CA2173844A1 - N-alkyl peptide chelate formers, their metal complexes with radionuclides, processes for producing them and radio-pharmaceutical compositions containing these compounds - Google Patents
N-alkyl peptide chelate formers, their metal complexes with radionuclides, processes for producing them and radio-pharmaceutical compositions containing these compoundsInfo
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- CA2173844A1 CA2173844A1 CA002173844A CA2173844A CA2173844A1 CA 2173844 A1 CA2173844 A1 CA 2173844A1 CA 002173844 A CA002173844 A CA 002173844A CA 2173844 A CA2173844 A CA 2173844A CA 2173844 A1 CA2173844 A1 CA 2173844A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D457/00—Heterocyclic compounds containing indolo [4, 3-f, g] quinoline ring systems, e.g. derivatives of ergoline, of the formula:, e.g. lysergic acid
- C07D457/04—Heterocyclic compounds containing indolo [4, 3-f, g] quinoline ring systems, e.g. derivatives of ergoline, of the formula:, e.g. lysergic acid with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 8
- C07D457/06—Lysergic acid amides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C327/00—Thiocarboxylic acids
- C07C327/20—Esters of monothiocarboxylic acids
- C07C327/26—Esters of monothiocarboxylic acids having carbon atoms of esterified thiocarboxyl groups bound to carbon atoms of six-membered aromatic rings
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D457/00—Heterocyclic compounds containing indolo [4, 3-f, g] quinoline ring systems, e.g. derivatives of ergoline, of the formula:, e.g. lysergic acid
- C07D457/10—Heterocyclic compounds containing indolo [4, 3-f, g] quinoline ring systems, e.g. derivatives of ergoline, of the formula:, e.g. lysergic acid with hetero atoms directly attached in position 8
- C07D457/12—Nitrogen atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
- C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
- C07J41/0072—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the A ring of the steroid being aromatic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/57536—Endothelin, vasoactive intestinal contractor [VIC]
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
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Abstract
The invention relates to N-alkyl peptide chelate formers, their metal complexes with radionuclides, processes for their production and radio-pharmaceutical compositions containing these compounds. The invention also relates to radio-pharmaceuticals containing these chelates in metal-complexed form, diagnostic kits in which the chelate may be present in non-complexed form and is complexed by the addition of either technetium or rhenium ions, and the use of such preparations for diagnostic and therapeutic purposes.
Description
~ 1 7 ~;p~4 Summary This invention relates to N-alkyl peptide chelating agents, their metal complexes with radionuclides, methods for their production, and radiopharmaceuticals containing these compounds.
~his invention further relates to radiopharmaceuticals containing said chelates in complexed form with metals and their diagnostic kits in which the chelate may not be contained in non-complexed form and may either be complexed by adding technetium or rhenium ions, and the use of such preparations for diagnostic and pharmaceutical purposes.
ID651R .DOC
1 ~ 2 l ~B 44 Description This inve~tion relates to N-alkyl peptide chelating agents, their metal complexes with radionuclides, methods for their production, and radiopharmaceuticals containing these compounds.
This invention further relates to radiopharmaceuticals containing said chelates in complexed form with metals, and their diagnostic kits in which the chelate may be contained in non-complexed form and may either be complexed by adding technetium or rhenium ions, and the use of such preparations f~r diagnostic and pharma-ceutical purposes.
Radiolabelled substances are mainly used in medical diagnostics to detect deformities and functional changes of internal organs or changes in pathologic processes in the body. The patient is given a formulation, for exam-ple, an injection solution that contains the radioactivesubstance. Images of organs, pathologic processes, and their changes in the body can be obtained by recording the emitted radiation using appropriate detectors (e.g. a gamma camera); when concentrations in organs or vessels are particularly high they may be used for therapeutic purposes.
The demand has grown in recent years for specific radio-labelled chemical compounds that can be coupled with so-called supporting molecules (e.g. steroids, antibodies,lipids, proteohormons, etc.) and act according to the "drug targeting" principle. As for tumour diagnosis and therapy as well as for receptor visualization (steroids, antibodies, etc.), substances capable of passing through the cell wall and the blood-cerebral barrier and sub-stances suitable for checking organic functions, e.g.
~D65311 . DO~
~ 2 ~ 1`738~4 before and after a transplantation, and for detecting vascular lesions, are of particular interest. More and more complexing agents are developed for many radio-nuclides and coupled with tissue-related or metabolism-related supporting molecules in order to achieve higher selectivity of the radiopharmaceutical and the signifi-cant concentration of the radionuclide in the target organ that goes with it. A radioactive isotope of rhenium is tried for use as a therapeutic agent.
Technetium-99m is the most frequently employed radio-nuclide as it is particularly suitable as an isotope for in-vivo diagnostics due to its favourable physical prop-erties (no corpuscular radiation, favourable physical half-life, ~-radiation 140 keV) and the low exposure to radiation resulting from them. Technetium-99m is easily gained from nuclide generators in the form of [99mTc]-pertechnetate. To recover technetium-99m chelates, the pertechnetate is reduced by means of suitable reductants (e.g. SnCl2, S2042-, etc.) to a lower oxidation number at which technetium-99m forms complexes with chelating agents or proteins. Specific methods have been developed and described for labelling potential chelating agents with technetium-99m and for manufacturing kits to cater for everyday clinical needs. Thus, proteins can be labelled directly with technetium-99m using donor groups (amino, amide, thiol, etc.) of the protein (J. Nucl. Med.
1986, 27, 685) or by introducing complexing agents (US
Patent 4 479 930 and Fritzberg, A. R. et al., J. Nucl.
Med. 1986, 27, 957).
Complexing agents have been developed from the group of cyclic amines (Troutner, D. E. et al.; J. Nucl. Med.
1980, 21, 443 and Macke, H.; DE-3 911 816), cyclames (Ketring, A. R.; Troutner, D. E. et al.; J. Nucl. Med., 1980, 21, 443-448, Int. J. Nucl. Med. Biol. 1984, 11, ID653e . DOc - ~2 1 ~3844 113, Volkert et al., Applied Radiat. Isot., 1982, 33, 891-896~, N22 systems (Pillai, M. R. A.; Troutner, D. E.
et al.; Inorg. Chem. 1990, 29, 1850); N2S2 systems (Bormans, G. et al.; Nucl. Med. Biol. 1990, 17, 499) and N3S systems (Fritzburg, A.; EP-A-0 173 424 and EP-A-0 250 013) have been described as well.
But all these complexing agents have considerable disadvantages. Thus, pH values of 10-13 are required for labelling the cyclames as well as the N2O2 systems known from literature (Pillai, M. R. A. et al; Inorg. Chem.
1990, 29, 1850-1856). Moreover, these systems do not have any reactive groups that facilitate coupling with bio-molecules or proteohormones. N2S2 and N3S systems may be sufficiently stable in individual cases but are suitable only for simple excretory stages such as diuresis, EP-A-0 250 013. Attempts to simplify the manufacture of the MAG3 known from literature (EP-A-0 250 013) that requires heating to 100C to be radiolabelled (Bannister, K. M. et al.; J. Nucl. Med. 1990, 31, 1568-1573) are being made (WO-91-16076) but there has been no clinical access to such procedures as yet.
The stability of N2S2 complexes (Bormans, G. et al.;
Nucl. Med. Biol. 1990, 17, 499) between preparation and application is less than 1 h which is a substantial dis-advantage. Efforts are being made - as yet with no relevance to clinical practice - to remove shortcomings (Merryn, W. et al., Applied. Radiat. Isot. vol. 42 (7), 607-612).
The complexing agents that have become known as yet have a homogeneous structure and are therefore suited for just one diagnostic purpose. Molecule variations are restricted to substituents of the skeleton and thus do not allow for the wider medical applications that would ID5401~ DX
.
~his invention further relates to radiopharmaceuticals containing said chelates in complexed form with metals and their diagnostic kits in which the chelate may not be contained in non-complexed form and may either be complexed by adding technetium or rhenium ions, and the use of such preparations for diagnostic and pharmaceutical purposes.
ID651R .DOC
1 ~ 2 l ~B 44 Description This inve~tion relates to N-alkyl peptide chelating agents, their metal complexes with radionuclides, methods for their production, and radiopharmaceuticals containing these compounds.
This invention further relates to radiopharmaceuticals containing said chelates in complexed form with metals, and their diagnostic kits in which the chelate may be contained in non-complexed form and may either be complexed by adding technetium or rhenium ions, and the use of such preparations f~r diagnostic and pharma-ceutical purposes.
Radiolabelled substances are mainly used in medical diagnostics to detect deformities and functional changes of internal organs or changes in pathologic processes in the body. The patient is given a formulation, for exam-ple, an injection solution that contains the radioactivesubstance. Images of organs, pathologic processes, and their changes in the body can be obtained by recording the emitted radiation using appropriate detectors (e.g. a gamma camera); when concentrations in organs or vessels are particularly high they may be used for therapeutic purposes.
The demand has grown in recent years for specific radio-labelled chemical compounds that can be coupled with so-called supporting molecules (e.g. steroids, antibodies,lipids, proteohormons, etc.) and act according to the "drug targeting" principle. As for tumour diagnosis and therapy as well as for receptor visualization (steroids, antibodies, etc.), substances capable of passing through the cell wall and the blood-cerebral barrier and sub-stances suitable for checking organic functions, e.g.
~D65311 . DO~
~ 2 ~ 1`738~4 before and after a transplantation, and for detecting vascular lesions, are of particular interest. More and more complexing agents are developed for many radio-nuclides and coupled with tissue-related or metabolism-related supporting molecules in order to achieve higher selectivity of the radiopharmaceutical and the signifi-cant concentration of the radionuclide in the target organ that goes with it. A radioactive isotope of rhenium is tried for use as a therapeutic agent.
Technetium-99m is the most frequently employed radio-nuclide as it is particularly suitable as an isotope for in-vivo diagnostics due to its favourable physical prop-erties (no corpuscular radiation, favourable physical half-life, ~-radiation 140 keV) and the low exposure to radiation resulting from them. Technetium-99m is easily gained from nuclide generators in the form of [99mTc]-pertechnetate. To recover technetium-99m chelates, the pertechnetate is reduced by means of suitable reductants (e.g. SnCl2, S2042-, etc.) to a lower oxidation number at which technetium-99m forms complexes with chelating agents or proteins. Specific methods have been developed and described for labelling potential chelating agents with technetium-99m and for manufacturing kits to cater for everyday clinical needs. Thus, proteins can be labelled directly with technetium-99m using donor groups (amino, amide, thiol, etc.) of the protein (J. Nucl. Med.
1986, 27, 685) or by introducing complexing agents (US
Patent 4 479 930 and Fritzberg, A. R. et al., J. Nucl.
Med. 1986, 27, 957).
Complexing agents have been developed from the group of cyclic amines (Troutner, D. E. et al.; J. Nucl. Med.
1980, 21, 443 and Macke, H.; DE-3 911 816), cyclames (Ketring, A. R.; Troutner, D. E. et al.; J. Nucl. Med., 1980, 21, 443-448, Int. J. Nucl. Med. Biol. 1984, 11, ID653e . DOc - ~2 1 ~3844 113, Volkert et al., Applied Radiat. Isot., 1982, 33, 891-896~, N22 systems (Pillai, M. R. A.; Troutner, D. E.
et al.; Inorg. Chem. 1990, 29, 1850); N2S2 systems (Bormans, G. et al.; Nucl. Med. Biol. 1990, 17, 499) and N3S systems (Fritzburg, A.; EP-A-0 173 424 and EP-A-0 250 013) have been described as well.
But all these complexing agents have considerable disadvantages. Thus, pH values of 10-13 are required for labelling the cyclames as well as the N2O2 systems known from literature (Pillai, M. R. A. et al; Inorg. Chem.
1990, 29, 1850-1856). Moreover, these systems do not have any reactive groups that facilitate coupling with bio-molecules or proteohormones. N2S2 and N3S systems may be sufficiently stable in individual cases but are suitable only for simple excretory stages such as diuresis, EP-A-0 250 013. Attempts to simplify the manufacture of the MAG3 known from literature (EP-A-0 250 013) that requires heating to 100C to be radiolabelled (Bannister, K. M. et al.; J. Nucl. Med. 1990, 31, 1568-1573) are being made (WO-91-16076) but there has been no clinical access to such procedures as yet.
The stability of N2S2 complexes (Bormans, G. et al.;
Nucl. Med. Biol. 1990, 17, 499) between preparation and application is less than 1 h which is a substantial dis-advantage. Efforts are being made - as yet with no relevance to clinical practice - to remove shortcomings (Merryn, W. et al., Applied. Radiat. Isot. vol. 42 (7), 607-612).
The complexing agents that have become known as yet have a homogeneous structure and are therefore suited for just one diagnostic purpose. Molecule variations are restricted to substituents of the skeleton and thus do not allow for the wider medical applications that would ID5401~ DX
.
2`~ r3~4~
be enabled by a complex variation. So they are costly regarding both the manufacture and the theoretical com-prehension of the individual complexes.
It is the problem of the invention to provide short-chain N-alkyl peptide chelating agents complexing at room temperature with technetium and rhenium across a wide pH
range, and forming both ionic and non-ionic complexes, and thus to provide compounds that do not have the disadvantages of known 99mTC_ and 180,188Re_CompleXeS
Requirements regarding exposure to radiation, stability, and solubility for applying these compounds to humans must herewith be met; at the same time, a method of representing them that is as simple as possible should be created, and a kit formulation of the compounds/con-jugates of the invention and their metal complexes be provided for clinical application.
This problem is solved according to the invention in that it provides compounds of the general formula I
R3-S-CH2-Co-NR1-[CH2-Co-NH]n-CH2-Co-R2 (I) with the exception of N-[2-(benzoylthio)acetyl]-N-cyclohexyl glycine wherein n represents the numbers 0,1 or 2, R1 is a straight-chain or branched alkyl residue contain-ing 1 to 20 carbon atoms which are optionally interrupted or replaced by one to three oxygen atoms, and which may include a terminal -COOH-, -OH- or -NH2 group that is optionally esterified or etherified by means of glycolic acid or its esters or ethers, ID54013 .DOC
the esters or ethers being formed using carboxylic acids or alcohols containing up to 18 carbon atoms, or represents a phenyl or cyclohexyl residue having at its 4-position, if required, a COOH, NH2 or OH group that is esterified, etherified or amidated using carboxylic acids or alcohols containing up to 6 carbon atoms, or replaced by a halogen atom, R2 is a halogen atom, a halogen methyl, methyl carboxyl, trifluoromethyl carboxyl, an NH2 or an OH group, the carboxyl group formed when R2 = OH being esterified or amidated either immediately, or after etherification with an a,~-hydroxycarboxylic acid containing up to 8 carbon atoms through its terminal carboxyl group, with a biomolecule, a steroid, an ergoline derivative, a benzodiazepine derivative, a cholecystokinin, a peptide, a protein, a proteo-hormone, an amino sugar, an endotheline, an endo-theline derivative, an endotheline antagonist or an endotheline fragment, is a residue of the general formula II
/\~ OH
¦ CH=CH--R4 ( I I ) /~/\/\/
R80~ ~
or the general formula II a " ~ ,OH
R O ~ C C R4 ID54 0~ . DOC
~ 6 where R4 is a methylene, propenylene amino, propinylene amino, methylene amino or methylenoxy group and R8 is a hydrogen atom or a methyl group, is a residue of the general formula III
Rs ,N ~ 7 or of the general formula III a ~, ~ ~, N ` R7 wherein are:
R5 an -NH-, -NH-CO-N<, -NH-CO-NH- or methylenoxy group, R6 a hydrogen atom, a halogen atom or a methyl group and ID5401~ .DOC
R7 a hydrogen atom or a straight-chain or branched alkyl residue containing up to 6 carbon atoms, R3 is a hydrogen atom, an acetyl, benzoyl, p-methoxybenzyl, acetamidomethyl, benzamidomethyl, trimethyl acetamidomethyl, hydroxyacetyl, ethoxyethyl, ethylthio, trityl or an easily separable sulfur-protective group and their salts formed from pharmaceutically acceptable acids or bases.
Surprisingly, a class of compounds was found that does not only avoid the disadvantages mentioned above but as 99mTc+5 complexes comprises a transition from N3S -> N2SO
-> S2O2 -> N2S2-N-alkyl peptide complex system and can therefore be used as complexing agents at a wide range.
Formula IV shows an N3S-N alkyl peptide complex in the 99mTc core (neutral) from R3S- CH2-CO-NR1[CH2-CO-NH]2-CH2-CO-R
~ "~
,Tc ) ~IV) ~""' ""`~
~R2 formula V shows a N2OS-N alkyl peptide complex in the 99mTc core (neutral) from R3S-CH2-Co-NRl[CH2-CO-NH]l-CH2-COOH, ID653E . DOC
8 .2 1.7 3 8 4 4 ~ ,~ (V) ~ ,T c J
~"' "`~
formula VI shows an O2S2_N alkyl peptide complex in the 99mTc core (having a negative charge) from R3S-CH2-Co-NRl[CH2-CO-NH]o--CH2-COOH, ~ O ,Q~
( Vl ) R1 _ ~ ,Tc N Rl ` J
`S' ~
and formula VII eventually shows a N2S2-N alkyl peptide complex in the 99mTc core (having a positive charge) from R3S-CH2-Co-NRl[CH2-Co-NH]o-CH2-Co-R2.
~ /R1 R1 ~
O ~1~ G ,N~ O
, Tc ~ ( Vll \\S"""'"""`~//
ID653E.DX
~ 21 73844 `
The complexes that result from the molecule variation show a surprising range and correspondence with regard to their biological behaviour. Thus compounds of this type, when coupled with a bioligand, are suited for receptor visualization, which has not yet been described for 99mTc compounds, and excellently suited for plaque visualiza-tion with vascular lesions and for measuring the kidney function, with the option that the compounds may be coupled with bioactive ligands.
Compounds are preferred in which R1 is a -CH2-(CH2)m-CH3 group with m = 1 - 14, or R1 is a phenyl, a p-phenyl amine, a cyclohexyl amine, p-hydroxyphenyl, 4-hydroxycyclohexyl, p-halogen phenyl or 4-halogen cyclohexyl group.
Furthermore, compounds of the general formula I are preferred in which R1 equals ~~CH2)q~OOC~CH2~OOC~(CH2)r~CH
q and r being integers from 1 to 16.
Such compounds of the general formula I are particularly preferred in which R1 is a straight-chain or branched alkyl residue containing 1 to 6 carbon atoms, or a P-bromophenyl residue.
Furthermore, compounds of the general formula I are pre-ferred in which R2 is a residue of the general formula II
ID540B .D0~
2 1;7f3~8;4:4 OH
~CH=CH--R4 ( I I ) R ~o~/
or of the general formula II a " ~ ~OH
R O ~ C _ C R4 (ll where R4 is a methylene, propenylamino, propinylamino, methylen~m;no or methylenoxy group and R8 is a hydrogen atom or a methyl group.
17a-ethinyl estradiols or their 3-methyl ethers are preferred among these steroids.
A 17a-ethinyl group of the estradiol may also be coupled with an ethene. The order of ethyne and ethene groups can be changed; doubling the ethyne or ethene group is useful as well. A minimum distance between the chelating agent and the estradiol molecule, preferably a rigid ethyne or ethene group, is important to prevent folding of the lateral chain.
If I is bonded to this preferred 17a-position of the estradiol skeleton, little or no reduction of bioactivity can be expected from the resulting chelate complex-ID540B . DOC
11 21 7~844 bioligand compounds as documented, for example, inEpperly, M. W. et al [J. Steroid Biochem. Molec. Biol.
vol. 39, no. SA, 729-734, 1991].
The outstanding significance of these estrogen chelate complexes is based on their ability to penetrate through the cell wall and become attached to the estrogen recep-tor. This applies, in particular, to complexing agents according to the general formula I with n - 2. These N3S
complexes are neutral at 99mTc+5 as a result of N1 alky-lation in the Tc core (cf. formula IV). While charged complexes such as the N3S complex as 17a-ethinyl chelate steroid of the thioacetyl-glycyl-glycyl-glycyl-0-ester type (own tests) are not able to penetrate the cell wall, the Nl alkylated complexes (N1 = methyl) of the invention penetrate through the cell wall and attach themselves in the uterus after an i.v. injection. Their concentration, in terms of the blood/uterine quotient, is 0.2 and can be varied depending on the length of the N1 alkyl chain or substituents. Thls facilitates early detection of mamma-ca according to the SPECT method. The compound according to Example 10 is particularly preferred here.
Furthermore, compounds of the general formula I are preferred in which R2 is a residue of the general formula III R5 ID653e . DOC
or of the general formula III a N~R7 where R5 is an -NH-, -NH-CO-N<, -NH-CO-NH- or methylenoxy group, R6 a hydrogen atom, a halogen atom or a methyl group and R7 is a straight-chain or branched alkyl residue containing up to 6 carbon atoms.
These compounds of the general formula III and III a are ergoline derivatives.
8a-amino-6-methyl-ergoline having, if required, alkyl substituents at the 2- and/or 6-positions with a chain length of C1 at position 2 or C1_3 at position 6 and/or a methyl- or halogen substituent at position 2, is pre-ferred among these ergolines. The substituent at position8 of the ergoline may, apart from aNH2, also be ~-CH2O-or aNH-CO-N< or aNH-co-NH-.
ID653E .DOC
~ 13 21 73844 The compounds according to the invention of the general formula I carry a R3 residue at the terminal sulfur atom.
This R3 residue is a sulfur protective group which is required during the synthesis of the compounds according to the invention of general formula I. Such a protective group should be easily separable. Appropriate R3 residues are, for example, acetyl, benzoyl, p-methoxybenzyl, acetamidomethyl, benzamidomethyl, trimethyl acetamido-methyl, hydroxyacetyl, ethoxyethyl, or trityl groups. The benzoyl group is particularly preferred.
Another object of the present invention are the metal-chelate complexes that radioactive metal ions form with the compounds according to the invention of the general formula I wherein R1, R2 and R3 are as defined above.
Suitable radioactive metal ions are, for example, radionuclide ions of Tc, Re, Cu, Ga, Gd, Y and In. The radionuclide is selected according to the desired appli-cation of the metal-chelate complexes according to the invention of the general formula I.
Different radionuclides are used for radiodiagnostics or radiotherapy.
Radioactive metal ions of Tc and Re isotopes are preferred here.
The radionuclide technetium-99m is particularly preferred.
Another object of the present invention are conjugates containing compounds of the general formula I or metal-chelate complexes ~ormed by radioactive metal ions of Tc, Re, Cu, Ga, Gd, Y and In and compounds of the general formula I, and substances that accumulate selectively in ID65~e .DOC
~ 14 2 1 73844 diseased tissue or in tumours, with a covalent bonding existing between said substances that is amidic for substances containing carboxy or amino groups such as peptides, proteins, antibodies or their fragments, ester-S like for substances containing hydroxy groups such asfatty alcohols, and imidic for substances containing aldehyde groups.
Conjugates are preferred having peptides that accumulate in diseased tissue such as endothelines, partial endo-theline sequences, endotheline analogues, endotheline derivatives, or endotheline antagonists.
Particularly preferred are such conjugates containing compounds of the general formula I and peptides that accumulate selectively in diseased tissue in which the peptides comprise the following sequences:
c ~ er-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, c ~ er-cys-ser-ser-trp-leu-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, c ~ hr-cys-phe-thr-tyr-lys-asp-lys-glu-cys-val-tyr-tyr-cys-his-leu-asp-ile-ile-trp, c ~ er-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, c ~ er-cys-asn-ser-trp-leu-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, ID65 31~ . DOC
cys-ser-cys-lys-asp-met-thr-asp-lys-glu-cys-leu-asn-phe-cys-his-gln-asp-val-ile-trp, ala-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, ala-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, cys-ser-cys-ser-ser-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, or the partial 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).
Another object of the present invention is a method for manufacturing the compounds according to the invention of the general formula I
ID5401~.DOC
- ` 21 73844 R3-S-CH2-Co-NR1-[CH2-Co-NH]n-CH2-Co-R2 (I) with the exception of N-[2-(benzoylthio)acetyl]-N-cyclohexyl glycine wherein n represents the numbers 0,1 or 2, R1 is a straight-chain or branched alkyl residue contain-ing 1 to 20 carbon atoms which may optionally be interrupted or replaced by one to three oxygen atoms, and which may optionally include a terminal -COOH, -OH or -NH2 group that may optionally be esterified or etherified by means of glycolic acid or its esters or ethers, these esters or ethers being formed using carboxylic acids or alcohols containing up to 18 carbon atoms, or represents a phenyl or cyclohexyl residue having an optional COOH, NH2 or OH group at its 4-position that is optionally esterified, etherified or amidated using carboxylic acids or alcohols containing up to 6 carbon atoms, or carries an additional halogen atom, R2 is a halogen atom, a halogen methyl, methyl carboxyl, trifluoromethyl carboxyl, an NH2 or an OH group, the carboxyl group formed when R2 = OH being esteri-fied or amidated either immediately, or after etherification with an a,~-hydroxycarboxylic acid containing up to 8 carbon atoms through its terminal carboxyl group, with a biomolecule, a steroid, an ergoline derivative, a benzodiazepine derivative, a cholecystokinin, a peptide, a protein, a proteo-hormone, an amino sugar, an endothelin, an endothelin ID54 OB . DOC
derivative, an endothelin antagonist or an endothelin fragment, is a residue of the general formula II
S
CH = CH--R4 ( I I ) ,o J~
or of the general formula II a ~____,OH
R ~o~J c=c_ R~ ( ~
where R4 is a methylene, propenylen~m;no, propinylen-amino, methylen~m;no or methylenoxy group and R8 is a hydrogen atom or a methyl group, is a residue of the general formula III
Rs or of the general formula III a ID54011 .DOC
21 ?3844 ~ 18 ¢~N ~ R7 wherein R5 is a -NH-, -NH-CO-N<, -NH-CO-NH- or methylenoxy group, R6 is a hydrogen atom, a halogen atom or a methyl group and R7 is a hydrogen atom or a straight-chain or branched alkyl residue containing up to 6 carbon atoms, R3 is a hydrogen atom, an acetyl, benzoyl, p-methoxy-benzyl, acetamidomethyl, benzamidomethyl, trimethyl acetamidomethyl, hydroxyacetyl, ethoxyethyl, ethylthio, trityl or an easily separable sulfur protective group and their salts formed from pharmaceutically acceptable acids or bases characterized in that a~ a diketo piperazine derivative of glycine or b) glycine ID65 3E . DOC
is first reacted with a haloacid halogenide and then with an alkyl amine or aryl amine of the general formula VI
R1 - NH2 (VI) where R1 is as defined above, is reacted again with a haloacid halogenide and then reacted with a compound of the general formula IV
R3 - SH (IV) where R3 is as defined above, or a compound of the general formula V
Rl-NH-CH2-CO-R2 (V) where R2 is as defined above, is reacted with a haloacid halogenide and then with an alkyl amine or aryl amine of the general formula VI
R1 - NH2 (VI) where R1 is as defined above, and reacted again with a haloacid halogenide and then with a compound of the general formula IV
R3 - SH (IV), where R3 is as defined above, and that these compounds are converted, if required, into a salt by means of a pharmaceutically acceptable acid or base.
The compounds according to the invention of the general formula I may also be manufactured when starting from a ID6531~ . DOC
compound according to the invention of the general formula I, where n is 0 or 1, and reacting it with a compound comprising terminal groups thereby forming a residue R2 after coupling with the above compound. This yields compounds according to the invention of general formula I in which n is 1 or 2. This means that part of the chain of compounds of the general formula I according to the invention is provided by the R2 residue added by coupling.
Another object of this invention is the manufacture of the metal-chelate complexes according to the invention consisting of radioactive metal ions and compounds of the general formula I according to the invention.
Compounds of the general formula I are reacted in a generally known way, optionally with prior or simultaneous splitting off of the R3 residue.
Metal-chelate complexes of radioactive metal ions of Tc and Re and compounds of the general formula I are manu-factured by reacting technetium-99m or Re in the form of pertechnetate or perrhenate in the presence of a reduc-tant and, optionally, an auxiliary ligand with a compound of the general formula I
R3-S-CH2-Co-NR1-[CH2-Co-NH]n-CH2-Co-R2 (I) with the exception of N-[2-(benzoylthio)acetyl]-N-cyclohexyl glycine where R1, R2 and R3 are as defined above.
The surprising variability of the compounds and their complexes is of particular relevance to the invention.
An N3S system is obtained for compounds according to the ID5401~ DX
2t 73844 general formula I with n = 2. When changing from n = 2 to n = 1, an N2SO-N-alkyl system can form that has a surprising complex stability. Finally, a chelate complex may form during complexing at n = 0 from the NlS1-N-alkyl system which, on the one hand, presents a N2S2-N-alkyl system or, as another dimer, is an O2S2-N-alkyl complex, without requiring a chemically covalent bridge to connect the complex.
The new compounds also have a wide range of clinical applications due to their chemical variability and their complexing variants.
Until today there is no method for visualizing in vivo the formation of plaques as a sign of beginning athero-sclerosis. Atherosclerosis is a widespread disease and an early stage of coronary heart disease which causes the death of about 40 ~ of the population in modern indus-trial nations. It is therefore particularly desirable tovisualize vascular lesions using a simple device. The N-alkyl chelates according to the invention may thus be used for early detection of atherosclerotic vascular diseases.
The quotient of activity in the plaque and the intact vessel obtained in an animal test (WHHL-type rabbits with artificially generated atherosclerotic changes of the vessels; WHHL rabbits show high levels of LDL in the blood due to a lacking or defective LDL receptor and thus develop atherosclerotic changes of the vessels) after i.v. application of compounds according to the general formula I wherein R2 is an endotheline, partial endo-theline sequence, endotheline analogue, endotheline derivative, or endotheline antagonist, was 1 : 5 (cf.
figures 1 and 2).
~ ~ 21 73844 21 a It was highly surprising that excretion had taken place after only 3 hours despite the lipophilia of the complex resulting from the R1 alkyl chain with a length of C6.
IDS~OI~ .DOC
Lesions in the aortic area can be visualized in auto-radiograms of an amazingly good quality. There is an af-finity for the plaques or their receptors due to the fatlike molecules which is strong enough to maintain adhesion to the plaques in spite of the falling concen-tration of the compound in the blood due to its excretion via the liver.
The affinity for plaques in atherosclerotically changed vessels may be influenced by varying the R1 alkyl group.
Lipophilia may thus be controlled through the character-istics of the residue in R1 while R2, as a free carboxyl group, provides the conditions for good solubility in water. As an alternative to R2 being a free carboxylic acid, amidation of this carboxyl group with an amino hydrocarbon can contribute to improving water solubility.
It is known that proteins, in particular, short-chain proteohormones such as endothelines lose their potency when coupled with a chelating agent via a covalent bonding. The behaviour of the compounds according to the invention after covalent bonding to endothelines, their antagonists or fragments therefore came completely unex-pected (Examples 11, 12).
The way of generating the N-alkyl complexing agents of the general formula I according to the invention using endothelines from preliminary stages of chelating shown herein (Examples 11 and 12) is a new method of generating ~h complexing positions. The new N-alkyl complexes of the general formula I have a similar stability as that of known N3S complexes.
Non-invasive techniques to diagnose atherosclerosis have been described, especially techniques involving radio-iodine labelling. Antibodies labelled with radionuclides ID653E.DOC
r 23 or labelled "low density lipoproteins" (LDL) were pre-sented that bond to atherosclerotic wall areas (Lees et al. 1983, J. Nucl. Med. 24, 154 -156, Kaliman et al.
1985, Circulation, 72, 300; Virgolini et al. 1991, Eur.
J. Nucl. Med., 18, 944-947). These methods, however, still retain major disadvantages as, for example, the antigenicity of the antibodies in the system or the long time required to isolate, clean, and label the LDL from the patient's blood (several days). Most disadvantageous of all, these big molecules have a long half-life in the blood which, in conjunction with high background radia-tion in the body as a whole, makes localization of the atherosclerotic lesions difficult if not impossible. Shih et al. (1990, Proc. Natl. Acad. Sci., 87, 1436 - 1440) synthetized partial sequences of the LDL protein portion (apo-B-100) that still bond to the atherosclerotic plaques but have a much shorter half-life in the blood and an improved signal-to-noise ratio. Due to too low an affinity to the plaque and/or low density of the bonding places of these peptides in the plaque, successful in vivo diagnosis of atherosclerosis could not be demon-strated using these apo-B-peptides.
These compounds share, in general, the disadvantage of radioactive iodine-123. They are not readily available as they are produced in a cyclotron and have a physical half-life of 13 hours, and they are easily separated in the system. So it was surprising to find that the compounds of the general formula I according to the invention, after covalent bonding to NH groups of endotheline and complexing with 99mTc showed good accumulation in atherosclerotic plaques. An advantage that is particularly striking is that the n = 1 and n = 0 compounds generate new N-alkyl peptide complexing agents after covalent bonding to endothelines by means of an amidation, involving the NH and SH groups, if any, of the ID653E.DOC
respective endotheline that are located in appropriate positions.
When reacting the compounds according to the invention with dopamine-like bioactive molecules, surprisingly receptor affinity of these compounds from the ergoline family is not lost; in preliminary stages, both with and without D2 receptor affinity, there was even achieved sufficient brain passage to facilitate visualization of the D2 receptors. Endeavours to visualize receptors in the brain using 99mTc+5 complexes have failed so far. For example, when 8a-amino-6-methyl ergoline is reacted with mercapto-acetyl-glycyl-glycyl-glycin to yield 8a-amide, and complexed using 99mTc+5 after i.v. injection to the rat (Wistar), there is no uptake in the brain.
But if this reaction is carried out using the compounds of the general formula I according to the invention where R1 = methyl and amidation or esterification takes place via R2, 0.1 to 0.5% of the dose are accumulated in the brain in analogous conditions.
O [NH--CO--CHz]2--N--CO--CH2--S--R3 ~/ I
¢~ CH~
HN
New structures of N-alkyl-peptide complexes are formed with n = 1 and n = 0 which, interacting with the bioactive molecule, surprisingly imitate the structures of dopamine-like compounds and accumulate in the brain in large quantities that can be used to visualize the D-receptors (formula IX).
ID653e .DOC
~/ I
HN
The compounds of the invention are manufactured according to a method known in the art [A Specialist Periodical Report; Amino-acids, Peptides and Proteins;
The Chemical Society, Burlington House, London, WlVOBN].
Manufacture of compounds with R1 = CH3 and R2 = OH
starts from sarcosine that is reacted with chloroacetyl chloride. The resulting chloroacetyl sarcosine is reacted with thiobenzoic acid in the common way, thus yielding structures with n = 0 R S-cH2-co-N-(cH2-co-NH)o-cH2-coR2 Compound A
Compounds with n = 2 are manufactured by reacting diketo-piperazine of glycine with chloroacetyl chloride. The resulting chloroacetylglycyl glycine is subsequently reacted with N-methyl amine or, to insert longer resi-dues, with N-alkyl amines, phenyl amines, alkyl-oxa-alkyl amines, cycloalkyl amines or glycine glycolate. Re-reacting with chloroacetyl chloride and subsequent thiobenzoylation results in compounds of the formula n =
2.
ID653E.DOC
R3S-CH2-Co-N-(CH2-CO-NH)2-CH2-COR2 /
Rl Compound B
Rl represents the variations in the sarcosyl-nitrogen atom The respective compounds with n = 1 are obtained by reacting glycine salts with N-protected sarcosyl com-pounds or by reacting glycine with chloroacetyl chloride and subsequent aminolysis of the chloromethyl group. Re-reacting with chloroacetyl chloride and subsequent thiobenzylation yields the title compounds with n = 1.
R3S-CH2-Co-N-(CH2-CO-NH)l-CH2-COR2 /
Rl Compound C
Compounds A, B, C do not react in the usual way with coreactants carrying amino groups or hydroxyl groups. For this purpose, A to C are dissolved in N-methyl pyrroli-done, pre-activated using BOP (B. Castro et al.
Tetrahedron Letters 14 (1975) 1219) or TBTU (Knorr et al.
Tetrahedron Letters 30 (1989), 1927), and then reacted with the amino component. Esterification is preferably carried out using dicyclohexyl carbodiimide in the presence of dimethyl aminopyridine. For the synthesis of compounds having an estrogen as bioligand, one starts from an estrogen that already carries an unsaturated substituent in 17a such as a propargyl amine or propar-gylyalcohol residue and reacts it with compounds A to C
in the way described.
ID653E.DOC
Compounds A to C are reacted with peptides, especially with endotheline components, according to the methods common in peptide chemistry, either conventionally in solution, preferably in dimethyl formamide, dimethyl acetamide, N-methyl pyrrolidone, dimethyl sulfoxide, or mixtures of these components. When using the solid-phase method, the amino component may be acylated in the usual conditions with the synthesis resin. Splitting off the synthesis resin yields the desired compounds. Preparative cleaning, if required, is carried out by chromatographing on an RP 18 column in a water / acetonitrile gradient containing 0.1% trifluoroacetic acid.
Ergolines carrying amino or hydroxy groups are reacted analogously to the peptide reactions in solution. Reac-tions were carried out in N-methyl pyrrolidone. Prepara-tive cleaning is carried out as described for peptides.
The other R4 residues are obtained after building the protected dipeptides A - C by reacting Rl, with R2 = OH, following activation using NH2-CH2Cl, NH2-CH2-CH=CHJ or NH2-CH2-C=CJ
and, after the palladium-O reaction, C-C-bonding the 17 -ethinyl steroid with the desired complexing agent.
Complexes are preferably formed by reducing Na pertechne-tate with, for example, tin(II) chloride or dithionide in an acqueous medium at a pH value between 6 and 9 and at room temperature; if required, using an auxiliary ligand ID653e.DOC
such as sodium citrate or sodium tartrate, with the protective group R3 being split off either prior to the reaction or in situ.
Activation of the R2 carboxyl group that is formed when R2 = OH, or its replacement by an NCS or similar reactive group results in covalent bonding of peptide chelates to a bigger protein or proteohormone, endotheline, endo-theline analogue, or endotheline antagonist, endotheline derivative, partial endotheline sequence, and subsequent complexing.
As documented in the manufacturing instructions, the 99mTc and 186~188Re complexes of the invention can be produced at room temperature, neutral to weakly alkaline pH, and with or without re-chelating (e.g. via a hepta-gluconate-99mTc complex) at yields of 95%. Their easy salification via a free cation ensures good solubility in water.
Manufacture of the metal complexes: The protective group is split off according to the methods known in the art (see "Protective groups in organic synthesis" T.N.
Greene, John; Wiley and Sons 1981).
The usual quantity employed for applications in human diagnostics is between 370 MBq and 1850 MBq, preferably 740 to 1480 MBq. The compounds of the invention are pro-vided in the form of a kit designed for application to human beings. This kit contains at least one chelating agent according to the general formula I, either free or bonded to ligands.
Another object of the invention is a cold kit containing a chelating agent of the general formula I that is either free or bonded to a bioactive molecule such as an ergo-ID653E~ . DOC
line, estradiol or endotheline derivative, and is capable of bonding metal atoms.
Splitting off the protective groups that were introduced intermediately to carry out the synthesis often makes difficulties. As splitting off the -S- protective group (R3) is accompanied by simultaneous splitting of the pep-tide chain, this process results in serious damaging or useless fragments especially in the case of chelating agents with peptide bondings; so it was all the more sur-prising that up to 80% were split off in the presence of hydrogen/Pd/CaC03 together with the usual alkaline, i.e.
protective group separation in hydrogenation conditions.
This decisive improvement of free chelate complex synthe-sis is important because protective groups can be split off before formulating the kit which is a major improve-ment in the technique of gaining chelating agents that are free from protective groups.
Example 3 describes the separation method according to the invention. While the conventional method with alka-line yielded only a mixture that was hard to separate, protective group separation with alkaline in the presence of hydrogen/Pd/CaC03 resulted in a ~ 100% yield contain-ing 80% of the desired material.
It shall be mentioned, for example, that the reaction with amines or alcohols is carried out by activating the carboxylic acid R2 = OH applying methods known in the art.
Activated groups are, for example: acid chlorides, mixed anhydrides [Org. Prep. Proc. Int. 1975, 7, 215], acti-vated esters [Adv. Org. Chem. Part B, 472]. These can be linked with biomolecules through linkers, for example 2~ 7~844 using the carbodiimide method (Fieser, Reagents for Organic Synthesis 10, 142). Linking takes place in such a way that a covalent bond is formed between the chelate and the biomolecule.
Where steroids are used as biomolecules, estradiol derivatives are preferred that have substituents in the 17a position. Synthesis of such compounds has been described, for example, by Blickenstaff for the 17~-ethinyl derivatives of estradiol. (Blickenstaff A.;
Steroids 46, 889 (1985); USP 462.426). Synthesis of estradiol derivatives with 17a-propargyl substituents having a terminal NH2 or OH function is described by Blickenstaff, Brandes and Poirier. (Blickenstaff et al.;
Steroids 48, 223 (86); Brandes, A. et al. Dissertation 87-01441, 1986, University of Illinois, D. Poirier et al.
J. Steroids. Biochem. Molec. Biol. 38, no. 6, 759-774, 1991) .
A survey of linking triple and double bonds, e.g. after a palladium-0 reaction, is given in Aldrichchimica Acta, vol. 15, no. 1, 1982; Shun-ichi Murahashi et al., Stephen A. Godleski, Tetrahedron Letters, vol. 22, no. 24, pp 2247-2250, 1981; Tetrahedron Letters, vol. 29, no. 24, 2973-2976, 1988. For example, I with R2 = - NH-CH2-C-CH
is reacted during coupling with l7a[2-halogen-ethenyl]
estradiol according to the references given.
It is known from literature that ergot alkaloids of the 6-methyl-8-substituted ergoline type have a wide range of chemical variation without a clearly recognizable connec-tion between an effect and the substituents at position 8 [Ergot Alkaloids and Related Compounds, Editors: B. Berde and H.O. Schild, Springer-Verlag, Berlin, Heidelberg, New York, 1978].
ID653E . DOC
Ergoline is a preferred dopamine-like ligand. A survey of ways for synthesizing 8-substituted ergolines is given in Ergot Alkaloids and Related Compounds, Editors B. Berde and H.O. Schild, Springer-Verlag Berlin, Heidelberg, New York, 1978, Chapter II Chemical Background, J. Ruisemmann and P.A. Stadler.
Reaction with 8a-amino- or 8~-hydroxy-methyl ergoline is preferred. It takes place via an activated group in R2, for example, benzotriazol-1-yl-tetra-methyl-uronium-tetrafluoroborate, or according to the known carbodiimide method.
New complexes are generated when the n = 1 and n = 0 complexing or partial complexing agents are applied according to the invention including the nitrogen in the 8a-amino and 6-N-methyl groups of ergoline.
Another object of the invention is the use of metal chelates for diagnostic and/or therapeutical purposes as well as pharmaceuticals containing at least one chelate of the general formula I according to the invention, and, if required, the additives common in galenics.
For application to human beings, the compounds according to the invention are provided in the form of a cold or hot kit. The kit contains at least one chelating agent according to formula I in free or bound form, with ligands from the classes of ergolines, estradiols, endothelines, short-chain synthetic peptides, cholecystokinins being preferred.
ID6S3E . DOC
Examples General complexing with sodium gluconate 99mTc-gluconate:
2 ml of 99mTc generator eluate are added to 2 ml of 0.1 M
sodium gluconate solution and mixed with 10 ul of 0.01 M
SnCl2 (in 0.01 M HCl). The quantitative pertechnetate reduction is then checked using thin-layer chromatog-raphy.
TLC (silica gel/acetone): 99mTc-gluconate Rf: 0-0.1;
99mTCO4- Rf 0-9 Example 1 [99mTc]-mercaptoacetyl sarcosine Step 1: chloroacetyl sarcosine:
0.07 mol of sarcosine are dissolved in 50 ml of lN soda lye, and 0.08 mol of chloroacetyl chloride and 110 ml of lN soda lye are added in portions at 0C. Adding ends after 45 min, and the reaction solution is acidified using 20 ml of 5N hydrochloric acid.
The product is evaporated to dryness under reduced pres-sure, and the residue is three times extracted, each time using 50 ml of cold acetone. The acetone is removed under reduced pressure and the residue extracted with 100 ml of ether. The ether is evaporated and the remaining oil taken up with some ether and crystallized out under heavy stirring.
Yield: 77% of theor. q'ty TLC:
silica gel// butanol/ glacial acetic acid/ water 2:1:1 Rf: 0.65 (iodine vaporization) ID653e.DOC
~_ 33 21 73844 Step 2: benzoylmercaptoacetyl sarcosine:
0.02 mol of chloroacetyl sarcosine are dissolved under stirring in 750 ml of methanol in a protective gas atmosphere at room temperature. A solution of 0.041 mol of thiobenzoic acid dissolved in 20 ml of methanol and neutralized with sodium methylate, is slowly added by dropping; the batch is agitated for 12 more hours.
The solvent is removed under reduced pressure and the residue taken up with 2N hydrochloric acid. The reaction product precipitates as a brown oil that is separated from the supernatant solution and extracted with chloro-form. The chloroform is removed under reduced pressure,and ether is added to the residue. The reaction product is precipitated by cooling and intense agitation, fil-tered off, and dried on a porous plate.
pH titration and IR spectrum showed that the reaction product consisted of a mixture of 60% methyl ester and 40% of free amino acid.
Step 3: Separation of the protective group:
0.08 mmol of the mixture obtained are suspended in 2 ml of absolute methanol, then 2 mg of Pd/CaCO3 (5%) are added and reacted under stirring with a solution of 0.13 mmol of sodium methylate in 1 ml of absolute methanol at a hydrogen pressure of 66 kPa. The batch is once more agitated for 15 min and then neutralized with methanolic Dowex 50WX8. The catalyst and the resin are filtered off, the substance lyophilized and the impurities extracted with 2 ml of benzene. The benzene is subsequently sepa-rated, and the substance lyophilized from ethanol.
ID653E . DOC
The mixture obtained of ester and free acid is separatedthrough Sephadex G10. (Column 14 x 1000, 20 ml/h, RI-detector) Mercapto-acetyl-sarcosine-methyl ester is split off as second fraction, cf. Example 1 a).
Mercaptoacetyl sarcosine :
TLC:
silica gel//butanol/glacial acetic acid/water 2:1:1 Rf: 0.7 (ninhydrin) RP 18/methanol Rf: 0.85 (ninhydrin) H-NMR (DMSO) TMS ~2.9 (SH, lH), ~3.1 (N-CH3, 3H), ~3.9 (-CH2, 2H) Labelling of N-methyl-MAG1 Ca. 1 mg N-methyl-MAG1 dissolved in 200 ul water is added to 2 ml 99mTc gluconate solution. After 15-20 min reac-tion time purity is tested using TLC on silica gel 60/ 95 % ethanol.
Most activity takes place at an Rf = 0.1 (ca.80%); other peaks are Rf = 0.2 (ca.10%) and Rf = 0.4(ca.10%).
The electropherogram shows an anionic Tc-MAG1 complex having a relative mobility Upertechn. / UTcM~Gl of 0.2.
Example 1 a:
When separating the reaction mixture from Example 1, Step
be enabled by a complex variation. So they are costly regarding both the manufacture and the theoretical com-prehension of the individual complexes.
It is the problem of the invention to provide short-chain N-alkyl peptide chelating agents complexing at room temperature with technetium and rhenium across a wide pH
range, and forming both ionic and non-ionic complexes, and thus to provide compounds that do not have the disadvantages of known 99mTC_ and 180,188Re_CompleXeS
Requirements regarding exposure to radiation, stability, and solubility for applying these compounds to humans must herewith be met; at the same time, a method of representing them that is as simple as possible should be created, and a kit formulation of the compounds/con-jugates of the invention and their metal complexes be provided for clinical application.
This problem is solved according to the invention in that it provides compounds of the general formula I
R3-S-CH2-Co-NR1-[CH2-Co-NH]n-CH2-Co-R2 (I) with the exception of N-[2-(benzoylthio)acetyl]-N-cyclohexyl glycine wherein n represents the numbers 0,1 or 2, R1 is a straight-chain or branched alkyl residue contain-ing 1 to 20 carbon atoms which are optionally interrupted or replaced by one to three oxygen atoms, and which may include a terminal -COOH-, -OH- or -NH2 group that is optionally esterified or etherified by means of glycolic acid or its esters or ethers, ID54013 .DOC
the esters or ethers being formed using carboxylic acids or alcohols containing up to 18 carbon atoms, or represents a phenyl or cyclohexyl residue having at its 4-position, if required, a COOH, NH2 or OH group that is esterified, etherified or amidated using carboxylic acids or alcohols containing up to 6 carbon atoms, or replaced by a halogen atom, R2 is a halogen atom, a halogen methyl, methyl carboxyl, trifluoromethyl carboxyl, an NH2 or an OH group, the carboxyl group formed when R2 = OH being esterified or amidated either immediately, or after etherification with an a,~-hydroxycarboxylic acid containing up to 8 carbon atoms through its terminal carboxyl group, with a biomolecule, a steroid, an ergoline derivative, a benzodiazepine derivative, a cholecystokinin, a peptide, a protein, a proteo-hormone, an amino sugar, an endotheline, an endo-theline derivative, an endotheline antagonist or an endotheline fragment, is a residue of the general formula II
/\~ OH
¦ CH=CH--R4 ( I I ) /~/\/\/
R80~ ~
or the general formula II a " ~ ,OH
R O ~ C C R4 ID54 0~ . DOC
~ 6 where R4 is a methylene, propenylene amino, propinylene amino, methylene amino or methylenoxy group and R8 is a hydrogen atom or a methyl group, is a residue of the general formula III
Rs ,N ~ 7 or of the general formula III a ~, ~ ~, N ` R7 wherein are:
R5 an -NH-, -NH-CO-N<, -NH-CO-NH- or methylenoxy group, R6 a hydrogen atom, a halogen atom or a methyl group and ID5401~ .DOC
R7 a hydrogen atom or a straight-chain or branched alkyl residue containing up to 6 carbon atoms, R3 is a hydrogen atom, an acetyl, benzoyl, p-methoxybenzyl, acetamidomethyl, benzamidomethyl, trimethyl acetamidomethyl, hydroxyacetyl, ethoxyethyl, ethylthio, trityl or an easily separable sulfur-protective group and their salts formed from pharmaceutically acceptable acids or bases.
Surprisingly, a class of compounds was found that does not only avoid the disadvantages mentioned above but as 99mTc+5 complexes comprises a transition from N3S -> N2SO
-> S2O2 -> N2S2-N-alkyl peptide complex system and can therefore be used as complexing agents at a wide range.
Formula IV shows an N3S-N alkyl peptide complex in the 99mTc core (neutral) from R3S- CH2-CO-NR1[CH2-CO-NH]2-CH2-CO-R
~ "~
,Tc ) ~IV) ~""' ""`~
~R2 formula V shows a N2OS-N alkyl peptide complex in the 99mTc core (neutral) from R3S-CH2-Co-NRl[CH2-CO-NH]l-CH2-COOH, ID653E . DOC
8 .2 1.7 3 8 4 4 ~ ,~ (V) ~ ,T c J
~"' "`~
formula VI shows an O2S2_N alkyl peptide complex in the 99mTc core (having a negative charge) from R3S-CH2-Co-NRl[CH2-CO-NH]o--CH2-COOH, ~ O ,Q~
( Vl ) R1 _ ~ ,Tc N Rl ` J
`S' ~
and formula VII eventually shows a N2S2-N alkyl peptide complex in the 99mTc core (having a positive charge) from R3S-CH2-Co-NRl[CH2-Co-NH]o-CH2-Co-R2.
~ /R1 R1 ~
O ~1~ G ,N~ O
, Tc ~ ( Vll \\S"""'"""`~//
ID653E.DX
~ 21 73844 `
The complexes that result from the molecule variation show a surprising range and correspondence with regard to their biological behaviour. Thus compounds of this type, when coupled with a bioligand, are suited for receptor visualization, which has not yet been described for 99mTc compounds, and excellently suited for plaque visualiza-tion with vascular lesions and for measuring the kidney function, with the option that the compounds may be coupled with bioactive ligands.
Compounds are preferred in which R1 is a -CH2-(CH2)m-CH3 group with m = 1 - 14, or R1 is a phenyl, a p-phenyl amine, a cyclohexyl amine, p-hydroxyphenyl, 4-hydroxycyclohexyl, p-halogen phenyl or 4-halogen cyclohexyl group.
Furthermore, compounds of the general formula I are preferred in which R1 equals ~~CH2)q~OOC~CH2~OOC~(CH2)r~CH
q and r being integers from 1 to 16.
Such compounds of the general formula I are particularly preferred in which R1 is a straight-chain or branched alkyl residue containing 1 to 6 carbon atoms, or a P-bromophenyl residue.
Furthermore, compounds of the general formula I are pre-ferred in which R2 is a residue of the general formula II
ID540B .D0~
2 1;7f3~8;4:4 OH
~CH=CH--R4 ( I I ) R ~o~/
or of the general formula II a " ~ ~OH
R O ~ C _ C R4 (ll where R4 is a methylene, propenylamino, propinylamino, methylen~m;no or methylenoxy group and R8 is a hydrogen atom or a methyl group.
17a-ethinyl estradiols or their 3-methyl ethers are preferred among these steroids.
A 17a-ethinyl group of the estradiol may also be coupled with an ethene. The order of ethyne and ethene groups can be changed; doubling the ethyne or ethene group is useful as well. A minimum distance between the chelating agent and the estradiol molecule, preferably a rigid ethyne or ethene group, is important to prevent folding of the lateral chain.
If I is bonded to this preferred 17a-position of the estradiol skeleton, little or no reduction of bioactivity can be expected from the resulting chelate complex-ID540B . DOC
11 21 7~844 bioligand compounds as documented, for example, inEpperly, M. W. et al [J. Steroid Biochem. Molec. Biol.
vol. 39, no. SA, 729-734, 1991].
The outstanding significance of these estrogen chelate complexes is based on their ability to penetrate through the cell wall and become attached to the estrogen recep-tor. This applies, in particular, to complexing agents according to the general formula I with n - 2. These N3S
complexes are neutral at 99mTc+5 as a result of N1 alky-lation in the Tc core (cf. formula IV). While charged complexes such as the N3S complex as 17a-ethinyl chelate steroid of the thioacetyl-glycyl-glycyl-glycyl-0-ester type (own tests) are not able to penetrate the cell wall, the Nl alkylated complexes (N1 = methyl) of the invention penetrate through the cell wall and attach themselves in the uterus after an i.v. injection. Their concentration, in terms of the blood/uterine quotient, is 0.2 and can be varied depending on the length of the N1 alkyl chain or substituents. Thls facilitates early detection of mamma-ca according to the SPECT method. The compound according to Example 10 is particularly preferred here.
Furthermore, compounds of the general formula I are preferred in which R2 is a residue of the general formula III R5 ID653e . DOC
or of the general formula III a N~R7 where R5 is an -NH-, -NH-CO-N<, -NH-CO-NH- or methylenoxy group, R6 a hydrogen atom, a halogen atom or a methyl group and R7 is a straight-chain or branched alkyl residue containing up to 6 carbon atoms.
These compounds of the general formula III and III a are ergoline derivatives.
8a-amino-6-methyl-ergoline having, if required, alkyl substituents at the 2- and/or 6-positions with a chain length of C1 at position 2 or C1_3 at position 6 and/or a methyl- or halogen substituent at position 2, is pre-ferred among these ergolines. The substituent at position8 of the ergoline may, apart from aNH2, also be ~-CH2O-or aNH-CO-N< or aNH-co-NH-.
ID653E .DOC
~ 13 21 73844 The compounds according to the invention of the general formula I carry a R3 residue at the terminal sulfur atom.
This R3 residue is a sulfur protective group which is required during the synthesis of the compounds according to the invention of general formula I. Such a protective group should be easily separable. Appropriate R3 residues are, for example, acetyl, benzoyl, p-methoxybenzyl, acetamidomethyl, benzamidomethyl, trimethyl acetamido-methyl, hydroxyacetyl, ethoxyethyl, or trityl groups. The benzoyl group is particularly preferred.
Another object of the present invention are the metal-chelate complexes that radioactive metal ions form with the compounds according to the invention of the general formula I wherein R1, R2 and R3 are as defined above.
Suitable radioactive metal ions are, for example, radionuclide ions of Tc, Re, Cu, Ga, Gd, Y and In. The radionuclide is selected according to the desired appli-cation of the metal-chelate complexes according to the invention of the general formula I.
Different radionuclides are used for radiodiagnostics or radiotherapy.
Radioactive metal ions of Tc and Re isotopes are preferred here.
The radionuclide technetium-99m is particularly preferred.
Another object of the present invention are conjugates containing compounds of the general formula I or metal-chelate complexes ~ormed by radioactive metal ions of Tc, Re, Cu, Ga, Gd, Y and In and compounds of the general formula I, and substances that accumulate selectively in ID65~e .DOC
~ 14 2 1 73844 diseased tissue or in tumours, with a covalent bonding existing between said substances that is amidic for substances containing carboxy or amino groups such as peptides, proteins, antibodies or their fragments, ester-S like for substances containing hydroxy groups such asfatty alcohols, and imidic for substances containing aldehyde groups.
Conjugates are preferred having peptides that accumulate in diseased tissue such as endothelines, partial endo-theline sequences, endotheline analogues, endotheline derivatives, or endotheline antagonists.
Particularly preferred are such conjugates containing compounds of the general formula I and peptides that accumulate selectively in diseased tissue in which the peptides comprise the following sequences:
c ~ er-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, c ~ er-cys-ser-ser-trp-leu-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, c ~ hr-cys-phe-thr-tyr-lys-asp-lys-glu-cys-val-tyr-tyr-cys-his-leu-asp-ile-ile-trp, c ~ er-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, c ~ er-cys-asn-ser-trp-leu-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, ID65 31~ . DOC
cys-ser-cys-lys-asp-met-thr-asp-lys-glu-cys-leu-asn-phe-cys-his-gln-asp-val-ile-trp, ala-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, ala-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, cys-ser-cys-ser-ser-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, or the partial 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).
Another object of the present invention is a method for manufacturing the compounds according to the invention of the general formula I
ID5401~.DOC
- ` 21 73844 R3-S-CH2-Co-NR1-[CH2-Co-NH]n-CH2-Co-R2 (I) with the exception of N-[2-(benzoylthio)acetyl]-N-cyclohexyl glycine wherein n represents the numbers 0,1 or 2, R1 is a straight-chain or branched alkyl residue contain-ing 1 to 20 carbon atoms which may optionally be interrupted or replaced by one to three oxygen atoms, and which may optionally include a terminal -COOH, -OH or -NH2 group that may optionally be esterified or etherified by means of glycolic acid or its esters or ethers, these esters or ethers being formed using carboxylic acids or alcohols containing up to 18 carbon atoms, or represents a phenyl or cyclohexyl residue having an optional COOH, NH2 or OH group at its 4-position that is optionally esterified, etherified or amidated using carboxylic acids or alcohols containing up to 6 carbon atoms, or carries an additional halogen atom, R2 is a halogen atom, a halogen methyl, methyl carboxyl, trifluoromethyl carboxyl, an NH2 or an OH group, the carboxyl group formed when R2 = OH being esteri-fied or amidated either immediately, or after etherification with an a,~-hydroxycarboxylic acid containing up to 8 carbon atoms through its terminal carboxyl group, with a biomolecule, a steroid, an ergoline derivative, a benzodiazepine derivative, a cholecystokinin, a peptide, a protein, a proteo-hormone, an amino sugar, an endothelin, an endothelin ID54 OB . DOC
derivative, an endothelin antagonist or an endothelin fragment, is a residue of the general formula II
S
CH = CH--R4 ( I I ) ,o J~
or of the general formula II a ~____,OH
R ~o~J c=c_ R~ ( ~
where R4 is a methylene, propenylen~m;no, propinylen-amino, methylen~m;no or methylenoxy group and R8 is a hydrogen atom or a methyl group, is a residue of the general formula III
Rs or of the general formula III a ID54011 .DOC
21 ?3844 ~ 18 ¢~N ~ R7 wherein R5 is a -NH-, -NH-CO-N<, -NH-CO-NH- or methylenoxy group, R6 is a hydrogen atom, a halogen atom or a methyl group and R7 is a hydrogen atom or a straight-chain or branched alkyl residue containing up to 6 carbon atoms, R3 is a hydrogen atom, an acetyl, benzoyl, p-methoxy-benzyl, acetamidomethyl, benzamidomethyl, trimethyl acetamidomethyl, hydroxyacetyl, ethoxyethyl, ethylthio, trityl or an easily separable sulfur protective group and their salts formed from pharmaceutically acceptable acids or bases characterized in that a~ a diketo piperazine derivative of glycine or b) glycine ID65 3E . DOC
is first reacted with a haloacid halogenide and then with an alkyl amine or aryl amine of the general formula VI
R1 - NH2 (VI) where R1 is as defined above, is reacted again with a haloacid halogenide and then reacted with a compound of the general formula IV
R3 - SH (IV) where R3 is as defined above, or a compound of the general formula V
Rl-NH-CH2-CO-R2 (V) where R2 is as defined above, is reacted with a haloacid halogenide and then with an alkyl amine or aryl amine of the general formula VI
R1 - NH2 (VI) where R1 is as defined above, and reacted again with a haloacid halogenide and then with a compound of the general formula IV
R3 - SH (IV), where R3 is as defined above, and that these compounds are converted, if required, into a salt by means of a pharmaceutically acceptable acid or base.
The compounds according to the invention of the general formula I may also be manufactured when starting from a ID6531~ . DOC
compound according to the invention of the general formula I, where n is 0 or 1, and reacting it with a compound comprising terminal groups thereby forming a residue R2 after coupling with the above compound. This yields compounds according to the invention of general formula I in which n is 1 or 2. This means that part of the chain of compounds of the general formula I according to the invention is provided by the R2 residue added by coupling.
Another object of this invention is the manufacture of the metal-chelate complexes according to the invention consisting of radioactive metal ions and compounds of the general formula I according to the invention.
Compounds of the general formula I are reacted in a generally known way, optionally with prior or simultaneous splitting off of the R3 residue.
Metal-chelate complexes of radioactive metal ions of Tc and Re and compounds of the general formula I are manu-factured by reacting technetium-99m or Re in the form of pertechnetate or perrhenate in the presence of a reduc-tant and, optionally, an auxiliary ligand with a compound of the general formula I
R3-S-CH2-Co-NR1-[CH2-Co-NH]n-CH2-Co-R2 (I) with the exception of N-[2-(benzoylthio)acetyl]-N-cyclohexyl glycine where R1, R2 and R3 are as defined above.
The surprising variability of the compounds and their complexes is of particular relevance to the invention.
An N3S system is obtained for compounds according to the ID5401~ DX
2t 73844 general formula I with n = 2. When changing from n = 2 to n = 1, an N2SO-N-alkyl system can form that has a surprising complex stability. Finally, a chelate complex may form during complexing at n = 0 from the NlS1-N-alkyl system which, on the one hand, presents a N2S2-N-alkyl system or, as another dimer, is an O2S2-N-alkyl complex, without requiring a chemically covalent bridge to connect the complex.
The new compounds also have a wide range of clinical applications due to their chemical variability and their complexing variants.
Until today there is no method for visualizing in vivo the formation of plaques as a sign of beginning athero-sclerosis. Atherosclerosis is a widespread disease and an early stage of coronary heart disease which causes the death of about 40 ~ of the population in modern indus-trial nations. It is therefore particularly desirable tovisualize vascular lesions using a simple device. The N-alkyl chelates according to the invention may thus be used for early detection of atherosclerotic vascular diseases.
The quotient of activity in the plaque and the intact vessel obtained in an animal test (WHHL-type rabbits with artificially generated atherosclerotic changes of the vessels; WHHL rabbits show high levels of LDL in the blood due to a lacking or defective LDL receptor and thus develop atherosclerotic changes of the vessels) after i.v. application of compounds according to the general formula I wherein R2 is an endotheline, partial endo-theline sequence, endotheline analogue, endotheline derivative, or endotheline antagonist, was 1 : 5 (cf.
figures 1 and 2).
~ ~ 21 73844 21 a It was highly surprising that excretion had taken place after only 3 hours despite the lipophilia of the complex resulting from the R1 alkyl chain with a length of C6.
IDS~OI~ .DOC
Lesions in the aortic area can be visualized in auto-radiograms of an amazingly good quality. There is an af-finity for the plaques or their receptors due to the fatlike molecules which is strong enough to maintain adhesion to the plaques in spite of the falling concen-tration of the compound in the blood due to its excretion via the liver.
The affinity for plaques in atherosclerotically changed vessels may be influenced by varying the R1 alkyl group.
Lipophilia may thus be controlled through the character-istics of the residue in R1 while R2, as a free carboxyl group, provides the conditions for good solubility in water. As an alternative to R2 being a free carboxylic acid, amidation of this carboxyl group with an amino hydrocarbon can contribute to improving water solubility.
It is known that proteins, in particular, short-chain proteohormones such as endothelines lose their potency when coupled with a chelating agent via a covalent bonding. The behaviour of the compounds according to the invention after covalent bonding to endothelines, their antagonists or fragments therefore came completely unex-pected (Examples 11, 12).
The way of generating the N-alkyl complexing agents of the general formula I according to the invention using endothelines from preliminary stages of chelating shown herein (Examples 11 and 12) is a new method of generating ~h complexing positions. The new N-alkyl complexes of the general formula I have a similar stability as that of known N3S complexes.
Non-invasive techniques to diagnose atherosclerosis have been described, especially techniques involving radio-iodine labelling. Antibodies labelled with radionuclides ID653E.DOC
r 23 or labelled "low density lipoproteins" (LDL) were pre-sented that bond to atherosclerotic wall areas (Lees et al. 1983, J. Nucl. Med. 24, 154 -156, Kaliman et al.
1985, Circulation, 72, 300; Virgolini et al. 1991, Eur.
J. Nucl. Med., 18, 944-947). These methods, however, still retain major disadvantages as, for example, the antigenicity of the antibodies in the system or the long time required to isolate, clean, and label the LDL from the patient's blood (several days). Most disadvantageous of all, these big molecules have a long half-life in the blood which, in conjunction with high background radia-tion in the body as a whole, makes localization of the atherosclerotic lesions difficult if not impossible. Shih et al. (1990, Proc. Natl. Acad. Sci., 87, 1436 - 1440) synthetized partial sequences of the LDL protein portion (apo-B-100) that still bond to the atherosclerotic plaques but have a much shorter half-life in the blood and an improved signal-to-noise ratio. Due to too low an affinity to the plaque and/or low density of the bonding places of these peptides in the plaque, successful in vivo diagnosis of atherosclerosis could not be demon-strated using these apo-B-peptides.
These compounds share, in general, the disadvantage of radioactive iodine-123. They are not readily available as they are produced in a cyclotron and have a physical half-life of 13 hours, and they are easily separated in the system. So it was surprising to find that the compounds of the general formula I according to the invention, after covalent bonding to NH groups of endotheline and complexing with 99mTc showed good accumulation in atherosclerotic plaques. An advantage that is particularly striking is that the n = 1 and n = 0 compounds generate new N-alkyl peptide complexing agents after covalent bonding to endothelines by means of an amidation, involving the NH and SH groups, if any, of the ID653E.DOC
respective endotheline that are located in appropriate positions.
When reacting the compounds according to the invention with dopamine-like bioactive molecules, surprisingly receptor affinity of these compounds from the ergoline family is not lost; in preliminary stages, both with and without D2 receptor affinity, there was even achieved sufficient brain passage to facilitate visualization of the D2 receptors. Endeavours to visualize receptors in the brain using 99mTc+5 complexes have failed so far. For example, when 8a-amino-6-methyl ergoline is reacted with mercapto-acetyl-glycyl-glycyl-glycin to yield 8a-amide, and complexed using 99mTc+5 after i.v. injection to the rat (Wistar), there is no uptake in the brain.
But if this reaction is carried out using the compounds of the general formula I according to the invention where R1 = methyl and amidation or esterification takes place via R2, 0.1 to 0.5% of the dose are accumulated in the brain in analogous conditions.
O [NH--CO--CHz]2--N--CO--CH2--S--R3 ~/ I
¢~ CH~
HN
New structures of N-alkyl-peptide complexes are formed with n = 1 and n = 0 which, interacting with the bioactive molecule, surprisingly imitate the structures of dopamine-like compounds and accumulate in the brain in large quantities that can be used to visualize the D-receptors (formula IX).
ID653e .DOC
~/ I
HN
The compounds of the invention are manufactured according to a method known in the art [A Specialist Periodical Report; Amino-acids, Peptides and Proteins;
The Chemical Society, Burlington House, London, WlVOBN].
Manufacture of compounds with R1 = CH3 and R2 = OH
starts from sarcosine that is reacted with chloroacetyl chloride. The resulting chloroacetyl sarcosine is reacted with thiobenzoic acid in the common way, thus yielding structures with n = 0 R S-cH2-co-N-(cH2-co-NH)o-cH2-coR2 Compound A
Compounds with n = 2 are manufactured by reacting diketo-piperazine of glycine with chloroacetyl chloride. The resulting chloroacetylglycyl glycine is subsequently reacted with N-methyl amine or, to insert longer resi-dues, with N-alkyl amines, phenyl amines, alkyl-oxa-alkyl amines, cycloalkyl amines or glycine glycolate. Re-reacting with chloroacetyl chloride and subsequent thiobenzoylation results in compounds of the formula n =
2.
ID653E.DOC
R3S-CH2-Co-N-(CH2-CO-NH)2-CH2-COR2 /
Rl Compound B
Rl represents the variations in the sarcosyl-nitrogen atom The respective compounds with n = 1 are obtained by reacting glycine salts with N-protected sarcosyl com-pounds or by reacting glycine with chloroacetyl chloride and subsequent aminolysis of the chloromethyl group. Re-reacting with chloroacetyl chloride and subsequent thiobenzylation yields the title compounds with n = 1.
R3S-CH2-Co-N-(CH2-CO-NH)l-CH2-COR2 /
Rl Compound C
Compounds A, B, C do not react in the usual way with coreactants carrying amino groups or hydroxyl groups. For this purpose, A to C are dissolved in N-methyl pyrroli-done, pre-activated using BOP (B. Castro et al.
Tetrahedron Letters 14 (1975) 1219) or TBTU (Knorr et al.
Tetrahedron Letters 30 (1989), 1927), and then reacted with the amino component. Esterification is preferably carried out using dicyclohexyl carbodiimide in the presence of dimethyl aminopyridine. For the synthesis of compounds having an estrogen as bioligand, one starts from an estrogen that already carries an unsaturated substituent in 17a such as a propargyl amine or propar-gylyalcohol residue and reacts it with compounds A to C
in the way described.
ID653E.DOC
Compounds A to C are reacted with peptides, especially with endotheline components, according to the methods common in peptide chemistry, either conventionally in solution, preferably in dimethyl formamide, dimethyl acetamide, N-methyl pyrrolidone, dimethyl sulfoxide, or mixtures of these components. When using the solid-phase method, the amino component may be acylated in the usual conditions with the synthesis resin. Splitting off the synthesis resin yields the desired compounds. Preparative cleaning, if required, is carried out by chromatographing on an RP 18 column in a water / acetonitrile gradient containing 0.1% trifluoroacetic acid.
Ergolines carrying amino or hydroxy groups are reacted analogously to the peptide reactions in solution. Reac-tions were carried out in N-methyl pyrrolidone. Prepara-tive cleaning is carried out as described for peptides.
The other R4 residues are obtained after building the protected dipeptides A - C by reacting Rl, with R2 = OH, following activation using NH2-CH2Cl, NH2-CH2-CH=CHJ or NH2-CH2-C=CJ
and, after the palladium-O reaction, C-C-bonding the 17 -ethinyl steroid with the desired complexing agent.
Complexes are preferably formed by reducing Na pertechne-tate with, for example, tin(II) chloride or dithionide in an acqueous medium at a pH value between 6 and 9 and at room temperature; if required, using an auxiliary ligand ID653e.DOC
such as sodium citrate or sodium tartrate, with the protective group R3 being split off either prior to the reaction or in situ.
Activation of the R2 carboxyl group that is formed when R2 = OH, or its replacement by an NCS or similar reactive group results in covalent bonding of peptide chelates to a bigger protein or proteohormone, endotheline, endo-theline analogue, or endotheline antagonist, endotheline derivative, partial endotheline sequence, and subsequent complexing.
As documented in the manufacturing instructions, the 99mTc and 186~188Re complexes of the invention can be produced at room temperature, neutral to weakly alkaline pH, and with or without re-chelating (e.g. via a hepta-gluconate-99mTc complex) at yields of 95%. Their easy salification via a free cation ensures good solubility in water.
Manufacture of the metal complexes: The protective group is split off according to the methods known in the art (see "Protective groups in organic synthesis" T.N.
Greene, John; Wiley and Sons 1981).
The usual quantity employed for applications in human diagnostics is between 370 MBq and 1850 MBq, preferably 740 to 1480 MBq. The compounds of the invention are pro-vided in the form of a kit designed for application to human beings. This kit contains at least one chelating agent according to the general formula I, either free or bonded to ligands.
Another object of the invention is a cold kit containing a chelating agent of the general formula I that is either free or bonded to a bioactive molecule such as an ergo-ID653E~ . DOC
line, estradiol or endotheline derivative, and is capable of bonding metal atoms.
Splitting off the protective groups that were introduced intermediately to carry out the synthesis often makes difficulties. As splitting off the -S- protective group (R3) is accompanied by simultaneous splitting of the pep-tide chain, this process results in serious damaging or useless fragments especially in the case of chelating agents with peptide bondings; so it was all the more sur-prising that up to 80% were split off in the presence of hydrogen/Pd/CaC03 together with the usual alkaline, i.e.
protective group separation in hydrogenation conditions.
This decisive improvement of free chelate complex synthe-sis is important because protective groups can be split off before formulating the kit which is a major improve-ment in the technique of gaining chelating agents that are free from protective groups.
Example 3 describes the separation method according to the invention. While the conventional method with alka-line yielded only a mixture that was hard to separate, protective group separation with alkaline in the presence of hydrogen/Pd/CaC03 resulted in a ~ 100% yield contain-ing 80% of the desired material.
It shall be mentioned, for example, that the reaction with amines or alcohols is carried out by activating the carboxylic acid R2 = OH applying methods known in the art.
Activated groups are, for example: acid chlorides, mixed anhydrides [Org. Prep. Proc. Int. 1975, 7, 215], acti-vated esters [Adv. Org. Chem. Part B, 472]. These can be linked with biomolecules through linkers, for example 2~ 7~844 using the carbodiimide method (Fieser, Reagents for Organic Synthesis 10, 142). Linking takes place in such a way that a covalent bond is formed between the chelate and the biomolecule.
Where steroids are used as biomolecules, estradiol derivatives are preferred that have substituents in the 17a position. Synthesis of such compounds has been described, for example, by Blickenstaff for the 17~-ethinyl derivatives of estradiol. (Blickenstaff A.;
Steroids 46, 889 (1985); USP 462.426). Synthesis of estradiol derivatives with 17a-propargyl substituents having a terminal NH2 or OH function is described by Blickenstaff, Brandes and Poirier. (Blickenstaff et al.;
Steroids 48, 223 (86); Brandes, A. et al. Dissertation 87-01441, 1986, University of Illinois, D. Poirier et al.
J. Steroids. Biochem. Molec. Biol. 38, no. 6, 759-774, 1991) .
A survey of linking triple and double bonds, e.g. after a palladium-0 reaction, is given in Aldrichchimica Acta, vol. 15, no. 1, 1982; Shun-ichi Murahashi et al., Stephen A. Godleski, Tetrahedron Letters, vol. 22, no. 24, pp 2247-2250, 1981; Tetrahedron Letters, vol. 29, no. 24, 2973-2976, 1988. For example, I with R2 = - NH-CH2-C-CH
is reacted during coupling with l7a[2-halogen-ethenyl]
estradiol according to the references given.
It is known from literature that ergot alkaloids of the 6-methyl-8-substituted ergoline type have a wide range of chemical variation without a clearly recognizable connec-tion between an effect and the substituents at position 8 [Ergot Alkaloids and Related Compounds, Editors: B. Berde and H.O. Schild, Springer-Verlag, Berlin, Heidelberg, New York, 1978].
ID653E . DOC
Ergoline is a preferred dopamine-like ligand. A survey of ways for synthesizing 8-substituted ergolines is given in Ergot Alkaloids and Related Compounds, Editors B. Berde and H.O. Schild, Springer-Verlag Berlin, Heidelberg, New York, 1978, Chapter II Chemical Background, J. Ruisemmann and P.A. Stadler.
Reaction with 8a-amino- or 8~-hydroxy-methyl ergoline is preferred. It takes place via an activated group in R2, for example, benzotriazol-1-yl-tetra-methyl-uronium-tetrafluoroborate, or according to the known carbodiimide method.
New complexes are generated when the n = 1 and n = 0 complexing or partial complexing agents are applied according to the invention including the nitrogen in the 8a-amino and 6-N-methyl groups of ergoline.
Another object of the invention is the use of metal chelates for diagnostic and/or therapeutical purposes as well as pharmaceuticals containing at least one chelate of the general formula I according to the invention, and, if required, the additives common in galenics.
For application to human beings, the compounds according to the invention are provided in the form of a cold or hot kit. The kit contains at least one chelating agent according to formula I in free or bound form, with ligands from the classes of ergolines, estradiols, endothelines, short-chain synthetic peptides, cholecystokinins being preferred.
ID6S3E . DOC
Examples General complexing with sodium gluconate 99mTc-gluconate:
2 ml of 99mTc generator eluate are added to 2 ml of 0.1 M
sodium gluconate solution and mixed with 10 ul of 0.01 M
SnCl2 (in 0.01 M HCl). The quantitative pertechnetate reduction is then checked using thin-layer chromatog-raphy.
TLC (silica gel/acetone): 99mTc-gluconate Rf: 0-0.1;
99mTCO4- Rf 0-9 Example 1 [99mTc]-mercaptoacetyl sarcosine Step 1: chloroacetyl sarcosine:
0.07 mol of sarcosine are dissolved in 50 ml of lN soda lye, and 0.08 mol of chloroacetyl chloride and 110 ml of lN soda lye are added in portions at 0C. Adding ends after 45 min, and the reaction solution is acidified using 20 ml of 5N hydrochloric acid.
The product is evaporated to dryness under reduced pres-sure, and the residue is three times extracted, each time using 50 ml of cold acetone. The acetone is removed under reduced pressure and the residue extracted with 100 ml of ether. The ether is evaporated and the remaining oil taken up with some ether and crystallized out under heavy stirring.
Yield: 77% of theor. q'ty TLC:
silica gel// butanol/ glacial acetic acid/ water 2:1:1 Rf: 0.65 (iodine vaporization) ID653e.DOC
~_ 33 21 73844 Step 2: benzoylmercaptoacetyl sarcosine:
0.02 mol of chloroacetyl sarcosine are dissolved under stirring in 750 ml of methanol in a protective gas atmosphere at room temperature. A solution of 0.041 mol of thiobenzoic acid dissolved in 20 ml of methanol and neutralized with sodium methylate, is slowly added by dropping; the batch is agitated for 12 more hours.
The solvent is removed under reduced pressure and the residue taken up with 2N hydrochloric acid. The reaction product precipitates as a brown oil that is separated from the supernatant solution and extracted with chloro-form. The chloroform is removed under reduced pressure,and ether is added to the residue. The reaction product is precipitated by cooling and intense agitation, fil-tered off, and dried on a porous plate.
pH titration and IR spectrum showed that the reaction product consisted of a mixture of 60% methyl ester and 40% of free amino acid.
Step 3: Separation of the protective group:
0.08 mmol of the mixture obtained are suspended in 2 ml of absolute methanol, then 2 mg of Pd/CaCO3 (5%) are added and reacted under stirring with a solution of 0.13 mmol of sodium methylate in 1 ml of absolute methanol at a hydrogen pressure of 66 kPa. The batch is once more agitated for 15 min and then neutralized with methanolic Dowex 50WX8. The catalyst and the resin are filtered off, the substance lyophilized and the impurities extracted with 2 ml of benzene. The benzene is subsequently sepa-rated, and the substance lyophilized from ethanol.
ID653E . DOC
The mixture obtained of ester and free acid is separatedthrough Sephadex G10. (Column 14 x 1000, 20 ml/h, RI-detector) Mercapto-acetyl-sarcosine-methyl ester is split off as second fraction, cf. Example 1 a).
Mercaptoacetyl sarcosine :
TLC:
silica gel//butanol/glacial acetic acid/water 2:1:1 Rf: 0.7 (ninhydrin) RP 18/methanol Rf: 0.85 (ninhydrin) H-NMR (DMSO) TMS ~2.9 (SH, lH), ~3.1 (N-CH3, 3H), ~3.9 (-CH2, 2H) Labelling of N-methyl-MAG1 Ca. 1 mg N-methyl-MAG1 dissolved in 200 ul water is added to 2 ml 99mTc gluconate solution. After 15-20 min reac-tion time purity is tested using TLC on silica gel 60/ 95 % ethanol.
Most activity takes place at an Rf = 0.1 (ca.80%); other peaks are Rf = 0.2 (ca.10%) and Rf = 0.4(ca.10%).
The electropherogram shows an anionic Tc-MAG1 complex having a relative mobility Upertechn. / UTcM~Gl of 0.2.
Example 1 a:
When separating the reaction mixture from Example 1, Step
3, 20 ~ mercapto-acetyl-sarcosine-methyl ester in the form of a colourless oil are obtained.
ID653E . DOC
( 35 21 73844 Example 2 Mercapto-acetyl-hexylaminoacetic acid Step 1: Hexylaminoacetic acid 0.1 mol hexylamine is added to 0.021 mol chloroacetic acid and allowed to stand at room temperature for 5 days.
The thick syrup is then stirred into 500 ml of acetone and crystallized in the form of white lamellae. Yield 2.1 g (63 % of theor. q'ty) TLC:
silica gel//butanol/glacial acetic acid/water 2:1:1 Rf: 0.6 (ninhydrin) Step 2: Chloroacetyl-hexylaminoacetic acid:
0.01 mol hexylaminoacetic acid is dissolved in 10 ml of lN soda lye, cooled, then alternately mixed with 1.5 ml (1 ml = 0.012 mol) of chloroacetyl chloride and 20 ml of lN soda lye at 0C. The reaction is ended after 30 min.
The batch is stirred on for about 30 minutes while heating moderately. Then it is acidified using 3 ml of 5N
hydrochloric acid; the precipitating brown oil is sepa-rated.
The oil is several times extracted with hot water and used as white oil for the next step of the synthesis without further cleaning.
TLC:
silica gel//butanol/glacial acetic acid/water 2:1:1 Rf: 0.75 (iodine vaporation) 11~653i~ . DOC
21-738~4 Step 3: Benzoylmercaptoacetyl-hexylaminoacetic acid:
0.018 mol chloroacetyl-hexylamino-acid are stirred in 500 ml methanol in a nitrogen atmosphere. 0.036 mol thioben-zoic acid are neutralized with sodium methylate and added to the solution within 30 min. For 12 more hours the batch is agitated in a protective gas atmosphere. The methanol is removed under reduced pressure, the residue acidified using 2N hydrochloric acid, and the precipitat-ing yellow oil separated.
Yield: 370 mg TLC:
silica gel//butanol/glacial acetic acid/water 2:1:1 Rf: 0.5 Example 3 [99mTc] Mercaptoacetyl-sarcosyl-diglycine Step 1: Chloroacetyl diglycine:
10 g of glycine anhydride are dissolved at room tempera-ture in 50 ml of 2N soda lye in a 250 ml three-neck bottle. The solution is cooled down to 0 C after 45 min, and 11,1 g of chloroacetyl chloride and 24 ml of 5N soda lye are alternately dropped in under continuous agitating and cooling. Addition is ended after 45 min. Subsequent-ly, 40 ml of 5N hydrochloric acid are added which causes the solution to lose its colour while the substance begins to crystallize. It is allowed to stand for one more hour at 0 C and filtered off by suction. It is washed several times in cold water and recrystallized from hot water.
Yield: 5 g ID653E . DOC
r 37 21i3844 The mother liquor is evaporated under reduced pressure.
The precipitate is recrystallized from water.
Yield: 2.5 g Melting point: 173 C
TLC: silica gel//n-butanol/glacial acetic acid/water/
ID653E . DOC
( 35 21 73844 Example 2 Mercapto-acetyl-hexylaminoacetic acid Step 1: Hexylaminoacetic acid 0.1 mol hexylamine is added to 0.021 mol chloroacetic acid and allowed to stand at room temperature for 5 days.
The thick syrup is then stirred into 500 ml of acetone and crystallized in the form of white lamellae. Yield 2.1 g (63 % of theor. q'ty) TLC:
silica gel//butanol/glacial acetic acid/water 2:1:1 Rf: 0.6 (ninhydrin) Step 2: Chloroacetyl-hexylaminoacetic acid:
0.01 mol hexylaminoacetic acid is dissolved in 10 ml of lN soda lye, cooled, then alternately mixed with 1.5 ml (1 ml = 0.012 mol) of chloroacetyl chloride and 20 ml of lN soda lye at 0C. The reaction is ended after 30 min.
The batch is stirred on for about 30 minutes while heating moderately. Then it is acidified using 3 ml of 5N
hydrochloric acid; the precipitating brown oil is sepa-rated.
The oil is several times extracted with hot water and used as white oil for the next step of the synthesis without further cleaning.
TLC:
silica gel//butanol/glacial acetic acid/water 2:1:1 Rf: 0.75 (iodine vaporation) 11~653i~ . DOC
21-738~4 Step 3: Benzoylmercaptoacetyl-hexylaminoacetic acid:
0.018 mol chloroacetyl-hexylamino-acid are stirred in 500 ml methanol in a nitrogen atmosphere. 0.036 mol thioben-zoic acid are neutralized with sodium methylate and added to the solution within 30 min. For 12 more hours the batch is agitated in a protective gas atmosphere. The methanol is removed under reduced pressure, the residue acidified using 2N hydrochloric acid, and the precipitat-ing yellow oil separated.
Yield: 370 mg TLC:
silica gel//butanol/glacial acetic acid/water 2:1:1 Rf: 0.5 Example 3 [99mTc] Mercaptoacetyl-sarcosyl-diglycine Step 1: Chloroacetyl diglycine:
10 g of glycine anhydride are dissolved at room tempera-ture in 50 ml of 2N soda lye in a 250 ml three-neck bottle. The solution is cooled down to 0 C after 45 min, and 11,1 g of chloroacetyl chloride and 24 ml of 5N soda lye are alternately dropped in under continuous agitating and cooling. Addition is ended after 45 min. Subsequent-ly, 40 ml of 5N hydrochloric acid are added which causes the solution to lose its colour while the substance begins to crystallize. It is allowed to stand for one more hour at 0 C and filtered off by suction. It is washed several times in cold water and recrystallized from hot water.
Yield: 5 g ID653E . DOC
r 37 21i3844 The mother liquor is evaporated under reduced pressure.
The precipitate is recrystallized from water.
Yield: 2.5 g Melting point: 173 C
TLC: silica gel//n-butanol/glacial acetic acid/water/
4:1:1 Rf: 0.75 (iodine vaporation) IR (solid in KBr):
vc=ol636, 1676; ~NH 1550; VCOOH 1708 cm Step 2: Sarcosyl diglycine
vc=ol636, 1676; ~NH 1550; VCOOH 1708 cm Step 2: Sarcosyl diglycine
5 g of chloroacetyl diglycine are mixed with 15 ml of 33%
acqueous methylamine solution and allowed to stand for 2 days at room temperature. Then the solution is evaporated to syruplike consistency under reduced pressure, the syrup heated in a water-bath and mixed with 50 ml of hot ethanol. The precipitate is allowed to settle in the cold and filtered off by suction using a G4 fritted glass fil-ter. It is dissolved in 43 ml of hot water to which solu-tion 43 ml of hot ethanol are added. The product crystal-lizes , is filtered off by suction and lyophilized.
Yield: 3 g = 60 % d. Th.
Melting point.: 237 C
TLC: silufol//n-butanol/glacial acetic acid/water/ 4:1:1 Rf: 0.3 (ninhydrin) IR (solid in KBr):
vC=O 1620, 1650, ~NH1540; VCOOH 1672 cm 1H-NMR (DMSO) TMS ~2.8 (N-CH3, 3H), ~3.8-4.1 (-CH2-,6H) Step 3: Chloroacetyl-sarcosyl diglycine:
2 g of sarcosyl-diglycine are dissolved in 10 ml of lN
NaOH at room temperature and then mixed alternately with 1 ml of chloroacetyl chloride and 16 ml of lN NaOH within 30 min at 0C while keeping the solution agitated. The ID6$3e . DOC
solution is agitated for 30 more minutes, then acidified with 3 g of 5N HCl, and then the product is evaporated to dryness under reduced pressure. The crystals that develop are dissolved in hot water, and the solution mixed with ethanol. The sodium chloride precipitate is filtered off, and the filtrate evaporated under reduced pressure. The desired product is extracted by multiple treatment with hot acetone. The acetone is removed under reduced pres-sure, and the residue taken up with little water and lyo-philized.
Yield: 1,1 g = 53 % d. Th.
Melting point: 74 C
TLC: silufol//n-butanol/glacial acetic acid/water 4:1:1 Rf: 0.7 (iodine vaporation) IR (solid in KBr):
vC=O 1655; ~NH1550; vCOoH 1730 cm~1 Step 4: Benzoylmercaptoacetyl-sarcosyl diglycine:
1 g of chloroacetyl-sarcosyl-diglycine is dissolved in 140 ml of reagent-grade methanol under stirring and in a protective gas atmosphere at room temperature.
In an Erlenmeyer flask, 0.8 g of thiobenzoic acid are dissolved in 3 ml methanol and neutralized with sodium methylate. This solution is slowly added by dropping, under stirring and in a protective gas atmosphere, to the parent solution and kept agitated for 12 more hours. The solvent is removed under reduced pressure and the residue taken up with 2N HCl. It is filtered off by suction and washed neutrally in warm water. The residue is then washed with 20 ml of chloroform, 20 ml of acetonitrile and 20 ml of ether and dried under reduced pressure.
Yield: 0.83 g = 61 % of theor. q'ty Melting point: 134 C
ID653e .DOC
TLC: silufol//n-butanol/glacial acetic acid/water 4:1:1 Rf: 0.8 (iodine vaporation) IR (solid in KBr): Vc=ol620;~NHl550 cm 1,VcooH1720 cm~
Step 5: Mercaptoacetyl-sarcosyl diglycine Alkaline separation of the protective group:
When the protective group is separated according to the conventional method, the final product becomes highly impure. It contains only about 25% mercaptoacetyl-sarcosyl-diglycine. Apart from an unidentified by-product, the main products that develop during this saponification are mercaptoacetyl-sarcosine and diglycine. There is only a low yield of the desired product.
Alkaline separation under hydrogenation conditions:
0.08 mmol substance are suspended in 2 ml of anhydrous methanol, mixed with 2 mg of Pd/CaCo3 (5 %) and reacted under stirring with a solution of 0.13 mmol sodium methy-late in 1 ml anhydrous methanol at a hydrogen pressure of 60 kPa. The solution is agitated for 15 min and then neu-tralized with methanolic Dowex 50WX8. Catalyst and resin are filtered off, the substance lyophilized and extracted with 2 ml of benzene. The benzene is subsequently sepa-rated, and the substance lyophilized from ethanol.
Yield: 10. 2 mg = 61 % d. Th., colourless oil TLC: silufol//n-butanol/glacial acetic acid/water 4:1:1 Rf: 0.4 (ninhydrin) RP 18 // methanol Rf = 0. 8 (ninhydrin) IR (solid in KBr):
vc=ol650, 1680; ~NH1550, VCooHl745 cm 1H-NMR (DMSO) TMS ~ 2.8 (SH, lH), ~3.0 (N-CH3, 3H), ~ 3. 5-4.1 (-CH2,8H), ~8.1-8.2 (CO-NH-,2H) ID653E . DOC
-[99mTc] Mercaptoacetyl-sarcosyl diglycine 1 ml of 99mTc gluconate solution is mixed with a solution of 0.4 mg pure substance from preliminary step 4 in 0.5 ml water. The reaction is completed after 30 minutes.
TLC:
(silica gel 60, 95 % ethanol):
Two components Rf 0-0.1 (40 %), Rf 0.8 (60 %) Electrophoresis (pH 7.0):
Both components migrate as anions.
Example 4 Synthesis of [99mTc] mercaptoacetyl hexylglycyl diglycine Step 1: Hexylglycyl diglycine 0.0048 mol chloroacetyl diglycine are mixed with 0.05 mol hexyl amine and 5 ml of ethanol, and allowed to stand.
The solvent is removed under reduced pressure after 6 days. The oily residue is added to 50 ml of acetone and heated. After cooling, the yellow solution is separated and the white residue washed once again with ca. 5 ml of acetone. The product is dried on a porous plate.
Yield: 610.5 mg (47 % d. Th.) TLC:
silufol//n-butanol/glacial acetic acid/water 2:1:1 Rf: 0.3 (ninhydrin) RP 18//methanol, Rf: 0.7 (ninhydrin) ID653E . DOC
Step 2: Chloracetyl hexylglycyl diglycine 0.0022 mol hexylglycyl diglycine are dissolved in 5 ml of lN soda lye and agitated at room temperature for 30 min.
Then the solution is cooled. At 0C, 0.3 ml of chloro-acetyl chloride and 4 ml of lN soda lye are added alter-nately within 30 min. The solution is agitated without cooling for another 30 min.
After acidification with 5N hydrochloric acid, the sol-vent is removed under reduced pressure, and the product gained in the form of a yellow oil. After extracting several times with 100 ml of hot acetone, the actone is removed under reduced pressure, and the residue suspended lS with 30 ml of acetone. The white sediment is dried on a porous plate.
Yield: 311 mg (40 % of theor. q'ty) TLC: silufol//n-butanol/glacial acetic acid/water 2:1:1 Rf: 0.75 (iodine vaporation) Step 3: Benzoylmercaptoacetyl hexylglycyl diglycine 0.00089 mol chloroacetyl hexylglycyl diglycine are dissolved in 70 ml of methanol (special pure grade for microelectronics) and agitated in a protective gas atmosphere (N2). 0.002 mol thiobenzoic acid are dissolved in 5 ml of methanol and neutralized with 0.4 ml of Na-methanolate. This solution is slowly added in a protec-tive gas atmosphere and agitated for 12 more hours. The methanol is removed under reduced pressure and the residue taken up with 2N HCl. The hydrochloric acid is removed under reduced pressure as well, the residue washed with water, and the washing water removed by decanting. The thick residue is mixed with little methanol, and the product precipitates. It is washed with acetonitrile and dried on a porous plate.
ID653E . DOC
Yield: 253. 3 mg (67 % d. Th.) TLC: silufol//butanol/glacial acetic acid/water 2:1:1 Rf: 0.8 (UV) Step 4: Mercaptoacetyl hexylglycyl diglycine 0.064 (28,8 mg) mmol benzoylmercaptoacetyl hexylglycyl diglycine are suspended in 2 ml of methanol, 2,7 mg of 5 % Pd/CaCO3 are added and filled into the hydrogenating apparatus. 0.04 ml of Na-methanolate and 1 ml of methanol are filled in an angle flask. The apparatus is evacuated and subsequently rinsed with hydrogen.
The methanolate is added and agitated for 15 min at a hydrogen pressure of 60 kPa. The solution is acidified with methanolic Dowex 50WX8, the resin is filtered off using a plaited filter (argon bell jar), washed with methanol and then lyophilized. The residue is three times extracted, each time using 4 ml of benzene, the benzene is removed by decanting, the residue taken up in methanol again and lyophilized. The product was cleaned above a sephadex column.
Yield: 9.8 mg (42 % d. Th.) TLC: silufol//butanol/glacial acetic acid/water 2:1:1 Rf:0.5 (ninhydrin) H-NMR (DMSO) TMS ~1.2 (-CH3, 3H), ~3.1 (SH, lH), ~3.2 (N-CH2,10H), ~3.7 (-CH2, 6H), ~3.8 (CH2-SH, 2H) ~8.2-8.5 (-NH, 2H) [99mTc] Mercaptoacetyl hexyl glycyl diglycine:
1 ml of 99mTc gluconate solution is mixed with 400 ,ul of 0.1 N NaOH and 30 ~1 pre-stage 4 solution - 1.63 mg/100 ~1 water. ~fter allowing the batch to stand for 40 min-utes, it is neutralized with 2 ml of a 0.1 M sodium [D653e .DOC
2 1 ~3844 phosphate buffer solution (pH 7.0). The reaction mixturewill be stable for at least 2 hours.
TLC (silica gel 60; 95 % ethanol): Rf 0.7 (95 %) Electrophoresis (pH 7.0):
Migration as anion Example 4 a Accumulation of [99mTc] Mercaptoacetyl hexyl glycine diglycine in atherosclerotic plaques of aortas in WHHL
rabbits.
99.9 GBq (2.7 mCi) of the substance labelled according to Example 4 were diluted to 1 ml with phosphate-buffered saline and applied to narcotized Rompun/Ketavet WHHL
rabbits (1:2) through an ear vein. The rabbit was killed 5 hours after application, and an autoradiogram of the aorta as well as a Sudan(III) dyeing were carried out to visualize the atherosclerotic plaques (Fig. 1). The enrichment factor between normal and atherosclerotic wall areas was between 3 and 5 depending on the formation of the plaques (Sudan III dyeing). See Fig. 2 for an in-vivo image of the rabbit.
Example 5 S-bzl-acetyl-sar-gly-gly-[8a-ergolinylamide]
200 mg of S-bzl-acetyl-sar-gly-gly-OH and 161 mg of ben-zotriazol-1-yl-tetramethyl-uronium-tetrafluoroborate were dissolved in 1 ml of N-methyl pyrrolidone, and 172 ,ul of diisopropylethylamine were added. The colourless solution is dropped in a stirred solution of 120 mg of 8a-amino-ergoline after 3 minutes. The reddish brown solution is stirred on for 4 hours at room temperature while the fur-ther reaction course is tracked using HPLC-chromatography ID653E.DO~
~_ 44 on a VYDAC C18 column (4,6 x 250 mm) applying a gradient from 10 % to 60 % B within 25 min (eluent A 1000 ml water/2 ml trifluoroacetic acid anhydride, eluent B 500 ml acetonitrile/100 ml water/1 ml trifluoroacetic acid anhydride).
The reaction batch is preperatively separated without further pre-treatment on a VYDAc column (40 x 300 mm) applying a gradient from 20 % to 30 % B within 20 min (eluent composed as above). Then it is lyophilized.
Yield: 75 mg A mass spectrogram showed the expected molecular weight.
Example 6 S-bzl-acetyl-[N1-hexyl]-gly-gy-gly-[8a-ergolinylamide]
60 mg of S-bzl-acetyl-[N1-Hexyl]-gly-gy-gly-OH and 40 mg benzotriazol-1-yl-tetramethyl-uronium-tetrafluoroborate were suspended in 0.6 ml of N-methyl pyrrolidone and put into solution by adding 17,2 ,ul of diisopropylethyl amine. This solution was added by dropping to a solution of 30 mg 8a-amino-ergoline in 0.4 ml N-methyl pyrroli-done. The reaction solution was processed after 4 hours.
The crude reaction mixture was preparatively chromato-graphed (as described in Example 5). Subsequent lyophili-zation' yielded the desired product.
Yield: 30 mg of pseudocrystalline product.
A mass spectrogram showed the expected molecular weight.
Example 7 S-bzl-acetyl-sar-gly-[8a-ergolinylamide]
180 mg S-bzl-acetyl-sar-gly-OH and 161 mg benzotriazol-1-yl-tetramethyl-uronium-tetrafluoroborate were dissolved ID653E . DOC
ln 1 ml of N-methyl pyrrolidone, and 172 ul of diisopro-pylethyl amine added. After 3 minutes, the colourless solution is added by dropping and under stirring to a solution of 120 mg 8a-amino ergoline. The reddish brown solution is agitated at room temperature for 4 more hours, and processed and chromatographed as described in Example 5.
Yield: 70 mg (pseudocrystalline) An MS showed the expected peak.
Example 8 S-bzl-acetyl-sar-[8a-ergolinyl amide]
120 mg of S-bzl-acetyl-sar-OH and 161 mg of benzotriazol-1-yl-tetramethyl-uronium-tetrafluoroborate were dissolved in 1 ml of N-methyl pyrrolidone, and 172 ul of diisopro-pylethyl amine added.
The solution is reacted as in Example 5; after processing and separating, 52 mg of a pseudocrystalline compound are gained.
An MS shows the expected mass peak.
Example 9 3,17~-dihydroxy-17a-[5(S-benzoylthio-acetyl-sarcosyl-glycyl)-amino-pent-1-en-3-in]-1,3,5-estratriene 0.35 g of ~S-benzoyl-thioacetyl-sarcosyl-glycyl-propargylamide], 11.0 mg of benzyl triethyl ammonium chloride, 11.5 mg of tetrakis-(triphenyl phosphine) palladium (0), 9 mg of copper(I) iodide suspended in 5 ml of toluene are added to a suspension of 130.0 mg 17~-Hydroxy-17a-iodvinyl-1,3~5-estratrien-3-tetrahydropyranyl ether, and stirred for 90 hours at roomtemperature. The suspension is mixed with water, , 46 21 73844 extracted with toluene and dried. The solvent is removed under reduced pressure, the residue taken up in 10 ml of tetrahydrofurane, mixed with 190 mg of pyridinium-para-toluene-p-sulfonic acid in 5 ml ethanol, and refluxed for 3 hours. Then the solvent is removed under reduced pressure and cleaned using methylene chloride/methanol (8:1) on a silica gel column.
Yield: 30 % d. Th.
The substance shows an Rf of 0.4 using TLC (CH2Cl2/MeOH]
5 : 5.
Example 10 3,17~-dihydroxy-17a-[N-(benzoyl-thio-acetyl-sarcosyl-glycyl)-amino-propin-1]-1,3,5 estratriene 350 mg S-benzoyl-thioacetyl-sarcosyl-glycine in 3 ml DMF
are added to a suspension of 110 mg 17~-Hydroxy-17a-propargyl-amino-1,3,5-estratrien-3-tetrahydropyranylether in 3 ml DMF. The batch is stirred for 24 hours at 100C
in a protective gas atmosphere.
After adding 10 ml of tetrahydrofurane, the crystallizate is filtered off by suction and evaporated under reduced pressure. The residue is taken up in 20 ml of tetrahydro-furane, dissolved with 100 mg of pyridinium-paratoluene-p-sulfonic acid in 3 ml of ethanol, mixed, and refluxed for 1 hour. After removing the solvent, the substance is cleaned on a silica gel low-pressure column in a metha-nol/methylene chloride system (1:8).
The substance showed an Rf of 0.6 using TLC (CH2Cl2/MeOH) 5 : 5.
Yield: 71 mg ~D653E.DOC
Example 11 0.001 mol S-bzl-acetyl-sar-gly-OH and 322 mg benzotriazol-1-yl-tetramethyl-uroniumtetrafluoroborate are dissolved in 3 ml of DMF while adding 344 ul of diisopropylethyl amine. The solution is pre-activated for 5 minutes to form benzotriazolyl ester, and then the clear solution is added to 33 mmol protected his(trt)-leu-asp(Obut)-ile-ile-trp resin. The suspension is stirred for one hour, then washed several times with DMF, filtered off by suction, and dried. The dried resin is treated as usual with TFA/Scavanger to free the product from the protective groups. Cleaning is carried out as described in Example 5.
The compound obtained according to Example 10 is complexed with 99mTc according to the procedure given as general method (Example 1).
Example 12 0.010 mol of his(trt)-leu-asp(Obut)-ile-ile-trp-OH, produced on sasrin resin are dissolved in a mixture of DMF/NMP while adding 0.010 mol diisopropylethyl amine, and mixed under stirring with 0.010 mol S-bzl-acetyl-sar-OH that is prepared as in Example 11 and dissolved in 3 ml of DMF. The solution is agitated for 2 hours, then the solvent is removed. The remaining residue is stirred up with water and filtered off by suction, washed and dried.
The protective groups are removed as usual, and the product is obtained by pouring-in in ether. Cleaning is carried out as described in Example 5.
S-bzl-acetyl-[N14-bromophenyl]-gly-gly-gly-OH
I D6 5 ~ . DOC
Step 1: Chloroacetyl diglycine 10 g of glycine anhydride are dissolved at room tempera-ture in 50 ml of 2N soda lye in a 250 ml three-neck flask. After 45 min, the still somewhat cloudy solution is cooled down to about 0 C and 11.1 g (7.8 ml) chloroacetyl chloride and 24 ml of 5N soda lye are alter-nately dropped in under stirring. Addition is completed after 45 min. The solution takes on a slightly pink col-ouring. 40 ml of 5N hydrochloric acid are added which causes the solution to decolourize, and the substance begins to crystallize. The batch is allowed to stand for about 1 hour at 0 C, and the residue filtered off by suction. It is washed several times with cold water and recrystallized from hot water.
Yield: 5 g Melting point: 173-174 C
The parent lye is evaporated under reduced pressure, and the residue recrystallized from water as well.
Yield: 2.5 g Melting point: 173 C
Total yield: 7.5 g = 41 % of theor. q'ty TLC:
silufol//n-butanol/glacial acetic acid/water 4:1:1 Step 2: 4-bromophenyl triglycine 0.0144 mol chloroacetyl diglycine are refluxed with 0.029 mol 4-bromoaniline in 20 ml of ethanol and 20 ml of lN
NaOH for 7 hours. After removing the solvent under reduced pressure, the remaining residue is three times extracted, each time with 50 ml of heated acetone. The ID653E . DOC
remaining product is recrystallized from hot water. The crude product is introduced in Step 3.
Yield: ca. 60 %.
Step 3: Chloroacetylation of 4-bromophenyl triglycine 0.01 mol 4-bromophenyl triglycine are dissolved in 10 ml of lN NaOH at room temperature and mixed under stirring within 30 min, and at 0C , alternately with 0.012 mol chloroacetyl chloride and 20 ml of lN NaOH. The solution is agitated for 30 more minutes, acidified with 5N HCl, and the product is evaporated to dryness under reduced pressure. The developing crystals are dissolved in hot water, and the solution mixed with ethanol. The precipi-tating sodium chloride is filtered off, and the filtrate is evaporated using the rotary evaporator. The desired product is extracted by treating the filtrate several times, each time with 30 ml of hot acetone, then the acetone is removed under reduced pressure, the residue taken up with little water and lyophilized.
Yield: 50% of theor. q'ty TLC:
silufol//n-butanol/glacial acetic acid/water 2:1:1 Rf: 0.6 Step 4: S-bzl-acetyl-[N1-bromophenyl]-gly-gly-gly-OH
0.01 mol of the chloroacetyl product are dissolved at room temperature, under stirring and in a protective gas atmosphere, in 140 ml methanol. For this, 0.02 mol thiobenzoic acid are dissolved in 20 ml of methanol and neutralized with sodium methylate, slowly dropped in under stirring and in a protective gas atmosphere, and kept agitated for another 12 hours. The solvent is removed under reduced pressure and the residue taken up with 2N HCl. The resulting crystals are filtered off by ID653E . DOC
' 50 21 73844 suction and washed neutrally with heated water. Subse-quently, they are washed with 20 ml of chloroform, 20 ml of acetonitrile and 20 ml of ether, and the residue is dried under reduced pressure.
Yield: 60% of theor. q'ty Step 5: Thio-acetyl-[N1-bromophenyl]-gly-gly-gly-OH
0.08 mmol of the substance from Step 4 are suspended with 2 ml of anhydrous methanol, then mixed with 2 mg of Pd/CaCO3(5%) and reacted, under stirring and at a hydro-gen pressure of 66 kPa, with a solution of 0.13 mmol sodium methylate in 1 ml of absolute methanol. The solu-tion is agitated for another 15 min and neutralized with methanolic Dowex 50WX8. The catalyst and the resin are filtered off, the substance is lyophilized and extracted with 2 ml of benzene. The benzene is then separated, and the substance lyophilized from ethanol. It is cleaned using preparative HPLC.
TLC: silufol//butanol/glacial acetic acid/water 2:1:1 Rf: 0.8 Yield: 40% of theor. q'ty 99mTc [Thioacetyl-[N1-4-bromophenyl]-gly-gly-gly-OH
As described in the general remarks on complexing pre-ceding Example 1, thio-acetyl-[N1-bromophenyl]-gly-gly-gly-OH is reacted with a [99mTc] gluconate preparation.
ID653E: . DOC
acqueous methylamine solution and allowed to stand for 2 days at room temperature. Then the solution is evaporated to syruplike consistency under reduced pressure, the syrup heated in a water-bath and mixed with 50 ml of hot ethanol. The precipitate is allowed to settle in the cold and filtered off by suction using a G4 fritted glass fil-ter. It is dissolved in 43 ml of hot water to which solu-tion 43 ml of hot ethanol are added. The product crystal-lizes , is filtered off by suction and lyophilized.
Yield: 3 g = 60 % d. Th.
Melting point.: 237 C
TLC: silufol//n-butanol/glacial acetic acid/water/ 4:1:1 Rf: 0.3 (ninhydrin) IR (solid in KBr):
vC=O 1620, 1650, ~NH1540; VCOOH 1672 cm 1H-NMR (DMSO) TMS ~2.8 (N-CH3, 3H), ~3.8-4.1 (-CH2-,6H) Step 3: Chloroacetyl-sarcosyl diglycine:
2 g of sarcosyl-diglycine are dissolved in 10 ml of lN
NaOH at room temperature and then mixed alternately with 1 ml of chloroacetyl chloride and 16 ml of lN NaOH within 30 min at 0C while keeping the solution agitated. The ID6$3e . DOC
solution is agitated for 30 more minutes, then acidified with 3 g of 5N HCl, and then the product is evaporated to dryness under reduced pressure. The crystals that develop are dissolved in hot water, and the solution mixed with ethanol. The sodium chloride precipitate is filtered off, and the filtrate evaporated under reduced pressure. The desired product is extracted by multiple treatment with hot acetone. The acetone is removed under reduced pres-sure, and the residue taken up with little water and lyo-philized.
Yield: 1,1 g = 53 % d. Th.
Melting point: 74 C
TLC: silufol//n-butanol/glacial acetic acid/water 4:1:1 Rf: 0.7 (iodine vaporation) IR (solid in KBr):
vC=O 1655; ~NH1550; vCOoH 1730 cm~1 Step 4: Benzoylmercaptoacetyl-sarcosyl diglycine:
1 g of chloroacetyl-sarcosyl-diglycine is dissolved in 140 ml of reagent-grade methanol under stirring and in a protective gas atmosphere at room temperature.
In an Erlenmeyer flask, 0.8 g of thiobenzoic acid are dissolved in 3 ml methanol and neutralized with sodium methylate. This solution is slowly added by dropping, under stirring and in a protective gas atmosphere, to the parent solution and kept agitated for 12 more hours. The solvent is removed under reduced pressure and the residue taken up with 2N HCl. It is filtered off by suction and washed neutrally in warm water. The residue is then washed with 20 ml of chloroform, 20 ml of acetonitrile and 20 ml of ether and dried under reduced pressure.
Yield: 0.83 g = 61 % of theor. q'ty Melting point: 134 C
ID653e .DOC
TLC: silufol//n-butanol/glacial acetic acid/water 4:1:1 Rf: 0.8 (iodine vaporation) IR (solid in KBr): Vc=ol620;~NHl550 cm 1,VcooH1720 cm~
Step 5: Mercaptoacetyl-sarcosyl diglycine Alkaline separation of the protective group:
When the protective group is separated according to the conventional method, the final product becomes highly impure. It contains only about 25% mercaptoacetyl-sarcosyl-diglycine. Apart from an unidentified by-product, the main products that develop during this saponification are mercaptoacetyl-sarcosine and diglycine. There is only a low yield of the desired product.
Alkaline separation under hydrogenation conditions:
0.08 mmol substance are suspended in 2 ml of anhydrous methanol, mixed with 2 mg of Pd/CaCo3 (5 %) and reacted under stirring with a solution of 0.13 mmol sodium methy-late in 1 ml anhydrous methanol at a hydrogen pressure of 60 kPa. The solution is agitated for 15 min and then neu-tralized with methanolic Dowex 50WX8. Catalyst and resin are filtered off, the substance lyophilized and extracted with 2 ml of benzene. The benzene is subsequently sepa-rated, and the substance lyophilized from ethanol.
Yield: 10. 2 mg = 61 % d. Th., colourless oil TLC: silufol//n-butanol/glacial acetic acid/water 4:1:1 Rf: 0.4 (ninhydrin) RP 18 // methanol Rf = 0. 8 (ninhydrin) IR (solid in KBr):
vc=ol650, 1680; ~NH1550, VCooHl745 cm 1H-NMR (DMSO) TMS ~ 2.8 (SH, lH), ~3.0 (N-CH3, 3H), ~ 3. 5-4.1 (-CH2,8H), ~8.1-8.2 (CO-NH-,2H) ID653E . DOC
-[99mTc] Mercaptoacetyl-sarcosyl diglycine 1 ml of 99mTc gluconate solution is mixed with a solution of 0.4 mg pure substance from preliminary step 4 in 0.5 ml water. The reaction is completed after 30 minutes.
TLC:
(silica gel 60, 95 % ethanol):
Two components Rf 0-0.1 (40 %), Rf 0.8 (60 %) Electrophoresis (pH 7.0):
Both components migrate as anions.
Example 4 Synthesis of [99mTc] mercaptoacetyl hexylglycyl diglycine Step 1: Hexylglycyl diglycine 0.0048 mol chloroacetyl diglycine are mixed with 0.05 mol hexyl amine and 5 ml of ethanol, and allowed to stand.
The solvent is removed under reduced pressure after 6 days. The oily residue is added to 50 ml of acetone and heated. After cooling, the yellow solution is separated and the white residue washed once again with ca. 5 ml of acetone. The product is dried on a porous plate.
Yield: 610.5 mg (47 % d. Th.) TLC:
silufol//n-butanol/glacial acetic acid/water 2:1:1 Rf: 0.3 (ninhydrin) RP 18//methanol, Rf: 0.7 (ninhydrin) ID653E . DOC
Step 2: Chloracetyl hexylglycyl diglycine 0.0022 mol hexylglycyl diglycine are dissolved in 5 ml of lN soda lye and agitated at room temperature for 30 min.
Then the solution is cooled. At 0C, 0.3 ml of chloro-acetyl chloride and 4 ml of lN soda lye are added alter-nately within 30 min. The solution is agitated without cooling for another 30 min.
After acidification with 5N hydrochloric acid, the sol-vent is removed under reduced pressure, and the product gained in the form of a yellow oil. After extracting several times with 100 ml of hot acetone, the actone is removed under reduced pressure, and the residue suspended lS with 30 ml of acetone. The white sediment is dried on a porous plate.
Yield: 311 mg (40 % of theor. q'ty) TLC: silufol//n-butanol/glacial acetic acid/water 2:1:1 Rf: 0.75 (iodine vaporation) Step 3: Benzoylmercaptoacetyl hexylglycyl diglycine 0.00089 mol chloroacetyl hexylglycyl diglycine are dissolved in 70 ml of methanol (special pure grade for microelectronics) and agitated in a protective gas atmosphere (N2). 0.002 mol thiobenzoic acid are dissolved in 5 ml of methanol and neutralized with 0.4 ml of Na-methanolate. This solution is slowly added in a protec-tive gas atmosphere and agitated for 12 more hours. The methanol is removed under reduced pressure and the residue taken up with 2N HCl. The hydrochloric acid is removed under reduced pressure as well, the residue washed with water, and the washing water removed by decanting. The thick residue is mixed with little methanol, and the product precipitates. It is washed with acetonitrile and dried on a porous plate.
ID653E . DOC
Yield: 253. 3 mg (67 % d. Th.) TLC: silufol//butanol/glacial acetic acid/water 2:1:1 Rf: 0.8 (UV) Step 4: Mercaptoacetyl hexylglycyl diglycine 0.064 (28,8 mg) mmol benzoylmercaptoacetyl hexylglycyl diglycine are suspended in 2 ml of methanol, 2,7 mg of 5 % Pd/CaCO3 are added and filled into the hydrogenating apparatus. 0.04 ml of Na-methanolate and 1 ml of methanol are filled in an angle flask. The apparatus is evacuated and subsequently rinsed with hydrogen.
The methanolate is added and agitated for 15 min at a hydrogen pressure of 60 kPa. The solution is acidified with methanolic Dowex 50WX8, the resin is filtered off using a plaited filter (argon bell jar), washed with methanol and then lyophilized. The residue is three times extracted, each time using 4 ml of benzene, the benzene is removed by decanting, the residue taken up in methanol again and lyophilized. The product was cleaned above a sephadex column.
Yield: 9.8 mg (42 % d. Th.) TLC: silufol//butanol/glacial acetic acid/water 2:1:1 Rf:0.5 (ninhydrin) H-NMR (DMSO) TMS ~1.2 (-CH3, 3H), ~3.1 (SH, lH), ~3.2 (N-CH2,10H), ~3.7 (-CH2, 6H), ~3.8 (CH2-SH, 2H) ~8.2-8.5 (-NH, 2H) [99mTc] Mercaptoacetyl hexyl glycyl diglycine:
1 ml of 99mTc gluconate solution is mixed with 400 ,ul of 0.1 N NaOH and 30 ~1 pre-stage 4 solution - 1.63 mg/100 ~1 water. ~fter allowing the batch to stand for 40 min-utes, it is neutralized with 2 ml of a 0.1 M sodium [D653e .DOC
2 1 ~3844 phosphate buffer solution (pH 7.0). The reaction mixturewill be stable for at least 2 hours.
TLC (silica gel 60; 95 % ethanol): Rf 0.7 (95 %) Electrophoresis (pH 7.0):
Migration as anion Example 4 a Accumulation of [99mTc] Mercaptoacetyl hexyl glycine diglycine in atherosclerotic plaques of aortas in WHHL
rabbits.
99.9 GBq (2.7 mCi) of the substance labelled according to Example 4 were diluted to 1 ml with phosphate-buffered saline and applied to narcotized Rompun/Ketavet WHHL
rabbits (1:2) through an ear vein. The rabbit was killed 5 hours after application, and an autoradiogram of the aorta as well as a Sudan(III) dyeing were carried out to visualize the atherosclerotic plaques (Fig. 1). The enrichment factor between normal and atherosclerotic wall areas was between 3 and 5 depending on the formation of the plaques (Sudan III dyeing). See Fig. 2 for an in-vivo image of the rabbit.
Example 5 S-bzl-acetyl-sar-gly-gly-[8a-ergolinylamide]
200 mg of S-bzl-acetyl-sar-gly-gly-OH and 161 mg of ben-zotriazol-1-yl-tetramethyl-uronium-tetrafluoroborate were dissolved in 1 ml of N-methyl pyrrolidone, and 172 ,ul of diisopropylethylamine were added. The colourless solution is dropped in a stirred solution of 120 mg of 8a-amino-ergoline after 3 minutes. The reddish brown solution is stirred on for 4 hours at room temperature while the fur-ther reaction course is tracked using HPLC-chromatography ID653E.DO~
~_ 44 on a VYDAC C18 column (4,6 x 250 mm) applying a gradient from 10 % to 60 % B within 25 min (eluent A 1000 ml water/2 ml trifluoroacetic acid anhydride, eluent B 500 ml acetonitrile/100 ml water/1 ml trifluoroacetic acid anhydride).
The reaction batch is preperatively separated without further pre-treatment on a VYDAc column (40 x 300 mm) applying a gradient from 20 % to 30 % B within 20 min (eluent composed as above). Then it is lyophilized.
Yield: 75 mg A mass spectrogram showed the expected molecular weight.
Example 6 S-bzl-acetyl-[N1-hexyl]-gly-gy-gly-[8a-ergolinylamide]
60 mg of S-bzl-acetyl-[N1-Hexyl]-gly-gy-gly-OH and 40 mg benzotriazol-1-yl-tetramethyl-uronium-tetrafluoroborate were suspended in 0.6 ml of N-methyl pyrrolidone and put into solution by adding 17,2 ,ul of diisopropylethyl amine. This solution was added by dropping to a solution of 30 mg 8a-amino-ergoline in 0.4 ml N-methyl pyrroli-done. The reaction solution was processed after 4 hours.
The crude reaction mixture was preparatively chromato-graphed (as described in Example 5). Subsequent lyophili-zation' yielded the desired product.
Yield: 30 mg of pseudocrystalline product.
A mass spectrogram showed the expected molecular weight.
Example 7 S-bzl-acetyl-sar-gly-[8a-ergolinylamide]
180 mg S-bzl-acetyl-sar-gly-OH and 161 mg benzotriazol-1-yl-tetramethyl-uronium-tetrafluoroborate were dissolved ID653E . DOC
ln 1 ml of N-methyl pyrrolidone, and 172 ul of diisopro-pylethyl amine added. After 3 minutes, the colourless solution is added by dropping and under stirring to a solution of 120 mg 8a-amino ergoline. The reddish brown solution is agitated at room temperature for 4 more hours, and processed and chromatographed as described in Example 5.
Yield: 70 mg (pseudocrystalline) An MS showed the expected peak.
Example 8 S-bzl-acetyl-sar-[8a-ergolinyl amide]
120 mg of S-bzl-acetyl-sar-OH and 161 mg of benzotriazol-1-yl-tetramethyl-uronium-tetrafluoroborate were dissolved in 1 ml of N-methyl pyrrolidone, and 172 ul of diisopro-pylethyl amine added.
The solution is reacted as in Example 5; after processing and separating, 52 mg of a pseudocrystalline compound are gained.
An MS shows the expected mass peak.
Example 9 3,17~-dihydroxy-17a-[5(S-benzoylthio-acetyl-sarcosyl-glycyl)-amino-pent-1-en-3-in]-1,3,5-estratriene 0.35 g of ~S-benzoyl-thioacetyl-sarcosyl-glycyl-propargylamide], 11.0 mg of benzyl triethyl ammonium chloride, 11.5 mg of tetrakis-(triphenyl phosphine) palladium (0), 9 mg of copper(I) iodide suspended in 5 ml of toluene are added to a suspension of 130.0 mg 17~-Hydroxy-17a-iodvinyl-1,3~5-estratrien-3-tetrahydropyranyl ether, and stirred for 90 hours at roomtemperature. The suspension is mixed with water, , 46 21 73844 extracted with toluene and dried. The solvent is removed under reduced pressure, the residue taken up in 10 ml of tetrahydrofurane, mixed with 190 mg of pyridinium-para-toluene-p-sulfonic acid in 5 ml ethanol, and refluxed for 3 hours. Then the solvent is removed under reduced pressure and cleaned using methylene chloride/methanol (8:1) on a silica gel column.
Yield: 30 % d. Th.
The substance shows an Rf of 0.4 using TLC (CH2Cl2/MeOH]
5 : 5.
Example 10 3,17~-dihydroxy-17a-[N-(benzoyl-thio-acetyl-sarcosyl-glycyl)-amino-propin-1]-1,3,5 estratriene 350 mg S-benzoyl-thioacetyl-sarcosyl-glycine in 3 ml DMF
are added to a suspension of 110 mg 17~-Hydroxy-17a-propargyl-amino-1,3,5-estratrien-3-tetrahydropyranylether in 3 ml DMF. The batch is stirred for 24 hours at 100C
in a protective gas atmosphere.
After adding 10 ml of tetrahydrofurane, the crystallizate is filtered off by suction and evaporated under reduced pressure. The residue is taken up in 20 ml of tetrahydro-furane, dissolved with 100 mg of pyridinium-paratoluene-p-sulfonic acid in 3 ml of ethanol, mixed, and refluxed for 1 hour. After removing the solvent, the substance is cleaned on a silica gel low-pressure column in a metha-nol/methylene chloride system (1:8).
The substance showed an Rf of 0.6 using TLC (CH2Cl2/MeOH) 5 : 5.
Yield: 71 mg ~D653E.DOC
Example 11 0.001 mol S-bzl-acetyl-sar-gly-OH and 322 mg benzotriazol-1-yl-tetramethyl-uroniumtetrafluoroborate are dissolved in 3 ml of DMF while adding 344 ul of diisopropylethyl amine. The solution is pre-activated for 5 minutes to form benzotriazolyl ester, and then the clear solution is added to 33 mmol protected his(trt)-leu-asp(Obut)-ile-ile-trp resin. The suspension is stirred for one hour, then washed several times with DMF, filtered off by suction, and dried. The dried resin is treated as usual with TFA/Scavanger to free the product from the protective groups. Cleaning is carried out as described in Example 5.
The compound obtained according to Example 10 is complexed with 99mTc according to the procedure given as general method (Example 1).
Example 12 0.010 mol of his(trt)-leu-asp(Obut)-ile-ile-trp-OH, produced on sasrin resin are dissolved in a mixture of DMF/NMP while adding 0.010 mol diisopropylethyl amine, and mixed under stirring with 0.010 mol S-bzl-acetyl-sar-OH that is prepared as in Example 11 and dissolved in 3 ml of DMF. The solution is agitated for 2 hours, then the solvent is removed. The remaining residue is stirred up with water and filtered off by suction, washed and dried.
The protective groups are removed as usual, and the product is obtained by pouring-in in ether. Cleaning is carried out as described in Example 5.
S-bzl-acetyl-[N14-bromophenyl]-gly-gly-gly-OH
I D6 5 ~ . DOC
Step 1: Chloroacetyl diglycine 10 g of glycine anhydride are dissolved at room tempera-ture in 50 ml of 2N soda lye in a 250 ml three-neck flask. After 45 min, the still somewhat cloudy solution is cooled down to about 0 C and 11.1 g (7.8 ml) chloroacetyl chloride and 24 ml of 5N soda lye are alter-nately dropped in under stirring. Addition is completed after 45 min. The solution takes on a slightly pink col-ouring. 40 ml of 5N hydrochloric acid are added which causes the solution to decolourize, and the substance begins to crystallize. The batch is allowed to stand for about 1 hour at 0 C, and the residue filtered off by suction. It is washed several times with cold water and recrystallized from hot water.
Yield: 5 g Melting point: 173-174 C
The parent lye is evaporated under reduced pressure, and the residue recrystallized from water as well.
Yield: 2.5 g Melting point: 173 C
Total yield: 7.5 g = 41 % of theor. q'ty TLC:
silufol//n-butanol/glacial acetic acid/water 4:1:1 Step 2: 4-bromophenyl triglycine 0.0144 mol chloroacetyl diglycine are refluxed with 0.029 mol 4-bromoaniline in 20 ml of ethanol and 20 ml of lN
NaOH for 7 hours. After removing the solvent under reduced pressure, the remaining residue is three times extracted, each time with 50 ml of heated acetone. The ID653E . DOC
remaining product is recrystallized from hot water. The crude product is introduced in Step 3.
Yield: ca. 60 %.
Step 3: Chloroacetylation of 4-bromophenyl triglycine 0.01 mol 4-bromophenyl triglycine are dissolved in 10 ml of lN NaOH at room temperature and mixed under stirring within 30 min, and at 0C , alternately with 0.012 mol chloroacetyl chloride and 20 ml of lN NaOH. The solution is agitated for 30 more minutes, acidified with 5N HCl, and the product is evaporated to dryness under reduced pressure. The developing crystals are dissolved in hot water, and the solution mixed with ethanol. The precipi-tating sodium chloride is filtered off, and the filtrate is evaporated using the rotary evaporator. The desired product is extracted by treating the filtrate several times, each time with 30 ml of hot acetone, then the acetone is removed under reduced pressure, the residue taken up with little water and lyophilized.
Yield: 50% of theor. q'ty TLC:
silufol//n-butanol/glacial acetic acid/water 2:1:1 Rf: 0.6 Step 4: S-bzl-acetyl-[N1-bromophenyl]-gly-gly-gly-OH
0.01 mol of the chloroacetyl product are dissolved at room temperature, under stirring and in a protective gas atmosphere, in 140 ml methanol. For this, 0.02 mol thiobenzoic acid are dissolved in 20 ml of methanol and neutralized with sodium methylate, slowly dropped in under stirring and in a protective gas atmosphere, and kept agitated for another 12 hours. The solvent is removed under reduced pressure and the residue taken up with 2N HCl. The resulting crystals are filtered off by ID653E . DOC
' 50 21 73844 suction and washed neutrally with heated water. Subse-quently, they are washed with 20 ml of chloroform, 20 ml of acetonitrile and 20 ml of ether, and the residue is dried under reduced pressure.
Yield: 60% of theor. q'ty Step 5: Thio-acetyl-[N1-bromophenyl]-gly-gly-gly-OH
0.08 mmol of the substance from Step 4 are suspended with 2 ml of anhydrous methanol, then mixed with 2 mg of Pd/CaCO3(5%) and reacted, under stirring and at a hydro-gen pressure of 66 kPa, with a solution of 0.13 mmol sodium methylate in 1 ml of absolute methanol. The solu-tion is agitated for another 15 min and neutralized with methanolic Dowex 50WX8. The catalyst and the resin are filtered off, the substance is lyophilized and extracted with 2 ml of benzene. The benzene is then separated, and the substance lyophilized from ethanol. It is cleaned using preparative HPLC.
TLC: silufol//butanol/glacial acetic acid/water 2:1:1 Rf: 0.8 Yield: 40% of theor. q'ty 99mTc [Thioacetyl-[N1-4-bromophenyl]-gly-gly-gly-OH
As described in the general remarks on complexing pre-ceding Example 1, thio-acetyl-[N1-bromophenyl]-gly-gly-gly-OH is reacted with a [99mTc] gluconate preparation.
ID653E: . DOC
Claims (18)
1. Compounds of the general formula I
R3-S-CH2-CO-NR1-[CH2-CO-NH]n-CH2-CO-R2 (I) with the exception of N-[2-(benzoylthio)acetyl]-N-cyclohexyl glycine wherein n represents the numbers 0,1 or 2, R1 is a straight-chain or branched alkyl residue con-taining 1 to 20 carbon atoms which may optionally be interrupted or replaced by one to three oxygen atoms, and which may optionally include a terminal -COOH, -OH
or -NH2 group that may optionally be esterified or etherified by means of glycolic acid or its esters or ethers, these esters or ethers being formed using carboxylic acids or alcohols containing up to 18 carbon atoms, or represents a phenyl or cyclohexyl residue having an optional COOH, NH2 or OH group at its 4-position that may optionally be esterified, etherified or amidated using carboxylic acids or alcohols containing up to 6 carbon atoms, or carries an additional halogen atom, R2 is a halogen atom, a halogen methyl, methyl carb-oxyl, trifluoromethyl carboxyl, an NH2 or an OH group, the carboxyl group formed when R2 = OH being esterified or amidated either immediately or after etherification with an .alpha.,.omega.-hydroxy-carboxylic acid containing up to 8 carbon atoms through its terminal carboxyl group, with a biomolecule, a steroid, an ergoline derivative, a benzodiazepine derivative, a cholecystokinin, a peptide, a protein, a proteohormone, an amino sugar, an endothelin, an endothelin derivative, an endothelin antagonist or an endothelin fragment, is a residue of the general formula II
(II) or of the general formula II a (IIa) where R4 is a methylene, propenylene amino, propinylene amino, methylene amino or methylenoxy group and R8 is a hydrogen atom or a methyl group, is a residue of the general formula III
(III) or of the general formula III a (IIIa) wherein are:
R5 an -NH-, -NH-CO-N<, -NH-CO-NH- or methylenoxy group, R6 a hydrogen atom, a halogen atom or a methyl group and R7 a hydrogen atom or a straight-chain or branched alkyl residue containing up to 6 carbon atoms, R3 is a hydrogen atom, an acetyl, benzoyl, p-methoxy-benzyl, acetamidomethyl, benzamidomethyl, trimethyl acetamidomethyl, hydroxyacetyl, ethoxyethyl, ethylthio, trityl, or an easily separable sulfur-protective group, and their salts formed from pharmaceutically accept-able acids or bases.
R3-S-CH2-CO-NR1-[CH2-CO-NH]n-CH2-CO-R2 (I) with the exception of N-[2-(benzoylthio)acetyl]-N-cyclohexyl glycine wherein n represents the numbers 0,1 or 2, R1 is a straight-chain or branched alkyl residue con-taining 1 to 20 carbon atoms which may optionally be interrupted or replaced by one to three oxygen atoms, and which may optionally include a terminal -COOH, -OH
or -NH2 group that may optionally be esterified or etherified by means of glycolic acid or its esters or ethers, these esters or ethers being formed using carboxylic acids or alcohols containing up to 18 carbon atoms, or represents a phenyl or cyclohexyl residue having an optional COOH, NH2 or OH group at its 4-position that may optionally be esterified, etherified or amidated using carboxylic acids or alcohols containing up to 6 carbon atoms, or carries an additional halogen atom, R2 is a halogen atom, a halogen methyl, methyl carb-oxyl, trifluoromethyl carboxyl, an NH2 or an OH group, the carboxyl group formed when R2 = OH being esterified or amidated either immediately or after etherification with an .alpha.,.omega.-hydroxy-carboxylic acid containing up to 8 carbon atoms through its terminal carboxyl group, with a biomolecule, a steroid, an ergoline derivative, a benzodiazepine derivative, a cholecystokinin, a peptide, a protein, a proteohormone, an amino sugar, an endothelin, an endothelin derivative, an endothelin antagonist or an endothelin fragment, is a residue of the general formula II
(II) or of the general formula II a (IIa) where R4 is a methylene, propenylene amino, propinylene amino, methylene amino or methylenoxy group and R8 is a hydrogen atom or a methyl group, is a residue of the general formula III
(III) or of the general formula III a (IIIa) wherein are:
R5 an -NH-, -NH-CO-N<, -NH-CO-NH- or methylenoxy group, R6 a hydrogen atom, a halogen atom or a methyl group and R7 a hydrogen atom or a straight-chain or branched alkyl residue containing up to 6 carbon atoms, R3 is a hydrogen atom, an acetyl, benzoyl, p-methoxy-benzyl, acetamidomethyl, benzamidomethyl, trimethyl acetamidomethyl, hydroxyacetyl, ethoxyethyl, ethylthio, trityl, or an easily separable sulfur-protective group, and their salts formed from pharmaceutically accept-able acids or bases.
2. Compounds according to Claim 1, characterized in that R1 is a straight-chain or branched alkyl residue con-taining 1 to 6 carbon atoms or a p-bromophenyl resi-due.
3. Compounds according to at least one of Claims 1 and 2, characterized in that R2 is a CH3-O group.
4. Compounds according to at least one of Claims 1 and 2, characterized in that R2 is a residue of the general formula II
(II) or of the general formula II a (IIa) wherein R4 is a methylene or propinylene amino group and R8 is a hydrogen atom.
(II) or of the general formula II a (IIa) wherein R4 is a methylene or propinylene amino group and R8 is a hydrogen atom.
5. Compounds according to at least one of Claims 1 and 2, characterized in that R2 is a residue of the general formula III
(III) or of the general formula III a (IIIa) where R5 is an NH group, R6 is a hydrogen atom or a methyl group and R7 is a methyl, ethyl or n-propyl residue.
(III) or of the general formula III a (IIIa) where R5 is an NH group, R6 is a hydrogen atom or a methyl group and R7 is a methyl, ethyl or n-propyl residue.
6. Compounds according to at least one of Claims 1 to 5, characterized in that R3 is a hydrogen atom or a benzoyl residue.
7. Compounds according to Claim 1, namely 17.alpha.-[5-(Mercapto-acetyl-sarcosyl-glycyl-amino-1-pentene-3-inyl] estra-1,3,5(10)-triene-3,17.beta.-diol, 6-N-methyl-8.alpha.-amino-[8.alpha.-N-(thio-acetyl-sarcosyl-glycyl-glycyl)]ergoline, 6-N-methyl-8.alpha.-amino-[8.alpha.-N-(thio-acetyl-sarcosyl)]
ergoline, 6-N-methyl-8.beta.-hydroxymethylene-[O-(thio-acetyl-sarco-syl-glycyl-glycyl)]ergoline and 6-N-methyl-8.beta.-hydroxymethylene-[O-(thio-acetyl-sarco-syl-glycyl]ergoline.
ergoline, 6-N-methyl-8.beta.-hydroxymethylene-[O-(thio-acetyl-sarco-syl-glycyl-glycyl)]ergoline and 6-N-methyl-8.beta.-hydroxymethylene-[O-(thio-acetyl-sarco-syl-glycyl]ergoline.
8. Metal chelate complexes of radioactive metal ions of the elements Tc, Re, Cu, Ga, Gd, Y, and In with compounds of the general formula I
R3-S-CH2-CO-NR1-[CH2-CO-NH]n-CH2-CO-R2 (I) with the exception of N-[2-(benzoylthio)acetyl]-N-cyclohexyl glycine wherein n represents the numbers 0,1 or 2, R1 is a straight-chain or branched alkyl residue con-taining 1 to 20 carbon atoms which may optionally be interrupted or replaced by one to three oxygen atoms, and which may include a terminal -COOH, -OH, or -NH2 group that may optionally be esterified or etherified by means of glycolic acid or its esters or ethers, these esters or ethers being formed using carboxylic acids or alcohols containing up to 18 carbon atoms, or represents a phenyl or cyclohexyl residue having an optional COOH, NH2 or OH group at its 4-position that may optionally be esterified, etherified or amidated using carboxylic acids or alcohols containing up to 6 carbon atoms, or carries an additional halogen atom, R2 is a halogen atom, a halogen methyl, methyl carb-oxyl, trifluoromethyl carboxyl, an NH2 or an OH group, the carboxyl group formed when R2 = OH being esterified or amidated either immediately or after etherification with an .alpha.,.omega.-hydroxycar-= boxylic acid containing up to 8 carbon atoms through its terminal carboxyl group, with a biomolecule, a steroid, an ergoline derivative, a benzodiazepine derivative, a cholecystokinin, a peptide, a protein, a proteohormone, an amino sugar, an endothelin, an endothelin derivative, an endothelin antagonist or an endothelin fragment, is a residue of the general formula II
(II) or of the general formula II a (IIa) where R4 is a methylene, propenylene amino, propinylene amino, methylene amino or methylenoxy group and R8 is a hydrogen atom or a methyl group, is a residue of the general formula III
(III) or of the general formula III a (IIIa) wherein are:
R5 an -NH-, -NH-CO-N<, -NH-CO-NH- or methylenoxy group, R6 a hydrogen atom, a halogen atom or a methyl group and R7 a hydrogen atom or a straight-chain or branched alkyl residue containing up to 6 carbon atoms, R3 is a hydrogen atom, an acetyl, benzoyl, p-methoxybenzyl, acetamidomethyl, benzamidomethyl, trimethyl acetamidomethyl, hydroxyacetyl, ethoxyethyl, ethylthio, trityl, or an easily separable sulfur-protective group, and their salts formed from pharmaceutically accept-able acids or bases.
R3-S-CH2-CO-NR1-[CH2-CO-NH]n-CH2-CO-R2 (I) with the exception of N-[2-(benzoylthio)acetyl]-N-cyclohexyl glycine wherein n represents the numbers 0,1 or 2, R1 is a straight-chain or branched alkyl residue con-taining 1 to 20 carbon atoms which may optionally be interrupted or replaced by one to three oxygen atoms, and which may include a terminal -COOH, -OH, or -NH2 group that may optionally be esterified or etherified by means of glycolic acid or its esters or ethers, these esters or ethers being formed using carboxylic acids or alcohols containing up to 18 carbon atoms, or represents a phenyl or cyclohexyl residue having an optional COOH, NH2 or OH group at its 4-position that may optionally be esterified, etherified or amidated using carboxylic acids or alcohols containing up to 6 carbon atoms, or carries an additional halogen atom, R2 is a halogen atom, a halogen methyl, methyl carb-oxyl, trifluoromethyl carboxyl, an NH2 or an OH group, the carboxyl group formed when R2 = OH being esterified or amidated either immediately or after etherification with an .alpha.,.omega.-hydroxycar-= boxylic acid containing up to 8 carbon atoms through its terminal carboxyl group, with a biomolecule, a steroid, an ergoline derivative, a benzodiazepine derivative, a cholecystokinin, a peptide, a protein, a proteohormone, an amino sugar, an endothelin, an endothelin derivative, an endothelin antagonist or an endothelin fragment, is a residue of the general formula II
(II) or of the general formula II a (IIa) where R4 is a methylene, propenylene amino, propinylene amino, methylene amino or methylenoxy group and R8 is a hydrogen atom or a methyl group, is a residue of the general formula III
(III) or of the general formula III a (IIIa) wherein are:
R5 an -NH-, -NH-CO-N<, -NH-CO-NH- or methylenoxy group, R6 a hydrogen atom, a halogen atom or a methyl group and R7 a hydrogen atom or a straight-chain or branched alkyl residue containing up to 6 carbon atoms, R3 is a hydrogen atom, an acetyl, benzoyl, p-methoxybenzyl, acetamidomethyl, benzamidomethyl, trimethyl acetamidomethyl, hydroxyacetyl, ethoxyethyl, ethylthio, trityl, or an easily separable sulfur-protective group, and their salts formed from pharmaceutically accept-able acids or bases.
9. Metal chelate complexes according to Claim 8, charac-terized in that the radioactive metal ions are iso-topes of Tc and Re.
10. Metal chelate complexes according to at least one of Claims 8 and 9, characterized in that the radio-nuclide is technetium-99m.
11. Compounds according to Claim 8, namely [99mTc]-Mercaptoacetylsarcosine, [99mTc]-Mercaptoacetyl-sarcosyl-diglycine, [99mTc]-Mercaptoacetyl-hexyglycyl-diglycine and [99mTc]-Mercaptoacetyl-N1-[4-bromophenyl]-glycyl-glycyl-glycine.
12. Conjugates containing compounds of the general formula I or metal chelate complexes of radioactive metal ions of the elements Tc, Re, Cu, Ga, Gd, Y and In with compounds of the general formula I and sub-stances that accumulate selectively in diseased tis-sues, with a covalent bonding existing between said substances that is amidic for substances containing carboxy or amino groups such as peptides, proteins, antibodies or their fragments, ester-like for sub-stances containing hydroxy groups such as fatty alcohols, and imidic for substances containing alde-hyde groups.
13. Conjugates according to Claim 12, characterized in that the substances accumulating in the diseased tissue are peptides such as endothelins, partial endothelin sequences, endothelin analogues, endothelin derivatives or endothelin antagonists.
14. Conjugates according to Claim 12, characterized in that the peptides comprise the following sequences:
, .
, .
15. Method for manufacturing a compound of the general formula I, characterized in that a) a diketo piperazine derivative of glycine or b) glycine is first reacted with a haloacid halogenide and subsequently with an amine of the general formula VI
R1 - NH2 (VI) where R1 is as defined in Claim 1 above, then is reacted again with a haloacid halogenide and then reacted with a compound of the general formula IV
R3 - SH (IV) where R3 is as defined in Claim 1 above, or a compound of the general formula V
NH2 - CH2 - CO - R2 (V) where R2 is as defined in Claim 1 above, is reacted with a haloacid halogenide and then with an amine of the general formula VI
R1 - NH2 (VI), where R1 is as defined in Claim 1 above, and reacted again with a haloacid halogenide and then with a compound of the general formula IV
R3 - SH (IV) where R3 is as defined in Claim 1 above, and that these compounds are optionally converted into a salt by means of a pharmaceutically acceptable acid or base.
R1 - NH2 (VI) where R1 is as defined in Claim 1 above, then is reacted again with a haloacid halogenide and then reacted with a compound of the general formula IV
R3 - SH (IV) where R3 is as defined in Claim 1 above, or a compound of the general formula V
NH2 - CH2 - CO - R2 (V) where R2 is as defined in Claim 1 above, is reacted with a haloacid halogenide and then with an amine of the general formula VI
R1 - NH2 (VI), where R1 is as defined in Claim 1 above, and reacted again with a haloacid halogenide and then with a compound of the general formula IV
R3 - SH (IV) where R3 is as defined in Claim 1 above, and that these compounds are optionally converted into a salt by means of a pharmaceutically acceptable acid or base.
16. Method for manufacturing metal chelate complexes of radioactive metal ions of Tc and Re with compounds of the general formula I, characterized in that techne-tium-99m or Re in the form of pertechnetate or per-rhenate are reacted in the presence of a reductant and, optionally, an auxiliary ligand, with a compound of the general formula I
R3-S-CH2-CO-NR1-[CH2-CO-NH]n-CH2-CO-R2 (I) with the exception of N-[2-(benzoylthio)acetyl]-N-cyclohexyl glycine wherein R1, R2 and R3 are as defined in Claim 1 above.
R3-S-CH2-CO-NR1-[CH2-CO-NH]n-CH2-CO-R2 (I) with the exception of N-[2-(benzoylthio)acetyl]-N-cyclohexyl glycine wherein R1, R2 and R3 are as defined in Claim 1 above.
17. A kit for manufacturing radiopharmaceuticals, con-sisting of a compound of the general formula I ac-cording to any one of Claims 1 to 7, or a conjugate according to any one of Claims 12 to 14, as well as a reductant and, optionally, an auxiliary ligand, either dry or in solution, and instructions for use, including instructions for reacting the compounds described with technetium-99m or Re in the form of a pertechnetate or perrhenate solution.
18. A radiopharmaceutical preparation for the non-invasive in-vivo visualization of receptors and tissue containing receptors and/or of atherosclerotic plaques and/or for renal function checking, characterized in that it contains a compound according to any one of Claims 8 to 10 or a conjugate according to any one of Claims 12 to 14 and, optionally, additives common in galenics, said compound being prepared in a kit according to Claim 17 with technetium-99m or Re in the form of a pertechnetate or perrhenate solution.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4337600.2 | 1993-11-01 | ||
| DE4337600A DE4337600A1 (en) | 1993-11-01 | 1993-11-01 | N-alkyl peptide chelating agents, their metal complexes with radionuclides, processes for their preparation and radiopharmaceutical compositions containing these compounds |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2173844A1 true CA2173844A1 (en) | 1995-05-11 |
Family
ID=6501750
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002173844A Abandoned CA2173844A1 (en) | 1993-11-01 | 1994-10-27 | N-alkyl peptide chelate formers, their metal complexes with radionuclides, processes for producing them and radio-pharmaceutical compositions containing these compounds |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0726909A1 (en) |
| JP (1) | JPH09508351A (en) |
| KR (1) | KR960705842A (en) |
| CN (1) | CN1134158A (en) |
| AU (1) | AU681919B2 (en) |
| CA (1) | CA2173844A1 (en) |
| DE (1) | DE4337600A1 (en) |
| HU (1) | HUT74881A (en) |
| NO (1) | NO961743D0 (en) |
| WO (1) | WO1995012610A1 (en) |
| ZA (1) | ZA948411B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6241963B1 (en) * | 1995-10-19 | 2001-06-05 | The Trustees Of The University Of Pennsylvania | Dopamine and serotonin transporter ligands and imaging agents |
| US6162378A (en) * | 1999-02-25 | 2000-12-19 | 3D Systems, Inc. | Method and apparatus for variably controlling the temperature in a selective deposition modeling environment |
| US6171578B1 (en) | 1999-04-14 | 2001-01-09 | Diatide, Inc. | Benzodiazepine derivatives for imaging thrombi |
| CA2781858C (en) | 2000-05-12 | 2015-03-31 | Genzyme Corporation | Modulators of tnf-.alpha. signaling |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ZA811340B (en) * | 1980-06-09 | 1982-03-31 | Morton Norwich Products Inc | N-(cycloalkyl)amino acid compounds |
| WO1992019274A1 (en) * | 1991-05-08 | 1992-11-12 | Mallinckrodt Medical, Inc. | Technetium chelates to be used for determining the renal function |
| JPH0570484A (en) * | 1991-09-12 | 1993-03-23 | Hitachi Chem Co Ltd | Peptide and its salt |
| JPH07503732A (en) * | 1992-02-06 | 1995-04-20 | マリンクロッド・インコーポレイテッド | Ligands to increase the rate of metal chelate formation |
| JP2941057B2 (en) * | 1992-05-21 | 1999-08-25 | ダイアテク，インコーポレイテッド | Technetium-99m labeled peptide for thrombus imaging |
| DE4311021A1 (en) * | 1993-03-31 | 1994-10-27 | Diagnostikforschung Inst | Bifunctional chelating agents, their technetium and rhenium complexes, processes for their preparation and radiopharmaceutical compositions containing these compounds |
-
1993
- 1993-11-01 DE DE4337600A patent/DE4337600A1/en not_active Withdrawn
-
1994
- 1994-10-26 ZA ZA948411A patent/ZA948411B/en unknown
- 1994-10-27 CN CN94193990A patent/CN1134158A/en active Pending
- 1994-10-27 AU AU81038/94A patent/AU681919B2/en not_active Ceased
- 1994-10-27 WO PCT/DE1994/001295 patent/WO1995012610A1/en not_active Application Discontinuation
- 1994-10-27 JP JP7512959A patent/JPH09508351A/en active Pending
- 1994-10-27 EP EP95900059A patent/EP0726909A1/en not_active Withdrawn
- 1994-10-27 CA CA002173844A patent/CA2173844A1/en not_active Abandoned
- 1994-10-27 HU HU9601140A patent/HUT74881A/en unknown
-
1996
- 1996-04-30 NO NO961743A patent/NO961743D0/en unknown
- 1996-04-30 KR KR1019960702247A patent/KR960705842A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO1995012610A1 (en) | 1995-05-11 |
| NO961743L (en) | 1996-04-30 |
| NO961743D0 (en) | 1996-04-30 |
| HUT74881A (en) | 1997-02-28 |
| JPH09508351A (en) | 1997-08-26 |
| AU8103894A (en) | 1995-05-23 |
| CN1134158A (en) | 1996-10-23 |
| DE4337600A1 (en) | 1995-05-04 |
| KR960705842A (en) | 1996-11-08 |
| HU9601140D0 (en) | 1996-07-29 |
| EP0726909A1 (en) | 1996-08-21 |
| ZA948411B (en) | 1995-06-30 |
| AU681919B2 (en) | 1997-09-11 |
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