Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~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-
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~'~
-- 8 --
~ 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~
:
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' ;;
~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~
- 12 -
~ 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~
- 16 -
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~
- 18 -
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~
- 19 -
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~
- 21 -
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~.
.:
- 22 -
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.
.
'' ~ ~ ` :'.' '
'~
- 23 -
.. . . .
.
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 ~
;'..
