Note: Descriptions are shown in the official language in which they were submitted.
~OQS;~l
O.Z. 0050/40383
NOVEL TNF PEPTIDES
The present invention relates to novel peptides derived
from tumor necrosi~ factor (TNF), the preparation thereof
and the use thereof as drugs.
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 strain of Mycobacteria (BCG) brought
about hemorrhagic necrosi~ in various mouse tumors. This
activity was ascribed to tumor necrosis factor. TNF also
has a cytostatic or cytotoxic effect on a large number of
tran~formed cell lines in vitro, wherea~ 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 gene for human
TNF have been described (Nature 312 (1984) 724; J. Biol.
Chem. 260 (1985) 2345; Nucl. Acid~ Res. 13 (1985) 6361).
It is possible to deduce from this data the following
protein structurs for mature human TNFs
V IAqO~ f ~ g-~--e~--~ysprovaL~sHisvalvaLuJA~o
ValGl--~ u ~valv~rpn~incluGly~o~r~aale~br
GlnV~L~ eLysGly~ yCy~o6e~n~f~JV~LL~ulhdOk~SrIle
S~gIleAlaVal~5DG~nirLysVa~ b~leLy~Pro
Cyd~nxq~uThrknx~uGlyA~uAlaLyu?~nrprpcluproIle~leu
GayG~yValPhoG~IK~luLysGlyAypb~euS3LU4~luIleA~YrgPn~sp
Tyri~4pPh~ u&~yGlnValTyrPh~yIleIle~k~eu
The TNF genes of cattle, rabbits and mice have also been
de~cribed (Cold Spring Harbor Symp. Quant. Biol. 51
(1936) 597~.
Besides its cytotoxic properties, TNF i~ one of the main
2(~0~2~1.
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subctances involved in inflammatory reaction-q (Pharmac.
R~ . 5 (1988) 129). AnLmal models have shown that TNF i8
involved in septic shock (Science 229 (1985) 869) and
graft-versus-ho~t disease (J. Exp. Med. 166 (1987) 1280).
we have now found that peptide~ with a con~iderably lower
molecular weight have beneficial propertie~.
The present invention relates to peptides of the
formula I
X-A-Asn-B-Y I,
where
A is Asp or Asn,
B i~ Gln or Ser,
X iY G-NH-CHM-CO-, G-NH-CHM-CO-W-, G-R-NH-CHN-CO- or
G-R-NH-CHM-CO-W- and5 Y i8 -Z, -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 i8 OH or NH2 or a carboxyl-protective group or
G and Z together are also a covalent bond or
-CO-(CH2),-NH-, where a i8 from 1 to 12,
R, U, V and W are peptide chains composQd of 1-4 natu-
rally occurring ~-amino acids and
N and Q aro hydrogens or one of the following groups
CH(CH3) 2, -CH~CH3)-C2H5, -C5H~, -CH(OH)-CH3,
--CH2~ CH2~ or --ICH2)b--T
(with b being from 1 to 6 and T being hydrogan or
OH, CH30, CH3S, (CH3)2CH, C6H5~ p-HO-C~H~, HS, H2N,
HO-CO, H2N-CO, H2N-C(=NH)-NH or
M and Q together are a -(CH2)c-S-S-(CH2) d- ~ - ( CHz).-CO-NH-
(CH2)r~ or -(CHZ).-NH-co-(cH2)s-NH-co-(cH2)r- 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),
Z005Z~l
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a well as the salt~ thereof with physiologically tolera-
ted acids.
The peptides of the formula I are constructed of L-amino
acids, but they can contain 1 or 2 D-amino acid~. The
side-chain3 of the trifunctional amino acids can carry
protective groups or be unprotected.
Particularly preferred physiologically tolerated acids
are: hydrochloric acid, citric acid, tartaric acid,
lactic acid, phosphoric acid, methanesulfonic acid,
acetic acid, formic acid, maleic acid, fumaric acid,
malic acid, succin~c acid, malonic acid, sulfuric acid,
L-glutamic acid, L-aspartic acid, pyruvic acid, mucic
acid, benzoic acid, glucuronic acid, oxalic acid, ascor-
bic acid and acetylglycine.
The novel paptides can be open-chain (G = H, amino-
protective group; Z = OH, NH2, carboxyl-protective group,
and Q not connected together) and, in particular, have
a disulfide bridge (G = H, amino-protective group;
Z = OK, NH2, carboxyl-protective group; M + Q = -(CH23c~
S-S~(CH2)d-) or a side-chain bridge (G = H, amino-protec-
tive group, Z = OH, NH2~ carboxyl-protective group, M + Q
5 - ( CH2 ) ~-NH-CO- (CH2)~- or -(CH2).-NH-CO-(CH2)s-N~-CO-
(CH2)~-) or be linked head-to-tail (G + Z = covalent bond
or -CO-(CH2),-NH-).
The novel compounds can be prepared by conventional
methods of peptide chemistry.
Thus, the peptides can be con~tructed sequentially from
a~ino acid~ or by linking together suitable smaller
peptide fragments. In the ~equential construction, the
peptide chain i8 extended stepwise, by one amino acid
each tlme, starting at the C terminus. In the ca~e of
fragments coupl;ng, it i8 possible to link together
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fragment~ of different lengths, these in turn being
obtainable by sequential con~truction from amino acid~ or
coupling of other fragments. The cyclic peptides are
obtained, after synthesic of the open-chain peptides, by
a cyclization reaction carried out in high dilution.
In the case both of sequential construction and of
fragment coupling, it i~ necessary for the building
blocks to be linked by formation of an amide linkage.
Enzymatic and chemical methods are suitable for this.
Chemical methods for forming amide linkages are dealt
with in detail by M~ller, Methoden der Organischen Chemie
(~ethods of Organic Chemistry) Vol. XV/2, pp 1-364,
Thieme Verlag, Stuttgart, 1974; Stewart, Young, Solid
Phase Peptide Synthesis, pp 31-34, 71-82, Pierce Chemical
Company, Rockford, 1984; Bodanszky, Rlausner, Ondetti,
Peptid~ Synthe0is, pp 85-128, John Wiley & Sons, New
York, 1976 and other standard works of peptide chemi~try.
Particularly preferred are the azide method, the symmetr-
ical and mixed anhydride method, active esters generated
in situ or preformed and the formation of amide linkages
using coupling reagents (activators), in particular
dicyclohexylcarbodiimide ~DCC), diisopropylcarbodiimide
(DIC), l-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
(EEDQ), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDCI), n-propanephosphonic anhydride
(PPA), N,N-bis(2-oxo-3-oxazolidinyl)amidophosphoryl
chloride (~OP-Cl), diphenylphosphoryl azide (DPPA),
Ca~tro's re~gont (BOP), O-benzotriazolyl-N,N,N~,N'-tetra-
methyluronium salts (HBTU), 2,5-diphenyl 2,3-dihydro-3-
oxo-4-hydroxythiophene dioxide (Stoglich~s reagent;
HOTDO) and l,l'-carbonyldiimidazole (CDI). The coupling
r~agents can be employed alone or in combination with
additive~ such a~ N,N~-dimethyl-4-aminopyridine (DMAP),
N-hydroxybenzotriazole (HOBt), N-hydroxybenzotriazine
(HOOBt), N-hydroxy~uccinimide (HOSu) or 2-hydroxy-
'~05'~8~.
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pyridine.
Whereas it is normally possible to dispense with protec-
tive groups in enzy~atic peptide synthesis, for chemical
synthe~is it is necessa~y for there to be reversible
protection of the reactive functional groups which are
not involved in the formation of the amide linkage on the
two reactants. Three conventional protective group
techniques are preferred for chemical peptide qynthQses:
the benzyloxycarbonyl (Z), the t-butyloxycarbonyl (~oc)
and the 9-fluorenylmethyloxycarbonyl (Fmoc) technique~.
In each case the protective group on the ~-amino group of
the chain-extending building block is identif ied. The
side-chain protactive groups on the trifunctional amino
acids are chosen 8e that they are not necessarLly elimin-
ated together with the ~-amino protective group. A
detailed review of amino acid protective groups is given
by Muller, Methoden der Organischen Chemie Vol XV/l,
pp 20-906, Thiemo Verlag, Stuttgart, 1974.
The building blocks used to construct the peptide chain
can be react2d in solution, in ~uspension or by a method
similar to that described by Merrifield in J. Amer. Chem.
Soc. 85 (1963) 2149. Particularly preferred methods are
tho~e in which peptides are constructed sequentially or
by fragment coupling by u3e of ths Z, Boc or Fmoc protec-
tive group technique, in which case the reaction takesplace in solution, as well as those in which, similar to
the Nerrifield technique, one reactant is bound to an
insoluble polymeric support ~also called resin herein-
after). Thi~ typically entails the peptide belng con-
structed sequentially on the polymeric support, by use ofthe Boc or Fmoc protective group technique, with the
growing pQptide chain being covalently bonded at the
C terminus to tha insoluble resin particles (cf. Figures
1 and 2). This procedure allows reagents and byproducts
to be removed by filtration, and thus recry~tallization
~005~81.
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of intermediates i-~ superfluous.
The protected amino acids can be bonded to any ~uitable
polymers which merely need to be insoluble in the sol-
vent~ used and to have a 3table 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 i3 suitable for this purpose, for example
cellulose, polyvinyl alcohol, polymethacrylate, sulfon-
ated poly~tyrene, chloromethylated copolymer of styreneand divinylbenzene (Merrifield resin), 4-methylbenz-
hydrylamine-xesin (MBHA-resin), phenylacetamidomethyl-
resin (Pam-resin), p-benzyloxybenzyl alcohol-resin,
benzhydrylamine-resin (BHA-resin), 4-hydroxymethyl-
benzoyloxymethyl-resin, the re~in u~ed by Breipohl et al.
(Tetrahedron Lett. 28 (1987) 565; from BACHEM), HYCRAM
re~in (from ORPEGEN) or SASRIN resin (from BACHEM).
Solvents suitable for peptide synthesis in solution are
all tho~e which are inert under the reaction conditions,
in particular water, N,N-dimethylformamide (DMF),
dimethyl sulfoxidQ (DNS0), acetonitrile, dichloromethane
(DC~), 1,4-dioxane, tetrahydrofuran (lnr), N-methyl-2-
pyrrolidone ~N~P) and mixture~ of the said solvents.
Peptide syntheai3 on polymeric supports can be carried
out in all inert organic solvent~ which dissclve the
amino acid derivatives used; however, solvents which also
have resin-swelling properties are preferred, such as
DMF, DCN, NNP, acetonitrile and DMS0, as well as mixtures
of the~e solvents.
After the peptide has been synthesized it i8 cleaved off
the polymeric support. The cleavage conditions for the
various types of resins are disclosed in the literature.
The cleavage reactions most commonly use acid and
palladium catalysis, in particular cleavagQ in anhydrous
2 0 0 S ~
- 7 - O.Z. 0050/40383
liquid hydrogen fluoride, in anhydrous trifluoromethane-
sulfonic acid or in dilute or concentrated trifluoro-
acetic acid, or palladium-catalyzed cleavage in THF or
THF-DCM mixtures in the presence of a weak base ~uch as
morpholine. The protective groups may, depending on the
choice thereof, be retained or likewi~e cleaved off under
the cleavage conditions. Partial deprotection of the
peptide may also be worthwhile if the intention is to
carry out certain derivatization reactions or a
cyclization.
Some of the novel peptides have good cytotoxic proper-
ties. Some others of the peptide~ have high affinity for
the cellular TNF receptor without, however, having
cytotoxic activity. They are therefore TNF antagonists.
They compete with natural TNF for binding to the cellular
TNF receptor and thus suppress the TNF effect. The novel
peptides are valuable drugs which can be employed for
treating neoplastic diseases and autoimmune diseases a~
well as for controlling and preventing infections,
inflammations and tran~plant re~ection reactions. Simple
experLments can be used to elucidate the mode of action
of the individual peptides. The cytotoxicity of the
peptide i8 determined by incubating a TNF-sensitive cell
line in the pre~ence of the peptide. In a second experi~
mental approach, the cell line iB incubated with the
relevant peptide in the presence of a lethal amount of
TNF. It is possible in this way to detect the
TNF-antagoni~tic effect. In addltion, the affinity of the
psptide for the cellular TNF receptor is determined in an
in vitro binding experiment.
The following te~t ~y~t~ms were used to characterize the
agoni~tic and antagonistic effects of the novel peptides:
I. Cytotoxicity test on TNF-~ensitive indicator cells,
II. Cytotoxicity antagonism test on TNF-sensitive
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indicator cells,
III. Competitive receptor-binding test on indicator cells
expressing TNF receptor.
I. Cytotoxicity test
The agonistic effect~ of the novel peptide~ are
assessed on the basis of their cytotoxic effect on
TNF-sQnsitive cell~ (e.g. L929, MCF-7, A204, U937).
The test with L929 and MCF-7 was carried out as
follow8 5
1. 100 ~1 of culture medium containing 3 to 5 x 103
freshly tryp~inized, exponentially growing, L929
cell~ (mouse) or MCF-7 cells (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 was ~aturated
with water vapor and contained 5% CO2 by volume.
The L929 culture medium contained 500 ml of lx
Earle~s M~M (Boehringer Mannheim), 50 ml of heat-
inactivated (56C, 30 min) fetal calf serum
(FC5), 50 ml of L-glutamine (200 mM), 5 ml of
lOOx non-es~ential amino acids, 3 ml of 1~ HEP~S
buffer pH 7.2,and 50 ml of gentamicin (50 mg/ml).
The NCF-7 culture medium contained 500 ml of lx
Dulbecco~s ME~ (Boehringer Mannheim), 100 ml of
heat inactivated (56-C, 30 min) FCS, 5 ml of
~-glutamine and 5 ml of lOOx non-essential amino
acids ~
2. The next day 100 ~1 of the peptide solution to be
te~t~d ware added to the cQll culturQs and
sub~ected to serial 2-fold dilution. In addition,
qom~ cell controls (i. Q . CQll culture~ not
treated with peptide dilution) and some rhu-TNF
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~ 9 - O.Z. 0050/40383
controls (i.e. cell cultures treated with recom-
binant human TNF) were also made up. The culture
plate waB incubated at 37C in an atmosphera of
air saturated with water vapor and containing 5%
C02 by volume for 48 h.
3. The percentage of surviving cells in the cultures
treated with peptide dilution was determinad by
staining with crystal violet. For this purposQ,
the liquid was removed from the wells 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
was as followss
3.75 g of cry~tal violet
lS 1.75 g of NaC1
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 likewise removed by tapping.
~he plate~ were then wa~hed 5 times by immersion
in water in order to remove dye not bound to the
cell~. The dye bound to the cells was e~tracted
by adding 100 ~1 of reagent solution (504 eths-
nol, 0.1% glacial acetic acid, 49.9~ water) to
each well.
4. Th~ plates were shaken for 5 min to obtain a
solution of uniform color in each well. The
surviving cells were determined by measuring the
ext~nction at 540 nm of the colored ~olution in
the individual well~.
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5. Subs~quently, by relating to the cell control,
the 50% cytotoxicity value was defined, and the
reciprocal of the ~ample dilution which resulted
in 50~ cytotoxicity was calculated as the cyto-
toxic activity of the te~t sample.
II. Cytotoxieity antagoniqm test
The antagonistic effect of the peptides was a~se~ed
on the basis of their property of antagonizing the
cytotoxie effeet of rhu-TNF on TNF-sensitive cells
(~.q. L929, MCF-7, A204, U937). Th~ cytotoxicity
antagonism teat with L929 and MCF-7 eells was
earried out as follow~t
1. 100 ~1 of eulture medium eontaining 3 to 5 x 103
freshly trypsinized, exponentially growing, L929
cells (mou~e) or MCF-7 eell~ (human) were
pipatted in~o the wells of a 96-well flat-bottom
eulture plate. The plate was ineubated at 37C
overnight. The air in the ineubator was saturated
with water vapor ~nd eontained 5% CO2 by volume.
The L929 elllture medium eontained 500 ml of lx
Earle's M~M (Boehringer Mannheim), 50 ml of heat-
inaetivated (56C, 30 min) PCS, 5 ml of L-gluta-
mine (200 m~), 5 ml of lOOx non-es~ential amino
aeids, 3 ml of lM HEPES buffer pH 7.2, and 500 ~1
of gentamiein (50 m~/ml).
The NCF-7 eulture medium eontained 500 ml of lx
Dulbeeeo~s NEM (Boehringer Mannheim), 100 ml of
heat inaetivated (56C, 30 min) FCS, 5 ml of
L-glutamine (200 mM) and S ml of lOOx non-essen-
tial amino aeids.
2. ~he next day 100 ~1 of the peptide solution to be
teEted were added to the cell eulture~ and
sub~eet2d to ser~al 2-fold dilution. Then, 100 ~1
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~ O.Z. 0050/40383
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 addition, some
cell controls (i.e. cell culture~ not treated
with peptide solution or with rhu-TNF solution)
and some rhu-TNF controls (= cell cultures
treated only with rhu-TNF solution) were also
made up. The culture plate was then incubated at
37C in an atmosphere of air saturated with water
vapor and containing 5% C02 by volume for 48 h.
3. The percentage of surviving cells in the culture~
treated with sub~tance solution was determined by
~taining with crystal violet. 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 crystal violet solution had the composition
specifi~d in I.3
The crystal violet solution was left in the well~
for 20 min and then likewise removed by tapping.
The plate~ were then washed 5 times by immersion
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 reagsnt solution ~50% etha-
nol, 0.1~ glacial acetic acid, 49.9% water) to
each well.
4. The plates were shaken for 5 min to obtain a
301ution of uniform color in each well. The
sur~iving cells were determined by measuring the
extinction at 540 nm of the colored solution in
the individual wells.
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- 12 - O.Z. 0050/~0383
5. Subsequently, by relating tO the cell control and
the rhu-TNF control, the 50~ antagonism value was
defined, and the ~ample concentration which
regulted in 50~ antagonism of rhu-TNF cytotox-
icity at the rhu-TNF concentration used was
calculated as antagonistic activity of the sample
te~ted.
III. Competitive receptor-binding test
Both the agonistic and antagonistic effect~ of
peptides are conditional on the iatter binding to
the TNF receptor. Thi~ means that peptides with an
agonistic or antagonistic effect compete with
rhu-TNF for binding to the TNF receptor on TNF-
sensitive indicator cells (e.g. U937). The competi-
tive receptor-binding te~t was carried out as
followss
1. 100 ~1 of medium containing various concentra-
tions of the peptide to be te~ted and of rhu-TNF
(= control) were pipatted into the reaction
ve~sels. The medium comprised 500 ml of PBS
(Boehringer Mannheim), 10 ml of heat-inactivated
(56UC, 30 min) FCS and 100 mg of sodium a2~ide.
2. Subsequently, 100 ~1 of medium containing 1 ng of
1Z5I-labsled rhu-TNF (Bolton lactoperoxida~e
method) were placed in the reaction vessels and
mixed. The non-specific b~nding (NSB) was deter-
mined by mixing in the reaction vessel~ the
125I-laboled rhu-TNF (1 ng of125I-rhu-TNF in 100 ~1
of medium) with a 200-fold excess of unlabaled
rhu-TNF ~200 ng of rhu-TNF in 100 ~1 of medium)~
3. Then 100 ~1 of medium contsining 2 x loB U937
cells (human) wsre pipetted into the reaction
ve~8el8 and mixed. The reaction ves~al~ (test
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volume 300 ~1) were incubated at 0C for 90 min.
The reaction mixture~ were remixed after 45 min.
4. After the incubation, the cell~ were centrifuged
at 1800 rpm and 4C for 5 min, wa~hed 3 times
with medium and tran~ferred quantitatively into
cou~ting vials, and the cell-bound radioactivity
was determined in a Clini gamma counter 1272
(LRB Wallac).
5. After the measurement~ had been corracted for the
non-~pecific binding, the 50~ competition value
was defined by relating to the overall binding,
and the sample concentration which led to 50%
competition ofl25I-rhu-TNF binding at the125I-rhu-
TNF concentration used wa~ calculated as the
competitive activity of the sample tested.
The Examples which follow ar~ intended to explain the
invention in more detail. The proteinogenous amino acids
are abbreviated in the Examples u~ing the conventional
three-letter code. Other meanings ares
Abs = 4-æminobutyric acid, Ac = acetic acid,
Ahp - 7-aminoheptanoic acid, Ahx = 6-aminohexanoic acid,
Bal = ~-alanine, Hcy = homocysteine, Hly = homolysine,
Orn - ornithine.
A. General procedures
I. The peptides claimed in claim 1 were synthesized
u~ing standard methods of solid-phase peptide
synthe~is in a completely automatic model 430A
peptide synthesizer from APP~IED BIOSYSTEMS. The
apparatus use~ different ~ynthesis cycles for the
Boc and Fmoc protective group techniques.
a) Synthesis cycle for th~ Boc protective group
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- 14 - O.Z. 0050/40383
technique
1. 30% trifluornacetic acid in DCM 1 x 3 min
2. 50% trifluoroacetic acid in DCM1 x 17 min
3. DCM washing 5 x l min
4. 5% dilsoprcpylethylamine in DCM l x 1 min
5. 5% di~3aprcpylethylamine in NMP 1 x 1 min
6. NMæ wzshing 5 x 1 min
7. A~dition of preactivated pGctocted amino
acid (activation by 1 equival~L of DCC
and 1 Q~ivalent of HOBt in NMP/DCM);
peptide coupling (lst part) 1 x 30 min
8. A~dition of DMSO to the reacticn nixturs
u~rtil it ccntains 20% ~90 }~y vellm~
9. Peptide co~pling (2~ part) 1 x 16 min
10. A~iti~ of 3.8 ffa!liV~ of ~ii~
p~letl~ylan~ to tho ~n nL~
11. P~e ca~pling (3rd part) ~ x 7 min
12. DCM washirbg 3 x 1 min
13. If reactian i~ i~lote, rE~etiticsn
of ca~lir~ (retu~n to 5. )
14. 10% acetic ar~ride, 5% dii~pyl-
~ ylamine in DCM 1 x 2 min
15. 10S acetic ar~ydride in DC~ 1 x 4 min
16. DCM wa~hing 4 x 1 min
17 Reburn to 1.
b) SyntheBiR cycle for the Fmoc protective group techndquo
1. ~IP wa~ 1 x 1 min
2. 20~ piperidine im NMP 1 x 4 min
3. 20% piperidine in N~P 1 x 16 n~n
4. N~P w ~ g 5 x 1 m~n
5. A~dition of preactivated proeeltod amino
acid (activatian ky 1 eq~ivalent of DOC
and 1 equivalent of HLBt in NMP/DCM);
~ e ooupling 1 x 61 n~n
6. NNP washing 3 x 1 min
7. I~ reacti~n i8 inocmple~e, repekiti~n of
Z ~ 0 S~ 8~
- 15 -O.Z. OOS0/4~383
o~ling (return to 5.~
8. 10~ ic ~ride in NMP1 x 8 min
9. NMP washing 3 x 1 min
10. R~l~n to 2.
II. Working up of peptide-resins obtained as in Ia
The peptide-resin obtained as in Ia wa~ dried under
reduced pressure and transferred into a reaction
vessel of a Teflon HF apparatus (from PENIN~ULA).
Addition of a scavenger, preferably anisole (1 ml/g
ld of resin), and of a thiol in the case of tryptophan-
containing peptides, to remove the indole formyl
group, preferably ethanedithiol (O.S ml/g of resin),
was followed by condensation in of hydrogen fluoride
(10 ml/g of resin) while cooling with liquid N2. The
lS mixture wa8 allowed to warm to O-C, and was stirred
at this temperature for 45 min. The hydrogen fluor-
ide was then ~tripped off under reduced pressure and
the residue was wa~hed with ethyl acetate in order
to removs 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 ~errifield resin) was suspended in DME
(15 ml/g of resin), hydrazine hydrate (20 equiva-
~5 lents) w~s added, and the mixture was stirred at
room temperature for 2 days. To work up, the resin
wa~ filtered off and the filtrate was evaporated to
dryness. The re~idue was crystallized from DME/Et20
or Me0H/Et20~
III. Working up of the peptide-resins obtained as in Ib
The peptide-resin obtained as in Ib was dried under
reduced pressure and subsequQntly sub~ectad to one
~005~81.
- 16 - O.Z. 0050/40383
of the following cleavage procedures, depending on
the amino acid composition (Wade, Tregear, Howard
Florey Fmoc-Workshop Manual, Melbourne 1985).
Peptide containing Cleavage conditionY
__ _ _
Arg(Mtr~ Met Trp TFA Scavenger Reaction
tLme
. . _ _
no no no 95% 5% H2O 1.5 h
yes no no 95% 5% thioani~ole 2 3 h
no yes no 95% 5% ethyl methyl 1.5 h
sulfide
no no yes 95% 5% ethanedithiol/ 1.5 h
anisols (1:3)
no yes yas 95~ 5~ ethanedithiol/ 1.5 h
anisole/ethyl methyl
sulfide (1:3sl)
yes yes yes 93% 7% ethanedithiol/ 2 3 h
anisole/ethyl methyl
sulfide (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 re~in was 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
tha precipitate wa~ filtered off, taken up in 30%
acetic acid and freeze-dried.
IV. Purification and characterization of the peptides
Purification was by gel chromatography (SEPHADEX~
G-10, G-15/10% HQAc; SEPHADEX~ LH20/MeOH) and sub-
Z~O~X81
- 17 - O.Z. 0050/40383
~equent medium pressure chromatography (~tationary
pha~e: HD-SIL C-18, 20-45 ~, 100 A; mobile phases
gradient with A = 0.1% TFA/MeOH, B = 0.1% TFAtH2o).
The purity of the final products was determined by
analytical HPLC (~tationary phase: 100 x 2.1 mm
YYDAC C-18, 5 ~, 300 A; mobile phase = CH3CN/HzO
gradient buffered with 0.1% TFA, 40C). Charac-
terization was by means of amino acid analy~i~ and
fast atom bombardment ma~s spectrometry.
B. Specific procedures
EXANPLE 1
H-Arg-A8p-A~n-Gln-Leu-NH2
1.1 g of Boc-Leu-p-MBHA-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-Gln-OH
Boc-A~p(OChx)-OH
Boc-Asn-OH
Boc-Arg(Tos)-OH
After the ~ynthe~is was complete, ths peptide-resin
underwent N-terminal deprotection (steps 1-3 as in AIa)
and subsequent drying under reduced pre~sure; the yield
was 1.45 g.
0.73 g of the resin obtained in this way was sub~ected to
HF cleavage a~ in AII. The crude product (125 g) was
purified by gel filtration (SEPHADEX G-10) and medium
pressure chromatography (cf. ~IV; 10-25 ~ A; 0.25 %
min~1). 89 mg of pure product wsrQ obtained.
- 18 -
~ 5æI~ a 2005~1
Ac~eu~ A8p-~n~ ln~u-0~
3.43 g o~ u-p-alkox~an~yl a~ohol-~e~ln (suhs~i-
~u~on ~.5g mms:~l/g~ ~orresponding ~o a batch ~i~e ~f
S 0.25 ~nol, wa~ ~eaotsd a~ in ~Ib with 1 mmol each of
Fmoc--t ;ln-OK E~oc -Arg ( Mtr ) -O~
Fmoc-A4n-O~ Fmoc-L~u~
Fmoc~ 0t~u ) -0~1
Aft~sr the ~ he~i~ w~ co~ te, the N te~minu~ waQ
ac~tyl~ted ~te~s ~-J, ~d 8-9 as in AIb)~ T~e re~ultir~g
~eptide-re~in wa~ d uz~Ldar ~educed pressure; the yielci
wa8 0-6 ~T-
~Q c:rude ~ptLdo ~143 ~g) sbtained after ~FA cleavage as
in AIII wa~ pu~ ed ~y ~el ~iltrat~on (5~P~E~ 10)
and m~diu3n pre~ure ch~omatography (ef. AIV; 15-25 ~ At
O.25 96 m~n ~j . 107 mg o~ pure product were obtained~
~h~ ~ollow~n~ e ~rQp~r~ Ln a ~nilar ~a~uter to
~x~nples 1 and 2
*~ GESRI`lTSE I TEN 0132 `~"
Z005~81.
- 19 - O.Z. 0050/40383
3. H-Arg-Asp-Asn-Gln-Leu-OH
4. Ac-Arg-Asp-Asn-Gln-Leu-OH
5. Ac-Arg-Asp-Asn-Gln-Leu-NH2
6. H-Leu-Ar9-Asp-Asn-Gln-Leu-oH
7. ~-Leu-Arg-ASp-ASn-Gln-Leu-NH2
8. Ac-Leu-Arg-Asp-Asn-Gln-Leu-NH2
9. H-Glu-Leu-Arg-Asp-Asn-Gln-Leu-OH
10. Ac-Glu-Leu-Arg-Asp-Asn-G1n-Leu-OH
Il. H-Glu-Leu-Arg-Asp-Asn-Gln-Leu-NH2
12. Ac-Glu-Leu-Arg-Asp-Asn-Gln-Leu-~H2
13. H-Glu-Leu-Arg-Asp-Asn-Gln-Leu-Val-OH
14. Ac-Glu-Leu-Arg-Asp-Asn-Gln-Leu-Val-NH2
15. ~-Val-Glu-~eu-Arg-Asp-Asn-Gln-Leu-Val-OH
16. Ac-Val-Glu-Leu-Arg-Asp-Asn-Gln-Leu-Val-NH2
17. H-Gly-Val-Glu-Leu-lrg-Asp-~Sn-Gln-L~u-Val-Val-OH
18. Ac-Gly-Val-Glu-Leu-Arg-Asp-Asn-Gln-Leu-Val-Val-NH2
19. Ac-Leu-Arg-Asn-Asn-G~n-Leu-NH2
20. Ac-Glu-Leu-Arg-~sp-Asn-Ser-Leu-NH2
21. ~-Val-Glu-L~u-~rg-~sn-Asn-Gln-Lou-Val-OH
22. H-61y-~hr-Glu-Leu-~rg-Asp-Asn-Gln-Leu-Val-Val-OH
EX~PL~ 23
Ac -Cys -Arg-A~p-Asn-Gln-Cys -NH2
1.16 g of Boc-Cys (pMB) -p-MBHA-resin ( substitution
0 . 43 mmol~g), corresponding to a batch size of 0 . 5 mmol
were reacted as in AIa with 2 mmol each of
Boc-Gln-OH Boc-Arg ( Tos ) -OH
Boc-Asn-OH Boc-Cys ( pMB ) -OH
Boc-Asp ( OChx) -OH
The resulting peptide-resin was dried under reduced
pressure; the yield was 1.45 g.
0.73 g of the resin obtained in this way was ~ub~ected to
HF cleavaga as in AII. The freeze-dried crude product wa~
talcen up in 2 1 of 0.195 strength acetic acid, and the pH
wa~ then ad~usted to 8 . 4 with aqueou~ ammonia . Under an
ZOO~
- 20 - O.Z. 0050/40383
argon atmosphere, 0.01 N K3rFe(CN)6] solution was slowly
added dropwise until the yellowish-green color per~i~ted
for at lea~t 15 min. The mixture was then stirred for 1 h
and then acidified to p~ 4.5 with glacial acetic acid,
and 15 ml of an aqueou~ ~u~pen~ion of an anion exchanger
(BIORAD~ 3 x 4A, chloride form) were added. After 30 min,
the ion exchanger resin was filtered off, and the filt-
rate was concentrated to 100 ml in a rotary evaporator
and subsequently freeze-dried.
All the solvent~ used had previously been saturated with
nitrogan in order to prevent any oxidation of the free
cysteine residues.
The crude product was purified by gel chromatography
(SEPHADEX G-15) and medium pressure chromatography (cf.
AIV; 10-30 % A; 0.25 ~ min1). 71 mg of pure product were
obtained.
The following can be prepared in manner similar to
Example 23 (Pam-recin was used to prepare the peptide
acid~)s
24. H-Cys-AFg-Asp-Asn-GIn-Cjs-OH
25. Ac-Cys-Arg-Asp-Asn-Gln-Cys-OH
26. AC-CyS-Arg-O-Asp-Asn-Gln-Cjs-NH2
27. H-cys-Ar9-Asp-Asn-Gln-cys-NH2
28. Ac-CjS-Glù-ASp-ASn-Gln-CyS-NH2
29. H-Cys-Glu-Asp-Asn-Gln-Cys-OH
30. H-Cys-Thr-Asp-Asn-Gln-Cys-OH
31. Ac-Cys-Thr-Asp-Asn-Gln-Cys-NH2
;~OSZ~l.
- 21 - O.Z. 005~/40383
32. H-Cys-Ly5-A5p-Asn-Gln-Cj5-OH
r
33. Ac-Cys-Lys-Asp-Asn-Gln-Cy5-NH2
34. H-Cys-Ser-Asp-Asn-Gln-Cys-OH
35. Ac-Cys-Ser-Asp-Asn-Gln-Cys-NH2
36. Ac-Hcy-Arg-Asp-Asn-Gln-Cys-NH2
37. Ac-Cys-Arg-Asp-Asn-Gln-Hcy-NH2
38. Ac-Hcy-Arg-Asp-Asn-Gln-Hcy-NH2
39. Ac-Cys-Leu-Arg-~sp-Asn-Gln-Cys-NH2
40. Ac-Hcy-Leu-Arg-Asp-Asn-Gln-Cys-NH2
41. Ac-Cy5-Arg-A5p-Asn-Gln-Leu-Cjs-NH2
42. Ac-Hcy-Arg-Asp-Asn-Gln-Leu-Cjs-NH2
43. H-Cys-Leu-Arg-Asp-Asn-Gln-Leu-Cys-OH
44. Ac-Cys-Leu-Arg-Asp-Asn-Gln-Leu-Cys-OH
45. H-Cy5-Leu-Arg-Asp-~sn-Gln-Leu-Cys-NH2
46. Ac-Cys-Leu-Arg-Asp-Asn-Gln-Leu-Cys-NH2
47. Ac-Hcy-Leu-~rg-Asp-~sn-Gln-Leu-Cys-NH2
48. Ac-CyS-Leu-Arg-Asp-Asn-GI n-Leu-Hcy-NH2
49. Ac-Hcy -Leu-Arg-Asp-ASn-Gln-Leu-Hcy-NH2
50. H-Cys-Arg-Asp-Asn-Gln-Leu-Cys-NHz
51. Ac-Cy5-Arg-Asn-Asn-Gln-Cys--~H2
52. Ac-Cys-Arg-D-Asp-Asn-Gln-CyS-NH2
53. Ac-Val-Glu-Cjs-Arg-Asp-Asn-Gln-Cjs-Val-Val-Pro-NH2
54. Ac-Cys-Lys-Asp-Asn-Ser-Cys-NH2
55. Ac-Asn-Gly-Yal-Cys-Leu-Arg-Asp-Asn-Gln-Leu-CjS-Val-Pro-NH2
OS~81
- 22 - O.Z. 0050/40383
EXAMPLE 56
Ac-Glu-Arg-Asn-Asn-Gln-Ly~-NH2
1.16 g of Boc-Lys(Cl-Z)-MBHA-resin (substitution 0.43
mmol/g), corre~ponding to a batch size of 0.5 mmol, were
reacted as in AIa with 2 mmol each of
Boc-Gln-OH Boc-Arg( TOB ) -OH
Boc-Asn-OH Boc-Glu(OBzl)-OH
Boc-Asn-OH
After the synthesi~ was complete, the N terminus was
acetylated (steps 1-6 and 14-16 as in AIa). The resulting
peptide-re~in was dried under reduced pressure; the yield
was 1.6 g.
~ he crudo product (317 mg) obtained after HF cleavage a~
in AII was dissolved in 500 ml of degassed DNF, and
0.2 ml of triethylamine and (at -25C) 0.20 ml of di-
phenylphosphoryl azide were added. The mixture was
stirred at -25C for 2 h, ~tored at -20C for 2 ~ays~ at
4C for 2 days and at room tamperature for 2 day~ and
~ubsequently evaporated to drynes~. The crude peptide was
purified by gel chromatography (SEPHADEX LH 20) and
medium pre~ure chromatography (cf. AIV; 10-25% A;
0.25% min~l). 101 mg of pure product were obtained.
Examplo 57
Ac-Orn-Arg-Asp-Asn-Gln-ABp-NH2
2.5 g of reQin de~cribed by Breipohl et al. (from
BACHEM), corresponding to a bstch size of 1.25 mmol, was
reacted a~ in AIb with 5 mmol each of
200$Z~
- 23 - O.Z. 0050/40383
Fmoc-A8p(Ot~u)-OH Fmoc-Asp(OChx)-OH
Fmoc-Gln-OH Fmoc-Arg(~os)-OH
Fmoc-Asn-OH Fmoc-Orn(Boc)-OH
After the synthesis was complete, the N terminus was
acetylated (step~ 2-4 and 8-9 as in AIb). The peptide-
resin was dried under reduced pressure; yield 1.4 g.
The crude product (1.22 g) obtained after TFA cleavage as
in AIII was purified as in AIV (414 mg). 200 mg were
dissolved in 250 ml of degassed DMF. 0.24 ml of NEt3 and,
at -25C, 0.24 ml of diphenylphosphoryl azide were added
and then the mixture was stirred at -25C for 2 h. It was
subsequently stored at -20C for 2 days, at 4C for 2
days and at room temperature for 2 day~. It was then
evaporated to dryness, and ths crude peptide was purified
by gel chromatography (SEPHADEX LH 20). The i~olated
monomer (143 mg) was deprotected with HF as in AII and
purified by medium pressure chromatography (cf. AIV;
5-25~ A; 0.25% min1). 89 mg of pure prod~ct werQ
obtained.
~xample 58
Ac-Glu-Leu-Arg-Asp-A~n-Gln-Leu-Lys-OH
3.2 g of Fmoc-Lys(Boc)-Merrifield resin (substitution
0.38 mmol/g), corresponding to a batch si~Q of 1.0 mmol,
were reacted as in AIb with 4 mmol each of
Fmoc-Leu-OH Fmoc-Arg(To~)-OH
Fmoc-Gln-OH Fmoc-Leu-OH
Fmoc-Asn-OH Fmoc-Glu(OtBu)-OH
Fmoc-Asp(OChx)-OH
Subsequently N-terminal deprotection and acetylation
(step~ 2-4 and 8-9 as in AIb) were carried out, and the
X~OS~81.
- 24 - O.Z. 0050/40383
t-butyl and Boc protective groups were cleaved off (~teps
1-6 as in AIa). The cycliz~tion on thr re~in took place
in NMP with addition of 1.77 g of BOP and 1.74 ml of
diisopropylethylamin~ (40 h). The peptide-resin was dried
under reduced pressure. Thr yield was 3.g5 g. The crude
product obtained after HF cleavage a~ in AII was purified
by gel filtration (Sephadex G-15) and medium pre 3ure
chromatography twice (cf. AIV; 5-25% A; 0.25% min~').
17 mg of pure product were obtained.
The following can be prepared in a sLmilar manner to
Examples 56, 57 and 58:
59. Ac-GIu-Asp-Asn-GIn-LyS-NH2
60. bc-GIu-Arg-Asp-Asn-GIn-Lys-NH2
61. Ac-Asp-Arg-Asp-Asn-GIn-Lys-NH2
62. H-ASp-Arg-Asp-Asn-GIn-Ljs-NH2
63. Ac-Asp-Arg-Asp-ASn-Gln-Orn-NH2
64 . AC -Lys-Arg-Asp-Asn-Gln-G~u-OH
65. H-Lys-Arg-Asp-Asn-Gln-Glu-OH
66. Ac-G~u-Arg-Asp-Asn-Gln-Hty-NH2
67. H-0rn-Arg-A5p-ASn-GlU-NH2
68. Ac-Giu-Asp-Asn-Ser-Lyjs-NH2
69. Ac-Lys-Arg-Asp-Asn-Gln-Asp-NH2
70. Ac-Lys-Arg-Asp-Asn-Gtn-~sp-OH
71. Ac-Glu-Arg-Asp-Asn-Gln-LjS-OH
72. H-G~u-~rg-Asp-Asn-Gln-Ljs-OH
73. Ac Lys-Arg-A5p-~sn-Gln-Glu-N~2
Z0052~
- 25 - O.Z. 0050/40383
74. Ac-Glu-Leu-Arg-Asp-Asn-Gln-Leu-Lys-OH
75. Ac-Glu-Arg-Asp-Asn-Gln-Leu-Lys-NH2
~6. Ac-Glu-Arg-Asp-Asn-Gln-Leu-Orn-OH
1-- ~
77. Ac-Orn-Arg-Asp-Asn-Gln-Leu-Asp-NH2
78. Ac-Glu-Leu-Arg-Asp-Asn-Gln-Leu-Lys-NH2
79. Ac-G~u-Leu-Arg-Asp-A5n-Gln-Leu-Hiy-NH2
80. Ac-Asp-Leu-Arg-Asp-Asn-Gln-Leu-Lys-NH2
81. Ac-Asp-Leu-Arg-Asp-Asn-Gln-Leu-Orn-NH2
82 . AC -Orn-Lcu-Arg-Asp-Asn-61n-~eu-Asp-NH2
83. Ac-Lys-L~u-Arg-Asp-Asn-Gln-Leu-Asp-NH2
84. Ac-Lys-Leu-Arg-Asp-Asn-Gln-Leu-Asp-OH
85. Ac-Lys-Leu-Arg-Asp-Asn-Gln-Leu-Glu-NH2
86. Ac-Glu-Asp-Asn-61n-Leu-Ljs-NH2
87. ~-G1u-Asp-~sn-Gln-Leu-Lys-OH
88. Ac-Lys-Asp-Asn-Gln-Leu-G u-NHz
r
89. Ac-Gly-Val-61u-Orn-~rg-Asp-~sn-Gln-~sp-Val-Val-NH2
90. ~c-Asp-~5-Asp-~n-Gln-L~s-NH2
1. ~c-Gtu-Leu-~rg-D-~sp-~sn-Gln-L~u-Lys-Nt~2
2. ~c-~n-~ly-Val-Orn-Leu-~rg-~sp-~sn-Gln-Leu-~sp-Val-Pro-NH
~0~
- 26 - O.~. 0050/40383
Exampl~ 93
IBal-Arg-Asn-Asn-Gln-Leul
0.56 ~ of Boc-Leu-Merrifield re~in (Yubstitution
0.9 mmol/g), corre~ponding to a batch size of 0.5 mmol,
wa~ reacted as in AIa with 2 mmol each of
Boc-Gln-OH Boc-Arg(To~)-OH
Boc-Asn-OH Boc-Bal-OH
Boc-Asn-OH
After the synthesis was complete, the peptide-resin
underwent N-terminal deprotection (steps 1-3 as in AIa)
and subsequent dryinq under reduced pressure. The yield
was 0.85 g.
The crude product (267 mg) obtained after HP cleavage a~
in AII was di3solved in 500 ml of degassed DNF. 210 mg of
NaHCO3 and, at -25C, 0.20 ml of diphenylphosphoryl azide
were added and then the mixture wa~ ~tirred at -25C for
2 h and at room temperature for 2 days. It was then
evaporated to dryness, and the crude peptide was purified
by gel chromatography (SEPHADEX0 LH 20) and medium pres-
sure chromatography (cf. AIV; 20-50~ A; 0.25% min~l).
75 mg of pure product were obtained.
Example 94
~Glu-Leu-Arg-Asp-Asn-Gln-Leu-Val
0.88 g of Fmoc-Glu(OtBu)-p-alkoxybenzyl alcohol-re~in
(substitution 0.57 mmol/g), corre~ponding to a batch sizQ
of 0.5 iol, wa3 reacted as in AIb with 2 mmol ~ach of
;~005;~
- 27 - O.Z. 0050~403~3
Fmoc-Val-OH Fmoc-Asp(OBzl)-OH
Fmoc-Leu-OH Fmoc-~rg(Tos)-OH
Fmoc-Gln-OH Fmoc-Leu-OH
Fmoc-As~-OH
After the ~ynthesi~ was complete, the peptide-re-~in
underwent N-terminal deprotection (steps 2-4 a~ in AIb)
and subsequent drying under r~duced pressure. The yi~ld
was 1.2 g.
The crude peptide obtained after TFA cleavag~ a~ in AIII
was diRsolved in 500 ml of degassed DMF 210 mg of NaHCO3
and, at -25C, 0.24 ml of diphenylphosphoryl azide were
added, and the mixture was stirred at -25C for 2 hours
and at room temperature for 2 days. It was then evapora-
ted to dryness, and the crude peptide was purified by gel
chromatography (SEPHADEX LH 20). The isolated monomer
(87 mg) was deprotected with HF as in AII and purified by
medium pre~sure chromatography (cf. AIV; 25-45% A; O.25%
min~~. 49 mg of pure product were obtained.
The following can be prepared in a similar m~nner to
Examples 93 and 94s
-
95. rArg-Asp-Asn-Gln-Ahx
96. Asp-Asn-Gln-Leu-~h~
r _ l
5~. rArg-asp-Asn-Gln-~hp
'?3. rArg-Asp-~sn-GIn-Leu-3al
99. ~y-~rg-~sp-Asn-Gln-Leu
lOO.rLeu-Arg-ASp-Asn-Gln-LCU-Ah
101.rGlu-L~u-Arg-Asp-~sn-Gln-Leu-Bal
102. rLeu-Arg-D-Asp-Asn-Gln-L
103. r~eu-Lys-Asp-~sn-Gln-Leu-~h
104 . r~bs-~rg-~p-~ n-61n-Leul
;~oos~
- 28 - O.Z. ~050/40383
lQ5.rLeu-~rg-~sp-Asn-GIn-D-Leu
106. rLeu-Arg-~sp-Asn-GIn-0-Pro
107. rLeu-Arg-Asp-Asn-GIn-GIy
108. rLeu-Arg-Asp-Asn-Gln-D-Ala
