Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02232620 1998-03-18
Bifunctional 8ulfide-Containing 8ulfon~mide-Chelating Agents
8uch as X8NY 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 the 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 02232620 1998-03-18
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 02232620 1998-03-18
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 complete 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 properties 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 02232620 1998-03-18
.
(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~, ~ 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 the 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, radionuclide-containing
radiopharmaceutical agents are produced by the ligand first being
synthesized and then being reacted 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 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 quick complexing.
To form technetium-99m complexes, pertechnetate is first
obtained from a nuclide generator and shifted, with the aid of
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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 pH > 9. N202
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 (~Brechbiel, M. W. et al.; Inorg.
Chem. 1986, 25, 2772). It is therefore necessary to produce
bifunctional complexing agents that carry both functional groups
for binding the desired metal ion and a (another, several)
functional group(s) for binding the selectively accumulating
molecule, or to configure complexing agents in such a way that
the desired complexing agent structure is formed only by coupling
to a selectively accumulating substance and thus weakening of the
complex stability is prevented. Such ligands make possible a
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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 according to the invention in that
new chelating agents that contain bifunctional sulfonamide groups
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and their coupling products with specifically accumulating
compounds are made available.
The object of the inventio:n is compounds of general formula
(I)
M - :L (I)
in which
M means a radioisotope of Tc or Re and L is a ligand of
general formula (II)
B-CR1R2-(CR3R~)~12-8-CHR5-CHR6-802-NH-CR7R8-(CR9R10) Fl 2-D
(II)
in which
R1, R2, R3, R4, R5, R6 and R7 are the same or different and in
each case stand for a hydrogen atom and/or for a
branched or unbranched C16 alkyl radical,
R8 stands for a hydrogen atom, for a branched or
unbranched C16 alkyl radical or a radical -Co-R15
in which
R15 represents a hydroxyl group, a branched or
straight-chain, cyclic or polycyclic C130 alkoxy,
alkenyloxy, polyalkenyloxy, alkinyloxy,
polyalkinyloxy, aryloxy, alkylaryloxy or
arylalkyloxy group, which optionally is
substituted with hydroxy, oxy, oxo, carboxy,
aminocarbonyl, alkoxycarbonyl, amino, aldehyde or
alkoxy groups with up to 20 carbon atoms and/or is
optionally inter:rupted and/or substituted by one
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or more heteroatoms from the series O, N, S, P,
As, Se or an N(RaRb) group,
whereby Ra and Rb are the same or different
and/or represent 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, Se,
R9 and R10 are the same or different and in each case stand
for a hydrogen atom and/or for a branched or unbranched
C1-6 alkyl radical,
B stands for a radical -SR11, -NHR1Z or -oR13,
in which
R11 stands for a hydrogen atom, for a branched or
unbranched C16 alkyl radical or for a sulfur
protective group,
R12 stands for a hydrogen atom, an amino protective
group or a branc:hed or straight-chain cyclic or
polycyclic C130 alkyl, alkenyl, polyalkenyl,
alkinyl, polyalkinyl, aryl, alkylaryl or arylalkyl
group, which optionally is substituted with
CA 02232620 l998-03-l8
11
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 0, N, S, P, As, Se,
R13 stands for a hydrogen atom or for an alcohol
protective group,
D stands for a radical -SR14, in which
R14 stands for a hydrogen atom, for a branched or
unbranched C16 alkyl radical or for a sulfur
protective group,
or if B represents a radical SR11, also for a radical
-NHR16 or -oR17, in which
R16 represents a hydrogen atom, an amino
protective group or a branched or straight-
chain, cyclic or polycyclic C1,30 alkyl,
alkenyl, polyalkenyl, alkinyl, polyalkinyl,
aryl, alkylaryl or arylalkyl group, 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 or substituted by one or more
heteroatoms from the series 0, N, S, P, As,
Se,
R17 stands for a hydrogen atom or for an alcohol
protective group.
12
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, R8 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, R3, R4, R5, R6, R8, R9 and R10 are hydrogen atoms and R7
stands for a radical -Co-R15
in which
R15 represents a hydroxyl group, a branched or
straight-chain Cl30 alkoxy group or an N(RaRb)
group, whereby
Ra and Rb are the same or different and/or
represent a hydrogen atom, a branched or
straight-chain, C130 alkyl radical, which
optionally is substituted with carboxy,
aminocarbonyl, alkoxycarbonyl or amino groups
with up to 20 carbon atoms and/or optionally
is interrupted and/or is substituted by one
or more heteroatoms from the series O, N, S.
Especially preferred are compounds according to the
invention in which n and m in each case stand for 1.
Especially preferred are also compounds according to the
invention, in which R1, R2, R5, R6, R8, R9 and R10 represent
hydrogen atoms.
Especially preferred are compounds according to the
invention in which R3 and R4 in each case stand for a hydrogen
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atom and R7 stands for a hydrogen atom, a branched or unbranched
C1-6 alkyl radical or a radical -Co-Rl5~
in which R15 represents a hydroxyl group, a branched or
straight-chain C130 alkoxy group or an N(RaRb) group,
whereby Ra and Rb are the same or different and/or
represent a hydrogen atom, a branched or straight-
chain, cyclic or polycyclic C130 alkyl radical,
which optionally is substituted with hydroxy, oxy,
oxo, carboxy, aminocarbonyl, alkoxycarbonyl, amino
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.
Another subject of the invention relates to new bifunctional
sulfur atom-interrupted sulfonamide ligands of general formula
(II)
B-CR1R2-(CR3R~ 2-S-CHR5-CHR6-go2-NH-CR7R8-(CR9R1~)~2-D
~II)
in which R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, n, m, B and D
in each case have the meaning that is indicated above.
Preferred compounds of general formula (II) are
distinguished in that in each case l stands for n and m.
Other preferred compounds of general formula (II) are
distinguished in that R1, R2, R5, R6, R8, R9 and R10 are hydrogen
atoms.
Especially preferred compounds of general formula (II) are
distinguished in that in each case l stands for n and m and in
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14
that R1, R2, R3, R4, R5, R6, R8, R9 and Rl~ are hydrogen atoms and R7
stands for a radical -Co-R15
in which
R15 represents a hydroxyl group, a branched or straight-
chain C1 30 alkoxy group or an N(RaRb) group, whereby
Ra and Rb are the same or different and/or represent ahydrogen atom, a branched or straight-chain C130
alkyl radical, which optionally is substituted
with carboxy, aminocarbonyl, alkoxycarbonyl or
amino groups with up to 20 carbon atoms or
optionally is interrupted and/or substituted by
one or more heteroatoms from the series 0, N, S.
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
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
CA 02232620 l998-03-l8
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
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16
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-
~ I .
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-Ile-Trp,
Ala-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-
Phe-Ala-His-Leu-Asp-Ile-Ile-Trp,
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,
I
Cys-Val-Tyr-Phe-Cys-His-Gln-Asp-Val-Ile-Trp,
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N-Acetyl-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-Hi
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,
D-Trp-Leu-Asp-Ile-Ile-Trp,
Phe-D-Trp-Leu-Asp-Ile-Ile-Trp,
Val-Tyr-Ile-His-Pro-Phe,
Val-Tyr-Ile-His-Pro,
[Key:]
die Teilsequenzen = the partial sequences
CA 02232620 l998-03-l8
18
or the cyclic amino acid sequences
cyclo-(DTrp-DAsp-Pro-DVal-Leu),
cyclo-(DGlu-Ala-alloDIle-Leu-DTrp).
Another subject of this invention is also compounds of
general formula (II)
B-CR1RZ-~CR3R~ z-S-CHR5-CHR6-Soz~NH~CR7R8~(CR9R1~)~1z~D
~II)
in which
R1, RZ, R3, R4, R5, R6 and R7 are the same or different and in
each case stand for a hydrogen atom and/or for a
branched or unbranched C16 alkyl radical,
R8 stands for a hydrogen atom, for a branched or
unbranched C16 alkyl radical or a radical -Co-R15,
in which
R15 represents a hydroxyl group, a branched or
straight-chain , cyclic or polycyclic C130 alkoxy,
alkenyloxy, polyalkenyloxy, alkinyloxy,
polyalkinyloxy, aryloxy, alkylaryloxy or
arylalkyloxy group, which optionally is
substituted with hydroxy, oxy, oxo, carboxy,
aminocarbonyl, alkoxycarbonyl, amino, aldehyde or
alkoxy groups with up to 20 carbon atoms and/or is
optionally interrupted and/or substituted by one
or more heteroatoms from the series O, N, S, P,
As, Se, or an N(RaRb) group,
whereby Ra and Rb are the same or different
and/or represent a hydrogen atom, a branched
CA 02232620 1998-03-18
19
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, Se,
R9 and R10 are the same or different and in each case stand
for a hydrogen atom and/or for a branched or unbranched
C1-6 alkyl radical,
B stands for a radical -SR11, -NHR12 or -oR13
in which
R11 stands for a hydrogen atom, for a branched or
unbranched C16 a:lkyl radical or for a sulfur
protective group,
R12 stands for a hydrogen atom, an amino protective
group or branched or straight-chain cyclic or
polycyclic C130 alkyl, alkenyl, polyalkenyl,
alkinyl, polyalkinyl, aryl, alkylaryl or arylalkyl
group, 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
CA 02232620 1998-03-18
.
interrupted and/or substituted by one or more
heteroatoms from the series 0, N, S, P, As, Se,
R13 stands for a hydrogen atom or for an alcohol
protective group,
D stands for a radical -SR14, in which
R14 stands for a hydrogen atom, for a branched or
unbranched C16 alkyl radical or for a sulfur
protective group,
or if B represents a radical SR11, also for a
radical -NHR16 or -oR17~ in which
R16 represents a hydrogen atom, an amino
protective group or a branched or straight-
chain, cyclic or polycyclic C130 alkyl,
alkenyl, polyalkenyl, alkinyl, polyalkinyl,
aryl, alkylaryl or arylalkyl group, 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 i5
interrupted or substituted by one or more
heteroatoms from the series 0, N, S, P, As,
Se,
R17 stands for a hydrogen atom or for an alcohol
protective group,
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
CA 02232620 l998-03-l8
21
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 or Re.
Another subject of this invention is a process for the
production of a compound of general formula (I), characterized in
that technetium-99m or Re in the form of pertechnetate or
perrhenate is reacted in the presence of a reducing agent and
optionally an auxiliary ligand with a compound of general formula
(II)
B-CR1R2-~CR3R~ 2-8-CHR5-CHR6-~02-NH-CR7R8-(CR9R1~)F~ 2-D
in which R1 R2 R3 R4, R5, R6, R7, R8, R9, R10, n, m, B and D
have the meaning that is indicated above.
The production of the compounds of general formula (II)
according to the invention is carried out in that 2-
chloroethanesulfonic acid chloride that is optionally substituted
with Rs and R6 is reacted in a way known in the art in an aprotic
solvent with the addition of a suitable base with compounds of
general formula (III)
H2N-CR7R8-~CR9R1~)F1 2-D
(III)
CA 02232620 l998-03-l8
22
in which R7, R8, R9, R10, m and D have the meaning that
is indicated above,
to compounds of general formula (IV)
R5HC=CR2-So-NH-cR7R8-(cR9R1o)Flz-D
(IV)
in which R5, R6, R7, R8, R9, R10, m and D 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~ to 120~C optionally 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 in a way known in the art with compounds of general formula
(V)
BCR1-R2-~CR3R~)~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
CA 02232620 1998-03-18
23
a compound of general formula (II) or compounds of general
formula ~I and/or II) that contain a conjugate according to the
invention and substances that accumulate selectively in tissues,
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
CA 02232620 1998-03-18
24
.
substance according to the invention (Example 2), which was
coupled to a fatty alcohol, no decomposition product could be
observed after 24 hours. It was also found by 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 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
CA 02232620 1998-03-18
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 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 N2Sz systems were described in EP-0200492. Other
metabolic products, such as saccharides, deoxyglucose, lactate
and amino acids (leucine, methyl methionine, glycine) were used
CA 02232620 1998-03-18
26
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
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 fatty alcohols, fatty
alcohol derivatives or with fatty alcohol amines or their
derivatives have proven 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.
CA 02232620 1998-03-18
27
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,
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 lx10-5 to 5x104 nmol/kg of body weight, preferably in
CA 02232620 1998-03-18
28
.
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.
CA 02232620 1998-03-18
29
Example 1
8-~4-Nethoxybenzyl)cy~teinethyle~ter ~1)
In a solution of 27.7 g of S-4-methoxybenzylcysteine in 250
ml of absolute EtOH, HCl is introduced until saturation is
achieved, and it is heated to boiling. After the reaction has
been completed, it is filtered after cooling to room temperature,
and the mother liquor is concentrated by evaporation again.
28.4 g of white crystals remains.
Yield: 93%
Analysis:
Cld: C 51. 06 H 6. 59 N 4.58 O 15.70 S 10.49
Fnd: C 50.88 H 6.83 N 4.45 S 10.15
N-Vinylsulfon~Yl-8-(4-methoxYbenzyl)-cysteinethYle~ter ~2)
An ice-cooled solution of 3.06 g (10 mmol) of S-protected
cysteine derivative 1 and 1.79 g of chloroethanesulfonyl chloride
(11 mmol) in 10 ml of dichloromethane is mixed slowly with dry
pyridine (44 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 20 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). 2. 37 g of a slowly crystallizing oil remains.
Yield: 66%
CA 02232620 l998-03-l8
Analysis:
Cld: C 50.12 H 5.89 N 3.80 0 22.26 8 17.84
Fnd: C 49.97 H 6.01 N 3.62 S 17.56
N-~5-[(N-tert-ButYloxycarbonyl)-~mino]-3-thi~-pentylsulfonYl}-8-
~4-methoxYbenzYl~-cysteinethylester (3)
9.8 mmol of vinylsulfonamide 2 (35. 2 g) is added in drops to
100 mmol of N-Boc-2-mercaptoethylamine ( 17.6 g) and 500 ~1 of
piperidine within 45 minutes while being stirred, whereby 50~C iS
not exceeded. During the reaction, 500 ~1 of piperidine is added
in several portions. Then, the mixture is heated for several
minutes to 50~C, contaminants are filtered out and washed with
water. After drying, the solvent is drawn off, and the residue
is chromatographed (silica gel, EtOAc).
Yield: 41%
Analysis:
Cld: C 49. 23 H 6.76 N 5.22 O 20.87 8 17.92
Fnd: C 49.11 H 6.87 N 5.08 8 17.82
N-~5-Amino-3-thiaPentylsulfonYl~-8-(4-methoxYbenzYl)-
cysteinetbYlester (4)
8.5 g of 3 (15.8 mmol) is dissolved in 200 ml of 3 M HCl in
ethyl acetate, and it is stirred for 2 hours at room temperature.
After the solvent is drawn off, 7.25 g of white crystals remains.
Yield: 97%
CA 02232620 1998-03-18
31
.
Analysis:
Cld: C 43.16 H 6 .18 N 5.92 0 16.91 S 20.34
Fnd: C 42.97 H 6.41 N 5.73 S 20.19
N-~5-Amino-3-thia-pentylsulfonyl~-cYsteinethyle~ter (S~
10 ml of HF in a 100 ml teflon round-bottom flask is
condensed down to 2.87 g of 4 (5 mmol) and one drop of anisole at
0~C in a moisture-free environment. It is stirred for 30 minutes
at 0~C, and then hydrogen fluoride is carefully distilled off.
The residue is taken up in dichloromethane, washed with sodium
bicarbonate solution and water, dried and concentrated by
evaporation. The oily residue is crystallized by trituration
with diethyl ether.
Yield: 59%
Analysis:
Cld: C 34.16 H 6.37 N 8.85 O 20.22 S 30.40
Fnd: C 33.99 H 6.52 N 8.74 S 30.25
N-{5-Amino-3-thiaPentyl~ulfonyl~-cysteinethyl e~ter, technetium-
99m complex
10 mg of compound 5 iS dissolved in 1.0 ml of ethanol. 50
~1 of this ligand solution is mixed with 100 ,ul of ethanol, 150
~1 of phosphate buffer with a pH of 8.5, 50 ~1 of a deoxygenated
aqueous citrate solution (50 mg/ml), 2.5 ~1 of a deoxygenated
tin(II) chloride solution (S mg/ml of O.lN HCl) and 100 ~1 of a
pertechnetate solution (400-1000 ~Ci). After an incubation time
of 10 minutes, the reaction mixture is examined by HPLC to
CA 02232620 1998-03-18
.
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/min. The radiochemical purity is > 97%.
Example 1~
N-Vinylsulfonyl-8-(4-methoxYbenzyl)-cysteine ~6)
3.59 g (10 mmol) of 2 is stirred into 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 and concentrated by evaporation.
Yield: 80%
Analysis:
Cld: C 47.12 H 5.17 N 4.23 0 24.14 S 19.35
Fnd: C 46.99 H 5.28 N 4.09 S 19.18
N-~5- r (N-tert-ButoxycarbonYl)-amino]-3-thia-Pentylsulfonyl~-8-~4-
methoxYbenzyl)-cysteine ~7)
9.8 mmol of vinylsulfonamide 6 (3.25 g) is added in drops to
10 mmol of N-Boc-2-mercaptoethylamine (1.76 g) and 50 ~l of
piperidine within 45 minutes while being stirred, whereby 50~C is
not exceeded. During the reaction, another 500 ~l of piperidine
is added in several portions. Then, the mixture is heated for
several minutes to 50~C, contaminants are filtered out and washed
CA 02232620 1998-03-18
33
with water. After drying, the solvent is drawn off, and the
crystalline residue is recrystallized from MeOH/CH2Cl2.
Yield: 37%
Analysis:
Cld: C 47.23 H 6.34 N 5.51 O 22.02 S 18.91
Fnd: C 47.11 H 6.77 N 5.38 S 18.82
N-~5-Amino-3-thiapentylsulfonYl~-8-(~-methoxYbenzyl)-CYs-His-Leu-
Asp-Ile-Ile-Trp ~8)
2 mmol of acid 7 (1.02 g) and 2 mmol of NEt3 are mixed in 10
ml of dichloromethane under nitrogen atmosphere at 0~C with 2.2
mmol (560 mg) of BOP-Cl, and it is stirred for 2 hours at room
temperature. Then, the solution of 2 mmol of the peptide H2N-
Leu-Asp-Ile-Ile-Trp (1.32 g) (produced analogously to Barany and
Nerrifield, The Peptides: Analysis, Biology, Academic Press, New
York, 1980; Stewart and Young, Solid Phase Peptides Syntheses,
2nd ed., Pierce Chemical W., Rockford, II, 1984) is added in
drops to anhydrous DMF, and it is stirred overnight. It is mixed
with a little water, acidified and freeze-dried. The residue is
taken up in 50 ml of anhydrous trifluoroacetic acid, and it is
stirred for 1 hour at room temperature. Then, the
trifluoroacetic acid is drawn off in a vacuum, the residue is
mixed three times with dimethylformamide and in each case
concentrated by evaporation. While being stirred with diethyl
ether, a solid precipitates, which is filtered off and is
recrystallized for purification from DMF/diethyl ether mixtures.
Yield: 39%
CA 02232620 1998-03-18
34
.
Analysis:
Cld: C 54.67 H 6.71 N 12.99 0 17.53 S 8.11 H 6.71
Fnd: C 54.39 H 6.92 N 12.68 S 7.77
N- r 5-Amino-3-thi~p~ntyl~ulfonyll-Cy~-His-Leu-A~p-Ile-Ile-Trp (9)
10 ml of HF in a 100 ml teflon round-bottom flask is
condensed down to 1. 18 g of 8 (1 mmol) and one drop of anisole at
o~C in a moisture-free environment. It is stirred for 30 minutes
at 0~C, and then hydrogen fluoride is carefully distilled off.
The residue is crystallized by trituration with diethyl ether.
Yield: 26%
Analysis:
Cld: C 51.82 H 6.71 N 14. 45 0 18.01 S 9.02
Fnd: C 51.35 H 6.99 N 14.28 S 8.76
L~belin~ with Tc99m
10 mg of compound 9 is dissolved in 1.0 ml of ethanol. 50
,ul of this ligand solution is mixed with 250 ~1 of phosphate
buffer with a pH of 8.5, 50 ,ul 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 ~1 of a pertechnetate
solution (400-1000 ~LCi). 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);
CA 02232620 1998-03-18
.
eluant B: acetonitrile/Na-phosphate 5 mmol, pH 2.0 (75/25); 1
ml/min. The radiochemical purity is > 97%.
~x~mple 2
CysteinoctYlester (10)
Dry HCl gas is introduced for 1.5 hours into the mixture of
12.1 g (100 mmol) of L-cysteine and 100 ml of octanol while being
stirred (heating to 140-150~C). After the reaction has been
completed, it is allowed to cool, mixed with 300 ml of anhydrous
ether and stirred for 30 minutes. After filtration, it is
recrystallized from a mixture of ether and ethanol.
Yield: 72%
Analysis:
Cld: C 48.97 H 8.97 N 5.19 0 11.86 S 11.88
Fnd: C 48.84 H 9.08 N 5.23 S 12.12
8-Bis-(4-methoxyphenyl)-methylcy~teinoctylester (11)
2.70 g of anhydrous cysteinoctylester-hydrochloride 10 (10
mmol) is suspended in 10 ml of glacial acetic acid and about 2.76
g of S-bis-(4-methoxyphenyl)-methanol (15 mmol) and 2.1 ml of
BF3-diethyletherate (15 mmol) are added to it. It is stirred for
2 hours at room temperature, whereby almost all is dissolved in
clear form. Then, it is concentrated by evaporation in a rotary
evaporator at a bath temperature of 40~C.
The oily residue is dissolved in ethyl acetate. By shaking
with saturated sodium acetate solution, the protected cysteine
CA 02232620 l998-03-l8
36
derivative, which is suctioned off and easily washed with water
and acetone, precipitates.
Yield: 73%
Analysis:
Cld: C 62.95 H 7.72 N 2.82 0 12.90 S 6.46
Fnd: C 62.71 H 7.96 N 2.84 S 6.64
N-VinylsulfonYl-S-bis(4-methoxY~henyl)-methylcysteinoctylester
~12)
An ice-cooled solution of 4.96 g (10 mmol) of S-protected
cysteine derivative 11 and 1. 79 g of choroethanesulfonyl chloride
(11 mmol) in 10 ml of dichloromethane is slowly mixed with dry
pyridine (44 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 20 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, CH2C12)-
Yield: 61%
Analysis:
Cld: C 61.18 H 7 .15 N 2. 55 0 17.46 S 11.67
Fnd: C 60.89 H 7.30 N 2.64 S 11.33
CA 02232620 1998-03-18
N-~5-r~N-tert-ButoxYcarbonyl)-amino~-3-thia-pentYlsulfonyl~-S-
bi~(4-methoxyPhenYl~-methylcysteinoctylester ~13)
9.8 mmol of vinylsulfonamide 12 (5.39 g) in THF is added in
drops to 10 mmol of N-Boc-2 mercaptoethylamine (1.76 g) and 50 ~1
of piperidine within 45 minutes while being stirred, whereby 50~C
is not exceeded. During the reaction, another 50 ~1 of
piperidine is added in several portions. Then, the mixture is
heated for several minutes to 50~C, contaminants are filtered out
and concentrated by evaporation. The residue is taken up in
ethyl acetate and washed with water. After drying, the solvent
is drawn off, and the residue is chromatographed (silica gel,
cH2cl2) -
Yield: 59%
Analysis:
Cld: C 57.83 H 7.49 N 3.85 0 17.61 S 13.23
Fnd: C 57.67 H 7.70 N 3.68 S 13.50
N-~5-Amino-3-thiapentYlsulfonYl~-cYsteinoctylester (14)
727 mg of protected cysteine derivative 13 (1 mmol) and a
trace of anisole are added to 10 ml of trifluoroacetic acid in an
oxygen-free environment at room temperature, and it is refluxed
for 1 hour. Then, the trifluoroacetic acid is drawn off in a
vacuum, and the residue is taken up in dichloromethane. After
washing with saturated sodium bicarbonate solution and water, it
is dried with sodium sulfate and concentrated by evaporation.
The oily residue is crystallized by trituration with diethyl
ether.
CA 02232620 l998-03-l8
38
Yield: 67%
Analysis:
Cld: C 44.97 H 8.05 N 6.99 0 15.98 S 24.01
Fnd: C 44.75 H 8. 40 N 7.11 S 24.23
N-~5-Amino-3-thiapentYlsulfonYl~-cysteinoctylester, technetium-
99m complex
10 mg of compound 14 iS dissolved in 1.0 ml of ethanol. 50
~l of this ligand solution is mixed with 100 ~l of ethanol, 150
~1 of phosphate buffer with a pH of 8.5, 50 ,ul 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 1~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 ,11, 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 (lOt90); eluant B: acetonitrile/Na-phosphate 5 mmol, pH
2.0 (75/25); 1 ml/min. The radiochemical purity is > 95%.
Example 3
8-Tritylcysteine methyl eQter (15)
The solution of 2.79 g of triphenylchloromethane in DMF is
slowly added in drops to a solution of 4. 71 g of the cysteine
methyl ester (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 alkalized with saturated sodium bicarbonate
CA 02232620 1998-03-18
solution and extracted with dichloromethane. The organic phase
is dried and concentrated by evaporation. The remaining residue
is chromatographed (silica gel, CH2Cl2).
Yield: 67
Analysis:
Cld: C 73.18 H 6.14 N 3.71 O 8.48 S 8.49
Fnd: C 73.46 H 5.96 N 3.44 S 8.59
N-VinylsulfonYl-S-tritYlcysteine methYl ester (16)
An ice-cooled solution of 7.55 g (20 mmol) of S-
tritylcysteine derivative 15 and 4.89 g of chloroethanesulfonyl
chloride (30 mmol) in 50 ml of dichloromethane is mixed slowly
with 20 ml of dry pyridine while being cooled with ice, and it is
stirred for 6 hours at 0~C. It is allowed to heat to room
temperature, and after the reaction has been completed, it is
mixed with dilute HCl, and the dichloromethane phase is
separated. The aqueous phase is extracted several times with
dichloromethane, washed with water, dried and concentrated by
evaporation.
Yield: 58%
Analysis:
Cld: C 64.22 H 5.40 N 3.00 O 13.69 S 13.72
Fnd: C 64.02 H 5.75 N 3.09 S 13.52
CA 02232620 1998-03-18
N-~5-rtN-tert-Butyloxycarbonyl)-aminol-3-thia-pentylQulfonYl~-8
tritylcYsteine methYl ester (17)
9.8 mmol of vinylsulfonamide 16 (4. 58 g) in THF is added in
drops to 10 mmol of N-Boc-2 mercaptoethylamine (1. 76 g) and 50 ~1
of piperidine within 45 minutes while being stirred, whereby 50~C
is not exceeded. During the reaction, another 50 ~1 of
piperidine is added in several portions. Then, the mixture is
heated for several minutes to 50~C, contaminants are filtered
out, and concentrated by evaporation. The residue is taken up in
ethyl acetate and washed with water. After drying, the solvent
is drawn off, and the residue is chromatographed (silica gel,
CH2Clz/PE) ~
Yield: 45%
Analysis:
Cld: C 59.60 H 6.25 N 4.34 0 14.89 S 14.92
Fnd: C 59.37 H 6.46 N 4.43 8 14.77
N-{5-~(N-tert-ButyloxYcarbonYl)-amino~-3-thia~entylsulfonYl~-8-
tritylcysteine-rN-t2-~minoethyl)amidel tl8)
A solution of 6.45 g of cysteine ester 17 (10 mmol) in 50 ml
of toluene is slowly added in drops to a solution of 25 g of
ethylenediamine (240 mmol) at 95~C, and it is refluxed for 2
hours. After cooling to room temperature, it is concentrated by
evaporation in a vacuum, and the residue is recrystallized from
methanol/CH2C12.
Yield: 85%
CA 02232620 1998-03-18
Analysis:
Cld: C 58.90 H 6.59 N 8.33 0 11.89 S 14.30
Fnd: C 58.69 H 6.74 N 8.37 S 14.15
For-Met-Leu-Phe-~N-{N'-{5- r (N''-tert-butoxycarbonYl)-~mino]-3-
thia-Pentylsulfonyl~-8-tritylcysteinyl~-2-aminoethyl}~mide~ (19
2.11 g of EDC (11 mmol) in 10 ml of anhydrous
dimethylformamide is added in drops to a solution of 6.72 g of
cysteine derivative 18 (10 mmol), 1.01 g of triethylamine and
1.15 g of N-hydroxysuccinimide (10 mmol) in 50 ml of anhydrous
dimethylformamide while being stirred at -10~C, and it is stirred
for 2 hours at 0~C. Then, a solution of For-Met-Leu-Phe (11
mmol) in DMF is added in drops within 60 minutes. It is first
stirred for another 2 hours at 0~C, and stirred for 12 hours at
room temperature. The product is filtered off from N,N'-
dicyclohexylurea, and the filtrate is concentrated by evaporation
in a vacuum and taken up in dichloromethane. After filtration
has again been 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: 35%
Analysis:
Cld: C 59.37 H 6.74 N 8.98 0 13.18 S 11.74
Fnd: C 59.09 H 6.91 N 8.77 S 12.01
CA 02232620 l998-03-l8
42
For-Net-Leu-Phe-~N-[N'-(5-amino-3-thia-PentylsulfonYl)-
cysteinyll-2-aminoethgl~amide~ ~20)
1.09 g of peptide 19 (1 mmol) is treated for 45 minutes at
0~C with 10 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 180
mg of an oil.
Yield: 24%
Analysis:
Cld: C 48. 04 H 6. 85 N 13. 07 O 14.93 S 17.10
Fnd: C 47.82 H 6. 89 N 12.85 S 16. 84
For-Met-Leu-Phe-~N-rN'-(S-amino-3-thia-PentYlsulfonyl)-
cystQinyl]-2-aminoethyl}~mide}, technetium-99m complex
10 mg of compound 20 iS dissolved in 1.0 ml of ethanol. 50
~1 of this ligand solution is mixed with 100 ,ul of ethanol, 150
,ul of phosphate buffer with a pH of 8.5, 50 ~Ll of a deoxygenated
aqueous citrate solution (50 mg/ml), 2.5 ~ of a deoxygenated
tin(II) chloride solution (5 mg/ml of O.lN HCl) and 100 ~1 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~, 125 X 4. 6 mm; gradient elution of 100% A after 100% B
within 15 minutes (eluant A: acetonitrile/Na-phosphate 5 mmol,
CA 02232620 l998-03-l8
43
.
pH 2.0 (10/90); eluant B: acetonitrile/Na-phosphate 5 mmol, pH
2.0 (75/25); 1 ml/min. The radiochemical purity is > 98%.
Ex~mple ~
N-~S-Hydroxy-3-thiaDentylsulfonyl~-S-tritylcysteine methyl ester
(21~
4.65 g of vinylsulfonic acid 15 (10 mmol) is added to a
stirred solution of 7. 81 g of mercaptoethanol (100 mmol) from 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: 46%
Analysis:
Cld: C 59.43 H 5.73 N 2.57 0 14.66 S 17.63
Fnd: C 59.09 H 5.95 N 2.41 S 17.43
N-~5-HydroxY-3-thiapentylsulfonyl~-cYsteine methyl e~ter ~22)
2.73 g of cysteine derivative 21 (5 mmol) is treated for 45
minutes at 0~C with 35 ml of anhydrous HF in the presence of 13
ml of anisole and 7 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.
Yield: 75%
CA 02232620 1998-03-18
. 44
.
Analysis:
Cld: C 31.67 H 5.65 N 4.62 0 26.37 S 31.71
Fnd: C 31.32 H 5.89 N 4.40 S 31.81
N-~5-Hydroxy-3-thi~DentylsulfonYl~-cysteino methyl ester.
technotium-9sm complex
lo mg of compound 22 is dissolved in 1.0 ml of ethanol. 50
~1 of this ligand solution is mixed with 100 ~1 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 ~1 of a deoxygenated
tin(II) chloride solution (5 mg/ml of O.lN HCl) and 100 ~1 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/min. The radiochemical purity is > 97%.
Example 5
N-~5-Chloro-3-thiaPentylsulfonyl~-8-trityl-cysteine methyl oster
(23)
A solution of 5.46 g of cysteine derivative 21 (10 mmol) in
50 ml of anhydrous carbon tetrachloride is mixed under nitrogen
atmosphere with 3.14 g of pulverized triphenylphosphine (12 mmol)
and refluxed. After cooling, it is diluted with 50 ml of hexane
and stored for some time at -20~C. The precipitate is suctioned
CA 02232620 l998-03-l8
.
off, and the procedure as above is repeated until precipitate no
longer settles out. Then, it is freeze-dried and concentrated by
evaporation.
Yield: 89%
Analysis:
Cld: C 57.48 H 5.36 N 2.48 0 11.34 S 17.05
Fnd: C 57.16 H 5.56 N 2.33 S 16.84
N-~5-Thiouronyl-3-tbiapentYlsulfonYl}-S-trityl-cysteine methYl
ester hydrochloride (24)
The solution of 11. 3 g of 23 (20 mmol) in ethanol is added
in drops to a solution of 1. 52 g of thiourea in ethanol, and then
it is refluxed for 2 hours. After the solvent is drawn off, a
crystalline residue remains, which is recrystallized from
ethanol.
Yield: 60%
Analysis:
Cld: C 55.29 H 5.63 N 6.91 0 10.52 S 15.82
Fnd: C 54.97 H 5.82 N 6.77 S 15. 80
N-~5-Mercapto-3-thiaPentyl~ulfonyl~-~-trityl-cY~teine ~25)
6.08 g (10 mmol) of 24 iS stirred into 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.
CA 02232620 1998-03-18
46
Yield: 52%
Analysis:
Cld: C 57.01 H 5.34 N 2.56 O 11.68 S 23.42
Fnd: C 56.87 H 5.55 N 2.48 S 23.81
N-~5-MercaPto-3-thiapentylsulfonYl~-cY~teine (26~
2.74 g of cysteine derivative 25 (5 mmol) is treated for 45
minutes at 0~C with 35 ml of anhydrous HF in the presence of 13
ml of anisole and 7 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. The
residue is crystallized by renewed trituration with diethyl
ether.
Yield: 23%
Analysis:
Cld: C 27.53 H 4.95 N 4.59 O 20.95 S 41.99
Fnd: C 27.11 H 4.78 N 4.40 S 42.21
N-~5-Mercapto-3-thiaPentYl~ulfonyl}-cysteine, technetium-99m
complex
10 mg of compound 26 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 ,ul 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
CA 02232620 l998-03-l8
47
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 S mmol,
pH 2.0 (lO/9O); eluant B: acetonitrile/Na-phosphate S mmol, pH
2.0 (75/25); 1 ml/min. The radiochemical purity is > 96%.
Example 6
N,N'-tert-ButyloxYc~rbonyl-vinylsulfonyl-ethylenedi~mine (27
An ice-cooled solution of 1. 60 g (10 mmol) of N-tert-
butyloxycarbonylvinylsulfonylethylene diamine and 1.79 g of
chloroethanesulfonyl chloride (11 mmol) in 10 ml of
dichloromethane is mixed slowly with 20 ml of dry pyridine (44
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 20 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). 1. 75 g of
white crystals remain.
Yield: 70%
Analysis:
Cld: C 43.19 H 7.25 N 11.19 0 25.57 S 12.81
Fnd: C 42.97 H 7.41 N 11. 32 S 12.56
CA 02232620 l998-03-l8
48
N,N'-tert-ButyloXYcarbonyl-~5-hydroxY-3-thi~PFntylsulfonyl)
ethYlenediamine (28)
2.50 g of vinylsulfonic acid 27 (lO mmol) is added to a
stirred solution of 7. 81 g of mercaptoethanol (100 mmol) of 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: ~7%
Analysis:
Cld: C 40.23 H 7.37 N 8.53 O 24.36 S 19.53
Fnd: C 40.59 H 7.75 N 8.41 S 19.43
N,N'-tert-ButYloxyc~rbonYl-(5-chloro-3-thiapentylsulfon~Yl)-
ethylenedi~mine (29)
A solution of 3. 28 g of ethylenediamine derivative 28 (lO
mmol) in 50 ml of anhydrous carbon tetrachloride is mixed under
nitrogen atmosphere with 3.14 g of pulverized triphenylphosphine
(12 mmol) and 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: 69%
CA 02232620 1998-03-18
49
Analysis:
Cld: C 38.09 H 6.68 N 8.09 O 18.45 S 18.49
Fnd: C 38.16 H 6.56 N 8. 33 S 18.24
N,N'-tert-Butyloxycarbonyl-~5-thiouronYl-3-thiaPentYlsulfonYl)-
ethYlenediamine hydrochloride ~30)
The solution of 6. 94 g of 29 (20 mmol) in ethanol is added
in drops to a solution of 1. 52 g of thiourea in ethanol, and it
is then refluxed for 2 hours. After the solvent is drawn off, a
crystalline residue remains, which is recrystallized from
ethanol.
Yield: 71%
Analysis:
Cld: C 34.07 H 6.43 N 13.25 0 15.13 S 22.74
Fnd: C 33.97 H 6.82 N 13.34 S 22.80
N,N'-tert-ButyloxYcarbonyl-~5-mercapto-3-thiapentYlsulfonyl)-
ethYlene~iamine ~31)
4.23 (10 mmol) of 30 iS stirred into 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: 59%
CA 02232620 l998-03-l8
Analysis:
Cld: C 38.35 H 7.02 N 8.13 0 18.58 S 27.92
Fnd: C 38.17 H 7.35 N 8.48 S 27.61
N-(5-Mercapto-3-thi~pentyl~ulfonyl)-ethylenediamine t32)
5.44 g of 31 (15.8 mmol) is dissolved under protective gas
in 200 ml of 3 M HCl in ethyl acetate, and it is stirred for 2
hours at room temperature. After the solvent is drawn off, 3.55
g of white crystals remains.
Yield: 92%
Analysis:
Cld: C 29.49 H 6.60 N 11. 46 O 13.09 S 39.36
Fnd: C 28.17 H 6.41 N 11.73 S 39.19
N-tS-Merc~Pto-3-thiapentYlsulfonYl)-ethylenediamine, technetium-
99m complex
10 mg of compound 32 iS dissolved in l.0 ml of ethanol. 50
,ul of this ligand solution is mixed with 100 ,ul of ethanol, 150
1 of phosphate buffer with a pH of 8.5, 50 ~1 of a deoxygenated
aqueous citrate solution (50 mg/ml), 2.5 ~1 of a deoxygenated
tin(II) chloride solution (5 mg/ml of 0.lN HCl) and lO0 ~Ll 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,
CA 02232620 1998-03-18
51
pH 2~0 (10/90); eluant B: acetonitrile/Na-phosphate 5 mmol, pH
2.0 ~75/25); 1 ml/min. The radiochemical purity is > 94%.
