Note: Descriptions are shown in the official language in which they were submitted.
a
! n $ ~~ .; ~
,., .. .; _ ~. . r zs ~
HOECHST ARTIENGESELLSCHAFT HOE 90/F 131 Dr.WI/je
Description
Peptides with a bradykinin-antagonistic action
The invention relates to novel peptides with a brady
kinin-antagonistic action and to a process for the
preparation thereof.
Bradykinin-antagonistic peptides are described in
WO 86/07263, where, inter alia, L-Pro in position 7 of
the peptide hormone bradykinin or other bradykinin
analogs is replaced by a D-amino acid such as D-Phe,
D-Thia, D-Pal, CDF, D-Nal, MDY, D-Phg, D-His, D-Trp,
D-Tyr, D-hPhe, D-Val, D-Ala, D-His, D-Ile, D-Leu and
DOMT.
The object of the invention is to find novel active
peptides with a bradykinin-antagonistic action.
This object is achieved by the peptides of the formula I
A-B-C-E-F-R-(D)-Tic~G-M-F'-I (I)
in which
A is al ) hydrogen,
2 O ( C1-Cg ) -alkyl ,
( C1-CB ) -alkanoyl ,
(Cl-CB)-alkoxycarbonyl or
(C1-CB)-alkylsulfonyl,
in each of which 1, 2 or 3 hydrogen
atoms are optionally replaced by l, 2 or
3 identical or different radicals from
the series comprising
carboxyl,
amino,
( C1-C4 ) -alkyl,
d Y ~'Z
W v! .: _Y. I d ~1
- 2 -
( Cl-C4 ) -alkylamino,
hydroxyl,
( C1-C4 ) -alkoxy,
halogen,
di- ( C1-C, ) -alkylamino,
carbamoyl,
sulfamoyl,
( C1-C, ) -alkoxycarbonyl ,
( Cs-Ciz ) -aryl and
( Cs-Ciz ) -aryl- ( Cl-Cs ) -alkyl ,
or in each of which 1 hydrogen atom is
optionally replaced by a radical from
the series comprising
(C3-CB)-cycloalkyl,
(C1-C,)-alkylsulfonyl,
(C1-C,)-alkylsulfinyl,
( Cs-Clz ) -aryl- ( C1-C, ) -alkylsul fonyl ,
( Cs-Clz ) -aryl- ( C1-C4 ) -alkylsulf inyl,
( C8-Cl2 ) -a~lOXy,
(C3-C9)-heteroaryl and
( C3-Ce ) -heteroaryloxy
and
1 or 2 hydrogen atoms are replaced by 1
or 2 identical or different radicals
from the series comprising
carboxyl,
amino,
( C1-C4 ) -alkylamino,
hydroxyl,
( C1-C4 ) -alkoxy,
halogen,
di- ( C1-C4 ) -alkylamino,
carbamoyl,
sulfamoyl,
(C1-C,)-alkoxycarbonyl,
C6-Clz ) -aryl and
( Cs-Ciz ) -aryl- ( Cl-Cs ) -alkyl ,
az) (C3-Cs)-cycloalkyl,
carbamoyl which can optionally be substituted
.,
,- ,.
- 3 -
on the nitrogen by ( C1-C6 ) -alkyl or ( C6-Cla ) -
aryl,
( C6-C12 ) -a~l ~
( C?-C13 ) °aroyl,
( C6-C12 ) -arylsulfonyl ,
( C3-Ca ) -heteroaryl or ( C3-C9 ) -heteroaroyl ,
where in each of the radicals defined under al)
and a2) aryl, heteroaryl, aroyl, arylsulfonyl
and heteroaroyl is optionally substituted by 1,
2, 3 or 4 identical or different radicals from
the series comprising
carboxyl,
amino,
vitro,
( C,-C, ) -alkylamino,
hydroxyl,
( C1-C4 ) -alkyl ,
( C1-C4 ) -alkoxy,
halogen,
cyano,
di- ( C1-C,, ) -alkylamino,
carbamoyl,
sulfamoyl and
( C~-C4 ) -alkoxycarbonyl
or
a3) a radical of the formula II
(II)
R1 _ N _ CH _ H _
R2 R3 O
in which
R1 is defined as A under al ) or a2 ) ,
~0 R2 is hydrogen or methyl,
R3 is hydrogen or
(C1-C6)-alkyl, preferably (Cl-C4)-alkyl,
which is optionally monosubstituted by
amino,
cl~ f~ . ~ .'i ;, n
' -t !.
i1 J ~.; _.. m .. L
- 4 -
substituted amino,
hydroxyl,
carboxyl,
carbamoyl,
guanidino,
substituted guanidino,
ureido,
mercapto,
methylmercapto,
phenyl,
4-chlorophenyl,
4-fluorophenyl,
4-nitrophenyl,
4-methoxyphenyl,
4-hydroxyphenyl,
phthalimido,
4-imidazolyl,
3-indolyl,
2-thienyl,
3-thienyl,
2-pyridyl,
3-pyridyl or
cyclohexyl,
where substituted amino is -NH-A and substituted
guanidino is -NH-C(NH)-NH-A in which
A is as defined under al) or a2) ;
B is a basic amino acid which is in the L or D con
figuration and can be substituted in the side chain;
C is a linkage of the formula IIIa or IIIh
3 0 G' -G' -Gly G' -NH- ( CH2 ) p CO
(IIIa) (IIIb)
in which
G' is, independently of one another, a radical of the
formula IV
n , ~ ,; -~ ,-W7
_ ,. . . t~
- 5 -
R4 R5 p (IV)
in which - N - CH - C -
R' and RS form, together with the atoms carrying
them, a heterocyclic mono-, bi- or tricyclic ring
system with 2 to 15 carbon atoms, and
n is 2 to 8;
E is the residue of a neutral, acidic or basic alipha-
tic or alicyclic-aliphatic amino acid;
F is, independently of one another, the residue of a
neutral, acidic or basic, aliphatic or aromatic
amino acid which can be substituted in the side
chain, or is a direct bond;
(D)-Tic is the radical of the formula V;
H O
(V)
N
G is defined as G' above or is a direct bond;
F° is defined as F, is a radical -NH-(CHZ)a- with n = 2
to 8, or can be a direct bond if G is not a direct
bond, and
I is -OH, -NHZ or -NHCZHS,
R is the radical -NH-(CHZ)x CO- with x = 1-4 or is a
direct bond, and
M is defined as F,
and the physiologically tolerated salts thereof.
Unless stated otherwise, the abbreviation of an amino
acid residue without a stereodescriptor represents the
residue in the L form (cf. Schrdder, Liibke, The Peptides,
Volume T, New York 1965, pages XXII-XXIII; Houben-Weyl,
;o~1 ~> ~. ;~.J '' i '~
- 6 -
Methoden der Organischen Chemie (Methods of Organic
Chemistry), Volume XV/1 and 2, Stuttgart 1974), such as,
for example,
Aad, Abu, yAbu, ABz, 2ABz, eAca, Ach, Acp, Adpd,Ahb,
Aib, ~Aib,Ala, liAl.a, eAla, Alg, All, Ama, Apm,
Amt, Ape,
Apr, Arg, Asn, Asp, Asu, Aze, Azi, Bai, Bph, Cit,
Can,
Cys, Cyta,Daad, Dab, Dadd, Dap, Dapm, Dasu, Dpa,
Djen,
Dtc, Fel, Gln, Glu, Gly, Guv, hAla, hArg, hCys,hGln,
hGlu, His, hIle, hLeu, hLys, hMet, hPhe, hero, hSer,
hThr, hTrp, Kyn,
hTyr,
Hyl,
Hyp,
3Hyp,
Ile,
Ise,
Iva,
Lant, Lcn, Leu, Lsg, Lys, ~9Lys, ALys, Met, iMim,Min,
nArg, Nle, Nva, Oly, Orn, Pan, Pec, Pen, Phe, Pic,
Phg,
Pro, nPro,Pse, Pya, Pyr, Pza, Qin, Ros, Sar, Sem,
Sec,
Ser, Thi, ~Thi, Thr, Thy, Thx, Tia, Tle, Tly, Trta,
Trp,
Tyr, Val.
Suitable as radical of a heterocyclic ring system of the
formula IV are, in particular, radicals of heterocycles
from the following group:
pyrrolidine-2-carboxylic acid; piperidine-2-carboxylic
acid; 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;
decahydroisoquinoline-3-carboxylic acid;octahydroindole-
2-carboxylic acid; decahydroquinoline-2-carboxylic
acid; octahydrocyclopenta[b]pyrrole-2-carboxylic acid;
2-azabicyclo[2.2.2]octane-3-carboxylic acid; 2-azabicyclo-
[2.2.1]heptane-3-carboxylic acid; 2-azabicyclo[3.1.0]-
hexane-3-carboxylic acid; 2-azaspiro[4.4]nonane-3-
carboxylic acid; 2-azaspiro[4.5]decane-3-carboxylic acid;
spiro[(bicyclo[2.2.1]heptane)-2,3-pyrrolidine-5-
carboxylic acid]; spiro[(bicyclo[2.2.2]octane)-2,3-
pyrrolidine-5-carboxylic acid];2-azatricyclo[4.3Ø18~H]-
decane-3-carboxylic acid; decahydrocyclohepta[b]pyrrole-
2-carboxylic acid; decahydrocycloocta[b]pyrrole-2-
carboxylic acid; octahydrocyclopenta[c]pyrrole-2-
carboxylic acid; octahydroisoindole-1-carboxylic acid;
2,3,3a,4,6a-hexahydrocyclopenta[b]pyrrole-2-carboxylic
'~ t''. ; _i ,::~ r: a j
7 _
acid; 2,3,3a,4,5,7a-hexahydroindole-2-carboxylic acid;
tetrahydrothiazole-4-carboxylic acid; isoxazolidine-3
carboxylic acid; pyrazolidine-3-carboxylic acid; hydroxy
proline-2-carboxylic acid; all of which can optionally be
substituted:
~~CO- ; ~ .
CO-
CO- CO-
w
N ; ~~~~~~~~ ' ~ CO ~ C O- i
I I
I rC0- i ~ ~CO_
~N ~C0- , CO- ~ ~N i
I I
CO-
~CO- ; NCO- ; N i
I I : I
~ CO- ~~CO- ; ,-CO- i
~:~N i ~N N
~CO- j
~ CO- ;
I CO- ;
j I
i ~~-CO- ; ~~CO- .
~~''//''~~N N r
HO
O- j ~CO- s ~CO- i
I
The heterocycles which form the basis for the above-
mentioned radicals are disclosed, for example, in
US-A 4,344,949, US-A 4,374,847, US-A 4,350,704,
l 1!
~a ~ n a
- 8 -
EP-A 29 488, EP-A 31 741, EP-A 46 953,EP-A 49 605,
EP-A 49 658, EP-A 50 800, EP-A 51 020,EP-A 52 870,
EP-A 79 022, EP-A 84 164, EP-A 89 637,EF-A 90 341,
EP-A 90 362, EP-A 105102, EP-A 109 020,EP-A 111 873,
EP-A 271865 and 344 682.
EP-A
Unless otherwise stated in the specific case, alkyl can
be straight-chain or branched. The corresponding state-
ment applies to radicals derived therefrom, such as
alkoxy, aralkyl or alkanoyl.
(C6-C12)-Aryl is preferably phenyl, naphthyl or biphenyl-
yl. Radicals derived therefrom, such as aryloxy, aralkyl
or aroyl, are to be formulated correspondingly.
Halogen is fluorine, chlorine, bromine or iodine, prefer-
ably chlorine.
Particularly suitable salts are alkali metal or alkaline
earth metal salts, salts with physiologically tolerated
amines and salts with inorganic or organic acids such as,
for example, HC1, HBr, HZS04, H3P04, malefic acid, fumaric
acid, citric acid, tartaric acid and acetic acid.
Preferred peptides of the formula I are those in which
B is Arg, Lys, Orn, 2,4-diaminobutyryl or an L-homo-
arginine residue where, in each case, the amino or
the guanidino group of the side chain can be substi-
tuted by A as described under al) or a2);
E is the residue of an aliphatic or alicyclic-ali-
phatic amino acid which is in the L or D configura-
tion and which contains 1 to 14 carbon atoms in the
side chain, such as alanine, serine, threonine,
O- ( C1-C6 ) -alkyl- or 0- ( C6-Clo ) -aryl-protected serine
or threonine, valine, norvaline, leucine,isoleucine,
norleucine, neopentylglycine, tert-butylglycine or
( C~-C7 ) -cycloalkyl- ( C1-C3 ) -alkylglycine;
'~ , i 'i '" =j C7
_ _.
_ g _
F' is the residue of a basic amino acid in the L or D
configuration, such as Arg or Lys, where the guani-
dine group or amino group of the side chain can be
substituted by A as described under al) or a2), or is
a radical -NH-(CHZ)p - with n = 2 to 8;
R is the radical -NH-(CHZ)= CO- with x = 2-4 or is a
direct bond.
Particularly preferred peptides of the formula I are
those in which
B is Arg, Orn or Lys, where the guanidine group or the
amino group of the side chain is unsubstituted or
can be substituted by ( C1-Ce ) -alkanoyl , ( C~-Cls ) -
aroyl, ( C3-Ce ) -heteroaroyl , ( Cl-Ce ) -alkylsulfonyl or
(Cs-C12)-arylsulfonyl, where the aryl, heteroaryl,
aroyl, arylsulfonyl and heteroaroyl radicals can be
substituted with, where appropriate, 1, 2, 3 or 4
identical or different radicals as described under
as )
E is leucine, isoleucine, norleucine, tert-butyl-
glycine, serine, threonine or cyclohexylalanine;
R is a direct bond, and
M is a direct bond.
Very particularly preferred peptides of the formula I are
those in which
2 5 A is hydrogen, ( D ) - or ( L ) -H-Arg, ( D ) - or ( L ) -H-Lys or
(D)- or (L)-H-Orn,
B is Arg, Orn or Lys, where the guanidine group or the
amino group in the side chain can be substituted by
( C1-C8 ) -alkanoyl , ( C~-C13 ) -aroyl , ( C3-C9 ) -heteroaroyl ,
(C1-C8)-alkylsulfonyl or (C6-C12)-arylsulfonyl, where
'l ~ 'i ~~ ~ ~ :7
,.. .. _ . ': t3
- 10 -
the aryl, heteroaryl, aroyl, arylsulfonyl and
heteroaroyl radicals can optionally be substituted
with 1, 2, 3 or 4 identical or different radicals
from the series comprising methyl, methoxy and
halogen;
C is Pro-Pro-Gly, Hyp-Pro-Gly or Pro-Hyp-Gly,
E is Leu, Ile, Tbg or Cha,
F is Ser, Hser, Lys, Leu, Val, Nle, Ile or Thr,
R is a direct bond,
M is a direct bond,
G is the radical of a heterocyclic ring system of the
formula IV, where the radicals of the heterocycles
pyrrolidine-2-carboxylic acid; piperidine-2-
carboxylic acid;tetrahydroisoquinoline-3-carboxylic
acid, cis- and trans-decahydroisoquinoline-3
carboxylic acid; cis-endo-, cis-exo-, trans-octa
hydroindole-2-carboxylic acid; cis-endo-, cis-exo-,
trans-octahydrocyclopenta[b]pyrrole-2-carboxylic
acid or hydroxyproline-2-carboxylic acid are pre
ferred,
F' is Arg and
I is OH.
Examples of very particularly preferred peptides of the
formula I are:
H-(D)-Arg-Arg-Pro-Hyp-Gly-Leu-Ser-(D)-Tic-Oic-Arg-OH
H-(D)-Arg-Arg-Pro-Hyp-Gly-Cha-Ser-(D)-Tic-Oic-Arg-OH
H-(D)-Arg-Arg-Pro-Hyp-Gly-Tbg-Ser-(D)-Tic-Oic-Arg-OH
The invention also relates to a process for preparing
peptides of the formula I, which comprises
. ~ a i,;)
- 11 - ,. .
a) reacting a fragment with a C-terminal free carboxyl
group or its activated derivative with a correspond-
ing fragment with an N-terminal free amino group, or
b) synthesizing the peptide stepwise,
eliminating one or more protective groups temporar-
ily introduced into the compound obtained as in (a)
or (b) to protect other functions where appropriate,
and converting the compounds of the formula I which
have been obtained in this way into their physio-
logically tolerated salt where appropriate.
The peptides of the present invention were prepared by
generally known methods of peptide chemistry, see, for
example, Houben-Weyl, Methoden der organischen Chemie,
Volume 15/2, preferably by solid-phase synthesis as
described, for example, by B. Merrifield, J. Am. Chem.
Soc. 85, 2149 (1963) or R.C. Sheppard, Int. J. Peptide
Protein Res. 21, 118 (1983) or by equivalent known
methods. Used as a-amino protective groups are
urethane protective groups such as, for example, the
tert-butyloxycarbonyl(Boc) or
fluorenylmethyloxycarbonyl(Fmoc) protective group.
If necessary to prevent side reactions or for synthesiz-
ing specific peptides, the functional groups in the side
chain of amino acids are additionally protected by
suitable protective groups (see, for example,
T.W. Greene, "Protective Groups in Organic Synthesis"),
employing primarily
Arg(Tos), Arg(Mts), Arg(Mtr), Arg(PMC), Asp(oszl),
Asp(OBut), Cys(4-MeBzl), Gys(Acm), Cys(SBut), Glu(OBzl),
Glu(OBut), His(Tos), His(Fmoc), His(Dnp), His(Trt),
Lys(C1-Z), Lys(Boc), Met(O), Ser(Bzl), Ser(But),
Thr(Bzl), Thr(But), Trp(Mts), Trp(CHO), Tyr(Br-Z),
Tyr(Bzl) or Tyr(But).
The solid-phase synthesis starts at the C-terminal end of
the peptide with the coupling of a protected amino acid
onto an appropriate resin. Starting materials of this
- 12 - .: . . _. . , .. ;_s
type can be obtained by linking a protected amino acid,
via an ester or amide linkage, to a polystyrene or
polyacrylamide resin modified with a chloromethyl,
hydroxymethyl, benzhydrylamino(BHA) or methylbenzhydryl-
amino (MBHA) group. The resins used as support material
are commercially available. BHA- and I~HA-resins are
usually employed when the synthesized peptide is to
contain a free carbamoyl group at the C terminus. If the
peptide is to contain a secondary carbamoyl group at the
C-terminal end, a chloromethyl- or hydroxymethyl-resin
is used and the cleavage off is carried out~with the
appropriate amines. If, for example, it is wished to
obtain the ethylamide, the peptide can be cleaved off the
resin with ethylamine, in which case the side-chain
protective groups are subsequently eliminated by other
suitable reagents. If the tert-butyl protective groups in
the amino acid side chain are to remain in the peptide,
the synthesis is carried out with the Fmoc protective
group for temporary blocking of the a-amino group of the
amino acid using the method described, for example, by
R.C. Sheppard, J. Chem. Soc., Chem. Comm. 1982, 587, in
which case the guanidino group of the arginine is protec-
ted by protonation with pyridinium perchlorate, and the
other amino acids functionalized in the side chain are
protected with benzyl protective groups which can be
eliminated by catalytic transfer hydrogenation (A. Felix
et al. J. Org. Chem. 13, 4194 (1978) ) or by sodium in
liquid ammonia (W. Roberts, J. Am. Chem. Soc. 7~, 6203
(1954)).
After elimination of the amino protective group of the
amino acid coupled to the resin using a suitable reagent
such as, for example, trifluoroacetic acid in methylene
chloride in the case of the Boc protective group or a 20%
strength solution of piperidine in dimethylformamide in
the case of the Fmoc protective group, the subsequent
protected amino acids are coupled on successively in the
required sequence. The peptide-resins with N-terminal
protection which are the intermediate products are
- 13 -
deblocked by the reagents described above for the linkage
to the subsequent amino acid derivative.
It is possible to use as coupling reagent all possible
activating reagents used in peptide synthesis, see, for
example, Houben-Weyl, Methoden der organischen Chemie,
Volume 15/2, but especially carbodii.mides such as, for
example,N,N'-dicyclohexylcarbodiimide,N,N'-diisopropyl-
carbodiimide or N-ethyl-N'-(3-dimethylaminopropyl)carbo-
diimide. The coupling can, moreover, be carried out
directly by addition of amino acid derivative with the
activating reagent and, where appropriate, with an
additive suppressing racemization, such as, for example,
1-hydroxybenzotriazole (HOBt) (W. Rbnig, R. Geiger, Chem.
Ber. 103, 708 (1970)) or 3-hydroxy-4-oxo-3,4-dihydro-
benzotriazine (HOObt) (W. Ronig, R. Geiger, Chem. Ber.
103, 2054 (1970)) to the resin, or else the preactivation
of the amino acid derivative as symmetrical anhydride or
HOBt or HOObt ester can take place separately, and the
solution of the activated species in a suitable solvent
can be added to the peptide-resin ready for coupling.
The coupling or activation of the amino acid derivatives
with one of the abovementioned activating reagents can be
carried out in dimethylformamide, N-methylpyrrolidone or
methylene chloride or a mixture of the said solvents . The
activated amino acid derivative is normally employed in
a 1.5- to 4-fold excess. In cases where incomplete
coupling occurs, the coupling reaction is repeated
without previously carrying out the deblocking, which is
necessary fox the coupling of the amino acid next in
sequence, of the a-amino group of the peptide-resin.
The success of the coupling reaction can be checked using
the ninhydrin reaction as described, for example, by
E. Raiser et al. Anal. Biochem. 34 595 (1970). Automated
synthesis is also possible, for example using a model
430A peptide synthesizer from Applied Biosystems, in
which case it is gossible to use either the synthesis
..
- 14 -
programs provided by the manufacturer of the apparatus or
else those drawn up by the user himself. The latter are
employed especially when amino acid derivatives protected
by the Fmoc group are used.
After the peptides have been synthesized in the manner
described above, the peptide can be cleaved off the resin
using reagents such as, for example, liquid hydrogen
fluoride (preferably for the peptides prepared by the Boc
method) or trifluoroacetic acid (preferably for the
peptides synthesized by the Fmoc method). These reagents
cleave not only the peptide off the resin but also the
other side-chain protective groups of the amino acid
derivatives. The peptide is obtained in the form of the
free acid in this way, except when HHA- and MBHA-resins
are used. In the case of the BHA- and MBIiA-resins, the
cleavage with hydrogen fluoride or trifluoromethane-
sulfonic acid yields the peptide as amide. Further
processes fox preparing peptide amides are described in
EP-A 28? 882 and EP-A 322 348. In this case the peptide
amides are cleaved off the resin by treatment with
moderately strong acids conventionally used in peptide
synthesis (for example trifluoroacetic acid), adding as
cation traps substances such as phenol, cresol, thio-
cresol, anisole, thioanisole, ethanedithiol, dimethyl
sulfide, ethyl methyl sulfide or similar cation traps
conventional in solid-phase synthesis, singly or a
mixture of two or more of these aids. The trifluoroacetic
acid can also be used diluted by suitable solvents such
as, for example, methylene chloride in this case.
If the tert-butyl or benzyl side-chain protective groups
on the peptides are to be retained, the peptide which has
been synthesized on a specially modified sugport resin is
cleaved off with 1~ trifluoroacetic acid in methylene
chloride as described, for example, by R.C. Sheppard
J. Chem. Soc., Chem. Gomm. 1982, 587. If individual tert-
butyl or benzyl side-chain protective groups are to be
retained, a suitable combination of the synthesis and
...
- 15 -
cleavage off methods is used.
The modified support resin described by Sheppard is also
used for synthesizing peptides with a C-terminal
carbamoyl group or an w-amino- or w-guanidinoalkyl group.
After the synthesis, the peptide which is completely
protected in the side chain is cleaved off the resin and
then reacted in classical solution synthesis with the
appropriate amine or w-aminoalkylamine or w-guanidino-
alkylamine, in which case it is possible where approp-
riate to protect temporarily other functional groups
which are present in a known manner.
Another process for preparing peptides with an w-amino-
alkyl group is described in EP-A 264 802.
The peptides of the present invention were preferably
synthesized using the solid-phase technique with two
general protective group tactics:
The synthesis was carried out with a model 430 A auto-
matic peptide synthesizer from Applied Biosystems using
Boc and Fmoc protective groups for temporary blocking of
the a-amino group.
When the Boc protective group was used, the synthesis
cycles preprogramed by the manufacturer of the apparatus
were used for the synthesis.
The peptides with a free carboxyl group at the C-terminal
end were synthesized on a 4-(hydroxymethyljphenylacet-
amidomethylpolystyrene resin functionalized with the
appropriate Boc-amino acid (R. B. Merrifield, J. Org.
Chem. 43, 2845 (1978jj from Applied Biosystems. An MBHA-
resin from the same company was used to prepare peptide
amides. The activating reagents used were
N,N~-dicyclohexylcarbodiimide or
N,N~-diisopropylcarbodiimide. The activation took place
as symmetrical anhydride, as HOBt ester or HOObt ester in
- 16 -
CH2C12, CHZC12/DMF mixtures or NMP . 2-4 equivalents of
activated amino acid derivative were employed for the
coupling. In cases where the coupling was incomplete, the
reaction was repeated.
When the Fmoc protective group was used for temporary
protection of the a-amino group, our own synthesis
programs were entered for the synthesis with the model
430A automatic peptide synthesizer from Applied Bio-
systems. The synthesis was carried out on a p-benzyloxy-
benzyl alcohol-resin (S. Wang, 3. Am.Chem.Soc. ,9~, 1328
(1973)) from Bachem, which was esterified with the
appropriate amino acid by a known method (E. Atherton et
al. J.C.S.Chem. Comm. 19 1, 336). The activation of the
amino acid derivatives as HOBt or HOObt ester took place
directly in the amino acid cartridges supplied by the
manufacturer of the apparatus, by addition of a solution
of diisopropylcarbodiimide in DMF to the mixture of amino
acid derivative and HOBt or HOObt which had previously
been weighed in. It is likewise possible to employ Fmoc-
amino acid OObt esters prepared in bulk, as are described
in EP-A 247 573. The F'moc protective group was eliminated
using a 20~ strength solution of piperidine in D1~ in the
reaction vessel. The excess of reactive amino acid
derivative used was 1.5 to 2.5 equivalents. If the
coupling was incomplete, it was repeated as for the Boc
method.
The peptides according to the invention have, singly or
in combination, a bradykinin-antagonistic action which
can be tested in various models (see Handbook of Exp.
Pharmacol. Vol. 25, Springer Verlag, 1970, pp. 53 - 55),
for example on the isolated rat uterus, on the guinea pig
ileum or on the isolated guinea pig pulmonary artery.
To test the peptides according to the invention in the
isolated pulmonary artery, guinea pigs (Dunkin Hartley)
weighing 400 - 450 g are sacrificed by a blow to the back
of the neck.
1 . ~ i .~ ~ .1
i
- 17 - , . .... .,°
The thoracic cavity is opened and the pulmonary artery is
carefully dissected out. The surrounding tissue is
carefully removed and the pulmonary artery is cut open
helically at an angle of 45°.
The strip of vessel which is 2.5 cm long and 3 - 4 mm
wide is fixed in an organ bath with a capacity of 10 ml
which is filled with Ringer solution.
Composition of the solution in mmol/1
NaCl 154
RC1 5.6
CaClz 1. 9
NaHC03 2 . 4
Glucose 5.0
95% OZ and 5% C02 is bubbled through the solution, which
is heated to 37°C. The pH is 7.4, and the load on the
strip of vessel is 1.0 g.
The changes in the isometric contractions are detected
with a lever attachment and an HF modem (path-measuring
device) from Hugo Sachs and recorded on a potentiometric
recorder (BEC, Goerz Metrawatt SE 460).
After equilibration for 1 hour the experiment is started.
Once the strips of vessel have reached their maximum
sensitivity to 2 x 10-' mol/1 bradykinin - bradykinin
causes the strips of vessel to contract - the peptides
are allowed to act in the doses 5 x 10-e - 1 x 10-5 mol/1
for 10 minutes each and, after renewed addition of
bradykinin, the decrease in the effect of bradykinin is
compared with the control.
To detect a partial agonistic effect, the peptides are
used in the doses 1 x 10-5 - 1 x 10-3 mol/1.
The ICsg values of the peptides according to the invention
- lg - .. , ,;1~ ,,
calculated from the dose-effect plots are listed in
Table 1.
Table 1:
Compound ICso [M]
H-(D)-Arg-Arg-Pro-Hyp-Gly-Leu-Ser-(D)-Tic-Oic-Arg-OH 5.9x10-8
H-(D)-Arg-Arg-Pro-Hyp-Gly-Cha-Ser-(D)-Tic-Oic-Arg-OH 3,7x10-$
H-(D)-Arg-Arg-Pro-Hyp-Gly-Tbg-Ser-(D)-Tic-Oic-Arg-OH 6,0x10-6
The therapeutic uses of the peptides according to the
invention embrace all pathological states which are mediated,
induced or assisted by bradykinin and peptides related to
bradykinin. These comprise, inter alia, traumata such as
wounds, burns, rashes, erythemas, edemas, tonsillitis,
arthritis, asthma, allergies, rhinitis, shock, inflammations,
low blood pressure, pain, pruritus and altered sperm motility.
The invention therefore also relates to the use of peptides of
the formula I as medicines and to pharmaceutical products
which contain these compounds.
Pharmaceutical products contain an effective amount of
the active compound of the formula I - singly or in
combination - together with an inorganic or organic
pharmaceutically utilizable excipient.
Administration can be enterally, parenterally - such as,
for example, subcutaneously, i.m. or i.v. -, sublingu-
ally, epidermally, nasally, rectally, intravaginally,
intrabuccally or by inhalation. The dosage of the active
compound depends on the warm-blooded species, the body
weight, age and on the mode of administration.
The pharmaceutical products of the present invention are
prepared by dissolution, mixing, granulation or coating
processes known per se.
:)
- 19 - ~. " . . . '..~ ,°~
For the form for oral administration or for administra-
tion onto the mucosa, the active compounds are mixed with
the additives customary for this purpose, such as excipi-
ents, stabilizers or inert diluents, and converted by
customary methods into suitable dosage forms such as
tablets, coated tablets, hard gelatin capsules, aqueous,
alcoholic or oily suspensions or aqueous, alcoholic or
oily solutions. Examples of inert vehicles which can be
used are gum arabic, magnesia, magnesium carbonate,
potassium phosphate, lactose, glucose, magnesium stearyl
fumarate or starch, especially corn starch. This prepara
tion can take place both as dry and wet granules.
Examples of suitable oily excipients or solvents are
vegetable or animal oils, such as sunflower oil and fish
liver oil.
A product for topical administration can be in the form
of an aqueous or oily solution, lotion, emulsion or
jelly, ointment or fatty ointment or, if possible, in
spray form, it being possible where appropriate to
improve adhesion by adding a polymer.
For the form for intranasal administration, the compounds
are mixed with the additives customary for this purpose,
such as stabilizers or inert diluents, and converted by
the customary methods into suitable dosage forms such as
aqueous, alcoholic or oily suspensions or aqueous,
alcoholic or oily solutions. It is possible to add
chelating agents, ethylenediamine-N,N,N~,N~-tetraacetic
acid, citric acid, tartaric acid or salts thereof to
aqueous intranasal preparations. The nasal solutions can
be administered using a metering atomizer or as nose
drops with a viscosity-increasing content, or nasal gels
or nasal creams.
It is possible to use for administration by inhalation
atomizers or compressed gas packs employing inert carrier
gases.
..
- 2 0 - ,.. .. r. , ''
For intravenous, subcutaneous, epicutaneous or intra-
dermal administration, the active compounds or the
physiologically tolerated salts thereof are converted, if
required with the pharmaceutically customary auxiliaries,
for example for isotonicity or adjusting the pH, and
solubilizers, emulsifiers or other auxiliaries, in
solution, suspension or emulsion.
The short half-lives of some of the described pharma-
ceutical substances in body fluids mean that it is
worthwhile to use injectable depot preparations. Examples
of pharmaceutical forms which can be used are oily
crystal suspensions, microcapsules, rods or implants, it
being possible for the latter to be composed of tissue-
compatible polymers, especially biodegradable polymers
such as, for example, based on polylactic acid/poly-
glycolic acid copolymers or human albumin.
A suitable dose range for forms for administration
topically and by inhalation are solutions containing
0.01-5 mg/ml, while 0.01-10 mg/kg are suitable for
systemic administration forms.
List of abbreviations:
The abbreviations used for amino acids correspond to the
three-letter code customary in peptide chemistry, as
described in Europ. J. Biochem. 1~$, 9 (19$4). Other
abbreviations used are listed below.
Acm acetamidomethyl
E-AhX E-aminohexanoyl
Aoc cis, endo-2-azabicyclo[3.3.OJoctane-3-S-
carbonyl
Boc tert-butyloxycarbonyl
But tert-butyl
Bzl benzyl
CDF chloro-(D)-phenylalanyl
Cha cyclohexylalanyl
Chg cyclohexylglycyl
- 21 -
:a n '~ L~ i.
r~: 1~.:~ _',. i ,r '., i
C1-Z 4-chlorobenzyloxycarbonyl
DMF dimethylformamide
DOyIT 0-methyl-(D)-threonyl
Dnp 2,4-dinitrophenyl
Fmoc 9-fluorenylmethyloxycarbonyl
MDY 0-methyl-(D)-tyrosyl
Me methyl
4-Mebzl 4-methylbenzyl
Mtr 4-methoxy-2,3,6-trimethylphenylsulfonyl
Mts mesitylene-2-sulfonyl
Nal 1- or 2-naphthylalanyl
NMP N-methylpyrrolidine
Npg neopentylglycyl
Oic cis-endo-octahydroindole-2-carbonyl
Opr isoxazolidin-3-ylcarbonyl
Pal 2- or 3-pyridylalanyl
Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl
Tbg tert-butylglycyl
TFA trifluoroacetic acid
Thia 2-thienylalanyl
Tcs 4-methylphenylsulfonyl
Tic 1,2,3,4-tetrahydroisoquinolin-3-ylcarbonyl
Trt trityl
The examples which follow are intended to illustrate the
preferred methods for the solid-phase synthesis of the
peptides according to the invention without restricting
the invention thereto.
The following amino acid derivatives were used:
Fmoc-Arg(Mtr)-OH, Hoc-(D)-Arg-OH, Fmoc-Arg(Pmc)-OH,
Fmoc-Hyp-OH, Fmoc-Pro-OObt, Fmoc-Gly-OObt, Fmoc-Phe-OObt,
Fmoc-Ser(tBu)-OObt, Fmoc-(D)-Tic-OH, Fmoc-Gln-OH,
Fmoc-Aoc-OH, Fmoc-Thia-OH, Fmoc-Opr-OH, Fmoc-(D)-Asn-OH,
Fmoc-~S-Ala-OH, Fmoc-Oic-OH.
CA 02041268 2000-10-20
- 22 -
gaample 1:
H-(D)-Arg-Arg-Hyp-Pro-Gly-Leu-Ser-(D)-Tic-Oic-Arg-OH
was synthesized stepwise using a model 430 A peptide
synthesizer from Applied Hiosystems and using the Fmoc
method on a p-benzyloxybenzyl alcohol-resin esterified
with Fmoc-Arg(Mtr)-OH from Novabiochem (loading about
0.5 mmol/g of resin). 1 g of the resin was employed, and
the synthesis was carried out using a synthesis program
modified for the Fmoc method.
In each case, 1 mmol of the amino acid derivatives with
free carboxyl group were weighed together with 0.95 mmol
of HOObt into the synthesizer cartridges. These amino
acids were preactivated directly in the cartridges by
dissolving in 4 ml of DMF and adding 2 ml of a 0.55 mol/1
solution of diisopropylcarbodiimide in DMF. The HOObt
esters of the other amino acids were dissolved in 6 ml of
NMP and then coupled just like the in situ preactivated
amino acids to the resin which had previously been
deblocked with 20% piperidine in DMF. After the synthesis
was complete, the peptide was cleaved off the resin with
simultaneous removal of the side-chain protective groups
with trifluoroacetic acid using thioanisole and ethanedi-
thiol as cation traps. The residue obtained after stripp-
ing off the trifluoroacetic acid was digested several
times with ethyl acetate and centrifuged. The remaining
residue was chromatographed On ~Sephadex~ LH 20 with 10%
strength acetic acid. The fractions containing the pure
peptide were combined and freeze-dried.
MS(FAB) s 1264 (M+H)
The following examples were prepared in analogy to
Example 1.
-23- c:n;.~s; ,n
yi ~J ~: .:~ 1'/ i,~ O
$sample 2:
H-(D)-Arg-Arg-Hyp-Pro-Gly-Cha-Ser-(D)-Tic-Oic-Arg-OH
MS (FAB): 1304 (M+H)
Baample 3:
H-(D)-Arg-Arg-Hyp-Pro-Gly-Tbg-Ser-(D)-Tic-Oic-Arg-OH
MS (FAB): 1264 (M+H)
