WO2011073137A2 - Composés pour l'imagerie de la mort cellulaire apoptotique - Google Patents

Composés pour l'imagerie de la mort cellulaire apoptotique Download PDF

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WO2011073137A2
WO2011073137A2 PCT/EP2010/069501 EP2010069501W WO2011073137A2 WO 2011073137 A2 WO2011073137 A2 WO 2011073137A2 EP 2010069501 W EP2010069501 W EP 2010069501W WO 2011073137 A2 WO2011073137 A2 WO 2011073137A2
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connectivity
defines
point
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compound
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PCT/EP2010/069501
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WO2011073137A3 (fr
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Andre Müller
Sabine Zitzmann-Kolbe
Niels Böhnke
Purnama DEWI-WÜLFING
Doerte Oltmanns
Michael Eisenhut
Ulrike Bauder-Wuest
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Bayer Schering Pharma Aktiengesellschaft
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Publication of WO2011073137A2 publication Critical patent/WO2011073137A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

  • the invention pertains to compounds for detecting and imaging of apoptotic cell death in vitro and in vivo, as well as to a method for detecting the progression or regression of a tumor in a patient after treatment. Furthermore, the invention relates to pharmaceutical compositions, comprising such compounds, precursors suitable for obtaining them, methods for obtaining them and to kit for detecting and imaging of apoptotic cell death.
  • Radiotherapy control of tumors is one of the main areas of interest during the follow up of cancer patients. This information is currently acquired by a series of conventional investigations including morphological changes, symptomatic responses and clinical chem istry.
  • Rad iolog ical techn iques l i ke X-ray, computed tomography (CT), ultrasonography (US) and magnetic resonance (MR) are imaging techniques which cover the size and morphology of tumor masses and to some degree alterations of tumor perfusion.
  • [ 18 F]FDG represents the main protagonist which is acting as a surrogate marker used to show the influence of e.g. cytostatic drugs on the glucose transport and hexokinase activity in tumors.
  • a decrease in tracer accumulation is expected to ind icate treatment response and a better su rvival .
  • interpretation of the 18 F-FDG-signal might be complicated by inflammatory reactions, for example early after radiation therapy.
  • tumor response is shown via a negative signal, i.e. a decrease in tracer uptake. This may be problematic in cases of a moderate accumulation of the tracer prior to therapy. In this respect, tracers providing a positive (increasing) signal are more desirable.
  • Tc labeled annexin V was presented as a new agent capable to image cell death in vivo. Apoptosis was visualized in several model systems including myocardial infarction, Fas antibody treated liver and tumors after treatment (Blankenberg et al. (1997) J Nucl Med. 38(suppl):268P, Blankenberg et al. (1998) Proc Natl Acad Sci U SA. 95:6349-6354). Annexin V has first been isolated from human placenta (Bohn & Kraus (1979) Arch Gynecol.227:125-134) demonstrating an anticoagulant effect by inhibiting e.g. prothrombinase activity.
  • Annexins are ubiquitous homologous proteins that bind to phospholipids in the presence of calcium.
  • Annexin V binds together with Ca 2+ on externalized phosphatidylserine (PS) present on the cell membranes early after the onset of apoptosis.
  • PS externalized phosphatidylserine
  • phosphatidylserine resides to about 100 % on the interior leaflet of the bi- layered cell membrane where it functions as an anchor for e.g. annexin I, which is involved in transmembrane signaling and internalization processes.
  • proteolytic inactivation of proteins occurs which maintain under normal conditions the asymmetric distribution of lipids within the cell membrane (e.g. an ubiquitous expressed Mg 2+ -ATP dependent aminophospholipid flippase that selectively catalyzes the inward transport of phosphatidylserine and phosphatidylethanolamine, and an ATP dependent floppase that moves phospholipids outward with little headgroup specificity).
  • an ubiquitous expressed Mg 2+ -ATP dependent aminophospholipid flippase that selectively catalyzes the inward transport of phosphatidylserine and phosphatidylethanolamine, and an ATP dependent floppase that moves phospholipids outward with little headgroup specificity.
  • annexin V for cells which expose phosphatidylserine, exhibiting a K d of ⁇ 10 "10 mol/l, is the basis of detecting apoptosis in vivo using radiolabeled annexin derivatives.
  • Annexin V is typically labeled with a fluorescent dye for in vitro assays. Although it is utilized extensively in molecular biology, the labeled protein is expensive and moderately unstable. The dye as well as the radiolabeled annexin V is a useful apoptosis sensor, but it has a number of limitations especially the slow pharmacokinetics, which makes this protein-based approach difficult for in vivo application using radiolabels with a short half-life like F18. Therefore it exists a need for small molecule agents that are cheap, robust, and show a favourable pharmacokinetic.
  • Zn 2+ bis-DPA zinc bis-(2,2-dipicolylamine) coordination complexes. It was designed to mimic the apoptosis sensing function of annexin V (Hanshaw et al. (2005) ChemBioChem 12, 2214, Lakshmi & Hanshaw, (2004) Tetrahedron 60: 11307-11315).
  • annexin V One phosphatidylserine head group is coordinated to two bridging Ca 2+ ions. Compared to annexin V, these complexes also depend on high Ca 2+ -concentrations for binding and are not taken up by apoptotic cells.
  • a macrocyclic Zn-fluorophore as a detector of apoptosis was described recently (Kimura et al. (2003) PNAS 100(7): 3731-3736). Unfortunately, this compound crosses the cell membrane of apoptotic cells only if not complexed with Zn. The intracellular complexation of Zn in a second step is then necessary for obtaining a fluorescent signal.
  • the problem underlying the present invention was to provide a molecule that would show improved characteristics compared to available apoptosis imaging reagents, i n pa rticu l a r, to provid e a mol ecu l e th at wou ld show i m proved characteristics as an apoptosis sensor over annexin V or caspase inhibitors.
  • the aim of the invention is to provide improved molecule enabling the in vivo and in vitro detection of apoptotic cell death.
  • the improved molecule is a marker specifically being taken up by apoptotic cells.
  • the inventors solved the problem by discovering small molecules which show improved characteristics as an apoptosis sensor over annexin V or caspase inhibitors.
  • the small molecules are robust, are rapidly being taken up, are Zn 2+ and Ca 2+ -independent and surprisingly show high selectivity over apoptotic cells.
  • the inventors used a compound with only one cyclic polyamine unit, in particular cyclen (12[ane]N ) or cyclam (14[ane]N ) instead of DPA and observed uptake into apoptotic cells.
  • the invention is directed to compounds of formula I, la, lb, lc, II, lla, Mb, l ie, III, Xa, Xb or Xc for imaging apoptotic cell death in vitro and in vivo, as well as to a method for detecting the progression or regression of a tumor in a patient. Furthermore, the invention relates to compositions, comprising such compounds, precursors of formula I I I suitable for obtaining them, methods for obtaining them and to kits for imaging apoptotic cell death.
  • FIG. 1 FACS-analysis of compound 2a using treated and control Jurkat cells and costained with Propidium iodide.
  • FL-I-Plot shows the green fluorescence from the te st co m po u n d a n d th e
  • F L-l/FL-lll-pl ot shows th e co m pen sat ion of g ree n fluorescence (FL-I) and red fluorescence (FL-I I I).
  • FL-I g ree n fluorescence
  • FL-I I I red fluorescence
  • FIG. 2 FACS-analysis of compound 3b using treated and control Jurkat cells.
  • FL-I- Plot shows the green fluorescence from the test compound .
  • 56 % of cells were stained with the green compound 3b (quarter Q1 ).
  • FIG. 3 FACS-analysis of compound 4b using treated and control Jurkat cells.
  • FL-I- Plot shows the green fluorescence from the test compound . 54.5 % of cells were stained with the green compound 4b (quarter Q1 ).
  • Figure 4 FACS-analysis of compound 5b using treated and control Jurkat cells.
  • FL-I- Plot shows the green fluorescence from the test compound . 66.6 % of cells were stained with the green compound 5b (quarter Q1 ).
  • Figure 5 (A) Phase contrast of Jurkat cells treated with Staurosporine for 4 h.
  • B Staining with test-compound (2a). Cells which have taken up the test compound and show green fluorescence are marked with a black circle in Figure 5.
  • C Co-staining of staurosporine-treated Jurkat cells with compound (2a) and Annexin V, labeled with Phycoerythrin (Annexin-PE). Circled cells have taken up Compound 2a, boxed cells were stained with Annexin V-PE.
  • Figure 8 Compound 13 HPLC (t R 3.1 min, column ACE 3 ⁇ C18 50x4.6 mm, 2 ml/min (Agilent), solvent A: H 2 O + 0.1 %TFA, solvent B: MeCN + 0.1 % TFA, gradient: 0%B to 95%B in 7 min), ⁇ detector (up), coinjection with compound 11 b,UV 254 nm (bottom).
  • Figure 11 Fluorination of compound 15 to give compound 16, reaction mixture.
  • HPLC t R 4.8 min, column ACE 3 ⁇ C18 50x4.6 mm, 2 ml/min (Agilent), solvent A: H 2 O + 0.1 %TFA, solvent B: MeCN + 0.1 % TFA, gradient: 0%B to 95%B in 7 min), ⁇ detector.
  • Figure 12 FACS-analysis of compound 1 a using treated and control Jurkat cells.
  • FL- l-Plot shows the green fluorescence from the test compound. 42 % of cells were stained with the green compound. Description of the invention
  • the invention is directed to compounds of the general formula (I)
  • A is c clen of formula IV or cyclam of formula V
  • ** defines the point of connectivity to L
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • T 1 is a fluorescence-tag selected from the group of
  • E is selected from the group of hydrogen and halogen
  • B 1 is selected from the group of O, NH and N(Ci-C3-alkyl);
  • B 2 is selected from the group of hydroxy, amino, (Ci-C3-alkyl)amino and di(Ci-C3-alkyl)amino;
  • # ' defines the point of connectivity to T 2 ;
  • Formula (I) encompasses single isomers, tautomers, diastereomers, enantiomers, mixtures thereof, and suitable salts thereof.
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • r 1 -2.
  • T 2 is CH2"CH2- ⁇ F.
  • r 1 -2.
  • T 2 is CH2"CH2- ⁇ F.
  • T 2 is hydrogen
  • both substituents E are either hydrogen or halogen, halogen being selected from bromo, iodo or chloro.
  • T 1 is a fluorescence-tag selected from
  • B 1 is selected from the group of O, NH and N(Ci-C3-alkyl);
  • B 2 is selected from the group of hydroxy, amino, (Ci-C3-alkyl)amino and di(Ci-C3-alkyl)amino;
  • # ' defines the point of connectivity to T 2 .
  • B 1 is oxygen
  • B 2 is hydroxy
  • A is a c clen of formula IV
  • the invention compound is a compound of general formula (I)
  • A is cyclen of formula IV or cyclam of formula V
  • ** defines the point of connectivity to L
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • T 1 is a fluorescence-tag selected from
  • B 1 is selected from the group of O, NH and N(Ci-C3-alkyl);
  • B 2 is selected from the group of hydroxy, amino, (Ci-C3-alkyl)amino and di(Ci-C3-alkyl)amino;
  • Formula (I) encompasses single isomers, tautomers, diastereomers, enantiomers, mixtures thereof, and suitable salts thereof.
  • the invention compound is a compound of general formula (I)
  • A is c clen of formula IV or cyclam of formula V
  • ** defines the point of connectivity to L
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • T 1 is a fluorescence-tag selected from
  • the invention compound is a compound of general formula (I) wherein
  • T 1 is a fluorescence-tag selected from the group of
  • E is selected from the group of hydrogen and halogen
  • T 1 is a fluorescence-tag selected from the group of
  • T 1 is a fluorescence-tag selected from the group of
  • r 1 -2.
  • T 2 is CH2"CH2- ⁇ 8 F.
  • the invention compound is a compound of general formula (I) wherein
  • T 1 is a fluorescence-tag selected from the group of
  • E is selected from the group of hydrogen and halogen
  • T 1 is a fluorescence-tag selected from the group of
  • T 1 is a fluorescence-tag selected from the group of
  • r 1 -2.
  • T 2 is CH2"CH2- ⁇ 9 F.
  • the invention compound is a compound of general formula (I) wherein
  • T 1 is a fluorescence-tag selected from the group of
  • E is selected from the group of hydrogen and halogen
  • T 2 is hydrogen
  • T 1 is a fluorescence-tag selected from the group of
  • T 2 is hydrogen.
  • T 1 is a fluorescence-tag selected from the group of
  • T 2 is hydrogen. Preferred features disclosed above for L and T 1 and compound of general formula (I) are herein enclosed.
  • Invention compounds are selected from but not limited to 3',6'-Dihydroxy-5-[(1 ,4,7,10-tetraazacyclododecan-1 -yl)carbonyl]-spiro[isobenzofuran- 1 (3H),9'-[9H]xanthen]-3-one
  • the invention is directed to compounds of the general formula (II)
  • A' is tris-nitrogen-protected cyden of formula VI or VII or cyclam of formula VIII or IX
  • R 2 is a nitrogen protecting group
  • L is a linker moiety attached to one of the cyden or cyclam ring nitrogen atoms and selected from the group of
  • T 3 is an optionally protected fluorescence-tag selected from
  • E is individually and independently selected from the group of hydrogen and halogen
  • B 1 is selected from the group of O, NH and N(Ci-C3-alkyl);
  • B 3 is selected from the group of O-R 3 , OH, NH 2 , NHR 2 , (C1-C3- alkyl)amino, R 2 (Ci-C3-alkyl)amino and di(Ci-C3-alkyl)amino; R 3 is a hydroxy protecting group;
  • # ' defines the point of connectivity to T 2 ;
  • Formula (II) encompasses single isomers, tautomers, diastereomers, enantiomers, mixtures thereof, and suitable salts thereof.
  • R 2 is an nitrogen-protecting group selected from the group comprising Carbobenzyloxy (Cbz), p-Methoxybenzyl carbonyl ( M oz o r M eOZ ) , tert- Butyloxycarbonyl (BOC), 9-Fluorenylmethyloxycarbonyl (FMOC), Benzyl (Bn), p- Methoxybenzyl (PMB), 3,4-Dimethoxybenzyl (DM PM), Triphenylmethyl , p-methoxyphenyl (PMP) and p-Methylbenzenesulfonyl (Tos).
  • Cbz Carbobenzyloxy
  • M oz o r M eOZ tert- Butyloxycarbonyl
  • FMOC 9-Fluorenylmethyloxycarbonyl
  • Benzyl Bn
  • PMB p- Methoxybenzyl
  • DM PM 3,4-Dimethoxybenz
  • R 2 is selected from the group comprising Carbobenzyloxy (Cbz), tert-Butyloxycarbonyl (BOC), p-Methylbenzenesulfonyl (Tos), 9-Fluorenylmethyloxycarbonyl (FMOC), Benzyl (Bn).
  • R 2 is selected from the group comprising tert-Butyloxycarbonyl (BOC) and Carbobenzyloxy (Cbz). Even more preferably, R 2 is tert-Butyloxycarbonyl (BOC).
  • R 4 is methyne (CH).
  • A' is tris-nitrogen-protected cyclen of formula VI or tris-nitrogen-protected cyclam of formula VIII
  • ** defines the point of connectivity to L.
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • L is a linker moiety attached to one of the cyclen or cycla ring nitrogen atoms and selected from the group of
  • r 1 -2.
  • T 2 is CH2"CH2- ⁇ F.
  • r 1 -2.
  • T 2 is CH2"CH2- ⁇ F.
  • T 2 is hydrogen.
  • both substituents E are either hydrogen or halogen, halogen being selected from bromo, iodo or chloro.
  • T 3 is a fluorescence-tag selected from
  • B 1 is selected from the group of O, NH and N(Ci-C3-alkyl);
  • B 3 is selected from the group of O-R 3 , OH, NH 2 , NHR 2 , (C1-C3- alkyl)amino, R 2 (Ci-C3-alkyl)amino and di(Ci-C3-alkyl)amino;
  • R 3 is a hydroxy protecting group
  • # ' defines the point of connectivity to T 2 .
  • B 1 is oxygen
  • B 3 is O-R 3 or OH. Even more preferably, B 3 is OH.
  • R 3 is a hydroxy protecting group.
  • R 3 is tert-butoxycarbonyl, 2,2- dimethylpropanoyl, methoxymethyl, allyl, terf-butyl, or acetyl. More preferably, R 3 is 2,2-dimethylpropanoyl.
  • the invention compound is directed to a compound of the general formula
  • A' is tris-nitrogen-protected cyclen or tris-nitrogen-protected cyclam selected from the grou of
  • ** defines the point of connectivity to L
  • R 2 is a nitrogen protecting group
  • L is a linker moiety attached to one of the cyclen- or cyclam-ring nitrogen atoms and selected from the group of
  • T 3 is an optionally protected fluorescence-tag selected from
  • B 1 is selected from the group of O, NH and N(Ci-C3-alkyl) ;
  • B 3 is selected from the group of O-R 3 , OH, NH 2 , NHR 2 , (C1-C3- alkyl)amino, R 2 (Ci-C3-alkyl)amino and di(Ci-C3-alkyl)amino;
  • R 3 is a hydroxy protecting group
  • # ' defines the point of connectivity to T 2 ;
  • Formula (II) encompasses single isomer, tautomers, diastereomers, enantiomers, mixtures thereof, and suitable salts thereof. Even more preferably, the invention compound is directed to a compound of the general formula (II)
  • A' is tris-nitrogen-protected cyclen or tris-nitrogen-protected cyclam selected from the group of
  • ** defines the point of connectivity to L
  • R 2 is a nitrogen protecting group
  • L is a linker moiety attached to one of the cyclen- or cyclam-ring nitrogen atoms and selected from the group of
  • T 3 is an optionally protected fluorescence-tag selected from
  • B 3 is selected from the group of O-R 3 and OH;
  • R 3 is a hydroxy protecting group
  • # ' defines the point of connectivity to T 2 ;
  • the invention compound is a compound of general formula (II) wherein T 3 is a fluorescence-tag selected from the group of
  • E is selected from the group of hydrogen and halogen
  • r 1 -6.
  • r 1 -2.
  • T 2 is CH 2 -CH 2 - 18 F.
  • the invention compound is a compound of general formula (II) wherein
  • T 3 is a fluorescence-tag selected from the group of
  • E is selected from the group of hydrogen and halogen
  • T 3 is a fluorescence-tag selected from the group of
  • r 1 -2.
  • T 2 is CH 2 -CH 2 - 19 F.
  • the invention compound is a compound of general formula (II) wherein
  • E is selected from the group of hydrogen and halogen
  • T 3 is a fluorescence-tag selected from the group of
  • T 2 is hydrogen
  • Invention compounds are selected from but not limited to
  • the invention is directed to compounds of the general formula (X)
  • A' is tris-nitrogen-protected cyclen of formula VI or VII or cyclam of formula VIII or IX
  • R 2 is a nitrogen protecting group
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • T 5 is a fluorescence-tag selected from the group of
  • E is selected from the group of hydrogen and halogen
  • # ' defines the point of connectivity to T 2 ;
  • Formula (X) encompasses single isomers, tautomers, diastereomers, enantiomers, mixtures thereof, and suitable salts thereof.
  • R 2 is an nitrogen-protecting group selected from the group comprising Carbobenzyloxy (Cbz), p-Methoxybenzyl carbonyl ( M oz o r M eOZ ) , tert- Butyloxycarbonyl (BOC), 9-Fluorenylmethyloxycarbonyl (FMOC), Benzyl (Bn), p- Methoxybenzyl (PMB), 3,4-Dimethoxybenzyl (DMPM), Triphenylmethyl, p-methoxyphenyl (PMP) and p-Methylbenzenesulfonyl (Tos).
  • Cbz Carbobenzyloxy
  • M oz o r M eOZ tert- Butyloxycarbonyl
  • FMOC 9-Fluoreny
  • R 2 is selected from the group comprising Carbobenzyloxy (Cbz), tert-Butyloxycarbonyl (BOC), p-Methylbenzenesulfonyl (Tos), 9-Fluorenylmethyl- oxycarbonyl (FMOC), Benzyl (Bn).
  • Cbz Carbobenzyloxy
  • BOC tert-Butyloxycarbonyl
  • Tos p-Methylbenzenesulfonyl
  • FMOC 9-Fluorenylmethyl- oxycarbonyl
  • Bn Benzyl
  • R 2 is selected from the group comprising tert-Butyloxycarbonyl
  • R 2 is tert-Butyloxycarbonyl (BOC).
  • R 4 is methyne (CH).
  • A' is tris-nitrogen-protected cyclen of formula VI or tris-nitrogen-protected cyclam of formula VIII
  • ** defines the point of connectivity to L.
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • r 1 -2.
  • T 2 is CH 2 -CH 2 - 18 F.
  • r 1 -2.
  • T 2 is CH 2 -CH 2 - ⁇ F.
  • T 2 is hydrogen.
  • T 5 is a fluorescence-tag selected from the group of
  • # ' defines the point of connectivity to T 2 .
  • the invention compound is directed to a compound of the general formula
  • A' is tris-nitrogen-protected cyclen or tris-nitrogen-protected cyclam selected from the grou of
  • R 2 is a nitrogen protecting group
  • L is a linker moiety attached to one of the cyclen- or cyclam-ring nitrogen atoms and selected from the group of
  • T 5 is a fluorescence-tag selected from the group of
  • Formula (X) encompasses single isomer, tautomers, diastereomers, enantiomers, mixtures thereof, and suitable salts thereof.
  • the invention compound is directed to a compound of the general formula (X)
  • A' is tri -nitrogen-protected cyclen of formula
  • ** defines the point of connectivity to L
  • R 2 is a nitrogen protecting group
  • L is a linker moiety attached to one of the cyclen- or cyclam-ring nitrogen atoms and selected from the group of
  • T 5 is selected from
  • # ' defines the point of connectivity to T 2 ;
  • the invention compound is a compound of general formula (X) wherein
  • T 5 is a fluorescence-tag selected from the group of
  • E is selected from the group of hydrogen and halogen B 1 is O;
  • T 5 is a fluorescence-tag selected from the group of
  • T 5 is a fluorescence-tag selected from the group of
  • r 2-3. More preferably, T 2 is CH 2 -CH 2 - ⁇ F.
  • the invention compound is a compound of general formula (X) wherein T 5 is a fluorescence-tag selected from the group of
  • E is selected from the group of hydrogen and halogen
  • T 5 is a fluorescence-tag selected from the group of
  • r 2-3.
  • T 2 is CH 2 "CH 2 - ⁇ F.
  • the invention compound is a compound of general formula (X) wherein
  • T 5 is a fluorescence-tag selected from the group of
  • E is selected from the group of hydrogen and halogen
  • T 2 is hydrogen
  • T 5 is a fluorescence-tag selected from the group of
  • T 2 is hydrogen
  • T 5 is a fluorescence-tag selected from the group of
  • T 2 is hydrogen.
  • Invention compounds are selected from but not limited to
  • the invention is directed to compounds of the general formula (III)
  • A' is tris-nitrogen-protected cyclen of formula VI or VII or cyclam of formula VIII or IX
  • ** defines the point of connectivity to L
  • R 2 is a nitrogen protecting group
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • T 4 is selected from the group of
  • E is selected from the group of hydrogen and halogen
  • Formula (III) encompasses single isomers, tautomers, diastereomers, enantiomers, mixtures thereof, and suitable salts thereof.
  • A' is tris-nitrogen-protected cyclen of formula VI or cyclam of formula VIII
  • ** defines the point of connectivity to L.
  • A' is a tris-nitrogen-protected cyclen of formula VI
  • R 2 is a nitrogen-protecting group selected from the group comprising
  • R 2 is selected from the group comprising Carbobenzyloxy (Cbz), tert- Butyloxycarbonyl ( B O C ) , p-Methylbenzenesulfonyl (Tos), 9-Fluorenylmethyl- oxycarbonyl (FMOC), Benzyl (Bn).
  • Cbz Carbobenzyloxy
  • B O C tert- Butyloxycarbonyl
  • Tos p-Methylbenzenesulfonyl
  • FMOC 9-Fluorenylmethyl- oxycarbonyl
  • Bn Benzyl
  • R 2 is selected from the group comprising tert-Butyloxycarbonyl
  • R 2 is tert-Butyloxycarbonyl (BOC).
  • R 4 is methyne (CH).
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • L is a linker moiety attached to one of the cyclen or cyclam ring nitrogen atoms and selected from the group of
  • PM is CH 2 -CH 2 -Y.
  • Y is chloro, bromo, iodo, (4-Methylphenyl)sulfonyloxy, Methylsulfonyloxy, (4-Nitrophenyl)sulphonyloxy, trifluormethylsulfonyloxy or nonafluorbutylsulfonyloxy. More preferably, Y is (4-Methylphenyl)sulfonyloxy, Methylsulfonyloxy or (4- Nitrophenyl)sulphonyloxy. Even more preferably, Y is (4-Methylphenyl)sulfonyloxy.
  • the invention compound is directed to a compound of the general formula
  • A' is tris-nitro en-protected cyclen or cyclam selected from the group of
  • R 2 is a nitrogen protecting group
  • L is a linker moiety attached to one of the cyclen- or cyclam-ring nitrogen atoms and selected from the group of
  • T 4 is selected from
  • Formula (III) encompasses single isomers, tautomers, diastereomers, enantiomers, mixtures thereof, and suitable salts thereof.
  • the invention compound is directed to a compound of the general formula
  • A' is a tris-nitro en- rotected cyclen of formula wherein * defines the point of connectivity to L and
  • R 2 is a nitrogen protecting group
  • L is a linker moiety attached to one of the cyclen- or cyclam-ring nitrogen atoms and selected from the group of
  • T 4 is selected from
  • the invention compound is directed to a compound of the general formula (III)
  • A' is a tris-nitro en- rotected cyclen of formula wherein * defines the point of connectivity to L and
  • R 2 is a nitrogen protecting group
  • L is a linker moiety attached to one of the cyclen- or cyclam-ring nitrogen atoms and selected from the group of
  • T 4 is selected from
  • Formula (III) encompasses single isomers, tautomers, diastereomers, enantiomers, mixtures thereof, and suitable salts thereof.
  • Invention compounds are selected from but not limited to
  • the invention is directed to compositions comprising compounds of the general formula (I), (la), (lb), (Ic), (II), (lla), (Mb), (lie), (Xa), (Xb), (Xc) (III), or mixtures thereof and pharmaceutically acceptable carrier or diluent.
  • the composition is pharmaceutically acceptable.
  • auxiliaries, vehicles, excipients, diluents, carriers or adjuvants which are suitable for the desired pharmaceutical formulations, preparations or compositions on account of his/her expert knowledge.
  • compositions from the current invention containing a fluorescence-label are suitable for in vitro detection of apoptotic cells, either e.g. by confocal microscopy or FACS-analysis.
  • the pharmaceutical compositions containing 18 F according to the invention can be administered into a patient such that the dose of the active compound is in the range of 37 MBq (1 mCi) to 740 MBq (20 mCi). In particular, a dose in the range from 150 MBq to 370 MBq will be used.
  • composition comprises compounds of the general formula (la), (lb), (Ic) or (Ma), (Mb), (Mc) or (Xa), (Xb), (Xc) or (III) or mixtures thereof and pharmaceutically acceptable carrier or diluent.
  • the invention is directed to method for obtaining fluorine containing compounds of the general formula (I), or (II) mixtures thereof.
  • the method of the invention is a fluoro-labeling method.
  • the method concerns a method for labeling invention compounds with a Fluorine atom (F) containing moiety wherein the Fluorine atom (F) containing moiety preferably comprises 18 F or 19 F.
  • Compounds of the general formula (I) mean compounds of preferred embodiment in respect of ( la), (l b), and ( Ic) .
  • Compounds of the general formula (II) mean compounds of preferred embodiment in respect of (I la), (lib), and (lie).
  • the method is a direct labelling method for obtaining compound of formula (I) wherein T 2 comprises Fluorine atom (F), (la), (lb), (II) wherein T 2 comprises Fluorine atom (F), (I la) or (Mb) or mixtures thereof.
  • the fluoro-labeling method comprises the steps Reacting compound of general Formula (I I I) with a Fluorine atom (F) containing moiety to give compound of formula (X) comprising a Fluorine atom (F), (Xa) or (Xb)
  • the method is a direct labelling method for obtaining compound of formula (la) or (I la) or mixtures thereof.
  • Fluorine atom (F) is 18 F.
  • the method is a direct labelling method for obtaining compound of formula
  • the Fluorine atom (F) is 19 F. More preferably, the Fluorine atom (F) containing moiety comprises 18 F. Even more preferably, the Fluorine atom (F) containing moiety is 4,7,13,16,21, 24-Hexaoxa-1, 10- diazabicyclo[8.8.8]-hexacosane K 18 F (crownether salt Kryptofix K 18 F), K 18 F, H 18 F, KH 18 F 2 , Cs 18 F, Na 18 F or tetraalkylammonium salt of 18 F (e.g.[F-18] tetrabutylammonium fluoride). Most preferably, the Fluorine atom (F) containing moiety is K 18 F, H 18 F, or KH 18 F 2.
  • the Fluorine atom (F) containing moiety comprising 19 F is a reagent suitable for the conversion of -OH or -OS(0) 2 R into an organic 19 F fluoride.
  • reagents are exemplified by but not limited to inorganic salts and/or adducts of hydrofluoric acid, e.g. sodium fluoride, potassium fluoride, potassium hydrogen difluoride, or cesium fluoride as such or in combination with chelating reagents, e.g.
  • aminopolyether 2.2.2 K2.2.2
  • organic salts and/or adducts of hydrofluoric acid such as tetra n-butylammonium fluoride (TBAF) or triethylamine tris-hydrofluoride
  • hypervalent fluorosilicates e.g. tetrabutylammonium triphenyldifluorosilicate
  • sulfur fluorides e.g.
  • DAST diethylaminosulfur trifluoride
  • sulfonyl fluorides e.g.1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulfonyl fluoride
  • /V-fluoropyridinium salts e.g. N-fluoropyridinium triflate
  • N-fluorosulfonimides e.g. /V-fluorobenzenesulfonimide
  • aliphatic N-fluoroamines derivatives e.g.
  • the method is an indirect labelling method for obtaining compound of formula (I) comprising a Fluorine atom (F), (la), (lb), (II) comprising a Fluorine atom (F), (I la) or (Mb) or mixtures thereof.
  • the method is an indirect labelling method for obtaining compound of formula
  • Fluorine atom (F) is 18 F.
  • the method is an indirect labelling method for obtaining compound of formula
  • the solvents used in the present method is DMF, DMSO, acetonitrile, DMA, or mixture thereof, preferably the solvent is DMF.
  • E is hydrogen; B 1 is O;
  • # ' defines the oint of connectivity to T 2 .
  • # ' defines the point of connectivity to T 2 .
  • the invention is related to a compound of general formula (I), ( l a ) o r ( I c) mixtures thereof as described here i n u sed fo r b i n d i ng a nd imaging/detecting apoptotic cell death (dying cells), both in vitro and in vivo.
  • I n particular, such use makes apoptosis imaging in vivo possible, by using positron emission tomography (PET).
  • PET positron emission tomography
  • the invention relates to the use of invention cyclic polyamine compounds as described herein or compositions thereof for in vivo or in vitro detection of cell death resulting from apoptosis.
  • the use is for in vitro detection .
  • In vitro detection can be conducted by FACS, microspectrometry, fluorescence microscopy or ELISA. Even more preferably, the use is for in vivo detection. In vivo detection can be conducted by PET.
  • such a method allows for the assessment of a patient suffering from a tumor, from an infection, and/or from an inflammation, from injury, from stroke, from heart attack, allows for the assessment of a patient responding to an anti-tumor therapy, to anti-infection therapy and/or to anti-inflammatory therapy, respectively, measuring the amount of apoptotic cells which is directly correlated to disease progression or regression and also allows monitoring of treatment efficacy.
  • the compounds of the invention are advantageous as the increase in their uptake is readily detectable.
  • the use is for the manufacture of pharmaceutical composition or use as a radiopharmaceutical agent for detecting apoptosis.
  • a method for in vivo or in vitro detection of apoptotic cell death and thereby the progression or regression of a tumor, of an infection, and/or of an inflammation in a tissue of a patient as well as a method for in vivo or in vitro detection of stroke, heart attack or injury in a tissue of a patient is also provided .
  • Such a method comprises or contains the following steps:
  • the patient can be any mammal and is preferably a human being.
  • the measurement of the amount and/or the concentration of the compound of general formula I in the tissue of interest, i.e. the diseased tissue, can be performed using PET.
  • the compound of general formula (I) or (lc) is useful as dyes in FACS experiments suitable for imaging/detecting apoptotic cell death (dying cells).
  • the suitable compound is a compound formula (lc) that is suitable as dyes for FACS experiments.
  • the invention is related to a compound of general formula I comprising F1 8 as described above for use as radiopharmaceutical.
  • the invention is related to the use of a compound of general formula I comprising F18 as described above for the manufacture of a radiopharmaceutical for binding and imaging/detecting apoptotic cell death (dying cells), both in vitro and in vivo, preferably, imaging/detecting apoptotic cell death (dying cells) .
  • the invention is related to a compound of general formula I comprising F18 (la) as described above for binding and imaging/detecting apoptotic cell death (dying cells), both in vitro and in vivo, preferably, imaging/detecting apoptotic cell death (dying cells).
  • the invention is directed to the use of compounds of general formula (I), (lb),for conducting biological assays and chromatographic identification. More preferably, the use relates to compounds of general formula (I) wherein the fluorine isotope is 18 F or 19 F, more preferably 19 F, (la) or (lb) .
  • the present invention provides a kit comprising a sealed vial containing a predetermined quantity of a compound having general chemical Formula (lc), (lie), (Xc) or (III) and suitable salts of inorganic or organic acids thereof, hydrates, complexes, and solvates thereof.
  • the kit comprises a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • the kit comprises compound having general chemical Formula (Ic), (lie), (Xc) or (III).
  • kit comprising compound of formula (Ic), (lie) or (Xc) when the kit is suitable for conducting FACS experiment.
  • kit comprises compound of formula (Ic).
  • kit comprising compound of formula (Xc) or (III), when the kit is suitable for conducting PET imaging .
  • kit comprises compound of formula (III).
  • the invention is directed to a method for therapy monitoring of processes associated with apoptotic cell death (dying cells).
  • the method for therapy monitoring of therapeutic treatment of diseases wherein the therapeutic treatment leads to apoptotic cell death in a patient comprising monitoring over a period of time the radioactivity uptake of compounds of general formula (I), or (la), preferably (la), by a patient, wherein the radioactivity uptake, which is measured over the period of time towards a control level, is indicative of response of said disease to therapeutic treatment.
  • chiral centers or other forms of isomeric centers are present in a compound according to the present invention, all forms of such stereoisomers, including enantiomers and diastereoisomers, are intended to be covered herein.
  • Compounds containing chiral centers may be used as racemic mixture or as an enantiomerically enriched mixture or as a diastereomeric mixture or as a diastereomerically enriched mixture, or these isomeric mixtures may be separated using well-known techniques, and an individual stereoisomer maybe used alone.
  • compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
  • preferred suitable salts are pharmaceutically acceptable salts of the compounds according to the invention.
  • the invention also comprises salts which for their part are not suitable for pharmaceutical applications, but which can be used, for example, for isolating or purifying the compounds according to the invention.
  • Pharmaceutically acceptable salts of the compounds according to the invention include acid addition salts of mineral acids, carboxylic acids and sulphonic acids, for example salts of hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, toluenesulphonic acid, benzenesulphonic acid, naphthalene disulphonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.
  • hydrochloric acid hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, toluenesulphonic acid, benzenesulphonic acid, naphthalene disulphonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid,
  • Pharmaceutically acceptable salts of the compounds according to the invention also include salts of customary bases, such as, by way of example and by way of preference, alkali metal salts (for example sodium salts and potassium salts), alkaline earth metal salts (for example calcium salts and magnesium salts) and ammonium salts, derived from ammonia or organic amines having 1 to 16 carbon atoms, such as, by way of example and by way of preference, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine and N-methylpiperidine.
  • customary bases such as, by way of example and by way of preference, alkali metal salts (for example sodium salts and potassium salts), alkaline earth metal salts (
  • Ci-C3-alkyl used herein on its own or as part of another group, refers to linear saturated carbon chains, in particular to methyl, ethyl and n-propyl.
  • Halogen as used herein refers to fluoro, chloro, bromo or iodo.
  • nitrogen-protecting group as employed herein by itself or as part of another group is known or obvious to someone skilled in the art, which is chosen from but not limited to a class of protecting groups namely carbamates, amides, imides, N-alkyl amines, N-aryl amines, imines, enamines, boranes, N-P protecting groups, N-sulfenyl, N-sulfonyl and N-silyl, and which is chosen from but not limited to those described in the textbook Greene and Wuts, Protecting groups in Organic Synthesis, third edition, page 494-653, included herewith by reference. Nitrogen-protecting groups are selected from the group comprising
  • Carbobenzyloxy (Cbz), p-Methoxybenzyl carbonyl (Moz or MeOZ), tert- Butyloxycarbonyl (BOC), 9-Fluorenylmethyloxycarbonyl (FMOC), Benzyl (Bn), p- Methoxybenzyl (PMB), 3,4-Dimethoxybenzyl (DMPM), p-Methylbenzenesulfonyl (Tos) or p-methoxyphenyl (PMP).
  • leaving group as employed herein by itself or as part of another group is known or obvious to someone skilled in the art, and means that an atom or group of atoms is detachable from a chemical substance by a nucleophilic agent. Examples are given e.g . in Synthesis (1 982), p. 85-125, table 2 (p.86; (the last entry of th is table 2 needs to be corrected: "n-C 4 F 9 S(O) 2 -O- nonaflat” instead of "n-C 4 H 9 S(O) 2 -O- nonaflat”), Carey and Sundberg, Organische Synthese, (1995), page 279-281 , table 5.8; or Netscher, Recent Res. Dev. Org .
  • Fluorescein and many of its derivatives - as for example the compounds of present invention - may display a complex pH- dependent ionic equil ibrium, and emission from the various ionic forms. Only the monoanion and dianion of fluorescein are fluorescent.
  • the tautomeric lactone form (ring-chain tautomerism : S. Du tt a n d J. Field Thorpe, J. Chem. Soc, Trans., 1924, 125, 2524-2538; Paul R. Jones, Chem. Rev., 1963, 63 (5), pp 461 -487) is usually found in organic solvents and is not formed in aqueous solutions above pH 5 (J. R. Lakowicz, "Principles of fluorescence spectroscopy", p. 637 ff, 2006, Springer, Berlin).
  • the present invention includes all of the hydrates, salts, and complexes.
  • the radiofluorination reaction can be carried out, for example in a typical reaction vessel (e.g . Wheaton vial) which is known to someone skilled in the art or in a microreactor.
  • the reaction can be heated by typical methods, e.g. oil bath, heating block or microwave.
  • the radiofluorination reactions are carried out in dimethylformamide with potassium carbonate as base and "kryptofix" as crown-ether.
  • solvents can be used which are well known to experts. These possible conditions include, but are not limited to: dimethylsulfoxid and acetonitril as solvent and tetraalkyi ammonium and tetraalkyi phosphonium carbonate as base.
  • Radiofluorination reactions are conducted for one to 60 minutes. Preferred reaction times are five to 50 minutes. Further preferred reaction times are 10 to 40 min. This and other conditions for such radiofluorination are known to experts (Coenen, Fluorine-18 Labeling Methods: Features and Possibilities of Basic Reactions, (2006), in: Schubiger P.A., Friebe M . , Lehmann L , (eds), PET-Chemistry - The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pp.15-50).
  • the radiofluorination can be carried out in a "hot-cell” and/or by use of a module (review: Krasikowa, Synthesis Modules and Automation in F-18 labeling (2006), in : Schubiger P.A., Friebe M., Lehmann L, (eds), PET-Chemistry - The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pp. 289-316) which allows an automated or semi-automated synthesis.
  • the 18 F-fluoroalkylation can be carried out, for example in a typical reaction vessel (e.g. Wheaton vial) or in an module which is known to someone skilled in the art according to the methods known in the art (Block, et al . (1988), J. Label . Compd. Radiopharm, 25, 2, 201 -216; Bauman et al ., (2003) Tetrahedron Lett, 44, 51 , 9165- 9167; Erlandsson, et al ., (2008) J. Label. Compd. Radiopharm, 51 , 5, 207-212).
  • a typical reaction vessel e.g. Wheaton vial
  • an module which is known to someone skilled in the art according to the methods known in the art (Block, et al . (1988), J. Label . Compd. Radiopharm, 25, 2, 201 -216; Bauman et al ., (2003) Tetrahedron Lett, 44, 51 , 9165- 9167; Erlandsson, et al
  • Precursors for al kyl-F-18 compounds of genera l form u l a I a re e . g . tosylates, brosylates, nosylates, mesylates, inflates, nonaflates etc. (formula II I) which can be synthesized from the respective hydroxy compounds according to methods known in the art (J. March, Advanced Organic Chemistry, 4 th ed. 1992, John Wiley & Sons, pp 352ff). More specifically, a hydroxy group being attached to a sp 3 hybridized carbon atom can be converted to a leaving group by an activating agent like thionyl chloride (e.g.
  • Chem ical Com m u n ications (Cambridge, United Kingdom); 1 ; (2008); 120 - 122), bromine / triphenylphosphine (e.g. European Journal of Organic Chemistry; 9; (2007); 1510 - 1516), mesylchloride, tosylchloride, trifluormethylsulfonylchloride, nona-fluorobutylsulfonyl- chloride, (4-bromo-phenyl)sulfonylchloride, (4-nitro-phenyl)sulfonylchloride, (2-nitro- phenyl)sulfonylchloride, (4-isopropyl-phenyl)sulfonylchloride, (2,4,6-tri-isopropyl- phenyl)sulfonylchloride, (2,4,6-trinnethyl-phenyl)sulfonylchloride, (4-tert
  • F-18 compounds of general formula I are e.g. iodides and bromides and the like whose conversion to the respective fluorides is also known in the art (J. March, see above).
  • the 19 F-compounds can be synthesized according to the syntheses for the 18 F- compounds (see above), by deoxofluorination reactions (M. Hudlicky, Org. React. 1988, 35, 513; H. Vorbruggen, Synthesis 2008, 8, 1 165-1 174.), by electrophilic fluorinations (T. Suzuki et al ., J. Org. Chem. 2007, 72, 246-250.), or by employment of fluorine-bearing building blocks, (see e.g. Example 1 b).
  • An additional method which is applicable to the synthesis of those O(CH 2 ) r -Y-chains in formula (III) comprises the al kylation of the unprotected hydroxy or am ino compounds (I la) by suitable bis(arylsulfonates) or bis(alkylsulfonates) and the like, e.g. bis(tosylates) TsO-(CH 2 ) r -OTs (see Scheme 1 ).
  • the ester compounds of formula (lla) are accessible by alkylation of unprotected lactone compounds of formula (II).
  • the unprotected compounds of formula (I) are also accessible from compounds of formula (I I) by deprotection of the polyazamacrocyl ic moiety.
  • the free fluorescein derivatives (II) bearing unprotected hydroxy su bstituents are accessi bl e by deprotecting the related O-protected compounds (3) by means known to those skilled in the art (e.g . see P. J . Kociensky, Protecting Groups, 3rd Edition, 2005, Georg Th ieme Verl ag Stuttgart, p . 1 87-350 and p. 487-631 ).
  • Th e cou pl i ng of the fluorescein derivative (2) with the linker L or the cyclen or cyclam derivative (1 ), respectively, to give (3) can be achieved by reacting the free amine functionality of A, A', or L with the activated carboxylate moiety or with the free carboxylate of (2) employing amide coupling agents known to those skilled in the art to give the corresponding amides (see e.g. R. C. Larock, Comprehensive Organic Transformations, VCH Publishers 1989, p.972-976; E. Valeur et al., Chem Soc. Rev.2009, 38, 606-631; M. Adamczyk, J. Org. Chem.2000, 65, 596-601; M.
  • (3) can be synthesized by employing unprotected cyclen or cyclam (A) in the reaction with (2) followed by protection of the remaining free ring-nitrogen-atoms of the polyazamacrocyclic moiety by method known to those skilled in the art (e.g. see P. J. Kociensky, Protecting Groups, 3rd Edition, 2005, Georg Thieme Verlag Stuttgart, p. 187-350 and p.487-631).
  • fully protected (3) is accessible by coupling unprotected fluorescein moiety (2) with with unprotected polyazamacrocyclic moiety to give compounds of formula (I) followed by full protection of polyazamacrocyclic amine and phenolic hydroxyl groups.
  • the corresponding thioureas (5) can be synthesized by reacting the free amine functionality of A, or L with the isothiocyanate derivative of the fluorescence moiety (6) (see e.g. S. Boonyarattanakalin et al., J. Am. Chem. Soc. 2004, 126(50), 16379- 16386.) (see Scheme 2).
  • N-aminoalkyl substituted cyclen or cyclam derivatives (see Scheme 4) is possible by reacting an optionally protected cyclen or cyclam derivative with an alkylnitrile bearing a leaving group at the ⁇ -position followed by reduction of the nitrile functionality to the corresponding amino functionality (see e.g. T. Koike et al., J. Am. Chem. Soc. 1996, 1 18, 12696-12703 .
  • N-aminoalkyl-aminooxoalkyl substituted cyclene derivatives (Scheme 5) is possible by reacting an optionally protected cyclen or cyclam derivative with an appropriately protected carboxlic acid derivative bearing a leaving group Y at the ⁇ -position of the carboxylate carbon chain by means of a nucleophilic substitution reaction known to those skilled in the art followed by an amidation of the carboxylate moiety with an monoprotected alkylene diamine followed by a deprotection step (see e.g. M. Kalesse et al., Lieb. Ann. 1996, 935-939); Jeon et al., Org. Lett. 2002, 4(23), 4155-4158).
  • N-aminoalkylcarbonyl substituted cyclen and cyclam derivatives are accessible by acylation of an optionally protected cyclen or cyclam derivative with an N- protected ⁇ -amino carboxylic acid derivative by means known to those skilled in the art to give the corresponding amides (see e.g. R. C. Larock, Comprehensive Organic Transformations, VCH Publishers 1989, p. 972-976; E. Valeur et al., Chem Soc. Rev. 2009, 38, 606-631 ) (see Scheme 6).
  • Apoptosis in Jurkat cells was induced by adding 1 ⁇ staurosporine (Stepczynska (2001 ) Oncogene 20, 1 193) to 10 6 cells/ mL and incubating for 3-4 h. Subsequently the cells were washed with PBS (140 mM NaCI, 10 mM KCI , 6.4 mM Na 2 HPO 4 , 2 mM KH 2 PO ) buffer. After centrifugation 10 6 cells were resuspended in 100 ⁇ TES (5 mM TES, 145 mM NaCI) buffer. 1 ⁇ of a 0.5 mM aqueous solution of the test compound was added and the cells were incubated for 15 minutes at 37 °C.
  • the cells were washed twice with 500 ⁇ TES-buffer and finally resuspended in 400 ⁇ TES-buffer.
  • 100 ⁇ _ of the suspension were diluted with 900 ⁇ _ TES buffer directly before the flow cytometry measurement.
  • Example 2 3'-(2-Fluoroethoxy)-6'-hydroxy-5-(1 ,4,7,10-tetraazacyclododecan-1 -ylcarbonyl)- spiro[isobenzofuran-1 (3H),9'-[9H]xanthen]-3-one tris(trifluoroacetate)
  • the prod uct was purified by semipreparative HPLC (Chromolith® SemiPrep RP-18e column (1 00-1 0 mm) with water (+0.1 % TFA) and acetonitrile (+0.1 % TFA) as eluent with a gradient of 0-100% acetonithle in 10 min.)
  • Tri-teff-butyl 1 ,4,7,10-tetraazacyclododecane-1 ,4,7-tricarboxylate 160 mg, 339 ⁇ was dissolved in anhydrous DMF (3 mL) and DIPEA (40 ⁇ _) was added.
  • DIPEA 40 ⁇ _ was added.
  • a solution of fluorescein isothiocyanate (30 mg, 85 ⁇ ) in anhydrous DMF (1 mL) was added dropwise. The reaction mixture was stirred at r.t. for 4 hours. The solvent was removed / ' . vac. and the product was used in the next step without further purification.
  • the product was purified by semipreparative HPLC (Chromolith® SemiPrep RP-18e column (100-10 mm) with water (+0.1 % TFA) and acetonitrile (+0.1 % TFA) as eluent with a gradient of 0-100% acetonitrile in 10 min.)
  • Tri-teff-b u tyl 1 0-(2-aminoethyl)-1 ,4,7,10-tetraazacyclododecane-1 ,4,7-tricarboxylate (prepared according to Reichenberg-Klinke et al, J. Am. Chem. Soc, 2002, 124, 44, 12999-13007) (100 mg, 194 ⁇ ) was dissolved in anhydrous DMF (2 mL). A solution of 5-/6-carboxyfluorescein-pentafluorophenylester (100 mg, 184 ⁇ ) in anhydrous DMF (1 mL) was added . The reaction mixture was stirred at r.t. for 3 hours. The solvent was removed / ' . vac. The product was used in the next step without further purification.
  • Tri-terf-b u ty l 1 0-(2- ⁇ 2-[2-( ⁇ [3',6'-dihydroxy-3-oxo-spiro(isobenzofuran-1 (3H),9'- [9H]xanthen)-5-yl]carbonyl ⁇ amino)ethoxy]ethoxy ⁇ ethyl)-1 ,4,7,10-tetraazacyclodo decane-1 ,4,7-tricarboxylate;
  • Tri-terf-b u t y I 1 0-(2- ⁇ 2-[2-( ⁇ [3',6'-dihydroxy-3-oxo- spiro(isobenzofuran-1 (3H),9'-[9H]xanthen)-6-yl]carbonyl ⁇ amino)ethoxy]ethoxy ⁇ ethyl)- 1 ,4,7,10-tetraazacyclododecane-1 ,4,7-tricarboxylate
  • Tri-teff-butyl 1 0- ⁇ 2-[2-(2-aminoethoxy)ethoxy]ethyl ⁇ -1 ,4,7,10-tetraazacyclododecane- 1 ,4,7-tricarboxylate (prepared according to Jiang et al., Synthesis 2008, 2, 215-220) (88 mg, 146 ⁇ ) was dissolved in anhydrous DMF (1 mL). A solution of 5-/6- carboxyfluorescein-pentafluorophenylester (70 mg, 129 ⁇ ) in anhydrous DMF (1 mL) was added. The reaction mixture was stirred at r.t. for 4 hours. The solvent was removed / ' . vac. The product was used in the next step without further purification.
  • the FL-I/FL-I I l-plot shows that the majority of stained cells only green indicating that the cells have bound the cyclen compound (with green fluorescence, FL-I). Only also the necrosis marker Propidium iodide (red, R L- III).
  • FACS measurements were performed with a DAKO Galaxy Flow Cytometry System using the following experimental setup: Jurkat-cells were grown to a density of about 10 6 cells/ml . Apoptosis was induced by adding 1 ⁇ staurosporine (Stepczynska et al . (2001 ) Oncogene (2001 ) 20, 1 193). After incubation for 4 h at 37 °C, the cells were washed with cold PBS-buffer and resuspended in cold TES-buffer. 10 6 cells in 100 ⁇ buffer with 1 ⁇ of a 0.5 mM aqueous solution of compound 2a were incubated for 15 minutes at 37 °C.

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Abstract

L'invention concerne des composés pour la détection et l'imagerie de la mort cellulaire apoptotique in vitro et in vivo, ainsi qu'un procédé de détection FACS, de microscopie par fluorescence et d'imagerie TEP de la progression ou de la régression d'une tumeur chez un patient après un traitement. L'invention concerne également des compositions pharmaceutiques, comprenant de tels composés, des précurseurs appropriés pour les obtenir, des procédés pour les obtenir et un kit pour la détection et l'imagerie de la mort cellulaire apoptotique.
PCT/EP2010/069501 2009-12-18 2010-12-13 Composés pour l'imagerie de la mort cellulaire apoptotique WO2011073137A2 (fr)

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Publication number Priority date Publication date Assignee Title
CN102898392A (zh) * 2012-09-06 2013-01-30 中国工程物理研究院核物理与化学研究所 N-乙酰氧苯基-3-[三叔丁氧甲酰基-四氮杂环十二烷基]中间体及其制备方法
CN102898392B (zh) * 2012-09-06 2015-08-12 中国工程物理研究院核物理与化学研究所 N-乙酰氧苄基-3-[-三叔丁氧甲酰基-四氮杂环十二烷基]中间体及其制备方法
EP3050886A1 (fr) * 2015-02-02 2016-08-03 Bürkert Werke GmbH Colorants fluorescents et précurseurs de colorants
EP3493855A4 (fr) * 2016-08-02 2020-04-01 ISI Life Sciences, Inc. Méthode de détection de cellules cancéreuses.
CN110041315A (zh) * 2019-05-06 2019-07-23 济南大学 一种检测细胞凋亡的荧光探针及其制备方法和应用

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