addition, the
affinity of the peptide for the cellular TNF receptor is
d~termined in an in vitro binding experiment.
The following te~t ~ystem~ were used to charac
terize the agoni~tic and antagoni~tic effect~ of the
novel peptides:
I. Cytotoxicity te~t on TNF-sen~itive indicator cell~,
II. Cytotoxicity antagonism test on TNF-sensitive
indicator cell~,
III. Competitive receptor-binding test on indicator cell~
expressing TNF receptor.
I. Cytotoxicity te~t
The agoni~tic effect~ of the novel peptides are
asses~ed on the ba~i~ of their cytotoxic effect on
TNF-sensitive cells (e.g. ~929, NCF-7, A204, U937).
The te~t with L929 and MC~-7 wa~ carried out a~
followss
1. 100 ~1 of culture medium containing 3 to 5 x 103
fre~hly tryp~inized, exponentially growing Lg29
cell~ (mouse) or NCF-7 cells (human) were
pipstted into the well8 of a 96-well flat-~ottom
culture plate. The plate was incubated at 37-C
overnight. The air in the i~cubator wa~ saturated
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with water vapor and contained 5~ CO2 by volumQ.
The L929 culture medium contained S00 ml of lx
Earle'~ ME~ (Boehringer Mannheim), 50 ml of heat-
inactivated (56C, 30 min) fetal calf qerum
S (FCS), 50 ml of L-glutamine (200 mM), S ml of
lOOx non-e~sential amino acids, 3 ml of lM HEPES
buffer pH 7.2,and 50 ml of ge~tamicin (50 mg/ml).
The MCF-7 culture medium contained 500 ml of lx
Dulbecco'3 MEM (Boehringer Nannheim), 100 ml of
heat-inactivated (56C~ 30 min) FCS, 5 ml of
L-glutamine and S ml of lOOx nones~ential am~no
acld~.
2. Th~ next day 100 ~1 of the peptide solution to be
te6ted were added to the cell culture~ and
sub~ected to serial 2-fold dilu~ion. In addition,
some cell control~ (i.e. cell culture~ not
treated with peptide dilution) and some rhu-TNF
controls (i.e. cell culture~ treated with recom-
binant hu~an INP) were al80 made up. The culture
plate wa~ incubated at 37-C in an atmo%phere of
air saturated with wator vapor and containing 5%
CO~ by volume for 48 h.
3. The percentage of surv~ving cells in the culture~
treat2d with peptide dilution wa~ ~etermined by
~taining with cry~t~l v~olet. For thi~ purpo~e,
the liquid~ wer~ remo~d from the wells of the
te~t plate by tapping it~ gO ~1 of crystal violet
~olution wera pipetted into each well.
The compoRition of the crystal violet ~olu~ion
wa~ 88 follow~ t
3.75 g of cry~tal violet
1.75 g of NaCl
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161.5 ml of ethanol
43.2 ml of 37% formaldehyde
water ad 500 ml
The crystal violet solution was left in the well~
for 20 min and then likewi~e removed by tapping.
The plates were then washed 5 times by immer~ion
in water in order to remove dye not bound to the
cells. The dye bound to the cell~ wa~ extracted
by adding 100 ~1 of reagent solution (50% 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 w911. The
surviving cells were deter~ined by measuring the
lS extinction at 540 nm of ths colored solution in
the individual well~.
5. Subsequently, by relating to the cell control,
tha 50~ cytotoxicity value wa~ defined, and the
reciprocal of the ~smple dilution which re~ulted
in 50~ cytotoxicity was calculated as the cyto-
toxic activity of the te~ ~ample.
II. Cytotoxicity antagoni~m te~t
The ~ntagonistic effect of the peptide~ wa~ a~seRsed
on tho ba~is of their prop~rty of antagonizing the
cytotoxic effect of rhu-TNF on TNF-~ensitive cell~
(e.g. ~929, MCF-7, A204, U937). The cytotoxicity
antagonis~ ta~t with L929 and ~C~-7 cells wa~
carried out as followss
1. 100 ~1 of culture mediu~ containinq 3 to 5 x 103
freshly tryp~inizsd, exponentially gr~wing L929
cell~ (mou3e) or ~CF-7 cells (human) were
pipetted into the well~ of a 96-wall flat-bottom
culture plat~. Tha plste was incubated at 37C
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overnight. The air in the incubator wa~ saturated
with water vapor and contained S% CO2 by volume.
The L929 culture medium contained 500 ml of lx
Earle's MEM (Boehringer M~nnheim), 50 ml of heat-
inactivated ~56C, 30 min) FCS, 5 ml of L-gluta-
mine (200 mM), 5 ml of lOOx non-e~sential amino
acid~, 3 ml of 1~ HEPES buffer pH 7.2, and 500 ~1
of gentamicin (50 mg/ml).
The MCF-7 culture medium contained 500 ml of lx
Dulbecco's MEM (Boehringer Nannheim), 100 ml of
heat-inactivated (56-C, 30 min) FCS, 5 ml of
L-glutamine (200 mM) and 5 ml of lOOx nonessen-
tlal amlno acids.
2. The next day 100 ~1 of the peptide solution to be
tested were added to ~he cell culture~ and
sub~ected to 3erial 2-fold dilution. Then, 100 ~1
of a rhu-TNF dilution in ~uiture medium, which
dilution had an 80-100~ cytstox~c effect in the
final concentration in the cell culture, were
added to the~e coll cultures. In addition, ~ome
cell controls (i.e. cell culture~ not treated
with p~ptide ~olution or with rhu-TNF solution)
and some rhu-~NF controls (- cell culture~
treated only with rhu-~NF solution) were also
~de up. The culture plate wa~ then lncuba~ed at
37-C in an atmosphere of air saturated with water
vapor and containing 5% CO2 by volume for 48 h.
3. Th~ percentage of surviving cell~ in the culture~
treated with ~ubstancQ solution was det~rmined by
staining with crystal violet. For this purpo5e,
th~ liquids were removed from the wells of the
te~t plate by tapping it. 50 ~1 of cry~tal violet
~nlution wer~ pipstted into each well.
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The crystal violet solution had the composition
specified in I.3
The crystal violet solution wa~ left in the wells
for 20 min and then likewise removed by tapping.
The plates were then washed 5 times by i~mersion
in water in order to remove dye not bound to the
cell~. The dye bound to the cells was extracted
by adding 100 ~1 of reagent solution (S0~ etha-
nol, 0.1% glacial acetic acid, 49.9% water) to
each well.
4. The plates were shaken for 5 min to obtain a
~olution of uniform color in each well. The
~urviving cells were determined by mea~uring the
extinction at 540 n~ of the colored solution in
the individual well~.
5. Sub3equently, by relating to the cell control and
the rhu-TNF control, the 50% antagonis~ value was
defined, and the sample concentration which
re~ulted in 50% antagoni~m of rhu-TNF cytotox-
icity at the rhu-TNF concen~ration u~ed W8~
calcul~ted a~ antagoni~tic activity of the ~ample
te~ted.
III. Competltive receptor-binding teet
Both the agoni~tic and antagoni~tic effects of
peptides are conditional on ths latter binding to
the ~NF receptor. Thi~ mean~ that peptide~ with an
agoni~tic or antagoni~tic effect compete with
rhu-TNF for binding to the TNF receptor on TN~-
sens$tive indicator cell~ (e.g. U93?). The competi-
tive receptor-binding te~t was csrried out a~
follow~
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1. 100 ~1 of medium containing various concentra-
tions of the peptide to be tested and of rhu-TNF
(= control) were pipetted into the reaction
ves~el~. The medium comprised 500 ml of PBS
(Boehringer Mannheim3, 10 ml of heat-inactivated
(56DC, 30 min) FCS and 100 mg of sodium azide.
2. Subsequently, 10a ~1 of medium containing 1 ng of
l25I-labeled rhu-TNF (Bolton lactoperoxida~e
method) were placed in the reaction ve~els and
mixed. The non-specific binding (NSB~ was deter-
mined by mixing in the reaction vessel~ the
l25I-labeled rhu-TNF ~1 ng ofl25I-rhu-TNF in 100 ~1
of medium) with a 200-fold exress of unlabeled
rhu-TNF (200 ng of rhu-TNF i~ 100 ~1 of medium).
3. ~hen 100 ~1 of medium containing 2 x 108 V937
cell~ (human) were pipetted into the reaction
ve~sels and mixed. The reaction ve~sel~ (te~t
volume 300 ~1) were incubated at 0C for 90 min.
The reaction mixtures were remixed after 45 min.
4. After the incubation the cell~ wcre centrifuged
at 1800 rp~ and 4-C for 5 min, washed 3 tim~s
with medium and tran~ferrsd qua~titatively into
counting vial~, and the cell-bound radioact~vity
wa~ determined in a Clini gamma COuntQr 1272
(L~B Wellac).
5. After the ~easurement~ had been corrected for the
non-~pecific binding, the 50% competition value
was defined by relation to the ovQrall b~nding,
and tho 9ample concentration which led to 50%
competition of ~5I-rhu-TNF binding at the ~I-rhu-
TNF concentration u~ed wa~ c~lculated a~ the
compet~tive ac~ivity of the sample tested.
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The Example~ which follow are intended to explain the
invention in more detail. The proteinogenou~ amino acids
ars abbreviated in the Examples using t~P conventional
three-letter code. Other meanings are:
Aad = ~-aminoadipic acid, Abs=4-aminobutyric acid,
~c = acetic acid, Ahp = 7-aminoheptanoic acid,
Aoc = 8-aminooctanoic acid, Ape = 5-aminopentanoic acid,
Hcy = homocystaine, Orn = ornithine.
A. General pro~edure
0 I. The peptides claimed in claim 1 were synthesized
u~ing ~tandard me~hods of solid-phase peptide
synthe~is in a completely automatic model 430
peptide ~ynthesizer from APP~IED BIOSYSTEMS. ~he
apparatu~ use~ different ~ynthe~i3 cycles for the
Boc and Fmoc protective group techniques.
a) Synthe~i~ cycle for the Boc prot~ctive group
technique
1. 30~ triflooro~Ydic acid in DCK 1 x 3 min
2. 50% trifl~on~YYdic aci~ in DCM 1 x 17 min
3. DoMwa~ng 5 x l min
4. 5~ tLiK~pylethylam~nP in DCM l x l min
5. 5% diix~nnpylethyla~ne in NMP 1 x 1 min
6. NNP wæ~ng 5 x l min
?. ~diticn of pn#Ytivated ~rJboted 3~0
~c~d (activation ky 1 eqyivalent of DoC
and l eq~i ~ t of HaBS in NMP~DaM);
pq~b ooupling (lst part) l x 30 min
8. A~dit~on of D~gO to the reY~on m~dIIe
u~l it o~u~n~ 20% D~gO ky ~lume
9. P~ e coupling (2nd part) 1 x 16 min
10.A~it~on of 3.8 eqlivalerts of di~
propy~ la~l~ to t~e ~3Y~i~n m~xh~e
ll.Pg~ coy~ng (3cd part) 1 x 7 min
12.DCHw~ng 3 x 1 min
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of ca~lin~ (reb~rn to 5. )
14. 10~ acetic ar~id~, 5% di~so~l-
et~ylamine in K:M 1 x 2 min
15. 10% acetic anly~i~ in DCM 1 x . min
16. DCM wa~hir~ 4 x 1 min
17 Return to 1.
b) Ss~ cycle f~ the h~c ~ive ~up tec~i~
1. ~P ~g 1 x 1 min
2. 209~ p;E~ridil# in 2~P 1 x 4 min
3. 20% pip~idinE~ in r~P 1 x 16 min
4, ~e w~shi~ 5 x 1 min
5. Addition o~ pn#~ivated prrux~ed ~
a~id (~ivation by 1 eqyivzlent of DCC
and 1 equivalent of HOBt in NMP/DaM);
p~ e ooupl;n~ 1 x 61 min
6. N~æ wo~hing 3 x 1 min
7. If re~icn is Lnool~te, nq~ltiDn of
ooupling (n~h~n to 5.)
8. 10% ~o~ic ~y~kide in NMP 1 x 8 min
9. NMP wsshing 3 x 1 min
10. R~lxn t~ 2.
II. Working up of peptide-resins o~tained aa in Ia
Tha pept~de-resin obtained as in Ia was dried under
reduced pressure and transferred lnto a reaction
ve~el of a Teflon HF apparatus (from PENIN~ULA).
Addit~on of a scavenger, preferably ani~ole (1 ml/g
of re~in), and of a thiol, in the c~se of tryptophan-
containing peptides, to remove the indole formyl
group, preferably ethan~dithiol (0.5 m~/q of re~in),
was followsd by condensation in of hydrogen fluoride
(10 ml/g of resin) while cooling with liquid N2. The
mixture w~ allowed to warm to O-C~ and was ~tirred
at thi~ temperature for 45 min. Tho hydrog~n fluor-
ide w~ then ~tripped off under reduced preasure and
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the residue wa3 washed with ethyl acetate in order
to remove remaining scavenger. The peptide was
extracted with 30~ strength acetic acid and
filtered, and the filtrate was freeze-dried.
To prepare peptide hydrazides, the peptide-resin
Pam- or Merrifield re~in) wa~ suspended in DMF
(15 ml~g of re~in~, hydrazine hydrate (20 equiva-
lents) was added, and ths mixture wa~ ~tirred at
room temperature for 2 day3. To work up, the resin
was filtered off and the filtrate wa~ evaporated to
dryne~. The residue was crystalli2ed from DMF/Et2O
or MeOH/Et2O.
III. Working up of the peptide-re~ins obtainsd as in Ib
The peptide-resin obtained aff in Ib was dried under
reduced pres~ure and ~ubsequently sub~ected to one
of the following cleavage procedures, depending on
the amino acid composition (Wade, Tregear, Howard
Florey Fmoc-WorX~hop Nanual, Nelbourne 1985).
Peptide containing Cleavage condition~
. - .
Arg(Mtr) ~et Trp TFA Scavenger Reaction
Time
25 no no - no 95% 5% H2O 1.5 h
yes no no 95% 5~ thioanisole ^ 3 h
no ye~ no 35% 5% ethyl methyl 1.5 h
sulfide
no no ye~ 95~ 5% ethanedithiol/ 1.5 h
ani~ole (1:3)
no yes yes 95% 5% ethanedithiol/ 1.5 h
an~sole/ethyl methyl
sulfide (1:3:1)
ye~ yea yes 93% 7% ethanedithiol/ ^ 3 h
ani~ole/ethyl methyl
sulfide ~ls3s3)
_ _.
Z00506~.
- 16 - O.Z. 0050/40385 CA
The suspension of the peptide-re~in in the suitable
TFA mixture was stirred at room temperature for the
~tated time and then the re~in wa~ filtered off and
washed with TFA and with DCM. The filtrate and the
washings were extensively concentrated, and the
peptide was precipitated by addition of diethyl
ether. The mixture was cooled in an ice bath, and
the precipitate was filtered off, taken up in 30%
acetic acid and freeze-dried.
IV. Purification and charactarization of the peptide~
Purification wa~ by gel chromatography tSEPHADEX~
G-10, G-15~10% HOAC; SEPHADEX~ I,H20/MeOH) and sub-
sequent medium pressure chromatography (stationary
pha~e: HD-SI~ C-18, 20-45 ~, 100 A; mobile pha~es
gradient with A = 0.1% ~F~/MeOH, B = 0.1~ TFA/H2O)o
The purity of the final product~ wa~ determined by
analytical HPLC (~tationary pha~es 100 x 2.1 mm
VYDAC C-18, 5 ~, 300 ~; mobile phase = CH3CN/H20
gradient buffered with 0.1% TFA, 40 DC ) . Charac-
terization wa~ by mean~ of ~mino acid analy~is and
fast atom bombardment mas~ ~pectrometry.
B. Specific procedure~
EXANPL~ 1
Ac-Gly-Gln-G'y-Thr-Pro-Ser-Thr-His-NH2
1.30 g of Boc-~ic(z)-NBHA-resin (sub~titution
0.39 mmol/g), corr~ponding to a b~tch ~ize of 0.5 mmol,
were reacted a~ in AIa (steps 14-16 wer~ dispan~ed with
in all the coupling~ followin~ Hi~) with 2 mQol each of
Boc-~hr(Bzl)-OH Boc-Gly-OH
Boc-Ser(Bzl)-OH Bo~-Gln-OH
Boc-Pro-~ Boc-Gly-O~
8Oc-Thr(Bzl)-OH
2C~050fil.
- 17 - O.Z. 0050/40385 CA
After the synthe i~ was complete the peptide-
re~in underwent N terminal deprotection and acetylation
(steps 1-5 and 14-16 a~ in AIa) and subsequently dried
under reduced pre~ure; the yield wa3 1.65 g.
0.80 g of the resin obtained in thi~ way was
subjected to HF cleavage a~ in AII. The crude product
(152 mg) wa~ purified by gel filtration (SEPHADEX0 G-10)
and medium pre~sure chromatography (cf. AIV; 30-50~ A
0.25% min~1). 87 mg of pur~ product were obtained.
EXAMPLE 2
~-Ly~-Gly-Gln-Gly-Thr-Pro-Ser-Thr-His-Val-Leu-Leu-OH
O.46 g of Fmoc-Leu-p-alkoxybenzyl alcohol-re~in
(sub~titution 0.55 mmol/g), corresponding to a batch siz~
of 0.25 ~mol, was r~acted as in AIb with 1 mmol ~ach of
Fmo~-Leu-OH Fmoc-Thr-(tBu~-OH
Fmoc-Val-OH Fmoc-Gly-OH
Fmoc-His(Trt)-OH Fmoc-Gln-OH
Fmoc-Thr(tBu)-OH Fmoc-Gly-OH
Fmoc-Ser(tBu)-OH Fmoc-Ly~(Boc)-OH
Fmoc-Pro-OH
After the synth~is was complete, the peptide-
re~in underwent ~-~erminal deprotection (~teps 2-4 a~ in
AIb). The resultin~ peptide-r~in wa~ dried under reduced
pres~ura; th~ yi~ld wa~ 0.7 g.
The crude peptid~ (217 mg) obtained after TFA
cleav~ge 2~ in AIII wa~ purified by gel filtration
(SEPHADBX~ G-10) ~nd medium pressur~ chromatography (cf.
AIV; 30-50~A; 0.25% min~'). 107 mg of pure product were
obtained.
The following can bQ preparsd in a similar manner
to Examplo~ 1 and 2:
3. Ac-Lys-Gay~ ly~Thr-Pn~br-Thr-His-VAl-LQu-Ieu~
4. Ac-Ly~Gly~Gk~ Thr- ~ r~hr~s~Val-L~u-Ieu~N~
5. H~Lys-Gly~G~41y~Iyr-P~brJThr~-Val-Ieu-~eu-aH
Z005061.
- 18 - O. Z . 0050/40385 CA
6 . Ac-Lys-Gly-Gln-GlyTyr-Pro~-mr-Elis-VA 1 -Ieu-Lsu-NH~
7. H-Gly-Gln-Gly-~r-Pro-Ser-q~r-His~
8. H-Leu-Phe-Lys-Gly Gln-Gly-qhr-Pro Ser-~r-His-Val-Ieu-Ieu{~l
9. A~-Ieu-Phe-Ly~-Gly-&lll-Gly-q~7r-Pro-S~r-~lhr-His-VAl-Leu-Ieu-NH~
S 10 . A~-Lys-Gly-Gln-Ala-qhr-E~Ser-~r-T~ R-Val-leu-L~u~
E~E 12
~ ~ - i
Ac-Cys-Lys-Gly-Gln-Gly-Thr-Pn~br-Thr-Hi~-Val-Leu~ N~
0.98 g of Boc-Cys(pMB)-MBHA-resin (sub~titution
0.51 mmol/g), corre~ponding to a batch ~ize of 0.5 mmol,
were reacted a~ in AIa (steps 14-16 were dispensed with
in all couplings following His) with 2 mmol each of
Boc-Leu-OH Boc-Thr(Bzl)-O~
Boc-Val-OH Boc-Gly-OH
Boc-His(Z)-O~ Boc-~ln-OH
Boc-Thr(Bzl)-OH Boc-Gly-OH
Boc-Ser(Bzl)-OH Boc -LYB ( Cl-Z)-O~
Boc-Pro-OH Boc-Cys(pMB)-OH
After the synthesis wa~ complete, the N terminu~
was acetylated (steps 2-4 and 8-9 a~ in AIb). The re~ul-
ting peptide-re~in wa~ dried under reduced pre~sure; and
the yield w~ 1.82 g.
9.9 g of the re~in obtained in this way wa~
sub~ected to HF clesvage a~ in AII. The freeze-dried
crude product was taken up in 2 1 of 0.1% strength acetic
acid, and the pH Wa8 then ad~usted to 8.4 with aqueou~
ammonia. Under an argon atmosphere, 0.01 N R3tFe(CN)~
~olution was ~lowly added dropwise until the yellowi~h-
green color per~isted for at least 15 min. The mix~ure
wa~ then stirred for 1 h and then acidified to p~ 4.5
with glacial ~cetic acid, and 15 ml of an aqueou~ ~u~pen-
~ion of an anion exchanger (BIORAD 3 x 4A, chloride form)
X~)0506~
- 19 - O.Z. 0050/40385 CA
were added. After 30 min, the ion exchanger re~in wa~
filtered off, and the filtrate was concentrated to 100 ml
in a rotary evaporator and subsequently freeze-dried.
All the solvents used had previously been satura
ted with nitrogen in order to prevent any oxida~ion of
the free cy~teine re~idues.
The crude product wa~ purified by gel chromato-
graphy (SEPHADEX~ G-15) and medium pressure chromato-
graphy (cf. AIV; 30-50~ A; 0.25~ minl)~ 65 mg of pure
product were obtained.
The following can be prepared in a similar manner
to Example 12 (Pam-resin wa~ u~ed for the preparation of
the peptide acid~):
13. H~ Lys-Gly-Gln-Gly-Thr-Pn~Ser-lhr-Hi~-Val-Leu~ oH
14. Ac~-Ly~-Gay ~ n-Gly-Thr-Pn~br-Thr-Hi~-Val-Leu~-N~
- i
15. E~}~:y-Ly~ly~ Gly-qbr-Pr~Ser~-His-VAl-Ieu{~s{~
, -- --
16. ~c-~y-Ly~Ly~y-~r-E~r~r-}~-V7.
r
17. ~-Ly~y~ lyJ~yr- ~ r-Thr~ Val-Leu-Hcy-CH
18. Ac~Ly~ay-Gan-Gly-Tyr-Pn~r~ Val-Leu-~cy-N~
19. H~ y~4Qy~ ly~r- ~ r~Ihr~-Val-Leu-H~y-off
20. Ac~H~y-~y8-Gly~ ayJTyr-Pn}~er~Thr~ Val-Leu-Hcy~
21. H~-Phe-~ys-&ly~ y-Ihr-Pn~br-$hr~-Val-Leu~-UH
22. Ac~ ~Lys-Gly~Gk~41y~1hr-P~br~Thri~-Val-Le~
23. H~H~ ~Lys-Gl~ ay-Ihr-Pn~br-lhr~ Val-Ieu~-oH
200~061.
- 20 - O . Z . 0050/40385 CA
_
24. Ac-E~-Phe-Ly8-Gly-Gln~ly-Thr-Pro~-q~r-Hig-Val-Ieu~y8-NH~
2 5 . H~ys-Ly 3~1y-Glll-Gly-Tyl:-P~Ser-mr-Hi8-V~ 1 -leu~l
26. Ac~ys-Ly3-Gly~ Gly-q~-P~-qhr-His-Val-Leu~
27. H-~y-Lys-Gly~ Gly-q~-Pro~-~lr-Hi8-Val-T~I
_. _
28. A~ L~-Gly-G~ y-Tyr-p~-q~-Hi~-va~
29. H~ Ly8-Gly-Gln~y~ -~S~-~-EI~-Val-I~Il-E~I
r - i
30 ~ Ly~3~Gly-Gln-Gly-T~?r-Pro~-q~ 8-Val-L~u-E~-N~z
31 . H-Hcy-Ly~3-Gly~lll-Gly-q~-P3:0-Ser~-~i5-Val-L~3ll-E~{~
_
32 . AC-E~ y-Ly8-Gly~l~y~ q~-Hi8-Val-Ieu-E~y-N~
33. Ac-H~y-Ly8~Ly~l-Ala-q~-P~S~-q~-His-V;~
34. AC~-Lyf3~1y~Tyr-Pr~P~-Hi8-V~31-I~aa-~:y-N~2
35. Ac~OE~Val-Ieu~-Ly~-Gay~G~ y~rhr-Pn~b¢-Thr-His-Val-
2S
.
36. ~c-Val~ H~y-Lys-Gly-Gln-Gly-Ihr-Pn~b¢JThr~ Val-Leu-
C?~-~H2
EXAMPLE 37
.
Ac-Glu-Gly-Gln-Gly-Thr-Pro-Ser-Thr-Hi~-V~l-Leu-Ly8 -NH2
1.38 g of Boc-Ly~(Cl-~)-MBHA-re~in (sub~t~tution
0.36 mmol/q), corre~ponding to a batch ~ize of 0.5 mmol,
ware roacted a~ in AIa (steps 14-16 were di~pensed with
200506~.
- 21 - ~.Z. 00S0/40385 CA
in all coupling~ following Hi~) with 2 mmol each of
Boc-Leu-OH Boc-Pro-OH Boc-Glu(OBzl)-OH
Boc-Val-OH Boc-Thr(Bzl)-O~
Boc-Hi (Z)-OH Boc-Gly-OH
5Boc-Thr(Bz~)-OH Boc-Gln-OH
Boc-Ser(Bzl)-OH Boc-Gly-OH
After the synthe~i~ was complete, the N terminu~
was acetylated (steps 1-5 and 14-16 a~ in AIa). The
resulting peptide-re~in was dried under reducsd pressure;
the yield waq 2 g.
The crude product (450 mq) obtained after HF
cleavage as in AII wa~ dissolved ~n 500 ml of degassed
DMF, and 210 mg of NaHCO3 and 660 mg of BOP were addQd.
ThQ mixture was stirred at room temperature for 6 days
and aub~equently evaporated ~o dryne~ he crude peptide
wa~ purified by gel chromatography lSEPHADEX~ LH 20) and
sub~equent medium pressure chromatography (cf. A IV,
40-60% A; 0.25S min~~). 117 mg of pure product were ob-
tained.
EXAMPL~ 38
--i
Ac-Gau-Lys-Gly~4~y~1hr-Pn~br'lhr~s~al-Le~-Ly~
1 g of re~in described by Breipohl et al. (from
BASHEM), correfiponding to ~ batch aize of 0.5 mmol, was
reacted a8 in AIb with 2 mmol each of
Fmoc-Lys(BQc)-OH Fmoc-Thr(t~u)-OH
Fmoc-Leu-OH Fmoc-Gly-OH
Fmoc-Val-O~ Fmoc-&ln-OH
Fmoc-His(Trt)-OH Fmoc-Gly-OH
Fmoc-Thr~tBu)-OH Fmoc-Ly~(Z)-OH
Fmoc-Ser(tBu)-OH Fmoc-Glu(OtBu)-OH
F~oc-Pro-OH
Z00506~.
- 22 - O.Z. 0050/40385 CA
After the synthe~i~ wa~ complete, the N terminus
wa~ acetylated (~teps 2-4 and 8-9 a~ in AIb). The pep-
tide resin was dried under reduced pressure; yield 1.8 g.
The crude product (~43 mg) obtained after TFA
5cleavage as in AIII was di~olved in 500 ml of degassed
DMF. After addition of 0.43 ml of triethylamine and (at
-25C) 0.43 ml of diphenylphosphoryl azide, the mixture
wa~ ~tirred at -25C for 2 h and ~ub~equently ~tored at -
25~C for 2 day~, at 4-C for 2 days and at room temp~ra-
10ture-for 2 day~. It was then evaporated to dryne~, and
the crude peptide wa~ purified by gel chromatography
(SEPHADEX~ LH 20).
Ths i~olated monomer (205 g) wa~ deprotected with
HF as in AII and purified by medium pressure chromato-
15graphy (cf. AIV 40-60% A; 0.25~ min~1). 78 mg of pure
product were obtained.
EXANPLE 39
i
H-~y~-Gly-Gln-Gly-Thr-Pro-Ser-Thr-His-Val-Leu-Glu-OH
202.9 g of Fmoc-~lu(OtBu3-Narrifield re~in (sub0ti-
tution about 0.34 mmol/g), corre~ponding to a batch 8ize
of 1.0 mmol, were re~cted a~ in AIb with 4 ~mol each of
Fmoc-Leu-O~ F~oc-Thr(Bzl) OH
Fmoc-Va1-0~ - Fmoc-Gly-OH
FmQC-Hi~ (TOB) -OH Fmoc-Gln-OH
Fmoc-Thr(Bzl)-OH Fmoc-Gly-OH
Fmo~-Pro-O~ Fmoc-Ly~(Boc)-OH
The ~-butyl and Boc protective group~ were then
cleaved off (step~ 1-6 as in AIa). The cycliz~tion en the
re~in took place in NMP with the addition of 1.77 g of
BOP and 1.74 ml of diisopropyl~thylamine (24 h). ~hs
peptide-re~in underwent ~tarminal deprotection (~t2p8
2-4 as i~ AIb) and drying under reduced pre~sure. The
2100S06~
- 23 - O.Z. 0050/40385 CA
yield wa~ 3.75 g. The crude product obtained after EF
cleavage as in AII wa~ purified by gel filtration
(Sephadex~ G-25) and medium pressure chromatography ~wice
(cf . AIV; ~0-40% A; 0.25% min1). 17 mg of pure product
were obtained.
The following can be prepared in a similar manner
to Examples 37,38 and 39:
40. Ac-Glu-Ly8~Gly~n~Gly-Thr-Pn~r-Thr-His-Val-Leu-Ly~-N~
41. Ac-&lu-Lys ~ y~n-Gly-lhr-kn~b~-Thr-Hi~-V l-Leu-Ly8-oH
42. Ac-Asp-Lys-Gly ~ n-Gly-Thr-Pn~br-Thr-~ val-Leu~-N~
43. H-Asp-Ly8-Gly-Gln-&ly-Thr-Pn}~br-Thr-Hi8-Val-T~u-orn-~
r
44. Ac-Ly8-Gly-Gl~-Gly-Thr-P~br-Thr-His-Val-Leu-Glu-~
45. Ac-Crn-Gay-Gln-GlyJThr-Pn~br-qhr~ Val-Leu-AsF-
~
46. H-orn-Çay~n-Gly-Thr-Pr~br~ ~-Val-Leu-h~H
-
47. Ac-Aa~-Gly-Gln-Gly~Thr- ~ r-Thr~-VAl-Leu-Lys-N~
I
48. Ac~lu-L~-Gly~l~Ly-lhr-Pro-P~lhr-}Ii8-V~ Ly~s-NH~
r
49. Ac-Ly~y~G~ Tyr-Pn~br~Thr~s-Val-Leu-Gau~N~
50. Ac-V~ R~Gly~ {iay~-Pro~-V2l1-Leu-
Aqp~r-His-N~
E~A~PL13 5 1
rLys-Gly-Gln-Gly-Thr-pro-ser-Thr-His-val-L~u-A
20050~i1.
- 24 - O.Z. 0050i40385 CA
1.22 g of Fmoc-Ly~(Z)-p-alkoxybenzyl alcohol-
resin (substitution 0.41 mmol/g), corresponding to a
batch size of 0.5 mmol, were reacted a~ in AIb with
2 mmol each of
S Fmoc-Aoc-OH Fmoc-Pro-OH
Fmoc-Leu-OH Fmoc-Thr(tBU)-OH
Fmoc-Val-OH Fmoc-Gly-OH
Fmoc-Hi~(Trt)-OH Fmoc-Gln-OH
Fmoc-Thr~tBu)-OH Fmoc-Gly-OH
Fmoc-Ser(tBu3-O~
After the ~ynthe~is wa~ complete, the peptide-
resin underwent N-terminal deprotection (~tep~ 2-4 as in
AIb) and subsequent drying under reduced pres6ure. The
yield wa~ 1.63 g.
The crud~ peptide obtained after TFA cleavage as
in AIII wa~ dis~olved in 300 ml of dega~sed DMF. 147 mg
of NaHCO3 and 462 mg of BOP were added and the mixture
was stirred at room temperature for 5 days. It was then
evaporated to dryne~s, and the crude peptide was purified
by gel chromstography (SEPHADEX LH 20). The isolated
monomer (105 mg) was deprotected with HF as in AII and
purified by medium pres~ure chrom~tography (cf. AIV; 35-
55% A; 0.25~ min~l). 44 mg of pure product were obtained.
The following can be prepared in a manner similar
to ~xample 51s
52. r Ly~-Gly-Gln-Gly-Thr-Pro-Ser-Thr-Hi~-Val-Leu-Ahp ~
53. Phe-~ys-Gly-Gln-Gly-Tyr-Pro-Ser-Thr-~is-Val-Leu-Abs
r
54- r Phe-Ly~-Gly-Gl~-Gly-Thr-Pro-Ser-~hr-His-Val-Leu-Ape
55 r Leu-Phe-Ly -Gly-Gln-Gly-Thr-Pro Ser-Thr-Hi -Val-Leu
~:005061.
- 25 - O.Z. 0050~40385 CA
56. r Lys-Gly-Gln-Gly-Thr-Pro-Ser-Thr-His-Val-Leu-Aoc
57.rLeu-Phe-Lys-Gly-Gln-Gly-Tyr-Pro-Pro-Thr-His-Val-Leu~
