EP4127069A1 - Fluorochromes à large déplacement de stokes à base de sels de benzopyrylium pontés - Google Patents

Fluorochromes à large déplacement de stokes à base de sels de benzopyrylium pontés

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Publication number
EP4127069A1
EP4127069A1 EP21716703.0A EP21716703A EP4127069A1 EP 4127069 A1 EP4127069 A1 EP 4127069A1 EP 21716703 A EP21716703 A EP 21716703A EP 4127069 A1 EP4127069 A1 EP 4127069A1
Authority
EP
European Patent Office
Prior art keywords
dimethyl
amino
ium
xanthene
ethyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21716703.0A
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German (de)
English (en)
Inventor
Bernd Schweder
Frank Lehmann
Matthias Wenzel
Peter Czerney
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dyomics GmbH
Original Assignee
Dyomics GmbH
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Filing date
Publication date
Application filed by Dyomics GmbH filed Critical Dyomics GmbH
Publication of EP4127069A1 publication Critical patent/EP4127069A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C07D311/84—Xanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
    • C07D311/86—Oxygen atoms, e.g. xanthones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/44—Radicals substituted by doubly-bound oxygen, sulfur, or nitrogen atoms, or by two such atoms singly-bound to the same carbon atom
    • C07D213/46—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C07D311/84—Xanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
    • C07D311/88—Nitrogen 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
    • C07D405/04—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 directly linked by a ring-member-to-ring-member bond
    • 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
    • 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
    • C07D405/14—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 three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered
    • 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
    • C07D493/04—Ortho-condensed systems
    • 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
    • C09B57/00—Other synthetic dyes of known constitution
    • 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
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/02—Coumarine dyes

Definitions

  • Fluorescence-based markers have been used for decades in biological, biotechnological and medical research as well as in medical diagnostics [Brinkley M., A Brief Survey of Methods for Preparing Protein Conjugates with Dyes, Flaptens and Cross-Linking Reagents, Bioconjugate Chem, 3 (1992) 2-12; Wagoner A Covalent Labeling of Proteins and Nucleic Acids with Fluorophores, Meth. Enzymol., 246 (1995) 362-373; Flermanson G.T., Bioconjugate Techniques, Academic Press 2013; Springer Series on Fluorescence 18, 2019 (Pedras B.
  • An alternative approach for multiplex applications is the use of dyes that allow a spectral differentiation between donor and acceptor without energy transfer. Examples of this can be found in the MegaStokes dyes known since about 2002 (EP 1318 177 B1, EP 1 535 969 B1). These are preferably tailored to an excitation wavelength between 470 nm (blue-green LED) and 500 nm (488 nm Ar-ion laser), since they thus best corresponded to the state of the art for excitation light sources at the time.
  • the aim of the invention is to make fluorescent markers based on bridged benzopyrylium compounds accessible, whereby molecules K to be marked can be bound via the linker L and the reactive group A, and the fluorescent marker has as many of the following properties as possible: Shift, high photo and storage stability, solubility in aqueous media and high fluorescence quantum yields.
  • the invention describes compounds (in particular bridged benzopyrylium salts) of the general formula 1 1 and salts and solvates thereof, where
  • Rll and R12 are each independently hydrogen or alkyl, where alkyl is preferably Ci-C4-alkyl, particularly preferably methyl,
  • R2 is hydrogen, alkyl, preferably Ci-C4-alkyl, or alkenyl, hydrogen being preferred in one embodiment,
  • R3 is hydrogen, alkyl, preferably Ci-C4-alkyl, particularly preferably methyl, aryl, hydroxy or oxo, alkoxy, preferably methoxy or ethoxy, particularly preferably ethoxy, aryloxy, NR18R19 or a group Q, where R18 and R19 are each independently of one another from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bonded via a linker L, (vi) alkyl, preferably C 1 -C 4 -alkyl, more preferably ethyl , (vii) w-sulfonic acid-alkyl (- (CH2) X -SO3), where x is preferably 1-5, especially is preferably 3, (viii) w-carboxylic acid alkyl (- (CH 2 ) V -C0 2 H), where y is preferably 1-8, particularly preferably 6, and
  • R21, R22, R23, R24 are each independently hydrogen, a sulfonic acid or a sulfonic acid derivative
  • R25 is hydrogen, alkyl, preferably Ci-Cg-alkyl, w-sulfonic acid-alkyl (- (CI- -SO), where x preferably 1-5, particularly preferably 3, or a reactive group A bonded via a linker L, or R2 and R3 to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring together with the carbon atoms to which they are attached are bound, are bridged, whereby the ring may contain further substituents,
  • R4 is hydrogen, bromine, chlorine, sulfonic acid or a sulfonic acid derivative, alkyl, aryl or heteroaryl, with hydrogen, sulfonic acid and pyridine (4-pyridyl) being preferred, or R3 and R4 to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring are bridged together with the carbon atoms to which they are bound, it being possible for the ring to contain further substituents, in particular a sulfonic acid or a sulfonic acid derivative,
  • R5 is hydrogen, sulfonic acid or a sulfonic acid derivative, hydrogen being preferred
  • R6 is hydrogen, bromine, chlorine, hydroxy, alkoxy (for example 6-oxyhexanoic acid), aryloxy or NR29R30, where R29 and R30 are each independently hydrogen, alkyl, aryl or a reactive group A bonded via a linker L, where R29 and R30, independently of one another, in each case preferably from (i) Ci-C4-alkyl, preferably ethyl, (ii) w-sulfonic acid-alkyl (- (CH2) X -S03), where x is preferably 1-5, particularly preferably 3, (iii) w-Carboxylic acid alkyl (- (C ⁇ J y -CC H), where y is preferably 1-8, particularly preferably 6, and (iv) ethyl esters of (iii) are selected, or R5 and R6 are selected to form a saturated one Ring, partially unsaturated ring, aromatic ring or heteroaromatic ring are bridged together with the
  • R7 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy, NR31R32, sulfonic acid or a sulfonic acid derivative, with hydrogen, sulfonic acid or a sulfonic acid derivative being particularly is preferred, where R31 and R32, independently of one another, each selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bonded via a linker L, (vi) alkyl, are preferred Ci-C4-alkyl, more preferably ethyl, (vii) w-sulfonic acid-alkyl (- (CH2) X -SO3), where x is preferably 1-5, particularly preferably 3, (viii) w-carboxylic acid alkyl (- (C ⁇ J y -CC H), where y is preferably 1-8, particularly preferably 6, and (ix) ethyl esters of (
  • R8 is hydrogen, methyl or ethyl, with hydrogen being preferred, or R7 and R8 are bridged to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring together with the carbon atoms to which they are bonded, the ring being further May contain substituents, in particular a sulfonic acid or a sulfonic acid derivative,
  • R9 is hydrogen, alkyl, preferably Ci-C 6 -alkyl, or 2-carboxyphenyl, hydrogen being preferred,
  • L is a linker selected from - (CH2) S - and - [(CH 2 ) m -0] p - (CH 2 ) m -, where m is an integer from 2-5 and p and s are each independently one represent an integer from 1-10, each compound containing no linker or one linker L with a reactive group A bonded to L for covalent bonding to a molecule K to be labeled, wherein
  • A is an amine (-NH2), hydroxy (-OH) or phosphoramidite (-0-P- [0-CH 2 -CH 2 -CN] -N [(CH (CHB) 2 ] 2 ) function, a Carboxylic acid (-COOH), an alkyl or active ester derived therefrom (NHS ester, sulfo-NHS ester, tetrafluoro-phenyl ester, p-sulfo-tetrafluoro-phenyl ester), a carboxylic acid hydrazide (- CONHNH2) or a carboxylic acid -Amid (-CONHR28) where R28 is equal to - (CH2) t -Y, where
  • Y is -OH, -NH 2 , -NH 3 + , maleimide (-N [CO-CH] 2 ), -NCS, -NCO, -NH-CO-CH 2 -l, -NH-CO-CH 2 - Br, -azide (-N3), -alkine (-CCH) or -phosphoramidite (-0-P- [0-CH 2 -CH 2 -CN] -N- [CH- (CHB) 2 ] 2 ) and t is an integer from 1-10, and
  • haptens molecules that represent an incomplete antigen and only show the effect of an antigen when they bind to proteins or cell structures
  • proteins proteins, antibodies (proteins that are formed in response to antigens)
  • low-molecular drugs effective components in drugs that, due to their relatively low molar mass up to about 800 g / mol, in contrast to proteins as very large molecules, are able to penetrate cells
  • peptides small or short-chain proteins up to about 100 linked amino acids
  • nucleotides basic building blocks of nucleic acids like DNA or RNA, which consist of a phosphate part, a monosaccharide part and a nucleobase part like adenine, guanine, cytosine, thymine or uracil
  • nucleosides basic building blocks of nucleic acids like DNA or RNA that do not have a phosphate part but only consist of a monosaccharide part and a nucleobase part
  • DNA oligomers on the contrary
  • Aryl substituents and / or heteroaryl substituents (as in R3, R4, R6, R7, R18, R19, R29, R30, R31, R32) preferably contain further substituents such as sulfonic acids or sulfonic acid derivatives and / or alkoxy groups and / or substituted amino groups.
  • a compound according to the invention preferably contains at least one group selected from a sulfonic acid group, a sulfonic acid derivative, an alkoxy group and an amino group, preferably a sulfonic acid group.
  • sulfonic acid also includes the term sulfonate and the term sulfonate also includes the term sulfonic acid.
  • carboxylic acid also includes the term carboxylate and the term carboxylate also includes the term carboxylic acid.
  • the compound according to the invention preferably exhibits fluorescence.
  • the compound according to the invention is preferably a fluorescent dye.
  • the compound is preferably suitable as a fluorescent dye.
  • R3 hydroxy
  • the bridged compounds are, depending on the pH value, at low pH values as 3-hydroxy-xanthenium salts 1 and at higher pH values as 3-oxo-2H-xanthenes 4 with a neutral body.
  • R3 is preferably hydroxy, the compound being 3-oxo-2H-xanthene 4_ as a neutral base, depending on the pH.
  • one or more selected from R2-R3, R3-R4, R5-R6, R6-R7 and R7-R8 can be bridged by forming saturated rings, partially unsaturated rings, aromatic rings or heteroaromatic rings, which are independent of one another contain further substituents, in particular sulfonic acids or sulfonic acid derivatives.
  • At least one pair is selected from R3 with R4, R5 with R6, R6 with R7 and R7 with R8 to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring together with the carbon atoms to which they are attached, bridged, it being possible for each ring, independently of one another, to contain further substituents, in particular sulfonic acids or sulfonic acid derivatives.
  • R3 and R4 are not bridged together in such a way that, together with the carbon atoms to which they are attached, they form any ring selected from a saturated ring, partially unsaturated ring, aromatic ring, or heteroaromatic ring.
  • R3 and R4 are not bridged to one another in such a way that, together with the carbon atoms to which they are attached, they form an aromatic ring.
  • compounds according to the invention are indicated by the formula 5 (chromenoxanthenium compounds), to which, for example, a bridging of R2-R3 leads.
  • R13 is hydrogen, alkyl, preferably Ci-C4-alkyl, or 2-carboxyphenyl, hydrogen being particularly preferred,
  • R14 is hydrogen or alkyl, preferably Ci-C4-alkyl, with hydrogen being particularly preferred,
  • R15 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy, NR33R34, sulfonic acid or a sulfonic acid derivative or bridged to R16, hydrogen being particularly preferred, where R33 and R34 are each independently selected from (i) hydrogen, (ii) benzyl, ( iii) aryl, (iv) heteroaryl, (v) a reactive group A bonded via a linker L, (vi) alkyl, preferably C1-C4-alkyl, more preferably ethyl, (vii) w-sulfonic acid-alkyl (- (CH2) X -S03), where x is preferably 1-5, particularly preferably 3, (viii) w-carboxylic acid alkyl (- (CH 2 ) y -C0 2 H), where y is preferably 1-8, particularly preferably 6, and (ix) ethyl esters of (viii) are selected, or R14 and R15 are
  • R16 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy or NR35R36, where NR35R36 is particularly preferred, where R35 and R36 are each independently selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) a reactive group A bonded via a linker L, (vi) alkyl, preferably C1-C4-alkyl, more preferably ethyl, (vii) w-sulfonic acid-alkyl (- (CH2) X —SO3), where x is preferably 1 -5, particularly preferably 3, is (viii) w-carboxylic acid alkyl (- (CH 2 ) y -C0 2 H), where y is preferably 1-8, particularly preferably 6, and (ix) ethyl esters of (viii) are selected it is particularly preferred that R35 and R36, independently of one another, are preferably selected from Ci-C4
  • R17 is hydrogen, sulfonic acid or a sulfonic acid derivative, with hydrogen being particularly preferred, or R16 and R17 are bridged to form a saturated ring, partially unsaturated ring, aromatic ring or heteroaromatic ring together with the carbon atoms to which they are bonded, the Ring may contain further substituents, in particular a sulfonic acid or a sulfonic acid derivative.
  • Aryl substituents and / or heteroaryl substituents (as in R4, R6, R7, R15, R16, R18, R19, R33, R34, R35, R36) preferably contain further substituents such as sulfonic acids or sulfonic acid derivatives and / or alkoxy groups and / or substituted amino groups.
  • the absorption maxima are in the entire range of visible light, with a focus on 500-530 nm.
  • the compounds have a high Stokes shift in the range of 80 nm.
  • the absorption is measured, for example, at 25 ° C. in aqueous or ethanolic solutions with an extinction of 1.0 using a Specord 205 from Analytik Jena, the aqueous solutions preferably being phosphate-buffered saline solutions (PBS buffer; approx. 100 mM sodium chloride and 100 mM total phosphate) and have a pH of 7.5.
  • PBS buffer phosphate-buffered saline solutions
  • the emission is measured, for example, at 25 ° C in dilute aqueous or ethanolic solutions with an extinction of 0.1 using an FP-6600 Spectrofluorometer from Jasco, the dilute aqueous solutions preferably being phosphate-buffered saline solutions (PBS buffer; approx. 100 mM sodium chloride and 100 mM total phosphate) and have a pH of 7.5.
  • PBS buffer phosphate-buffered saline solutions
  • a compound according to the invention is preferably at least characterized in that the compound is a fluorescent compound.
  • the compound according to the invention has an absorption maximum in the wavelength range from 400 nm to 650 nm, preferably from 500 nm to 550 nm.
  • the compound according to the invention has an absorption maximum in the wavelength range from 630 nm to 700 nm, preferably from 650 nm to 690 nm.
  • the Stokes shift is preferably at least 40 nm, preferably 50 nm to 120 nm, even more preferably 70 nm to 90 nm.
  • R3 is NR18R19, where R18 and R19 are each independently selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) one via one Linker L-bonded reactive group A, (vi) alkyl, preferably C 1 -C 4 -alkyl, more preferably ethyl, (vii) w-sulfonic acid-alkyl (- (CH2) X -SO3), where x is preferably 1-5, especially is preferably 3, (viii) w-carboxylic acid alkyl (- (CH 2 ) y -C0 2 H), where y is preferably 1-8, particularly preferably 6, and (ix) ethyl esters of (viii) are selected, and where NR18R19 is particularly preferably selected from 3-aminopropane sulfonate, N-methyl aniline, 4- (methylamino)
  • a compound of the invention is preferably water-soluble.
  • a compound according to the invention is particularly preferably characterized at least by the fact that at 25 ° C. at least 1 mg, preferably 2 mg to 4 mg, of the compound is soluble in 1,000 g (1000 mg) of water.
  • the sulfonic acid groups influence the aggregation behavior and reduce non-covalent dimer formation and non-covalent attachment to biomolecules and surfaces.
  • Sulphonic acid substituents in particular which are bound directly to the dye base, also have an effect on the physico-chemical properties of the dyes in such a way that the absorption and emission wavelengths are shifted and a significant increase in the quantum yield is usually achieved.
  • the compound contains at least one sulfonic acid group.
  • a compound according to the invention preferably contains at least one group selected from a sulfonic acid group, a sulfonic acid derivative, a hydroxyl group, an amino group, a carboxylic acid and a carboxylic acid derivative.
  • the invention does not preferably include compounds in which R7 is methoxy and R3 is an amino group, in particular NR18R19, in particular NH (4-HOOCCeH 4 ), NH (4-C 2 H 5 COOC 6 H 4 ), NH (2- C2H5COOC6H4) , NH (C 6 H 5 ), N (C 2 H 5 ) 2, or N (CH 2 ) 2 (CH 2 ) 20.
  • the invention does not preferably include compounds in which R4 is bromine and R6 and R16 are N (CH 3 ) 2 .
  • the invention does not preferably include compounds in which R6 and R16 are present and one of R6 and R16 is a hydroxyl group.
  • the bridging gives stable benzopyrylium compounds which, depending on the substituents, achieve surprisingly high fluorescence quantum yields in aqueous solution.
  • the fluorescence quantum yield of a compound according to the invention is preferably 0.1 to 0.95, more preferably 0.5 to 0.9. Fluorescence quantum yields are preferably determined at 25 ° C in dilute aqueous or ethanolic solutions with an extinction of 0.1 using a Hamamatsu Absolute Photoluminescence Quantum Yield Measurement System C-9920, the dilute aqueous solutions preferably being phosphate-buffered saline solutions (PBS buffer; approx. 100 mM sodium chloride and 100 mM total phosphate) and have a pH of 7.5.
  • PBS buffer phosphate-buffered saline solutions
  • the compounds complement the already commercially available dyes with a high Stokes shift or represent better fluorescent alternatives.
  • the photo stability of the connections is very good. It is significantly higher than that of comparable coumarin-based dyes, for example as DY-510XL (see Fig. 1).
  • a compound according to the invention is preferably selected from the following compounds and their salts and solvates thereof (in the case of ions, the counterion, optionally the counterions, is preferably selected from tetrafluoroborate, chloride and sodium): 6-ethoxy-N, N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amine,
  • a compound according to the invention is particularly preferably selected from 6-ethoxy-N, N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amine tetrafluoroborate, 6 - [[6- (diethylamino) -l, l -dimethyl-2H-xanthen-10-ium-3-yl] amino] hexanoic acid chloride salt,
  • a compound according to the invention is particularly preferably selected from the following compounds and their salts and solvates thereof: 3 - [[6- [5-carboxypentyl (3-sulfonatopropyl) amino] -1, l-dimethyl-2H-xanthene-10-ium-3-yl] amino] propane-1-sulfonate,
  • a compound according to the invention is particularly preferably selected from
  • the compounds according to the invention can be used as dyes for the optical marking of organic or inorganic recognition units, e.g. B. of amino acids, peptides, proteins, antibodies, antigens, haptens, enzyme substrates, enzyme cofactors, biotin, carotenoids, hormones, neurohormones, neurotransmitters, growth factors, lectins, toxins, carbohydrates, oligosaccharides, polysaccharides, dextrans, DNA, nucleic acids , RNA, biological cells, lipids, receptor-binding pharmaceuticals or organic or inorganic polymeric carrier materials can be used.
  • organic or inorganic recognition units e.g. B. of amino acids, peptides, proteins, antibodies, antigens, haptens, enzyme substrates, enzyme cofactors, biotin, carotenoids, hormones, neurohormones, neurotransmitters, growth factors, lectins, toxins, carbohydrates, oligosaccharides, polysaccharides, dextrans,
  • the identification units can be marked by the formation of ionic or van der Waals interactions between the markers (compounds according to the invention) and the materials to be marked.
  • Both the compounds according to the invention and systems derived therefrom can be used in optical, in particular fluorescence-optical, qualitative and quantitative determination methods for diagnosing cell properties, in biosensors (point of care measurements), for researching the genome (DNA sequencing) and in miniaturization technologies.
  • Typical applications are in cytometry and cell sorting, fluorescence correlation spectroscopy (FCS), in ultra-high throughput screening (UHTS), in multicolor fluorescence in situ hybridization (FISH) and in microarrays (DNA- and protein chips).
  • a receptor is a molecule that has an affinity for a given ligand.
  • Receptors can be naturally occurring or man-made molecules. Receptors can be in pure form or bound to other species can be used. Receptors can be linked to a binding partner covalently or non-covalently either directly or through certain coupling mediators.
  • a ligand is a molecule that is recognized by a specific receptor.
  • ligands that can be detected by this invention include agonists and antagonists for cell membrane receptors, toxins and other toxins, viral epitopes, hormones such as opiates and steroids, hormone receptors, peptides, enzymes, enzyme substrates, agents acting as cofactors, Lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins and antibodies include, but are not limited to, the substances listed.
  • one of the compounds disclosed herein is also used as a fluorescent dye and / or in a fluorescent probe or as a fluorescent probe for labeling one or more compounds selected from amino acids, peptides, proteins, antibodies, antigens, haptens, enzyme substrates, enzyme cofactors , Biotin, carotenoids, hormones, neurohormones, neurotransmitters, growth factors, lectins, toxins, carbohydrates, oligosaccharides, polysaccharides, dextrans, nucleic acids, oligonucleotides, DNA, RNA, lipids, receptor-binding drugs and cells.
  • Compounds according to the invention can have at least one reactive group A in the form of an active ester, the active ester preferably being an NHS ester (N-hydroxysuccinimidyl ester), a sulfo-NHS ester (sulfo-hydroxysuccinimidyl ester), a TFP ester ( Tetrafluoro-phenyl ester) or an STP-ester (p-sulfo-tetrafluoro-phenyl ester), as indicated in the table below.
  • an NHS ester N-hydroxysuccinimidyl ester
  • a sulfo-NHS ester sulfo-hydroxysuccinimidyl ester
  • TFP ester Tetrafluoro-phenyl ester
  • STP-ester p-sulfo-tetrafluoro-phenyl ester
  • the compounds according to the invention can have A in the form of a carboxylic acid derivative, the carboxylic acid derivative preferably being a hydrazide, an amine, an iodoacetamide, a maleimide, an alkyne or an azide, as indicated in the table below.
  • the invention also relates to a process for the preparation of a compound of the formula 1, preferably for the preparation of a compound according to the invention according to the formula 1.
  • the method comprises the reaction of (E) - (3-ethoxy-5,5-dimethyl-cyclohex-2-en-1-ylidene) -ethyl-oxonium or (E) - [3-ethoxy-5,5-dimethyl -2- (4-pyridyl) cyclohex-2-en-l-ylidene] -ethyl-oxonium with a benzaldehyde compound, the benzaldehyde compound having a benzaldehyde group and the benzaldehyde compound having a flydroxy group in the ortho position to the benzaldehyde group .
  • the reaction is preferably carried out in an organic solvent, the organic solvent preferably being triethyl orthoformate.
  • the reaction is carried out at 70 ° C to 200 ° C, more preferably at 90 ° C to 130 ° C.
  • E) - [3-ethoxy-5,5-dimethyl-2- (4-pyridyl) cyclohex-2-en-1-ylidene] -ethyl-oxonium tetrafluoroborate is used.
  • the benzaldehyde compound is preferably selected from 2-flyroxy-4-diethylaminobenzaldehyde, 3- (N- (6-ethoxy-6-oxo-hexyl) -4-formyl-3-hydroxy-anilino) propane-1-sulfonate, ethyl 6- (N-ethyl-4-formyl-3-hydroxy-anilino) hexanoic acid, 2,4-dihydroxybenzaldehyde, 3- (6-formyl-7-hydroxy-2,2,4-trimethyl-l-quinolyl) propane l-sulfonate and 6- (6-formyl-7-hydroxy-2,2,4-trimethyl-l-quinolyl) hexanoic acid.
  • the process can include the conversion of the compound 5,5-dimethylcyclohexane-1,3-dione or 3-flydroxy-5,5-dimethyl-2- (4-pyridyl) cyclohex-2-en-1-one
  • the invention also relates to a process for the preparation of a compound of the formula 5, preferably for the preparation of a compound according to the invention according to the formula 5.
  • the process comprises the reaction of a compound of formula 1, preferably 6-ethoxy-N, N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amine or 6 - [(6-ethoxy-8 , 8-dimethyl-7H-xanthen-10-ium-3-yl) -ethyl-amino] ethyl hexanoate, with a benzaldehyde compound, the benzaldehyde compound having a benzaldehyde group and the benzaldehyde compound in the ortho position to the benzaldehyde group Has hydroxyl group.
  • the reaction is preferably carried out in an organic solvent, the organic solvent preferably being glacial acetic acid.
  • the reaction is carried out at 70 ° C to 200 ° C, more preferably at 90 ° C to 110 ° C.
  • 6-Ethoxy-N, N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amine tetrafluoroborate or 6 - [(6-ethoxy-8,8-dimethyl-7H-xanthene-10 -ium-3-yl) -ethyl-amino] hexanoic acid ethyl ester tetrafluoroborate is used.
  • the benzaldehyde compound is preferably selected from 2- [4- (diethylamino) -2-hydroxy-benzoyl] benzoic acid, 4- (diethylamino) -2-hydroxy-benzaldehyde and 2-hydroxy-4-methoxy-benzaldehyde.
  • the processes according to the invention for preparing a compound of the formula 1 or 5 preferably comprise the introduction of at least one group selected from a sulfonic acid group, a sulfonic acid derivative, an alkoxy group and an amino group, particularly preferably a sulfonic acid group.
  • Fig. 1 Photostability of compounds 5, 8, 11 and 38 compared to the MegaStokes dye DY-510XL.
  • Fig.2 Emission spectra of selected compounds according to the invention in PBS.
  • UV-Vis in ethanol l, ⁇ c: 531 nm; 605 nm
  • 125 pmol of compound 5 are dissolved in 3 ml of DMF.
  • TSTU N, N, N ', N'-Tetramethyl-Q- (N-succinimidyl) uronium tetrafluoroborate
  • DIPEA diisopropyl-ethylamine
  • Connection 14 6- [5-carboxypentyl (ethyl) amino] -1, l-dimethyl-3- (4-sulfonatoanilino) -2H-xanthene-10-ium-4-sulfonate
  • mitioI compound 13 are dissolved in 2 ml oleum (20% SO3) and stirred at RT for 2 hours. The mixture is poured onto ice and stirred for a further 1 hour at RT. After blunting with sodium carbonate, purification is carried out by RP chromatography.
  • the mixture is warmed to RT and stirred at RT for a further 1 hour.
  • the organic solvents are distilled off and the product is isolated from the aqueous phase by RP chromatography.
  • UV-Vis in water pH 3 X m3x - x nm; x nm (available as Hydroxy-BPS)
  • UV-Vis in PBS l TM c : 409 nm; 485 nm UV-Vis in buffer pH 9: X max : 462 nm; X em : 536 nm
  • FIG. 1 shows the results of the irradiation of aqueous solutions (PBS pH 7.5, 100 mM & 100 mM NaCl plus 5 mM NaNs) of compounds 5, 8, 11 and 38 in comparison with the MegaStokes dye DY-510XL.
  • the solutions were adjusted to an absorbance of "1" in the absorption maximum with a layer thickness of 1 cm and irradiated with white light from the 150W Xe lamp of a fluorescence spectrometer (JASCO FP-6600, monochromator at 0 nm, column position L: 10 nm) and the Absorption was monitored at a maximum in 5 minute intervals over one hour.
  • FIG. 2 shows the fluorescence maxima of selected compounds.
  • the invention relates to novel, water-soluble fluorescent dyes with a high fluorescence quantum yield based on oxygen-containing heterocycles, their reactive derivatives and dye conjugates and their use for marking samples and detecting analytes.
  • the compounds of the new class of dyes are compatible with commercial excitation light sources and are characterized by Stokes shifts of more than 50 nm.
  • Rll and R12 are each independently hydrogen or alkyl, R2 is hydrogen, alkyl or alkenyl,
  • R4 is hydrogen, bromine, chlorine, sulfonic acid or a sulfonic acid derivative, alkyl, aryl or heteroaryl and
  • R5 is hydrogen, sulfonic acid or a sulfonic acid derivative
  • R6 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy or NR29R30, where R29 and R30, independently of one another, are each hydrogen, alkyl, aryl or a reactive group A bonded via a linker L, where R29 and R30, independently of one another, are each preferably composed of ( i) Ci- C4-alkyl, (ii) w-sulfonic acid-alkyl (- (CH 2 ) X -S0 3 _ ), where x is 1-5, (iii) co-carboxylic acid alkyl (- (C ⁇ J y -CC H), where y is 1-8 and (iv) ethyl esters of (iii) are selected,
  • R7 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy, NR31R32, sulfonic acid or a sulfonic acid derivative, where R31 and R32 are each independently selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, ( v) a reactive group A bonded via a linker L, (vi) alkyl, preferably Ci-C4-alkyl, (vii) co-sulfonic acid-alkyl (- (CH2) X -S03 _ ), where x is 1-5, (viii) co-carboxylic acid alkyl (- (CH 2 ) y -C0 2 H), where y is 1-8, and (ix) ethyl esters of (viii) are selected,
  • R8 is hydrogen, methyl or ethyl
  • R9 is hydrogen, alkyl or 2-carboxyphenyl
  • L is a linker selected from - (CH2) s - and - [(CH 2 ) m -0] p - (CH 2 ) m -, where m is an integer from 2-5 and p and s are each independently one represent integers from 1-10, each compound containing no linker or one linker L with a reactive group A bonded to L for covalent bonding to a molecule K to be labeled, where A is an amine (-NH2), hydroxy (-OH) or phosphoramidite (-0-P- [0-CH 2 -CH 2 -CN] -N [(CH (CHB) 2 ] 2 ) function, a Carboxylic acid (-COOH), an alkyl or active ester derived therefrom (NHS ester, sulfo-NHS ester, tetrafluoro-phenyl ester, p-sulfo-tetrafluoro-phenyl ester), a carboxylic acid hydra
  • Y is -OH, -NH 2 , -NH 3 + , maleimide (-N [CO-CH] 2 ), -NCS, -NCO, -NH-CO-CH 2 -l, -NH-CO-CH 2 - Br, -azide (-N3), -alkine (-CCH) or -phosphoramidite (-0-P- [0-CH 2 -CH 2 -CN] -N- [CH- (CHB) 2 ] 2 ) and t is an integer from 1-10,
  • haptens molecules that represent an incomplete antigen and only show the effect of an antigen when they bind to proteins or cell structures
  • proteins proteins, antibodies (proteins that are formed in response to antigens), low-molecular drugs (effective components in drugs, which due to their relatively low molar mass up to about 800 g / mol, in contrast to proteins as very large molecules, are able to penetrate cells
  • peptides small or short-chain proteins up to about 100 Amino acids
  • nucleotides basic building blocks of nucleic acids such as DNA or RNA, which consist of a phosphate part, a monosaccharide part and a nucleobase part such as adenine, guanine, cytosine, thymine or uracil
  • nucleosides basic building blocks of nucleic acids such as DNA or RNA that do not have a phosphate part , but only consist of a monosaccharide part and a nucleobase part
  • R3 hydroxyl, the compound being 3-oxo-2H-xanthene 4 as a neutral base, depending on the pH is present.
  • Compound according to aspect 1 or 2 characterized in that at least one selected from R2-R3, R3-R4, R5-R6, R6-R7 and R7-R8 is bridged by saturated rings, partially unsaturated rings, aromatic rings or heteroaromatic rings be formed which, independently of one another, contain further substituents, in particular sulfonic acids or sulfonic acid derivatives.
  • R13 is hydrogen, alkyl or 2-carboxyphenyl
  • R14 is hydrogen, alkyl or 2-carboxyphenyl
  • R15 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy, NR33R34, sulfonic acid or a sulfonic acid derivative or bridged to R16, where R33 and R34 are each independently selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv ) Heteroaryl, (v) a reactive group A bonded via a linker L, (vi) alkyl, preferably Ci-C4-alkyl, (vii) co-sulfonic acid-alkyl (- (CH2) X -S03), where x 1- 5 is, (viii) co-carboxylic acid alkyl (- (C ⁇ J y -CC H), where y is 1-8, and (ix) ethyl esters of (viii) are selected,
  • R16 is hydrogen, bromine, chlorine, hydroxy, alkoxy, aryloxy or NR35R36, where R35 and R36 are each independently selected from (i) hydrogen, (ii) benzyl, (iii) aryl, (iv) heteroaryl, (v) one via one Linker L bonded reactive group A, (vi) alkyl, preferably Ci-C4-alkyl, (vii) co-sulfonic acid alkyl (- (CH2) X -SO3 _ ), where x is 1-5, (viii) w-carboxylic acid alkyl (- (C ⁇ J y -CC H), where y is 1-8, and (ix) ethyl esters of (viii) are selected,
  • R17 is hydrogen, sulfonic acid or a sulfonic acid derivative and one or more selected from R5-R6, R6-R7, R7-R8, R14-R15, R15-R16 and R16-R17 can be bridged by saturated rings, partially unsaturated rings, aromatic Rings or heteroaromatic rings are formed which, independently of one another, can contain further substituents, in particular sulfonic acids or sulfonic acid derivatives, and preferably aryl substituents and / or heteroaryl substituents (as in R4, R6, R7, R15, R16, R18, R19, R33, R34, R35, R36) contain further substituents such as sulfonic acids or sulfonic acid derivatives and / or alkoxy groups and / or substituted amino groups.
  • R3 is NR18R19, where R18 and R19 are each independently hydrogen, alkyl, benzyl, aryl, heteroaryl or a reactive group A bonded via a linker L, where NR18R19 preferably consists of 3-aminopropanesulfonate , N-methylaniline, 4- (methylamino) benzene sulfonate, aniline, 5-carboxypentylamine and 3-carboxypropyl (methyl) amine.
  • a compound according to any one of the preceding aspects characterized in that at 25 ° C at least 1 mg of the compound is soluble in 1,000 g of water.
  • the compound is a fluorescent compound which has an absorption maximum in the wavelength range from 400 nm to 650 nm, preferably from 500 nm to 550 nm.
  • the Stokes shift is at least 40 nm, preferably 50 nm to 120 nm, even more preferably 70 nm to 90 nm.
  • connection according to any of the previous aspects, characterized in that the connection is selected from
  • a process for the preparation of a compound of formula 1, which process comprises the reaction of (E) - (3-ethoxy-5,5-dimethyl-cyclohex-2-en-1-ylidene) -ethyl-oxonium or (E) - [ 3-ethoxy-5,5-dimethyl-2- (4-pyridyl) cyclohex-2-en-l-ylidene] ethyl oxonium with a benzaldehyde compound, the benzaldehyde compound having a benzaldehyde group and the benzaldehyde Compound in ortho position to the benzaldehyde group has a hydroxyl group.
  • a compound of formula 1 preferably 6-ethoxy-N, N-diethyl-8,8-dimethyl-7H-xanthene-10-ium-3-amine or 6 - [(6-ethoxy-8,8-dimethyl-7H-xanthene-10-ium-3

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Abstract

L'invention concerne de nouveaux fluorochromes hydrosolubles à haut rendement quantique de fluorescence à base d'hétérocycles oxygénés, leurs conjugués de colorants et dérivés réactifs, ainsi que leur utilisation pour le marquage d'échantillons et la détection d'analytes. Les composés de la nouvelle classe de colorants sont compatibles avec des sources de lumière d'excitation commerciales et se caractérisent par des déplacements de Stokes supérieurs à 50 nm.
EP21716703.0A 2020-04-03 2021-04-01 Fluorochromes à large déplacement de stokes à base de sels de benzopyrylium pontés Pending EP4127069A1 (fr)

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DE102020109362.4A DE102020109362A1 (de) 2020-04-03 2020-04-03 Fluoreszenzfarbstoffe mit hohem Stokes Shift auf der Basis von verbrückten Benzopyryliumsalzen
PCT/EP2021/058647 WO2021198433A1 (fr) 2020-04-03 2021-04-01 Fluorochromes à large déplacement de stokes à base de sels de benzopyrylium pontés

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DE2942931A1 (de) 1979-10-24 1981-05-07 Basf Ag, 6700 Ludwigshafen Fluoreszierende verbindungen
DE10160524A1 (de) 2001-12-05 2003-06-26 Dyomics Gmbh Neue Fluoreszenzmarker mit großem Stokes-Shift
DE10356130A1 (de) 2003-11-28 2005-06-23 Dyomics Gmbh Neue Polymethinfarbstoffe auf Cumarin-Basis mit einstellbarem Stokes-Shift
EP1928822B1 (fr) 2005-09-26 2013-02-27 Life Technologies Corporation Colorants excitables au laser violet et leur methode d'utilisation
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