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WO2018206126A1 - Agents et procédés d'imagerie - Google Patents

Agents et procédés d'imagerie Download PDF

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Publication number
WO2018206126A1
WO2018206126A1 PCT/EP2017/062048 EP2017062048W WO2018206126A1 WO 2018206126 A1 WO2018206126 A1 WO 2018206126A1 EP 2017062048 W EP2017062048 W EP 2017062048W WO 2018206126 A1 WO2018206126 A1 WO 2018206126A1
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Prior art keywords
compound
group
alkyl
formula
imaging
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Inventor
Vladimir Kral
Martin Havlik
Robert Kaplanek
Tomas Briza
Zdenek Kejik
Pavel Martasek
Lucie KRCOVA
Jarmila Kralova
Tomas Ruml
Silvie Rimpelova
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Matematicko-Fyzikalni Fakulta University Karlovy V Praze
Vysoka Skola Chemicko Technologicka V Praze
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Matematicko-Fyzikalni Fakulta University Karlovy V Praze
Vysoka Skola Chemicko Technologicka V Praze
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Publication of WO2018206126A1 publication Critical patent/WO2018206126A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
    • C09B23/083—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines five >CH- groups
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/12—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain being branched "branched" means that the substituent on the polymethine chain forms a new conjugated system, e.g. most trinuclear cyanine dyes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the invention relates, in one aspect to methods of imaging biological samples in vitro and in vivo, to methods of imaging and diagnosis, and of compounds useful in such methods.
  • Fluorescence imaging is used as a non-destructive way of tracking or analysing biological molecules, both in vitro and in vivo.
  • Certain small molecules or proteins present in cells are intrinsically fluorescent (such as NADH and tryptophan).
  • biological molecules can be "labelled" with an extrinsic fluorophore.
  • extrinsic fluorophores include fluorescent antibodies, proteins and small molecule probes.
  • fluorescence resonance energy transfer where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.
  • Two-photon fluorescence microscopy is a technique that allows imaging of living tissue to a greater depth than conventional (single photon) fluorescence imaging. Being a type of multiphoton fluorescence, it employs red-shifted excitation light which is capable of exciting fluorescent dyes.
  • Two-photon excitation is a phenomenon in which two photons are simultaneously absorbed by the same fluorophore by exposure to high photon density per unit volume and time by irradiation with a suitable light source. The use of infrared light minimizes scattering in the tissue. The background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced phototoxicity.
  • Probes for use in two-photon microscopy are known. These include coumarin, rhodamine, xanthene, and cyanine dyes. Examples of rhodamine dyes include Alexa Fluor® 488 (J. Biol. Chem., 2004, 279, 37544-37550). Examples of cyanine dyes include Alexa Fluor® 647, available from Thermo Fisher Scientific of Waltham, Massachusetts, USA. Mitochondria, an organelle found in almost all eukaryotic cells, play a vital role in the life and death of cells. The function of mitochondria is to produce ATP.
  • ROS reactive oxygen species
  • Fluorescent probes which selectively stain mitochondria are powerful tools for monitoring and studying cellular processes. Such probes must be photostable under the conditions of light exposure from fluorescent microscopes. Fluorescent dyes for mitochondrial staining have been developed, including those mentioned above. The photostability of known dyes is, however, rather poor. Moreover, known probes display significant systemic toxicity, rendering them less useful for in vivo imaging.
  • the ideal characteristics of such an agent would include one or more of the following: i) narrow excitation and fluorescence emission spectra; ii) minimal fluorescence in an aqueous environment, iii) maximal fluorescence after incorporation in the lipophilic environment; and iv) high binding selectivity for live cells and tissue; v) high binding photostability, and vi) low systemic toxicity.
  • Fluorescent probes displaying selectivity for cancer cells are extremely desirable. Improved visualization of tumours in vivo would aid diagnosis, facilitate surgical resection, investigate therapeutic efficacy, and improve prognosis. Fluorescence imaging has high specificity and sensitivity and has been utilized for medical imaging.
  • the present invention addresses these, and other, problems.
  • Dyes and Pigments, 2014, 107, 51 -59 discloses symmetrical ⁇ -substituted pentamethinium fluorescent probes.
  • Chem. Comm. 2008, 16, 1901-1903 discloses polymethinium salts as visualization agents for heparin.
  • J. Phys. Chem. B., 201 1 , 1 15, 1 1530-1 1535 discloses two photon optical properties of certain near infra-red dyes, including Indocyanine green, 3,3'- diethylthiatricarbocyanine perchlorate, 5,5'-Dichloro-1 1 -diphenylamino-3,3'-diethyl- 10,12-ethylenethiatricarbocyanine perchlorate (IR-140), and 2-([3-(2-[4- (Dimethylamino)phenyl]ethenyl)-5,5-dimethyl-2-cyclohexen-1 -ylidene]methyl)-3- methylbenzothiazolium perchlorate (Styryl 9M).
  • Figure 1 shows mitochondrial localization of compound 1 in opossum kidney cells in vitro.
  • Figure 2 shows mitochondrial localization of compound 2 in opossum kidney cells in vitro.
  • Figure 3 shows a time-lapse image performed by multiphoton fluorescence microscopy of podocytes in glomeruli of a living mouse labelled by compound 1.
  • Figure 4 shows a time-lapse image performed by multiphoton fluorescence microscopy of podocytes in glomeruli of a living mouse labelled by compound 2.
  • Figure 5 shows an excitation scan of compound 1 at a penetration depth of 25 ⁇ .
  • Figure 6 shows an excitation scan of compound 2 at 25 ⁇ penetration depth.
  • Figure 7 shows podocyte labelling in glomeruli in a living mouse by compound 1.
  • Figure 8 shows accumulation of compounds of the invention in mitochrondria of U-2
  • OS human osteosarcoma
  • FIG. 9 shows localization after intratumoral (top) and intravenous application
  • the invention provides compounds of Formula I,
  • Y is selected from the group consisting of
  • a and A' are independently selected substituents
  • B is an optionally substituted aromatic or heteroaromatic group
  • R1-R12 are independently selected from hydrogen and a substituent
  • Q and Q' independently selected from NH, N(Ci-C6 alkyl), oxygen, sulphur, selenium and di(Ci-C6 alkyl)-methylene,
  • m is an integer 1 , 2 or 3;
  • p is an integer 1 , 2 or 3;
  • Z is an anion having a negative charge of n
  • n is an integer 1 , 2 or 3;
  • q is an integer 1 , 2 or 3;
  • the invention provides an imaging method comprising bringing a biological specimen into association with a compound of formula I, irradiating the specimen with light, and observing two-photon excited fluorescence images emitted from the compound of formula I.
  • the invention provides a method for imaging live cells, the method comprising administering to an animal a compound of formula I, irradiating a portion of the animal with light, and observing two-photon excited fluorescence images emitted from the compound of formula I.
  • the invention provides a class of compounds of formula I as defined wherein B is phenyl substituted with from one to three groups independently selected from the group consisting of fluoro, and C1-C3 perfluoroalkyl, preferably trifluoromethyl.
  • the invention relates to two-photon fluorescent dyes based of Formula I, which, in one embodiment, are employed as fluorescent probes for deep multiphoton imaging.
  • the probes enable long-term real-time monitoring of in vivo samples and selective labelling of podocytes in glomerulus in living animals.
  • the two-photon fluorescent dyes of the present invention can be employed for imaging of living samples to exceptionally large depth of up to 160 ⁇ for periods of 90 minutes or longer.
  • polymethinium salts of Formula I which show very strong fluorescence intensity, high target affinity, low systemic toxicity, high photostability and enable imaging to a significant penetration depth.
  • the polymethinium salts of Formula I of the present invention can be used as two-photon probes for deep real-time imaging for podocytes in glomerulus, and cellular and subcellular visualization in general. These systems are based on the utilization of a structural motif of pentamethinium salts prepared from corresponding malondialdehydes.
  • the preparation of polymethinium salts of Formula I is based on condensation of a suitable aromatic malondialdehyde with a salt of the corresponding heteroaromatic compound.
  • Preferred compounds for uses of the invention are those wherein X is a group
  • Preferred compound for uses of the invention are those wherein Y is a group
  • A', Q', * , and R7 to R10 are as defined elsewhere herein.
  • B is a group selected from phenyl, pyridyl, thienyl, furanyl, naphthyl, quinolyl and isoquinolyl, optionally substituted as defined. More preferably, B is a pyridyl group. Still more preferably, B is a 4-pyridyl group. Most preferably, B is an unsubstituted 4-pyridyl group.
  • B is substituted with at least an electron- withdrawing group.
  • electron-withdrawing group Preferred electron withdrawing groups are cyano, nitro, -CO(Ci-C 6 alkyl), -COO(Ci-C 6 alkyl), C1-C3 haloalkyl (especially perfluoroalkyl), halogen (especially F and CI), -SO(Ci-C6 alkyl), -SC>2(Ci-C6 alkyl), and -SC>3(Ci-C6 alkyl).
  • B is substituted with from 1 to 3 such substituents.
  • B is preferably phenyl.
  • B is phenyl substituted with from one to three groups independently selected from the group consisting of fluoro, and C1-C3 perfluoroalkyl.
  • B is phenyl substituted with from one to three fluoro groups.
  • B is phenyl substituted with from one to three (preferably one) trifluoromethyl groups.
  • Specifically preferred values of B are 4-fluorophenyl, 3,5-difluorophenyl, 4-trifluoromethyl and 3-trifluoromethyl.
  • A is selected from a C1-C18 alkyl group, or a group having the formula
  • n1 is an integer from 1 to 6 (preferably 2), n2 is an integer from 1 to 8, and R13 is selected from the group consisting of C1-C18 alkyl, hydrogen, a sulphonic acid group SO3H or a lithium, sodium, potassium, caesium or rubidium salt thereof, benzyl, allyl, a cobalt bis(dicarbollide) preferably substituted in the 8-position or a cyclodextrin, preferably an ⁇ , ⁇ or ⁇ cyclodextrin.
  • A is selected from a C1-C18 alkyl group. More preferably, A is a Ci- C6 alkyl group, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert- butyl, pentyl, isopentyl, 2-methylbutyl, neopentyl, 1 -ethylpropyl, hexyl, isohexyl, 4- methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2- dimethylbutyl, 1 ,1-dimethylbutyl, 1 ,2-dimethylbutyl, 1 ,3-dimethylbutyl, 2,3- dimethylbutyl, and 2-ethylbutyl. Most preferably, A is n-butyl.
  • A' is selected from a C1-C18 alkyl group, or a group having the formula wherein n1 is an integer from 1 to 6 (preferably 2), n2 is an integer from 1 to 8, and R13 is selected from the group consisting of C1-C18 alkyl, hydrogen, a sulphonic acid group SO3H or a lithium, sodium, potassium, caesium or rubidium salt thereof, benzyl, allyl, a cobalt bis(dicarbollide) preferably substituted in the 8-position or a cyclodextrin, preferably an ⁇ , ⁇ or ⁇ cyclodextrin.
  • A' is selected from a C1-C18 alkyl group. More preferably, A is a Ci- C6 alkyl group, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert- butyl, pentyl, isopentyl, 2-methylbutyl, neopentyl, 1 -ethylpropyl, hexyl, isohexyl, 4- methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2- dimethylbutyl, 1 ,1-dimethylbutyl, 1 ,2-dimethylbutyl, 1 ,3-dimethylbutyl, 2,3- dimethylbutyl, and 2-ethylbutyl. Most preferably, A' is n-butyl.
  • groups R1-R12 are independently selected from hydrogen, halide, C1-C6 alkyl, C3-C6 cycloalkyl, d-Ce alkoxy, C1-C6 thioalkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-C 6 haloalkyl, Ci-C 6 trialkylsilyloxy, phenyl, NH 2 , NH(Ci-C 6 alkyl), N(Ci-C 6 alkyl)2, N + (Ci-C6 alkyl)3, carboxylic acid, C1-C6 carboxyalkyl, hydroxy, azide, nitro, nitroso, nitrile, cyanate, isocyanate, thiocyanate, isothiocyanate, thiol, or a group having the formula
  • n1 is an integer from 1 to 6 (preferably 2), n2 is an integer from 1 to 8, and R13 is selected from the group consisting of C1-C18 alkyl, hydrogen, a sulphonic acid group SO3H or a lithium, sodium, potassium, caesium or rubidium salt thereof, benzyl, allyl, a cobalt bis(dicarbollide) preferably substituted in the 8-position or a cyclodextrin, preferably an ⁇ , ⁇ or ⁇ cyclodextrin.
  • R1-R12 are selected from fluorine and hydrogen. More preferably R1-R12 are hydrogen.
  • Z is selected from the group consisting of chloride, bromide, iodide, sulfate, phosphate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, oxalate, succinate, benzoate and p-toluenesulfonate. More preferably, Z is selected from the group consisting of chloride, bromide and iodide. Most preferably, Z is iodide.
  • compounds of the formula I are partially fluorinated, that is at least some of the hydrogen atoms are replaced with fluorine. In other embodiments, the compounds are perfluorinated, i.e. all of the hydrogen atoms are replaced with fluorine. These compounds display enhanced photostability.
  • a preferred class of compounds is those having the formula
  • a very preferred compound for use in the invention is compound 1 , (2Z)-3-propyl-2- [(2Z,4E)-5-(3-propyl-1 ,3-benzothiazol-3-ium-2-yl)-3-(4-pyridyl)penta-2,4-dienylidene]- 1 ,3-benzothiazole, having the formula
  • Z is chloride, bromide or iodide, most preferably iodide.
  • An alternative, very preferred compound for use in the invention is compound 2, 2- [(1 E,3Z,5E)-5-(3,3-dimethyl-1 -propyl-indolin-2-ylidene)-3-(4-pyridyl)penta-1 ,3-dienyl]- 3,3-dimethyl-1 -propyl-indol-1 -ium
  • Z is chloride, bromide or iodide, most preferably iodide.
  • the compounds of the invention have utility in two-photon imaging methods.
  • the methods of the invention are applicable to both in vitro and in vivo imaging.
  • the invention relates to a method for imaging living cells, the method comprising administering to an animal a compound of formula I, irradiating a portion of the animal with light, and observing two-photon excited fluorescence images emitted from the compound of formula I.
  • the irradiating light has a wavelength of from 600 to 1300 nm.
  • the irradiating light has a wavelength of between 600 and 700 nm, more preferably between 640 and 680 nm, such as about 640 nm, about 655 nm, or about 680nm.
  • the irradiating light has a wavelength of from 680 to 1300 nm, more preferably from 1 100 to 1200 nm, such as about 1 180 nm.
  • Compounds of formula I are suitably administered to a live animal (including human) subject in an aqueous vehicle.
  • Compounds of formula I are preferably present at a concentration of between 10 ⁇ and 1 mM, more preferably between 100 ⁇ and 500 ⁇ , still more preferably 200 ⁇ and 300 ⁇ , such as about 250 ⁇ .
  • compounds of the invention are employed at working concentrations of from 1 to 100 nM, preferably from 10 to 50 nM.
  • mitochondrial probes known in the art, such as MitoTrackers® (available from Thermo Fisher Scientific of Waltham, Massachusetts, USA), typically used at concentrations of 100-200 nM, and rhodamine dyes, typically used at concentrations of 500-1000 nM.
  • Compounds of formula I are suitably administered to a live animal via intravenous injection. However, other modes of administration are contemplated.
  • the methods of the invention can be used to image any organ, organelle or cellular structure in living tissue.
  • the methods are used for imaging the kidney, and in particular the glomerulus.
  • the compounds of the invention show high affinity for the glomerulus, and in particular the podocyte of the glomerulus.
  • the methods of the invention are capable of providing images of living tissue to a substantial depth compared with known methods.
  • the methods of the invention are capable of providing images at a depth of at least 50 ⁇ , more preferably at least 100 ⁇ , still more preferably at least 150 ⁇ .
  • a problem with known mitochondrial probes is that they exhibit an undesirable degree of nonspecific accumulation, and are frequently redistributed within the endoplasmic reticulum.
  • the probes of the invention namely the compounds of formula I, exhibit an extremely high degree of selectivity for mitochondria.
  • imaging agent coumarin, rhodamine, xanthene and cyanine dyes
  • coumarin, rhodamine, xanthene and cyanine dyes are their toxicity, which limits their utility in diagnostic applications.
  • the compounds of formula demonstrate low cytotoxicity, and may display lower systemic toxicity.
  • Compounds of formula I are particularly suitable for forming conjugates with a variety of species.
  • the compounds may be attached (covalently, e.g. via a linker or otherwise) to various substrates such as biomolecules (including antibodies, oligonucleotides, enzymes, proteins, saccharides, etc.), aptamers, nanoparticles, drug molecules and polymers.
  • Conjugation of the compounds of formula I to the substrate may suitably be achieved via a chemical linker.
  • Various linking strategies are known, and the skilled person will be able to select the appropriate one.
  • a preferred class of linkers is the N- hydroxysuccinimide or succinimidyl ester.
  • Alternative linkers are described e.g. in WO2004018493, which is incorporated herein by reference.
  • the compounds of formula I of the present invention under appropriate conditions, are selectively sequestered in mitochondria.
  • substituent refers to a group other than hydrogen.
  • Preferred substituents include alkyl (such as C1-C6), alkenyl (such as C2-C6), alkoxy (such as Ci- Ce), aryl (such as C6-C12), aryloxy (such as C6-C12), heteroaryl, fluoroalkyl (such as Ci- Ce), perfluoroalkyl (such as C1-C6) and perfluoroaryl (such as C6-C12), unless otherwise stated.
  • aromatic group group that contains any carbon-based aromatic group preferably having from 6 to 14 ring carbon atoms.
  • Preferred aromatic groups are phenyl, naphthyl, biphenyl, and anthracenyl, with phenyl most preferred.
  • heteromatic group refers to a 5- or 6-membered monocyclic aromatic group wherein 1 , 2, 3, 4 ring heteroatoms independently selected from O, S and N; or to a 8- to 1 1 -membered bicyclic aromatic group wherein 1 , 2, 3, 4 or 5 ring heteroatoms independently selected from O, S and N.
  • Examples of 5- or 6-membered monocyclic heteroaromatic groups include pyrrolinyl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyrimidinyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl and pyrimidinyl.
  • Examples of 8- to 1 1 - membered bicyclic heteroaromatic groups include 6H-thieno[2,3-b]pyrrolyl, imidazo[2,1-b][1 ,3]thiazolyl, imidazo[5,1- b][1 ,3]thiazolyl, indolyl, isoindolyl, indazolyl, benzimidazolyl, benzoxazolyl e.g.
  • benzoxazol-2-yl benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzothienyl, benzofuranyl, naphthridinyl, quinolyl, quinoxalinyl, quinazolinyl, cinnolinyl and isoquinolyl.
  • substituents are from one to three groups independently selected from C1-C6 alkyl, C3- C6 cycloalkyl, C1-C6 alkoxy, C1-C6 thioalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, Ci-C 6 trialkylsilyloxy, phenyl, NH 2 , NH(Ci-C 6 alkyl), N(Ci-C 6 alkyl) 2 , N + (Ci- C6 alkyl)3, carboxylic acid, C1-C6 carboxyalkyl, halide, hydroxy, azide, nitro, nitroso, nitrile and thiol.
  • alkyl refers to a saturated straight or branched hydrocarbon chain of typically Ci to C6, and specifically includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3- methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl, and the like.
  • alkoxy refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, t-butoxy, pentoxy, isopentoxy, neopentoxy, hexyloxy, isohexyloxy, cyclohexyloxy, 2,2-dimethylbutoxy, and 2,3-dimethylbutoxy, and the like.
  • haloalkyl refers to an alkyl as defined herein, which is substituted by one or more halo groups as defined herein.
  • the haloalkyl can be monohaloalkyl, dihaloalkyl, trihaloalkyl, or polyhaloalkyi including perhaloalkyi.
  • a monohaloalkyl can have one iodo, bromo, chloro or fluoro within the alkyl group. Chloro and fluoro are preferred.
  • Dihaloalkyl and polyhaloalkyi groups can have two or more of the same halo atoms or a combination of different halo groups within the alkyl.
  • haloalkyl examples include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl.
  • a perhaloalkyi refers to an alkyl having all hydrogen atoms replaced with halo atoms, e.g, trifluoromethyl.
  • haloalkoxy refers to a haloalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
  • examples include fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, pentafluoroethoxy, heptafluoropropoxy, difluorochloromethoxy, dichlorofluoromethoxy, difluoroethoxy, difluoropropoxy, dichloroethoxy and dichloropropoxy and the like.
  • a labeling Solution Compounds of formula I generally have low solubility in water.
  • a stock solution is prepared dissolving the reagent in an organic solvent (suitably DMSO, dimethylformamide, acetonitrile, dioxane, tetrahydrofuran etc).
  • the labeling solution is prepared by diluting the stock solution into aqueous buffer to the desired labeling concentration.
  • the amount of dye in the labeling solution is the minimum amount required to yield detectable staining in the sample, without significant background fluorescence or undesired staining of other organelles.
  • the amount of reagent required for staining eukaryotic mitochondria depends on the sensitivity required for staining of intracellular vs. cell-free mitochondria, the number of cells present, and the permeability of the cell membrane to the reagent.
  • the cells Before or after exposure to the inventive methods, the cells may optionally be treated with solvents to fix, and optionally permeabilize, the membranes.
  • Various fixatives and fixation conditions are suitable for achieving this, for example formaldehyde, methanol, and ethanol.
  • fixation is accomplished by incubating in a 4% solution of formaldehyde for 15 minutes.
  • Methods according to the present invention may be combined with the use of an additional visualization reagent.
  • One or more additional visualization reagents may be used in conjunction with the compounds of Formula I .
  • the additional visualization reagent may be used to stain the entire cell, or a cellular substructure by selection of an appropriate reagent with the desired degree of selectivity, such as a labeled antibody, labeled oligonucleotide, an enzymic activity, or other indicator for a specific cellular component or substructure such as the cytoplasm, nucleus, membrane, lysosome, or Golgi apparatus.
  • the imaging methods of the present invention and the response of the additional visualization reagent may be observed simultaneously or sequentially.
  • nucleic acid stains One class of appropriate additional detection reagents is fluorescent nucleic acid stains.
  • a wide variety of appropriate nucleic acid stains are known, such as thiazole orange, propidium iodide, ethidium homodimer, and DAPI.
  • an appropriate additional detection reagent is any probe that selectively stains a cellular organelle such as the cell membrane, nucleus, Golgi apparatus, endoplasmic reticulum, lysosomes, or a second mitochondrial stain, such as MitoTracker, Alexa-647 or Alexa-488.
  • Betama-substituted polymethinium salts 1 and 2 were tested for in vitro and in vivo fluorescence microscopy applications in conventional wide-field, confocal and multiphoton fluorescence microscopy techniques. Both compounds showed selective localization in mitochondria of various cell lines, stated by the example of opossum kidney cells.
  • these polymethinium salts are cell-permanent, with compound 1 being retained in mitochondria even after treatment with an uncoupling reagent, e.g. carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), which enables media exchange, fixation or even permeabilization of cells without loss of the staining (see Example 1 ).
  • FCCP carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
  • Compound 2 is not retained after FCCP staining, nevertheless it offers a broad application in long-term live-cell imaging. Both compounds did not show any signs of cytotoxicity in in vitro imaging experiments.
  • the newly prepared, positively charged symmetric polymethinium salts have convenient spectroscopic properties: narrow excitation and fluorescence emission spectra, minimal fluorescence in water environment, maximal fluorescence after incorporation in the lipophilic environment of mitochondrial membranes. Moreover, these dyes show extraordinary photostability, the significantly outperform commercially available xanthylium dyes broadly used for mitochondrial labelling.
  • both dyes can serve for in vivo imaging.
  • Compounds 1 and 2 are two-photon fluorescent probes, which were successfully employed for podocyte labelling in glomeruli in living mice of C57BI/6J-Rj (see Examples 7-1 1 ).
  • Compound 1 enables in vivo imaging for 90 minutes or longer, compound 2 up to several hours and was very well tolerated for several hours, and also with reinjection.
  • compound 1 outperforms commercially available Alexa-647-albumin for in vivo imaging, compound 1 enables imaging up to 160 ⁇ (in contrast to Alexa-647 conjugate to 120 ⁇ )
  • Compound 1 is suitable for deep tissue imaging and was superior to Alexa 647- Albumin.
  • Compound 2 was able to demonstrate long term imaging for several hours.
  • Opossum kidney cell (OK) line was used for in vitro evaluation of the localization and fluorescence microscopy performance of compound 1.
  • Compound 1 was incubated at 50 nM concentration with the cells for 30 minutes at physiological conditions and washed with phosphate buffered saline, then the cells were imaged using a Leica SP8 confocal fluorescence microscope equipped with a white light laser. The excitation wavelength employed for compound 1 was 655 nm. HyD-Detectors were used for image acquisition. Uncoupling was performed by 30 second treatment with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 100 nM - 10 ⁇ ) disrupting ATP synthesis.
  • FCCP carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
  • Figure 1 shows mitochondrial localization of compound 1 in Opossum kidney cells in vitro, specifically:
  • Compound 1 did not show any obvious signs of cytotoxicity.
  • Opossum kidney cell (OK) line was used for in vitro evaluation of the localization and fluorescence microscopy performance of compound 2.
  • Compound 2 was incubated at 50 nM concentration with the cells for 30 minutes at physiological conditions and washed with phosphate buffered saline, then the cells were imaged by Leica SP8 confocal fluorescence microscope equipped with a white light laser. The excitation wavelength used for compound 2 was 640 nm. HyD-Detectors were used for image acquisition. Uncoupling was performed by a 30 second treatment with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 100 nM - 10 ⁇ ) disrupting ATP synthesis.
  • FCCP carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
  • Figure 2 shows mitochondrial localization of compound 2 in opossum kidney cells in vitro, specifically:
  • FIG. 2 B 30 second co-treatment with a mitochondrial uncoupler FCCP, according to the decreased fluorescence signal (the same manner as for commercial TMRM mitochondrial dye).
  • Model mouse strain C57BI/6J-Rj was used for this experiment.
  • the mice were catheterized with a jugular-vein catheter for dye application.
  • Compound 1 was used at 250 ⁇ concentration in 0.9% NaCI (volume 120 ⁇ ).
  • the imaging was performed immediately after dye application using a custom-built multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used.
  • the excitation wavelength used was 1 180 nm and the frame rate was 1 fps.
  • Figure 3 shows a time-lapse image performed by multiphoton fluorescence microscopy of podocytes in glomeruli of a living mouse labelled by compound 1
  • the target structure was stained 6 minutes after application of compound 1.
  • Model mouse strain C57BI/6J-Rj was used for this experiment.
  • the mice were catheterized with a jugular-vein catheter for dye application.
  • Compound 2 was used at 250 ⁇ concentration in 0.9% NaCI (volume 120 ⁇ ).
  • the imaging was performed immediately after dye application using a custom-built Multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used.
  • the excitation wavelength used was 1 180 nm and the frame rate was 1 fps.
  • Figure 4 shows a time-lapse image performed by multiphoton fluorescence microscopy of podocytes in glomeruli of a living mouse labelled by compound 2
  • Excitation wavelength scan Multiphoton fluorescence microscopy in C57BI/6J-Rj mouse by Compound 1
  • Model mouse strain C57BI/6J-Rj was used for this experiment.
  • the mice were catheterized with a jugular-vein catheter for dye application.
  • Compound 2 was used at 250 ⁇ concentration in 0.9% NaCI (volume 120 ⁇ ).
  • the imaging was performed using a custom-built Multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used.
  • the excitation wavelength scan was performed at 25 ⁇ depth (px size 228 nm).
  • Figure 5 shows an excitation scan of compound 1 at a penetration depth of 25 ⁇ .
  • Selected wavelengths maximal fluorescence intensities: 820 (max 4000), 1 100 (max 2500), 1 150 (max 5000), 1200 (max 4000), 1300 (max 5000).
  • Podocyte labelling in the glomerulus in a living mouse was achieved by the methods of the invention.
  • Excitation wavelength scan Multiphoton fluorescence microscopy in C57BI/6J-Rj mouse by Compound 2
  • Mouse model strain C57BI/6J-Rj was used for this experiment.
  • the mice were catheterized with a jugular-vein catheter for dye application.
  • Compound 2 was used at 250 ⁇ concentration in 0.9% NaCI (volume 120 ⁇ ).
  • the imaging was performed using a custom-built Multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used.
  • the excitation wavelength scan was performed at 25 ⁇ depth (px size 381 nm).
  • Multiphoton fluorescence in vivo microscopy in C57BI/6J-Ri mouse by Compound 1 Mouse model strain C57BI/6J-Rj was used for this experiment.
  • the mice were catheterized using a jugular-vein catheter for dye application.
  • Compound 1 was used at 250 ⁇ concentration in 0.9% NaCI (volume 120 ⁇ ).
  • the imaging was performed using a custom-built Multiphoton fluorescence microscope setup equipped with a Spectra-Physics Insight Deep Sea laser (680 to 1300 nm) and the emission-BP-filter at 700/75 nm and a GaAsP-detector were used.
  • Figure 7 shows podocyte labelling in glomeruli in a living mouse by polymethinium salt 1 at A) 133 ⁇ and B) 160 ⁇ penetration depth.
  • the penetration depth exceeds the best-performing Alexa-647-Albumin, in which case only 120 ⁇ penetration depth can be achieved.
  • MitoTracker Green A mitochondria-specific probe, MitoTracker Green, was used to confirm the selective accumulation in mitochrondria of U-2 OS (human osteosarcoma) cells. Cells were incubated with the compounds for 30 minutes in the presence of 100 nM MitoTracker Green, washed with phosphate buffer saline, and then imaged. The images were acquired using an inverse fluorescent microscope (Olympus IX-81 ).
  • mice bearing subcutaneously growing human pancreatic carcinoma received Compound 7 intravenously.
  • Compound 7 was also injected intratumorally in mice bearing human glioblastoma (U-87). Mice were anaesthetised and subjected to fluorescence imaging. Imaging was performed immediately after dye application, and then after 1 and 72 hours using Bruker In-Vivo Xtreme II.
  • Figure 9 shows localization after intratumoral (top) and intravenous application (bottom) of Compound 7 in nu/nu mice.

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Abstract

L'invention concerne des procédés d'imagerie de tissus biologiques. Les sels de polyméthénium décrits dans la présente description présentent une activité en tant qu'agents d'imagerie à deux photons, ayant une plus grande profondeur d'imagerie et une plus grande photostabilité que les agents connus, tout en ayant une toxicité inférieure. Les procédés présentent une grande sélectivité pour les mitochondries. Les procédés sont utiles à la fois dans l'imagerie in vitro et in vivo, par exemple, l'imagerie de tumeurs.
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CN111574499A (zh) * 2020-06-09 2020-08-25 河南大学 Cy5-650及其在制备抗肿瘤药物中的应用
CN111675921A (zh) * 2020-06-09 2020-09-18 河南大学 Cypy及其在制备抗肿瘤药物中的应用
CN113429335A (zh) * 2021-06-25 2021-09-24 安徽大学 一种溶酶体靶向的双响应双光子荧光探针及其制备方法和用途
CN113773666A (zh) * 2021-08-11 2021-12-10 大连理工大学 一类三线态系间窜越菁染料、其制备方法及应用
CN114045045A (zh) * 2021-10-26 2022-02-15 大连理工大学 一类单光子上转换五甲川菁类光敏染料、其制备方法和应用
CN114736200A (zh) * 2022-03-22 2022-07-12 大连理工大学 一类基于硫代五甲川菁染料的无重原子三重态光敏剂、其制备方法和应用
CN114874638A (zh) * 2022-06-23 2022-08-09 西安建筑科技大学 一种中位取代的五甲川菁染料及其制备方法和应用及一种荧光探针
WO2023160737A1 (fr) 2022-02-23 2023-08-31 Univerzita Karlova Sels de polyméthinium utilisés comme inhibiteurs de la dihydroorotate déshydrogénase
CN116947823A (zh) * 2023-07-25 2023-10-27 李哲 一种具有长余辉发光性质的有机小分子及其生物应用
CN117049992A (zh) * 2023-08-23 2023-11-14 济南大学 一种同时监测线粒体和溶酶体的荧光探针及其制备和应用
CN117263928A (zh) * 2023-09-20 2023-12-22 南通大学 一种荧光探针及其制备方法

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CN111675921A (zh) * 2020-06-09 2020-09-18 河南大学 Cypy及其在制备抗肿瘤药物中的应用
CN111675921B (zh) * 2020-06-09 2021-12-14 河南大学 Cypy及其在制备抗肿瘤药物中的应用
CN113429335A (zh) * 2021-06-25 2021-09-24 安徽大学 一种溶酶体靶向的双响应双光子荧光探针及其制备方法和用途
CN113429335B (zh) * 2021-06-25 2023-05-16 安徽大学 一种溶酶体靶向的双响应双光子荧光探针及其制备方法和用途
CN113773666A (zh) * 2021-08-11 2021-12-10 大连理工大学 一类三线态系间窜越菁染料、其制备方法及应用
CN114045045B (zh) * 2021-10-26 2022-11-04 大连理工大学 一类单光子上转换五甲川菁类光敏染料、其制备方法和应用
CN114045045A (zh) * 2021-10-26 2022-02-15 大连理工大学 一类单光子上转换五甲川菁类光敏染料、其制备方法和应用
WO2023160737A1 (fr) 2022-02-23 2023-08-31 Univerzita Karlova Sels de polyméthinium utilisés comme inhibiteurs de la dihydroorotate déshydrogénase
CZ310199B6 (cs) * 2022-02-23 2024-11-13 Univerzita Karlova Polymethiniové soli jako inhibitory dihydroorotát dehydrogenázy
CN114736200A (zh) * 2022-03-22 2022-07-12 大连理工大学 一类基于硫代五甲川菁染料的无重原子三重态光敏剂、其制备方法和应用
CN114874638A (zh) * 2022-06-23 2022-08-09 西安建筑科技大学 一种中位取代的五甲川菁染料及其制备方法和应用及一种荧光探针
CN114874638B (zh) * 2022-06-23 2024-01-19 西安建筑科技大学 一种中位取代的五甲川菁染料及其制备方法和应用及一种荧光探针
CN116947823A (zh) * 2023-07-25 2023-10-27 李哲 一种具有长余辉发光性质的有机小分子及其生物应用
CN117049992A (zh) * 2023-08-23 2023-11-14 济南大学 一种同时监测线粒体和溶酶体的荧光探针及其制备和应用
CN117263928A (zh) * 2023-09-20 2023-12-22 南通大学 一种荧光探针及其制备方法

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