The present invention relates to compounds which are useful for the protection of
organic compounds comprising at least one guanidino moiety and/or at least one
amino group. The invention further relates to a process for the preparation of these
compounds and to their use as protecting reagents. The invention also relates to the
process for the protecting reaction and to the protected compounds thereof.
Suitable protection of a guanidine moiety is still an unsolved problem in chemistry
because of the difficulty to remove the known protecting groups. This applies particularly
to peptide chemistry as the natural amino acid arginine, bearing a guanidine
moiety, is of great importance for the preparation of numerous drug substances. C> During the coupling reaction, guanidine protection of arginine is necessary to avoid
acylation potentially followed by deguanidation, thus rendering undesired ornithine and
o-lactam formation.
Depending on the coupling strategy, the most commonly used protecting groups for
arginine are p-toluenesulfonyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc)
and 2,2,4,6,7 -pentamethyldihydrobenzofuran-5-sulfonyl (Pbf). However, these protecting
groups are too acid-stable, thus requiring harsher removal conditions and a
longer removal time. Therefore, the known guanidine protecting groups are prone to
form by-products on their removal. Particularly problematic is their cleavage in
peptides with multiple arginine residues or in peptides containing tryptophan.
Carpino et al. (Tetrahedron Letters 1993, Vol. 34, No. 49, 7829-7832} compare the
Pbf protecting group with the Pmc protecting group when used for arginine side chain
protection.
WO 01/57045 discloses tricyclic sulfames obtained via benzofuran-, benzothiopheneand
indole-intermediates.
Lowe et al. describe the synthesis of heterocyclic sulfonylureas, which for example
comprise an indole moiety (J. Heterocyclic Chern., 1996, 33, 763-766).
It is an object of the present invention to provide a compound which easily protects the
guanidine moiety of an organic compound and which can be easily removed.
The object described above is achieved by the compounds of claim 1, which can be
prepared by the process of claim 5 and which is used according to claim 9 in the
protection process of claim 12 affording compounds of claim 16.
In one aspect, the present invention relates to a compound of formula
(II),
wherein R1 is hydrogen, C1-a alkyl, C1-a alkoxy or C1-a alkylthio; R2 is C1-a alkyl,
C1-a alkoxy or C1-a alkylthio; or R1 and R2 together form a moiety of formula -{CH2)n-.
wherein n is an integer from 3 to 5; R3 is hydrogen, halogen, C1-a alkyl, C1-a alkoxy,
C1-e alkylthio, phenyl or benzyl; and X is chlorine or bromine.
~ Here and as follows, the term "C1-n alkyl" is to be understood to mean any linear or
branched alkyl group containing 1 to n carbon atoms. For example the term "C1-a alkyl"
comprises groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,
terl-butyl, pentyl, isopentyl (3-methylbutyl), neopentyl (2,2-dimethylpropyl), hexyl,
isohexyl (4-methylpentyl) and the like.
Accordingly, the term "C1-n alkoxy" means a group composed of a C1-n alkyl group as
defined above and an oxygen atom linked by a single covalent bond.
In the same manner, the term "C1-s alkylthio" means a group composed of a C1-s alkyl
group as defined above and an sulfur atom linked by a single covalent bond.
Here and as follows the term "halogen" means fluorine, chlorine, bromine and iodine.
Favourably, the present invention relates to a compound of formula (II), wherein R1
,
R2, R3 and X are as defined above with the exception of 1-methylindole-3-sulfonyl
chloride.
In a preferred embodiment, R1 and R2 are independently C1-4 alkyl, C1-4 alkoxy or
C1-4 alkylthio; R3 is hydrogen or halogen; and X is chlorine or bromine.
In a particular embodiment, both R1 and R2 are methyl; R3 is hydrogen; and X is
chlorine being 1 ,2-dimethylindole-3-sulfonyl chloride. For the sake of convenience, this
compound will be abbreviated as MIS-CI.
In a further aspect of the present invention, the compound of formula (II) is prepared by
a process comprising the step of reacting a compound of formula
,
(I),
wherein R1
, R2 and R3 are as defined above, or a salt thereof, with oxalyl chloride or
oxalyl bromide.
In a preferred embodiment, R1 and R2 are independently C1-4 alkyl, C1-4 alkoxy or
C1-4 alkylthio, and R3 is hydrogen or halogen.
In a more preferred embodiment, both R1 and R2 are methyl and R3 is hydrogen.
In the process for the preparation of the compound of formula (II) both the acidic form
and the salt form of the compound of formula (I) may be used as reagent. Any salt
form of the compound of formula (I) can be applied. Suitable salts are e.g. the sodium
salt, the potassium salt, the calcium salt and the pyridinlum salt.
In a preferred embodiment, the reaction is performed with the pyridinlum salt of the
compound of formula (I).
Preferably, oxalyl chloride is applied for the reaction.
For the preparation process, any suitable solvent or mixtures of suitable solvents may
be applied. Suitable solvents are solvents which do not react with the reactants or with
the product and which dissolve the reactants to a sufficient extent. Examples of
suitable solvents are dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran
and 1 ,4-dioxane. Preferably, dichloromethane is used as solvent.
The reaction may also be performed without a solvent.
Expediently, the reaction is performed in the presence of N,N-dimethylforrnamide.
In an additional aspect of the present invention, the compound of formula
{II),
wherein R1 is hydrogen, C,~ alkyl, C,~ alkoxy or c,~ alkylthio; R2 is C,~ alkyl,
C1~ alkoxy or C1~ alkylthio; or R1 and R2 together form a moiety of formula -{CH2),-,
wherein n is an integer from 3 to 5; R3 is hydrogen, halogen, C,~ alkyl, C1~ alkoxy,
c,~ alkylthio, phenyl or benzyl; and X is chlorine or bromine,
is used for the protection of an organic compound which comprises at least one guani-
C) dina moiety and/or at least one amino group.
In a preferred embodiment, the organic compound is an optionally resin-bound peptidic
compound which is optionally side chain protected and/or protected at a free terminus;
R1 and R2 are independently c,-4 alkyl, c,-4 alkoxy or c,-4 alkylthio, and R3 is
hydrogen or halogen.
Here and in the following, the term "peptidic compound" is to be interpreted in a wide
manner as defined hereafter. Therefore, the term "peptidic compound" is to be
understood to mean any compound of one of the following categories {a) to {d):
(a) A peptide, i.e. a compound produced by formation of an amide bond between a
carboxyl group of one amino acid and an amino group of another. The amide
bond is typically formed between C-1 of one amino acid and N-2 of another
(eupeptide bond), but a compound with residues linked by other amide bonds
(isopeptide bonds) is also meant to be covered by the term "peptidic
compound". Oligopeptides consisting of two to fifteen amino acid residues and
polypeptides consisting of sixteen to about fifty amino acid residues are typical
peptides of this category. The amino acid residues may be any natural or
unnatural amino acids. An example for a peptide is H-Arg-Vai-OH.
(b) An amino acid, which may not only be an amino acid commonly found in
proteins {natural a-amino acid) but also any unnatural amino acid. Examples are
H-Aia-OH (natural amino acid) and homoarginine (unnatural amino acid).
(c) A derivative of a peptide, meaning a peptide in which one or more of the amino
acid residues have been chemically modified, e.g. by acylation, alkylation, ester
formation or amide formation. Examples are Ac-Phe-Arg-Giy-Aia-Vai-OH {SEQ
5
ID NO 5), H-Phe-Arg-Giy-Aia-Vai-NH2 (SEQ ID NO 6) and H-Arg-Giy-Aia-GiyGiy-
Lys(Ne-tetradecanoyi)-Aia-Giy-Giy-OH (SEQ 10 NO 7).
(d) A derivative of an amino acid, meaning an amino acid which has been
chemically modified, e.g. by acylation, alkylation, ester formation or amide
formation. An example is H-Aia-OMe.
In another preferred embodiment, the peptidic compound comprises at least one
guanidine moiety and optionally at least one amino group, the guanidine moiety being
part of an arginine, homoarginine or norarginine residue. More preferably, said
guanidine moiety is part of an arginine or homoarginine residue. Even more preferably,
the peptidic compound is Z-Arg-OH.
The prefix "homo" to the name of a common amino acid (like homoarginine) means
that said amino acid contains one additional methylene group in the carbon chain.
In contrast, the prefix "nor" to the name of a common amino acid (like norarginine)
denotes removal of one methylene group in the carbon chain.
In another preferred embodiment, the peptidic compound comprises at least one
amino group and optionally at least one guanidine moiety, said at least one amino
group(s) being the N-terminal amino group or part of the side chain of an amino acid
residue.
More preferably, the amino group to be protected is the N-terminal amino group of said
peptidic compound. Most preferably, the peptidic compound is H-Aia-OMe.
Also more preferably, the amino group to be protected is part of the side chain of an
amino acid residue of said peptidic compound. Even more preferably, said amino
group is part of a lysine, homolysine or norlysine residue. Most preferably, said
peptidic compound is Z-Lys-OH.
In another aspect, the present invention relates to a process for the protection of an
organic compound, which comprises at least one guanidine moiety and/or one amino
group,
said process comprising the reaction of said compound with the compound of formula
X
0, I
'S"
R3~'0R1 V-N/ k2 (II),
wherein R1 is hydrogen, C1-6 alkyl, C1-6 alkoxy or C1-6 alkylthio; R2 is C1-6 alkyl,
C1-s alkoxy or C1-6 alkylthio; or R1 and R2 together form a moiety of formula -(CH2)n-.
wherein n is an integer from 3 to 5; R3 is hydrogen, halogen, C1-6 alkyl, C1-0 alkoxy,
~ C1-s alkylthio, phenyl or benzyl; and X is chlorine or bromine,
thus affording a compound, which comprises at least one moiety of the formula
(Ill),
wherein R1
, R2 and R3 are as defined above and m is 0 or 1.
In one embodiment, m is 1, thus affording a compound, which comprises at least one
moiety of the formula
(IV),
wherein R1
, R2 and R3 are as defined above.
In another embodiment, m is 0, thus affording a compound, which comprises at least
one moiety of the formula
7
,
i
i
(V),
wherein R1
, R2 and R3 are as defined above.
In a preferred embodiment, R1 and R2 are independently c,-4 alkyl, C1-4 alkoxy or
C1-4 alkylthio, R3 is hydrogen or halogen. In one embodiment, m is 1 and in another
embodiment, m is 0.
In a more preferred embodiment, R1 and R2 are methyl, R3 is hydrogen and X is
chlorine. In one embodiment, m is 1; and in another embodiment, m is 0.
In another preferred embodiment, the organic compound is an optionally resin-bound
peptidic compound which is optionally side chain protected and/or protected at a free
terminus,
thus affording said peptidic compound, which comprises at least one moiety of the
formula
(Ill),
wherein R1 is hydrogen, C1-s alkyl, C1-s alkoxy or C1-s alkylthio; R2 is C1-s alkyl,
C,-a alkoxy or C,-s alkylthio; or R1 and R2 together form a moiety of formula -(CH2)n-.
wherein n is an integer from 3 to 5; and R3 is hydrogen, halogen, C1-s alkyl, C1-s
alkoxy, C,-e alkylthio, phenyl or benzyl; and m is 0 or 1.
z f I
l
Preferably, R1 and R2 are independently Ct-4 alkyl, Ct-4 alkoxy or Ct-4 alkylthio and R3
is hydrogen or halogen. Most preferably, R1 and R2 are methyl and R3 is hydrogen.
In a preferred embodiment, said peptidic compound comprises at least one guanidine
moiety being part of an arginine, homoarginine or norarginine residue, preferably being
part of an arginine or homoarginine residue,
thus affording said peptidic compound, which comprises at least one moiety of the
formula (Ill), wherein R1 is hydrogen, C1-e alkyl, Ct-e alkoxy or C1-e alkylthio; R2 is
Ct-a alkyl, Ct-a alkoxy or C1-a alkylthio; or R1 and R2 together form a moiety of formula
-(CH2)n-. wherein n is an integer from 3 to 5; and R3 is hydrogen, halogen, C1-e alkyl,
Ct-a alkoxy, C1-a alkylthlo, phenyl or benzyl; and m is 1;
preferably, wherein R1 and R2 are independently C1-4 alkyl, Ct-4 alkoxy or C1-4 alkylthio
and R3 is hydrogen or halogen; and
more preferably, wherein R1 and R2 are methyl and R3 is hydrogen.
Most preferably, said peptidic compound to be reacted comprises only one guanidino
moiety.
In a preferred embodiment, said peptidic compound to be reacted is N-a.-benzyloxycarbonyi-
L-arginine (Z-Arg-OH).
In an even more preferred embodiment, the peptidic compound to be reacted is
Z-Arg-OH; R1 and R2 of the compound of formula (II) are methyl, R3 of the compound
of formula (II) is hydrogen and X of the compound of formula (II) is chlorine,
thus affording N-a.-benzyloxycarbonyi-N-ro-( 1,2-dimethylindole-3-sulfonyi)-L -arginine.
For the sake of convenience this compound is abbreviated as Z-Arg(MIS)-OH in the
following.
In another preferred embodiment, said peptidic compound comprises at least one
amino group, said at least one amino group(s) being the N-terminal amino group or
being part of the side chain of an amino acid residue,
thus affording said peptidic compound, which comprises at least one moiety of the
formula (Ill), wherein R1 is hydrogen, Ct-a alkyl, C1-e alkoxy or C1-e alkylthio; R2 is
Ct-a alkyl, Ct-a alkoxy or Ct-a alkylthio; or R1 and R2 together form a moiety of formula
-(CH2)n-. wherein n is an integer from 3 to 5; and R3 is hydrogen, halogen, C1-a alkyl,
Ct-a alkoxy, C1-a alkylthio, phenyl or benzyl; and m is O;
preferably, wherein R1 and R2 are independently C1-4 alkyl, Ct-4 alkoxy or C1-4 alkylthio
and R3 is hydrogen or halogen; and
9
more preferably, wherein R1 and R2 are methyl and R3 is hydrogen.
Most preferably, said peptidic compound to be reacted comprises only one amino
group which is the N-terminal amino group or which is part of the side chain of an
amino acid residue.
Even more preferably, the amino group of said peptidic compound to be reacted is the
N-terminal amino group.
In a preferred embodiment, said peptidic compound to be reacted is L-alanine methyl
ester (H-Aia-OMe ).
In an even more preferred embodiment, the peptidic compound is H-Aia-OMe; R1 and
R2 of the compound of formula (II) are methyl, R3 of the compound of formula (II) is
hydrogen and X of the compound of formula (II) is chlorine,
thus affording N-cx-(1,2-dimethylindole-3-sulfonyi)-L-alanine methyl ester. For the sake
of convenience this compound is abbreviated as MIS-Aia-OMe in the following.
Also even more preferably, the amino group of said peptidic compound to be reacted is
part of the side chain of an amino acid residue. Most preferably, said amino group is
part of a lysine, homolysine or norlysine residue.
As solvent for the protection process, any inert liquid solvent which can dissolve the
reactants may be used. Applicable solvents include halogenated hydrocarbons such
as dichloromethane, carbon tetrachloride and dichloroethane; ethers such as diethyl
ether, tetrahydrofuran, 2-methyltetrahydrofuran: carboxylic esters and lactones such as
ethyl acetate, methyl acetate and valerolactone; and organic solvents containing
heteroatoms such as acetone, acetonitrile, dimethylformamide and dimethyl sulfoxide.
The solvents can be used alone or as mixtures. Optionally, the solvent or solvent
mixture may contain water if the solubility of the reactants requires the presence of
water. Preferred solvents are dichloromethane and acetone, alone or in the presence
of water.
Optionally, the reaction mixture may contain inorganic or organic bases. Examples for
inorganic bases are sodium hydroxide, potassium hydroxide, lithium hydroxide and
sodium carbonate. Examples for organic bases are diisopropylethylamine, pyridine and
triethylamine. Preferred bases are sodium hydroxide and diisopropylethylamine.
tO
The amount of the compound of formula (II) varies with the reactor volume and can be
at a molar ratio from 0.9:1 to 4:1, preferably from 1:1 to 3:1, relative to the organic
compound, which comprises at least one guanidine moiety and/or one amino group.
The compound of formula (II) may be added in portions to the reaction mixture.
The protection process may be carried out at low or slightly elevated temperatures. For
example, a suitable temperature range is from -10 octo 30 oc, preferably from 0 octo
room temperature.
The reaction time depends on different factors like the temperature or the molar ratio of
the compound of formula (II) and the organic compound, which comprises at least one
guanidine moiety and/or one amino group. Therefore, the reaction may be completed
within a few minutes or several hours.
In a further aspect, the present invention relates to an organic compound comprising at
least one moiety of the formula
(Ill),
wherein R1 is hydrogen, C1-e alkyl, C1-e alkoxy or C1-e alkylthio; R2 is C1-e alkyl,
C1-e alkoxy or C1-e alkylthio; or R1 and R2 together form a moiety of formula -(CH2)n-.
wherein n is an integer from 3 to 5; R3 is hydrogen, halogen, C1-e alkyl, C1-e alkoxy,
C1-6 alkylthio, phenyl or benzyl; and m is 0 or 1.
In one embodiment, m is 1, so that the organic compound comprises at least one
moiety of the formula
(IV),
I (
wherein R1
, R2 and R3 are as defined above.
In another embodiment, m is 0, so that the organic compound comprises at least one
moiety of the formula
(V),
wherein R1
, R2 and R3 are as defined above.
In a preferred embodiment, R1 and R2 are independently C1-4 alkyl, C1-4 alkoxy or
C1-4 alkylthio and R3 is hydrogen or halogen. In one embodiment, m is 1 and in another
embodiment, m is 0.
In a more preferred embodiment, R1 and R2 are methyl, R3 is hydrogen and X is
chlorine. In one embodiment, m Is 1 and in another embodiment, m is 0.
In another preferred embodiment, the organic compound is an optionally resin-bound
peptidic compound which is optionally side chain protected and/or protected at a free
terminus and which comprises at least one moiety of the formula
(Ill),
wherein R1 is hydrogen, C1-a alkyl, C1-e alkoxy or C1-a alkylthio; R2 is C1-a alkyl,
C1-a alkoxy or C1-e alkylthio; or R1 and R2 together form a moiety of formula -{CH2),,
wherein n is an integer from 3 to 5; R3 is hydrogen, halogen, C1-e alkyl, C1-a alkoxy,
C1-e alkylthio, phenyl or benzyl; and m is 0 or 1, preferably m is 1.
Preferably, R1 and R2 are independently C1-4 alkyl, C1-4 alkoxy or C1-4 alkylthio and R3
is hydrogen or halogen. Most preferably, R1 and R2 are methyl and R3 is hydrogen.
\2-
In a preferred embodiment, said peptidic compound comprises at least one guanidine
moiety being part of an arginine, homoarginine or norarginine residue, preferably being
part of an arginine or homoarginine residue, comprising at least one moiety of the
formula (Ill), wherein R1 is hydrogen, C1-e alkyl, C1-e alkoxy or C1-e alkylthio; R2 is
C1-s alkyl, C1-s alkoxy or C1-s alkylthio; or R1 and R2 together form a moiety of formula
-(CH2)n-. wherein n is an integer from 3 to 5; and R3 is hydrogen, halogen, C1-6 alkyl,
C1-6 alkoxy, C1-6 alkylthio, phenyl or benzyl; and m is 1;
preferably, wherein R1 and R2 are independently C1-4 alkyl, C1-4 alkoxy or C1-4 alkylthio
and R3 is hydrogen or halogen; and
more preferably, wherein R1 and R2 are methyl and R3 is hydrogen.
Most preferably, said peptidic compound comprises only one guanidine moiety.
Even more preferably, said peptidic compound is Z-Arg(MIS)-OH.
In another preferred embodiment, said peptidic compound comprises at least one
amino group, said at least one amino group(s) being theN-terminal amino group or
being part of the side chain of an amino acid residue, comprising at least one moiety of
the formula (Ill), wherein R1 is hydrogen, C1-6 alkyl, C1-s alkoxy or C1-6 alkylthio; R2 is
C1-s alkyl, C1-s alkoxy or C1-s alkylthio; or R1 and R2 together form a moiety of formula
-(CH2)n-. wherein n is an integer from 3 to 5; and R3 is hydrogen, halogen, C1-e alkyl,
C1-s alkoxy, C1-s alkylthio, phenyl or benzyl; and m is 0;
preferably, wherein R1 and R2 are independently C1-4 alkyl, C1-4 alkoxy or C1-4 alkylthio
and R3 is hydrogen or halogen; and
more preferably, wherein R1 and R2 are methyl and R3 is hydrogen.
Also more preferably, said peptidic compound comprises only one amino group which
is the N-terminal amino group or which is part of the side chain of an amino acid
residue.
Even more preferably, the amino group of said peptidlc compound is the N-terminal
amino group.
Most preferably, said peptidic compound is MIS-Aia-OMe.
Also even more preferably, the amino group of said peptidic compound is part of the
side chain of an amino acid residue. Most preferably, said amino group is part of a
lysine, homolysine or norlysine residue.
A further aspect of the invention is modifying and/or coupling in following step(s) the
organic compound obtained according to the present invention.
As the organic compounds obtained according to the present invention are important
building blocks, they can be applied to form organic compounds being useful as e.g.
drug substances.
According to the present invention, the organic compound which comprises at least
one moiety of the formula
(Ill),
wherein R1 is hydrogen, C1-s alkyl, C1-s alkoxy or C1-e alkylthio; R2 is C1-s alkyl,
C1-e alkoxy or C1-s alkylthio; or R1 and R2 together form a moiety of formula -(CH2)n-.
wherein n is an integer from 3 to 5; R3 is hydrogen, halogen, C1-s alkyl, C1-s alkoxy,
C1-e alkylthio, phenyl or benzyl; and m is 0 or 1,
is chemically modified in a following step.
In a preferred embodiment, the organic compound to be modified in a following step is
an optionally resin-bound peptidic compound which is optionally side chain protected
and/or protected at a free terminus and which comprises at least one moiety of the
formula (Ill), wherein R1
, R2
, R3 and m are as defined above.
Preferably, said peptidic compound comprises at least one guanidine moiety being part
of an arginine, homoarginine or norarginine residue, preferably being part of an arginine
or homoarginine residue, comprising at least one moiety of the formula (Ill),
wherein R1
, R2
, R3 and m are as defined above;
preferably, wherein R1 and R2 are independently C1-4 alkyl, C1-4 alkoxy or C1-4 alkylthio
and R3 is hydrogen or halogen; and
more preferably, wherein R1 and R2 are methyl and R3 is hydrogen.
Most preferably, said peptidic compound comprises only one guanidine moiety.
Even more preferably, said peptidic compound is Z-Arg(MIS)-OH.
Modification comprises any organic reaction which complies with the protecting group
of the present invention. As an example, Z-Arg(MIS)-OH may be modified by
deprotection of the Z group, thus forming H-Arg(MIS)-OH.
Optionally, the modified compound thus obtained is at least once further modified. As
an example, H-Arg(MIS)-OH as obtained by a first modification may be further
modified by protection of its N-terminus, thus forming e.g. Fmoc-Arg(MIS)-OH.
The deprotection and protection steps can be carried out using reaction conditions
known in the art of peptide synthesis.
Also according to the present invention, the organic compound which comprises at
least one moiety of the formula
(Ill),
wherein R1 is hydrogen, C1-s alkyl, C1-s alkoxy or C1-s alkylthio; R2 is C1-s alkyl,
C1-a alkoxy or C1-a alkylthio; or R1 and R2 together form a moiety of formula -(CH2)n-.
wherein n is an integer from 3 to 5; R3 is hydrogen, halogen, C1-a alkyl, C1-a alkoxy,
C1-s alkylthio, phenyl or benzyl; and m is 0 or 1,
is coupled to an organic compound Q in a following step.
Optionally, said coupling step may be repeated at least once and/or at least one further
coupling with a suitable coupling compound may be performed. The compound thus
obtained may be chemically modified and in case said compound is resin-bound,
cleavage from the resin may follow.
\5
In a preferred embodiment, the organic compound to be coupled in a following step to
the organic compound a is an optionally resin-bound peptidic compound which is
optionally side chain protected and/or protected at a free terminus and which
comprises at least one moiety of the formula (Ill), wherein R\ R2
, R3 and mare as
defined above.
Preferably, said peptidic compound comprises at least one guanidine moiety being part
of an arginine, homoarginine or norarginine residue, preferably being part of an arginine
or homoarginine residue, comprising at least one moiety of the formula (Ill),
wherein R1
, R2
, R3 and mare as defined above;
preferably, wherein R1 and R2 are independently C1-4 alkyl, C1-4 alkoxy or C1-4 alkylthio
and R3 is hydrogen or halogen; and
more preferably, wherein R1 and R2 are methyl and R3 is hydrogen.
Most preferably, said peptidic compound comprises only one guanidine moiety.
Even more preferably, said peptidlc compound is Fmoc-Arg(MIS)-OH or
Z-Arg(MIS)-OH.
The organic compound a is preferably an optionally resin-bound peptidic compound
which is optionally side chain protected and/or protected at a free terminus. Most
preferably, the organic compound Q is a peptidic compound which is optionally side
chain protected and which is optionally resin-bound.
In a preferred embodiment, said peptidic compound isH-Val-resin or H-Trp(Boc)-AiaGiy-
resin, preferably the resin originates from the Sieber amide resin (9-Fmoc-aminoxanthen-
3-yloxy-Merrifield resin).
In an even more preferred embodiment, the peptidic compound to be coupled is FmocArg(
MIS)-OH and the peptidic compound to which said peptidic compound is coupled
is H-Vai-NH-xanthen-3-yloxy-Merrifield resin, thus affording Fmoc-Arg(MIS)-Vai-NHxanthen-
3-yloxy-Merrifield resin.
Preferably, said coupling reaction is repeated three times thus affording FmocArg(
MIS)-Arg(MIS)-Arg(MIS)-Arg(MIS)-Vai-NH-xanthen-3-yloxy-Merrifield resin (SEQ
10 NO 8). Also preferably, Fmoc-Phe-OH is coupled after N-terminal deprotection of
said resin-bound peptide, thus affording Fmoc-Phe-Arg(MIS)-Arg(MIS)-Arg(MIS)Arg(
MIS)-Vai-NH-xanthen-3-yloxy-Merrifield resin (SEQ 10 NO 1 ). More preferably, the
N-terminus is deprotected and acetylated, thus affording Ac-Phe-Arg(MIS)-Arg(MIS)Arg(
MIS)-Arg(MIS)-Vai-NH-xanthen-3-yloxy-Merrifield resin (SEQ 10 NO 1 ). Most
preferably said resin-bound peptide is cleaved from the resin, thus affording Ac-PheArg(
MIS)-Arg(MIS)-Arg(MIS)-Arg(MIS)-Vai-NH2 (SEQ 10 NO 2).
In an also even more preferred embodiment, the peptidic compound to be coupled is
Z-Arg(MIS)-OH and the peptidic compound to which said compound is coupled is
H-Trp(Boc)-Aia-Giy-NH-xanthen-3-yloxy-Merrifield resin, thus affording Z-Arg(MIS)·
Trp(Boc)-Aia-Giy-NH-xanthen-3-yloxy-Merrifield resin (SEQ 10 NO 3). Preferably, said
resin-bound peptide is cleaved from the resin, thus affording Z-Arg(MIS)-Trp(Boc)-AiaGiy-
NH2 (SEQ 10 NO 3).
C The coupling procedure to the organic compound Q may follow according to any
coupling method known to the person skilled in the art. In case the organic compound
to be coupled is a peptidic compound, coupling is preferably performed in solid or
liquid phase. Most preferably, optionally preceding and optionally following coupling
steps are also performed In solid and/or liquid phase.
The term "solid phase" is to be understood to mean solid phase peptide synthesis
(SPPS). In SPPS an amino acid or peptide group, optionally protected, is bound to a
solid support resin. Then, successive amino acids or peptide groups, optionally
protected, are attached to the support-bound peptide until the peptide material of
interest is formed. The support-bound peptide is then typically cleaved from the
support and subject to further processing and/or purification. In some cases, solid
phase synthesis yields a mature peptide product; in other cases the peptide cleaved
from the support, i.e. a "peptide intermediate fragment", is used in the preparation of a
larger, mature peptidic product.
The term "solution phase" is to be understood to mean solution phase peptide
synthesis. In solution phase peptide synthesis, two peptide intermediate fragments,
optionally protected, or a peptide intermediate fragment and a reactive amino acid,
both optionally protected, are coupled in an appropriate solvent, usually in the
presence of additional reagents that promote the efficiency and quality of the coupling
reaction. The peptide intermediate fragments are reactively arranged so that the
N-terminal of one fragment becomes coupled to the C-terminal of the other fragment,
or vice versa. In addition, side chain protecting groups, which are present during solid
phase synthesis, are commonly retained on the fragments during solution phase
coupling to ensure the specific reactivity of the terminal ends of the fragments. These
\7
side chain protecting groups are typically not removed until a mature peptidic
compound has been formed.
A further aspect of the invention is the cleavage of an organic compound, comprising
at least one moiety of the formula
(Ill),
wherein R1 is hydrogen, C1....s alkyl, C,....s alkoxy or C1....s alkylthio; R2 is C,....s alkyl, C1....s
alkoxy or C,....s alkylthio; or R1 and R2 together form a moiety of formula -(CH2)n-.
wherein n is an integer from 3 to 5; R3 is hydrogen, halogen, C1....s alkyl, C1-e alkoxy,
c,-6 alkylthio, phenyl or benzyl; and m is 0 or 1,
said cleavage optionally taking place in the presence of at least one scavenger.
In a preferred embodiment, the cleavage procedure is performed by use of an acid,
preferably by use of trifluoroacetic acid (TFA). The acid is applied neat or as mixture
with an inert solvent.
An example for a suitable inert solvent is dichloromethane (DCM). Preferably, the
molar ratio of acid and solvent is in the range from 1 :0 and 1 :2, preferably from 1 :0 and
1 :1.
After cleavage, the formed sulfonylium compound may be trapped by any suitable
scavenger. Examples for scavengers are triisopropylsilane (TIS), water, dimethyl
sulfide, C1-4 alkoxybenzenes such as 1,3,5-trimethoxybenzene (TMB) and c, ..... alkoxyphenols
such as 3,4-dimethoxyphenol and 3,5-dimethoxyphenol. The scavenger may
be used alone or as mixture such as waterffiS.
Preferably, c, ..... alkoxybenzenes and C1-4 alkoxyphenols are used as scavengers as
they are less polar nucleophiles compared to e.g. water. As a consequence, the
adducts thus obtained are easier to separate from the target compound in the following
work-up procedure.
\Z
In a preferred embodiment, said organic compound to be cleaved is an optionally
resin-bound peptidic compound which is optionally side chain protected and/or
protected at a free terminus. Preferably, R1 and R2 are independently C1-4 alkyl, C1-4
alkoxy or C1-4 alkylthio and R3 is hydrogen or halogen, most preferably, R1 and R2 are
methyl and R3 is hydrogen.
In a more preferred embodiment, said peptidic compound to be cleaved is Ac-PheArg(
MIS)-Arg(MIS)-Arg(MIS)-Arg(MIS)-Vai-NH-xanthen-3-yloxy-Merrifield resin (SEQ
10 NO 1 ), thus affording Ac-Phe-Arg-Arg-Arg-Arg-Vai-NH2 (SEQ 10 NO 2). Preferably,
TFA/OCM/TIS/water is applied as cleavage solution, most preferably in a molar ratio of
50:45:2.5:2.5. Also preferably, TFA/OCM/3,4-dimethoxyphenol is applied as cleavage
solution, most preferably in a molar ratio of 50:40:10. Also preferably, TFA/OCM/3,5-
dimethoxyphenol is applied as cleavage solution, most preferably in a molar ratio of
50:40:10. Also preferably, TFA/OCM/TMB is applied as cleavage solution, most
preferably in a molar ratio of 50:40:10.
In an also more preferred embodiment, said peptidic compound to be cleaved is
Z-Arg(MIS)-Trp(Boc)-Aia-Giy-NH-xanthen-3-yloxy-Merrifield resin (SEQ 10 NO 3), thus
affording Z-Arg-Trp-Aia-Giy-NH2 (SEQ 10 NO 4). Preferably, TFA/DCM/TMB is applied
as cleavage solution, most preferably in a molar ratio of 50:40:10.
In an also more preferred embodiment, said peptidic compound to be cleaved is
MIS-Aia-OMe, thus affording H-Aia-OMe. Preferably, TFA/dimethyl sulfide is applied
as cleavage solution, most preferably in a molar ratio of 90:10.
Examples
The following examples further illustrate this invention but are not intended to limit it in
any way. Examples 1 to 8 refer to preparation procedures and Examples 9 to 20 refer
to removal assays.
If not indicated otherwise, the L-enantiomer of the amino acid residue was used and all
reagents were obtained commercially.
Abbreviations:
Boc = terl-butoxycarbonyl
DIG = diisopropylcarbodiimide
DIPEA = diisopropylethylamine
ESMS = electrospray mass spectrometry
Fmoc = fluoren-9-ylmethoxycarbonyl
HOAt = N-hydroxy-7 -azabenzotriazole
HOBt = N-hydroxybenzotriazole
HRMS (CI) =high resolution mass spectrometry (chemical ionization)
MALO I-TOF = matrix-assisted laser desorption ionization-time of flight
MIS = 1 ,2-dimethylindol-3-sulfonyl
PyBOP = benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate
TF A = trifluoroacetic acid
TIS = triisopropylsilane
TMB = 1 ,3,5-trimethoxybenzene
Z-OSu = N-(benzyloxycarbonyloxy)succinimide
Example 1: Preparation of pyridinium 1 ,2-dimethylindole-3-sulfonate
1 ,2-0imethylindole (19. 7 g, 135.9 mmol) and sulfur trioxide pyridine complex (20.4 g,
128.3 mmol) were dissolved in pyridine (100 ml) under argon atmosphere. The
reaction mixture was refluxed for 40 hours and then cooled down to room temperature.
After addition of water (400 ml), the resulting solution was washed four times with
diethyl ether (each 250 ml). The aqueous phase was evaporated to dryness and dried
in the vacuum desiccator to render pyridinium 1 ,2-dimethylindole-3-sulfonate as red oil
(37.6 g, 96% yield).
1H NMR (400 MHz, 020): 0 = 8.44 (d, 2H, J = 5.8 Hz), 8.31 (m, 1H), 7.75 (m, 2H), 7.67
(d, 1H, J = 7.7 Hz), 7.14 (d, 1H, J = 7.4 Hz), 7.05 (m, 2H), 3.38 (s, 3H), 2.41 (s, 3H).
13C NMR (100 MHz, 020): 0 = 147.0, 140.9, 139.2, 135.6, 127.3, 124.1, 122.0, 121.0,
119.2, 112.8, 109.9, 29.2, 10.4.
HRMS (CI): m/z calc. for C1oH1oN03S [M - Ht 224.0386, found 224.0388.
Example 2: Preparation of 1 ,2-dimethylindole-3-sulfonyl chloride (MIS-CI)
Pyridinium 1 ,2-dimethylindole-3-sulfonate of Example 1 ( 16.4 g, 53.7 mmol) was
suspended in dry dichloromethane (120 ml) under nitrogen atmosphere. The solution
was cooled in an ice bath and oxalyl chloride (14 ml, 161 mmol) was slowly added.
Then, N,N-dimethylforrnamide (0.5 ml) was very slowly added under stirring. The
reaction mixture was stirred for further 30 minutes in the ice bath and then at room
temperature. After 6 hours, the solution was cooled down in an ice bath. Extra oxalyl
chloride (4 ml, 46 mmol) and N,N-dimethylformamide (0.4 ml) were added and the
reaction mixture was stirred at room temperature for further 15 hours. After addition of
oxalyl chloride (2 ml, 23 mmol) and further stirring for 4 hours, the reaction was
completed (measured by HPLC; prior measurement, treatment of a small aliquot with
methanol for 20 minutes).
The reaction mixture was evaporated to dryness at room temperature. N,N-dimethylformamide
(200 mL) was added, followed by water (100 mL). The mixture was stirred
for 5 minutes to remove the oxalyl chloride. Then, the phases were separated and the
organic phase was washed three times with water (each 100 mL). The organic phase
was dried over anhydrous magnesium sulfate and evaporated to dryness affording
1 ,2·dimethylindole-3-sulfonyl chloride. (MIS-CI) as purple solid (10.2 g, 78% yield).
1H NMR (400 MHz, DMSO): 0 = 7.82 (d, 1H, J = 7.8 Hz), 7.36 (d, 1H, J = 8.0 Hz), 7.08
(m, 2H), 7.00 (m, 2H), 3.63 (s, 3H), 2.56 (s, 3H).
13C NMR (100 MHz, DMSO): 6 = 137.2, 135.9, 125.5, 121.4, 120.8, 120.1, 109.7, 30.0,
11.3.
HRMS (CI): m/z calc. for C1oH10N02S [M - Cit 208.0426, found 208.0427.
Example 3: Preparation of Z-Arg(MIS)·OH
Z-Arg-OH (2 g, 6.5 mmol) was dissolved in acetone (65 mL) and 3N aqueous sodium
hydroxide solution (18 mL, 54 mmol). The reaction was cooled in an ice bath and
MIS-CI of Example 2 (1.59 g, 6.5 mmol), dissolved in acetone (50 mL), was added
during 10 minutes. The reaction mixture was stirred for 1 hour at 0 °C. Then, additional
MIS-CI (0.95 g, 3.9 mmol) in acetone (20 mL) was added, followed by stirring for
90 minutes at 0 °C. Finally, a last portion of MIS-CI (0.95 g. 3.9 mmol) in acetone
(15 mL) was added. The reaction mixture was stirred for additional 30 minutes at 0 °C
and further 3 hours at room temperature, until no MIS-CI was detected by TLC
(hexane:ethyl acetate 1:1 ). The pH of the reaction was neutralized with 1 0% aqueous
citric acid. After evaporation of the acetone in vacuo, water (100 mL) was added and
the pH was acidified to pH 3 with 10% aqueous citric acid. Then, the solution was three
times extracted with ethyl acetate (each 100 mL). The organic phases were put together,
washed three times with water (each 75 mL), dried over magnesium sulfate
and finally evaporated to dryness. The crude obtained was purified twice by column
chromatography (dichloromethane, methanol, acetic acid). The pure fractions were
combined and the solvent was removed in vacuo yielding an oil. For precipitation, the
minimum amount of a mixture of ethyl acetate, dichloromethane and methanol was
added followed by addition of hexane until no further precipitation was observed. The
solvent was decanted and the solid was washed four times with a mixture of dichloromethane
and hexane and finally dried over magnesium sulfate yielding 18% (0.61 g) of
Z-Arg(MIS)-OH.
I '
'
I
I
I
'
1
~I
~· I i
1H NMR (400 MHz, DMSO): 6 = 7.85 (d, 1H, J = 7.6 Hz), 7.52 (d, 1H, J = 8.0 Hz), 7.43
(d, 1 H, J = 8.0 Hz), 7.30 (m, 5H), 7.10 {m, 2H), 5.01 (s, 2H), 3.87 (m, 1H), 3.66 (s, 3H),
3.0 (m, 2H), 2.60 {s, 3H), 1.64 (m, 1 H), 1.49 (m, 1 H), 1.41 (m, 2H).
13C NMR (100 MHz, DMSO): 6 = 174.4, 157.0, 156.8, 139.4, 137.7, 135.9, 129.0,
128.5, 128.4, 125.2, 122.11 121.1 1 120.11 11 0.4, 66.1 1 54.3, 40.0, 30.2, 28.9, 26.4,
11.4.
HRMS (CI): rn/z calc. for c24H30NsOsS [M + Ht 516.1911 I found 516.1911.
Example 4: Preparation of Fmoc-Arg(MIS)·OH
1. Preparation of H-Arg(MIS)-OH
A mixture of Z-Arg(MIS)-OH as obtained from Example 3 {486 mg, 0.94 mmol) and
10% Pd/C {110 mg) in methanol {60 mL) was hydrogenated overnight at atmospheric
pressure. As the reaction was still incomplete (measured by TLC; dichloromethane:
methanol:acetic acid, 90:9:1), 10% Pd/C (100 mg) was added and the reaction was
hydrogenated for further 24 hours till completeness {measured by TLC).
The reaction mixture was filtered over celite and evaporated to dryness yielding 98%
(352 mg) of H-Arg(MIS)-OH.
1H NMR (400 MHz, DMSO): 6 = 7.83 (d, 1H, J = 7.6 Hz), 7.47 (d, 1H, J = 8.1 Hz), 7.42
(d, 1H, J = 8.1 Hz), 7.11 (m, 2H), 3.65 {s, 3H), 3.17 (m, 1H), 3.00 {m, 2H), 2.60 (s, 3H),
1.65 (m, 1 H), 1.54 (m, 1 H), 1.42 (m, 2H).
2. Preparation of Fmoc-Arg(MISl-OH
Fmoc-CI {84 mg, 0.32 mmol) was dissolved In 1 ,4-dioxane (0.5 mL). Sodium azide
(25 mg, 0.39 mmol) in water (0.4 mL) was added and the resulting emulsion was
stirred for 2 hours at room temperature. Then, the emulsion was slowly added to a
solution of H-Arg(MIS)-OH as obtained in the previous step (136 mg, 0.36 mmol) in a
1:1 mixture of water and dioxane at pH 9, which was controlled by addition of 10%
aqueous sodium carbonate. The reaction mixture was stirred while keeping the pH at
9. Once the pH was stabilized, the mixture was stirred overnight. Then, water (30 ml)
was added and the mixture was washed three times with tert-butyl methyl ether (each
20 mL).The aqueous phase was acidified with 1 N HCI to a pH of 2 to 3 and was then
quickly extracted three times with ethyl acetate {30 ml). The organic phases were
combined and dried over magnesium sulfate. After evaporation to dryness, an oil
(115 mg) was obtained, which was dissolved in a minimum of acetone. Then, aqueous
sodium carbonate (20 mL) was added at pH 9 and the aqueous solution was washed
three times with tert-butyl methyl ether (each 30 ml). The aqueous solution thus
obtained was acidified with 1 N HCI to a pH of 2 to 3, then extracted three times with
ethyl acetate (each 20 ml), dried over magnesium sulfate and finally evaporated to
dryness yielding 34.3% (67.4 mg) of Fmoc-Arg(MIS)-OH.
1H NMR (400 MHz, DMSO): 6 = 7.86 (m, 3H), 7.70 (d, 2H, J = 7.4 Hz), 7.59 (d, 1H,
J = 7.9 Hz), 7.42 (d, 1H, J = 8.1 Hz), 7.39 (m, 2H), 7.30 (m, 2H), 7.10 (m, 2H), 4.27 (m,
2H), 4.20 (m, 1 H), 3.86 (m, 1 H), 3.66 (s, 3H), 3.01 (m, 2H), 2.61 (s, 3H), 1.65 (m, 1 H),
1.52 (m, 1 H), 1.38 (m, 2H).
13C NMR (100 MHz, DMSO): 6 = 174.4, 157.0, 156.8, 144.5, 141.4, 139.4, 135.9,
128.3, 127.8, 126.0, 125.2, 122.1, 121.1, 120.8, 120.1, 110.4, 66.3, 55.6,47.3,40.0,
30.2, 28.8, 26.5, 11.4.
HRMS (CI): mlz calc. for C31H34NsOaS [M + Ht 604.2224, found 604.2222.
Example 5: Preparation of Ac-Phe-Arg(MIS)-Arg(MIS)-Arg(MIS)-Arg(MIS)-Vai-NHz
(SEQ 10 NO 2)
Sieber amide resin (25 mg, 0.42 mmol/g, 9-Fmoc-aminoxanthen-3-yloxy-M~rrifield
resin) was placed in a 2 ml polypropylene syringe fitted with a polyethylene filter disk.
The resin was swollen with dichloromethane. Subsequently, washings with
dichloromethane and N,N-dimethylformamide were carried out and the Fmoc group
was removed by treatment with a 2:8 mixture of piperidine and N,N-dimethylformamlde
(once for 1 minute, and two times for 10 minutes). Fmoc-Vai-OH (14.3 mg, 42.1 IJmol)
was coupled using HOBt (5.7 mg, 42.1 j.lmol) and DIC (6.71JL, 42.1 j.lmol) in
N,N-dimethylformamide for 1.5 hours. The Fmoc group was removed In the usual way,
and Fmoc-Arg(MIS)-OH as obtained from Example 4 (15.8 mg, 26.3 1Jmol) was
coupled using PyBOP (13.7 mg, 26.31JmOI), HOAt (3.6 mg, 26.31Jmol) and DIPEA
(13.4 IJL, 78.9 1Jmol) in N,N-dimethylformamide for 90 minutes. The resin was
acetylated by treatment with acetic anhydride (50 eq) and DIPEA (50 eq) in DMF for
25 min in order to do the capping of the unreacted amines, the Fmoc group was
removed and the same procedure was repeated three more times including acetylation
of the resin prior to Fmoc removal. After the last Fmoc removal, Fmoc-Phe-OH
(13.6 mg, 351Jmol) was coupled using PyBOP (18.3 mg, 35J,Jmol), HOAt (4.8 mg,
35 IJmol) and OIPEA (17.91JL, 105.2 J,~mol) in N,N-dimethylformamide for 90 min. The
Fmoc group was removed, and the resulting free amino group was acetylated in the
same way as described above. The protected, resin-bound peptide Ac-Phe-Arg(MIS)Arg(
MIS)-Arg(MIS)-Arg(MIS)-Vai-NH-xanthen-3-yloxy-Merrifield resin thus obtained
was washed with N,N-dimethylformamide, dichloromethane and diethyl ether, dried in
vacuo and then divided into five aliquots.
One aliquot was used for the preparation of the target compound of this example and
the other aliquots were used as starting material for the removal assays of e.g. Example
9.
Thus, one aliquot was swollen with dichloromethane and treated with 1.5 ml of a
mixture of TFA, dichloromethane, TIS and water (2:93:2.5:2.5) for 20 minutes in order
to cleave the protected peptide from the resin. The resin was filtered and the collected
solution was diluted with dichloromethane and neutralized by adding DIPEA (80 J.IL,
1.2 eq per eq of TFA). The solvent was removed in vacuo. After addition of water and
acetonitrile, the solution was lyophilized obtaining Ac-Phe-Arg(MIS)-Arg(MIS)Arg(
MIS)-Arg(MIS)-Vai-NH2 (SEQ 10 NO 2).
The product was characterized by LC-MS and HRMS (CI): rn/z calc. for
CsoH1o1N2301sS4 [M + Nat 1780.7092, found 1780.7152.
Example 6: Preparation of Z·Arg(MIS)· Trp(Boc)·Aia-Giy-NH2 (SEQ ID NO 4)
Sieber amide resin (70 mg, 0.40 mmol/g) was placed in a 2 ml polypropylene syringe
fitted with a polyethylene filter disk. The resin was swollen with dichloromethane,
washings with dichloromethane and N,N-dimethylformamide were carried out and the
Fmoc group was removed. Fmoc-Giy-OH {33.3 mg, 112 J.lmol), Fmoc-Aia-OH
(34.9 mg, 112 J.lmol) and Fmoc-Trp(Boc)-OH (59.0 mg, 112 J.lmol) were sequentially
coupled using PyBOP (58.3 mg, 112 J.lmol) HOAt (15.2 mg, 112J,Jmol) and DIPEA
(57.4 JJL, 336 J.lmol) in N,N-dimethylformamide for 1.5 hours. The resin was divided
into two equal parts. One part was used for the preparation of the target compound of
this example and the other part for the preparation of Z-Arg(Pbf)-Trp(Boc)-Aia-Giy-NH2
(see Example 8.2).
Thus, Z-Arg(MIS)-OH (28.9 mg, 56 J,Jmol) was coupled with one resin part using
PyBOP (29.2 mg, 561Jmol), HOAt (7.6 mg, 561Jmol) and DIPEA (28.7 J.IL, 1681Jmol) in
N,N-dimethylformamide for 1.5 hours. The protected, resin-bound peptide Z-Arg(MIS)Trp(
Boc)-Aia-Giy-NH-xanthen-3-yloxy-Merrifield resin thus obtained was washed with
N,N-dimethylformamide, dichloromethane and diethyl ether, dried in vacuo and then
divided into aliquots of 4 mg. One aliquot was used for the preparation of the target
compound of this example and the other aliquots were used as starting material for the
removal assays of e.g. Example 19.
Thus, one aliquot was swollen with dichloromethane and treated with 1.5 ml of a
mixture of TFA, dichloromethane, TIS and water (2:93:2.5:2.5) for 20 minutes in order
to cleave the protected peptide from the resin. The resin was filtered and the collected
solution was diluted with dichloromethane and neutralized by adding DIPEA (80 !JL,
1.2 eq per eq of TFA). The solvent was removed in vacuo. After addition of water and
acetonitrile, the solution was lyophilized obtaining Z-Arg(MIS)-Trp(Boc)-Aia-Giy-NH2
with 95% purity (by HPLC). The product obtained was characterized by LC-MS.
Example 7: Preparation of MIS·Aia-OMe
H-Aia-OMe (95 mg, 0.68 mmol, 1 eq) was dissolved in dry dichloromethane and
DIPEA (3 eq) was added. A solution of MIS-CI (200 mg, 1.2 eq), as obtained from
Example 2, in dry dichloromethane was added, and the reaction mixture was stirred for
1.5 hours at room temperature. Work up in the usual way yielded 85.4 mg (40%) of
MIS-Aia-OMe.
Example 8: Preparation of the Pbf protected comparison compounds
8.1 Preparation of Ac-Phe-Ara(Pb0-Ara(Pb0-Arg(Pb0-Ara(Pbfl-Vai-NH2
(SEQ 10 NO 2)
The same procedure as for the preparation of Ac-Phe-Arg(MIS)-Arg(MIS)-Arg(MIS)Arg(
MIS)-Vai-NH2 was applied (see Example 5) except for replacing Fmoc-Arg(MIS)OH
by Fmoc-Arg(Pbf)-OH (17.1 mg, 26.3 J.Jmol). The product obtained was
characterized by LC-MS and HRMS (CI): m/z calc. for Cg2HnsN19019S4 [M + Ht
1938.9137, found 1938.9202.
8.2 Preparation of Z-Ara(PbQ-TrpCBoc)-Aia-Giy-NHz (SEQ ID NO 4)
Fmoc-Arg(Pbf)-OH (36.3 mg, 56 ~mol) was coupled with the other resin part from
Example 6 using PyBOP (29.2 mg, 56 J,Jmol), HOAt (7.6 mg, 56 ~mol) and DIPEA
(28.7 JJL, 168 ~mol) in N,N~imethylformamide for 1.5 hours. The Fmoc group was
removed and the free amine was protected with the Z group by treatment with Z-OSu
(14.0 mg, 56 J,Jmol) and DIPEA (35.9 JJL, 210 J.Jmol). The protected, resin-bound
peptide Z-Arg(Pbf)-Trp(Boc)-Aia-Giy-NH-xanthen-3-yloxy-Merrifield resin thus obtained
was washed with N,N-dimethylformamide, dichloromethane and diethyl ether, dried in
vacuo, and then divided into aliquots of 4 mg.
One aliquot was used for the preparation of the target compound of this example and
the other aliquots were used as starting material for the removal assay of e.g. Example
20.
Thus, one aliquot was cleaved in the same way as described in Example 5.
Z-Arg(Pbf)-Trp(Boc)-Aia-Giy-NH2 was obtained with 96% purity (by HPLC). The
product was characterized by LC-MS.
8.3 Preparation of Pbf-Aia-OMe
Preparation was performed analogous to Example 7 except for Pbf-CI (1.2 eq) instead
of MIS-CI. Yield: 209 mg (82%) of Pbf-Aia-OMe.
Examples 9 to 12: Removal assays of MIS versus Pbf protected, resin-bound
peptides
General procedure
The protected, resin-bound peptide (3 mg) was treated with cleavage solution (50 ~L).
After the cleavage time, the solution was poured into water (4 ml). Then, TFA and
dichloromethane were evaporated. The resulting aqueous solution was washed six
times with dichloromethane (each 1 ml) and lyophilized. The resulting solid was
analyzed by HPLC (A= 220 nm) and ESMS or MALDI-TOF.
Table 1: Examples 9 to 12 with cleavage solution TFA/DCMITIS/water (50:45:2.5:2.5)
(water and TIS as scavengers)
Example Protected, resin Cleavage time Ac-Phe-Arg-Arg-Arg-Arg-Vai-NH2
bound peptide (SEQ 10 NO 2)
9 a 30 min 100%
10 a 60 min 100%
11 b 30 min 4%
12 b 60 min 38%
a = Ac-Phe-Arg(MIS)-Arg(MIS)-Arg(MIS)-Arg(MIS)-Vai-NH-xanthen-3-yloxy-
Merrifield resin (SEQ 10 NO 1) as obtained from Example 5.
b = Ac-Phe-Arg(Pbf)-Arg( Pbf)-Arg(Pbf)-Arg(Pbf)-Vai-N H-xanthen-3-yloxyMerrifield
resin (SEQ 10 NO 1) as obtained from Example 8.1 (comparison
example).
DCM = Dichloromethane.
Examples 13 to 15: Removal assays of MIS protected, resin-bound peptide& with
different scavengers
The general procedure as described in Examples 9 to 12 was followed. As protected,
resin-bound peptide Ac-Phe-Arg(MIS)-Arg(MIS)-Arg(MIS)-Arg(MIS)-Vai-NH-xanthen-3-
yloxy-Merrifield resin (SEQ 10 NO 1 ), as obtained from Example 5, was used. The
cleavage time was 60 minutes. A mixture of TFA, dichloromethane and scavenger
(50:40:10) was used as cleavage solution.
The scavengers tested were 3,4-dimethoxyphenol (Example 13), 1,3,5-trimethoxybenzene
(TMB) (Example 14) and 3,5-dlmethoxyphenol {Example 15).
As a result, the amount of MIS-OH was reduced by more than 10 times compared to
Example 9, in which water (2.5%) and TIS (2.5%) were used as scavengers. In the
case of Tmb, a reduction by more than 40 times was even observed.
Examples 16 and 17: Removal assays of MIS versus Pbf protected, resin·bound,
Trp-containing peptides
The general procedure as described in Examples 9 to 12 was followed. The resulting
crudes were characterized by LC-MS and its purity was analyzed by HPLC
(A= 220 nm).
The purity of the resulted crude was higher for the MIS protected starting material
compared to the Pbf protected starting material. For both protected peptides c and d,
neither undesired Trp alkylation nor sulfonation was observed in the formed product.
Table 2: Examples 16 and 17; with cleavage solution TFAIDCMITMB (50:40:10) (TMB
as scavenger)
Example Protected, resin Cleavage time Z-Arg-Trp-Aia-Giy-NH2
bound peptide (SEQ 10 N04}
16 c 60min 83.4%.
17 d 60min 63.4%*
c = Z-Arg(MIS}-Trp(Boc}-Aia-Giy-NH-xanthen-3-yloxy-Merrifield resin (SEQ ID
NO 3) as obtained from Example 6.
d = Z-Arg(Pbf)-Trp(Boc)-Aia-Giy-NH-xanthen-3-yloxy-Merrifield resin (SEQ 10
NO 3) as obtained from Example 8.2 (comparison example).
DCM = Oichloromethane- TMB = 1,3,5-trimethoxybenzene- • Z-Arg{MIS)-TrpAia-
Giy-NH2 (SEQ 10 NO 4) was not detected in the resulting crude (by LC-MS)*
20.4% of Z-Arg{Pbf}-Trp-Aia-Giy-NH2 (SEQ ID NO 4) was detected in the resulting
crude (by HPLC}.
Examples 18 to 20: Removal assays of MIS versus Pbf as N"-amino protecting
groups
The protected amino acids were treated with the cleavage solution at room temperature
and the deprotection was followed by TLC.
MIS removal was slightly faster, presenting a positive Kaiser test (indicating the
presence of free amines) after 5 min, whereas for the case of Pbf the positive Kaiser
test was in the next control (10 min).
Table 3: Examples 18 to 20; with cleavage solution TFA/dimethyl sulfide (90:10)
(dimethyl sulfide as scavenger)
Example Protected peptide Cleavage time H-Aia-OMe•
18 e 5 min detected
19 f 5 min not detected
20 f 10 min detected
e = MIS-Aia-OMe as obtained from Example 7.
f = Pbf-Aia-OMe as obtained from Example 8.3 (comparison example).
• Presence or absence of H-Aia-OMe was detected by the Kaiser test.
I

We Claim:
1. A process for the preparation of the compound of formula (II),
X
0" I
s~
0
(II),
wherein R1 is hydrogen, C1-s alkyl, C1-s alkoxy or C1-s alkylthio; R2 is C1-s alkyl,
C1-s alkoxy or C1-s alkylthio; or R 1 and R2 together form a moiety of formula
-(CH2)n-, wherein n is an integer from 3 to 5; R3 is hydrogen, halogen,
C1-s alkyl, C1-s alkoxy, C1-s alkylthio, phenyl or benzyl; and X is chlorine or
bromine;
comprising the step of reacting a compound of formula
(I),
wherein R 1, R2 and R3 are as defined above, or a salt thereof,
with oxalyl chloride or oxalyl bromide.
2. The process of claim 1, wherein R1 and R2 are independently C1-4 alkyl,
C1-4 alkoxy or C1-4 alkylthio, and R3 is hydrogen or halogen.
3. The process of claim 1 or 2, wherein R1 and R2 are methyl and R3 is hydrogen
and X is chlorine.
4. The process of any of claims 1 to 3, wherein the reaction is performed with the
pyridinium salt of the compound of formula (I).
5. Use of the compound of formula (II), with the compound of formula (II) being as
defined in claim 1, as a protective reagent for the protection of a peptidic
compound which comprises at least one guanidino moiety and/or at least one
amino group.
6. The use of claim 5, wherein the peptidic compound is a resin-bound peptidic
compound; R1 and R2 are independently C1-4 alkyl, C 1-4 alkoxy or C 1-4 alkylthio,
and R3 is hydrogen or halogen.
7. The use of claim 5, wherein the peptidic compound is side chain protected
and/or protected at a free terminus; R1 and R2 are independently C1-4 alkyl, C1-4
alkoxy or C1-4 alkylthio, and R3 is hydrogen or halogen.
8. The use of claim 5, wherein the guanidino moiety is part of an arginine or homoarginine
residue.
9. A process for the protection of a peptidic compound, the peptidic compound
being as defined in claim 5,
comprising the step of reacting said peptidic compound with the compound of
formula (II), with the compound of formula (II) being as defined in claim 1,
thus affording a compound, which comprises at least one moiety of the formula
(Ill),
wherein R 1
, R2 and R3 are defined as in claim 1, and m is 0 or 1.
r
t
-
10. The process of claim 9, wherein R1 and R2 are independently C1-4 alkyl,
C1-4 alkoxy or C1-4 alkylthio and R3 is hydrogen or halogen.
11. The process of claim 9 or 10, wherein R1 and R2 are methyl, R3 is hydrogen and
X is chlorine.
12. The process of any of claims 9 to 11, wherein the peptidic compound is a resinbound
peptidic compound.
13. The process of any of claims 9 to 12, wherein the peptidic compound is side
chain protected and/or protected at a free terminus.
14. A peptidic compound comprising at least one moiety of the formula (Ill), with the
moiety of formula (Ill) being as defined in claim 9.
15. The compound of claim 14, wherein the peptidic compound is resin-bound
peptidic compound.
16. The compound of claim 14 or 15, wherein the peptidic compound is side chain
protected and/or protected at a free terminus.
17. N-alpha-benzyloxycarbonyi-N-omega-(1 ,2-dimethylindole-3-sulfonyi)-L-arginine
18. N-alpha-Fmoc-N-omega-(1 ,2-dimethylindole-3-sulfonyi)-L-arginine
Dated this the [ ~ ~
31
I HA SINGH NAIR
Agent f4 r the Applicant [IN/PA
LEX 0 BIS
Intelle ual Property Practice
709171 0, Tolstoy House,
15-17, Tolstoy Marg,
New Delhi-It 0 00 I
1
l.