Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
HOECHST AKTIENGESELLSCHAFT HOE 89/F 261 Dr.wI/gm
Description
Peptides having bradykinin antagonist action
The invention relates to novel peptides having bradykinin
antagonist action and to a process for their preparation.
Bradykinin antagonist peptides are described in
WO 86/07263 in which, inter alia, L-Pro in position 7 of
the peptide hormone bradykinin or other bradykinin
analogs is replaced by a D-amina acid, such as D-Phe,
D-Thi, 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 invention is based on the object of finding novel
active peptides having bradykinin antagonist action.
This object is achieved by the peptides of the formula I
A-B-C-E-F-K-(D)-Phe-G-M-~"-I (I)~
in which
A al) denotes hydrogen,
(C1-C8)-alkyl,
2 0 ( Cl-C8 ) -alkanoyl ,
(C1-CB)-alkoxycarbonyl or
(C1-C8)-alkylsulfonyl,
in which in each case 1, 2 or 3 hydrogen atoms are
optionally replaced by 1, 2 or 3 identical or different
radicals from the series comprising
carboxyl,
amino,
( C1-C4 ) -alkyl ,
( Cl-C4 ) -alkylamino,
hydroxyl,
( C1-C4 ) -alkoxy,
halogen,
di- ( C1-Cr~ ) -alkylamino,
- 2 -
carbamoyl,
sulfamoyl,
( C1-C4 ) -alkoxycarbonyl,
( Cs-Clz ) -aryl and
(Cs-Cxz)-aryl-(C1-CS)-alkyl, or in which in each case
hydrogen atom is optionally replaced by a radical from
the series comprising
( C3-Ce ) -cycloalkyl,
( C1-C4 ) -alkylsul fonyl ,
(C1-C4)-alkylsulfinyl,
( C6-C12 ) -aryl- ( C1-C4 ) -alkylsulfonyl,
( Cs-Clz ) -aryl- ( Cl-C4 ) -alkylsulf inyl,
( Cs-Czz ) -aryloxy r
( C3-C9 ) -heteroaryl and
( C3-Cg ) -heteroaryloxy
and
1 or 2 hydrogen atoms are replaced by 1 or 2 identical
or different radicals from the series comprising
carboxyl,
amino,
( Cz-C,, ) -alkylamino,
hydroxyl,
( C1-C4 ) -alkoxy,
halogen,
di- ( C1-C,, ) -alkylamino,
carbamoyl,
sulfamoyl,
( C1-C4 ) -alkoxycarbonyl ,
( Cs-Clz ) -aryl and
f Cs-Czz) °a~'1- ( Ci-Cs) °alkyl,
az) denotes (C3-Cs)-cycloalkyl,
carbamoyl, which may be optionally substituted on
the nitrogen by ( C1-Cs ) -alkyl or ( Cs-Clz ) -aryl ,
( Cs-Ciz ) °aryl
( C~-Cl~ ) -aryloyl,
( Cs-Clz ) -arylsulfonyl ,
( C3-C9 ) -heteroaryl , or ( C3-C9 ) -heteroaryloyl ,
where in the radicals defined under al) and az) in each
case aryl, heteroaryl, aryloyl, arylsulfonyl and
- 3 -
heteroaryloyl is optionally substituted by 1, 2, 3 or 4
identical or different radicals from the series com-
prisrng
carboxyl,
amino,
nitro,
( ~1"~-4 ) -alkylamino,
hydroxyl,
( C1-C4 ) -alkyl,
( Cl-C4 ) -alkoxy,
halogen,
cyano,
di- ( C1-Cu ) -alkylamino,
carbamoyl,
sulfamoyl and
(C1-C4)-alkoxycarbonyl, or
a3) denotes a radical of the formula II
R1 - N - CH - C - (II)
R2
3 0
R
R1 is defined as A under al) or a2) ,
Rz denotes hydrogen or methyl,
R3 denotes hydrogen or
( C1-Cs ) -alkyl , preferably ( C1-C4 ) -alkyl
,
which is optionally monosubstituted by
amino,
substituted amino.,
hydroxyl,
carboxyl,
carbamoyl,
guanidino,
substituted guanidino,
ureido,
mercapto,
methylmercapto,
phenyl,
4-chlorophenyl,
4-fluorophenyl,
4-nitrophenyl,
4-methoxyphenyl,
- 4 -
4-hydroxyphenyl,
phthalimido,
4-imidazolyl,
3-indolyl,
2-thienyl,
3-thienyl,
2-pyridyl,
3-pyri.dyl or
cyclohexyl,
ZO where substituted amino stands for a compound -NH-A- and
substituted guanidino stands for a compound
-NH-C(NH)-NH-A, in which A is deffined as under al) or a2);
B stands for a basic amino acid in the I,- or D-con-
figuration, which may be substituted in the side
chain;
C stands for a compound of the formula IIIa or IITb
G'-G'-Gly . G'-NH-(CH2)n-CO
(IITa) (IIIb),
in which
G' independently of one another denotes a radical of
'the formula IV
4 5
_ N - CH - ~ _ (IV)
in which
R° and R5 together with the atoms carrying them form a
heterocyclic mono-, bi- or tricyclic ring system having
2 to 15 carbon atoms, and
n is 2 to 8;
E stands for the radical of an aromatic amino acid;
F independently of one another denotes the radical of
a neutral, acidic or basic, aliphatic or aromatic
amino acid which may be substituted in the side
chain, or stands for a direct bond;
(D)-Phe denotes D-phenylalanine which may be optionally
substituted in the phenyl moiety;
G is as defined above for G' or denotes a direct bond;
F' is as defined for F, denotes a radical -NH-(CHZ)"-,
with n = 2 to 8, or, if G does not denote a direct
- 5 -
bond, can stand for a direct bond, and
I is -0H, -NH2 Or -NHCZHS,
K denotes the radical -NH-(CHZ)=-CO- with x = 1-4 or
stands for a direct bond, and
M is as defined for F,
and their physiologically tolerable salts.
If not stated otherwise, the abbreviation of an amino
acid radical without a stereodescriptor stands for the
radical in the L-form (compare Schroder, Liibke, The
Peptides, Volume I, New York 1965, pages XXII-XXIII;
Houben-Tflleyl, Methoden der Organischen Chemie (Methods of
Organic Chemistry), Volume XV/1 and 2, Stuttgart 1974),
such as, for example,
Aad, Abu, 7Abu, ABz, 2ABz, eAca, Ach, Acp, Adpd, Ahb, Aib,
SAlb, Ala, SAla, ~Ala, Alg, All, Ama, Amt, Ape, Apm, Apr,
Arg, Asn, Asp, Asu, Aze, Azi, Bai, Bph, Can, Cit, Cys,
Cyta, Daad, Dab, Dadd, Dap, Dapm, Dasu, Djen, Dpa, Dtc,
Fel, Gln, Glu, Gly, Guv, hAla, hArg, hCys, hGln, hGlu, His,
hIle, hLeu, hLys, hMet, hPhe, hero, hSer, hThr, hTrp, hTyr,
Hyl, Hyp, 3Hyp, Ile, Ise, Iva, Kyn, Lant, Lcn, Leu, Lsg,
Lys, sLys, ~Lys, Met, Mim, Min, nArg, Nle, Nva, Oly, 0rn,
Pan, Pec, Pen, Phe, Phg, Pic, Pro, OPro, Pse, Pya, Pyr,
Pza, Qin, Ros, Sar, Sec, Sem, Ser; Thi, ~Thi, Thr, Thy,
Thx, Tia, Tle, Tly, Trp, Trta, Tyr, Val.
Suitable radicals of a heterocyclic ring system of the
formula TV are in particular radicals of heterocycles of
the group below:
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;
~~~~~~1~'C
- 6 -
2-aza-bicyclo[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)-2,3pyrrolidine-5-
carboxylic acid];
spiro[(bicyclo[2.2.2]octane)-2,3-pyrrolidine-5-carboxylic
acid];
2~azatricyclo[4.3Ø18'9]decane-3-carboxylic acid;
decahydrocyclohepta[b]pyrrole-2-carboxylic acid;
decahydrocycloocta[b]pyrrole-2-carboxylic acid;
octahydrocyclotenta[c]pyrrole-2-carboxylic acid;
octahydroisoindole-1-carboxylic acid;
2,3,3a,4,6a-hexahydrocyclopenta[b]pyrrole-2-carboxylic
acid;
2,3,3a,4,5,7a-hexahydroindole-2-carboxylic acid;
tetrahydrothiazole-4-carboxylic acid;
isoxalidine-3-carboxylic acid; pyrazolidine-3-carboxylic
acid;
hydroxyproline-2-carboxylic acid; all of which may be
optionally substituted:
~~CO- i
i ~ CO- '
CO- Co-
N\ ; ~ ~CO ~ ~~~~CO-
i
i
~CO-
' ?k # ~~C~- i
y-CO- ; ~-CO- ; N
i i
CO-
_ i Ni~CO_ ; ;
_N
i
co- ~~CO- ; ~~CO- ;
a ~N ~N
i i
CO i ~"-CO ;
C0- ; ~~CO- ° --"
--- N ~ N ~ ~ C o- ,
i N
HO
~~#CO- ; '~
CO- i ~~" ~~-CO- ;
~~~i~
g -
The heterocycles based on the abovementioned radicals are
known, for example, from
US-A-4,344,949, US-A-4,374,847, US-A-4,350,704,
EP-A-50,800, EP-A-31,741, EP-A-51,020, EP-A-49,658,
EP-A-49,605, EP-A-29,488, EP-A-46,953, EP-A-52,870,
EP-A-271,865, DE-A-3,226,768, DE-A-3,151,690,
DE-A-3,210,496, DE-A-3,211,397, DE-A-3,211,676,
DE-A-3,227,055, DE-A-3,242,151, DE-A-3,246,503 and
DE-A-3,246,757.
Some of these heterocycles are furthermore proposed in
DE-A-3,818,850.3.
If not stated otherwise in the individual case, alkyl can
be straight-chain or branched. The same applies to
radicals derived therefrom such as alkoxy, aralkyl or
alkanoyl.
(Cs-C12)-Aryl preferably denotes phenyl, naphthyl or
biphenylyl. Radicals derived therefrom, such as aryloxy,
aralkyl or aroyl, are to be formulated correspondingly.
Halo stands for fluorine, chlorine, bromine or iodine,
preferably fox chlorine.
Possible salts are, in particular, alkali metal or
alkaline earth metal salts, salts with physiologically
tolerable amines and salts with inorganic or organic
acids such as, for example, HC1, HBr, H2SOy, H3POp, malefic
acid, fumaric acid, citric acid, tartaric acid and acetic
acid.
Preferred peptides of the formula I are those in which
B denotes Arg, Lys, Orn, 2,4-diaminabutyr~yl or an L-
homoarginine radical, where in each case the amino
or guanidino group of the side chain may be sub-
stituted by A as described under al) or a2);
~~~3~.~1~
E stands for the radical of an aromatic amino acid in
the L- or D-configuration, which contains 6 to 14
carbon atoms in the aryl moiety as ring members,
such as phenylalanine which is optionally substi-
tuted by halogen in the 2-, 3-~ or 4-position,
tyrosine, 0-methyltyrosine, 2-thienylalanine, 2-
pyridylalanine or naphthylalanine;
F' denotes the radical of a basic amino acid in the L-
or D-configuration, such as Arg or Lys, where the
guanidino group or amino group of the side chain may
be replaced by A as described under al) or a2), or
denotes a radical -NFI- ( CHZ ) n - with n = 2 to 8 and
K stands for the radical -NH-(CHZ)X-CO- with x = 2-4
or denotes a direct bond;
(D)-Phe denotes D-phenylal_anine which may be optionally
substituted in the phenyl moiety by halogen or (C1-
C4 ) -alkoxy.
Particularly preferred peptides of the formula I axe
those in which
B denotes Arg, Orn or Lys, where the guanidino group
or the amino group of the side chain is unsubsti-
tuted or may be substituted by ( Ci-Ce ) -alkanoyl, ( C~-
C13 ) -aryloyl, ( C3-C9 ) -heteroaryloyl , ( Cl-CB ) -alkylsul-
fonyl or (Cs-C12)-arylsulfonyl, where the aryl,
heteroaryl, aryloyl, arylsulfonyl and heteroaryloyl
xadicals may optionally be substituted, as described
under az), with 1, 2, 3 or 4 identical or different
radicals.
E denotes phenylalanine, 2-chlorophenylalanine,
3-chlorophenylalanine, 4-chlorophenylalanine,
2-fluorophenylalanine, 3-fluorophenylalanine,
4-fluorophenylalanine, tyrosine, 0-methyltyrosine
or ~-(2-thienyl)alanine;
- 10
K stands for a direct bond and
M stands for a direct bond and
(D)-Phe denotes D-phenylalanine which may be optionally
substituted by fluorine, chlorine, bromine or methoxy.
Very particularly preferred peptides of the formula I are
those in which
A denotes hydrogen, (D)- or (L)-H-Arg, (D)- or (L)-H-
Lys or ( D ) -- or ( L ) -H-Orn,
B denotes Arg, Orn or Lys, where the guanidine group
or the amino group of the side chain may be sub-
stituted by hydrogen, ( Cl-Ce ) -alkanoyl, ( C~-Cls ) ~-
aryloyl, (C3-C9)-heteroaryloyl, (C1-CB)-alkylsulfonyl
or (C6-C12)-arylsulfonyl, where the aryl, heteroaryl,
aryloyl, arylsulfonyl and heteroaryloyl radicals may
optionally be substituted with 1, 2, 3 or 4 identi-
cal or different radicals from the series comprising
methyl, methoxy and halogen.
C denotes Pro-Pro-Gly, Hyp-Pro-Gly or Pro-Hyp-Gly
E denotes Phe or Thia
F denotes ,er, Hser, Lys, Leu, Val, Nle, Tle or Thr
K stands for a direct bond
M stands for a direct bond
G stands for the radical of a heterocyclic ring system
of the foranula xV, where the radicals of the hetero-
cycles pyrrolidine-2-carboxylic acid; piperidine-2-
carboxylic acid; 1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid, cis- and trans-decahydroisoquino-
line-3-carboxylic acid; cis-endo-,cis-exo-,trans-
- 11 -
octahydroindole-2-carboxylic acid, cis-endo-, cis-
exo-, traps-octahydrocyclopentano[b]pyrrole-2-
carboxylic acid or hydroxyproline-2-carboxylic acid
are preferred,
F' denotes .Arg
I stands for OH and
(D)-Phe denotes D-phenylalanine.
The invention furthermore relates to a process for the
preparation of peptides of the formula I, which comprises
a) reacting a fragment having a C-terminal free car-
boxyl group ~r its activated derivative with an
appropriate fragment having an N-terminal free amino
group or
b) synthesizing the peptide stepwise, optionally,
splitting off one or more protective groups temp-
orarily introduced for the protection of other
functions in the compound obtained according to (a)
or ( b ) and optionally converting the compounds of
the formula I thus obtained into their physiologi
cally tolerable salt.
The peptides of the present invention were prepared by
generally known methods of peptide chemistry, see, for
example, Houben-Weyl, Methoden der organischen Chemie
(Methods of Drganic Chemistry), Volume 15/2, preferably
by means of solid phase synthesis such as described, for
example, by H. Merrifield, ~.Am.Chem.Soc. 85, 2149 (1963)
or R. C. Sheppard, Int. ,7. Peptide Protein Res. 21, 118
(1983) or by equivalent known methods. Urethane protec-
tive groups such as, for example, the tent-butyloxy-
carbonyl(Boc) or fluorenylmethoxycarbonyl(Fmoc)
protective group are used as «-amino protective group. If
necessary for the prevention of side reactions or for the
- 12 -
synthesis of 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"), where
primarily,
Arg(Tos), Arg(Mts), Arg(Mtr), Arg(Pmc), Asp(OBzl),
Asp(OBut), Cys(4-MeBzl), Cys(Acm), Cys(SBut), Glu(OBzl),
Glu(OBut), His(Tos), His(Fmoc), His(Dnp), His(Trt),
Lys(C1-Z), Lys(Boc), Met(O), Ser(Bz1), Ser(But), Thr-
(Bzl), Thr(But), Trp(Mts), Txp(CHO), Tyr(Br-Z), Tyr(Bzl)
or Tyr(But) are employed.
Solid phase synthesis begins at the C-terminal end of the
peptide with the coupling of a protected amino acid to an
appropriate resin. Starting materials of this type may be
obtained by linking a protected amino acid via an ester
or amide bond to a polystyrene or polyacrylamide resin
modified with a chloromethyl, hydroxymethyl, benzhydryl-
amino(BHA) or methylbenzhydrylamino(MBHA) group. The
resins used as support materials are commercially obtain- .
able. BHA and MBHA resins are usually used if the peptide
synthesized is intended to have a free amide group at the
C-terminus. If the peptide is intended to have a second-
ary amide group at the C-terminal end, a chloromethyl or
hydroxymethyl resin is used and the splitting off is
carried out using the corresponding amines. If it is
wished to obtain, for example, the ethylamide, the
peptide can be split off from the resin using ethylamine,
the splitting off of the side chain protective groups
subsequently being carried out by means of other suitable
reagents. Tf it is intended to retain the tart-butyl
protective groups of the amino acid side chain in the
peptide, the synthesis is carried out using the Fmoc
protective group for temporary blocking of the a-$mino
group of the amino acid using 'the method described, for
example, in R.C. Sheppard, J.Chem.Soc., Chem.Comm 1982,
587, the guanidino function of the arginine being protec-
ted by protonation with pyridinium perchlorate and the
protection of the other functionalized amino acids in the
- 13 -
side chain being carried out using benzyl protective
groups which can be split off by means of catalytic
transfer hydrogenation (A. Felix et al. J. Org. Chem. 13,
4194 (1978) or by means of sodium in liquid ammonia
(W. Roberts, J.Am.Chem.Soc. 76, 6203 (1954)).
After splitting off 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 subsequently
protected amino acids are successively coupled in the
desired sequence. The intermediately resulting N-terminal
protected peptide resins are deblocked by means of the
reagents described above before linkage with the subse-
quent amino acid derivative.
All possible activating reagents used in peptide syn-
thesis can be used as coupling reagents, see, for ex-
ample, Houben-Weyl, Methoden der organischen Chemie
(Methods of Organic Chemistry), Volume 15/2, in par-
ticular, however, carbodiimides such as, for example,
N,N'-dicyclohexylcarbodiimide, N,N'-diisopropyl-carbodi-
imide or N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide.
The coupling can in this case be carried out directly by
addition of amino acid derivative and the activating
reagent and, if desired, a racemization-suppressing
additive such as, for example, 1-hydroxy-benzotriazole
(HOBt) (W. KBnig, R. Geiger, Chem. Ber. 103, 708 (1970))
or 3-hydroxy-4-oxo-3,4-dihydrobenzo-triazine (HOObt)
(W. Konig, R. Geiger, Chem.Ber. 103, 2054 (1970)) to the
resin or, however, the preactivation of the amino acid
derivative as symmetrical anhydride or HOBt or HOObt
ester can be carried out separately and the solution of
the activated species in a suitable solvent can be added
to the peptide resin capable of coupling.
- 14 -
The coupling or activation of the amino acid derivative
with one of the abovementioned activating reagents can
be carried out in dimethylformamide, N-methylpyrrolidone
or methylene chloride or a mixture of the solvents men-
tinned. The activated amino acid' derivative is
customarily employed in a 1.5 to 4 fold excess. In cases
in which an incomplete coupling takes place, the coupling
reaction is repeated without previously carrying out the
deblocking of the a-amino group of the peptide resin
necessary for the coupling of the following amino acid.
The successful course of the coupling reaction can be
monitored by means of the ninhydrin reaction, such as
described, for example, by E. Kaiser et al. Anal.
Biochem. 34 595 (1970). The synthesis can also be auto-
mated, for example using a peptide synthesizer model 430A
from Applied Biosystems, it being possible either to use
the synthesis program provided by the apparatus manufac-
turer or else, however, one set up by the user himself.
The latter are in particular employed in the use of amino
acid derivatives protected with the Fmoc group.
After synthesis of the peptides in the previously des-
cribed manner, the peptide can be split off from the
resin using reagents, such as, for example, liquid
hydrogen fluoride (preferably in the peptides prepared
according to the Boc method) or trifluoroacetic acid
(preferably in the peptides synthesized according to the
Fmoc method). These reagents not only cleave the peptide
from the resin but also the other side chain protective
groups of the amino acid derivative. In this manner, the
peptide is obtained in the form of the tree acid in
addition using BHA and MBHA resins. With the BHA or MBHA
resins, the peptide is obtained as acid amide when
splitting off is carried out using hydrogen fluoride or
trifluoromethanesulfonic acid. Additional processes for
the preparation of peptide amides are described in Euron_
Patent Applications Nos. 271 865 and 322 348. The
splitting off of the peptide amides from the resin here
- 15 -
is carried out by treatment with medium strength acids
(for example tzifluoroacetic acid) usually used in
peptide synthesis, cation entrainer substances such as
phenol, cresol, thiocresol, anisole, thioanisole, ethane-
dithiol, dimethyl sulfide, ethyl methyl sulfide or
similar cation entrainers customary in solid phase
synthesis being added individually or as a mixture of two
or more of these auxiliaries. In this case, the tri-
fluoroacetic acid can also be used diluted by suitable
solvents, such as, for. example, methylene chloride.
If the tart-butyl or benzyl side chain protective groups
of the peptides are to be retained, the splitting off of
the peptide synthesized on a particularly modified
support resin is carried out using 1~ trifluoroacetic
acid in methylene chloride, such as described, far
example, in R.C. Sheppard J.Chem. Soc., Chem. Comm. 1982,
587. If individual tart-butyl or benzyl side chain
protective groups are to be retained, a suitable
combination of synthesis and splitting off methods is
used.
For the synthesis of peptides having a C-terminal amide
grouping or an w-amino or w-guanidinoalkyl grouping, the
modified support resin described by Sheppard is likewise
used. After the synthesis, the peptide fully protected in
the side chain is split off from the resin and subse
quently reacted with the appropriate amine or w-amino
alkylamine or w-guanidinoalkylamine in classical solution
synthesis, it being possible for optionally present
additional functional groups to be temporarily protected
in a known manner.
An additional process for the preparation of peptides
having an w-aminoalkyl grouping is described in EP-A
2S4 802.
The peptides of the present invention were preferably
synthesized by two general protective group tactics using
- 16 -
the solid phase technique:
The synthesis was carried out using an automatic peptide
synthesizer model 430 A from Applied Biosystems, with Boc
or Fmoc protective groups for temporary blockage of the
a-amino group.
When using the Boa protective group, the synthesis cycles
pre-programmed by the manufacturer of the apparatus were
used for the synthesis.
The synthesis of the peptides having a free carboxyl
group on the C-terminal end was carried out on a 4-
(hydroxymethyl)phenylacetamidomethylpolystyrene resin
functionalized with the corresponding Boc amino acid
(R. B. Merrifield, J. Oxg. Chem. 43, 2845 (1978)) from
Applied Biosystems. An MBHA resin from the same firm was
used for the preparation of the peptide amides.
N,N'-Dicyclohexylcarbodiimide or N,N'-diisopropylcarbodi-
imide were used as activating reagents. Activation was
carried out as the symmetrical anhydride, as the HOBt
ester or HOObt ester in CHZC12, CHZC12 - DMF mixtures or
NMP. 2-4 equivalents of activated amino acid derivative
were employed for the coupling. zn cases in which the
coupling took place incompletely, the reaction was
repeated.
During the use of the Fmoc protective group for the
temporary protection of the a-amino group, our own
synthesis programs were used for synthesis using the
automatic peptide synthesizer model 430A from Applied
Biosystems. The synthesis was carried out on a p-ben-
zyloxybenzyl alcohol resin (S. Wang, J.Am.Chem.Soc. 9~,
1328 (1973)) from Bachem which was esterified by a known
method (E. Atherton et al. J.C.S. Chem. Comm. 1981, 336)
using the appropriate amino acid. The activation of the
amino acid derivatives as HOBt or HOObt esters was
carried out directly in the amino acid cartridges pro-
vided by the apparatus manufacturer by addition of a
- 17 -
solution of diisopropylcarbodiimide in DPZF' to the pre-
viously weighed-in mixture of amino acid derivative and
HOBt or HOObt. Fmoc-amino acid-OObt esters prepared in
substance can likewise be employed as described in EP-A-
247 573. The splitting off of the Fmoc~protective group
was carried out using a 20~ strength solution of
piperidine in DMF in the reaction vessel. The excess of
reactive amino acid derivative used was 1.5 to 2.5
equivalents. If the coupling was not complete, it was
repeated as in the Boc method.
The peptides according to the invention have, individu-
ally or in combination, a bradykinin antagonist action
which can be tested in various models (see Handbook of
Exp. Pharmaaol. Vol. 25, Springer Verlag, 1970, p. 53-
55), for example on the isolated rat uterus, on the
guinea pig ileum or on the isolated pulmonary artery of
the guinea pig.
For testing the peptides according to the invention on
the isolated arteria pulmonalis, guinea pigs (Dunkin
Hartley) having a weight of 400 - 450 g are sacrificed
by a blow to the back of the neck.
The thorax is opened and the arteria pulmonalis is
carefully dissected out. The surrounding tissue is
carefully removed and the arteria pulmonalis is cut
spixally at an angle of 45°.
The vessel strip of 2.5 cm length and 3-4 mm width is
fixed in a 10 ml capacity organ bath which is filled with
Ringer solution.
Composition of the solution in mmol/l
NaCl 154
KC1 5.6
CaCl2 Z . g
NaHC03 2 . 4
Glucose 5.0
2~~3~.~~~
- 18 -
955 OZ and 5~ COZ is bubbled through the solution, which
is warmed to 37°C. The pH is 7.4 and the preload on the
vessel strip is 1.0 g.
The isotonic contraction changes are 'detected using a
lever arrangement and an HF modem (position sensor) from
Hugo Sachs and recorded on a compensating recorder (BEC,
Goerz Metrawatt SE 460).
After equilibration for 1 hour, the experiment is begun.
After the vessel strips have achieved their maximum
sensitivity to 2 x 10~' mol/1 of bradykinin - bradykinin
leads to a contraction of the vessel strips - the pep-
tides axe allowed to act for 10 minutes in each case in
the doses 5 x 10-8 - 1 x 10-5 mol/1 and, after adding
bradykinin again, the decrease in the effect of bradykin-
in as opposed to the control is compared.
For the detection of a partial agonistic effect, the
peptides are used in the doses 1 x 10-5 - 1 x 10-3 mol/1.
The ICSO values of the peptides according to the invention
calculated from the dose-effect curves are shown in Table
1.
Table 1:
Compound IC54 ~M)
H-(D)-Arg-Arg-Pro-Myp-Gly-Phe-Ser-(D)-Phe-Oic-Arg-OH l.4xi0-8
The therapeutic utility of the peptides according to the
invention includes all pathological states which are
mediated, caused or supported by bradykinin and brady-
kinin-related peptides. This includes, inter alia,
traumas, such as wounds, burns, rashes,' erythemas,
edemas, angina, arthritis, asthma, allergies, rhinitis,
shock, inflammations, low blood pressure, pain, itching
and changed sperm motility.
19 -
The invention therefore also relates to the use of
peptides of the formula I as medicaments, and to pharma-
ceutical preparations which contain these compounds.
Pharmaceutical preparations contain an'effective amount
of the active substance of the formula T - individually
or in combination - together with an inorganic or organic
pharmaceutically utilizable excipient.
Administration can be carried out enterally, parenterally
- such as, for example, subcutaneously, i.m. or i.v. -,
sublingually, epicutaneausly, nasally, rectally, intra-
vaginally, intrabuccally or by inhalation. The dosage of
the active substance depends on the mammal species, the
body weight, age and on the manner of administration.
The pharmaceutical preparations of the present invention
are prepared in solution, mixing, granulating or tablet
coating processes known per se.
For oral administration or application to the mucosa, the
active compounds are mixed with the customary additives
for this, such as excipients, stabilizers or inert
diluents, and brought into suitable forms for admini-
stration, such as tablets, coated tablets, hard gelatin
capsules, aqueous, alcoholic or oily suspensions or
aqueous, alcoholic or oily solutions, by customary
methods. Tnert excipients which may be used are, for
example, gum arabic, magnesia, magnesium carbonate,
potassium phosphate, lactose, glucose, magnesium stearyl
fumarate or starch, in particular maize starch. In this
case, the preparation may be present both as dry and
moist granules. Suitable oily excipients or solvents are,
for example, vegetable or animal oils, such as sunflower
oil and cod liver oil.
A preparation for topical application may be present as
an aqueous or oily solution, lotion, emulsion or gel,
ointment or fatty ointment or, if possible, in spray
- 20 -
form, it being possible to improve the adhesion, if
desired, by addition of a polymer.
For the intranasal form of administration, the compounds
are mixed with the customary auxiliaries for this, such
as stabilizers or inert diluents, and brought into suit-
able forms for administration, such as aqueous, alcohol-
ic ox oily suspensions or aqueous, alcoholic or oily
solutions, by customary methods. Chelating agents,
ethylenediamine-N,N,N°,N°-tetraacetic acid, citric acid,
tartaric acid or their salts may be added to aqueous
intranasal preparations. Administration of the nasal
solutions can be carried out by means of metered atom-
izers or as nasal drops, having a viscosity-increasing
component, or nasal gels or nasal creams.
For administration by inhalation, atomizers or
pressurized gas packs using inert carrier gases can be
used.
For intravenous, subcutaneous, epicutaneous or intra-
dermal administration, the active compounds or their
physiologically tolerable salts, if desired with the
pharmaceutically customary auxiliaries, for example for
isotonisizing or adjusting pH, and solubilizers, emul-
sifiers or other auxiliaries, are brought into solution,
suspension or emulsion.
Because of the short half-lives of some of the medica-
ments described in body fluids, the use of injectable
sustained release preparations :i.s efficient. Medicament
forms which may be used are, for example, oily crystal
suspensions, microcapsules, rods or implants, it being
possible to synthesize the latter from tissue-compatible
polymers, in particular biodegradable polymers, such as,
for example, those based on polylactic acid/ polyglycolic
acid copolymers or human albumin.
A suitable dose range for forms for topical application
- 21 -
and administration by inhalation are solutions containing
0.01-5 mg/ml, and with forms for systemic administration
0.01-10 mg/kg is suitable.
List of abbreviations:
The abbreviations used for amino acids correspond to the
three-letter code customary in peptide chemistry as
described in Burop. J. Biochem. 138, 9 (1984). Addition-
ally used abbreviations are listed below.
Acm Acetamidomethyl
e-A.hx e-Aminohexanoyl
Aoc cis, endo-2-Azabicyclo[3.3.0]octane-3-S-
carbonyl
Boc tart-Butyloxycarbonyl
But tart-Butyl
Bzl Benzyl
CDF Chloro-(D)-phenylalanyl
Cha Cyclohexylalanyl
Chg Cyclohexylglycyl
C1-Z 4-Chlorobenzyloxycarbonyl
DMF Dimethylformamide
DOMT 0-Methyl-(D)-threonyl
Dnp 2,4-Dinitrophenyl
Fmoc 9-Fluorenylmethoxycarbonyl
MDY 0-Methyl-(D)-tyrosyl
Me Methyl
4-Mebzl 4-Methylbenzyl
Mtr 4-Methoxy-2,3,6-trimethylphenylsulfonyl
Mts Mesitylene-2-sulfonyl
Nal Napthylalanyl
NMP N-Methylpyrrolidine
Npg Neopentylglycyl
Oic cis-endo-.octahydroindol-2-ylcarbonyl
Opr Tsoxazolidin-3-ylcarbonyl
Pal Pyridylalanyl
Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl
Tbg tart-Butylglycyl
TFA Trifluoroacetic acid
~~~J~~G~
- 22
Tcs 4-Methylphenylsulfonyl
Thia 2-Thienylalanyl
Tic 1,2,3,4-Tetrahydroisoquinolin-3-ylcarbonyl
Trt Trityl
The following examples are intended to illustrate the
preferred methods for solid phase synthesis of the
peptides according to the invention, without limiting the
invention thereto.
The amino acid derivatives below were used:
Fmoc-Arg(Mtr)-OH, Boc-(D)-Arg-OH, Fmoc-Arg(Pmc)-OH,
Fmoc-Hyp-0H, Fmoc-Pro-OObt, Fmoc-Gly-OObt, Fmoc-Phe-OObt,
Fmoc-Ser{tBu)-OObt, Fmoc-(D)-Phe-OH, Fmoc-G1n-OH,
Fmoc-Aoc-OH, Fmoc-Thia-OH, Fmoc-Opr-OH, Fmoc- {D)-Asn-OH,
Fmoc-s-Ala-OH, Fmoc-Oic-OH.
H- (D)-Arg-Arg-Pro-Hyp-Gly-Phe-Ser- (D)-Phe-Oic-Arg-OH
Example 1:
H-(D)-Arg-Arg-Pro-Hyp-Gly-Phe-Ser-(D)-Phe-Oic-Arg-OH
was synthesized stepwise using a peptide synthesizer
model 430 A from Applied Biosystems by the Fmoc method on
a p-benzyloxybenzyl alcohol resin from Novabiochem
(loading about 0.5 mmol/g of resin) esterified with Fmoc-
Arg(Mtr)-OH. 1 g of the resin was employed and the
synthesis was carried out with the aid of a synthesis
program modified for the Fmoc method.
In each case 1 mmol of the amino acid derivative having
a free carboxyl group together with 0.95 rmnol of HOObt
was weighed into the cartridges of the synthesizer. The
preactivation of these amino acids was carried out
directly in the cartridges by dissolving in 4 ml of DMF
and adding 2 ml of a 0.55 mol solution of diisopropyl
carbodiimide in DMF.
The HOObt esters of the other amino acids were dissolved
in 6 ml of NMP and then similarly coupled to the resin
previously deblocked using 20~ piperidine in DMF, like
the amino acids preactivated in situ. After completion of
- 23 -
the synthesis, the peptide was split off from the resin
using thioanisole and ethanedithiol as cation entrainers,
with simultaneous removal of the side chain protective
groups using txifluoroacetic acid. The residue obtained
after stripping off the trifluoroacetic~acid was repeat-
edly digested with ethyl acetate and centrifuged. The
residue which remained was chromatographed on ~Sephadex
LH 20 using 10~ strength acetic acid. The fractions
containing the pure peptide were combined and freeze-
dried.
MS(FAH) . 1292.4 (M+H)
