Note: Descriptions are shown in the official language in which they were submitted.
ID-13317WO
2156620
This invention relates to new bifunctional chelating agents
with intermittent chalcogen atoms, pharmaceuticals
containing these compounds, their use in radiodiagnostics
and radiotherapy, and methods for the production of these
S compounds.
It has been known for a long time that complexing agents
for radioactive isotopes or their complexes with radio-
active metals can be applied in radiodiagnostics and radio-
therapy. Technetium-99m is the most frequently used radio-
nuclide in radiodiagnostics because it is particularly wellsuited for in-vivo applications due to its favourable
physical properties (no corpuscular radiation, low half-
life of 6.02 h, goo~ detectability by 140 keV y-radiation)
as well as its low biological half-life and easy availabil-
ity. The first step of forming complexes of technetium-99m
is to gain pertechnetate from a nuclide generator; it is
then converted to a lower oxidation number using appro-
priate reductants (such as SnCl2, S2O42-, etc.). This
oxidation number is stabilize,d by an appropriate chelating
agent. As technetium may have several oxidation numbers (+7
to -1) which may vehemently alter its pharmacological
properties by changing the charge of the complex, it is
necessary to provide chelating agents or complex ligands
for technetium-99m that are capable of binding technetium
2S in a specific oxidation number safely, firmly and stably to
prevent undesirable biodistribution due to in-vivo redox
processes or release of technetium from the radiodiagnostic
agent which would impede the safe diagnosis of the respec-
tive diseases.
For example, cyclic amines (Troutner, D.E. et al.: J. Nucl.
Med. ~1, 443 (1980)) are regarded as suitable complexing
agents for technetium and rhenium isotopes but their
disadvantage is that they are only capable of binding
technetium-99m in sufficient quantities from a pH value ~9.
N2O2 systems (Pillai, M.R.A., Troutner, D.E. et al.: Inorg.
~ ID-13317WQ
3 2156620
Chem., 29, 1850 (1990)) are in clinical use. Non-cyclic N4
systems such as HMPAO have the great disadvantage of low
complex stability. Tc-99m-HMPA0 has to be applied
immediately after labelling due to its low stability
s (Ballinger, J. R. et al., Appl. Radiat. Isot. ~, 315
(1991); Billinghurst, M. W. et al., Appl. Radiat. Isot. 42,
607 (1991)) to keep the portion of decomposition products
low which have different pharmacokinetic and excretion
properties. Such radiochemical impurities make detection of
the diseases to be diagnosed more difficult. Any coupling
of these chelates or chelating agents with other substances
that accumulate selectively in centres of diseases cannot
be broken by simple means so that these normally spread
unspecifically in the organism.
~,
N2S2 chelating agents (Bormans, G. et al.: Nucl. Med.
Biol., 17, 499 (1990)) such as ethylene dicysteine (EC;
Verbruggen, A.M. et al.; J. Nucl. Med. 33, 551 (1992)) meet
the requirement of sufficient stability of their respective
technetium-99m complex but form radiodiagnostic agents of a
purity greater than 69~ only~at pH values ~9 of the
complexing medium. N3S systems (Fritzburg, A.; EPA 0 173
424 and EPA 0 250 013) yield stable technetium-99m
complexes but have to be heated up to temperatures of about
100C to insert the radionuclide.
Another disadvantage of N2S2 and N3S systems is that they
are discharged partially too rapidly and without specific
accumulation by the organism. Thus they are only used
clinically, though to a limited extent, in renal function
diagnostics. In the last few years the demand has increased
for radiodiagnostics that accumulate specifically in
diseased tissues. This can be accomplished if one manages
to link complexing agents easily with selectively accumu-
lating substances while the latter retain their favourable
complexing properties. But as it happens quite frequently
that a certain reduction of complex stability is observed
ID-l33l7Wo 2 15 6 fi 2 ~
after coupling the complexing agent to such a molecule by
means of one of its functional groups, previous approaches
to coupling chelating agents with substances that accumu-
late selectively are hardly satisfactory because a quantity
5 of the isotope that is not tolerable with a view to
diagnostics is released in vivo from the conjugate
(Brechbiel, M. W. et al.; Inorg. Chem. 1986, ~, 2772). It
is therefore necessary to produce bifunctional complexing
agents that have functional groups to bind the desired
metallic ion and one (or several other) functional groups
to bind the select~vely accumulating molecule. Such bifunc-
tional ligands allow specific, chemically defined bonding
of technetium or rhenium isotopes to the most various bio-
logical materials even in cases in which pre-labelling is
~5 applied. Some chelating agents coupled with monoclonal
antibodies (e.g. EP Appl. 0 247 866 and EP Appl. 0 188 256)
or fatty acids (EP Appl. 0 200 492) have been described.
But these chelating agents were based on the N2S2 systems
mentioned above which are hardly appropriate due to their
low stability. As both the properties of the substances
that accumulate selectively and the mechanisms of
accumulation are quite varied, one should be able to vary
the chelating agent meant for coupling to adapt it to the
physiological requirements of its coupling partner with
regard to lipophilic or hydrophilic behaviour, membrane
permeability or impermeability, etc.
It is therefore an object of this invention to provide
stable complex compounds coupled with or capable of cou-
pling with various compounds that accumulate selectively,
and to provide such linkable chelating agents or complexes
whose substituents show a wider range of chemical variation
to be adaptable to the above requirements. It is another
object of this invention to provide such compounds and
pharmaceuticals containing these compounds, as well as
methods for their production.
' ID-13317wo
215662~
_ 5
This problem is solved by the invention, surprisingly, in
that the new, uncommon, bifunctional chelating agents with
intermittent chalcogen atoms and their coupling products
with compounds that accumulate selectively are excellently
suited for producing radiodiagnostic and radiotherapeutic
agents.
The subject matter of this invention are compounds of the
general formula (I)
M~- L (I)
wherein
M represents a radionuclide of Tc or Re
and L represents a ligand of the general formula (II)
B-Co-CRlR2-A-CR3R4-CooH (II)
wherein
A represents an O, S, or Se chalcogen atom,
Rl, R2, R3, and R4 are same or different and represent a
hydrogen atom and/or a branched or unbranched Cl-C6 alkyl
resldue,
B represents a residue
-NH-CR5R6- (CR7R8)n=1 2-S-R9,
wherein
R5 and R6 are same or different and represent a hydrogen
atom or an unbranched, branched, cyclic, or polycyclic Cl-
C60 alkyl, alkenyl, polyalkenyl, alkinyl, polyalkinyl,
25 aryl, alkylaryl, or arylalkyl residue which may optionally
be carry an additional hydroxy, oxo, oxy, carboxy,
aminocarbonyl, alkoxycarbonyl, amino, aldehyde, or alkoxy
groups containing up to 20 carbon atoms, and/or may
optionally be interrupted, and/or replaced, by one or
several heteroatoms from the series of O, N, S, P, As, Se,
ID-13317WO
215662~
R7 and R8 are same or different and represent a hydrogen
atom and/or a branched or unbranched Cl-C6 alkyl residue,
R9 represents a hydrogen atom, a branched or unbranched Cl-
C6 alkyl residue, or a sulfur protecting group,
with Rs and R6, together with the groups that connect them,
optionally forming a 4- to 8-membered ring which may
optionally carry additional hydroxy, oxo, oxy, or alkoxy
groups containing up to 6 carbon atoms.
Preferred compounds of the general formula (I) are
characterized in that Rl, R2, R3, and R4 are hydrogen
atoms, and that A is a sulfur atom.
Particularly prefer~ed compounds according to the invention
of the general formula (I) are characterized in that RS and
R6 are different, and that R6, R7 and R8 each represent a
hydrogen atom.
Another object of this invention is related to the new,
bifunctional ligands with intermittent chalcogen atoms of
the general formula (II)
B-CO-CR1R2-A- CR3R4-COOH (II)
wherein R1, R2, R3, R4, A and B have the meanings specified
above.
Such ligands according to the invention of the general
formula (II) are preferred in which A is a sulfur atom and
Rl, R2, R3 and R4 are hydrogen atoms.
Particularly preferred compounds according to the invention
of general formula (II) are characterized in that Rs and R6
are different, and that R6, R7 and Rs each represent a
hydrogen atom.
Yet another object of this invention are conjugates
containing compounds of the general formulae (I and/or II)
ID-l33l7wo
21S6620
-
and substances that accumulate selectively in diseased
tissues, with a covalent bond existing between these sub-
stances, said bond being amidic if the substances contain
carboxy or amino groups such as peptides, proteins,
antibodies or their fragments, ester-like if the substances
contain hydroxy groups such as fatty alcohols, and imidic
if the substances contain aldehyde groups.
Particularly preferred conjugates according to the
invention are characterized in that the substances that
accumulate in dise~sed tissue are peptides such as endo-
thelines, partial endotheline sequences, endotheline
analogues, endotheline derivatives, or endotheline
antagonists.
Other preferred embodiments of the conjugates according to
the invention are characterized in that the peptides com-
prise the following sequences or parts thereof:
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-
~ I
tyr-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,
i rD-13317WO
8 2156620
-
cys-ser-cys-asn-ser-trp-leu-asp-lys-glu-cys-val-tyr-
-
phe-cys-his-leu-asp-ile-ile-trp,
cys-ser-cys-lys-asp-met-thr-asp-lys-glu-cys-leu-asn-
i
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-trp-leu-asp-lys-glu-ala-val-tyr-
phe-ala-his-leu-asp-ile-ile-trp,
cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp,
N-acetyl-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-
asp-ile-ile-trp,
the partia~ sequence
his-leu-asp-ile-ile-trp
or the cyclic amino acid sequences
Cyclo-(Dtrp-Dasp-pro-Dval-leu),
Cyclo-(Dglu-ala-alloDile-leu-Dtrp).
ID-1~317Wo
9 21S6fi20
-
The compounds according to the invention of the general
formula (I) are produced by reacting technetium-99m in the
form of pertechnetate or Re in the form of perrhenate in
the presence of a reductant and, optionally, an auxiliary
ligand, with a compound of the general formula (II)
B-Co-CRlR2-A-CR3R4-CooH (II)
wherein Rl, R2, R3, R4, A, and B, have the meanings
specified above.
The ligands accordlng to the invention of the general for-
mula (II) are produced by reacting compounds of the general
formula (III) with compounds of the general formula (IV)
according to the following reaction scheme:
CR1R2-A_CR3R4
+ NH2 ~ CR5 R6 - ( CR7R8 ) n= l 2 ~ S - R9
1 5 C~- O CO
(III) (IV)
B-Co-CRlR2-A-CR3R4-CooH (II)
.
wherein
Rl R2 R3, R4, R5, R6, R7, R8, R9, A, and B have the
meanings specified above.
These reactions are carried out in polar and non-polar
aprotic solvents such as dichloromethane, tetrahydrofurane,
chloroform, 1,4-dioxane, DMF, or DMSO at temperatures
between -30 and +100C; an auxiliary base is added to trap
any acids that may be liberated. Among these bases could
be, for example: tertiary amines, alkali and alkaline-earth
hydroxides, alkali and alkaline-earth carbonates.
Another object of the present invention is a kit for pro-
ducing radiopharmaceuticals consisting of a compound of the
general formula (II) or a conjugate according to the
invention containing compounds of the general formulae (I
~ ID-13317wo
21~6620
-
and/or II) and substances that accumulate selectively in
tissues, a reductant and, optionally, an auxiliary ligand,
said agents being either dry or in solution, instructions
for use including instructions for reacting the compounds
described with technetium-99m or rhenium in the form of a
pertechnetate or perrhenate solution.
Another object of this invention is a radiopharmaceutical
formulation for non-invasive in-vivo visualization of
receptors and tissue containing receptors and/or athero-
sclerotic plaques.JIt contains a compound of the generalformula (I) or a conjugate according to the invention
containing compounds of the general formulae (I and/or II)
and substances that accumulate selectively in tissues,
optionally with the~adjuvants common in galenics; the
compound is prepared in a kit using technetium-99m or
rhenium in the form of a pertechnetate or perrhenate
solution.
Yet another object of this invention is a method for
carrying out radiodiagnostic examinations according to
which the radiopharmaceutical formulation is applied at
doses from 0.1 to 30 mCi, preferably from 0.5 to 10 mCi,
per 70 kg of a patient's body weight, and that radiation
emitted by the patient is recorded.
Many of the synthesized chelates that were labelled with
Tc-99m or Re surprisingly showed a greater stability than
comparable-N2S2 and N3S systems described in the litera-
ture. For example, no decomposition products were found of
a substance according to the invention (Examples 2a, 2b)
coupled with a fatty alcohol even after 23 hours. It was
also found in competition tests that the Tc-99m or-Re
chelating agents complex better than comparable N2S2, N3S
and propylene aminoxium systems. The chelates and chelating
agents described in the present invention are clearly bet-
ter suited for diagnostic and therapeutic purposes than the
systems known so far. It is a specific advantage of the
ID-13317WO
215662~
11
- chelating agents according to the invention that they may
be synthesized without sulfur protecting groups This makes
synthesis very simplei in addition, the compounds according
to the invention, when radiolabelled, do not contain any
other foreign molecules in the solutions used for radio-
diagnostics or radiotherapy, for example, solutions to be
administered intravenously. Biodistribution of the radio-
pharmaceutical and thus the value of diagnostic information
are frequently diminished by such foreign molecules. More-
over, such ligands~or their coupling products with sub-
stances that accumulate selectively in diseased tissues can
be labelled very gently. The ligands according to the in-
vention and their coupling products with substances that
accumulate selectively in diseased tissues can be labelled
at room temperature and at the physiological pH value
without having to split off protecting groups using bases,
acids, or other auxiliary substances known to a person
skilled in the art. This guarantees that the very sensitive
substances that accumulate selectively in diseased tissues
are not modified chemically by such auxiliary substances,
as such modification frequently reduces selective accumu-
lation in diseased tissue and diminishes the value of
radiodiagnostic information.
Sulfur protecting groups may be used here, of course, if
the disadvantages described can be accepted. The groups are
attached to sulfur atoms and split off according to methods
known to a-person skilled in the art. The ways in which the
substances that accumulate selectively in diseased tissues
are bonded are also known to a person skilled in the art
(e.g. Fritzberg et al.; J. Nucl. Med. ~, 7 (1987)), for
example, by a reaction of electrophilic groups of the
complex ligand with nucleophilic centres of the substances
that accumulate selectively in diseased tissues. Otherwise,
nucleophilic groups of the chelating agent are coupled with
electrophilic groups of the substances that accumulate
selectively in diseased tissues.
rD- 13317WO
21S66~0
12
The partners for coupling are, among others, various bio-
molecules, ligands that bond to specific receptors which
are capable of detecting tissue showing a modified receptor
density. This includes peptides, steroid hormones, growth
factors and neurotransmitters. Ways for improved diagnosis
of carcinomas of the breast and the prostata were shown
using ligands for steroid hormone receptors (S. J. Brandes
and J. A. Katzenellenbogen, Nucl. Med. Biol. lS, S3, 1988).
Tumour cells sometimes show a modified density of receptors
for peptide hormone~s or growth factors such as the epider-
mal growth factor (EGF). The differences in concentration
could be utilized for selective accumulation of cytostatic
agents in tumour cells (E. Aboud-Pirak et al., Proc. Natl.
Acad. Sci. USA 86; 3778, 1989). Ligands for neuroreceptors
labelled with positron-emitting isotopes were successfully
used for the diagnosis of various brain diseases (J. J.
Forst, Trends in Pharmacol. Sci., 7, 490, 1989). Other
biomolecules are metabolites that can be introduced into
the metabolism of cells to make changes visible; this
includes fatty acids, sacchar~des, peptides, and amino
acids. Fatty acids that were coupled with the more unstable
N2S2 chelating agents have been described in EPA 0 200 492.
Other metabolic products such as saccharides
(desoxyglucose), lactate, pyruvate, and amino acids
2S (leucine, methylmethionine, glycine) were used in the PET
technique for visualizing changes in metabolic processes
(R. Weinreich, Swiss Med., 8, 10, 1986). Likewise, non-
biological substances such as misonidazol and its
derivatives which bond irreversibly to cell components in
tissues or parts of tissues with a reduced oxygen
concentration, can be used for specific accumulation of
radioactive isotopes and thus for the visualization of
tumours or ischaemic regions (M. E. Shelton, J. Nucl. Med.
30; 3S1, 1989). Finally, bifunctional chelating agents may
be coupled with monoclonal antibodies or their fragments.
Coupling products of the chelating agents according to the
rD- 133 17WO
215fi620
~_ 13
invention or their technetium-99m or Re complexes with
fatty alcohols, fatty alcohol derivatives, or fatty amines
and their derivatives, or with endothelines, partial
endotheline sequences, endotheline analogues, endotheline
derivatives, or endotheline antagonists have proved
particularly favourable for the detection of
atherosclerotic vascular diseases. These derivatives were
applied to WHHL rabbits that had high LDL concentrations in
their blood - and thus atherosclerotic lesions - due to a
genetic defect of ~heir LDL receptor. Concentration
quotients from 3 to 40 were found in atheromatose plaques
as compared with undamaged tissue about 4 to 5 hours after
i.v. application of the derivatives to WHHL rabbits. This
allowed detection of atherosclerotic areas of vessels using
the common methods of radiodiagnostics (e.g. a gamma
scintillation camera). Only very late stages of
atherogenesis could up to now be diagnosed by using more
invasive methods (e.g. arteriography). The substances
according to the invention provide the decisive advantage
of being able to diagnose much earlier stages of athero-
sclerosis using non-invasive methods.
It is unimportant whether the chelating agent is labelled
with Tc-99m or Re before or after coupling with the selec-
tively accumulating molecule. But if coupling takes place
after complexing, it is a prerequisite that the reaction of
the radioactive complex with the accumulating compound is
rapid, gentle, and nearly quantitative, requiring no
subsequent purification.
The radiopharmaceuticals of the invention are produced in a
generally known way by dissolving the complexing agents
according to the invention in an aqueous medium and adding
a reductant, preferably tin(II) salts such as chloride or
tartrate, optionally adding the adjuvants common in
galenics, and subsequent sterile filtration. Among the
suitable additives are physiologically tolerable buffers
ID- 133 17WO
21~662~
14
(such as tromethamine), small quantities of electrolytes
(e.g. sodium chloride) or stabilizers (e.g. gluconate,
phosphate, or phosphonate). The pharmaceutical according to
the invention is either available as a solution or as
lyophilizate and is mixed shortly before application with a
solution of Tc-99m pertechnetate, eluated from commercial
generators, or a perrhenate solution.
For in-vivo applications in nuclear medicine, the agents
according to the invention are administered at doses from
1 x 10-5 to 5 x 104~nmol/kg of body weight, preferably from
1 x 10-3 to 5 x 102 nmol/kg of body weight. The amount of
radioactivity per application, based on an average body
weight of 70 kg, is between 0.05 and S0 mCi, preferably
between 5 and 30 mCi`, for diagnostic applications. For
therapeutic applications, doses applied are between 5 and
500 mCi, preferably from 10 to 350 mCi. Normally, 0.1 to
2 ml of a solution of the agents according to the invention
is applied by intravenous, intra-arterial, peritoneal or
intra-tumoral injection. The intravenous injection is
preferred.
The following examples shall explain the object of this
invention in greater detail.
rD-13317WO
21~6620
~_ 15
Example la
N-(2-mercapto-1-(methoXycarbonyl)-ethyl)-thiodiglycolic
acid monoamide
13.21 g (0.1 mol) of thiodiglycolic acid anhydride are
added by dropping, and in an argon atmosphere, to a
solution of 17.16 g (0.1 mol) of cysteine methyl ester
hydrochloride and 20.24 g (0.2 mol) of triethyl amine in
500 ml of anhydrous dichloromethane. This mixture is
stirred for 16 hourf.s at room temperature and washed with
aqueous 2~ citric acid. After drying above sodium sulfate,
the solvent is evaporated under reduced pressure, and the
oily residue crystallized by trituration with diethyl
ether.
Yield: 18.73 g (70.1%), white powder
15 Analysis referring to the anhydrous substance:
Calc.: C 35.95 H 4.90 N 5.24 0 29.93 S 23.99
Found: C 35.72 H 5.12 N 5.03 S 23.71
Example lb
Technetium-99m complex of N-(2-mercapto-1-(methoxy-
carbonyl)-ethyl)-thiodiglycolic acid monoamide
10 mg of the ligand produced according to Example la are
dissolved in 1.0 ml of 0.5 M phosphate buffer, pH 7.5.
50 ~l of this ligand solution are mixed with 250 ~1 of
phosphate buffer, pH 8.5, 50 ~l of a deoxygenated aqueous
citrate solution (50 mg/ml), 2.5 ~l of a deoxygenated
aqueous tin(II) chloride solution (5 mg/ml 0.05 N HCl), and
lO0 ~l of a pertechnetate solution (400-900 ~Ci). After an
incubation time of 10 minutes, the reaction mixture is
tested for purity of the Tc complex formed using HPLC:
Hamilton PRP-1 column, 5 ~m, 125 x 4.6 mm; gradient elution
from 100~ A to 100~ B within 7.5 minutes ~eluent A: sodium
hydrogenphosphate, 0.005 M, pH 7.4; eluent B:
ID-13317WO
16 21~66zo
-
acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4
(75/25); 2.0 ml/min. Radiochemical purity is ,98~.
Example 2a
S N-(2-mercapto-1-(decyloxycarbonyl)-ethyl)-thiodiglycolic
acid monoamide
1.32 g (10 mol) of thiodiglycolic acid anhydride dissolved
in 50 ml of anhydrcus-dichloromethane are added by
dropping, and in an argon atmosphere, to a solution of
2.98 g (10 mmol) of cysteine decyl ester hydrochloride and
2.02 g (20 mol) of triethyl amine in 250 ml of anhydrous
dichloromethane. This mixture is stirred for 16 hours at
room temperature and washed with aqueous 2~ citric acid.
After drying above sodium sulfate, the solvent is
evaporated under reduced pressure, and the oily residue
crystallized by trituration with diethyl ether.
Yield: 3.37 g (85.6~), white powder
Analysis referring to the anhydrous substance:
Calc.: C 51.88 H 7.94 N 3.56 O 20.33 S 16.29
Found: C 51.63 H 8.07 N 3.37 S 16.02
~D- 133 17WO
215 662 B
17
Example 2b
Technetium-99m complex of N-(2-mercapto-1-(decyloxy-
carbonyl)-ethyl-thiodiglycolic acid diamide
10 mg of the ligand produced according to Example 2a are
dissolved in 1.0 ml of O.S M phosphate buffer, pH 7.5.
50 ~1 of this ligand solution are mixed with 250 ~1 of
phosphate buffer, pH 7.5, 50 ~1 of a deoxygenated aqueous
ci~rate solution (50 mg/ml), 2.5 ~1 of a deoxygenated
aqueous tin(II) chlorlde solution (5 mg/ml 0.05 N HCl), and
100 ~1 of a pertechnetate solution (400-900 ~Ci). After an
incubation time of 10 minutes, the reaction mixture is
tested for purity of the Tc complex formed using HPLC:
Hamilton PRP-1 column, 5 ~m, 125 x 4.6 mm; gradient
eluation from 100% A to 100% B within 7.5 minutes (eluent
A: sodium hydrogenphosphate, 0.005 M, pH 7.4; eluent B:
acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4
(75/25); 2.0 ml/min. Radiochemical purity is ~98%.
Example 3a
N-(2-oxo-1-tetrahydrothiophene-3-yl)-thiodiglycolic acid
monoamide
13.21 g (0.1 mmol) of thiodiglycolic acid anhydride
dissolved in 250 ml of anhydrous dichloromethane are added
by dropping, and in an argon atmosphere, to a solution of
15.36 g (0.1 mol) of homocysteine thiolactone hydrochloride
and 20.24 g (0.2 mol) of triethyl amine in 500 ml of
anhydrous dichloromethane. This mixture is stirred for 16
hours at room temperature and washed with aqueous 2~ citric
acid. After drying above sodium sulfate, the solvent is
evaporated under reduced pressure, and the oily residue
crystallized by trituration with diethyl ether.
Yield: 22.73 g (91.2~), white powder
Analysis referring to the anhydrous substance:
ID-13317WO
18 21 S 6 ~ 2 0
Calc.: C 38.54 H 4.45 N 5.62 0 25.67 S 25.72
Found: C 38.37 H 4.68 N 5.41 S 25.47
Example 3b
N-(3-mercapto-1-(octylaminoCarbonyl)-propyl)-thiodiglycolic
mono~mide
30 ml of octyl amine are added in an argon atmosphere to a
solution of 2.49 g (10 mmol) of the thiolactone derivative
of the thiodiglyco~ic acid monoamide produced in Example 3a
in 30 ml of ethanol. The mixture is stirred at room
temperature for 4 hours and evaporated in a medium high
vacuum. The residue is mixed with 200 ml of aqueous 2~
citric acid and 200~ml of dichloromethane. The mixture is
stirred thoroughly, and the organic phase, after
separation, is dried above sodium sulfate. The solvent is
evaporated under reduced pressure, and the oily crude
product is crystallized by trituration with diethyl ether.
Yield: 878 mg (23.2~), white powder
Analysis referring to the anhydrous substance:
Calc.: C 50.77 H 7.99 N 7.40 0 16.91 S 16.94
Found: C 50.48 H 8.13 N 7.15 S 16.71
Example 3c
Technetium 99-m complex of N-(3-mercapto-1-(octylamino-
carbonyl)-propyl)-thiodiglycolic acid monsr ;de
10 mg of the ligand produced according to Example 3b are
dissolved in 1.0 ml of ethanol. 50 ~l of this ligand solu-
tion are mixed with 250 ~1 of phosphate buffer, pH 8.5, 50
~l of a deoxygenated aqueous citrate solution (50 mg/ml),
2.5 ~l of a deoxygenated aqueous tin(II) chloride solution
(5 mg/ml 0.05 N HCl), and 100 ~1 of a pertechnetate solu-
tion (400-900 ~Ci). After an incubation time of 10 minutes,
the reaction mixture is tested for purity of the Tc complex
ID-13317WO
lg 2ls662a
formed using HPLC: Hamilton PRP-1 column, 5 ~m, 125 x 4.6
mm; gradient eluation from 100~ A to 100~ B within 7.5
minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH
7.4; eluent B: acetonitrile/sodium hydrogenphosphate, O.oo5
M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is
,95~ .
Example 4a
N-(3-mercapto-1-(2,methoxyethylamino carbonyl)-propyl)-
thiodiglycolic acid monoamide
30 ml of 2-methoxy ethyl amine are added in an argon
atmosphere to a solution of 2.49 g (10 mmol) of the
thiolactone derivative of the thiodiglycolic acid monoamide
produced in Example 3a in 30 ml of ethanol. The mixture is
stirred at room temperature for 5 hours and evaporated
under reduced pressure. The residue is mixed with 200 ml of
aqueous 2~ citric acid and 200 ml of dichloromethane. The
mixture is stirred thoroughly, and the organic phase, after
separation, is dried above sodium sulfate. The solvent is
evaporated under reduced pressure, and the oily crude
product is crystallized by trituration with diethyl ether.
Yield: 734 mg (22.6~), white powder
Analysis referring to the anhydrous substance:
Calc. C 40.73 H 6.21 N 8.64 O 24.66 S 19.76
Found: C 40.47 H 6.49 N 8.38 S 19.51
ID-13317Wo
215662~
Example 4b
Technetium-99m complex o~ N-(3-mercapto-l-(2-methyleth
amino carbonyl)-propyl)-thiodiglycolic acid monoamide
10 mg of the ligand produced according to Example 4a are
S dissolved in 1.0 ml of ethanol. S0 ~1 of this ligand solu-
tion are mixed with 250 ~1 of phosphate buffer, pH 8.5, 50
~1 of a deoxygenated aqueous citrate solution (50 mg/ml),
2.5 ~1 of a deoxygenated aqueous tin(II) chloride solution
(S mg/ml O.OS N HCl), and 100 ~1 of a pertechnetate solu-
tion (400-900 ~Ci)~ After an incubation time of 10 minutes,
the reaction mixture is tested for purity of the Tc complex
formed using HPLC: Hamilton PRP-1 column, S ~m, 125 x 4.6
mm; gradient eluation from 100~ A to 100% B within 7.5
minutes (eluent A:-~odium hydrogenphosphate, 0.005 M, pH
lS 7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005
M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is
,95~ .
Example Sa
N-(3-mercapto-1-(2-hydroxy-ethylamino carbonyl)-propyl)-
thiodiglycolic acid monoamide
30 ml of 2-amino ethanol are added in an argon atmosphere
to a solution of 2.49 g (10 mmol) of the thiolactone
derivative of the thiodiglycolic acid monoamide produced in
Example 3a in 30 ml of ethanol. The mixture is stirred at
room temperature for 4 hours and evaporated in a medium
high vacuum. The residue is mixed with 200 ml of aqueous 2
citric acid and 200 ml of dichloromethane. The mixture is
stirred thoroughly, and the organic phase, after separa-
tion, is dried above sodium sulfate. The solvent is evapo-
rated under reduced pressure, and the oily crude product is
crystallized by trituration with diethyl ether.
Yield: 435 mg (14.0~), white powder
ID- 133 17WO , 2 1 5 6 6 2 0
21
Analysis referring to the anhydrous substance:
Calc.: C 38.70 H 5.85 N 9.03 0 25.77 S 20.66
Found: C 38.38 H 5.74 N 8.91 S 20.43
Example 5b
Technetium-99m complex of N-(3-mercapto-1-(2-hydroxy-
ethylaminocarbonyl)-propyl)-thiodiglycolic acid monoamide
10 mg of the ligand produced according to Example Sa are
dissolved in 1.0 ml of ethanol. 50 ~l of this ligand solu-
tion are mixed with 250 ~l of phosphate buffer, pH 8.5, 50~l of a deoxygenated aqueous citrate solution (50 mg/ml),
2.5 ~l of a deoxygenated aqueous tin(II) chloride solution
(5 mg/ml 0.05 N HCl), and 100 ~l of a pertechnetate solu-
tion (400-900 ~Ci). After an incubation time of 10 minutes,
the reaction mixture is tested for purity of the Tc complex
formed using HPLC: Hamilton PRP-1 column, 5 ~m, 125 x 4.6
mm; gradient eluation from 100~ A to 100~ B within 7.5
minutes (eluent A: sodium hydrogenphosphate, O.OOS M, pH
7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005
M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is
~95%.
Example 6a
N-(3-mercapto-1-~carbonyl-gly-his-leu-asp-ile-ile-trp)-
propyl)-thiodiglycolic acid monoamide
250 mg (1 mmol) of the N-(2-oxo-tetrahydrothiophene-3-yl)-
thiodiglycolic acid monoamide produced in Example 3a are
added in an argon atmosphere to a solution of 853 mg
(1 mmol) of NH2-gly-his-leu-asp-ile-ile-trp (produced in a
similar way as described by Barany and Merrifield, The
Peptides: Analysis, Biology, Academic Press, New York 1980;
Stewart and Young, Solid Phase Peptides Syntheses, 2nd ed.,
Pierce Chemical W., Rockford, II, 1984) and 404 mg (4 mmol)
ID-13317WO
_ 22 2156620
of triethyl amine in 100 ml of anhydrous dimethyl
formamide. The resulting reaction mixture is stirred at
room temperature for 12 hours. When the reaction is
finished, the solution is filtered and the solvent removed
under reduced pressure. The residual oiI is mixed three
times with 50 ml of dimethyl formamide and evaporated each
time. The residue is stirred up with 200 ml of anhydrous
diethyl ether. A white solid material settles down which is
filtered off. The material is recrystallized from mixtures
of dimethyl formamide and diethyl ether for purification.
Yield: 282 mg (25.6~), white powder
Analysis referring to the anhydrous substance:
Calc.: C 53.39 H 6.49 N 13.98 O 20.32 S 5.82
Found: C 53.17 H 6.63 N 13.74 S 5.61
Example 6b
Technetium-99m complex of N-(3-mercapto-1-(carbonyl-gly-
his-leu-asp-ile-ile-trp)-propyl)-thiodiglycolic acid
monoamide
10 mg of the ligand produced according to Example 6a are
dissolved in 1.0 ml of ethanol. 50 ~l of this ligand solu-
tion are mixed with 250 ~l of phosphate buffer, pH 8.5, 50
~1 of a deoxygenated aqueous citrate solution (50 mg/ml),
2.5 ~l of a deoxygenated aqueous tin(II) chloride solut-ion
(5 mg/ml 0. 05 N HCl), and 100 ~1 of a pertechnetate solu-
tion (400-900 ~Ci). After an incubation time of 10 minutes,
the reaction mixture is tested for purity of the Tc complex
formed using HPLC: Hamilton PRP-l column, 5 ~m, 125 x 4.6
mm; gradient eluation from 100~ A to 100~ B within 7.5
minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH
7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005
M, pH 7.4 (7S/25)i 2.0 ml/min. Radiochemical purity is
~97%.
rD-13317WO
23 215662~
-
Example 7
Accumulation of N-(2-mercapto-1-(decyloxycarbonyl)-ethyl)-
thiodiglycolic acid monoamide, technetium-99m complex, in
atherosclerotic vascular lesions of WHHL rabbit
N-(2-mercapto-I-(decyloxycarbonyl)-ethyl)-thiodiglycolic
acid monoamide (produced according to Example 2a) is
labelled as described in Example 3b. 99.9 GBq (2.7 mCi) of
the substance label~led according to Example 3b were diluted
to 1 ml with phosphate-buffered saline and administered via
the ear vein to a narcotized WHHL rabbit, Rompun/Ketavet
(1:2). The rabbit was killed 5 hours after the application,
and an autoradiogram of the aorta as well as a Sudan(III)
staining were carried out to visualize the atherosclerotic
plaques (Figure 1). The accumulation factor between normal
and atherosclerotic walls was between 3 and 8 depending on
the thickness of the plaques (Sudan(III) staining).
