Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
-- 1
RED~CING C~E~rING AGENTS,
THEIR TEC~NETI~M AND R}IENI~M COMPLEXES,
PROCESS FOR THEIR PROD~CTION
A8 WELL ~S T~EIR I~SE IN I)IAGNOSI~ AND TREATMENT
Backqround of the Invention
Radioactive metal ions, usually bound to a complexing
agent, have been used for in vivo diagnosis for some time.
of these, technetium-99m (Tc-99m), because of its almost
ideal physical properties for these purposes -- good
absorption of radiation in corresponding detection devices
(gamma camera, SPECT devices) relative to a low absorption
in the human organism and easy availability with a
molybdenum/technetium generator -- is the radionuclide most
often used in clinical nuclear medicine. Its short half-
life of 6.02 hours guarantees an only slight exposure of the
patient to gamma radiation, particularly since also the
secondary product technetium-99 has only an insignificant
residual radiation. But a drawbac~ of the technetium is its
complicated and not yet completely known complex chemistry.
Technetium can be present in a number of oxidation states
(+7 to -1), and the pharmacological properties can be
greatly changed by changing the charge of a complex. It is
therefore necessary to use complexes which bind the
technetium in a defined oxidation state and to prevent redox
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-- 2
reactions, which could lead to a redistribution of the
pharmaceutical agent.
For organ- or tissue-specific diagnosis, it is
necessary that the radiopharmaceutical agents be selectively
S concentrated in the desired target organs or tissues and
remain there for a while. This selectivity can be achieved,
on the one hand, by the formation of complexes, which on
their own show a specificity for certain tissues, or by
coupling the technetium complexes to selecti~e substances,
such as, e.g., monoclonal antibodies.
For labeling organ-specific substances with Tc~99m, the
pertechnetate eluted from the nuclide generator first has to
be converted to a lower oxidation state. In this reduced
form, technetium forms more or less stable compounds with
the selectively concentrated substances. The special
problem of labeling with Tc-99m consists in the fact that
normally tin(II) ions are present in the reaction solution
as reducing agents. Tin(II) is thus far the only reducing
agent which makes possible a quick and quantitative
conversion of the pertechnetate at room temperature to a
lower and thus reactive oxidation state. There, the added
tin(II) salts have to be used in a high excess (about 100:1)
relative to the pertechnetate. But the tin(II) and tin(IV)
ions present after the reduction has been completed, in
addition to the reduced Tc-99m, compete for the binding
sites of the ligands, so that either the complexing agent
again has to be used in excess relative to the tin, by which
the specific activity is greatly reduced or unbound Tc-99m
and tin as common colloid results in undesirable storage of
radioactivity in other organs. In both cases, the
diagnostic informative value is reduced.
This prcble~ can be avoided by the use of reducing
ligands. In the production of diagnostic agents according
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to this principle, a part of the added ligand excess acts as
reducing agent for pertechnetate, which reduces technetium
in an oxidation state lower than +7. In this way, reduced
technetium species are then complexed by the excess of the
unoxidized chelating agent. In this case, it is important
to obtain stable complexes in a defined oxidation state for
the technetium.
DeLearie et al. (L. A. deLearie, R. C. Haltiwanger, C.
G. Pierpont; J. Am. Chem. Soc. 111: 4324, 1989) showed that
3,5-di-tert-butylcatechols are suitable for reduction and
chelation of Tc-99 (half~life: 212,000 years). The
reduction took place by 24 hour-s of boiling in methanol.
For the labeling with the short-lived isotope of technetium
(Tc-99m; half-life: 6 hours), however, only substances are
usable as radiopharmaceutical agents which can be labeled
quickly and gently also in the clinic and for which no
subsequent purification after the labeling with, eOg., Tc-
99m is necessary. The compounds described by DeLearie are
thus unsuitable for the production of a clinically usable
radiopharmaceutical agent.
Summary of the In~ention
The present invention provides new reducing and tissue-
specific chelating agents, as well as their stable
technetium and rhenium complexes. Surprisingly, substances
were ound which reduce and completely complex Tc-99m under
mild conditions as well as quickly. ~oreover, they also can
be used, in contrast to the above-described compounds, for
coupling per se to selectively concentrated substances in
foci of disease or certain tissues.
Upon further study of the specification and appended
claims, further objects and advantages of this invention
will become apparent to those skilled in the art.
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According to the invention, this object is achieved by
compounds of general formula I,
in which X stands for -O-, -S-, -NR2- with R2 meaning a
hydrogen atom or a C16 alkyl radical,
Y and Z are the same or different and stand for the
radicals -OH, -NHR or -SR with R meaning a hydrogen atom
or a C1-6 alkyl radical and
U stands for a hydrogen atom, a branched or unbranched
C1-6 alkyl, a C16 alkoxy, a hydroxyl or a carboxyl radical,
n means the numbers 2 to 6 and
m means the numbers 2 or 3 and R is present only if m
stands for the number 2,
R1 stands for a hydrogen atom, a benzyl radical or a
branched or unbranched C06 alkyl radical optionally
substituted with one to three hydroxyl, carboxyl or amino
groups;
wherein said radicals optionally contain (i) a
functional group B, (ii) a compound T which is selectively
concentrated in lesions or certain tissues; or (iii) a
compound T bound to the radical through a functional group;
and B, i~ Y and/or Z stand for -NHR , stands for an
amino, a hydrazino or hydrazide, a carboxyl, a C16-alkynyl
or alkenyl, a hydroxyl, an aminophenyl, an oxiranyl, a
fluorinated phenoxycarbonyl or a biotin radical;
or, if Y and Z stand for -OH or -SR , B in addition
also stands for a halogen, a formyl, a nitrile, a
phenylisothiocyanato or a succinimidoxycarbonyl radical
.,
-- 5
optionally substituted with a sodium sulfate radical;
and T stands for monoclonal antibodies or their
~ragments, hormones, growth factors, ligands for cell
me~brane receptors, steroids, neurotransmitters, fatty
acids, saccharides, amino acids and oligopeptides, biotin,
as well as radiosensitizers, such as, e.g., misonidazole,
and optional]y present funct:ional groups optionally in
protected form or their precursors ar~ present in R1,
with the exception of compound N{CH2-CH2-CH2-O-C6H3-2,3-
(OH)2~3, their technetium and rhenium complexes, as well astheir salts with inorganic and organic acids.
The excluded compound N{CH2-CH2-CH2-O-C~3-2,3-(OH) 2)3
is known (B. Wolff, Angew. Chem. [Appl. Chem.] 98: 173,
1986) and was used for complexing germanium and silicon.
Physiologically compatible inorganic acids, such as,
e.g., hydrochloric or sulfuric acid, and organic acids, such
as, eOg., acetic or citric acid, are used as acids for salt
formation.
A C0 alkyl radical is to be understood to mean a direct
bond from the nitrogen atom in Formula I to one of the
optional substituents -B, -T or -B-T.
According to the invention, preferred are those
compounds in whose general formula I U represents a hydrogen
atom and X r~presents an oxygen atom, n is the number 3, Y
2S and Z are the same and stand for OH- or NH2- and R1
represents a hydrogen atom, a benzyl radical, an unbranched
C03 alkyl radical optionally substituted with a hydroxyl or
amino group, wherein said xadicals optionally contain a
functional group B or antibodies or their fragments,
steroids or misonidazole bound to the radical through a
functional group B.
The substances according to the invention surprisingly
have the advantages that they
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1. can reduce pertechnetate from a nuclide generator
under mild conditions, quickly and without adding reducing
agents to an oxidation state lower than +7,
2. form stable complexes with the thus reduced techne-
tium without further adding reducing agents at neutral pH,
3. are concentrated selectively in certain tissues or
lesions because of compounds T, which are coupled with the
help of functional group B, such as monoclonal antibodies or
their fragments, hormones, growth factors, ligands for cell
membrane receptors, steroids, neurotransmitters, fatty
acids, saccharides, amino acids and oligopeptides, biotin as
well as radiosensitizors, such as, e.g., misonidazole
or, without containing a group T, are concentrated in
certain tissues or lesions.
The formation of the technetium complexes of the above-
described chelates takes place with Tc04- from a nuclide
generator with neutral pH without adding reducing agents in
aqueous solution.
This property of the chelating agent described here
offers significant advantages in comparison with previously
known ligands. The incorporation of tin, which has to be
added as reducing agent in the known ligand systems, is
avoided in the chelate. The formation of the Tc-99m
SpeGies, which ~re not bound by the above-described chelates
(examples 6 and 7), was not observed. The chelates
according to the invention are thus definitely better suited
for diagnostic purposes than the previously known chelates.
Their production t~kes place in that amines of general
formula II
R --N-~-(CH2)n--Nu)~ (II)
in which Nu stands for a nucleofuge, such as, e.g., Cl, Br,
I, CH3C6H4S03-, CH3S03- or CF3S03-;
2.V~88~
and R1 stands for a substituent R1, whose optionally
present functional groups are present in protected form or
as their precursors, and which contains no selectively
concentrated compound T, and
aromatic substances of general formula III
U'
r~ '.
H-X ~ (lil)
in which U' stands for a substituent U, whose hydroxy or
carboxyl radical is present in protected form, and Y' and Z'
stand for Y and Z or their precursors or in protected form,
are reacted under base catalysis in polar solvents at
temperatures of 50-200 C within 6 hours to 6 days,
preferably 2 hours to 4 days,
and then functional group B optionally contai~ed in R
or desired aromatic substance substituents Y and Z are -
generated, optionally the thus obtained couplable or
complexable compounds are coupled with the respective
desired selectively concentrating compound T or complexed
~ith the respective desired technetium or rhenium isotope
-- and the sequence of the steps coupling on T and
complexing with the technetium or rhenium isotope can be
interchanged -- and then the still present protective groups
are removed or the precursors are converted to the finally
desired substituents.
As hydroxy protective groups, e.g., the benzyl,
4-methoxybenzyl, 4-nitrobenzyl, trityl, diphenylmethyl,
trimethylsilyl, dimethyl-t-butylsilyl and diphenyl t-
butylsilyl groups are suitable. In the case of polyols, the
hydroxy groups can also be protected in the form of ketals
-`` 20~$9~
with, e.g., acetone, acetaldehyde, cyclohexanone or
benzaldehyde. Further, the hydroxy groups also can be
present, e.g., as THP ether, ~-alkoxyethyl ether, MEM ether
or as esters with aromatic or aliphatic carboxylic acids,
such as, e.g., acetie aeid or benzoie acid.
The hydroxy proteetive groups ean be released according
to the methods in the literature known to one skilled in the
art, e.g., by hydrogenolysis, reductive cleavage with
lithium/ammonia, aeid treat~ent of ethers and ketals or
alkali treatment of the esters (see, e.g., "Protective
Groups in Organic Synthesis," T. W. Greene, John Wiley and
Sons 1981).
As acid protective groups, lower alkyl, aryl and
aralkyl groups, for example, the methyl, ethyl, propyl,
n-butyl, t-butyl, phenyl, benzyl, diphenylmethyl,
triphenylmethyl, bis(p-nitrophenyl)-methyl group, as well as
trialkylsilyl groups, are suitable.
The cleavage of the protective groups takes place
according to processes known to one skilled in the art, for
example, by hydrolysis, hydrogenolysis, alkaline
saponification of the esters with alkali in aqueous-
alcoholic solution at temperatures of 0 to 50C, acidic
saponification with mineral acids or in the case of, e.g.,
tert-butyl esters with the help of trifluoroacetic acid.
Th~ starting materials for the production of the
compounds of this invention are all either commercially
available or routinely synthesizable by one of ordinary
skill in the art using conventional synthetic methods. For
the production of compounds of general formula I with Y and
Z meaning SH groups, the starting materials are ligand
precursors of general formula III with Y' and Z' meaning SR3
radicals. The cleavage of the protective groups after the
reaction with the amines of general formula II takes place,
2~8~9
g
alternatively with alkali alkylthiolates, alkali alcoholates
or alkali metals, preferably with sodium methylthiolate in a
polar solvent, preferably in HMPT, DMF or dimethylacetamide.
As amino protective groups, e.g., trifluoroacetyl,
t-butoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzoxy-
carbonyl and acetyl groups are suitable. The amino
protective groups can be cleaved according to methods known
in the literature, e.g., by basic or acidic hydrolysis,
reductive cleavage with zinc in acetic acid or
hydrogenolysis.
If the ligand precursor of formula III contains
functionalized aromatic substances with Y' and Z' meaning
N02 groups, then the production of the chelating agent
according to claim 1 takes place by reduction, preferably
with tin in hydrochloric acid solution.
Radical R1 can be modified in compounds according to
general formula I, in which m means the number 2. If R ,
for example, is a benzyl group, it can be removed by
reaction with hydrogen under increased pressure and
increased temperature in the presence of a palladium
catalyst. Radical R is then a hydrogen atom.
Rhenium 186 has a physical half-life of 3.7 days and
emits beta particles with an energy of 1.1 MeV suitable for
the treatment of, e.g., tumors, and, at the same time, gamma
radiation with an energy of 137 keV (9% frequency). Rhenium
is found in periodic systems in group VII A directly under
technetium and exhibits a practically identical structural
and chelate chemistry as technetium. These properkies make
rhenium 186 into an ideal isotope for therapeutic uses
(damage of diseased tissue by particular beta radiation)
with the possibility at the same time for diagnostic study
of a concentration with the help of the portion of gamma
radiation. Rhenium 188, which also can be used for
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treatment of tumors, has a significantly shorter half-life
of 17 hours and a beta energy of 2.1 MeV. Also, rhenium 188
has a gamma radiation portion (155 keV; 15%) and can thus
also be used for treatment and, at the same time, detection
with a gamma camera, e.g., according to the methods
disclosed in Fritzburg et al., N. Nucl. Med. 30, 743 (1989).
If the chelating agents complexed with a radioactive
isotope show no selectivity for lesions or certain tissues,
it is necessary that they be coupled to a selective
substance. Radical R is suitable for this purpose, e.g.,
with the help of functional group B, to produce a stable
connection to proteins or other selectively accumulating
molecules. By the corresponding selection of the functional
group, the coupling is possible under mild reaction
conditions, which do not influence the biological function
and/or selectivity.
The coupling to the described compounds also takes
place according to methods known in the art (e.g., Fritzberg
et al.; J. Nucl. Med.: 26, 7 [1987]), for example, by
reaction of group B with nucleophilic groups of the
selectively accumulating molecule or, if a nucleophile is
involved in the case of group B itself, with activated
groups of the selectively accumulating molecule.
Group B represents every substituent which, on the one
hand, represents a functional group, which makes possible a
coupling to a selectively accumulating molecule under mild
conditions (e.g., by acylation or amidation) as well as
every activated group, which can react with nucleophilic
groups of proteins, antibodies, hormones or other
biomolecules, such as the amino, phenol, sulfhydryl,
aldehyde or imidazole group. By an activated group is to be
understood a function which is capable of reacting with the
foxmation of a conjugate with a nucleophilic substituent of
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a selective molecule or of the complex ligand itself in
aqueous solution within a suitably short time, under reac-
tion conditions which, result in neither denaturing nor loss
of the biological activity or selectivity. Examples in this
respect are imide esters, alkylimide esters, amidoalkylimide
esters, succinimide esters, acylsuccinimides, phenol esters,
substituted phenol esters, tetrafluorophenol esters,
anhydrides, hydrazides, alkyl halides and Michael acceptors.
B is preferably a monoanhydride, acid chloride, acid
hydrazide, mixed anhydride, activated ester (such as phenol
or imide ester), nitrene or isothiocyanate, in particular
for the coupling with nucleophilic groups of amino acids or
an aliphatic or aromatic primary amine for the coupling to
carbohydrate radicals of proteins.
If a nucleophile is involved in the case of group B
itself, it can react with activated groups of a selactively
accumulating molecule, and also reacted groups of the
selective molecule are enclosed with so-called "crosslinking
reagents." These can be homofunctional crosslinkers having
two identical functional groups, e.g., imidoester groups or
N-hydroxysuccinimide ester (NHS) groups. Alternatively,
these crosslinking reagents can be, for example,
heterobifunctional "crosslinkers," which contain two
different functional groups, for example, two of an NHS
ester, a pyridyl disulfide and an activated halogen, such as
an ~-keto-halide. Such crosslinkers can be obtained
commercially.
Compounds which selectively accumulate compounds in
certain tissues or lesions are used as coupling partners.
Often, the selective accumulating of these suhstances could
already be shown by labeling with positron-emitting isotopes
(PET technique), iodoisotopes or other coupling partners.
Compounds labeled with Tc-99m have already partially come
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into use. See, e.g., Chiton, ~.M. and Witcofski, R.L.,
Nuclear Pharmacy: An Introduction to the Clinical
Application of Radiopharmaceuticals; Lea & Febiger,
Philadelphia, PA (1986). But technetium-labeled compounds
in such a way have the drawback thAt tin(II) ions have to be
added as reducing agent, which results in the above-
described consequencss (reduced specific radioactivity and
the possibility that unbound Tc-99m together with tin as
colloid results in an undesirable storage of radioactivity
in other organs and a reduced diagnostic informative value).
Ligands which bind to specific receptors can recognize
a changed tissue in their receptor density; they include,
i.a., peptide and steroid hormones, growth factors and
neurotransmitters. With ligands for steroid hormone
receptors, the possibility of an improved diagnosis of
breast and prostate cancers was demonstrated (S. J. Brandes
& J. A. Katzenellenbogen, Nucl. Med. Biol. 15:53, 1988).
Often, ligands labeled with positron-emitting isotopes could
be used for neuroreceptors for the diagnosis of various
brain diseases (J. J. Forst, Trends in Pharmacol. Sci. 7:
490, 1987). Occasionally, tumor cells exhibit a changed
density of receptors for peptide hormones or growth factors,
such as, e.g., the "epidermal growth factor" (EGF). The
concentration differences could be used for selective
concentration of cytostatic agents in tumor cells (E. Aboud-
Pirak et al., Proc. Natl. Acad. Sci. USA 86: 3778, 1989).
Other biomolecules are metabolites that can be put in
the metabolism of cells which make a changed metabolism
recognizable; they include, for example, lipids (also in the
form of liposomes), saccharides, porphyrins, peptides and
amino acids. Fatty acids coupled with Tc chelating agents
were described in EPA 0 200 492. Other metabolic products
such as saccharides (deoxyglucose), lactate, pyruvate and
.
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~8~9
- 13 -
amino acids (leucine, methylmethionine, glycine) were used
with the help of the PET technique for graphic display of
changed metabolic processes (R. Weinreich/ Swiss Med. 8, 10,
1986). Certain porphyrins showed a concentration in tumors
(P. A. Scourides, Cancer Res. 47: 3439, l9B7).
Also, nonbiological substances such as misonidazole and
its derivatives, which are bound irreversibly to cell
components in tissues or tissue parts with reduced oxygen
concentration, can be used for specific concentration of
radioactive isotopes and thus graphic display of tumors or
ischemic regions (M. E. Shelton, J. Nucl. Med. 30: 351,
198g). Other suitable nonbiological substances include
cytostatic agents, such as bleomycin, which accumulate in
tumors. Also, suitable polymers such as dextrans,
polyethylenimines, polyamides, polyureas, polyethers and
polythioureas are suitable as coupling partners.
The compounds according to the invention containing
biotin make possible the binding of radioactive conjugates
to substances containing avidin or streptavidin. This can
be used to concentrate antibody-streptavidin conjugates on
the tumor and only later to apply the radioactive component
containing biotin, which results in a reduced exposure of
the patient to radiation ~D. J. Hnatowich et al., J. Nucl.
Med. 28: 1294, 1987). Finally, the direct coupling of the
bifunctional chelating agents to proteins, such as, e.g.,
monoclonal antibodies or their fragments/ albumin, enzymes
(e.g., urokinase, streptokinase), fibrin, fibrinogen or
myosin, is also possible.
By complexing the conjugates with Tc-99m or rhenium
iso~opes, a diagnosis and treatment of tumors or other
diseases is made possible. In this case, it is unimportant
whether a labeling of the chelating agents with Tc-99m or a
rhenium isotope is performed before or after the coupling to
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the selectively accumulating molecule. But for a coupling
to the selectively accumulating molecule after a complexing,
the re~uirement is that the reaction of the radioactive
complex with the accumulating compound occurs quickly and
almost quantitatively under mild conditions, and that no
subsequent purification is necessary.
The production of the pharmaceutical agents according
to the invention takes place in a way known in the art, in
which the complexing agents according to the invention are
dissolved --optionally by adding the additives usual in
galenicals -- in aqueous medium and then sterilized by
filtration. Suitable additives are, for example, physio-
logically harmless buffers (e.g., tromethamine~, small
additions of electrolytes (e.g., sodium chloride),
lS stabilizers (e.g., gluconate or phosphonate) and small
amounts of oxidizing or reducing agents (10-500
micrograms/dose). 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 the administration
with a Tc-99m-pertechnetate solution, eluted from
commercially obtainable generators, or a perrhenate
solution.
In the case of the nuclear medicinal in vivo use, the
agents according to the invention are administered in
amounts of 1 10 to 5 104 nmol/kg of body weight,
preferably in amounts between 1 103 and 5 102 nmol/kg of
body weight. Starting from an average body weight of 70 kg,
the amount of radioactivity for diagnostic uses is between
0.05 and 50 mCi, preferably 5 to 30 mCi per administration.
For therapeutic uses, between 5 and 500 mCi, preferably 10-
350 mCi, is administered. The administration is normally
performed by intravenous, intraarterial, peritoneal or
intratumoral injection of 0.1 to 2 ml of a solution of the
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agents according to the invention. The intravenous
administration is preferred.
The following examples are used to explain the object
of the invention in more detail.
Without further elaboration, it is believed that one
skilled in the art can, using the preceding description,
utilize the present invention to its fullest extent. The
following preferred specific embodiments are, therefore, to
be construed as merely illustrative, and not limitative of
the remainder of the disclosure in any way whatsoever.
In the foregoing and in the following ~xamples, all
temperatures are set forth uncorrected in degrees Celsius
and unless otherwise indicated, all parts and percentages
are by weight.
The entire disclosures of all applications, patents and
publications, cited above and below, and of corresponding
application Federal Republic of German P 40 25 788.6, filed
August 10, 1990, are hereby incorporated by reference.
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_xample 1:
Tri(B-Carbetho~yethyl)iamine, 1 and di~-
carbethoxyethyl)amin~, 2
300 ml of freshly distilled ethyl acrylate is brought
to reaction with 300 ml of liquid ammonia for one day in a
sealing tube and, after removal of the final ammonia
residues from the water bath, the resulting product mixture
is fractionated in a vacuum. The first fraction forms bis-
(B-carbethoxyethyl)amine, 2, (boiling point 97-110C/0.05
mbar), the main fraction consists of the desired product, 1,
(boiling point 120-133 C/0.05 mbar).
Yield: 144 g (4996) of tri(B-carhethoxyethyl)amine,
H--NMR (CDCl3, ~, ppm):
4.10 (q, 2H, C(O)OCHzCH3); 2.74 (t, 2H, NCH2CH2); 2.41
(t, 2H, CH2CH2C(O)O);
1.23 (t, 3H, OCH2CH3)
3C-NMR ( CDCl3, ~ , ppm):
172.2 (CH2C(O)O); 60.1 (C(O)OCH2CH3); 49.1 (NCH2CH3);
32.8 (CH2CH2C(O)O); 14.1 (OCHzCH3)
For di(B-carbethoxyethyl)amine 2, it was found:
H~ (CDC13, ~ , ppm):
4.12 (q, 4H, C(O)OCH2CH3); 2.88 (t, 4H, NC~2CH2); 2.47
- (t, 4H, CH2C~I2C(O)O); 1.60 (s, br, lH, HN(CH2)2); 1.24
(t, 6H, OCH2CH3)
Tris~3-Hydroxypropyl)~mlne, 3
18 g (0.7 mol) of lithium aluminum hydride in 900 ml of
absolute ether is suspended in a 2~1iter three-necked flask
with dropping funnel and reflux condenser. A solution of 77
g (0.24 mol) Oe ester l in 200 ml of absolute ether is
instilled in it within one hour so that the solution boils
moderately. After ~ive hours of stirring at 25 C and
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careful hydrolysis of excess hydride with water, the product
is separated by a Buchner funnel from precipitated
hydroxides. After removal of the solvent in a vacuum, the
residue is briefly boiled up in ethanol, additional
LiAl(OH)4 is filtered off by suction by a îrit (D3), the
alcohol is drawn off and the remaining liquid is taken up in
methylene chloride. Pressureless filtering yields a honey
yellow, highly viscous liquid by another frit (D4) and
removal of the solvent in a vacuum.
Yield: 27.3 g (59%)
H-N~DR (CDC13, ~, ppm)
3.78 (t, 2H, CH2CH2OH); 2.62 (t, 2H, NCH2CH2); 1.80 (q,
2H~ CHzCH2CH2);
C-N~rR (CDC13, ~, ppm):
1560.6 (CH2CH2OH); 51.3 (NCH2CH2); 2~-1 (C~IzCH2CH2)
Tri~(3-Chloropropyl)amine, 4
18.9 g (160 mmol) of thionyl chloride is added to 8.6 g
(45 mmol of tris(3-hydroxypropyl)amine, 3, dissolved in 80
ml of chloroform. Thus, an insoluble white mass results,
which slowly dissolves again. After the reaction mixture
has been refluxed for three hours, excess SOCl2 is
hydrolyzed with water after cooling off. The organic phase
is shaken out four times wi~h 50 ml of hot water, the
combined aqueous phases are made strongly alkaline with 40%
sodium hydroxide solution and then are extracted four times
with 80 ml of ether each. After drying on Na2SO4 and
removal of the ether in a vacuum, the yellowish residue is
fractionated, and the product goes over as colorless liquid
and crystallizes out after prolonged standing at room
temperature. Tris~3-chloropropyl)amine can be
recrystalli~ed from ethanol (3 g of 4 for 7 ml of ethanol).
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- 18 -
Yield 9.6 g (87~)
~elting point: 35C
Boiling point: 120 C/0.05 mbar
H-NMR (CDC13, ~, ppm):
3.60 ~t, 2H, CHzCH2Cl); 2.52 (-t, 2H, NCH2CH2); 1.88 ppm
(q, 2H, CHZCH2CH2)
C-NMR (CDCl3, ~, ppm):
50.5 (CH2CH2Cl); 43.0 (NCH2CHz); 30.1 (CH2C~2CH2)
2,2-Dimethyl-1,3-benæodioxol-4-ol, 5
77 g (0.61 mol) of pyrogallol in 250 ml of abso]ute
toluene is suspended and heated in a 500-ml two-necked flask
with dropping funnel and a Widmer spiral column. When the
solvent begins to boil, 75 ml (0.61 mol) of 2,2-
dimethoxypropane is added. After that, distillate
lS continuously passes over at the column head at about 60C.
After t~o hours, another 75 ml of dimethoxypropane is added.
After the temperature at the column head drops ~about six
hours), the reaction mixture is refluxed overnight for
completion of the reaction. The cooled solution is freed in
a vacuum from toluene and the viscous residue is distilled
over a bridge with large cross section. As a result, the
product already crystallizes out on the bridge wall and has
to be transferred to the receiver by heating. The
distillation temperature is selected so that existing
yellowish impurities distill over only to a limited extent.
The slightly yellowish product can be sublimated at 75 C,
0.2 mbar. The acetal is readily soluble in acetone and
methanol, slightly soluble in chloroform.
Variants for working up: After removal of the toluene,
the crude product is taken up in so much hot carbon
tetrachloride, that it just about dissolves. Wlth cooling
off to room temperature, white product 5 crystallizes out
'20~99
- 19 -
from the solvent. It is sublimated according to the same
conditions as described. A significant difference in the
yields is not observed.
Yieldi 50 g (50%)
Melting point: 90C
H-NMR (CDCl3, ~, ppm):
6.68 (t, lM, Ar-~); 6.46 (d, lH, Ar-H); 6.40 (d, lH,
Ar-H); 5.13 (s, lH, Ar-OH); 1.69 (s, 6H, CH3)
C-NMR (MeOD, ~, ppm):
149.7, 141.8, 135.3, 122.1 (Ar); 118.6 (C(CH3)2);
111.3 (Ar); 25.8 (C(CH3)2)
Tris~3-(2,2-Dimethyl-1,3-benzodio~ol-4-ylo~y1-propyl~-
amine, 6
20.1 g (121 mmol) of dry pyrogallol acetal 5 is intro-
duced in a stung-out 250-ml two-necked Schlenk flask with a
reflux condenser and dissolved in 100 ml of absolute (99%)
ethanol. For removal of the final oxygen residues, the
flask is alternately degassed five times and aerated with
argon. Then, 4.75 g (121 mmol) of potassium ~etal is added
in small pieces. First, white potassium phenolate preci-
pitates from the solution, which is at once dissolved again.
Now, a li~ewise degassed solution of 9.6 g (39 mmol) of tri-
(3-chloropropyl)amine and 20 ml of ethanol is sprayed in the
now extremely oxygen-sensitive solution. After the solution
has been xefluxed for four days, 4 ml of glacial acetic acid
is added, precipitated KCl is filtered off from the still
hot but no longer air-sensitive solution and the filter cake
is rewashed with a little hot ethanol. By allowing it to
stand at room temperature, the ligand precursor crystallizes
out slowly. The crystals are filtered off and rewashed with
ice-cold ethanol. A second fraction can be obtained by
concentrating the mother liquor by evaporation.
- ; .. . - , ,. , : : , ~:
- : , . :: . .
2 ~
- 20 -
Yield: 11.5 g (46%)
Melting point: 65 C
H-NMR (CDCl3, ~, ppm):
6.66 ~t, lH, Ar-H); 6.42 (cl, lH, Ar-H); 6.26 (d, lH,
Ar-H); 3.95 (t, 2H, CHzCH2O); 2.59 (t, 2H, NCHzCH2);
1.91 (q~ 2H, CH2CH2CH2); 1.69 (s, 6H, C(CH3)2)
3C-NMR (CDCl3, ~, ppm):
148.3 (Ar); 143.2 (Ar); 135.2 ~Ar); 121. (Ar); 117.9
~C(CH3)2); 107.9 (Ar); 102.0 (Ar); 66.8 (CHzCH2O); 49.8
(NCH2CH2); 26-9 (CH2CH2CH2); 25.7 (C(CH3)2
Tris-~3-~2,3-Dihydroxyphenoxy)propyl)aminohydrochloride, 7
39.6 g (62 mmol) 6 is dissolved under argon in 250 ml
of glacial acetic acid and heated to boiling. 200 ml of
a mixture of 50% glacial acetic acid, 20% water and 30%
fuming hydrochloric acid is instilled in it within
two hours. The solvent is distilled of~, iso that about
200 ml remains in the flask. The solution is slowly
cooled off. The precipitated yellowish crystals are
filtered off and recrystallized in a little hot glacial
acetic acid. The white powder thus obtained is dried at
90 C, 10 mbar for two days on an oil pump vacuum.
Yield: 31.7 g (92%)
Melting point: 190C
H-NMR([D6]-DMSO, ~, ppm):
10.47 (s, br, 1/3H, NH); 8~93 ~s, br, lH, OH); 8.19 (s,
br, lH, OH); 6.42-6.54 (m, 3H, Ar-H);
4.02 ~m, br, undissolved, 2H, CH2CH2O); 3.40 (m, br,
undissolved, 2H, NCH2CH2);
2.17 (m, br, undissolved, 2H, CH2CH2CH2)
13C NMR (tD6]-DMSO, ~, ppm):
147.5, 146.2, 134.7, 118.5, 109.6, 105.2 (Ar); 66.2
(CH2CH2O); 49.9 (NCH2CH2); 23.4 (CH2CH2CH2)
~4~X!~3
- 21 -
Example 2:
Benzyl ~ aarbeth~yethyl)amine, ~
172 g (1.60 mol) of benzylamine is introduced in 500 ml
of ethanol and mixed under ice cooling with 384 g ~3.84) mol
of acrylate. The reaction mixture is stirred for 5 days at
room temperature. Solvents and excess feedstocks are drawn
off in a rotary evaporator. The remaining solution is
fractionated in a vacuum.
Fraction 1: less than 140 degrees/0.05 mbar
Fraction 20 140-145 degrees/0.05 mbar
Fraction 3: 145-148 degrees/0.05 mbar
Fxaction 4: 145-150 degrees/0.05 mbar
Yield: 380 g (77%~ 8 from fraction 4
1~-NMR (CDCl3, ~, ppm):
7.27 (m, 5H, Ar-H); 4.10 (q, kH, C(O)OCH2CH3); 3.59 (s, -
2H, C6HsCH2N); 2.80 (t, 4H, NCH2CH2); 2.46 (t, 4H,
C~2CH2C(O)); 1-23 (t, 6H, OC~zCH
C-NMR (~D30D, ~, ppm):
173.7 (c~2c(O)O); 140.2, 129.7, 129.0, 127.9 (ar); 61.1
(OC~2CH3); 59-1 (C6HsC~ZN); 51.1 (NCH2CH2); 33.5
(CH2CH2C(O)); 14-5 (OCH2CH3)
Benzyl-bis(3-hydroxypropyl)ami~e, 9
1~ g (0~5 mol) of lithium aluminum hydride is
introduced in 900 ml of ether and 92 g (0.3 mol) of ester 8
is instilled slowly under ice cooling. The solution is
stirred for 12 hours at room temperature and then carefully
hydrolyzed with water. The ether and the aqueous phase are
decanted from precipitated LiAl(O~) 4 . The solid is washed
several times with ether. The combined organic phases
(ether and ethanol) are separated from water in a separating
unnel, dried on MgSO4 and filtered. After removal of the
,
8 ~ 3
-- 22 --
solvent in a vacuum, the product: remains as colorless
liquid.
Yield: 60.5 g (90%)
l~-NMR (CdCl3, ~, ppm~:
7.30 (m, 5H, Ar-H); 4.11 (s, br, 2H, C~2OH); 3.67 (t,
4H, CH2CH2OH); 3.56 (s, 2H, C6HsCH2N); 2.61 (t, 4H,
NCH2CHz); 1-75 (q, 4H, CH2CH2CH2
C-NMR (CD30D, ~, ppm):
137.8, 130.1, 129.2, 128.0 (Ar); 61.9 (CH2CH2O); 59.5
(C6H5CH2N); 52.3 (NCH2CH2); 30-2 (CH2CH2CH2)
Benzyl-bi~3-chloropropyl)amine, 10
143 g (0.64 mol) of 9 is introduced in 600 ml of
chloroform. 182 g (1.53 mol) of thionyl chloride, dissolved
in 100 ml of chloroform, is instilled in it at room
15 temperature. The addition has to take place so that the
solvent boils moderately. After completion of the addition,
it is refluxed for 3 hours. The cooled solution is
carefully hydrolyzed with water and washed t~,rice with 300 ml
of hot water. Now, the organic phase is strongly
20 concentrated by evaporation and further shaken out twice
with 250 ml of hot water each. After combining the aqueous
phases, the latter are made strongly alkallne with sodium
hydroxide solution (40%) and extracted twice with 400 ml of
ether each. The combined ether extracts are dried on sodium
2S sulfate. Then the solvent is drawn off on a rotary
evaporator. The crude product is fractionated.
Yield: 140 g (84%)
Boiling point: 114-125 C
1H-NMR (CDCl3, ~, ppm):
7.30 (m, 5H, Ar-H); 3.58 (t, 4H, CHzCHzCl); 3.55 (s,
2H, C6HsCH2N); 2.S~ (t, 4H, NCHzC~2); 1.92 (q, 4H,
CH2CH2CH2 )
:, . .
: . :~
.:
~ ,.. . .
" ~0'~8~9
- 23 -
C-NMR (CD30D, ~, ppm):
140.3, 129.9, 129.2, 128.0 (Ar); 59~8 (C6HSCH2N); 51.9
(NCH2CH2~; 43.8 (CH2CH2Cl); 31.3 (C~I2C~I2CH2)
senzyl-bi~3-~2,2-dimethyl-1,3-benzodioxol-4-
yloxy]propyl~amine, 11
13.3 g (80 mmol) of ketal 5 is dried for a half hour in
a high vacuum at 50 degrees in a stung-out 250 ml two-necked
Schlenk flask. Then 5 is dissolved in 100 ml of absolute
(99%) ethanol. The solution is evacuated several times
until boiling and aerated with argon~ Potassium (3.1 g, 80
mmol), which is cut until clear, is added and oxygen-free
chloride 10 (9.7 g, 37.3 mmol) is added to the now oxygen-
sensitive solution. The batch is refluxed for 3 days. 3 ml
of glacial acetic acid is added for working up and the
precipitated potassium chloride is filtered off still hot.
The filtrate is drawn dry and dissolved in a mixture of
about 30 ml of pentane/ether 1:1. The brown solution is
eluted on a short column (about 50 g of silica gel) with
ether/pentane 1:1. Here, attention must be paid that dark-
colored products are not coeluted. Aft~r the removal of the
mobile solvent, a yellow oil remains, which is
recrystallized from ethanol (16 g in 200 ml of ethanol). In
doing so, the product accumulates at room temperature as
colorless crystals.
Yield: 16 g (80%)
Melting point: 46-47C ;;
H-NMR ( [ D6] -acetone, ~, ppm):
7. 30 (dd, 2H, benzyl aromatic substance); 7 . 20 (m, 3H,
benzyl aromatic substance); 6.67 (dd, 2H, catechol);
6. 41 (dd, 4H, catechol); 4 . 07 (t, 4H, CHzCH20);
3 . 58 (s, 2H, C6HsCH2N); 2 . 61 (t, 4H, NCH2CEI2);
1.91 (q, 4H, CH2CH2CH2); 1.60 (s, 12H, C(CH3)2)
- . .
.
-- 24 --
C-NMR (CD30D, S, ppm):
149.3, 144.1 (catechol); 140.7 (benzyl aromatic
substance); 136.1 (catechol~; 129.4, 128.8, 127. 4
(benzyl aromatic su~stance); 121.9 (catechol); 118.4
(C(CH3)2); 109.4, 102.6 (catechol); 67.8 (CH2CH2O); 59.3
(C6H5CH2N); 50 7 (NCH2C~I2); 27.9 (CHzC~2CH2); 25.8
(C (CH3) 2)
Benzyl-bis t ~3- ~2,3-
dihydroxyphenoxy)propyl]aminohydrochloride, 12
14 g (27 mmol) of ligand precursor 11 is dissolved in
100 ml of glacial acetic acid and heated to boiling. 100 ml
of an acid mixture (50% glacial acetic acid, 20% water, 30%
fuming hydrochloric acid) is added to it within two hours,
and the liquid loss resulting from distilled-off solvent is
compensated for by acetone that is being liberated. A~ter
completion of the addition, so much solvent is distilled off
that about 50 ml remains in the flask. The hot solution is
slowly cooled off. But the product cannot be precipitated
in this way. If all solvent is removed, the ligand
accumulates as voluminous residue. This crude product is
liberated from the final acetic acid residues by washing
with ether and is dried in a high vacuum.
Yield: 12.4 g (96%)
H-NMR( [D6]-DMSO, ~, ppm):
10.85 (s, br, lH, NH); 8.98 (s, 2H, OH); 8.17 (s, 2H,
OH); 7.65 (d, 2H, benzyl aromatic compound); 7.43 (m,
3H, benzyl aromatic compound); 6.53 (t, 2H, catechol);
6.43-6.38 (m, 4H, catechol); 4.39 (s, br, 2H, C6HsCH2N);
~ .97 (t, br, CH2CH2O); 3.27 (t, br, NCH2CH2); 1 92 (q,
br, 4M, CH2CH2CH2)
. ,. . . . - . : ~.;: .
'',
' ~ .' ' , : ' ,:
': ' . , '
~ ~ i ' , ' ' ' , ' ;; ' ~
2 Q ~ 9
- 25 -
C-NMR (CD30D, ~, ppm):
148.3, 147.0, 135.7 (catechol~; 132.2, 131.1, 130.4,
130.4 (benzyl aromatic compound): 120.3, 110.6, 106.6
(catechol); 67. 9 (CH2CH20); 58. 5 (C6HsCH2N); 52.5
(NCH2CH2); 24~ (CH2CH2CH2)
bis~3-~2,3-Dihydro~yph~noxy)propyl)ami~ohydrochloride, 13
10 g (21 mmol) of 17 ~ is dissolved in 300 ml of
absolute methanol and mixed with 2 g of Pd (OH) 2/C (20~). In
a hydrogenation unit, the mixture was shaken with a hydrogen
pressure of 3 bars for six hours at room temperature. The
catalyst is filtered off and the solvent is drawn off. The
oily residue is dried ~or 24 hours at 50C in a high vacuum.
Yield: 7 g (70%)
H-NMR ([Ds~-pyridine, ~, ppm): ~
8.96 (s, 6~, OH and NH2); 6.90-6.30 (m, 6H, catechol); ::
3.97 (t, ~H~ CH2C~20); 3.17 (t, 4H~ NCH2CHz); 2-2~ (q~ ~`
4 H r CH2cH2cH2 )
C-NMR (~Ds]-pyridine, ~, ppm):
148.8~ 148.2~ 136.5, 119.7, 111.0, 106.0 (catechol);
67.6 (CH2CHzO); 46. 5 (NCH2CH2); 26. 8 (CH2C~2CH2)
, ~ .
Example 3:
bis~3-(2,2-Dimethyl-1,3-be~zodio~ol-4-~loxy)propyllamine, 14
17.9 g (34.5 mmol) of 11 is dissolved in a
hydrogenation flasX in 300 ml of absolute methanol, mixed
with 2.0 g (2.8 mmol) of catalyst (Pd(OH)2/C) and shaken for
four hours in a hydrogen hydrogenation apparatus at 3 bars ~:
of H2 pressure and 25 C. Then, the catalyst is filtered off
and the solvent as well as resulting toluene are removed in
a water j`et vacuum. The resulting oil is dried at 60C/0.05
mbar for six hours.
,, , , . : .
,
'. ' , ~ ' ; .
2~ 9
- 26 -
Yield: 12.9 g (87%)
H-N~DR(CDCl3. ~, ppm):
6.69 (dd, 2H, cat2chol); 6.46 (d, 2H, catechol); 6~43
(d, GH, catechol); 4.14 (t, 4H, CE~zCH20); 2. 83 (t, 4H,
NCH2CH2); 1.99 (q, 4H, CH2CH2CH2); 1.68 (s, 12H, C(CH3)2)
3_NMR([D6] -benzene, ~, ppm):
149.3, 143.9, 136.2, 121.6 (catechol); 117.9 (C(CH3)2);
109.1, 102. 6 (catechol); 6~.9 (CH2CH20); 46.6 (NCH2CH2);
29. 8 (CH2CH2CH2); 2 5. 7 (C( CH3)z
bis~3-(2,3-Dihydroxyphenoxy)propyl)aminohydrochloride, 13
8 g (18.6 mmol) of 14 is dissolved in 80 ml of glacial
acetic acid and mixed in boiling heat within two hours with
80 ml of an acid mixture (50% glacial acetic acid, 30%
water, 20% fuming hydrochloric acid). In this case, the
solvent distills off with acetone that is liberated as
azeotrope. After completion of the addition, it is
distilled for another half hour. Then, the residual solvent
is drawn off on a rotary evaporator. The remaining residue
is dried at 60C/O.OS mbar for six hours in a high vacuum.
The powdery product is washed with ether. Ether residues
are then removed on the oil pump.
Yield: 6.25 g (87~)
The analytical data is identical with that of 13
from 12.
Example 4:
2,3-Dinitrophenol, 15
15.0 g (108 mmol) of 3-nitrophenol is dissolved in 150
ml of ethanol. 30 g (124.5 mmol) of Cu(N03)2-~3 H20) is
added to it. The reaction mixture is then refluxed to
boiling for 20 hours. The solvent is drawn off at a rotary
evaporator. The solid residue is dissolved in 2 M HCl and
- 27 -
extracted four times with 50 ml of ether each. The combined
ether extracts are dried on Na2SO4 and liberated from
solvent. The orange solid (21 g) is chromatographed on a
short column on about 60 g of silica gel with petroleum
ether/ethyl~ First, 3,6-dinitrophenol is eluted, followed
~y 3-nitrophenol and 3,4-dinitrophenol. Finally, the
desired 2,3-dinitrophenol is obtained. The desired product
can be recrystallized from benzene/petroleum ether (7:93,
v:v) .
Yield: 2.9 g (14.6%)
Melting point: 146C
H-NMR (CDCl3, ~, ppm):
7.18-7.79 (m, 3H, ArH); 9.90 (s, br, lH, Ar-OH)
13C-NMR (CD30D~ ~ ppm):
151.87 (C-OH); 142.17 (C-NO7(m); 134.22 (C-NO2(o));
132.27 (C-H0; 124.54 (C-H); 116.12 (C-H)
Be~zyl-bis L (3-(2,3-dinitrophenoxy)propyl]amine, 16
0.92 g (5 mmol) of 2,3-dinitrophenol is dissolved in 10
ml of ethanol. 0.28 g (5 mmol) of KOH in 25 ml of ethanol
is added to it under argon. In doing so, the potassium salt
precipitates as red solid. 0.65 g (2.5 mmol) of benzyl-
bis(3-chloropropyl)amine, 10 in 5 ml of ethanol is added to
it, and the solid is partially dissolved. With subsequent
heating to the boiling temperature of the ethanol, the solid
is completely dissolved. The red solution is now refluxed
for 24 hours. A precipitate of KCl forms. The suspension
is filtered hot. With cooling, 16 crystallizes out in the
form of colorless feathers~ The product is recrystallized
from ethanol.
Yield: 0.76 g (55-~)
Melting point: 98-104C
~ :
..
g~'3
- 2~ -
H-NMR(cDcl3~ ~, ppm)O
1.91 (q, 4H, CH2CH2CH2); 2.60 (t, 4H, NCH2); 3.58 (s,
2H, C6HsCH2); 4.15 (t, 4H, OCH2); 7.21 (s, 5H, benzyl-
H); 7.22-7.81 (m, 6H, catechol-H)
C-NMR (CDCl3, ~, ppm):
26.70 (CH2CH2CH2); 49.81 (NCHz); 58.77 (C6HsCH2); 68.57
(OCH2); 116.08, 119.56 (CH(phenyl)): 126.89, 128.20,
128.74 (CH(benzyl)): 131.04 (CH(phenyl)): 134.99
tC(NO2-o)): 139.06 (C(benzyl)): 140.59 (C(NO2~m)),
151.37 (C(O-phenyl))
Benzyl-bi~C3-(2,3-diaminophenoxy)propyl]amine, 17
1.06 g of tin (8.93 ~mol) is added to 5 ml of
concentrated hydrochloric acid. A solution of 0.5 g (o.9
mmol) of 16 in 5 ml of methanol is sprayed to it. The
reaction mixture is heated for 30 minutes to 50C, and its
color becomes brown. Then the reaction mixture is poured
into a solution of 2.5 g of NaOH in 50 ml of water. This
mixture is extracted five times with 15 ml of ether each.
The ether extracts are washed with water and dried on
Na2SO4. After the removal of the ether, a brownish oil
remains, which is not further purified.
Yield: 0.275 g (70%)
H-NMR (CDCl3, ~, ppm):
1-99 (q, 4H, CH2CH2CH2); 2.69 (t, 4H, NCH2); 3.32 (s,
8H, NH2); 3.65 (s, 2H, benz-CH2); 4.04 (t, 4H, OCH2);
6.28 6.80 (m, 6H, phenol-H); 7.31 (s, 5H, benz-H)
3C-NMR (CD3Cl, ~, ppm)
27.14 (CH2CH2CHz); 50.24 (NCH2); 58.69 (C6HsCH2); 62.23
(OCH2); 103.38, 109.50, 118.99 (CH(phenol); 123.67
(C(NH2-o)); 126.69, 128.06, 128.59 (CH(benzyl)); 135.32
(C(NH2~m)); 139.59 (C(ben2yl)); 147.71 (C(O-phanol)
.
,.: :::,.'~ ' .:.,.:
~. . 1.
~ O ~ 9
- 29 -
bi~[3-(2,3-Diaminophenoxy)propyl~amine, 18
846 mg (1.66 mmol) of ligand 17 is dissolved in 50 ml
of methanol. 0.09 g of Pd(OH)2 (on carbon, 10%) and 5 ml of
hydrazine hydrate (80% in water) are added to it. The
reaction mixture is heated to boiling for 10 hours. The
resulting suspension is filtered and evaporated to dryness.
The product is soluble in methanol and, after adding diethyl
ether, precipitates as light greenish oil. This oil is
liberated from solvent residues on the oil pump, and a gray
powder is obtained.
Yield: 400 mg (69%)
H-NMR (CD30D, ~, ppm):
1.99 (q, 4H, CH2CH2CH2); 2.89 (t, 4H, NCH2); 4.05 (t,
4H, OCHz); 6.24-6.68 (m, 6H, Ar-H)
C--NMR (CD30D, ~ , ppm):
29.77 (CH2CH2CH2); 47.06 (NCH2); 67.70 (OCHz); 104.39,
111.13, 120~08 (CH(phenol)); 124.47 (C(NH2-o) ); 136.70
tC(NHz~m))~ 148.87 (C(O-phenol))
Example 5:
tris[3-(2,3-Dinitrophenoxy~propyl]amine, 19
1 g of 2,3-dinitrophenol 15 (5.43 mmol) is stirred
under reflux with 0.446 g of tris(3-chloropropyl)amine, 4
(1.81 mmol) and 0.305 g of KOH (5.43 mmol) in 50 ml of
ethanol for 10 hours. Then, the reaction mixture is allowed
to cool off and the precipitated solid is filtered off. The
solid is taken up in acetone. In this case, organic
components are dissolved while the formed KCl remains.
Insoluble components are separated by filtration. The
acetone solution is concentrated by evaporation to 20 ml and
cooled to 4C. After 24 hours, 19 can be isolated in the
form of colorless needles.
.
,
'' '.
~' . `, . . .
~4~9
- 30 -
Yield: 638 mg ~51%)
Melting point: 145-147C
H-NMR(~D6]-acetone, ~, ppm):
1.93 (q, 6H, C~2CH2CH2); 2.59 (t, 6H, NCHz); 4.31 (t,
6H, OCH2); 7.63-7.88 (m, 9H, Ar-H)
trist3 (2,3-Diaminophenoxy)propyl]amine, 20
638 mg of 19 t0.925 mmol) is added to a mixture of 5 ml
of HCl (conc.), 5 ml of methanol and 1.64 g (13.8 mmol) of
tin. The reaction mixture is stirred under reflux for 2
hours. The reaction is completed when the tin has
completely dissolved. After the cooling, the green reaction
solution is made strongly alkaline (pH 13) with an excess of
KOH and shaken out twice with 15 ml of acetone each. The
combined acetone extracts are mixed with 5 ml of water and
shaken out twice with 30 ml of ether each. The ether
extract is dried on sodium sulfate. The solvent is removed
in a pump vacuum. A yellowish oil is obtained which
solidifies with drying in a pump vacuum.
Yield: 638 mg ~51%)
1H-NMR (CDCl3, ~, ppm):
1.92 (q, 6H, CH2CH2CHz); 2.65 (t, 6H, NC~z); 3.36 (s,
12H, NH2); 3.97 (t, 6H, OCH2); 6.23-6.74 (m, 9H, Ar-H)
Example 6:
Productio~ of the technetium complexe~ o~ 12 and 13
Methanol solutions of ligands 12 and 13 (30 mmol/l) are
produced. 8 microliters of such a solution is mixed with
10-40 microliters of a saline solution made from a Tc/ Mo
reactor. The resulting solution is immediately examined by
thin-layer chromatography. This examination (mobile solvent
THF) shows a complete incorporation of 9mTc in the ligands.
The Rf values are 0.3 for Tc04, 0.65 for Tc 12 and 0.60
., . . :,, .: . . ., -
: :, ' ',, ; , ~ , :
. ,:.:. :.. : .
for ~c 13. Residues of pertechnetate can be easily
discovered in these various Rf ~alues, but are not ~ound.
Example 7:
Productio~ of the tech~etium complex of 17
A methanol solution of 17 with the concentration of 50
mmol/l is produced. One microliter of this solution is
mixed with 50 microliters of an eluate solution of a
Tc/ Mo generator. lO microliters of a 0.1 N NaOH and 10
microliters of phosphate buffer (ionic strength of 0.1, pH
lo 7) are added to it. The reaction mixture is allowed to
stand for 5 minutes at room temperature and then is
characterized by thin-layer chromatography (mobile solvent
THF). This analysis shows the complete incorporation of
~ c in the ligands. The Rf values are 0.15 for ~ Tc04 and
0.24 for Tc 17.
Example 8:
siotin ~HS
1.72 g ~8.19 mmol) of DCC is added to a solution of 2.0
g (8.19 mmol) of D(+)biotin and 1.23 g (10.66 mmol) of N-
hydroxysuccinimide in 25 ml of DMF. The resultingsuspension is stirred for 24 hours at room temperature. The
solid is filtered off and the solution is cooled for 4 hours
to -16 C. It is filtered off from the precipitated solid
and the solvent of the filtrate is drawn off in a vacuum.
The colorless residue is wash~d several times with ether and
finally dried.
Yield: 2.47 g (89~)
-" 2~8.~9
- 32 -
H-NMR([D6]-DMSO, ~, ppm 400 MHz):
6.4~, 6.37 (s, br, 2H, b and g); 4.29 (m, br, 1~, c);
4.13 (m, br, lH, f); 3.09 (m, br, lH, e); 2.82 (dd,
J=12 Hz, 6 Hz, lh, d); 2.80 (s, 4H, n); 2.66 (t, J=7
Hz, 2H, k); 2.57 (d, J=13 Hz, lH, d); 1.63-1.50 (m, 6H,
h~
C-NMR ([D6]-DMS0, ~, ppm):
170.1 (2C, m); 168.8 (l); 162.6 (a); 60.9 (f), 59.1
(c); 55.1 (e); 39.8 (d); 29.9 (k); 27.7, 27.5 (i and
j); 25.4 (2C, n); 24.2 (h)
b HN NH~
H~H o
S ~ ~o ~ ,
Diagram for the allocation of the NMR signals for
blotin NHS.
Coupling of biotin NHS to bis~3-(2,2-dimethyl-1,3-
benzodioxol-4-yloxy)propyl]amine
860 mg (2.0 mmol) of ligand precursor 14 is stirred in
30 ml of degassed DMF with 670 mg (2 mmol) of bioti~ NHS and
860 mg (8 mmol) of triethylamine for 80 hours at room
temperature. Then, the reaction mixture is mixed with 80 ml
of degassed watex and cooled for 1 hour to 4 C. I'he
~0 precipitated sol:id is isolated by filtration and taken up in
50 ml of acetone. After the removal of the solvent, the
.. . ~ . -
, . ,, . :
.. i~ . .:
-- 2 0 ~ 9
- 34 ~
Cleavage o tha protuoti~e groups of conjugat~ biotin-14
1 g of the biotin-14 conjugate is dissolved in 20 ml o~
methanol. 4 ml of fuming HCl is added and it is stirred at
room temperature for 3 days. The solvents are removed in a
S vacuum and the solid residue is dissolved in methanol. The
purification takes place chromatographically on SiO2 with
methanol/THF (1:1) as mobile solvent. ~he cleavage of the
protective groups in the absence of the signals for the
acetal unit is detected in the H-NMR spectrum. This
observation is also confirmed by the C-NMR spectrum.
Yield: 53%
Because of the problems in the cleavage of the
protective group, an alternative method of synthesis was
also examined.
:" .
~xample 8a:
Tetratdimethylltert-butyl)]~ilylether of 13
3.85 g (10 mmol) of 13 and 6.55 g (96 mmol) of
imidazole in 35 ml of DMF are introduced in a 100 ml Schlenk
flask and placed under protective gas. Then, solid TBDMSCI
(7.5 g, 50 mmol) is added, and heating takes place. It is
stirred for 20 hours at room temperature. Then, the
solution is mixed with 200 ml of ether. This mixture is
washed three times with water fremoval of the DMF) and dried
on sodium sulfate. After the filtering off of the drying
agent, the ether is drawn off on a rotary evaporator. A
~right green liquid remains. From this, the product can be
isolated on SiO2 by column chromatography (pentane:ether
1:2~.
Yield: 7 g (86%)
.
,, , . ~ "
.,
, ~ .
2 ~ 9
H--NMR (CDCl3, ~ , ppm):
6.71 (t, lH, catechol); 6 . 50 (d, lH, catechol); 6 . 48
(d, lX, catechol); 3 . 99 (t, 2H, CH2C~2O); 2 . 81 (t, 2H,
N(:H2CH2~; 2.01 (q, 2H, CH2CH2CH2); 1.01 and 0.97 (s, 18H,
((CH3)3C-Sio); 0.21 and 0.14 (s, 12H, (CH3)2 sio
C-NMR (CDC13, ~ , ppm):
151.9, 148.0, 136.3, 120.2, 113.7, 105.9 (catechol);
66. 8 (CH2CH20); 46 . 9 (NCHzCH2); 30. 0 (CH2CH2CH2); 26 . 1
and 26.0 (6C, (CH3)3CSio); 18 . 6 (2C, (CH3)3CSio); -3 . 8
and -4.0 (4C(CH3)2Sio)
MS (70 eV):
806 (2) M ; 749 (23) M -C(CH3)3;
452 ~100) M-C6H3(OH) (osi(cH3)2c(cH3)3)2
Coupling of tetra[dimethyl(tert-butyl]silylether of 13 to
biotin
1.64 g (4.84 mmol) of biotin NHS and 3.90 g (4 . 84 mmol)
of tetra[dimethyl(tert-butyl)]silylether of 13 are stirred
with 2.02 g (20 mmol) of triethylamine in 70 ml of DMF for 3
days under argon. The solvent is drawn off in an oil pump
vacuum and the remaining oil is washed several times with
water. The thus obtained crude product is
chromatographically (sio2~ methanol) purified. The identity
- of the product could be shown by nuclear resonance
spectroscopy.
Yield: 43%
Cleavage of the protective group~ of conjugate bi~tin- -
tetratdimethyl~tert-butyl)]silylether o~ 13
1.03 g (1 mmol) of (T8DMS)-13-biotin conjugate is
stirred for 24 hours in a mixture of 2 ml of HCl (conc.~ and
20 ml of THF. Then, it is mixed with 20 ml of water and the
THF is removed in a vacuum. The conjugate precipitates in
, :; i : : ,, :;- , ,
: ~ . . ! ; ' ;; ' , .; , ;
2 ~ 9
- 3G -
doing so. The hydrochloric water solution is decanted and
the residue is washed several times with water. The residue
can be recrystalli~ed from methanol.
Yield: 45%
lH~ CDCl3, ~, ppm)
6.70-6.20 (m, 6H, catechol); 4.25 (m, lH, NH-CH-CH2);
4.15 ~m, lH, CH2-CH-CH); 3.92 (m, undissolved, 4H, O-
CH2-CH2); 3.48 (m, undissolved, 4H, N-CH2-CH2); 3.10 (m,
lH, CH-CH(R)-S3; 2.75 (d, lH, CH-CH(H)-S); 2.61 (sbr,
lH, CH-CH(H)~S); 2.28 (tbr, 2H, NC(O)-CH2-CH2); l.g5
(m, undissolved, 4H, CH2-CH2-CH2-O); l.S5-1.30 (m, 6H,
biotin alkyl chain)
Example 9~
tert-Butyl ethyl acetate of 14
2.7 g (6.3 mmol) of 14 is dissolved in 40 ml of THF/X20
(9:1) and mixed with 0.67 g (1 eq.) of Na2CO3. 2.46 g (2
eq.) of bromoacetic acid-tert~butyl ester is added and
stirred at room temperature for 24 hours. Then, 80 ml of
CH2C12 is added and dried on ~qgSO4. After the filtering off
of the drying agent, the solution is rigorously concentrated
by evaporation. The residue is taken up several times in
hexane and decanted. The crude product is purified by
column chromatography (70 g of SiO2,~pentane/ether 3:1).
Yield: 3.3 g (95%)
1H-NMR (CDCl3, ~, ppm):
6.90-6.30 (m, 6H, catechol); 4.11 (t, 4H, OCH2CH2);
3.26 (s, 2H, OC(O3CH2N); 2.81 (t, 4H,~ NCH2CH2); 1.93 (q,
4H, CH2CH2CH2); 1.69 ~s, 12H, C(CH3)2); 1.45 (s, 9H,
(CH3)3COC(O))
.
~ o ~
Gly--13
3.10 g (5.7 mmol) of the product of the preceding
reaction is treated in 50 ml of methanol in boiling heak
with 50 ml of acid mixture analogously to the production of
13 from 12. After completion of the reaction, the solvent
is drawn off on a rotary evaporator. In this case, the
product precipitates so that the aqueous phase can be
decanted. The product is dissolved in acetone and liberated
from the solvent in a vacuu~. In this case, it accumulates
as white powder.
Yield: l.gO g (75~)
H-NMR (CDCl3, ~, ppm):
6.90-6.30 (m, 6H, catechol); 4.17 (t, 4H, O-CH2~CHz);
3.80 (t, 4H, NCH2CH2); 3.60 (s, 2H, HOOCCH2N); 2.5 (q,
4H, C~2CH2C~2)
Pr~duction o~ the NHS e~ter of gly-13
0.464 g (2.25 mmol) of DCC is added to a solution of 1
g (2.5 mmol) of gly-13 and 0.388 g (2.4 mmol) of N-
hydroxysuccinimide in 8 ml of DMF. After 24 hours of
stirring at room temperature, the precipitated solid is
filtered off and filtrate is cooled for 4 hours to 4C. The
additional precipitated solid is again filtered off and the
filtrate is liberated from the solvent in a vacuum. The
residue is rewashed several times with ether and dried in a
high vacuum.
Yield: 0.95 g (78~)
H-NMR tCDCl3, ~, ppm):
6.80-6.20 (m, 6H, catechol); 4013 (t, 4H, OCH2CH2);
3.80 (s, 2H, OC(O)CH2N); 3.58 (t, 4H, NCHzCH2); 2.80 (s,
4H, C(O)CH2CH2C(0)); 2.07 (q, 4H, CH2CH2CH2)
., . ...~ . . ,
: - , ., ,. ~ ,
,.,. . ~
` ~0~8~9
- 3~ -
Exam~ o:
4-Nitrophe~yl-bi 9 ~ ( 3-(2,2-dimethyl-1,3-be~zodioxol-~-
yloxy)propyl]amlne, 21
2.15 y (5 mmol) of 14 and 0.5 g (5 mmol) of
triethylamine are introduced in 10 ml of ethanol and mixed
with 1.41 g (10 mmol~ of 4-fluoronitrobenzene. The mixture
is stirred for 3 days. Then, the solvent is removed and the
crude product is purified chromatographically.
Yield: 1.50 g (55%)
1H-NMR (CDCl3, 5, ppm):
8.06 (d, 2H, nitrophenyl); 6.72 ~t, 2H, catechol); 6.71
(d, 2H, nitrophenyl); 6~47 (d, 2H, catechol); 6.46 (d,
2H, catechol); 4.13 (t, 4H, OCHzCH2); 3.67 (t, 4H,
NCH2CH2); 2.11 (q, 4H, CH2CH2CH2); 1.~1 (s, 12H, C(CH3)2)
4-Ami~ophenyl-bist~3-~2,2-dimethyl-103-benzodioxol-4-
yloxy)propyl]amine, 22
1.50 g (2.75 mmol) of 4-nitrophenyl-14 is dissolved in
30 ml of methanol and stirred with 150 mg of Pd/C (10~)
under a hydrogen atmosphere for 4 hours at room temperature.
The catalyst is filtered off and the solvent is removed. A
greenish oil is obtained.
Yield: 1.27 g (89%)
4-Aminophenyl bis[3-~2,3-
dihydroxyphe~oxy)propyl]aminohydrochlorid~, ~3
1.04 g (2 mmol) of 22, DIPACE is reacted under the
conditions for the productiorl of 13 from 12. A white powder
is obtained:
Yield: 0.965 g (94%)
. .
i ~. .
.~
2 0 ~ 9
- 39 -
4-Isothioc~anato-bist3-(2,3-
dihydroxyphe~o~y~propyl]aminohydroahloride, 24
0.51 g (1 mmol) of 23 of the preceding reaction ls
reacted with 0.267 g (1.5 mmol) of N,N'-
thiocarbonyldiimidazole. After completion of the reaction,imidazole is washed out with water. The product is obtained
as yellow oil.
Yield~ 360 mg (70%)
Example 11:
N,N-bist3-~2,2-Dimethyl-1,3-benzodioxol-4-
yloxy)propyl~-N-~2,3-epoxypropyl)amine, 25
1.15 g (48 mmol) of sodium hydride is suspended in 40
ml of DMF at room temperature under nitrogen. A solution of
18.0 g of amine 14 (42 mmol) in 20 ml of DMF is slowly in-
stilled in it and, after completion of the addition, it is
stirred for 1 hour. Then, a solution of 5.48 g of epibromo-
hydrin ~40 mm~l) in 20 ml of DMF is instilled and stirred
for another`24 hours. The mixture is diluted with 70 ml of
ice water, extracted several times with ethyl acetate and
the combined organic extracts are dried on potassium carbo-
nate. After removal of the solvent, a yellow oil remains.
Yield: 71%
H-NMR ~CDC13)
6.90-6.30 (m, 6H, catechol); 4.12 (t, 4H, OCH2CH2);
3.24 ~m, lH, epoxide); 2.80-2.60 (m, 8H, NC~z,
epoxide); 1.93 (q, 4H, CH2CH2CH2); 1.69 (s, 12H, C(CH3)
N,N-bis[3-~2,2-dimethyl-1,3-benzodio~ol-4-yloxy)propyl]~N
r2-hydroxy-3-(2-ni~roimidazolyl~propyl]amine, 26
A mixture of 2S0 mg of 2-nitroimidazole (2.2 mmol), 237
mg of 1,8-bis-(dimethylamino)naphthalene (1.1 mmol), 2.12 g
of 25 (4.4 mmol~ and 5 ml of DMSO is heated with exclusion
- . :.,
: . ~ . ,:
: , ..
2 Q ~
- 40 -
of moisture and stirring for 6 hours to 80C, the solvent is
drawn off in a vacuum and the residue is chromatographed
(silica gel, 230-400 mesh, 3 x 15 cm column, 20% CH3CN/CEICl3
to 8 0% CH3CH/CHCl3) . The individual fractions are examLned
by thin-layer chromatography and thin~layer chromatography
analogous fractions are combined. The solvent is drawn off
and the residue is dried in a vacuum.
Yield: 27%
H--N~R (CDCl3)
8.40 (s, lH, imidazole); 8.34 (s, lH, imidazole); 6.90-
6.30 (m, 6H, catechol); 4.20-4.00 (m, 5H, OCH2CHz,
CHOH); 2. 90-2. 60 (m, 8H, NCH2CH2); 1. 93 (q, 4H,
CH2CH2CHz); 1.69 (s, 12H, C(CH3)2)
Example 12:
3-~N,N-biqt3-(2,2-Dimethyl-1,3-b~nzodio~ol-4-
yloxy)propyl])ami~opropanoic acid ~thyl eYter, 27
0.898 g of potassium-tert-butylate (8.0 mmol) is
dissolved in 150 ml of anhydrous tert-butanol and a solution
of 27 . 24 g of 14 (llo mmol) in 30 ml of tert-butanol and 400
ml of ether are added. With stirring, 33.04 g of freshly
distilled ethyl acrylate (330 mmol) is slowly distilled and
the reaction mixture is left for 3 days at room temperature.
After removal of the solvent, the remaining oil is taken up
in ether. The ether phase is washed neutral with water and
dried on magnesium sulfate. After the concentration by
evaporation, a pale yellow oil remains.
Yield: 48%
1H-NMF~ ( CDCl3 )
6.90-6.30 (m, 6EI, catechol); 4.25-4.10 (m, 6H, OCH2CH2,
CO2CH2CH3); 2.80-~. 50 (m, 8~, NCH2CH2, CH2N, COCH2); 1. 93
(q, 4H, CEl2CH2CHz); 1.69 (s, 12H, C(CH3)2); 1-25 (t, 3H,
OCH2CH2 )
~ . .. . . .
. . . .
:. .
:: :
.
- 41 - 2~8~
3-(N,N-biq[3-~2,2-Dimethyl-1,3-be~zodio~ol-4-
yloxy}propyl])ami~opropanoic aci~ hydra~ide, 28
20 g of anhydrous hydrazine (624 mmol) is added to a
solution of 10.0 g of ester 27 (18.9 mmol) in 200 ml of
anhydrous pyridine and refluxed for 3 days. It is
concentrated by evaporation to 5() ml and then mixed with 200
ml of water, extracted several times with ethyl acetate, the
combined organic phases are washed with water~ dried on
sodium sulfate and concentrated by evaporation. A white
residue remains.
Yield: 69%
H-NMR (CDCl3)
6.90-6.30 (m, 6H, catechol); 4.08 (t, 4H, OCH2C~z);
2.80-2.50 (m, 8H, NCH2CH2, CH2N, COC~2~; 1.93 (q, 4H,
CH2CH2CH2); 1.69 (s, 12H, C(CH3)z)
Example 13:
3-~,N-hisr3-~2,2 Dimethyl-1,3-be~zodioxol ~-
yloxy)propyl~aminopropanoic acid, 29
14.5 g of ester 27 (27.4 mmol) is refluxed in a
solution of 5.00 g of potassium hydroxide (90.0 mmol) in 75
ml of 95% ethanol for 2 hours. The ethanol is drawn off in
a vacuum and the remaining residue is taXen up in 100 ml of
water. After shaking out with 50 ml of ether, the aqueous
phase is carefully acidified with dilute hydrochloric acid.
The free acid is extracted by shaking out several tim~s with
50 ml of ether each. The combined ether phases are washed
with saturated common salt solution and dried on magnesium
sulfate. After removal of the solvent, a colorless oil
remains.
Yield: 85
- : . '-
.:, ' ,.' ~ . ' ' .' ;:,. .
~ ' :
, ,: .
2 0 ~
- 42 -
H-NMR (CDC13)
6.90-6.30 (m, 6H, catechol); 4.11 (t, 4H, OCH2CH2);
2.80-2.50 (m, 8H, NCH2CH2, CH2N, COC~2), 1.93 (q, 4H,
CH2CHzCH2); 1.69 (s, 12H, C(CH3)2)
3-{N,N-bis[3-~2,2-Dimethyl-1,3-benzodio~ol-4-
ylo~y)propyl]}aminosuccinimidopropionate, 30
The solution of 12.38 g of dicyclohexylcarbodiimide (60
mmol) in 50 ml of tetrahydrofuran is instilled in a
solution, cooled to -5C, of 2S.1 g of carboxylie aeid 29
(50 mmol) and 5.75 g of N-hydroxysuccinimide (50 mmol) in
100 ml of anhydrous tetrahydrofuran within 20 minutes and is
stirred ~or another 2 hours at this temperature and then for
another 15 hours at room temperature. After adding 200
mieroliters of acetic aeid, it is stirred for another hour,
then filtered and the residue is extracted twice with hot
tetrahydrofuran. The combined filtrates are evaporated to
dryness and the residue is recrystallized from ethyl
aeetate.
Yield: 65~ -
lH-NMR (CDCl3)
6.90-6.30 (m, 6H, catechol); 4.11 (t, 4H, OCHzCH2);
2.80-2.50 (m, 8H, NCH2CH2, CH2N, COCH2); 2.76 (s, 4H,
COCH2CH2C0); 1.93 (q, 4H, CH2CH2CHz); 1-69 (s, 12H,
C(CH3)2)
E~am~le 14:
3-{NfN-bi~[3-~2,2-dimethyl-1,3-benzodioxol-4
yloxy)propyl]}aminosuccinimidopropionate, 30
The solution of 10.37 g of 1-(3-dimethylaminopropyl) 3-
ethylearbodiimide (54 mmol) in 100 ml of acetonitrile is
instilled in a solution, eooled to 0 C, of 25.1 g of
earboxylie aeid 29 (50 mmol) and 8.30 g o.f 2,3,5r6-
.
: ~4~9
- 43 -
tetrafluorophenol (50 mmol) within 5 minutes and heated for
2 hours to 75C. After adding 200 microliters of acetic
acid, it is stirred for another hour, then filtered and the
residue is extracted twice with hot acetonitrile. The
combined filtrates are evaporated to dryness and the residue
is recrystallized from ethyl acetate.
Yield: 65%
H-NMR (CDCl3)
6.90-5.30 (m, 7H, catechol, tetrafluorophenol); 4.11
(t, 4H, OCH2CHz); 2.80-2.50 (m, 8H, NCH2CH2, CH2N,
COCH2); 1.93 (q, 4H, CH2CH2CH2); 1.69 (s, 12H, C(CH3)2
Exampl~ 15:
3-{~l,N-bi~3t3-(2,2-Dimethyl-1,3-}~enzodioxol-4-yloxy~propyl]}
aminopropa~ol, 32
15The solution of 20 g of ester 27 (38 mmol) in 50 ml of
anhydrous ether is instilled in a suspension of 2.88 g of
lithium aluminum hydrida (76 mmol) in 150 ml of anhydrous
ether within one hour so that the solution boils moderately.
Then, it is refluxed for another 5 hours, cooled ta room
temperature and excess hydride is carefully hydrolyzed with
water. It is filtared off from precipitated hydroxide and
the filtrate is washed several times with warm ether. After ~ -
removal of the solvent in a vacuum, the residue is boiled up
briefly in ethanol, filtered again and the solvent is drawn
off. A highly viscous liquid remains.
Yield: 61%
1H~ ( CDC13 )
6.gO-6.30 (m, 6H, catechol); 4.12 (t, 4H, OCH2CH2);
3.54 (t, 2H, CH20H); 2.80-2.60 (m, 6H, NCH2); 2.00-1.80
(m, 6H, C~2CH2CH2); 1-69 (s, 12H, C(CH3)2)
., :
:: . :. .
, . ~
' ' ,
,
2 ~ 9
Example ~6:
bis~3-~2,2-Dimethyl 1,3 benzodioxol-4-yloxy~propyl3-N-~3-
chloropropyl)amine, 33
A solution of 10.1 g of alcohol 32 (20.7 mmol) in 50 ml
of anhydrous carbon tetrachloride is mixed under a nitrogen
atmosphere with 7.86 g of triphenylphosphine (30 mmol). It
is refluxed for several hours. After cooling off, it is
diluted with half the volume of petroleum ether and stored
for some time at -20 C. The precipitate is suctioned off
and washed with petroleum ether, after drying on sodium
sulfate and removal of the solvent, a yellow oil remains.
Yield: 78%
H-NMR (CDCl3~
6.90-6.30 (m, 6H, catechol); 4.11 (t, 4H, OCH2CH2);
3.62 (t, 2H, CH~Cl); 2.80-2.60 (m, 6H, NCHz); 2.00-1.80
(m, 6H, CH2CH2CH2); 1.66 (s, 12H, C(CH3)2)
Example 17:
3~N,N-bis r3- (2,2-Dimethyl-1,3-benzodioxol-4-
yloxy1propyl]~aminopropanol, 34
4.88 g of alcohol 32 (10 ~mol), dissolved in 20 ml of
dichloromethane, is added all at once to a well-stirred
suspension of 3.23 g of PCC in 25 ml of anhydrous
dichloromethane and the mixture is stirred for 90 minutes at
room temperature. After adding 50 ml of anhydrous ether, it
is decanted and the residue is washed three times with 20 ml
each, the combined ether solutions are filtered on 20 g of
silica gel. After removal of the solvent, a yellow oil
remains.
Yield: ~0%
, . . .
'
,: . . " : :
- , . : . .::
2 0 ~
-- ~5 --
1~--N2IR ( CDCl3 )
9.74 (s, lH, aldehyde); 6.90-6.30 (m, 6H, catechol);
4 . 12 (t, 4H, OCH2CH2); ~ . 80-2 . 60 (m, 6H, NCH2); 2 . 42 (t,
2H, CH2CH0); 2.00-1.70 (m, 6H, CH2CH2CH2); 1.66 (s, 12H,
C (CH3) 2
Example ~8:
3 r ~N,N-bis~3-~2-2-Dimethyl-1,3-be~zodio~ol-4-
yloxy)propyl]~aminopropio~ic acid ~itrile, 35
0.90 g of potassium-tert~butylate (8.0 mmol) in 150 ml
of anhydrous tert-butanol is dissolved and a solution of
27.2 g of 14 (110 mmol) in 30 ml of tert-butanol and 400 ml
of ether is added. With stirring, 17.5 g of freshly
distilled acrylonitrile (330 mmol) is slowly instilled and
the reaction mixture is refluxed for 12 hours. After
removal of the solvent, the remaining oil is taken up in
ether. The ether phase is washed neutral with water and
dried on magnesium sulfate. After the concentration by
evaporation; a pale yellow oil remains. `~
Yield: 62%
H-NMR (CDC13)
6.90-6.30 (m, 6H, catechol); 4.11 (5, 4H, OCH2CH2);
2.81 (t, 4H, NCH2CH2); 2.50-2.30 (m, 4H, NCHzCH2CN);
1.97 (q, 4H, CH2CH2CH2); 1.69 (s, l~H, C(CH3)2)
Example 19
N,N-bis~3-(2,2-Dimethyl-1,3-benzodio~ol-4-
yloxy)propyl]propylenediaminff, 36
39 . 0 g of 100~ sulfuric acid (0.40 mol) is slowly
instilled in a suspension of 30. 6 g of lithium aluminum
hydride (0.81 mol) in 500 ml of anhydrous ether under ice
cooling. Then, it is stirred ~or one hour at room
20l~8~9
- 46 -
temperature, then the solution of 12.55 g of nitri~e 35
(0.26 mmol) in 50 ml of anhydrouc; ether is instilled so that
the solution boils moderately. Then, it is refluxed for
another 8 hours, cooled to room temperature and excess
hydride is carefully hydrolyzed with water. A solution of
40 g of NaOH in 360 ml of water is added, ~iltered off from
the precipitated hydroxide and the filtrate is washed
several times with warm ether. The combined ether extracts
are dried on potassium carbonate and the solvent is drawn
off. A yellow oil remains.
Yield: 47
H-NMR (CDCl3)
6~90-6.30 (m, 6H, catechol); 4.12 (t, 4H, OCH2CH2);
2.80-2.50 (m, 8H, NC~2); 2.00-1.80 (m, 6H, CH2CH2CH2);
151.69 (s, 12H, C(CH3)2)
Example 20:
N~N-bisC3-~2,2 Dimethyl-1~3-benzodio~ol-4-yloxy)propyl]-N-
t4-(nitro~enzyl)]amine~ 37
1.15 g (4~ mmol) of sodium hydride is suspended in 40
ml of DMF at room temperature under nitrogen. A solution of
18.0 g of amine 14 (42 mmol) in 20 ml of DMF is slowly
instilled in it and after completion of the addition, it is
stirred for another hour. Then, a solution of 8.64 g of 4-
nitrobenzyl bromide (40 mmol) in 20 ml of DMF is instilled
and it is stirred for another 24 hours. The mixture is
diluted with 70 ml of ice water, extracted several times
with ethyl acetate and the combined organic extracts are
dried on potassium carbonate. After removal of the solvent,
a yellow oil remains.
30Yield: 71%
, ~ - : , '.
.
2 ~ 9
- 4~ -
H NMR ([D6]-acetone)
8.21 (d, 2H, nitroaryl); 7.53 (d, 2H, nitroaryl~; 6.60
6.40 (m, 6H, catechol); 4.10 (t, 4H, OC~2C~2); 3.63 (s,
2H, C6HsCH2N); 2.61 (t, 4H, NCH2CHz~; 1.91 (q, 4H,
CH2CH2CHz); 1.60 (s, 12H, C(CH3)2
N,N-bis[3-~2,2-Dimethyl-103-benZodio~ol-4-yloxy)propyl]-N
(4-aminoben~yl)amine, 38
200 mg of 10% Pd/C in 250 ml of methanol is suspended
in a 500 ml two-necked flask, cooled to -20C and saturated
with water. Then, the solution of 5.0 g of 37 (8.8 mmol) in
50 ml of methanol is quickly instilled and stirred at -20C.
After completion of the absorption of hydrogen, it is
separated from the catalyst and the solvent is drawn off in
a vacuum. Pale yellow crystals remain.
Yield: 85%
H-N~R ( CD6]-acetone)
7.04 (d, 2H, aminoaryl); 6.60-6.40 (m, 8H, aminoaryl,
catechol); 4.10 (t, 4H, OC~2CH2); 3.36 (s, 2H, C6H5CH2N);
2.63 (t, 4H, NCH2CH2); 1-89 ~q, 4H~ CH2CH2CH2); 1-59 (s,
12~, C(cH3)2
N~N-bis[3-~2~3-Dihydroxyphe~oxy)l?~
aminobenzyl)aminohydrochloride, 39
10.7 g (20 mmol) of 37 is dissolved in 80 ml of glacial
acetic acid and mixed in boiling heat within two hours with
80 ml of an acid mixture (50% glacial acetic acid, 30%
water, 20% fuming hydrochloric acid). In doing so, solvent
distills off with acetone that is being liberated as
azeotrope. After completion of the addition, it is
distilled for another 30 minutes. Then, residual solvent is
drawn off on a rotary evaporator. The remaining residue is
dried for 6 hours in a high vacuum.
,
,: : .: : : : ,,;.
,
- 4~ -
Yield: 79~
H-NMR ~[D5] pyridine)
8.93 (s, 6H, OH and NH2); 7.10-6.40 (m, 10H, aminoaryl,
catechol); 3.92 (t, 4H, OCH2CH2); 3.47 (s, 2H, C6HsC~2N);
3.12 (t, 4H, NCH2CH2); 2.25 (q, 4H, CH2CH2CH2)
N~N-bi:;r3~ 2-Dimethy~ 3-be~zodioxol-4-yloxy)pr
t~-isothiocyanatobenzyl)ami~ohydrochloride, 40
1.15 g of thiocarbonyldichloride (10 mmol) is added to
a solution of 1.10 g of aniline 39 (2.23 mmol) in 50 ml of
lQ 3M hydrochloric a~-id and 50 ml of chloroform under a
nitrogen atmosphere with a one-way spray and is intensively
stirred for 6 hours at room temperature. Then, it is
evaporated to dryness in a vacuum.
Yield: 78%
l_NMR ([D5]-pyridine)
8.96 (s, 4H, OH); 7.10-6.40 (m, 10H, aryl, catechol);
3.92 (t, 4H, OCH2CH2); 3.61 (s, 2H, C6HsCH2N); 3.21 (t,
4H~ NCH2CH2); 2 27 (q, 4H, CHzCH2CH2)
Example 21:
N,N-bis[3-(2,2-Dimethyl-1,3-benzodioxol-4-yloxy)propyl]-N-
2-propeD.yl ) amine, 41
1.20 g (50 mmol) of sodium hydride is suspended in 50
ml of DMF at room temperature under nitrogen. A solution of
20.6 g of amine 14 (48 mmol) in 40 ml of DMF is slowlv
instilled in it and, after completion of the addition, it is
stirred for another hour. Then, a solution of 6.04 g of
allyl bromide ~50 mmol) in 25 ml of DMF is instilled and
stirred for another 24 hours. The mixture is diluted with
70 ml of ice water, extracted several times with ethyl
acetate and the combined organic extracts are dried on
--`' 2~8g9
- 49 -
potassium carbonate. After removal of the solvent, a yellow
oil remains.
Yield: 75%
H-NMR tCDCl3)
6.90-6.30 (m, 6H, catechol); 5.62 (m, lH, CH-CH2); 4.93
(m, 2H, CH=CH2); 4 . 09 (t, 4H, OCH2CH2); 2.80 2.60 (m,
6H, NCH2~; 2.32 (m, 2H, CH2CH=CH2); 2 . 00-1 . 80 (m, 6H,
CEI2CX2CH2); 1.66 (s, 12H, C(CH3~2)
Example 22:
bis[3-(2,2-Dimethyl-1,3-benzodio~ol-~-ylo~y~propylJ t2-
propinyl)amine, 42
0.24 g (10 mmol) of sodium hydride i5 suspended in 50
ml of DMF at room temperature under nitrogen. A solution of
4.29 g of amine 14 (10 mmol) in 20 ml of D~F is slowly
instilled in it and, after completion of the addition, it is
stirred for another hour. Then, a solution of 1.43 g of
propargyl bromide (12 mmol) in 10 ml of DMF is instilled and
stirred for another 24 hours at 50C. After the cooling
off, the mixture is diluted with 70 ml of ice water,
extracted several times with ethyl acetate and the combined
organic extracts are dried on potassium carbonate. After
removal of the solvent, a yellow oil remains.
Yield: 66%
H--NMR ( CDCl3 ) :
6.90-6.30 (m, 6H, catechol), 4015 (t, 4H, OCH2CH2~;
2.80-2.60 tm, 8H, NCH2, CH2CC~); 2 . 00-1 . 80 (m, 7H,
CH2CH2CH2, CH2CCH); 1. 65 ~s, 12H, C(CH3)2)
88~9
- 50 -
Example 23:
Coupling of a Tc-99m complex, aontaining isothiocyanate~, to
prot~in~
The coupling of Tc-~9m complexes containing
isothiocyanate (example 10) to proteins is to be described
by the example of F(ab')z fragments of monoclonal antibody
17-lA. Instead of the antibody fragments, any other protein
or a substance containing amino groups can be used.
Monoclonal antibody 17-lA is obtained corresponding to
methods known in the literature after administration of 107
of the corresponding hybridoma cells in the abdominal cavity
of a Balb/c-mouse and aspiration of the ascitic liquid after
7-10 days. The purification takes place according to
methods also known in the literature by ammonium sulfate
precipitation and affinity chromatography on protein A-
sepharose. The purified antibody (10 mg/ml) is treated at
pH 3.5 for 2 hours with 25 micrograms/ml of pepsin and the
F(ab')2-fragments are then isolated by FPLC~ Before
coupling with the chelating agent, the fragments are
dialyzed at 4C for 12-24 hours from 0.1 M KH2P0~0.1 M
NaHC03, pH 8.5. The protein concentration is adjusted to 10
mg/ml. The complex containing NCS labeled analogously to
example 6 is added in a molar ratio of 1:10 (complex:
protein) to the protein solution. For conjugate formation.
the mixture is incubated for 1 hour at 37C.
F.xample 24:
Biodistribution of a Tc-99m complex aoupled to fragme~ts of
monoclo~al antibody 17-lA
The biodistribution of protein-bound Tc-99m complexes
is to be described by the example of a conjugate with
F(ab')2 fragments of monoclonal antibody 17-lA. The
antibody, from which the fragments are obtained, recognizes
`i ' ' ~ :: : '
: ' ' ' ' ~: , ~,: ' ~- , ,, : : . -
'
~ . . , ,: ', .,:
- 51 -
an antigen, which is expressed by the human carcinomic cell
line l'HT29". A control cell line, which also was obtained
from a human carcinoma (MX-1), does not express this
antigen. Isolated cells of both lines are administered
subcutaneously to immunodeficient nude mice. A~ter the
tumors have grown to a size of 300-800 mg, the mice are
intravenously administered 20 micrograms of the complex,
labeled with 200 microCi of Tc-99m, coupled to F(ab')2-
fragments (example 11). The i~mune reactivity of the
conjugates is determined in a parallel manner by the binding
to an excess of intracellular antigen and is 75-80%. The
biodistribution is determined 24 hours after administration
of the conjugate by killing the animals, removing the organs
and measuring the radioactivity in the organs. The
following table represents the found amounts of
radioactivity and shows a marked concentration of chelate in
the antigen-positive tumor.
Or~an % of_the administered dose per gram_of tissue
Spleen 0.4
Liver 1.1
Kidneys 2.8
Lung o.
~uscle 0.1 ;-
Blood 0.6
MX-l 1.9
HT29 8.8
Example 25:
Biodistribution o~ a Tc-99m complex aontaining biotin
20 microliters of a commercially available
streptavidin-coupled sepharose gel (corresponding to 20
- : . ,
..,
.~ . . :.
:-. .,
2 ~ 9
- 52 -
micrograms of streptavidin) is administered to a 200 g rat
in the muscle of the left hind leg. Then, about 30 minutes
later, the intravenous administration of 5 micrograms of the
complex containing biotin (example 8) labeled with 200
microCi of Tc-99m takes place according to example 6. The
determination of radioactivity in the individual organs of
the rat takes place after 4 hours. A 16-times higher radio-
activity, which is found in the left hind leg muscle in com-
parison with the right hind leg muscle, shows a clear spe-
cific concentration of the Tc complex by binding to strept-
avidin-sepharose. In all other organs, no activities over
1.4% of the administered dose per gram of tissue are de-
tected after 4 hours. The highest concentration after the
left hind leg muscle (1.4% of the administered dose per gram
of tissue~ is found in the kidneys with 0.6% of the admini-
stered dose per gram of tissue. About 89% of the admini-
stered radioactivity is found in the urine after 4 hours.
The exampl~ shows that the complexes containing biotin
in the organism can bind to streptavidin conjugates. In-
stead of a streptavidin-sepharose conjugate, selective sub-
stances, such as, e.g., monoclonal antibodies, enzymes or
hormones, can be used, which -- coupled to streptavidin --
can be detected after selective concentration in lesions or
certain tissues by Tc-99m complexes containing biotin.
The preceding examples can be repeated with similar
success by substituting the generically or specifically
described reactants and/or operating conditions of this
invention for those used in the preceding examples.
From the foregoing description, one skilled in the art
can easily ascertain the essential characteristics of this
invention, and without departing from the spirit and scope
t:hereof, can make various changes and modifications of the
invention to adapt it to various usages and conditions.
' ~
.:;, . ..
~'
'
