CA1270100A - Peptides having an immunostimulating action, processes for their preparation and their use - Google Patents
Peptides having an immunostimulating action, processes for their preparation and their useInfo
- Publication number
- CA1270100A CA1270100A CA000521657A CA521657A CA1270100A CA 1270100 A CA1270100 A CA 1270100A CA 000521657 A CA000521657 A CA 000521657A CA 521657 A CA521657 A CA 521657A CA 1270100 A CA1270100 A CA 1270100A
- Authority
- CA
- Canada
- Prior art keywords
- lys
- arg
- radicals
- val
- tyr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
The invention relates to peptides of the formula II
in which W represents Lys (R1), D-lys (R1), Arg (R2) or D-Arg (R2), X represents Lys (R1), D-Lys (R1), Arg (R2) or D-Arg (R2), Y represents Asp (R3), D-Asp (R3), Glu (R3) or D-Glu (R3), U represents Val or D-Val, D represents Tyr, D-Tyr, Trp, D-Trp, Phe or D-Phe, and in which R1 represents a urethane-protective group, R2 represents a protective group for the guanidino function and R3 represents an ester-protective group, and wherein (a) all five radicals a represent peptide bonds or (b) one of the radicals a represents a C-terminal OH together with an N-terminal H
(a=OH H-) of two aminoacid radicals and the remaining four radicals a each denote peptide bonds, and processes for their preparation and their use. The peptides of the invention can be used for the synthesis of peptides of the Formula I. The latter can be used for the treatment of immunodeficiencies, viral, fungoidal and chronic bacterial infections and autoimmune diseases and for the therapy of diseases which are caused by cells with immunologically relevant changes in the cell membrane characteristics (for example tumor cells).
The invention relates to peptides of the formula II
in which W represents Lys (R1), D-lys (R1), Arg (R2) or D-Arg (R2), X represents Lys (R1), D-Lys (R1), Arg (R2) or D-Arg (R2), Y represents Asp (R3), D-Asp (R3), Glu (R3) or D-Glu (R3), U represents Val or D-Val, D represents Tyr, D-Tyr, Trp, D-Trp, Phe or D-Phe, and in which R1 represents a urethane-protective group, R2 represents a protective group for the guanidino function and R3 represents an ester-protective group, and wherein (a) all five radicals a represent peptide bonds or (b) one of the radicals a represents a C-terminal OH together with an N-terminal H
(a=OH H-) of two aminoacid radicals and the remaining four radicals a each denote peptide bonds, and processes for their preparation and their use. The peptides of the invention can be used for the synthesis of peptides of the Formula I. The latter can be used for the treatment of immunodeficiencies, viral, fungoidal and chronic bacterial infections and autoimmune diseases and for the therapy of diseases which are caused by cells with immunologically relevant changes in the cell membrane characteristics (for example tumor cells).
Description
~2701~
This Application is a Divisional of Canadian Patent Application Serial Number 460,240, Filed August 2, 19840.
Se~eral pept;des ~for example thymosin and thymo-poiet;n II), wh;ch contr;bute to d;fferent;ation ("matu-rat;on') of thymus-dependent lymphocytes ~T cells) have been ;solated frGm thymus extracts. A part sequence of thymopo;etin II, Arg-Lys-Asp Val-Tyr, also exhib;ts a thymopo;et;n-l;ke action in the corresponding test sys-temsr for example the rosette test (Sc;ence t1979) 204, 1309 to 1310).
Bes;des the rosette test mentioned, the P;~A sti-- mulation test and the plaque-form;ng cell test (PFC test), ~hich is spec;fic for B lymphocytes, are also suitable for investigating the influence of smaller peptides on ~ymphocytes.
In the PHA stimulation test (P~A - phytohemagglu tinin~ or the lymphocyte transformation test, conclusions as to the number of mature, i.e. stimulatable, lympho-cytes are drawn not v;a invest;gat;on of the surface anti gens but by the function test for ability to be stimu!a-ted by the plani lectin PHA. The lymphocytes (T cells) are stimulated to vesicular transformation by the lectin, in the same way as by bacterial or viral antigens. This leads to prol;feration, either directly or by the release o, lymphokins. The incorporation of the radiolabeled thymidine within a certain time ;s then a measure of the number of stimulated cells. Only the mature or imMunoiog~
ically potent r cells are stimulated. The influence of 2 ,, ' ' ,' ~
: :.,~' :
lZ7C:~O() `
substance on lymphocyte maturation can therefore be m~ni-tored w;th th;s test. Ho~ever, the st;mu~at;on must be sub-optimum, since other sub-populations of lymphocytes are also st;mu~ated at h;gher concentrat;ons and the effect can no lonser be observed. The peptides to be ;nvest;gated have been added to the culture med;um ;n var;ous concentrations. They are present throughout the ent;re duration of the test ~up to 72 hours).
In the PFC test, ce~l cu~tures of freshLy dissec-ted spleens of mice (2 x 107 spleen cells per ml) areplaced in RPMI 1640 and 30 ~l of fetal caLf serum (FCS) per mL of cell culture. The in vitro immunization is effected ~;th 5 x 10~ sheep erythrocytestml. The test cell cultures are ;ncubated daily with the corresponding dose of the test substance.
After a running time of 5 days, the ce~s are centrifuged off and ~ashed ~ith medium RPMI 164û and the direct PFC test is carried out. For this, the cells are mixed ~ith a 10X sheep erythrocyte suspen-sion in an agarose solution and the mixture is poured ontoa flat surface. In the layer of gel fcrmed, the stimu~a-ted lymphocytes release antibodies during subsequent incù-bation, and these diffuse into the environment and attach themselves to the sheep erythrocytes therein. After addi-t;on of guinea pig comp~ement, the red b~ood ce~ls lyse.Light colored round spots which can be seen w;th the naked eye form in the reddish-brown gel, i.e. the hemo~ys;s areolas of the plaques. An antibody-producing cèll is found in the center of such hemolysis areo~as. The num-; ~ . `
~: . .... .
~` '- :
. .
.
.
.
- , - :
, ,: , ~- .
lZ701 0~
., .
ber of Lympho;d cells wh;ch form specific immunoglobu~ins can consequently be equated with the numbers of plaques found.
NoYel cyclic peptides have now been found which, surprisingly, have a superior action to the known action of the linear peptide Arg-Lys-Asp-Val-Tyr.
The invention relates to cyclopeptides of the formula I
- A - B - C - U D - ~I) in wh;ch A denotes Lys, D-Lys, Arg or D-Arg, 8 denotes Lys, D-Lys, Arg or D-Arg, C denotes Asp~ D-Asp, Glu or D-Glu, U denotes Val or D-Val and D denotes Tyr, D-Tyr, Trp, D-Trp, Phe or D-Phe, 1 to 4 of the radicals A, 8, C, U and D being ;n the L-configurat;on and the remainder being in the D-configu-ration, and to physiologically acceptable salts thereof.
Preferred cyclopeptides of the formula I are those in ~hich U denotes Val, and furthermore those in which, in formula I, A denotes D-Lys or Arg, 8 denotes Lys, D-Lys or Arg, C denotes Asp or Glu, U denotes D-Yal and D denotes Tyr, Trp or D-Phe.
The invention furthermore relates to a process for the preparation of these cyclopeptides, which com-prises cyclizing linear peptide derivatives of the for-''~
~2~
-- 5mula II
- W - a - X - a - Y - a - U - a - D - a - (II) in which W denotes Lys (R1), D-Lys ~R1), Arg (R2) or D-Arg tRZ), X denotes Lys ~R1), D-Lys (R1), Arg (R2) or D-Arg (R2) and Y denotes Asp (R3), D-Asp (R3)~ Glu (R3) or D-Glu (R3) and U and D are as defined above, and in which R1 represents a urethane-protective group, RZ represents a protective group fGr the guanidino function and R represen~s an ester-protective group, and one of the radicals a denotes the C-terminal OH together with the N-terminal H (a = -OH H-) of two aminoacid radi-cals and the remaining Four radicals a each denote pep-tide bonds, and then removing, in a manner which isknown per se, the protective groups from the resulting compounds of the formula II, in which ~, X, Y, U, D, R1, R2 and R3 are as defined above~ all the five radicals a represent peptide bonds and 1 to 4 of the radicals W,-X~ Y, U and D are in the L-con-figuration and the re-mainder are ;n the D-conf;gurat;on, and, ;f appropriate, converting the resulting cyclopeptides of the ,ormula I
into their physiologically acceptable salts.
~ The linear cyclization precursors of the formula II are understood as meaning peptides of the following formulae III to VII
H - ~1 - X - Y - U - D - OH (III) H - D -- ~1 X ~ Y - U - OH (IV) H - U - D - ~1 - X -- Y - OH (V ) ..
: , . , . ~ `
: :: .. ~: .
. ,, .:: :: : ,.
::
.
OiOO
~5 ` - H - Y - U - D - W - X - OH ~VI) X - X - Y - U - D - W - OH ~VII) ;n ~h;ch W, X, Y, U, D, R1, R2 and R3 are as defined above.
The peptides of the formulae II to VI are prefer-ably cycl;zed ;n the presence of coupl;ng reagents, such as DCC or EDCI, w;th addition of DMAP.
Examples of urethane-protective groups of the -amino function of the lysine are Fmoc, Fcbocr Z, Boc, Ddz, Bpoc, Z-(N02), Pyoc, Dobz, Moc, Mboc, Iboc, Adoc, Adpoc, Msc or Pioc; Z or ~oc is preferred. These amino-protecti~e groups are removed w;th ac;ds or bases or by reduct;on (cf.
Kontakte Merck 3/79, page 14 et seq.).
Examples of protect;ve groups for the guan;d;no group of the arg;nine are N02, tosyl, Boc, 7, mesitylene-
This Application is a Divisional of Canadian Patent Application Serial Number 460,240, Filed August 2, 19840.
Se~eral pept;des ~for example thymosin and thymo-poiet;n II), wh;ch contr;bute to d;fferent;ation ("matu-rat;on') of thymus-dependent lymphocytes ~T cells) have been ;solated frGm thymus extracts. A part sequence of thymopo;etin II, Arg-Lys-Asp Val-Tyr, also exhib;ts a thymopo;et;n-l;ke action in the corresponding test sys-temsr for example the rosette test (Sc;ence t1979) 204, 1309 to 1310).
Bes;des the rosette test mentioned, the P;~A sti-- mulation test and the plaque-form;ng cell test (PFC test), ~hich is spec;fic for B lymphocytes, are also suitable for investigating the influence of smaller peptides on ~ymphocytes.
In the PHA stimulation test (P~A - phytohemagglu tinin~ or the lymphocyte transformation test, conclusions as to the number of mature, i.e. stimulatable, lympho-cytes are drawn not v;a invest;gat;on of the surface anti gens but by the function test for ability to be stimu!a-ted by the plani lectin PHA. The lymphocytes (T cells) are stimulated to vesicular transformation by the lectin, in the same way as by bacterial or viral antigens. This leads to prol;feration, either directly or by the release o, lymphokins. The incorporation of the radiolabeled thymidine within a certain time ;s then a measure of the number of stimulated cells. Only the mature or imMunoiog~
ically potent r cells are stimulated. The influence of 2 ,, ' ' ,' ~
: :.,~' :
lZ7C:~O() `
substance on lymphocyte maturation can therefore be m~ni-tored w;th th;s test. Ho~ever, the st;mu~at;on must be sub-optimum, since other sub-populations of lymphocytes are also st;mu~ated at h;gher concentrat;ons and the effect can no lonser be observed. The peptides to be ;nvest;gated have been added to the culture med;um ;n var;ous concentrations. They are present throughout the ent;re duration of the test ~up to 72 hours).
In the PFC test, ce~l cu~tures of freshLy dissec-ted spleens of mice (2 x 107 spleen cells per ml) areplaced in RPMI 1640 and 30 ~l of fetal caLf serum (FCS) per mL of cell culture. The in vitro immunization is effected ~;th 5 x 10~ sheep erythrocytestml. The test cell cultures are ;ncubated daily with the corresponding dose of the test substance.
After a running time of 5 days, the ce~s are centrifuged off and ~ashed ~ith medium RPMI 164û and the direct PFC test is carried out. For this, the cells are mixed ~ith a 10X sheep erythrocyte suspen-sion in an agarose solution and the mixture is poured ontoa flat surface. In the layer of gel fcrmed, the stimu~a-ted lymphocytes release antibodies during subsequent incù-bation, and these diffuse into the environment and attach themselves to the sheep erythrocytes therein. After addi-t;on of guinea pig comp~ement, the red b~ood ce~ls lyse.Light colored round spots which can be seen w;th the naked eye form in the reddish-brown gel, i.e. the hemo~ys;s areolas of the plaques. An antibody-producing cèll is found in the center of such hemolysis areo~as. The num-; ~ . `
~: . .... .
~` '- :
. .
.
.
.
- , - :
, ,: , ~- .
lZ701 0~
., .
ber of Lympho;d cells wh;ch form specific immunoglobu~ins can consequently be equated with the numbers of plaques found.
NoYel cyclic peptides have now been found which, surprisingly, have a superior action to the known action of the linear peptide Arg-Lys-Asp-Val-Tyr.
The invention relates to cyclopeptides of the formula I
- A - B - C - U D - ~I) in wh;ch A denotes Lys, D-Lys, Arg or D-Arg, 8 denotes Lys, D-Lys, Arg or D-Arg, C denotes Asp~ D-Asp, Glu or D-Glu, U denotes Val or D-Val and D denotes Tyr, D-Tyr, Trp, D-Trp, Phe or D-Phe, 1 to 4 of the radicals A, 8, C, U and D being ;n the L-configurat;on and the remainder being in the D-configu-ration, and to physiologically acceptable salts thereof.
Preferred cyclopeptides of the formula I are those in ~hich U denotes Val, and furthermore those in which, in formula I, A denotes D-Lys or Arg, 8 denotes Lys, D-Lys or Arg, C denotes Asp or Glu, U denotes D-Yal and D denotes Tyr, Trp or D-Phe.
The invention furthermore relates to a process for the preparation of these cyclopeptides, which com-prises cyclizing linear peptide derivatives of the for-''~
~2~
-- 5mula II
- W - a - X - a - Y - a - U - a - D - a - (II) in which W denotes Lys (R1), D-Lys ~R1), Arg (R2) or D-Arg tRZ), X denotes Lys ~R1), D-Lys (R1), Arg (R2) or D-Arg (R2) and Y denotes Asp (R3), D-Asp (R3)~ Glu (R3) or D-Glu (R3) and U and D are as defined above, and in which R1 represents a urethane-protective group, RZ represents a protective group fGr the guanidino function and R represen~s an ester-protective group, and one of the radicals a denotes the C-terminal OH together with the N-terminal H (a = -OH H-) of two aminoacid radi-cals and the remaining Four radicals a each denote pep-tide bonds, and then removing, in a manner which isknown per se, the protective groups from the resulting compounds of the formula II, in which ~, X, Y, U, D, R1, R2 and R3 are as defined above~ all the five radicals a represent peptide bonds and 1 to 4 of the radicals W,-X~ Y, U and D are in the L-con-figuration and the re-mainder are ;n the D-conf;gurat;on, and, ;f appropriate, converting the resulting cyclopeptides of the ,ormula I
into their physiologically acceptable salts.
~ The linear cyclization precursors of the formula II are understood as meaning peptides of the following formulae III to VII
H - ~1 - X - Y - U - D - OH (III) H - D -- ~1 X ~ Y - U - OH (IV) H - U - D - ~1 - X -- Y - OH (V ) ..
: , . , . ~ `
: :: .. ~: .
. ,, .:: :: : ,.
::
.
OiOO
~5 ` - H - Y - U - D - W - X - OH ~VI) X - X - Y - U - D - W - OH ~VII) ;n ~h;ch W, X, Y, U, D, R1, R2 and R3 are as defined above.
The peptides of the formulae II to VI are prefer-ably cycl;zed ;n the presence of coupl;ng reagents, such as DCC or EDCI, w;th addition of DMAP.
Examples of urethane-protective groups of the -amino function of the lysine are Fmoc, Fcbocr Z, Boc, Ddz, Bpoc, Z-(N02), Pyoc, Dobz, Moc, Mboc, Iboc, Adoc, Adpoc, Msc or Pioc; Z or ~oc is preferred. These amino-protecti~e groups are removed w;th ac;ds or bases or by reduct;on (cf.
Kontakte Merck 3/79, page 14 et seq.).
Examples of protect;ve groups for the guan;d;no group of the arg;nine are N02, tosyl, Boc, 7, mesitylene-
2-su~fonyl ~Mts) and the like. The protective groups can be split off hydrolytically or hydrogenolytically (cf.
Kontakte Merck 1/80, pages 23 and 30).
The COOH side functions of Asp and Glu are blocked ;n the form of alkyl esters, preferably methyl, ethyl or tert.-butyl esters, or as benzy~ esters or modif;ed benzyl esters (p-N02, p-Cl, p-Br and the ~;ke~. Deblocking ;s effected by alkal;ne or acid hydrolysis or hydrogenation (cf. Kontakte Merck 3/79, pages 14 and 2~). The allyl ester, which is easy to remove by reaction Nith metal catalysts, is also suitable (cf. Angew. Chem. 96, ~9 (18l~)) The invention furthermore re~ates to peptides of the formula II
`
..
.. . .
: ' ' , ~ .
,-, ,, -~LZ7~
, - - W - a - X - a - Y - a - U - a - D - a - (II) in ~hich W, X, Y, U~ D, R1, ~2 and R3 have the mean;ngs def;ned above and a~ all five radicals a represent peptide bonds or b) one of the radicals a denotes a C-terminal OH together wi-h an N-term;nal H (a = -OH H-) of two aminoacid radi-cals W and X, X and Y, Y ancl U, U and D, or D and W, and the remaining four radicaLs a each denote peptide bonds.
The linear peptides of the formula II (one radi-cal a = -OH H~) can be prepared either conventionally Gr by means of sol;d phase synthesis.
In the conventional peptide synthesis of the abo~e linear pept;des, build-up through fragments (convergent) may be the most rational route. A linear, protected penta-peptide is thereby obtained, from ~hich the linear pep-tides of the formulae III - VII can be obtained by split-ting-off the C-'an'd N-terminal protective groups. In the first example, Trp occupies the advan,ageous N-terminal position and is thus exposed to a minimum number of split-tins off reactions on protective groups, whict) frequently lead to side reactions in the case of Trp. Tyr could also be used in un?rotected form ;n this position, since the minimum number of coupling steps or o-F splitting-ofF reac-t;ons on protective groups makes a reaction of the pheno-lic OH group ;mprobable.
The selection of the protective groups and the synthesis strategy are determine'd by the nature and con-fi~uration of the aminoacids and by the nature of the .:
. -. . .. .
'.. -.' '': ~ , :
~;; :
: , ~;~701~'~
~ coupling and cyclization conditions.
The peptides of the formula I accord;ng to th~
invention contain a few polyfunctional aminoacids wh;ch require an increased use of the protective group tech-nique (orthogonal protective group tact;cs?. In conven-tional peptide synthesis (by fragment condensation), three protective groups uhich can be split off selectively are therefore required to build up the linear precursors of the formulae III - VII. All the polyfunctional am;no-acids are used in orthogonally protected form, i.e. w;tha permanent protective group in the side cha;n and a C-terminal or N-terminal protective group which can be spl;t select;vely to th;s group, for bu;ld;ng up the peptide cha;n. The am;noacids w;thout s;de cha;n funct;onal;~y are used in C- or N-terminally protected form. The cor-respond;ng aM;noacid derivat;ves are linked using a CGUp-ling reagent, such as PPA or DCC. After selective split-ting off of one of the C- or N-terminal protect;ve groups of the dipeptide fragment~ the missing C- or N-terminally protected aminoacid derivatives or fragments prepared in the same manner are condensed on until the des;red, fully protected l;near pentapept;de has been prepared. Split-ting off o-f the terminal protective gruups in as far as possible one step then gives the linear precursors III -VII~
The C-terminal and N-terminal protective groups are chosen so that only one splitting-off step is neces-sary for the preparat;on of the l;near, de-protected penta-pept;des of the formulae III - VII from the fully protected .
'~ ~
. .,.. :' '.... ~ .' ~7~
_ 9 _ peptide. Such a combination is~ for example, the Boc protective group and the tert.-butyl ester, both of which can be split off in one step, for example by trifluoro-acetic acid.
Since slobal de-protection of the side chain func-tions appears advantageous after the cyclizat;on, protec-tive groups with the same instability towards particular splitting-off reagents are preferably used there. Thus, for example, the ~-Z-protective group for lysine, the ~-benzyl ester for Asp and the ~-N02 group on the Arg can be split off in one step hydrogenolytically with the aid of a hydrogenation catalyst tfor example PD-on-active charcoal).
For cyclization of the linear peptides according to formula III - ~II, a novel cyclization method has been developed~ since other cyclization methods gave only unsat;sfactory amounts of the pentapeptides of the for-mula I according to the invention. In the novel process, the linear precursors according to formulae III - VII are dissolved in aprotic solvents, such as DMF, CH2Cl2, CHCl3, THF, dioxane or ~ixtures thereof.
In the case of salts of the linear compounds of the formulae III - VII, these are neutralized by addition of one equivalent of a base. Cyclization reagents ~Jhich can be used are, for example, DCC or EDCI (about 20 equi-valents) in combination with about 10 equivalents of the acylation catalyst DMAP. Both substances are 2dded to the reaction solution at -15C, and this mixture is left at between -2C and ~2SC for 6 days. The crude pro~
:
'; . ''- , : :" .
' ;;
~Z7~
- 10 ~
duct can then be purified, for example, by gel chromato-graphy or the like.
After the cyclization, the remaining protective groups are split off from the resulting peptides of the formula II tall the rad;cals a denote pept;de bonds).
In the cyclizat;on of the linear peptides of the formulae III - VII, in the cases where the N- and C-terminaL aminoac;ds of these linear precursors have the same absolute configuration, a configuration reversal on the C~terminal aminoacid wiLl take place.
Equation:
N-Terminal C-Terminal Reversa~
A H ~ -~o~ -- ~ OH
.
No reversall ~
H ~ -For~ orm~OH ~ 4L-FormL--~o .
: reversal~ ~
C H ~ ~~o ~ OH ~ ~ ~ o!L-~or ~ J
Re v e r s a ( l ¦
D H~ ~ OH ~ L
The examples which follow ;llustrate this synthe-sis principle ~ith the aid of a few selected compounds.
7~
.
C .
o .~ ~ ~ ~ Q ~
~ ^ ^ ^ ^ N N ~ ^ ~ ^ ^ N
~ ~ ~ ,~ m ~
., N N N N O O :~ N N N N O
_ m m m m --~ ~, I m m m m --u O O O 0 3 ~ ~-- O O O O _ ~ :~ Q ~ U N ~ . 3 R. C:~
~ ~ I I m ~ v~
Q CO ¢ ~ ¢ ^ ^ O c~ ¢ C~) ¢ ^
C IIII~`--IIII~
" ~ ¢ ~ o o :~
_~ U) ~ u~ ) Z. Z ~1 ~, ~~ n ~ ~ ~ .~
f~^ ^ ¢ ¢ o ~ ~ ~ ~~ ~ ~ ~ ~ ~ l l z _l o o ~ ~ ` -~ Z ~; Z Z; Z ~ I :Z :z; ~ ~ hO
c~ `~ ' O O L
bD bD t~ Z ¢
S.. S.. S . S.. S~ S.~ O S~ S.~
O ¢ '1: ¢ ¢ ¢ C :Z ¢ ¢ o~ U) O
S~ L L S~ L S~ ~ L L
. ~ ~ ~ ~ ~, S~
~J .IIIIIIIII.I
O ~ l l l l l l l l l l l VOO'OOOOOOOOO
Q ~1 ~ I ~1 ~1 ~1 ~ ~ ~ ~ ~ ~ ~
` C~ VC~ V C) C) C~ Cl C>
.
. ~ O O O O O O . ' O O (~ O ~ --t O ~ ~ S~ S~
, ~1 ~1~1 ~ ~ L ~ ~ E~
I I I ~ ~ I I I n~ ~ ~
^ ~ N Nr-l--` ~ ~ ~ N
~ ~ ~ ~ m m ~ ~ ~ I I ~
N N N N O O :~ N N --` ~ O
m m m m ~ I m m ~ ~ _~
O O O 0 3 3 ^ O O N N 3
Kontakte Merck 1/80, pages 23 and 30).
The COOH side functions of Asp and Glu are blocked ;n the form of alkyl esters, preferably methyl, ethyl or tert.-butyl esters, or as benzy~ esters or modif;ed benzyl esters (p-N02, p-Cl, p-Br and the ~;ke~. Deblocking ;s effected by alkal;ne or acid hydrolysis or hydrogenation (cf. Kontakte Merck 3/79, pages 14 and 2~). The allyl ester, which is easy to remove by reaction Nith metal catalysts, is also suitable (cf. Angew. Chem. 96, ~9 (18l~)) The invention furthermore re~ates to peptides of the formula II
`
..
.. . .
: ' ' , ~ .
,-, ,, -~LZ7~
, - - W - a - X - a - Y - a - U - a - D - a - (II) in ~hich W, X, Y, U~ D, R1, ~2 and R3 have the mean;ngs def;ned above and a~ all five radicals a represent peptide bonds or b) one of the radicals a denotes a C-terminal OH together wi-h an N-term;nal H (a = -OH H-) of two aminoacid radi-cals W and X, X and Y, Y ancl U, U and D, or D and W, and the remaining four radicaLs a each denote peptide bonds.
The linear peptides of the formula II (one radi-cal a = -OH H~) can be prepared either conventionally Gr by means of sol;d phase synthesis.
In the conventional peptide synthesis of the abo~e linear pept;des, build-up through fragments (convergent) may be the most rational route. A linear, protected penta-peptide is thereby obtained, from ~hich the linear pep-tides of the formulae III - VII can be obtained by split-ting-off the C-'an'd N-terminal protective groups. In the first example, Trp occupies the advan,ageous N-terminal position and is thus exposed to a minimum number of split-tins off reactions on protective groups, whict) frequently lead to side reactions in the case of Trp. Tyr could also be used in un?rotected form ;n this position, since the minimum number of coupling steps or o-F splitting-ofF reac-t;ons on protective groups makes a reaction of the pheno-lic OH group ;mprobable.
The selection of the protective groups and the synthesis strategy are determine'd by the nature and con-fi~uration of the aminoacids and by the nature of the .:
. -. . .. .
'.. -.' '': ~ , :
~;; :
: , ~;~701~'~
~ coupling and cyclization conditions.
The peptides of the formula I accord;ng to th~
invention contain a few polyfunctional aminoacids wh;ch require an increased use of the protective group tech-nique (orthogonal protective group tact;cs?. In conven-tional peptide synthesis (by fragment condensation), three protective groups uhich can be split off selectively are therefore required to build up the linear precursors of the formulae III - VII. All the polyfunctional am;no-acids are used in orthogonally protected form, i.e. w;tha permanent protective group in the side cha;n and a C-terminal or N-terminal protective group which can be spl;t select;vely to th;s group, for bu;ld;ng up the peptide cha;n. The am;noacids w;thout s;de cha;n funct;onal;~y are used in C- or N-terminally protected form. The cor-respond;ng aM;noacid derivat;ves are linked using a CGUp-ling reagent, such as PPA or DCC. After selective split-ting off of one of the C- or N-terminal protect;ve groups of the dipeptide fragment~ the missing C- or N-terminally protected aminoacid derivatives or fragments prepared in the same manner are condensed on until the des;red, fully protected l;near pentapept;de has been prepared. Split-ting off o-f the terminal protective gruups in as far as possible one step then gives the linear precursors III -VII~
The C-terminal and N-terminal protective groups are chosen so that only one splitting-off step is neces-sary for the preparat;on of the l;near, de-protected penta-pept;des of the formulae III - VII from the fully protected .
'~ ~
. .,.. :' '.... ~ .' ~7~
_ 9 _ peptide. Such a combination is~ for example, the Boc protective group and the tert.-butyl ester, both of which can be split off in one step, for example by trifluoro-acetic acid.
Since slobal de-protection of the side chain func-tions appears advantageous after the cyclizat;on, protec-tive groups with the same instability towards particular splitting-off reagents are preferably used there. Thus, for example, the ~-Z-protective group for lysine, the ~-benzyl ester for Asp and the ~-N02 group on the Arg can be split off in one step hydrogenolytically with the aid of a hydrogenation catalyst tfor example PD-on-active charcoal).
For cyclization of the linear peptides according to formula III - ~II, a novel cyclization method has been developed~ since other cyclization methods gave only unsat;sfactory amounts of the pentapeptides of the for-mula I according to the invention. In the novel process, the linear precursors according to formulae III - VII are dissolved in aprotic solvents, such as DMF, CH2Cl2, CHCl3, THF, dioxane or ~ixtures thereof.
In the case of salts of the linear compounds of the formulae III - VII, these are neutralized by addition of one equivalent of a base. Cyclization reagents ~Jhich can be used are, for example, DCC or EDCI (about 20 equi-valents) in combination with about 10 equivalents of the acylation catalyst DMAP. Both substances are 2dded to the reaction solution at -15C, and this mixture is left at between -2C and ~2SC for 6 days. The crude pro~
:
'; . ''- , : :" .
' ;;
~Z7~
- 10 ~
duct can then be purified, for example, by gel chromato-graphy or the like.
After the cyclization, the remaining protective groups are split off from the resulting peptides of the formula II tall the rad;cals a denote pept;de bonds).
In the cyclizat;on of the linear peptides of the formulae III - VII, in the cases where the N- and C-terminaL aminoac;ds of these linear precursors have the same absolute configuration, a configuration reversal on the C~terminal aminoacid wiLl take place.
Equation:
N-Terminal C-Terminal Reversa~
A H ~ -~o~ -- ~ OH
.
No reversall ~
H ~ -For~ orm~OH ~ 4L-FormL--~o .
: reversal~ ~
C H ~ ~~o ~ OH ~ ~ ~ o!L-~or ~ J
Re v e r s a ( l ¦
D H~ ~ OH ~ L
The examples which follow ;llustrate this synthe-sis principle ~ith the aid of a few selected compounds.
7~
.
C .
o .~ ~ ~ ~ Q ~
~ ^ ^ ^ ^ N N ~ ^ ~ ^ ^ N
~ ~ ~ ,~ m ~
., N N N N O O :~ N N N N O
_ m m m m --~ ~, I m m m m --u O O O 0 3 ~ ~-- O O O O _ ~ :~ Q ~ U N ~ . 3 R. C:~
~ ~ I I m ~ v~
Q CO ¢ ~ ¢ ^ ^ O c~ ¢ C~) ¢ ^
C IIII~`--IIII~
" ~ ¢ ~ o o :~
_~ U) ~ u~ ) Z. Z ~1 ~, ~~ n ~ ~ ~ .~
f~^ ^ ¢ ¢ o ~ ~ ~ ~~ ~ ~ ~ ~ ~ l l z _l o o ~ ~ ` -~ Z ~; Z Z; Z ~ I :Z :z; ~ ~ hO
c~ `~ ' O O L
bD bD t~ Z ¢
S.. S.. S . S.. S~ S.~ O S~ S.~
O ¢ '1: ¢ ¢ ¢ C :Z ¢ ¢ o~ U) O
S~ L L S~ L S~ ~ L L
. ~ ~ ~ ~ ~, S~
~J .IIIIIIIII.I
O ~ l l l l l l l l l l l VOO'OOOOOOOOO
Q ~1 ~ I ~1 ~1 ~1 ~ ~ ~ ~ ~ ~ ~
` C~ VC~ V C) C) C~ Cl C>
.
. ~ O O O O O O . ' O O (~ O ~ --t O ~ ~ S~ S~
, ~1 ~1~1 ~ ~ L ~ ~ E~
I I I ~ ~ I I I n~ ~ ~
^ ~ N Nr-l--` ~ ~ ~ N
~ ~ ~ ~ m m ~ ~ ~ I I ~
N N N N O O :~ N N --` ~ O
m m m m ~ I m m ~ ~ _~
O O O 0 3 3 ^ O O N N 3
3 Q. 3 S, V C7 N 3 C~. O O C~
¢ C~:l ¢ ^ ^ O (~ 3 C~. ^
M ~ C~l ~J ~ ~n ~) ~ ~ I I
U~ u~ l I u~ U~ N N ,_~
O O
N
^ V) ^ ^ b~D ~ O
~ , ~ ~ S., S~ :Z
OOOOOO~OO5C`-'Z 2; Z 2; ~. Z I
. !~ L S~ S~ S~ ~ O S~ S~
cc¢¢¢¢¢z¢¢~
~ . S
S~ L C~ S~ bD S~ S~
~ ~ ~ S~ :~ ~ I I I
E~ E~E~ E~ E I E~ '1: ~ ~ ,q ~1 ~_ I I1. 1 a.) X
D . ~_ ~ _~
~s ~ ~ c) 'a a~ c~ bl~ S ~ t -. , . -:,:. ~::. , .
, ' ~; ' '"', :. ' ~,,'-, ' "
- , :,' :~.: ~
.. ~ ' .
- ~.Z7~
~ As can be seen from the above equation, the cyc-lization products have the same configurations on the cyc-lization site independently of ~rhether A or B ~as used as the starting substance. Corresponding statements apply to C and D.
The cyclopeptides listed above under a) - g) con-tain, after the cyclization ~rith DCC(EDCI)/DMAP, the C-- terminal aminoacid in the linear precursor as a D-amino-acid. The linear educts here contain no D-aminoacid or a D-aminoacid in the center of the sequence.
The chiral;ty of the C-terminal aminoacid has been determined as a D-aminoacid in the cyclopeptides under a) and f) by chiral aminoacid analysis~ The NMR
spectra leave no doubt that the cyclopeptides under b) -e) can also be concluded from these data. ~gnor1ng theconfiguration, the cyclopeptide under g) has the same sequence as that under b), but the other cyclization site leads to another stereoisomeric product.
Insertions of the above cyclopeptides already con-tain D-aminoacids in the linear educts in the C- or in the ~-terminal position. No racemiz3tion or inversion of the C-terminal aminoacids is found here. The cyclopep-tides under h) and i) correspond to the cyclopeptides under a) and b) prepared from all L-aminoacid precursors~
The cyclopeptide in position j) sho~s no D-Tyr in the chiral arninoacid analysis. The cyclopeptide in posit;on l~ shows clear similarities with the cyclopeptide in posi-tion 9~ in the 1H-NMR spectrum. The cyclopeptides under a), j) and l) are also obtained by azide cyclization ~low ~: :
.
:
,. .
.
~2~
~ -13 -degree of racemization~.
Physiologically acceptable salts of the cjclo-peptldes of the formula I according to the inven~ion are understood as meaning~ in particular, acid addition salts ~ith inorganic acidsr such as HCl, HN03, H2S04 or H3P04, or organic acids, such as tartaric acid, citric acid or maleic ac;d, or base add;t;on salts with alkali metal hydroxides or alkaline earth metal hydroxides or physio-logically acceptable am;nes.
The cyclopept;des accord;ng to the invention have been tested for their lyrnphocyte-stimulating act;on in the plaque-forming cell assay (PFC test) and in the phyto-hemagglu.;n;n stimulat;on test (PHA test).
Poss;ble degradation of the pep'tides by serum or lyrnphocyte proteases is'not taken into consideration in these investigations.
The compounds according to the invention can be used for the treatment of immunodeficiencies, viral, fungoidal and chronic bacter;al ;nfections and auto;mmune diseases and for the therapy of diseases which are caused by cells w;th irnmut70logically relevant changes in the cell membrane characteristics (for example tumor cells~.
The invent;on furthermore relates qu;te generally to -the use of the pept;des ment;oned for influenc;ng matu-ration of T lymphocytes, and to ayents wh;ch contain thesepeptides'as the active ;ngred;ent.
The peptides accord;ng to the invent;on can be used intravenously, subcutaneously or intranasally. In adults of norrnaL weiaht, the dosage on parenteral adminis-., . -;
-.
.
tration is 0.001 - 10 mg, and that on ;ntranasal adminis-tration is 0.01 - 10 mg, per individual dose. In serious cases, it can also be increased, since no tox;c proper-ties have as yet been observed. It is also possible to reduce the dose.
The compounds according to the ;nvention can be administered intranasally or parenterally in a correspon-ding pharmaceutical formulation For an intranasa~ use form, the compounds are mixed with the additives custom-ary for this purpose, such as stabilizers or inert dilu-ents, and are brought ;nto suitable adm;nistrat;on forms, such as aqueous, alcohol;c or oily suspens;ons or aqueous, alcoholic or oily solutions, by customary methods. Ex-amples of poss;ble oily exc;pients or solvents are vege-table or animal oils, such as sunflower Q;l or cod l;ver o; ~.
For subcutaneous or ;ntravenous administration~the active compounds or phys;olog;cally acceptable salts thereof are d;ssoLved, suspended or emulsi~ied, ;f desired with the substances customary for th;s purpose, such as solub;l;zing agents, emuls;f;ers or other add;tives~
Examples of possible solvents for the novel act-ive compounds and corresponding physiologically acceptable salts are~ ater, physiological saline solutions or alco-hols, for example ethanol, propaned;ol or glycerol, and ;n addit;on also sugar solut;ons, such as glucose or mannitol solutions, or also a mixtwre o~ the var;ous sol-vents mentioned~
The test results which follo~ (Table 2) g;ve a :
L27C~1~1i0 - selection of the biological activity of the peptides according to the invention in the PFC and PHA tests.
T'able 2: Dosage 100 ng~substance/1 ml of cell culture PFC/106 PHA stimulation cells index 10 ~9 of PHA/1 ml 72 hours Control (untreated) 221 + 30 1.00 Arg-Lys-Asp-Val-Tyr i'98 + 43 1.05 cyclo-(Arg-Lys-Asp-D-Val-Tyr) 401 + 64 1.69 10 cyclo-(Arg-Lys-Glu-D-Val-Tyr) 687 + 73 1r9Z
cyclo-(D-Lys-Arg-Glu-Val-Tyr) 317 + 41 1.15 cyclo-(Arg-Lys-Glu-D-Val-Trp) 369 + 54 1~25 cyclo-(Arg-Lys-Asp-D-Val-Trp) 312 + 58 cyclo tD-Lys-Arg-Asp-Val-Tyr) 341 + 43 ~-15 cyclo-tArg-Lys-Glu-Val-D-P'ne) 250 + 31 The following synthesis examples are intended for fur.her illustration, ~ithout the invention be;ng restr;c-ted to these examples:
Example 1 Pre'para-ion of cyclo-(Arg-Lys-Asp-D-Val-Tyr)'2HOAc t1) according to synthesis scheme I
a) Boc-ArgtN02)-LystZ)-OMe t3) 8.25 9 (25 mmol) of H-Lys~Z~-OMe-HCl and 7.98 9 t25 mmol) of Boc-Arg(N02)-OH are suspended in 100 ml of Z5 CH2Cl2, the suspension is cooled to -15C and 12.94 ml tO.118 mol) of NMM are added. 22 9 (50.5 mmol) of PPA
solution t50% in CH2Cl2, 0.88 g/mmol) are then added dropwise under an N2 atmosphere. After the mixture has been warmed slowly, it is stirred at room temperature for ~ .
..
-- ,. . .
... ..
' ~ I
. . .
7~
2 days.
The solvent ;s stripped off on a rotary evaporator and the residue is partitioned between 300 ml of EE and 150 ml of sat. NaHC03 solution. The organic phase is 5 washed twice with 80 ml of sat. NaHC03 solution each time, three times with 80 ml of 5X strength citric acid solu-tion each time and again with 80 ml of sat. NaCl solutionO
The combined EE phases are dried over MgS04, the solvent is stripped off and the residue is dried to give a sol;d 10 foam.
Yield: 13.8 9 (937~); Rf A 0.31; B 0.54; C 0.71;
~20 = _7.44 (c = 1, abs. MeOH) Cz6H21B709 (595.35) calculated C 52.44 H 6.94 N 16.47 found 52.33 6.8Z 16.20 15 b) Ddz-AsptO8zl~-Val-OBut (43 The peptide coupling is carried out by the PPA
method as described for a)O
Batch: 13.4 9 (30 mmol) of Ddz-Asp(OBzl)-OH and 10.36 g (30 mmo l~ of H-Val-OBut-TsOH.
The product is obtained as an oil.
Yield: 16 g (88'~); Rf A 0.68; B 0.69; C 0075 c) H-Arg(N02)-Lys(~)-OMeHCl ~5~
To split off the Boc protective group, 9.53 g (16 mmol) of 3 are stirred with 1bO ml of 2 N HCl/MeOH at 25 room temperature for 2 hours. The soLution is then con-centrated and the residue is taken up in MeOH and eva-porated again, th;s procedure being repeated several timesO
The product is recrystallized from EtOHfdiethyl etherO
Yield: 8.2 9 ~97%) , :. '' ' ~ ',:
:, , ~ ~
~27~
M.p.: 165-168C; Rf A 0.0, B 0.41; C 0.34;
[~]20 = +9.61 ~c - 1, abs. MeOH) C2lH32N7Q7Cl (529.74) calcuLated C 47.61 H 6.0B N 1~.51 found ~7.90 6.30 18~55 d~ H-Asp~oszl)-val-oBut TFA (6~
To spl;t off the Ddz protective group, 12 g (ZO
mmol) of 4 are d;ssolved in 130 ~l of CH2Cl2, and 4~6 ml (60 mmol) of TFA are added t3.5~ of TFA/CH2Cl2).
10 The solution ;s st;rred at room temperature for 30 minutes and the solvent is stripped off. The residue is recrystall;zed from d;ethyl ether/petroleum ether~
Yield: 9.15 9 (93%);
M.p.~ 113-116C; Rf A 0.25; B 0.68; C 0.70;
Cq~2o = -3~56 ~c = 1, abs. MeOH) e) Boc-Tyr-Arg~N02)-Lys~)-OMe t7~
The peptide coupl;ng ;s carr;ed out by the PPA
method as described under a).
Batch: 5.55 9 ~12 mmol) of Boc-Tyr-OH~DCHA and 6D35 g (12 mmol) of 5.
The product is recrystall;zed from EtOH/diethyl ether.
Yield: 8.65 9 (95%);
M.p.: 128-130C; Rf A 0.15; B 0.80; C 0085;
~]~Q = -6.8 (c = 1, abs. MeOH) f) Boc-Tyr-Arg(N02)-Lys(Z)-OH t8) To hydrolyz~ the methyl ester, B.5 9 (11 mmol) of 7 are dissoLved in 33 ml of MeOH, and 26 ml of 1 N NaOH
are added. The aqueous solution ;s acidified to pH 2 .: .
- , - ' : :
,;
': :~ '''~' 3LZ70~
w;th 2 N HCl and is extracted three times uith 80 ml of EE each time. The aqueous phase is saturated w;th NaCl and extracted a further three times with 80 ml of EE each time. The combined organ;c phases are dried over Na2S04.
The solvent is removed and the residue is dried to give a solid foam.
Yield: 7.4 g (90%); Rf A 0.19; ~ 0.57; 0.48;
~]g = -5.6~ ~c = 1, abs. MeOH)--g) Boc-Tyr-Arg(N02)-Lys(Z)-Asp(OBzl)-Val-OBut (9) The peptide coupling is carried out by the P?A
method as described for a).
Batch: 4.46 g (6 mmol) of 8 and 3.25 9 (6.6 mmol) of 6.
The crude product obtained after working up is purified by gel chromatography on Sephadex LH 20 with DMF as the 1S eluting agent. The product is recrystalLized from EtOH~
diethyl e~her.
Yield: 3.6 g (54X);
M~p.: 108-111S; Rf A 0.0; B 0.91; C 0.84;
~J20 = - 19.4 (c 1, abs~ MeOH) 20 C54H76N10015 (1105.18) caiculated C 58.69 H 6.92 N 12.67 found 58.44 6.7812.S4 h) H-Tyr-ArgtN02)-Lys~I)-Asp(OBzl)-Val-OH TFA (10) - For sin~ultaneous splitting off of the Boc and tert.-butyl ester protect;ve groups~ 7.4 ml (96 mmol) of TFA are added to 2.65 9 (2.4 mmol) of 9 and the mixture is stirred under an N2 atmosphere at room temperature for 10 minu.es. The solution is taken up in 50 ml of CH2Cl2 and the mixture is evaporated. The product ;s then recrystallized from MeOH/diethyl ether and dried .; ' ~Z7~
over KO~I.
Yield: 2.39 g (95~);
M.p.: 158--160C, Rf A O n O; B 0.63; C O.57 i) cyclo-(Tyr-Arg(N02)-Lys(Z)-AsptOBzl?-D-Val) (11) - 5 2.1 g (2.0 mmol) of 11 are dissolved in a mixture of 300 ml of DMF and 1 7 liters of CH2Clz (= 1 mmol of peptide per liter of solvent) and the solution is cooled to -15C. 0.18 ml (2 mmol) of NMM and 2.44 g t20 mmol) s~f DMAP are added, and 3.83 g (20 mmol) of EDCI dissolved in 25 ml of DMF, are added dropwise to the solution. The reaction solution is left to stand at -Z/5C for 3 days and is then cooled to -15C and subsequently activated ~ith 3.83 g (20 mmol) of EDCI. After two days at -2/+5C
and one day at room temperature, the react;on solution is evaporated in vacuo. 300 ml of H20 are added to the oi~y residue ~hich remains after concentration, and the preci-pitate ~hich has separated out is filtered off with suc-tion. The resulting crude product is purified by gel chromatography on Sephadex LH 20 with DMF as the eluting agent,. and the resulting product is recrystallized from DMF/MeOH/diethyl ether.
Yield: 558 mg (30%); m.p.: 238-242C (decomposition);
Rf A 0.0; B 0.88; C 0.92;
Co~]2~0 = -45.58 (c = 0.6 DMF) C45H58N10012 (930.95) calculated C 58.05 H 6.27 N 15.05 found 57.85 6.07 15.08 j) cy~clo-(Tyr-Arg-Lys-A~'~ 2HOAc= cyclo-tArg-Lys-Asp-D-Val-Tyr)~Z tlOAc (1) To spl;t off the side chain protective groups, ,~
' : , , ~ -- ::~ .:: . , :. . .
- ~z~a~
~ 0 93 mg (0.1 mmol) of 11 are dissolved in 2 ml of MeOH and 5 ml of HOAc, 80 mg of catalyst (10% strength Pd-on-active charco-al) are added and hydrogenation is carried' out at room temperature for 6 hours. The catalyst is fil-tered off, the solut;on is concentrated and the residueis dried over KOH.
Yield: 63 mg (81%); m~p.: 205C (decomposit;on);
Rf A 0.0; a 0.22; C 0.0; D 0.43;
aminoacid analysis: Asp 1.0; D-VaL 1~01; Tys 0.81; Lys 1.00; Arg 0.97 content: 97X
Example 2 - Preparation of cyclo-tArg-Lys-Glu-D Val-Tyr~^2 HOAc (2) according to synthesis scheme I
' The desired compound is prepared by the same ex-perimental procedures as desc.ibed for Exarnple 1. Compounds uhich are required for the preparation of 2 and have already been described in Example 1 are designated with the figures used therein.
a) Ddz-Glu(08zl)-Val-OBut t12) The peptide coupling is carried out by the PPA
method as described in Example 1 under a).
Batch: 15.1 9 (32 mmol) of Ddz-Glu(06zl)-OH and 11.4 g (32 mmol) of tl-Val-OBut~TsOH~ The product is obtained as an oil.
Yield: 18.3 g t~3X); Rf A 0.70; B 0~65; C 0~78.
b) H-GlutOBzl)-V~I-O~t~ (13~
Splitting off of the Ddz protective group is as described in Example 1 under d~
~, -. , , , ' ~.'.
;
~L~7~
Batch: 17.8 9 (29 mmol) of 12, 6.7 ml (87 mmoL) of TFA
and 188 mmol of CH2Cl2~
The product is recrystallized from d;ethyl ether/
petroleum ether.
Yield: 12~7 9 (86C~); m.p.: 132-136C; Rf A 0.2;
B 0.65; C On70;
~]Z- = -0.23 (c = 1, MeOH) C23H33N27F3 (506.49) calcuLated C 54.54 H ~.56 N 5.58 found 54.82 6.40 5.53 10 c) Boc-Tyr-Arg(N02)-L~/s(Z~-GlytOBzl)-Val-Osut t14) The peptide coupl;ng and purification of the crude product are carried out as described in Example 1 under g).
Batch: 5.96 9 (18 mmol) of 8 and 5.06 9 (10 mmol) of 13.
The product is recrystallized from MeOH/EE.
Yield: 6.1 9 ~68%~; m.pu: 149-153C;
Rf A t).1; B 0.85; C 0.,1;
rcy]2o = - 20.84 ~c = 1, MeOH) C55H7~N10015 (1119.2) calculated C 59.02 H 7.02 N 12.52 found 58.78 6.94 12.29 d) H-Tyr-Arg(N02-Lys(Z)-Glu(OBzl)-Val-OH~TFA (15) The procedure is as described in Example 1 under h).
Batch: 2.75 g t2.45 mmol') of 14 and 7.6 ml (98 mmol) of TFA.
Yield: 2.4 g (92%~;
Rf A 0.00; B 0.63; C 0.58.
e) cyclo-(Tyr-ArgtN02)-LystZ)-Glu(OBzl~-D-Va l? (16) ~he cycli ation ;s carried out as described in Example 1 under i~.
.:
Batch: 2.4 9 (2.2 mmol) of 15, O.Z ml t2.2 mmol) of NMM, 2.68 9 (22 mmol) of DMAP and 8.4 9 (44 mmol) of EDCI.
The product obtained after gel chromatography is recrystall;~ed from DMF/MeOH/diethyl e~her~
Yie~d: 686 mg (33%); m.p.: 234-238C (decomposition) Rf A 0.00; B 0.80; C 0.86;
[~20 = _ 58.46 (c = 0.26, DMF);
- C46H60N10o12 (944.98) calculated 58.47 H 6 39 N 14 8Z
found 57.916.52 14.94 - FAB-MS: 945 f) cyclo-(Tyr-Arg-Lys-Glu-D-Val)-2 HOAc = cyclo-(Arg-Lys-Glu-D-Val-Tyr)~2HOAc (2) Splitting off of the side chain protective groups is as described in Example 1 under j).
Batch: 94 mg (0.1 mmo;) of 16;
Yield: 65 mg ~82~); m.p.: 140-142C (decomposition);
Rf A 0.00; B 0.22; C 0.00; D 0.44.
A~linoacid analysis: Glu 0~98; D-Val 1.01; Tyr 0.84;
Lys 0.98; Arg 1.00 content: 92X.
.
'' ~ ~ ` :'.' ' '~
.. . . .
.
Tyr Arg Lys X'. ~al ~ ~ ' I I .
i l ~2 ~ Z
3 - Boc ~ OH H ~ OMe-HCl .; ~ I I ~
~ ~ ~N2 3 ~ Z ~ T
Boc ~/ - ----,~ OMe ¦ 3 .1 -- I I I ~ .
l ~M2 5 ¦ Z ¦ OBzl Boc ~ OH H ~ OMe~HCl Ddz ~ OH H ~OtBu TsOH
} 5~'2 7 ¦ Z ¦ OBzl 4/12 ¦
Boc ~ / O~e Ddz~/ 3- OtBu I
'' I ., ~ ' ' I ~ ..
¦NO2 8 ~ Z ~ OBzl 6~13 . Boc ~ ~ 5/ OH H-~ OtBuoTF~
.~ I :' 1, 1 ~
i I I 9/14 ¦ OBzl Boc _,r _ . r/ / ~ ~-OtBu ¦ ¦ N02 ¦ Z lO/15 ¦/OBZ
.H ~ f ~ OHTFQ
I ~/N2 ~Z 11/16¦~ OBzl cyclo-/ . _ D-~al /
~ /2 1 ~
t ~ ~
cyclo,~yr Ar~ Lys _ X' = ~2HOAc X ' = AsptG lu Synthes i s s cheme I
- . ; . ..
: -,- ~
.
~Z7~
Abbreviations for protective groups and reagents Boc = tert.-butoxycarbonyl Bzl = benzyl DCC = d;cyclohexylcarbodiimide DCHA = dicyclohexylamine Ddz = o~-dimethyL-3~5-d;methoxybpnzyloxycarbonyl DMAP = 4-dimethylaminopyridine DMF = N,N-o'imethylformamide EDCI = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EE = acetic ester (ethyl acetate) EtOH = ethanol HOAc = acetic acid MeOH = methanol NMM = N-methylmorphol;ne OMe = methoxy OBut = tert.-butoxy PPA = n-propylphosphon;c acid anhydride TsOH = p-toluenesulfonic acid ~ = carbobenzoxycarbonyl Abbreviations ;n the text abs. = absolute sat. = saturated m.p. = melt;ng po;nt Eluting agents ~or thin-layer chromatography A chloroformJmethanol/glacial acetic acid (95:5:3) B n-butanol/gtacial acetic ac;d/water C ethyl acetate/n-butanol/pyridine/water ~20:100305 D methanol/glacial acetic acid ~
;'..
¢ C~:l ¢ ^ ^ O (~ 3 C~. ^
M ~ C~l ~J ~ ~n ~) ~ ~ I I
U~ u~ l I u~ U~ N N ,_~
O O
N
^ V) ^ ^ b~D ~ O
~ , ~ ~ S., S~ :Z
OOOOOO~OO5C`-'Z 2; Z 2; ~. Z I
. !~ L S~ S~ S~ ~ O S~ S~
cc¢¢¢¢¢z¢¢~
~ . S
S~ L C~ S~ bD S~ S~
~ ~ ~ S~ :~ ~ I I I
E~ E~E~ E~ E I E~ '1: ~ ~ ,q ~1 ~_ I I1. 1 a.) X
D . ~_ ~ _~
~s ~ ~ c) 'a a~ c~ bl~ S ~ t -. , . -:,:. ~::. , .
, ' ~; ' '"', :. ' ~,,'-, ' "
- , :,' :~.: ~
.. ~ ' .
- ~.Z7~
~ As can be seen from the above equation, the cyc-lization products have the same configurations on the cyc-lization site independently of ~rhether A or B ~as used as the starting substance. Corresponding statements apply to C and D.
The cyclopeptides listed above under a) - g) con-tain, after the cyclization ~rith DCC(EDCI)/DMAP, the C-- terminal aminoacid in the linear precursor as a D-amino-acid. The linear educts here contain no D-aminoacid or a D-aminoacid in the center of the sequence.
The chiral;ty of the C-terminal aminoacid has been determined as a D-aminoacid in the cyclopeptides under a) and f) by chiral aminoacid analysis~ The NMR
spectra leave no doubt that the cyclopeptides under b) -e) can also be concluded from these data. ~gnor1ng theconfiguration, the cyclopeptide under g) has the same sequence as that under b), but the other cyclization site leads to another stereoisomeric product.
Insertions of the above cyclopeptides already con-tain D-aminoacids in the linear educts in the C- or in the ~-terminal position. No racemiz3tion or inversion of the C-terminal aminoacids is found here. The cyclopep-tides under h) and i) correspond to the cyclopeptides under a) and b) prepared from all L-aminoacid precursors~
The cyclopeptide in position j) sho~s no D-Tyr in the chiral arninoacid analysis. The cyclopeptide in posit;on l~ shows clear similarities with the cyclopeptide in posi-tion 9~ in the 1H-NMR spectrum. The cyclopeptides under a), j) and l) are also obtained by azide cyclization ~low ~: :
.
:
,. .
.
~2~
~ -13 -degree of racemization~.
Physiologically acceptable salts of the cjclo-peptldes of the formula I according to the inven~ion are understood as meaning~ in particular, acid addition salts ~ith inorganic acidsr such as HCl, HN03, H2S04 or H3P04, or organic acids, such as tartaric acid, citric acid or maleic ac;d, or base add;t;on salts with alkali metal hydroxides or alkaline earth metal hydroxides or physio-logically acceptable am;nes.
The cyclopept;des accord;ng to the invention have been tested for their lyrnphocyte-stimulating act;on in the plaque-forming cell assay (PFC test) and in the phyto-hemagglu.;n;n stimulat;on test (PHA test).
Poss;ble degradation of the pep'tides by serum or lyrnphocyte proteases is'not taken into consideration in these investigations.
The compounds according to the invention can be used for the treatment of immunodeficiencies, viral, fungoidal and chronic bacter;al ;nfections and auto;mmune diseases and for the therapy of diseases which are caused by cells w;th irnmut70logically relevant changes in the cell membrane characteristics (for example tumor cells~.
The invent;on furthermore relates qu;te generally to -the use of the pept;des ment;oned for influenc;ng matu-ration of T lymphocytes, and to ayents wh;ch contain thesepeptides'as the active ;ngred;ent.
The peptides accord;ng to the invent;on can be used intravenously, subcutaneously or intranasally. In adults of norrnaL weiaht, the dosage on parenteral adminis-., . -;
-.
.
tration is 0.001 - 10 mg, and that on ;ntranasal adminis-tration is 0.01 - 10 mg, per individual dose. In serious cases, it can also be increased, since no tox;c proper-ties have as yet been observed. It is also possible to reduce the dose.
The compounds according to the ;nvention can be administered intranasally or parenterally in a correspon-ding pharmaceutical formulation For an intranasa~ use form, the compounds are mixed with the additives custom-ary for this purpose, such as stabilizers or inert dilu-ents, and are brought ;nto suitable adm;nistrat;on forms, such as aqueous, alcohol;c or oily suspens;ons or aqueous, alcoholic or oily solutions, by customary methods. Ex-amples of poss;ble oily exc;pients or solvents are vege-table or animal oils, such as sunflower Q;l or cod l;ver o; ~.
For subcutaneous or ;ntravenous administration~the active compounds or phys;olog;cally acceptable salts thereof are d;ssoLved, suspended or emulsi~ied, ;f desired with the substances customary for th;s purpose, such as solub;l;zing agents, emuls;f;ers or other add;tives~
Examples of possible solvents for the novel act-ive compounds and corresponding physiologically acceptable salts are~ ater, physiological saline solutions or alco-hols, for example ethanol, propaned;ol or glycerol, and ;n addit;on also sugar solut;ons, such as glucose or mannitol solutions, or also a mixtwre o~ the var;ous sol-vents mentioned~
The test results which follo~ (Table 2) g;ve a :
L27C~1~1i0 - selection of the biological activity of the peptides according to the invention in the PFC and PHA tests.
T'able 2: Dosage 100 ng~substance/1 ml of cell culture PFC/106 PHA stimulation cells index 10 ~9 of PHA/1 ml 72 hours Control (untreated) 221 + 30 1.00 Arg-Lys-Asp-Val-Tyr i'98 + 43 1.05 cyclo-(Arg-Lys-Asp-D-Val-Tyr) 401 + 64 1.69 10 cyclo-(Arg-Lys-Glu-D-Val-Tyr) 687 + 73 1r9Z
cyclo-(D-Lys-Arg-Glu-Val-Tyr) 317 + 41 1.15 cyclo-(Arg-Lys-Glu-D-Val-Trp) 369 + 54 1~25 cyclo-(Arg-Lys-Asp-D-Val-Trp) 312 + 58 cyclo tD-Lys-Arg-Asp-Val-Tyr) 341 + 43 ~-15 cyclo-tArg-Lys-Glu-Val-D-P'ne) 250 + 31 The following synthesis examples are intended for fur.her illustration, ~ithout the invention be;ng restr;c-ted to these examples:
Example 1 Pre'para-ion of cyclo-(Arg-Lys-Asp-D-Val-Tyr)'2HOAc t1) according to synthesis scheme I
a) Boc-ArgtN02)-LystZ)-OMe t3) 8.25 9 (25 mmol) of H-Lys~Z~-OMe-HCl and 7.98 9 t25 mmol) of Boc-Arg(N02)-OH are suspended in 100 ml of Z5 CH2Cl2, the suspension is cooled to -15C and 12.94 ml tO.118 mol) of NMM are added. 22 9 (50.5 mmol) of PPA
solution t50% in CH2Cl2, 0.88 g/mmol) are then added dropwise under an N2 atmosphere. After the mixture has been warmed slowly, it is stirred at room temperature for ~ .
..
-- ,. . .
... ..
' ~ I
. . .
7~
2 days.
The solvent ;s stripped off on a rotary evaporator and the residue is partitioned between 300 ml of EE and 150 ml of sat. NaHC03 solution. The organic phase is 5 washed twice with 80 ml of sat. NaHC03 solution each time, three times with 80 ml of 5X strength citric acid solu-tion each time and again with 80 ml of sat. NaCl solutionO
The combined EE phases are dried over MgS04, the solvent is stripped off and the residue is dried to give a sol;d 10 foam.
Yield: 13.8 9 (937~); Rf A 0.31; B 0.54; C 0.71;
~20 = _7.44 (c = 1, abs. MeOH) Cz6H21B709 (595.35) calculated C 52.44 H 6.94 N 16.47 found 52.33 6.8Z 16.20 15 b) Ddz-AsptO8zl~-Val-OBut (43 The peptide coupling is carried out by the PPA
method as described for a)O
Batch: 13.4 9 (30 mmol) of Ddz-Asp(OBzl)-OH and 10.36 g (30 mmo l~ of H-Val-OBut-TsOH.
The product is obtained as an oil.
Yield: 16 g (88'~); Rf A 0.68; B 0.69; C 0075 c) H-Arg(N02)-Lys(~)-OMeHCl ~5~
To split off the Boc protective group, 9.53 g (16 mmol) of 3 are stirred with 1bO ml of 2 N HCl/MeOH at 25 room temperature for 2 hours. The soLution is then con-centrated and the residue is taken up in MeOH and eva-porated again, th;s procedure being repeated several timesO
The product is recrystallized from EtOHfdiethyl etherO
Yield: 8.2 9 ~97%) , :. '' ' ~ ',:
:, , ~ ~
~27~
M.p.: 165-168C; Rf A 0.0, B 0.41; C 0.34;
[~]20 = +9.61 ~c - 1, abs. MeOH) C2lH32N7Q7Cl (529.74) calcuLated C 47.61 H 6.0B N 1~.51 found ~7.90 6.30 18~55 d~ H-Asp~oszl)-val-oBut TFA (6~
To spl;t off the Ddz protective group, 12 g (ZO
mmol) of 4 are d;ssolved in 130 ~l of CH2Cl2, and 4~6 ml (60 mmol) of TFA are added t3.5~ of TFA/CH2Cl2).
10 The solution ;s st;rred at room temperature for 30 minutes and the solvent is stripped off. The residue is recrystall;zed from d;ethyl ether/petroleum ether~
Yield: 9.15 9 (93%);
M.p.~ 113-116C; Rf A 0.25; B 0.68; C 0.70;
Cq~2o = -3~56 ~c = 1, abs. MeOH) e) Boc-Tyr-Arg~N02)-Lys~)-OMe t7~
The peptide coupl;ng ;s carr;ed out by the PPA
method as described under a).
Batch: 5.55 9 ~12 mmol) of Boc-Tyr-OH~DCHA and 6D35 g (12 mmol) of 5.
The product is recrystall;zed from EtOH/diethyl ether.
Yield: 8.65 9 (95%);
M.p.: 128-130C; Rf A 0.15; B 0.80; C 0085;
~]~Q = -6.8 (c = 1, abs. MeOH) f) Boc-Tyr-Arg(N02)-Lys(Z)-OH t8) To hydrolyz~ the methyl ester, B.5 9 (11 mmol) of 7 are dissoLved in 33 ml of MeOH, and 26 ml of 1 N NaOH
are added. The aqueous solution ;s acidified to pH 2 .: .
- , - ' : :
,;
': :~ '''~' 3LZ70~
w;th 2 N HCl and is extracted three times uith 80 ml of EE each time. The aqueous phase is saturated w;th NaCl and extracted a further three times with 80 ml of EE each time. The combined organ;c phases are dried over Na2S04.
The solvent is removed and the residue is dried to give a solid foam.
Yield: 7.4 g (90%); Rf A 0.19; ~ 0.57; 0.48;
~]g = -5.6~ ~c = 1, abs. MeOH)--g) Boc-Tyr-Arg(N02)-Lys(Z)-Asp(OBzl)-Val-OBut (9) The peptide coupling is carried out by the P?A
method as described for a).
Batch: 4.46 g (6 mmol) of 8 and 3.25 9 (6.6 mmol) of 6.
The crude product obtained after working up is purified by gel chromatography on Sephadex LH 20 with DMF as the 1S eluting agent. The product is recrystalLized from EtOH~
diethyl e~her.
Yield: 3.6 g (54X);
M~p.: 108-111S; Rf A 0.0; B 0.91; C 0.84;
~J20 = - 19.4 (c 1, abs~ MeOH) 20 C54H76N10015 (1105.18) caiculated C 58.69 H 6.92 N 12.67 found 58.44 6.7812.S4 h) H-Tyr-ArgtN02)-Lys~I)-Asp(OBzl)-Val-OH TFA (10) - For sin~ultaneous splitting off of the Boc and tert.-butyl ester protect;ve groups~ 7.4 ml (96 mmol) of TFA are added to 2.65 9 (2.4 mmol) of 9 and the mixture is stirred under an N2 atmosphere at room temperature for 10 minu.es. The solution is taken up in 50 ml of CH2Cl2 and the mixture is evaporated. The product ;s then recrystallized from MeOH/diethyl ether and dried .; ' ~Z7~
over KO~I.
Yield: 2.39 g (95~);
M.p.: 158--160C, Rf A O n O; B 0.63; C O.57 i) cyclo-(Tyr-Arg(N02)-Lys(Z)-AsptOBzl?-D-Val) (11) - 5 2.1 g (2.0 mmol) of 11 are dissolved in a mixture of 300 ml of DMF and 1 7 liters of CH2Clz (= 1 mmol of peptide per liter of solvent) and the solution is cooled to -15C. 0.18 ml (2 mmol) of NMM and 2.44 g t20 mmol) s~f DMAP are added, and 3.83 g (20 mmol) of EDCI dissolved in 25 ml of DMF, are added dropwise to the solution. The reaction solution is left to stand at -Z/5C for 3 days and is then cooled to -15C and subsequently activated ~ith 3.83 g (20 mmol) of EDCI. After two days at -2/+5C
and one day at room temperature, the react;on solution is evaporated in vacuo. 300 ml of H20 are added to the oi~y residue ~hich remains after concentration, and the preci-pitate ~hich has separated out is filtered off with suc-tion. The resulting crude product is purified by gel chromatography on Sephadex LH 20 with DMF as the eluting agent,. and the resulting product is recrystallized from DMF/MeOH/diethyl ether.
Yield: 558 mg (30%); m.p.: 238-242C (decomposition);
Rf A 0.0; B 0.88; C 0.92;
Co~]2~0 = -45.58 (c = 0.6 DMF) C45H58N10012 (930.95) calculated C 58.05 H 6.27 N 15.05 found 57.85 6.07 15.08 j) cy~clo-(Tyr-Arg-Lys-A~'~ 2HOAc= cyclo-tArg-Lys-Asp-D-Val-Tyr)~Z tlOAc (1) To spl;t off the side chain protective groups, ,~
' : , , ~ -- ::~ .:: . , :. . .
- ~z~a~
~ 0 93 mg (0.1 mmol) of 11 are dissolved in 2 ml of MeOH and 5 ml of HOAc, 80 mg of catalyst (10% strength Pd-on-active charco-al) are added and hydrogenation is carried' out at room temperature for 6 hours. The catalyst is fil-tered off, the solut;on is concentrated and the residueis dried over KOH.
Yield: 63 mg (81%); m~p.: 205C (decomposit;on);
Rf A 0.0; a 0.22; C 0.0; D 0.43;
aminoacid analysis: Asp 1.0; D-VaL 1~01; Tys 0.81; Lys 1.00; Arg 0.97 content: 97X
Example 2 - Preparation of cyclo-tArg-Lys-Glu-D Val-Tyr~^2 HOAc (2) according to synthesis scheme I
' The desired compound is prepared by the same ex-perimental procedures as desc.ibed for Exarnple 1. Compounds uhich are required for the preparation of 2 and have already been described in Example 1 are designated with the figures used therein.
a) Ddz-Glu(08zl)-Val-OBut t12) The peptide coupling is carried out by the PPA
method as described in Example 1 under a).
Batch: 15.1 9 (32 mmol) of Ddz-Glu(06zl)-OH and 11.4 g (32 mmol) of tl-Val-OBut~TsOH~ The product is obtained as an oil.
Yield: 18.3 g t~3X); Rf A 0.70; B 0~65; C 0~78.
b) H-GlutOBzl)-V~I-O~t~ (13~
Splitting off of the Ddz protective group is as described in Example 1 under d~
~, -. , , , ' ~.'.
;
~L~7~
Batch: 17.8 9 (29 mmol) of 12, 6.7 ml (87 mmoL) of TFA
and 188 mmol of CH2Cl2~
The product is recrystallized from d;ethyl ether/
petroleum ether.
Yield: 12~7 9 (86C~); m.p.: 132-136C; Rf A 0.2;
B 0.65; C On70;
~]Z- = -0.23 (c = 1, MeOH) C23H33N27F3 (506.49) calcuLated C 54.54 H ~.56 N 5.58 found 54.82 6.40 5.53 10 c) Boc-Tyr-Arg(N02)-L~/s(Z~-GlytOBzl)-Val-Osut t14) The peptide coupl;ng and purification of the crude product are carried out as described in Example 1 under g).
Batch: 5.96 9 (18 mmol) of 8 and 5.06 9 (10 mmol) of 13.
The product is recrystallized from MeOH/EE.
Yield: 6.1 9 ~68%~; m.pu: 149-153C;
Rf A t).1; B 0.85; C 0.,1;
rcy]2o = - 20.84 ~c = 1, MeOH) C55H7~N10015 (1119.2) calculated C 59.02 H 7.02 N 12.52 found 58.78 6.94 12.29 d) H-Tyr-Arg(N02-Lys(Z)-Glu(OBzl)-Val-OH~TFA (15) The procedure is as described in Example 1 under h).
Batch: 2.75 g t2.45 mmol') of 14 and 7.6 ml (98 mmol) of TFA.
Yield: 2.4 g (92%~;
Rf A 0.00; B 0.63; C 0.58.
e) cyclo-(Tyr-ArgtN02)-LystZ)-Glu(OBzl~-D-Va l? (16) ~he cycli ation ;s carried out as described in Example 1 under i~.
.:
Batch: 2.4 9 (2.2 mmol) of 15, O.Z ml t2.2 mmol) of NMM, 2.68 9 (22 mmol) of DMAP and 8.4 9 (44 mmol) of EDCI.
The product obtained after gel chromatography is recrystall;~ed from DMF/MeOH/diethyl e~her~
Yie~d: 686 mg (33%); m.p.: 234-238C (decomposition) Rf A 0.00; B 0.80; C 0.86;
[~20 = _ 58.46 (c = 0.26, DMF);
- C46H60N10o12 (944.98) calculated 58.47 H 6 39 N 14 8Z
found 57.916.52 14.94 - FAB-MS: 945 f) cyclo-(Tyr-Arg-Lys-Glu-D-Val)-2 HOAc = cyclo-(Arg-Lys-Glu-D-Val-Tyr)~2HOAc (2) Splitting off of the side chain protective groups is as described in Example 1 under j).
Batch: 94 mg (0.1 mmo;) of 16;
Yield: 65 mg ~82~); m.p.: 140-142C (decomposition);
Rf A 0.00; B 0.22; C 0.00; D 0.44.
A~linoacid analysis: Glu 0~98; D-Val 1.01; Tyr 0.84;
Lys 0.98; Arg 1.00 content: 92X.
.
'' ~ ~ ` :'.' ' '~
.. . . .
.
Tyr Arg Lys X'. ~al ~ ~ ' I I .
i l ~2 ~ Z
3 - Boc ~ OH H ~ OMe-HCl .; ~ I I ~
~ ~ ~N2 3 ~ Z ~ T
Boc ~/ - ----,~ OMe ¦ 3 .1 -- I I I ~ .
l ~M2 5 ¦ Z ¦ OBzl Boc ~ OH H ~ OMe~HCl Ddz ~ OH H ~OtBu TsOH
} 5~'2 7 ¦ Z ¦ OBzl 4/12 ¦
Boc ~ / O~e Ddz~/ 3- OtBu I
'' I ., ~ ' ' I ~ ..
¦NO2 8 ~ Z ~ OBzl 6~13 . Boc ~ ~ 5/ OH H-~ OtBuoTF~
.~ I :' 1, 1 ~
i I I 9/14 ¦ OBzl Boc _,r _ . r/ / ~ ~-OtBu ¦ ¦ N02 ¦ Z lO/15 ¦/OBZ
.H ~ f ~ OHTFQ
I ~/N2 ~Z 11/16¦~ OBzl cyclo-/ . _ D-~al /
~ /2 1 ~
t ~ ~
cyclo,~yr Ar~ Lys _ X' = ~2HOAc X ' = AsptG lu Synthes i s s cheme I
- . ; . ..
: -,- ~
.
~Z7~
Abbreviations for protective groups and reagents Boc = tert.-butoxycarbonyl Bzl = benzyl DCC = d;cyclohexylcarbodiimide DCHA = dicyclohexylamine Ddz = o~-dimethyL-3~5-d;methoxybpnzyloxycarbonyl DMAP = 4-dimethylaminopyridine DMF = N,N-o'imethylformamide EDCI = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EE = acetic ester (ethyl acetate) EtOH = ethanol HOAc = acetic acid MeOH = methanol NMM = N-methylmorphol;ne OMe = methoxy OBut = tert.-butoxy PPA = n-propylphosphon;c acid anhydride TsOH = p-toluenesulfonic acid ~ = carbobenzoxycarbonyl Abbreviations ;n the text abs. = absolute sat. = saturated m.p. = melt;ng po;nt Eluting agents ~or thin-layer chromatography A chloroformJmethanol/glacial acetic acid (95:5:3) B n-butanol/gtacial acetic ac;d/water C ethyl acetate/n-butanol/pyridine/water ~20:100305 D methanol/glacial acetic acid ~
;'..
Claims (5)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A peptide of the formula II
(II) wherein W represents Lys (R1), D-Lys (R1), Arg (R2) or D-Arg (R2), X represents Lys (R1), D-Lys (R1), Arg (R2) or D-Arg (R2), Y represents Asp (R3), D-Asp (R3), Glu (R3) or D-Glu (R3), U represents Val or D-Val, D represents Tyr, D-Tyr, Trp, D-Trp, Phe or D-Phe, and wherein, R1 represents a urethane-protective group, R2 represents a protective group for the guanidino function and R3 represents an ester-protective group, and wherein (a) all five radicals a represent peptide bonds or (b) one of the radicals a represents a C-terminal OH together with an N-terminal H (a = -OH H-) of two aminoacid radicals and the remaining four radicals a each represent peptide bonds.
(II) wherein W represents Lys (R1), D-Lys (R1), Arg (R2) or D-Arg (R2), X represents Lys (R1), D-Lys (R1), Arg (R2) or D-Arg (R2), Y represents Asp (R3), D-Asp (R3), Glu (R3) or D-Glu (R3), U represents Val or D-Val, D represents Tyr, D-Tyr, Trp, D-Trp, Phe or D-Phe, and wherein, R1 represents a urethane-protective group, R2 represents a protective group for the guanidino function and R3 represents an ester-protective group, and wherein (a) all five radicals a represent peptide bonds or (b) one of the radicals a represents a C-terminal OH together with an N-terminal H (a = -OH H-) of two aminoacid radicals and the remaining four radicals a each represent peptide bonds.
2. A peptide as claimed in claim 1, wherein R1 is Fmoc, Fcboc, Z, Boc, Ddz, Bpoc, Z-(NO2), Pyoc, Dobz, Moc, Mboc, Iboc, Adoc, Adpoc, Msc or Pioc.
3. A peptide as claimed in claim 1, wherein R2 is NO2, tosyl, Boc, Z or mesitylene-2-sulfonyl (Mts).
4. A peptide as claimed in claim 1, wherein R2 is an alkyl ester, a benzyl ester or modified benzyl ester.
5. A peptide as claimed in claim 1, wherein R2 is a methyl ester, ethyl ester, tert-butyl ester, P-NO2 benzyl ester, p-Cl benzyl ester or p-Br-benzyl ester.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA000521657A CA1270100A (en) | 1983-08-03 | 1986-10-28 | Peptides having an immunostimulating action, processes for their preparation and their use |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE3327927 | 1983-08-03 | ||
DEP3327927.6 | 1983-08-03 | ||
CA000460240A CA1237713A (en) | 1983-08-03 | 1984-08-02 | Peptides having an immunostimulating action, processes for their preparation and their use |
CA000521657A CA1270100A (en) | 1983-08-03 | 1986-10-28 | Peptides having an immunostimulating action, processes for their preparation and their use |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA000460240A Division CA1237713A (en) | 1983-08-03 | 1984-08-02 | Peptides having an immunostimulating action, processes for their preparation and their use |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1270100A true CA1270100A (en) | 1990-06-05 |
Family
ID=25670462
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA000521657A Expired - Fee Related CA1270100A (en) | 1983-08-03 | 1986-10-28 | Peptides having an immunostimulating action, processes for their preparation and their use |
Country Status (1)
Country | Link |
---|---|
CA (1) | CA1270100A (en) |
-
1986
- 1986-10-28 CA CA000521657A patent/CA1270100A/en not_active Expired - Fee Related
Similar Documents
Publication | Publication Date | Title |
---|---|---|
FI92325B (en) | Process for the preparation of thymopentin analogs | |
US4190646A (en) | Polypeptide compositions and methods | |
US4261886A (en) | Peptides having thymopoietin-like activity | |
US4003884A (en) | Peptides having LH-RH/FSH-RH activity | |
EP0614913A2 (en) | Dynorphin derivatives | |
US4783442A (en) | B-cell differentiating peptides | |
FI67691C (en) | FRAMEWORK FOR THE FRAMEWORK OF THERAPEUTIC MEDICINAL PRODUCTS | |
US4650788A (en) | Novel peptides having an immunostimulating action, processes for their preparation and their use | |
GB1570210A (en) | Peptides | |
US4397842A (en) | Peptides having thymopoietin-like activity | |
EP0018182B1 (en) | Peptides having thymopoietin-like activity, therapeutic compositions containing them, and process for their preparation | |
CA1270100A (en) | Peptides having an immunostimulating action, processes for their preparation and their use | |
US4866121A (en) | B cell differentiating peptides and conjugates thereof | |
US3987014A (en) | Secretin intermediates and derivatives | |
GB1574492A (en) | D-ala2,d-lys4-somatostatin | |
US4487764A (en) | New peptides and a process for their preparation | |
US3896105A (en) | (n-propionyl) -gly-cys-lys-asn-phe-phe-trp-lys-thr-phe-thr-ser-cys-oh and intermediates | |
US4244947A (en) | Carba decapeptide derivatives of [TYR6 ]-somatostatin | |
US4308181A (en) | Polypeptide compositions | |
US4118380A (en) | Decapeptide analogs of somatostatin | |
CA1100485A (en) | Somatostatin analogs and intermediates thereto | |
HELLSTERN et al. | Continuous Flow Peptide Synthesis on Aminopolyoxyethylene-Polystyrene Graft Copolymer using Fmoc-Strategy | |
US4258152A (en) | Pentapeptide modified resin | |
EP0103858A2 (en) | New peptides, process for preparation thereof and pharmaceutical compositions containing them | |
US4081530A (en) | Extended chain derivatives of somatostatin |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
MKLA | Lapsed |