Note: Descriptions are shown in the official language in which they were submitted.
CA 0223231~ 1998-03-17
Bifunctional 8ulfide-Containing 8ulfonamide-Chelating Agents
8uch as 8~Y for Radioactive Isotopes
The invention relates to new chelating agents that contain
sulfonamide groups, pharmaceutical agents that contain these
compounds, their use in radiodiagnosis and radiotherapy, a
process for the production of these compounds and agents, and
conjugates of these compounds with substances that selectively
accumulate in diseased tissue, especially peptides.
The use of radiopharmaceutical agents for diagnostic and
therapeutic purposes has been known for a long time in the field
of biological and medical research. In particular,
radiopharmaceutical agents are used to visualize certain
structures, such as, for example, the skeleton, organs, or
tissue. Diagnostic application requires the use of radioactive
agents which, after administration, accumulate specifically in
the structures in patients that are to be examined. These
radioactive agents that accumulate locally can then be traced,
plotted, or scintigraphed using suitable detectors, such as, for
example, scintillation cameras or other suitable recording
processes. The dispersion and relative intensity of the detected
radioactive agent characterize 1:he site of a structure where the
radioactive agent is located and can show the presence of
anomalies in structure and function, pathological changes, etc.
Similarly, radiopharmaceutical agents can be used as therapeutic
agents to irradiate specific pathological tissues or regions.
Such treatment requires the production of radioactive therapeutic
CA 0223231~ 1998-03-17
agents that accumulate in specific structures, tissues, or
organs. By concentrating these agents, therapeutic radiation is
brought to bear directly on the! pathological tissue.
The use of both diagnostic and therapeutic
radiopharmaceutical agents requires compounds that can be
radiolabeled. In the case of metallic radionuclides, the metal
can be present in free form as an ion or in the form of a metal
complex with one or more ligands. Examples of metallic
radionuclides that can form complexes are technetium-99m and the
various rhenium isotopes. The former is used in diagnosis, and
the latter is employed in therapy. The radiopharmaceutical
agents contain suitable vehicles and additives that allow
injection, inhalation, or ingestion by patients, just like
physiological buffers, salts, etc.
The radionuclide that is used most often for tasks in
nuclear medicine is technetium-99m, which, owing to its
advantageous physical properties (no corpuscular radiation, 6
hours of physical half-life, 140 KeV of gamma-radiation) and the
low radiation exposure that results from it, is especially well
suited as a radioisotope for in vivo diagnosis. Technetium-99m
can easily be obtained from nuclide generators as pertechnetate
and can be used in this form directly for the production of kits
for routine clinical needs.
The production of radiopharmaceutical agents first requires
the synthesis of a suitable ligand. Then, the complex with the
radionuclide is visualized separately (labeling). To do this,
the ligand that is produced, invariably in the form of a freeze-
CA 0223231~ 1998-03-17
dried kit, is reacted under complexing conditions with a solution
that contains the radionuclide. If, for example, the production
of a technetium-99m radiopharmaceutical agent is desired, the
ligand that is produced is mixed with a pertechnetate solution
with the addition of a suitable reducing agent, and the
corresponding technetium complex is produced under suitable
reaction conditions. These complexes are then administered to
the patient in a suitable way by injection, inhalation, or
ingestion.
The solutions that contain the radionuclide can, as in the
case of technetium-99m, be obtained from an available Mo-99/Tc-
99m nuclide generator, or may be ordered from a manufacturer, as
in the case of rhenium-186. The complexing reaction is carried
out at suitable temperatures (e.g. 20~-100~C) within periods
ranging from a few minutes to several hours. To ensure complete
complexing, a large excess (more than a 100-fold excess in the
metal-radionuclide) of the ligand that is produced and enough
reducing agent to ensure complet:e reduction of the radionuclide
that is used are necessary.
Radiopharmaceutical agents are produced by combining the
radionuclide complex, in an amount that is sufficient for
diagnostic or therapeutic application, with pharmacologically
acceptable radiological vehicles. This radiological vehicle
should have advantageous propert:ies for the administration of the
radiopharmaceutical agent in the form of an injection,
inhalation, or ingestion. Examples of such vehicles are HSA,
aqueous buffer solutions, e.g., tris-(hydroxymethyl)aminoethanes
CA 0223231~ 1998-03-17
(or their salts), phosphate, citrate, bicarbonate, etc., sterile
water, physiological common salt solution, isotonic chloride or
dicarbonate-ionic solutions or normal plasma ions, such as Ca2~,
Na', K' and Mg2'.
Since technetium can be present in a number of oxidation
stages (+7 to -1), it is often necessary for radiopharmaceutical
agents to contain additional agents, which are known as
stabilizers. The latter keep t:he radionuclide in a stable form
until it has reacted with the ligand. These stabilizers can
contain agents that are known as transfer or auxiliary ligands,
which are especially useful for stabilizing and complexing the
metal in a well-defined oxidation stage until the target ligand
complexes the metal via a ligand exchange. Examples of this type
of auxiliary ligands are (including their salts) gluconoheptoic
acid, tartaric acid, citric acid, or other common ligands, as is
explained in more detail later.
In a standard fashion, raclionuclide-containing
radiopharmaceutical agents are produced by the ligand first being
synthesized and then being reac:ted with the metal-radionuclide in
a suitable way to form a corresponding complex, in which the
ligand necessarily must be present unchanged after complexing,
with the exception of the cleavage of optionally present
protective groups or hydrogen ions. The removal of these groups
facilitates the coordination of the ligand on the metal ion and
thus results in guick complexing.
To form technetium-99m complexes, pertechnetate is first
obtained from a nuclide generat:or and shifted, with the aid of
CA 0223231~ 1998-03-17
suitable reducing agents (e.g., SnCl2, S2042~, etc.), into a lower
oxidation stage, which then is stabilized by a suitable chelating
agent. Since technetium can be present in a number of oxidation
stages (+7 to -1), which can greatly alter the pharmacological
properties by altering the charge of a complex, it is necessary
to provide chelating agents or complex ligands for technetium-99m
that can bind technetium securely, tightly, and in a stable
manner to a defined oxidation stage to keep undesirable
biodistribution, which impedes reliable diagnosis of
corresponding diseases, from occurring due to in vivo redox
processes or technetium release-; from the corresponding
radiodiagnostic agents.
The efficiency of radionuclides in in vivo diagnosis and in
therapy depends on the specificity and selectivity of the labeled
chelates with respect to the target cell. These properties are
enhanced by coupling the chelates to biomolecules according to
the "drug-targeting" principle. Offered as biomolecules are
antibodies, their fragments, hormones, growth factors, and
substrates of receptors and enzymes. Thus, in British Patent
Application GB 2,109,407, the use of radiolabeled monoclonal
antibodies against tumor-associated antigens is described for in
vivo tumor diagnosis. Direct protein labelings via donor groups
(amino, amide, thiol, etc.) of the protein (Rhodes, B. A. et al.,
J. Nukl. Med. 1986, 27, 685-693~ or by introducing complexing
agents (US Patent 4,479,930 and Fritzberg, A. R. et al., Nucl.
Med. 1986, 27, 957) with technetium-99m have also been described.
These experimental methods are not available for clinical use,
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however, since, on the one hand, their selectivity is too low
and, on the other hand, the background activity in the organism
is too high to make in vivo imaging possible.
Regarded as suitable complexing agents for technetium and
rhenium isotopes are, e.g., cyclic amines as they are described
by Volkert et al. (Appl. Radiol. Isot. 1982, 33; 891) and
Troutner et al. (J. Nucl. Med. 1980, 21; 443), which have the
drawback, however, that they frequently are able to bind
technetium-99m in good yields only starting from a pH > 9. Nz02
systems (Pillai, M. R. A., Troutner, D. E. et al., Inorg. Chem.
1990, 29; 1850) are in clinical use. Non-cyclic N4 systems, such
as, e.g., the HMPA0, suffer from low complex stability as a major
disadvantage. Because of its instability (Ballinger, J. R. et
al., Appl. Radiat. Isot. 1991, 42; 315), Billinghurst, M. W. et
al., Appl. Radiat. Isot. 1991, 42; 607), Tc-99m-HMPA0 must be
administered within 30 minutes after it is labeled, so that the
portion of decomposition products that have a different
pharmacokinetics and separation can be kept small. Such
radiochemical contaminants hamper the detection of diseases that
are to be diagnosed. Coupling these chelates or chelating agents
to other substances that accumulate selectively in foci of
disease cannot be accomplished by simple means, so that the
latter are dispersed in general in an unspecific manner in the
organism.
N2S2 chelating agents (Bormans, G. et al.; Nucl. Med. Biol.
1990, 17; 499), such as, e.g., ethylenedicysteine (EC;
Verbruggen, A.M. et al.; J. Nucl. Med. 1992, 33; 551)
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specifically meet the requirement for sufficient stability of the
corresponding technetium-99m complex, but form radiodiagnostic
agents with a purity of greater than 69% starting only from a pH
of the complexing medium > 9. N3S systems (Fritzburg, A.; EP-
0173424 and EP-0250013) form stable technetium-99m complexes but
must be heated to temperatures of about 100~C to form a uniform
radiopharmaceutical agent.
In recent years, the demand for radiodiagnostic agents that
accumulate specifically in diseased tissue has increased. This
can be accomplished if complexing agents can be readily coupled
to selectively accumulating substances and, in so doing, do not
lose their advantageous complexing properties. Since it very
frequently happens, however, that after a complexing agent is
coupled to such a molecule with the aid of its functional groups,
a weakening of complex stability is observed, previous attempts
to couple chelating agents to selectively accumulating substances
do not appear to have been very satisfactory since a
diagnostically non-tolerable portion of the isotope from the
conjugate is released in vivo (}3rechbiel, M. W. et al.; Inorg.
Chem. 1986, 25, 2772). It is therefore necessary to produce
bifunctional complexing agents 1hat carry both functional groups
for binding the desired metal ion and a (another, several)
functional group(s) for binding the selectively accumulating
molecule, or configuring the complexing agent in such a way that
the desired complexing agent structure is formed only by coupling
to a selectively accumulating substance, and thus a weakening of
complex stability will not occur. Such ligands make possible a
CA 0223231~ 1998-03-17
specific, chemically defined binding of technetium or rhenium
isotopes to a wide variety of biological materials, even if a so-
called prelabeling is carried out. Several chelating agents,
coupled to monoclonal antibodies (e.g., EP-0247866 and EP-
0188256) or fatty acids (EP-0200492), have been described. As
chelating agents, however, the already mentioned N2S2 systems are
used, which are not very suitable owing to their low stability.
Since both the selectively accumulating substances are very
different in terms of their properties and also in terms of the
mechanisms according to which they are concentrated, it is
further necessary to vary the couplable chelating agent and to be
able to adapt the physiological requirements of the coupling
partner with respect to its lipophilia, membrane permeability,
etc.
The object of the invention is therefore to make available
stable complex compounds that are or can be coupled to various
selectively accumulating compounds, without their specificity and
selectivity being fundamentally affected. In addition, the
object exists of preparing such couplable chelating agents or
complexes that have a greater chemical variation range of the
substituents, in order to be able to match the latter to the
above-referenced requirements. In this case, the requirements
for the application of these compounds to humans must be met in
terms of the radiation dose taken up and the stability and
solubility of the compounds.
This object is achieved ac:cording to the invention in that
new chelating agents that contain bifunctional sulfonamide groups
CA 02232315 1998-03-17
and their coupling products with specifically accumulating
compounds are made available.
The subject of the invention is compounds of general formula
(I)
M - L (I)
in which
M means a radioisotope of Tc or Re and L means a ligand
of general formula (II)
Rl~-CR2R3- (CR4RS) n 1 2-8-CHR6-CHR7-So2-NH- (CR8R9) ~F1 2--CO-B
(II)
in which
R1 stands for a hydrogen atom, for a branched or
unbranched C16 alkyl radical or for a sulfur protective
group,
R2, R3, R4, R5, R6, R7, R8 and R9 are the same or different and
in each case stand for a hydrogen atom and/or for a
branched or unbranched C16 alkyl radical,
B stands for a hydroxyl group, a mercapto group or a
radical -NHRa, in which
Ra represents a hydrogen atom, a branched or
straight-chain, cyclic or polycyclic C130 alkyl,
alkenyl, polyalkenyl, alkinyl, polyalkinyl, aryl,
alkylaryl or ary:Lalkyl radical, which optionally
is substituted w:ith hydroxy, oxy, oxo, carboxy,
aminocarbonyl, a:lkoxycarbonyl, amino, aldehyde or
alkoxy groups with up to 20 carbon atoms and/or
optionally is interrupted and/or substituted by
CA 02232315 1998-03-17
one or more heteroatoms from the series O, N, S,
P, As and Se.
Preferred compounds of general formula (I) are distinguished
in that n and m in each case stand for 1, and in that R1, R2, R5,
R6, R7 and R4 are hydrogen atoms.
Especially preferred compounds of general formula (I) are
distinguished in that n and m in each case stand for 1 and in
that Rl, R2, R5, R6, R7 and R9 are hydrogen atoms, and B stands for
a hydroxyl group or a radical -NHRa,
in which
Ra represents a hydrogen atom, a branched or straight-
chain, cyclic or polycyclic C130 alkyl, alkenyl,
polyalkenyl, alkinyl, polyalkinyl, aryl, alkylaryl or
arylalkyl radical, which optionally is substituted with
hydroxy, oxy, oxo, carboxy, aminocarbonyl,
alkoxycarbonyl, amino, aldehyde or alkoxy groups with
up to 20 carbon atoms and/or optionally is interrupted
and/or substituted by one or more heteroatoms from the
series O, N, S, P, As and Se.
Another subject of the invention relates to new bifunctional
sulfur atom-interrupted sulfonamide ligands of general formula
(II)
R18-CR2R3- tCR~'R5) ,F1 z-B-CHR6-CHR7-Bo2-NH- (CR8R9) F1 2-CO-B
(II)
in which R1, R2, R3, R4, Rs, R6, R7, R8, R9, n, m, and B in
each case have the meaning that: is indicated above.
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Preferred compounds of general formula (II) are
distinguished in that in each case 1 stands for n and m and in
that R1, RZ, R5, R6, R7 and R9 are hydrogen atoms.
Especially preferred compounds of general formula (I) are
distinguished in that in each case 1 stands for n and m, and in
that R1, R2, R5, R6, R7 and R9 are hydrogen atoms and B stands for
a hydroxyl group or a radical -NHRa,
in which
Ra represents a hydrogen atom, a branched or straight-chain,
cyclic or polycyclic C130 alkyl, alkenyl, polyalkenyl,
alkinyl, polyalkinyl, aryl, alkylaryl, arylalkyl radical,
which optionally is substituted with hydroxy, oxy, oxo,
carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde or
alkoxy groups with up to 2~ carbon atoms and/or optionally
is interrupted and/or substituted by one or more heteroatoms
from the series 0, N, S, P, As and Se.
Another subject of the invention is conjugates that contain
a compound of general formula (:1 and/or II) and nucleotides such
as DNA and RNA, as well as substances that selectively accumulate
in diseased tissue, whereby between the latter, a covalent bond
exists and this is present in amide form in the case of
substances that contain carboxyl or amino groups, such as
naturally occurring or modified oligonucleotides, in which
degradation is prevented or hampered by naturally occurring
nucleases, peptides, proteins, antibodies or their fragments, or
is present in imide form in the case of substances that contain
CA 022323l~ l998-03-l7
12
hydroxyl groups, such as fatty alcohols that are in ester form or
in the case of substances that contain aldehyde groups.
Especially preferred conjugates are distinguished in that
the substances that accumulate in diseased tissue mean peptides
such as endothelins, partial sequences of endothelins, endothelin
analogs, endothelin derivatives, endothelin antagonists or
angiotensins, partial sequences of angiotensins, angiotensin
analogs, angiotensin derivatives and angiotensin antagonists, as
well as chemotactic peptides.
In other preferred conjugates according to the invention,
the peptides have the following sequences
Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr
Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,
Cys-Ser-Cys-Ser-Ser-Trp-Leu-Asp-Lys-Glu-Cys-Val-Tyr-
Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,
Cys-Thr-Cys-Phe-Thr-Tyr-Lys-Asp-Lys-Glu-Cys-Val-Tyr-
Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,
Cys-Ser-Ala-Ser-Ser-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-
-
Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,
Cys-Ser-Cys-Lys-Asp-Met-Thr-Asp-Lys-Glu-Cys-Leu-Asn-
Phe-Cys-His-Gln-Asp-Val-Il.e-Trp,
Ala-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-
Phe-Ala-His-Leu-Asp-Ile-Ile-Trp,
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13
Ala-Ser-Ala-Ser-Ser-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-
Phe-Ala-His-Leu-Asp-Ile-Ile-Trp,
Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-
Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,
Cys-Thr-Cys-Phe-Thr-Tyr-Lys-Asp-Lys-Glu-Ala-Val-Tyr-
Phe-Ala-His-Leu-Asp-Ile-Ile-Trp,
Cys-Val-Tyr-Phe-Cys-His-Gln-Asp-Val-Ile-Trp,
N-Acetyl-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Gln-
Asp-Val-Ile-Trp,
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu,
Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu,
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe,
Arg-Val-Tyr-Ile-His-Pro-Phe,
Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu,
Sar-Arg-Val-Tyr-Val-His-Pro-Ala,
For-Met-Leu-Phe,
For-Met-Leu-Phe-Lys,
die Teilsequenzen
His-Leu-Asp-Ile-Ile-Trp,
[Key:] D-Trp-Leu-Asp-Ile-Ile-Trp,
die Teilsequenzen = the partial sequences
CA 022323l5 l998-03-l7
14
Phe-D-Trp-Leu-Asp-Ile-Ile-Trp,
Val-Tyr-Ile-His-Pro-Phe,
Val-Tyr-Ile-His-Pro,
or the cyclic amino acid sequences
cyclo-(DTrp-DAsp-Pro-DVal-Leu),
cyclo-(DGlu-Ala-alloDIle-I.eu-DTrp).
Another subject of this invention is also compounds of
general formula (II)
R18-CR2R3- tCR~R5) n 1 2-8-CHR6-CHR7-802-NH- tCR8R9) ~F1 z~CO--B
tII)
in which
R1 stands for a hydrogen atom, for a branched or
unbranched C16 alkyl radical or for a sulfur protective
group,
R2, R3, R4, R5, R6, R7, R8 and R9 are the same or different and
in each case stand for a hydrogen atom and/or for a
branched or unbranched C16 alkyl radical,
B stands for a hydroxyl group, a mercapto group or a
radical -NHRa, in which
R~ stands for a hydrogen atom, a branched or
straight-chain, cyclic or polycyclic C130
alkyl, alkenyl, polyalkenyl, alkinyl, polyalkinyl,
aryl, alkylaryl or arylalkyl radical, which
optionally is substituted with hydroxy, oxy, oxo,
carboxy, aminocarbonyl, alkoxycarbonyl, amino,
CA 0223231~ 1998-03-17
aldehyde or alkoxy groups with up to 20 carbon
atoms and/or optionally is interrupted and/or
substituted by one or more heteroatoms from the
series 0, N, S, P, As, and Se,
their conjugates with substances that selectively accumulate
in diseased tissue, whereby between the latter, a covalent bond
exists and this is present in amide form in the case of
substances that contain carboxyl or amino groups, such as
naturally occurring or modified oligonucleotides, in which
degradation is prevented or hampered by naturally occurring
nucleases, peptides, proteins, antibodies or their fragments, or
is present in imide form in the case of substances that contain
hydroxyl groups, such as fatty alcohols that are in ester form or
in the case of substances that contain aldehyde groups, as well
as their complexes with radioisotopes of Tc and Re.
The production of the compounds of general formula (II)
according to the invention is carried out in that 2-
chloroethanesulfonic acid chloride that optionally is substituted
with R3 and R4 is reacted in a way that is known in the art in an
aprotic solvent with the addition of a suitable base with
compounds of general formula (III)
HzN-tCR8R9)~1z-Co-B
(III)
in which R8, R9, m and B have the meaning that is indicated
above,
CA 0223231~ 1998-03-17
to compounds of general formula (IV)
R4HC=CR5-802-NH-CR6R7-(CR8R9),F~ 2-CO-B
(IV)
in which R4, R5, R6, R7, R8, R9, m and B have the meaning that
is indicated above.
These reactions are implemented in polar and nonpolar
aprotic solvents, such as, for example, dichloromethane,
tetrahydrofuran, chloroform, 1,4-dioxane, pyridine, DMF or DMSO
at temperatures of between -40~ and 120~C with the addition of an
auxiliary base to recover acids that are liberated. Such
auxiliary bases can be, for example, tertiary amines, alkali and
alkaline-earth hydroxides, alkali and alkaline-earth carbonates.
The compounds of general formula (IV) that result from this
are reacted optionally with the addition of a suitable auxiliary
base, such as, for example, a t;ertiary amine, with compounds of
general formula (V)
R18-CR2R3-(CR4R5) 12-8H
(V)
in which R1, R2, R3, R4, n and B have the meaning that
is indicated above,
and optionally present protective groups are cleaved off in a way
that is known in the art.
Another subject of the invention is kits, which are used for
the production of radiopharmaceutical agents and which consist of
a compound of general formula lII) or compounds of general
formula (I and/or II) that contain a conjugate according to the
invention and substances that accumulate selectively in tissues,
CA 0223231~ 1998-03-17
a reducing agent and optionally an auxiliary ligand, which are
present in the dry state or in solution, as well as directions
for use with a set of reaction instructions for the reaction of
the described compounds with technetium-99m or Re in the form of
a pertechnetate solution or perrhenate solution.
The subject of the invention is also a radiopharmaceutical
composition for noninvasive in-vivo visualization of organs,
receptors and receptor-containing tissue and/or arteriosclerotic
plaque, which contains a compound of general formula (I) or
compounds of general formula (I and/or II) that contain a
conjugate according to the invention and substances that
accumulate selectively in tissues, optionally with the additives
that are commonly used in galenicals, whereby the compound is
prepared in a kit with technetium-99m or Re in the form of a
pertechnetate or perrhenate solution.
In a method for implementing a radiodiagnostic
investigation, the radiopharmaceutical composition is
administered to a patient in an amount of 0.1 to 30 mCi,
preferably 0.5 to 10 mCi per 70 kg of body weight, and the
radiation that is given off by the patient is recorded.
Surprisingly enough, many of the chelates that are
synthesized and labeled with technetium-99m or Re show higher
stability than comparable N2S2 and N3S systems, which are
described in the literature. Thus, e.g., in the case of a
substance according to the invention (Example 2), which was
coupled to an endothelin partial sequence, no decomposition
product could be observed after 24 hours. It was also found by
CA 0223231~ 1998-03-17
competitive tests that the Tc-99m or Re chelating agents
described in this invention complex better than the comparable
N2S2, N3S and propylenaminoxime systems. The chelates and
chelating agents that are described in this invention are thus
clearly better suited for diagnostic and therapeutic purposes
than the previously known systems. Special advantage lies in the
restrained labeling conditions. Thus, after the protective
groups are cleaved off, the labeling of the ligands according to
the invention as well as their coupling products on substances
that accumulate selectively in diseased tissue is possible at
room temperature and at physiological pH. By selecting suitable
protective groups, which can be cleaved off under different
reaction conditions depending on the coupling product, it is
always ensured that undesirable secondary reactions cannot occur
in the purification of the coupling products. This carries the
danger that no undesirable cross-linking reactions or oxidations
of free sulfhydryl groups to disulfides occur under purification
conditions. Such alterations often affect the labeling yield and
radiochemical purity and thus also the background by
unspecifically bound technetium in a disadvantageous manner. The
establishment of sulfur protective groups or their cleavage is
carried out according to methods that are known to one skilled in
the art. The coupling to substances that selectively accumulate
in diseased tissue is also carried out according to methods that
are known to one skilled in the art (e.g., Fritzberg et al.; J.
Nucl. Med. 26, 7 (1987)), for example by reaction of
electrophilic groups of the complex ligand with nucleophilic
CA 0223231~ 1998-03-17
19
centers of the substances that accumulate selectively in diseased
tissue or by reaction of nucleophilic groups of the chelating
agent with electrophilic groups of the substances that
selectively accumulate in diseased tissue.
As coupling participants, i.a., various biomolecules are
used. Thus, e.g., ligands that bind to specific receptors and
can thus detect alterations of the receptor thickness include,
i.a., peptides, steroid hormones, growth factors and
neurotransmitters. Coupling products with steroid hormone-
receptor-affine substances make possible an improved diagnosis of
breast and prostate cancer (S. J. Brandes and J. A.
Katzenellenbogen, Nucl. Med. Biol. 15, 53, 1988). On various
occasions, tumor cells exhibit an altered density of receptors
for peptide hormones or growth factors, such as, e.g., the
"epidermal growth factor" (EgF). The concentration differences
can be used for selective concentration of cytostatic agents in
tumor cells (E. Abound-Pirak et al.; Proc. Natl. Acad. Sci. USA
86, 3778, 1989). Other biomolecules are metabolites that can be
incorporated into the metabolism of cells, which show an altered
metabolism; these include, e.g., fatty acids, saccharides,
peptides and amino acids. Fatty acids that are coupled to the
less stable N2S2 systems were described in EP-0200492. Other
metabolic products, such as saccharides, deoxyglucose, lactate
and amino acids (leucine, methyl methionine, glycine), were used
with the aid of PET technology for graphic visualization of
altered metabolic processes (R. Weinreich, Swiss Med. 8, 10,
1986). Also, nonbiological substances such as misonidazole and
CA 0223231~ 1998-03-17
its derivatives, which bind irreversibly to cell components in
tissues or tissue parts at reduced oxygen concentration, can be
used for specific concentration of radioactive isotopes and thus
for graphic visualization of tumors or ischemic regions (M. E.
Shelton, J. Nucl. Med. 30, 351, 1989). Finally, the coupling of
new chelating agents to monoclonal antibodies or their fragments,
polysaccharides such as dextrans or starches, bleomycins,
hormones, enzymes, polypeptides such as polylysine and
nucleotides such as DNA or RNA is also possible. Coupling
products of the chelates according to the invention or their
complexes with technetium-99m or Re with endothelins, endothelin
derivatives or with partial sequences of endothelins or their
derivatives have proven especially advantageous for the detection
of arteriosclerotic vascular diseases. These derivatives were
administered to WHHL rabbits, which show high LDL concentrations
in the blood by a genetic defect of the LDL receptor and thus
have arteriosclerotic lesions. About 1 to 6 hours after the
derivatives are administered to WHHL rabbits, a large degree of
concentration in arteriosclerotic plaque was detected. Up until
now, only very late stages of artherogenesis could be diagnosed
with an invasive process. The compounds according to the
invention therefore offer the decisive advantage of diagnosing
many earlier stages of arteriosclerosis with a noninvasive
process. Coupling products of the chelates according to the
invention or their complexes with technetium-99m or Re with fatty
alcohols, fatty alcohol derivatives or with fatty alcohol amines
or their derivatives have proven advantageous for the detection
CA 0223231~ 1998-03-17
of arteriosclerotic vascular diseases. These derivatives were
administered to WHHL rabbits, which show high LDL concentrations
in the blood by a genetic defect of the LDL receptor and thus
have arteriosclerotic lesions. About 1 to 6 hours after the
derivatives are administered to WHHL rabbits, a large degree of
concentration in arteriosclerotic plaque was detected. Up until
now, only very late stages of artherogenesis could be diagnosed
with an invasive process. The compounds according to the
invention therefore offer the decisive advantage of diagnosing
many earlier stages of arteriosclerosis with a noninvasive
process.
It is unimportant whether a labeling of the described
chelating agent with technetium-99m is carried out before or
after coupling to the selectively accumulating molecule. For
coupling to the selectively accumulating molecule after
complexing, however, there is a precondition that the reaction of
the radioactive complex with the accumulating compound proceeds
quickly under conservative conditions and almost quantitatively,
so that subsequent purification is not necessary.
The production of the pharmaceutical agents according to the
invention is carried out in a way that is known in the art,
whereby the complexing agents according to the invention are
dissolved in aqueous medium with the addition of a reducing
agent, preferably tin(II) salts, such as -chloride,
-pyrophosphate or -tartrate -- and optionally with the addition
of the additives that are commonly used in galenicals -- and then
sterilized by filtration. Suitable additives are, for example,
CA 0223231~ 1998-03-17
physiologically harmless buffers (e.g., tromethamine), small
additions of electrolytes (e.g., sodium chloride), stabilizers
(e.g., gluconate, phosphates or phosphonates). The
pharmaceutical agent according to the invention is present in the
form of a solution or in freeze-dried form and is mixed shortly
before administration with a Tc-99m-pertechnetate solution,
eluted from commercially available MoTc generators or a
perrhenate solution.
In the case of nuclear-medicine in vivo use, the
pharmaceutical agents according to the invention are dosed in
amounts of lxlO-5 to 5x104 nmoltkg of body weight, preferably in
amounts of between lx10-3 to 5X102 nmol/kg of body weight.
Starting from an average body weight of 70 kg, the amount of
radioactivity for diagnostic applications is between 0.05 to 50
mCi, preferably 5 to 30 mCi per 70 kg of application. For
therapeutic uses, between 5 and 500 mCi, preferably 10 to 350
mCi, is administered. The administration is carried out normally
by intravenous, intraarterial, peritoneal or intertumoral
injection of 0.1 to 2 ml of a solution of the agent according to
the invention. Intravenous administration is preferred.
The following examples are used for a more detailed
explanation of the subject of the invention.
23
Example 1
N-Vinylsulfonyl-glycine methyl ester (1)
An ice-cooled solution of 8.91 g (100 mmol) of glycine
methyl ester and 17.9 g of chloroethanesulfonyl chloride (110
mmol) in 100 ml of dichloromethane is slowly mixed with dried
pyridine (400 mmol) while being cooled with ice. It is allowed
to heat to room temperature, and after the reaction has been
completed, it is mixed with 200 ml of dilute HCl, and the
dichloromethane phase is separated. The aqueous phase is
extracted several times with dichloromethane, washed with water,
dried, concentrated by evaporation and chromatographed (silica
gel CH2Cl2). 12.9 g of a slowly crystallizing oil remains.
Yield: 72%
Analysis:
Cld: C 33.52 H 5.06 N 7.82 O 35.72 S 17.90
Fnd: C 33.24 H 5.23 N 7.66 S 17.61
N-t5-Hydroxy-3-thiapentyl~ulfonYll-qlycine methyl ester (2)
1.79 g of vinylsulfonic acid 1 (10 mmol) is added to a
stirred solution of 7.81 g of mercaptoethanol (100 mmol) and 150
mg of triton-B solution while being cooled, and it is stirred for
20 hours in an oxygen-free environment at room temperature.
Then, it is mixed with water and extracted several times with
ethyl acetate. The combined organic extracts are washed with
bicarbonate solution, dried on sodium sulfate and concentrated by
evaporation. The residue is chromatographed (silica gel, EtOAc).
Yield: 63%
CA 022323l~ l998-03-l7
24
Analysis:
Cld: C 32.67 H 5.88 N 5. 44 O 31.09 S 24.92
Fnd: C 32. 59 H 5. 75 N 5. 41 S 24.86
N-(5-Chloro-3-thi~pentylsulfonYl)-glycine methYl ester (3)
A solution of 2.57 g of glycine derivative 2 (10 mmol) in 50
ml of anhydrous carbon tetrachloride is mixed under a nitrogen
atmosphere with 3.14 g of pulverized triphenylphosphine ( 12
mmol), and it is refluxed. After cooling, it is diluted with 50
ml of hexane and stored for some time at -20~C. The precipitate
is suctioned off, and the procedure as above is repeated until
precipitate no longer settles out. Then, it is dried and
concentrated by evaporation.
Yield: 62%
Analysis:
Cld: C 30.49 H 5.12 N 5. 08 O 23.21 S 23.26
Fnd: C 30.16 H 5.56 N 5. 33 S 23.24
N-(5-Thiouronyl-3-thi~pentylsulfonyl)-glycine methyl ester ~4)
The solution of 5.51 g of 3 (20 mmol) in ethanol is added in
drops to a solution of 1. 52 g of thiourea in ethanol and then
refluxed for 2 hours. After the solvent is drawn off, a
crystalline residue remains, which is crystallized from ethanol.
Yield: 57%
Analysis:
Cld: C 27.31 H 5.16 N 11. 94 O 18.19 S 27.34
Fnd: C 27.11 H 5.52 N 11. 54 S 27.80
CA 022323l~ l998-03-l7
N-(5-NercApto-3-thi~pentylsulfonYl~-glycine (5)
3.52 g (10 mmol) of 4 iS stirred under protective gas in
aqueous-methanolic potassium hydroxide solution for 3 hours at
50~C. After cooling, it is diluted with 400 ml of water, and the
undissolved material is filtered out. The filtrate is acidified
with HCl, extracted with dichloromethane, dried, concentrated by
evaporation and recrystallized.
Yield: 64%
Analysis:
Cld: C 27.79 H 5.05 N 5.40 O 24.68 S 37.09
Fnd: C 28.17 H 5.35 N 5. 48 S 36.61
N-(5-Merc~pto-3-thiapentYlsulfonyl)-glycine, technetium-99m
complex
10 mg of compound 5 iS dissolved in 1.0 ml of ethanol. 50
,ul of this ligand solution is mixed with 100 ,ul of ethanol, 150
,ul of phosphate buffer with a pH of 8.5, 50 ,lll of a deoxygenated
aqueous citrate solution (50 mg/ml), 2.5 ,ul of a deoxygenated
tin(II)-chloride solution (5 mg/ml of O.lN HCl) and 100 ~l of a
pertechnetate solution (400-1000 ,UCi). After an incubation time
of 10 minutes, the reaction mixture is examined by HPLC to
determine the purity of the Tc complex formed: LiChrospher RP-18
column, 5 ,U, 125 X 4.6 mm; gradient elution of 100% A after 100%
B within 15 minutes (eluant A: acetonitrile/Na-phosphate 5 mmol,
pH 2.0 (10/90); eluant B: acetonitrile/Na-phosphate 5 mmol, pH
2.0 (75/25); 1 ml/minute. The radiochemical purity is > 92%.
CA 022323l~ l998-03-l7
26
Example 2
N- r ( 5-Triphenylmethylmercapto)-3-thiapentylsulfonYl]-qlycine ~6)
The solution of 2.79 g of triphenylchloromethane in DMF is
slowly added in drops to a solution of 2.59 g of glycerine
derivative 5 (10 mmol) and triethylamine (10 mmol) in DMF, and it
is stirred for 12 hours at room temperature. Then, it is mixed
with water, weakly acidified with dilute hydrochloric acid and
extracted with dichloromethane. The organic phase is dried and
concentrated by evaporation. The remaining residue is
chromatographed (silica gel, CH2Cl2/MeOH).
Yield: 57%
Analysis:
Cld: C 59.86 H 5.43 N 2.79 O 12.76 S 19.14
Fnd: C 59.46 H 5.66 N 2. 44 S 19.
Hooc-Trp-Ile-Ile-Asp-Leu-D-Trp-phe-{N-~5
triPhenylmethylmercapto~-3-thiapentylsulfonyl]Gly~ t7
211 mg of EDC (1.1 mmol) in 5 ml of anhydrous
dimethylformamide is added in drops to a solution of 502 mg of
acid 6 (1 mmol), 280 ,ul of triethylamine and 115 mg of N-
hydroxysuccinimide (1.0 mmol) in 10 ml of anhydrous
dimethylformamide while being stirred at -10~C, and it is stirred
for 2 hours at 0~C. Then, a solution of 992 mg of H2N-Phe D-Trp-
Leu-Asp-Ile-Ile-Trp-COOH (1.0 mmol) in DMF is added in drops
within 30 minutes. It is first stirred for another 2 hours at
0~C, and then stirred for 12 hours at room temperature. The
product is filtered off from urea, and the filtrate is
27
concentrated by evaporation in a vacuum and taken up in
dichloromethane. After filtration was again performed, it is
washed twice with 0.5N HCl and saturated sodium bicarbonate
solution, dried on magnesium sulfate, and the solvent is drawn
off. The residue is crystallized by trituration with diethyl
ether.
Yield: 33%
Analysis:
Cld: C 63.48 H 6.42 N 9.49 O 14.09 S 6.52
Fnd: C 63.09 H 6.66 N 9.37 S 6.46
HOOC-Trp-Ile-Ile-Asp-Leu-D-Trp-Phe-rN-(5-mercapto-3-
thiabutyl~ulfonyl~Glyl ~8)
1.48 g of peptide 7 (1 mmol) is treated for 45 minutes at
0~C with 20 ml of anhydrous HF in the presence of 5 ml of anisole
and 3.5 ml of diethyl sulfide. After the acid is evaporated, the
remaining residue is taken up in 5% acetic acid, washed several
times with diethyl ether and freeze-dried. Chromatographic
purification on Sephadex G-10 with 0.2 M acetic acid yields 470
mg of an oil.
Yield: 38%
Analysis:
Cld: C 57.45 H 6.54 N 11.36 O 16.86 S 7.10
Fnd: C 57.34 H 6.43 N 11.40 S 7.92
CA 0223231~ 1998-03-17
28
HOOC-Trp-Ile-Ile-Asp-Leu-D-Trp-Phe-[N-t5-mercapto-3-
thiaPentylsulfonyl~GlY~ technetium-99m comPlex
10 mg of compound 8 is dissolved in 1.0 ml of ethanol. 50
~l of this ligand solution is mixed with 100 ~l of ethanol, 150
~l of phosphate buffer with a pH of 8. 5, 50 ~l of a deoxygenated
aqueous citrate solution (50 mg/ml), 2. 5 ~l of a deoxygenated
tin(II) chloride solution (5 mg/ml of O.lN HCl) and 100 ~l of a
pertechnetate solution (400-1000 ~Ci). After an incubation time
of 10 minutes, the reaction mixture is examined by HPLC to
determine the purity of the Tc complex formed: LiChrospher RP-18
column, 5 ~, 125 x 4.6 mm; gradient elution of 100% A after 100
B within 15 minutes (eluant A: acetonitrile/Na-phosphate 5 mmol,
pH 2.0 (10/90); eluant B: acetonitrile/Na-phosphate 5 mmol, pH
2.0 (75/25); 1 ml/minute. The radiochemical purity is > 96%.
