WO2012069859A1 - Fluorescent dyes sensitive to sodium ions and optionally to potassium ions, process for their preparation and their use - Google Patents
Fluorescent dyes sensitive to sodium ions and optionally to potassium ions, process for their preparation and their use Download PDFInfo
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- WO2012069859A1 WO2012069859A1 PCT/HU2011/000109 HU2011000109W WO2012069859A1 WO 2012069859 A1 WO2012069859 A1 WO 2012069859A1 HU 2011000109 W HU2011000109 W HU 2011000109W WO 2012069859 A1 WO2012069859 A1 WO 2012069859A1
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- compound
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- hydrogen
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- 229910001415 sodium ion Inorganic materials 0.000 title claims abstract description 23
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 19
- 229910001414 potassium ion Inorganic materials 0.000 title claims abstract description 13
- 238000000034 method Methods 0.000 title claims description 10
- 150000001875 compounds Chemical class 0.000 claims abstract description 108
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 35
- 239000001257 hydrogen Substances 0.000 claims abstract description 35
- 150000003839 salts Chemical class 0.000 claims abstract description 19
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 17
- 239000000203 mixture Substances 0.000 claims abstract description 12
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims abstract description 10
- 229910052736 halogen Inorganic materials 0.000 claims abstract description 9
- 150000002367 halogens Chemical class 0.000 claims abstract description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 7
- 125000000623 heterocyclic group Chemical group 0.000 claims abstract description 5
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 3
- 125000005842 heteroatom Chemical group 0.000 claims abstract description 3
- 150000002500 ions Chemical class 0.000 claims description 23
- 150000002431 hydrogen Chemical group 0.000 claims description 15
- 125000005843 halogen group Chemical group 0.000 claims description 10
- 125000004432 carbon atom Chemical group C* 0.000 claims description 9
- 125000001309 chloro group Chemical group Cl* 0.000 claims description 8
- 229920006395 saturated elastomer Polymers 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 150000003983 crown ethers Chemical class 0.000 claims description 7
- -1 nitro, hydroxy, carboxy Chemical group 0.000 claims description 6
- 125000004429 atom Chemical group 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 125000004317 1,3,5-triazin-2-yl group Chemical group [H]C1=NC(*)=NC([H])=N1 0.000 claims description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 125000003277 amino group Chemical group 0.000 claims description 4
- 125000004663 dialkyl amino group Chemical group 0.000 claims description 4
- 125000001183 hydrocarbyl group Chemical group 0.000 claims description 4
- 125000003545 alkoxy group Chemical group 0.000 claims description 3
- 150000007942 carboxylates Chemical class 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 3
- BDHFUVZGWQCTTF-UHFFFAOYSA-N sulfonic acid Chemical compound OS(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-N 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- 125000006239 protecting group Chemical group 0.000 claims description 2
- 125000004356 hydroxy functional group Chemical group O* 0.000 claims 2
- 125000003917 carbamoyl group Chemical class [H]N([H])C(*)=O 0.000 claims 1
- 125000003118 aryl group Chemical group 0.000 abstract 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 abstract 1
- 230000000236 ionophoric effect Effects 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 10
- 238000001514 detection method Methods 0.000 description 9
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 8
- 239000000975 dye Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000005259 measurement Methods 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 4
- 125000001424 substituent group Chemical group 0.000 description 4
- 239000003643 water by type Substances 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- IOJUPLGTWVMSFF-UHFFFAOYSA-N benzothiazole Chemical compound C1=CC=C2SC=NC2=C1 IOJUPLGTWVMSFF-UHFFFAOYSA-N 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 125000004417 unsaturated alkyl group Chemical group 0.000 description 3
- NAWXUBYGYWOOIX-SFHVURJKSA-N (2s)-2-[[4-[2-(2,4-diaminoquinazolin-6-yl)ethyl]benzoyl]amino]-4-methylidenepentanedioic acid Chemical compound C1=CC2=NC(N)=NC(N)=C2C=C1CCC1=CC=C(C(=O)N[C@@H](CC(=C)C(O)=O)C(O)=O)C=C1 NAWXUBYGYWOOIX-SFHVURJKSA-N 0.000 description 2
- 0 CC(C)C(C(C)=C1C(C)C)=CC(C([C@]2IC(*)=C(*)C3)(C(C*C(*)C(*)=C4)=C4[U+]C2*3=C)O2)=I1SCC2=[O+] Chemical compound CC(C)C(C(C)=C1C(C)C)=CC(C([C@]2IC(*)=C(*)C3)(C(C*C(*)C(*)=C4)=C4[U+]C2*3=C)O2)=I1SCC2=[O+] 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 238000004847 absorption spectroscopy Methods 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 244000309464 bull Species 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 125000000753 cycloalkyl group Chemical group 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 2
- 238000000295 emission spectrum Methods 0.000 description 2
- 238000000695 excitation spectrum Methods 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical class O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical group C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 1
- BCMCBBGGLRIHSE-UHFFFAOYSA-N 1,3-benzoxazole Chemical class C1=CC=C2OC=NC2=C1 BCMCBBGGLRIHSE-UHFFFAOYSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- GZOJTRITAZODJV-UHFFFAOYSA-N 3,1-benzoxazin-2-one Chemical class C1=CC=CC2=COC(=O)N=C21 GZOJTRITAZODJV-UHFFFAOYSA-N 0.000 description 1
- HWQQCFPHXPNXHC-UHFFFAOYSA-N 6-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound C=1C(O)=CC=C2C=1OC1=CC(O)=CC=C1C2(C1=CC=2)OC(=O)C1=CC=2NC1=NC(Cl)=NC(Cl)=N1 HWQQCFPHXPNXHC-UHFFFAOYSA-N 0.000 description 1
- OREQHNDPNFLPQG-PPFNPFNJSA-N C/C(/c(cccc1)c1Nc1nc(Cl)nc(Nc2ccc3OCCOCCOCCOCCOc3c2)n1)=N\c1c(C)cccc1 Chemical compound C/C(/c(cccc1)c1Nc1nc(Cl)nc(Nc2ccc3OCCOCCOCCOCCOc3c2)n1)=N\c1c(C)cccc1 OREQHNDPNFLPQG-PPFNPFNJSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 241001432959 Chernes Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 125000005599 alkyl carboxylate group Chemical group 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000007080 aromatic substitution reaction Methods 0.000 description 1
- 125000005392 carboxamide group Chemical group NC(=O)* 0.000 description 1
- 150000003857 carboxamides Chemical class 0.000 description 1
- ANTSCNMPPGJYLG-UHFFFAOYSA-N chlordiazepoxide Chemical compound O=N=1CC(NC)=NC2=CC=C(Cl)C=C2C=1C1=CC=CC=C1 ANTSCNMPPGJYLG-UHFFFAOYSA-N 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000002148 esters Chemical group 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 229960004279 formaldehyde Drugs 0.000 description 1
- 235000019256 formaldehyde Nutrition 0.000 description 1
- 125000001072 heteroaryl group Chemical group 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 125000001820 oxy group Chemical group [*:1]O[*:2] 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- UGJCNRLBGKEGEH-UHFFFAOYSA-N sodium-binding benzofuran isophthalate Chemical compound COC1=CC=2C=C(C=3C(=CC(=CC=3)C(O)=O)C(O)=O)OC=2C=C1N(CCOCC1)CCOCCOCCN1C(C(=CC=1C=2)OC)=CC=1OC=2C1=CC=C(C(O)=O)C=C1C(O)=O UGJCNRLBGKEGEH-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C09B11/00—Diaryl- or thriarylmethane dyes
- C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
- C09B11/06—Hydroxy derivatives of triarylmethanes in which at least one OH group is bound to an aryl nucleus and their ethers or esters
- C09B11/08—Phthaleins; Phenolphthaleins; Fluorescein
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
-
- 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
- C07D417/14—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 three or more hetero rings
-
- 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
- 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
- C09B11/00—Diaryl- or thriarylmethane dyes
- C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
- C09B11/10—Amino derivatives of triarylmethanes
- C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
-
- 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
Definitions
- the present invention relates to new fluorescent dyes, to a method for the preparation of the new compounds, to analytical reagent compositions and kits comprising said new compounds, as well as to the use of the new compounds in the detection of sodium and optionally potassium ion concentration.
- the size of the crown ether influences the quality of the complexable ion.
- the ion-sensitive fluorescent dye undergoes a conformational change, which also results in a change of its fluorescent properties (most frequently in the intensity of fluorescence).
- the change in fluorescent property should not only be well measurable but should also be suitable for the unambiguous detection of the ion concerned, i.e. the dye should not only indicate the presence of the ion in question but it should, by the aid of appropriate calibration curves, also enable one to measure the accurate concentration of the ion.
- the term "detection" is mentioned, it is used always in this more strict sense; ion-selective fluorescent dyes which enable only or primarily qualitative indication are termed here as "indicators”.
- CoroNa ⁇ R Red and CoroNa (R) Green are two fluorescent dyes which are nowadays widely used for the detection of sodium ions [see e.g. J. Biol. Chem. 264, 19449-19457 and 19458-19467 (1989); Cytometry 21 , 248-256 (1995); Physiol. Rev. 79, 1127-1 55 (1999); Am. J. Physiol. 262, C 047-C1055 (1992); J. Biol. Chem. 270, 672-678 (1995); Methods Enzymol. 192, 38-81 ( 990) J. Physiol. 498, 295-307 (1997); Am. ' J. Physiol. 276. H1581-H1 90 (1999)].
- SBFI and GBFO are also two widely used sodium ion detectors, which are much more. selective ; han those mentioned before [J. Biol, Chem. 264, 19449 es 19458 (1989); Methods Enzymol. 192, 38 (1990); Am. J. Physiol. 262, F462 (1992); Biochem. Biophys. Res. Commun. 164;' 212 (1989); Am. J. Physiol. 259, C 9 (1990); J. Biol. Chem. 265, 10522 es 19543 (1990); Biochem. Biophys. Res. Commun. 175, 611 (1991); J. Physiol. 448, 493 (1992); J. Physiol. 517, 135 (1999)].
- the present invention relates to fluorescent dyes of formula (I)
- R 1 and R 2 each, independently, represent
- R 5 and R 6 each, independently, represent hydroxy, alkyl-CO-O-, amino, monoalkylamino or dialkylamino group
- R 7 and R 8 each, independently, represent hydrogen or halogen
- symbol ⁇ — TM means that the group of formula (e) may be coupled to the rest of the molecule at any of .the three positions marked by this symbol, and in formula (e) the ketal ring may optionally also be present in opened form, i e. as a car oxy group; or
- R 9 and R 10 each . , independently, represent amino, monoalkylamino or dialkylamino group
- R y is hydrogen or alky! or HOOC-alkyl group
- R 3 represents
- R J is hydrogen, alky! group, alkyl-O-CO- group, an optionally substituted 1 ,3,5-triazin-2-yl group, or a fluorophoric moiety identical to the fluorophoric moiety attached to the second nitrogen of the crown ether;
- hydrocarbyl means saturated and unsaturated alkyl, saturated and unsaturated cycloalkyl and phenyl groups.
- substituted means that the group concerned may bear one or more (preferably only one) small size substituent(s), such as halo, nitro, carboxy, cyano, amino, lower alkoxy and/or lower alkyl, wherein the term “lower” signifies a group containing 1 -7 carbon atoms, preferably 1-4 carbon atoms.
- alky means saturated and unsaturated alkyl groups which are optionally substituted as given just in the previous paragraph.
- the alkyl groups are groups of not more than 25 carbon atoms, and are preferably unsubstituted.
- the compounds of formuia (I) are also built up from a fluorophoric moiety and from an ionophoric moiety, where group R 3 is the ionophoric moiety and the remainder of the molecule is the fluorophoric moiety; or when R 3 is a group of formula (c) wherein R' is a second fluorophoric moiety, the ionophoric moiety is only the crown ether, to which two fluorophoric moieties are bound.
- Fluorescein derivatives usable for similar purposes are disclosed in Spectrochimica Acta Part A 51 (1995) L7-L21 ; these compounds do not comprise ionophoric moieties, too.
- Neuroimage 58 (201 1 ) 572-578 a fluorescein derivative is disclosed for indicating the presence of sodium ions wherein an ionophoric moiety, different from those appearing in the compounds according to the invention, is attached to the fluorophoric moiety.
- R damine compounds usable as fluorescent marker dyes are disclosed in J. Med. C'bem. 1998, 41 , 1671 -1678; J Biol. Chem. 1 39, 8171-8178 and Bull. Chern. Soc. Jpn. 1958, 31 , 974-980; in the ⁇ . compounds neither a triazine ring nor an ionophoric moiety appears.
- R* may represent preferably a group of formula (d) or (e).
- R 1 and/or R 2 stands for an optionally sub ⁇ stituted group, it is preferred that said group is unsubstituted.
- R 1 and/or R 2 represents a saturated or unsaturated alkyl group or a saturated or unsaturated cycloalkyl group as hydrocarbyl group, the number of carbon atoms contained in these groups is selected in accordance with the desired polarity, optionally also taking into account whether the molecule is to be bound to a carrier. When it is required to prepare a molecule which can be bound to a carrier, longer carbon chains may be preferred; it is, however, practical when the number of carbon atoms is not greater than 25.
- R 1 and/or R 2 is a carboxylate (ester) group, this may be preferably an alkyl carboxylate, where the alkyl chain may optionally be substituted.
- R 1 and/or R 2 is a carbox- amide group, this means a -CONH 2 group wherein one of the two hydrogens may optionally be replaced by a hydrocarbyl or heteroaryl group.
- both R 1 and R 2 represent hydrogen, or one of them represents sulphonic acid group (S0 3 " H + ), which is poreferably converted into a salt.
- R x represents a group of formula (e)
- R and R 6 are preferably hydroxy groups, whereas both R 7 and R 8 are preferably hydrogen.
- R* represents a group of formula (a) or (e).
- R is alkyl-C(-O)- or HOOC-alkyl
- the alkyl part may be saturated or unsaturated, and may optionall be substituted as defined?, above.
- the alkyl part is an unsubstituted group of not more than 25 carbon atoms.
- R 3 represents a group of formula (c), in this formula R' is preferably alky! or a second fluorophoric moiety.
- R' is a 1 ,3,5-triazin-2-yl group, preferably one or two halo substituents, particularly preferably two chloro substituents are attached to this group.
- R 4 is hydrogen, halogen or -NH-alkyl-NH 2 , and in this latter group the alkyl part may be saturated or unsaturated, and contains preferably not more than 4 carbon atoms.
- R 4 is halogen, it is particularly preferably chloro.
- R x is a group of formula (d)
- R y is hydrogen
- R 3 is a group of formula (a) or (b)
- R 4 is halo, particularly preferably chloro
- R x is a group of formula (d), R y is hydrogen, R 3 is a group of formula (c), and R 4 is -NH-alkyl- H 2 ;
- R is a group of formula (e), R y is hydrogen, R 3 is a group of formula (a) or (c), and R 4 is halo;
- R is a group of formula (f), R y is alkyi or HOOC-alkyl-, R 3 is a group of formula (c), and R 4 is halo.
- R* is a group of formula (d) wherein both R 1 and R 2 are hydrogen, R y is hydrogeji, .
- R ?' is a group oi formula (c) wherein R' is a second fiuorophoric moiety, and R 4 :s,, v -NH-a!kyl-NH 2 ;
- Salts of compounds of formula (I) which contain a salt-forming group e.g. sulphonic acid, phosphoric acid, carboxy or amino group
- compounds of formula (I) may be obtained directly in the form of such salts.
- Preferred representatives of the compounds of formula (I) are those of formulae (1 ) to (17),
- the compounds of formulae (1 ) to (8) can be used for,iine detection of sodium ions, whereas the compounds -of formulae (9) to (17) can also be used for the detection of potassium ions.
- the invention also relates to the preparation of the compounds of formula (I) and salts thereof.
- R x is a group of formula (d) or (e)
- L is a suitable leaving atom or group
- R y and R 4 are as defined above, with a compound of formula R 3 -H, wherein R 3 is as defined above.
- R 9 and R 10 are as defined above and L is. a suitable leaving atom or group, with a compound of formula (IV),
- the resulting product is converted into its sait.
- any of the reactants comprises a group, such as an amino group, which is sensitive to or can interfere with the reaction, prior to the reaction this group is blocked with a suitable protecting group, which is removed after the reaction.
- the compounds of formula R 3 -H are well known, generally commercially available substances. Methods for preparing compounds of formulae (II) and (III) are discussed in the following publications: WO 2007/012905; RU 2.247 17; Spectrochimica Acta Part A 51 (1995) L7-L21 ; Journal of Luminescence 121 (2006) 159-172; Chem. Commun. 2000, 1913- 1914; Org, Biomol. Chem.
- the compounds of formula (IV) can be prepared by reacting a compound oi3 ⁇ 4 formula (V), 4
- the compounds of formula (V) are discussed widely in the literature, and are generally commercially available.
- the invention also relates to compounds of formula (I) and their salts for detecting sodium ions and optionally potassium ions.
- compositions and kits for detecting sodium ions and optionally potassium ions which comprise, beside the conventional components of such compositions and kits, compounds of formula (I) or salts thereof as ion-selective fluorescent dyes.
- composition relates to a reagent formulation wherein a!l of the components have already been mixed with one another; whereas the term “kit” refers to a reagent formulation supplied in the form of discrete components or component mixtures to be combined with one another before analysis.
- DMSO dimethyl sulphoxide
- DMF dimethyl formamide
- the invention also relates to the use of the compounds of formula (I) and their salts in detecting sodium ions and optionally potassium ions. Detection is performed in a manner known per se (e.g. as disclosed in the cited references) with the difference that compounds of formula (I) or their salts are used as ion-sensitive fluorescent dyes.
- LCMS analysis The measurements were performed according to the prescriptions of the manufacturers on a Waters chromatograph/ZMD type mass spectrometer equipped with a Waters 996 DAD UV detector and with a Waters 2700 automated sampler, or on a Micromass VMD type mass spectrometer coupled to a Waters Alliance 2795 type liquid chromatograph. A Supelco RPAmideC16 type or Supelco Discovery C18 type column was used in gradient mode.
- Extinction maxima were determined as follows: Stock solutions with a concentration of about 1 mM were prepared from the dyes in water or in a water-miscible organic solvent. For spectroscopic measurements about 100fold to l OOOfofd dilutions were prepared from the stock solutions with water or with a buffer (0.05 M Tris MCI, pH 7.5; or 0.1 M phosphate, 0.01 M EDTA, 0.15 M NaCI, pH 7.2), ana the UWVIS absorption spectra of the solutions were recorded. Thereafter the
- Emission maxima were determined as follows; In the fluorescence emission . measurements a known volume of water or of a buffer was filled into the c uvette.pf the spectrophotometer, and the required volume of dye stock solution was added, taking care that in the solution to be measured the absorbance which belongs to the excitation wavelength should not exceed 0.1. Emission spectra were recorded then under excitation at the selected wavelength.
- Fluorophoric absorption maxima were determined as follows: Increasing amounts of a 5 M NaCI solution (5-300 ⁇ / 2 ml) were added gradually to the solution of the dye, the samples were shaken, and the emission spectra were recorded. The intensities belonging to the emission maxima were red off, the values were corrected by the dilution factors, and then listed in the function of NaCI concentration.
- the sodium ion sensitivity of the compounds was tested as follows:
- the compound to be tested was dissolved in water, and the concentration was adjusted so that the absorbance of the solution in the wavelength range to be tested was lower than 1. Thereafter varying amounts of aCI were added to this solution and the absorption spectra were recorded. The equilibrium constant was determined with nonlinear regression method by a global fitting of the absorbances measured at different wavelengths, assuming that a complex with a 1 : 1 stoichio- metry is formed. Testing ofNa + binding by fluorescence spectroscopy.
- the wavelength was defined where an isobestic point is obtained during the variation of the NaCI concentration.
- the concentration of the solution of the compound to be tested was adjusted so that the absorbance was around 0.1 at the wavelength of the exciting light.
- the fluorescence excitation spectrum of the solution was recorded (basic form; zero NaCI concentration). Thereafter the NaCI concentration of the solution was stepwise increased from zero. , and fluorescence spectra were recorded again 15 minutes after the addition of the respective NaC! batch.
- the fiuorescence spectra recorded this way for the compound of formula (1) are shown in Fig. 1.
- the wavelength or the : exciting light was 345 nm, and the NaCI concentration of the solution was raised3 ⁇ 4 ; gradually from zero to 0.13 moles in 9 consecutive steps. It appears clearly from the figure that the intensity of fluorescence at 540 nm increases in a well measurable manner in parallel with the increase of the NaCS concentration; thus, after taking a calibration curve, the compound of formula (1 ) is highly suitable for the detection of Na + concentration.
- the fluorescence excitation spectrum of the sample was always compared to the absorption spectrum of the sample in order to check whether the two spectra are in harmony with one another.
- the extinction maximum was 540, and the emission maximum was 625 nm.
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Abstract
The invention relates to fluorescent dyes of formula (I) which at a sensitive to sodium ions and optionally also to potassium ions, and to.' salts, of those compounds which are capable to salt formation - in this formula Rx is a group of formula (d), (e) or (f), Ry is hydrogen or alkyl or HOOC-alkyl group;. R3 is a group of formula (a), (b) or (c), R4 is (i) hydrogen or halogen, (ii) amino, or (iii) a nitrogen-containing mono- or bicydic aromatic or nonaromatic heterocyclic group which optionally contains further hetero at.om(s), or a -NH-hydrocarbyl group, any of them being optionally substituted. The invention also relates to the preparation and use of these compounds, as well as to compositions and kits comprising them.
Description
FLUORESCENT DYES SENSITIVE TO SODIUM IONS AND OPTIONALLY TO POTASSIUM IONS, PROCESS FOR THEIR PREPARATION AND THEIR USE
The present invention relates to new fluorescent dyes, to a method for the preparation of the new compounds, to analytical reagent compositions and kits comprising said new compounds, as well as to the use of the new compounds in the detection of sodium and optionally potassium ion concentration.
Compounds which can detect selectively and unambiguously the concentration of cenain ions in their surroundings have become rather important in the last few years. Primarily ion-sensitive fluorescent dyes are used for this purpose. With regard to their sit uctures, the overwhelming majority of fluorescent dyes consist of two main parts: one of them is a fluorophoric moiety which ensures fluorescency, and the other is an ionophoric moiety* which binds the ion or ions to be detected, generally as a complex. In most of the ion-sensitive fluorescent dyes this ionophoric moiety r. a crown ether, and the number of the members of this crown ether (i.e. the size of the crown ether) influences the quality of the complexable ion. When the ionophoric moiety binds the metal ion in complexed form, the ion-sensitive fluorescent dye undergoes a conformational change, which also results in a change of its fluorescent properties (most frequently in the intensity of fluorescence). With respect to the suitability of ion-sensitive fluorescent dyes for analytical purposes nowadays it is fundamental that the change in fluorescent property should not only be well measurable but should also be suitable for the unambiguous detection of the ion concerned, i.e. the dye should not only indicate the presence of the ion in question but it should, by the aid of appropriate calibration curves, also enable one to
measure the accurate concentration of the ion. Where in this description the term "detection" is mentioned, it is used always in this more strict sense; ion-selective fluorescent dyes which enable only or primarily qualitative indication are termed here as "indicators".
CoroNa<R) Red and CoroNa(R) Green are two fluorescent dyes which are nowadays widely used for the detection of sodium ions [see e.g. J. Biol. Chem. 264, 19449-19457 and 19458-19467 (1989); Cytometry 21 , 248-256 (1995); Physiol. Rev. 79, 1127-1 55 (1999); Am. J. Physiol. 262, C 047-C1055 (1992); J. Biol. Chem. 270, 672-678 (1995); Methods Enzymol. 192, 38-81 ( 990) J. Physiol. 498, 295-307 (1997); Am.' J. Physiol. 276. H1581-H1 90 (1999)]. SBFI and GBFO are also two widely used sodium ion detectors, which are much more. selective ;han those mentioned before [J. Biol, Chem. 264, 19449 es 19458 (1989); Methods Enzymol. 192, 38 (1990); Am. J. Physiol. 262, F462 (1992); Biochem. Biophys. Res. Commun. 164;' 212 (1989); Am. J. Physiol. 259, C 9 (1990); J. Biol. Chem. 265, 10522 es 19543 (1990); Biochem. Biophys. Res. Commun. 175, 611 (1991); J. Physiol. 448, 493 (1992); J. Physiol. 517, 135 (1999)].
However, different branches of technics, primarily clinical chemistry, diagnostics, food industry and environmental protection raise increasingly diverse demands, therefore ion-sensitive fluorescent dyes should be developed which fit better for these specific purposes. The present invention contributes to the satisfaction of this need.
The present invention relates to fluorescent dyes of formula (I)
which are sensitive to sodium ions and optionally also to potassium ions, and to salts of those compounds which are capable to salt formation - in this formula
Rx is
a group of formula (d)
X is -S-, -0- or -C(=0)0-, and
R1 and R2 each, independently, represent
(i) hydrogen or halogen,
(ii) nitro, hydroxy, carboxy or amino group,
(iii) su!phonic acid or phosphoric acid group, or
(iv) hydrocarby!, aikoxy, carboxylate, carboxamide, -NH-hydrocarbyl or a monocyclic aromatic or nonaromatic heterocyclic group, any of them being optionally substituted; or
a group of formula (e),
( e )
wherein R5 and R6 each, independently, represent hydroxy, alkyl-CO-O-, amino, monoalkylamino or dialkylamino group, R7 and R8 each, independently, represent hydrogen or halogen, and symbol ■—™| means that the group of formula (e) may be coupled to the rest of the molecule at any of .the three positions marked by this symbol, and in formula (e) the ketal ring may optionally also be present in opened form, i e. as a car oxy group; or
a group of formula (f),
wherein R9 and R10 each., independently, represent amino, monoalkylamino or dialkylamino group;
Ry is hydrogen or alky! or HOOC-alkyl group;
R3 represents
a group of formula (a),
( a )
wherein R is hydrogen or alkyl, alkyl-C(=0)- or HOOC-alkyI group; or
a group of formula (b);
( k )
or
a group of formula (c).
( c )
wherein RJ is hydrogen, alky! group, alkyl-O-CO- group, an optionally substituted 1 ,3,5-triazin-2-yl group, or a fluorophoric moiety identical to the fluorophoric moiety attached to the second nitrogen of the crown ether; and
R4 is
(i) hydrogen or halogen,
(ii) amino, or
(iii) a nitrogen-containing mono- or bicyclic aromatic or nonaromatic heterocyclic group which optionally contains further hetero atom(s), or a -NH-hydrocarbyl group, any of them being optionally substituted.
In the above definitions of substituents the term "hydrocarbyl" means saturated and unsaturated alkyl, saturated and unsaturated cycloalkyl and phenyl groups. The term "substituted" means that the group concerned may bear one or more (preferably only one) small size substituent(s), such as halo, nitro, carboxy, cyano, amino, lower alkoxy and/or lower alkyl, wherein the term "lower" signifies a group containing 1 -7 carbon atoms, preferably 1-4 carbon atoms.
If not indicatad otherwise, in the above definitions of substituents the term "alky!" means saturated and unsaturated alkyl groups which are optionally substituted as given just in the previous paragraph. The same relates to the alkyl groups which appear in group combinations [e.g. in alkyi -C(=0)-, alkoxy, HQOC alkyl-, alky!-O-CO- and alkyl-CO -O- groups]. Preferably the alkyl groups (or group arts) are groups of not more than 25 carbon atoms, and are preferably unsubstituted.
Like the known ion-sensitive fluorescent dyes, the compounds of formuia (I) are also built up from a fluorophoric moiety and from an ionophoric moiety, where group R3 is the ionophoric moiety and the remainder of the molecule is the fluorophoric moiety; or when R3 is a group of formula (c) wherein R' is a second fluorophoric moiety, the ionophoric moiety is only the crown ether, to which two fluorophoric moieties are bound.
Compounds structurally related to those of formula (I) have already been disclosed in the literature. Such compounds are the unsubstituted benzoxazole, benzo- thiazole and benzo[d][1 ,3]oxazin- -one compounds and some substituted derivat-
ives thereof disclosed in WO 2007/012905 and RU 2 247 117; these compounds do not comprise ionophoric moieties. These substances are invisible in daylight but fluorescate in UV light, and can be used e.g. as markers on banknotes in order to separate forgeries. Fluorescein derivatives usable for similar purposes are disclosed in Spectrochimica Acta Part A 51 (1995) L7-L21 ; these compounds do not comprise ionophoric moieties, too. In Neuroimage 58 (201 1 ) 572-578 a fluorescein derivative is disclosed for indicating the presence of sodium ions wherein an ionophoric moiety, different from those appearing in the compounds according to the invention, is attached to the fluorophoric moiety. R damine compounds usable as fluorescent marker dyes are disclosed in J. Med. C'bem. 1998, 41 , 1671 -1678; J Biol. Chem. 1 39, 8171-8178 and Bull. Chern. Soc. Jpn. 1958, 31 , 974-980; in the^. compounds neither a triazine ring nor an ionophoric moiety appears.
Although all of the compounds listed above are able to fluoresce, it cannot be* predicted whether they will be suitable for the quantitative determination of ions when coupled with an ionophoric moiety. It cannot be concluded, either, that when an ionophoric moiety present in a compound capable of indicating the presence of ions is replaced by a different ionophoric moiety, what will be the ion sensitivity of the resulting product. Just to illustrate this fact: when the structure of the compound of formula (1 ) which, as discussed later in more detail, is an excellent detector, is changed to obtain a compound of formula (E), which differs from the former one in the structure of the ionophoric moiety, the sodium ion sensitivity of the resulting compound falls to the 1/14th (Kd = 560 mM vs. the original value of 40 mM).
Thus it could not be predicted that when a narrow range of specifically selected ionophoric moieties «?s defined at R3 are attached to the fluorophoric moieties present in the compounds according to the invention,- ion-selective fluorescent dyes are obtained which are suitable for detection purposes.
. 'n the compounds of formula (I) R* may represent preferably a group of formula (d) or (e).
Where in the group of formula (d) R1 and/or R2 stands for an optionally sub¬ stituted group, it is preferred that said group is unsubstituted. Where in the group of formula (d) R1 and/or R2 represents a saturated or unsaturated alkyl group or a saturated or unsaturated cycloalkyl group as hydrocarbyl group, the number of carbon atoms contained in these groups is selected in accordance with the desired polarity, optionally also taking into account whether the molecule is to be bound to a carrier. When it is required to prepare a molecule which can be bound to a carrier, longer carbon chains may be preferred; it is, however, practical when the number of carbon atoms is not greater than 25. The more polar molecule is to be prepared, the lower should be the number of carbon atoms contained in these groups. When R1
and/or R2 is a carboxylate (ester) group, this may be preferably an alkyl carboxylate, where the alkyl chain may optionally be substituted. When R1 and/or R2 is a carbox- amide group, this means a -CONH2 group wherein one of the two hydrogens may optionally be replaced by a hydrocarbyl or heteroaryl group. Particularly preferably, both R1 and R2 represent hydrogen, or one of them represents sulphonic acid group (S03 "H+), which is poreferably converted into a salt.
When Rx represents a group of formula (e), in this group both R and R6 are preferably hydroxy groups, whereas both R7 and R8 are preferably hydrogen. The group of formula (e) is attached to the rest of the molecule preferably at the meta position related to the -C(=0)- moiety.
Particularly preferably, R* represents a group of formula (a) or (e).
When in the group of formula (a) R is alkyl-C(-O)- or HOOC-alkyl, the alkyl part may be saturated or unsaturated, and may optionall be substituted as defined?, above. Preferably the alkyl part is an unsubstituted group of not more than 25 carbon atoms.
When R3 represents a group of formula (c), in this formula R' is preferably alky! or a second fluorophoric moiety. When R' is a 1 ,3,5-triazin-2-yl group, preferably one or two halo substituents, particularly preferably two chloro substituents are attached to this group.
Particularly preferably, R4 is hydrogen, halogen or -NH-alkyl-NH2, and in this latter group the alkyl part may be saturated or unsaturated, and contains preferably not more than 4 carbon atoms. When R4 is halogen, it is particularly preferably chloro.
The following group combinations proved to be preferred:
- Rx is a group of formula (d), Ry is hydrogen, R3 is a group of formula (a) or (b), and R4 is halo, particularly preferably chloro;
- Rx is a group of formula (d), Ry is hydrogen, R3 is a group of formula (c), and R4 is -NH-alkyl- H2;
- R is a group of formula (e), Ry is hydrogen, R3 is a group of formula (a) or (c), and R4 is halo;
- R is a group of formula (f), Ry is alkyi or HOOC-alkyl-, R3 is a group of formula (c), and R4 is halo.
The following group combinations proved to be particularly preferred:
- Rx is a group of formula (d) wherein both R1 and R2 are hydrogen or one of the?ry su!phonic acid group, Ry Is hydrogen, 3 is a group of formula (a) wherein R is . hydrogen or alkyl-C(=0)- or HOOC-alkyl-, and R4 is chloro;
- R* is a group of formula (d) wherein both R1 and R2 are hydrogen, Ry is hydrogeji, . R?' is a group oi formula (c) wherein R' is a second fiuorophoric moiety, and R4 :s,,v -NH-a!kyl-NH2;
- Rx is a group of formula (e) attached to the rest of the molecule at the meta position related to the -C(=O)- moiety, wherein both R5 and R6 are hydroxy and both R7 and R8 are hydrogen, Ry is hydrogen, R3 is a group of formula (a) wherein R is alkyi or HOOC-alkyl-, and R4 is halo;
- Rx is a group of formula (e) attached to the rest of the molecule at the meta position related to the -C(=O)- moiety, wherein both R5 and R6 are hydroxy and both R7 and R8 are hydrogen, Ry is hydrogen, R3 is a group of formula (c) wherein R' is alkyi, and R4 is halo.
Salts of compounds of formula (I) which contain a salt-forming group (e.g. sulphonic acid, phosphoric acid, carboxy or amino group) also belong to the compounds of the present invention. Depending on the reaction media and additives (e.g. pH-adjusting agents) used in their preparation, compounds of formula (I) may be obtained directly in the form of such salts.
Preferred representatives of the compounds of formula (I) are those of formulae (1 ) to (17),
( 1 ) ( 2 )
( 3 ) ( 4 )
( 16 )
of which the compounds of formulae (1 ), (5), (6), (7), (9), (10) and ( ) are particularly preferred. The compound?, of formulae (1 ), (10) arid (11 ) are
outstandingly preferred. The compounds of formulae (1 ) to (8) can be used for,iine detection of sodium ions, whereas the compounds -of formulae (9) to (17) can also be used for the detection of potassium ions.
The invention also relates to the preparation of the compounds of formula (I) and salts thereof.
Those compounds of formula (I) wherein Rx is a group of formula (d) or (e) are prepared by reacting a compound of formula (II),
( » )
wherein Rx is a group of formula (d) or (e), L is a suitable leaving atom or group and Ry and R4 are as defined above, with a compound of formula R3-H, wherein R3 is as defined above.
Those compounds of formula (I) wherein Rx is a group of formula (f) are prepared by reacting a compound of formula (III),
wherein R9 and R10 are as defined above and L is. a suitable leaving atom or group, with a compound of formula (IV),
( IV ) wherein Ry, R3 and R4 are as defined above.
If desired and possible, the resulting product is converted into its sait. When any of the reactants comprises a group, such as an amino group, which is sensitive to or can interfere with the reaction, prior to the reaction this group is blocked with a suitable protecting group, which is removed after the reaction.
Of the starting substances and reactants listed above the compounds of formula R3-H are well known, generally commercially available substances. Methods for preparing compounds of formulae (II) and (III) are discussed in the following publications: WO 2007/012905; RU 2.247 17; Spectrochimica Acta Part A 51 (1995) L7-L21 ; Journal of Luminescence 121 (2006) 159-172; Chem. Commun. 2000, 1913- 1914; Org, Biomol. Chem. 20 1 , 9, 4108-41 5; J. Med. Chem. 1998, 41 , 1671-1678; J. Chem. Soc. 1923, 122, 1984-1996; J. Chem. Soc. 1922, 545-552; Bull. Chem. Soc. Jpn. 1958, 31 , 974-980; J. Org. Chem. 2005, 70, 9051 -9053. Substituted derivatives which are not mentioned in the. cited references can be prepared in analogy to the methods discussed therein' from appropriately substituted starting materials. The substitusnts can also be formed on the basic skeleton by appropris known subsequent conversions (aromatic substitution reactions, interchange of substituents, etc.).
The compounds of formula (IV) can be prepared by reacting a compound oi¾ formula (V), 4
( V ) wherein R4 and Ry are as defined above and L is a suitable leaving atom or group, with a compound of formula R3-H, wherein R3 is as defined above.
The compounds of formula (V) are discussed widely in the literature, and are generally commercially available.
The invention also relates to compounds of formula (I) and their salts for detecting sodium ions and optionally potassium ions.
The invention also relates to reagent compositions and kits for detecting sodium ions and optionally potassium ions, which comprise, beside the conventional components of such compositions and kits, compounds of formula (I) or salts thereof as ion-selective fluorescent dyes. The term "composition" relates to a reagent formulation wherein a!l of the components have already been mixed with one another; whereas the term "kit" refers to a reagent formulation supplied in the form of discrete components or component mixtures to be combined with one another before analysis. Of the conventional' components of reagent compositions and kits water, dimethyl sulphoxide (DMSO), ethanol, dimethyl formamide (DMF) etc. are mentioned as examples.
The invention also relates to the use of the compounds of formula (I) and their salts in detecting sodium ions and optionally potassium ions. Detection is performed in a manner known per se (e.g. as disclosed in the cited references) with the difference that compounds of formula (I) or their salts are used as ion-sensitive fluorescent dyes.
The following Examples are given to elucidate the invention in more detail. EXAMPLES
The following analytical methods were used to identify the compounds:
LCMS analysis: The measurements were performed according to the prescriptions of the manufacturers on a Waters chromatograph/ZMD type mass spectrometer equipped with a Waters 996 DAD UV detector and with a Waters 2700 automated sampler, or on a Micromass VMD type mass spectrometer coupled to a
Waters Alliance 2795 type liquid chromatograph. A Supelco RPAmideC16 type or Supelco Discovery C18 type column was used in gradient mode.
1H NMR analysis: The spectra were recorded with a Bruker AC-300 type apparatus at 300 MHz and 25°C; DMSO-d6 was used as solvent.
Extinction maxima were determined as follows: Stock solutions with a concentration of about 1 mM were prepared from the dyes in water or in a water-miscible organic solvent. For spectroscopic measurements about 100fold to l OOOfofd dilutions were prepared from the stock solutions with water or with a buffer (0.05 M Tris MCI, pH 7.5; or 0.1 M phosphate, 0.01 M EDTA, 0.15 M NaCI, pH 7.2), ana the UWVIS absorption spectra of the solutions were recorded. Thereafter the
wavelengths' belonging to the absorption maxima were. recorded; .these waveiengi|Q¾ were used then as excitation wavelengths in the fluorescence measurements.
■ Emission maxima were determined as follows; In the fluorescence emission . measurements a known volume of water or of a buffer was filled into the c uvette.pf the spectrophotometer, and the required volume of dye stock solution was added, taking care that in the solution to be measured the absorbance which belongs to the excitation wavelength should not exceed 0.1. Emission spectra were recorded then under excitation at the selected wavelength.
Fluorophoric absorption maxima were determined as follows: Increasing amounts of a 5 M NaCI solution (5-300 μΙ/ 2 ml) were added gradually to the solution of the dye, the samples were shaken, and the emission spectra were recorded. The intensities belonging to the emission maxima were red off, the values were corrected by the dilution factors, and then listed in the function of NaCI concentration.
Example 1
Preparation and testing of compounds of formula (I) wherein Rx is a group of formula (d)
540 mg (1.10 mmoles) of 3-benzotriazoi-2-yl-4-(4,6-dichloro-[1 ,3,5]tnazin-2- yl-amino)-benzenesulphonic acid potassium salt were dissolved in 50 ml of water, and the pH of the solution was adjusted to 7 with solid potassium carbonate. Thereafter 311 mg ( 1.10. mmoles) of 6,7, 9, 10, 12, 13, 15, 16-octahydro-5, 8, 11 ,14,17-penta- oxa-benzocyclopentadecen-2-yl-amine were added to the solution, and the reaction mixture was stirred at room temperature for 3 hours. The solution was then evaporated, and the residue was crystallized from methanol 510 mg of 3-benzothi;, az0i«2"yl-4-»|4-chloro-6-(6,7,9,-10, 12, 3,15, 6-octahydro~5,8, 1 ,14, 17 -pentaoxa-. ^ benzocyclopentadecen-2-yl-amino)-[1 ,3,5jtriazin-2-y!-amino]-benzenesulphonic acid potassium salt were obtained as a white powder.
The compounds of formulae (2) - (8) were prepared in analogy to the above, with the difference that 0.55 mmoles of the crown ether reagent were used in the preparation of the compound of formula (2). The physical constants of the obtained compounds are listed in Table 1.
Table 1
Mol. LC tR MS
weight min. [M+H]+ H NMR spectrum, δ
g/mol or ΓΜ-Η1
(1 ) 739.28 4.44 739 8.23 (s, 1 H), 8.12 (s, 1 H), 7.55 (d, 3H),
6.80 (d, 1 H), 6.41 (d, 2H), 5.86 (s, 1 H) 3.79-4.11 (m, 10H), 3.54 (s, 8H)
(2) 941.16 3.08 941 8.20 (d, 2H), 8.12 (d, 4H), 7.58 (d, 4H),
7.29 (d, 2H), 6.58-6.87 (m, 6H), 4.0 (s, 4H), 2.88-3.62 (m, 28H), 2.3 (s, 4H)
For- Mol. LC tR MS
mu!a weight min. IM+H]+ H ' NMR spectrum, δ
g/mol ΟΓ ΓΜ-ΗΓ
(3) 675:23 4.49 676 7.55-8.13 (m, 6H), 6.80 (d, 1 H), 4.12 (s,
1 H), 3.24-3.60 (m, 20H)
(4) 621.12 3.44 621 8.13 (s, 1 H), 8.1 1 (s, 1 H); 7.65 (d, 3H),
5.85 (d, 1 H), 6.51 (d, 2H), 5.80 (s, 1 H), 3.59-4.01 (m, 10H), 3.55 (s, 8H)
(5) 723.21 2.70 724 8.22 (s, 1 H), 8.12 (s, 1 H), 7.55 (d, 3H),
6.77 (d, 1 H), 6.40 (d, 2H), 5.60 (s, 1 H),
3.54- 3.90 (m, 0H), 3.45 (s, 8H)
(6) 977.69 4.51 978 6.64-8.26 (m, 10H), 3.79 4.01 (m, 10H),
3.66 (s, 8H), 0.96-2.25 (m, 30H)
(7) 891 .51 3.95 89 7.55- 8.20 (m, 6H), 6,85 (d, 1 H), 6.40 (d.
1 H), 5,86 (d, 2H), 3.79-4.16 (m, 9H), 3.79 (m, 8H), 1.29-3.06 (m, 10H)
(8) 633.07 3.53 633 8.1 C id, 1 H), 7.37-7,55 (m, 4H), 6.41 - - 7.01 (m, 4H), 5.92 (d, 2H), 3.79-4.1 1 (m, 10H), 3.58 (m, 8H)
The sodium ion sensitivity of the compounds was tested as follows:
Testing of Na* binding by absorption spectroscopy.
The compound to be tested was dissolved in water, and the concentration was adjusted so that the absorbance of the solution in the wavelength range to be tested was lower than 1. Thereafter varying amounts of aCI were added to this solution and the absorption spectra were recorded. The equilibrium constant was determined with nonlinear regression method by a global fitting of the absorbances measured at different wavelengths, assuming that a complex with a 1 : 1 stoichio- metry is formed.
Testing ofNa+ binding by fluorescence spectroscopy.
Based on the results of the absorption spectroscopic method described above the wavelength was defined where an isobestic point is obtained during the variation of the NaCI concentration. When recording the fluorescence spectra the sample was excited with a light of a wavelength corresponding to the isobestic point. The concentration of the solution of the compound to be tested was adjusted so that the absorbance was around 0.1 at the wavelength of the exciting light. The fluorescence excitation spectrum of the solution was recorded (basic form; zero NaCI concentration). Thereafter the NaCI concentration of the solution was stepwise increased from zero., and fluorescence spectra were recorded again 15 minutes after the addition of the respective NaC! batch. The fiuorescence spectra recorded this way for the compound of formula (1) are shown in Fig. 1. In this instance the wavelength or the : exciting light was 345 nm, and the NaCI concentration of the solution was raised¾ ; gradually from zero to 0.13 moles in 9 consecutive steps. It appears clearly from the figure that the intensity of fluorescence at 540 nm increases in a well measurable manner in parallel with the increase of the NaCS concentration; thus, after taking a calibration curve, the compound of formula (1 ) is highly suitable for the detection of Na+ concentration.
The fluorescence excitation spectrum of the sample was always compared to the absorption spectrum of the sample in order to check whether the two spectra are in harmony with one another.
The equilibrium constant of Na+ binding was determined with nonlinear regression method by a computerized global fitting of the dependencies of fiuorescence intensities from NaCI concentration measured at different wavelengths. The
obtained results are listed in Table 2, where Kd is the reciprocal value of the equi librium constant.
Table 2
Compound (formula) Sodium ion sensitivity (Kd; mM)
(1 ) 40
(2) 100
(3) 150
(4) 80
(5) 50
(6) 60
40
(?>
(8) 150
From the data of Table 2 it appears clearly that those dyes [formulae ( ), (5), (6) and (7)] have t e smallest Kd values, i.e. are the most sensitive to sodium ions, wherein R3 is a group of formula (a) and at the same time R4 is chloro. The data also reflect that the removal of the su!phonic acid attached to the benzothiazole ring did not influence the sodium ion sensitivity of the dye [formula (5)].
Example 2
Preparation and jesting of compounds of formula (I) wherein Rx is a group of formula (e)
60 mg (0.12 mmoles) of 5-[(4,6-dichloro-triazin-2-yl)-amino]-fluorescein were dissolved in 2 ml of methanol, and 50 mg of potassium carbonate and 61.5 mg (0.12 mmoles) of hexadecyl-(6,7,9, 10, 2:23, 15, 16-octahydro-5,8, 1 1 , 14, 7-pentaoxa- benzocyclopentadecen-2-yl)-amine were added. The reaction mixture was stirred at
11 000109
23
room temperature for 3 hours, thereafter the carbonate was filtered off, and the mother liquor was evaporated to dryness in vacuo. 34 mg of 5-[(4-chloro-triazin-2- yl)-amino]-f luorescein~hexadecyl-(6,7,9, 10,12,13,15,16-octahydro-5,8, 11 ,14,17- pentaoxa-benzocyclopentadecen--2-yl)-amine of formula (9) were obtained. LCMS: tR = 2.95 min., 965 (M+H)+.
Similarly were prepared the following compounds: formula (10); LCMS: tR = 3.34 min., formula (1 1 ): LCMS: tR = 3.25 min.
For all of these three compounds the fluorophoric absorption maximum was 496 t m.
Thereafter the fluorescence spectra of the compounds were recorded as described in Example 1 in an ion-free medium and in media containing varying , amounts of Na+ or ions. The relative increases in intensity were calculated by dividing the intensities (F) measured in the presence of ions with the intensity (F0). measured in ion-free medium, and the results were plotted against the ion concentration. The curve of increase obtained for the compound of formula (9) is shown in Fig. 2; that obtained for the compound of formula (10) is shown in Fig. 3
In this measurement the fluorescence intensity of the compound of formula ( 1 ) increased from 1 to 3.2.8 upon adding 833 mM of NaCI, and increased from 1 to 4.95 upon adding 800 mM of KCI.
Example 3
Preparation and testing of compounds of formula (I) wherein Rx is a group of formula (f)
50 mg (0.09 mmoles) of [9-(4-chlorosulphonyl)-2-sulpho-phenyl]-6-diethyl- amino-xanthen-3-ylidene]-diethylammonium chloride were dissolved in 2 ml of pyridine, 28.5 g of [4-chloro-6-( 1 ,4,1 O-trioxa-7,13-diaza-cyclopentadec-7 -yl)- [1,3,5]iriazin-2-y!]-hexadecyl-amine were added, and the mixture was stirred at room temperature for 3 hours. Thereafter the solution was evaporated, the residue was washed with 1 N aqueous hydrochloric acid and dried. 43 mg of [9-(4-{[4-chloro-6- (1 ,4,10-trioxa-7,13~diaza"Cyclopentadec-7-ol)-[1 ,3,5]iriazin-2-yl]-hexadecyl- sulphamoyl} -2-sulpho-phenyl)-6-diethylamino-xanthen-3-ylidene]-diethylammonium chloride of formula (14) were obtained. LCMS: tR = 2.95 min., 1111 (M+H)*.
Similarly were prepared the following compounds from the appropriate starting subsiances:
formula (12); LCMS: tR = 3.10 min-. ,
formula (13); LCMS: tR = 2.54 min.,
formula (15); LCMS: tR = 4.32 min.,
formula (16); LCMS: tR =? 3.33 min.,
formula (17); LCMS: tR = 2.84 min.
For all of the above compounds the extinction maximum was 540, and the emission maximum was 625 nm.
Claims
1. Fluorescent dyes of formula (I)
which are sensitive to sodium ions and optionally also to potassium ions, and salts of those compounds which are capable to salt formation - in this formula
Rx is
a group of formula (d),
X is -S-, -0- or -C(=0)0-, and
R1 and R2 each, independently, represent
(i) hydrogen or halogen,
(ii) nitro, hydroxy, carboxy or amino group,
(iii) sulphonic acid or phosphoric acid group, or
(iv) hydrocarbyl, alkoxy, carboxylate, carboxamide, -NH-hydrocarbyl or a monocyclic aromatic or nonaromatic heterocyclic group, any of them being optionally substituted; or
a group of formula (e),
( e )
wherein R5 and R6 each, independently, represent hydroxy, alkyl-CO-O-, amino, monoalkylamino or dialkylamino group, R7 and R8 each, independently, represent hydrogen or halogen and symbol j . means that the group of formula (e) may be coup!ed to the rest of the molecule at any of the three positions marked by this symbol,' nd in formula (a) the ketal ring mey optionally also be present in opened form, i.e. as a carboxy group; or
a group of formula (f), .
( 0
wherein R and R each, independently, represent amino, monoalkylamino or dialkylamino group;
Ry is hydrogen or alkyl or HOOC-alkyl group;
R3 represents
a group of formula (a),
( a )
wherein R is hydrogen or alkyl, alkyl-C(=0)- or HOOC-alky! group; or
a group of formula (b);
( b )
or
a group cf formula (c).
( c )
wherein R' is hydrogen, alky! group, alkyl-O-CO- group, an optionally substituted 1 ,3,5-triazin-2-yl group, or a fluorophoric moiety identical to the fluorophoric moiety attached to the second nitrogen of the crown ether; and
R4 is
(i) hydrogen or halogen,
(ii) amino, or
(iii) a nitrogen-containing mono- or bicyclic aromatic or nonaromatic heterocyclic group which optionally contains further hetero atom(s), or a -NH-hydrocarbyl group, any of them being optionally substituted.
2. A compound as claimed in claim 1 or a salt thereof, wherein Rx is a group of formula (d) and Ry is hydrogen.
3. A compound as claimed in claim 2, wherein Rx is a group of formula (d), and in this formula both R1 and R2 are hydrogen, or one of them is hydrogen and the other is suiphonic acid group preferably in its salt form.
4. A compound as claimed in claim 2 or 3; wherein R3 is a group of formula (a), and in this formula R is as defined in claim 1.
•5. A compound as claimed in clsim 4, wherein R3 is a group of formula (a),, and in this formula R is hydrogen, alkyl-C(=0)- group or HOOC-aikyl- group.
6. A compound as claimed any of claims 2 to 5, wherein R4 is halo, preferably chloro.
7. A compound as claimed in claim 2 or 3, wherein R3 is a group of formula (c), and in this formula R' is a second fluorophor ic moiety.
8. A compound as claimed in any of claims 2, 3 and 7, wherein R4 is a
-NH-alkyl-NH2 group, and in this group the alkyl part is saturated or unsaturated and comprises preferably up to 4 carbon atoms.
9. A compound as claimed in claim 2, which is a compound of formula (1 ), (2), (3), (4), (5), (6), (7) or (8)
10. A compound as claimed in claim 9, which is a compound of formula (1 ), (5), (6) or .7).
11. A compound as claimed in claim 0, which is a compound of formulaf£|).
12. A compound as claimed in claim 1 or a salt thereof, wherein R is a group of formu'a (e) attached to the rest of the molecule preferably in the meta position related to the -C(=0)- moiety, and Ry is hydrogen.
13. A compound as claimed in claim 12, wherein in the group of formula (e) both R5 and R6 are hydroxy.
14. A compound as claimed in claim 12 or 13, wherein in the group of formula (e) both R7 and R8 are hydrogen.
5. A compound as claimed in any of claims 12 to 14, wherein R3 is a group of formula (a) or (c).
16. A compound as claimed, in any of claims 12 to 15, wherein R3 is a group of formula (a), and in this formula R is alkyl group or HOOC-alkyl- group.
17. A compound as claimed in any of claims 12 to 15, wherein R3 is a group of formula (c), and in this formula R' is alkyl.
18. A compound as claimed in any of claims 12 to 17, wherein R4 is halo, preferably chloro.
9. A compound as claimed in claim 12, which is a compound of formula (9), (10) or (11 )
( 11 )
20. A compound as claimed in claim 1 or a salt thereof, wherein R* is a group of formula (f) and Ry is alkyl group or HOOC-alkyl- group.
21. A compound as claimed in claim 20, wherein R3 is a group of formula (c).
22. A compound as claimed in claim 20 or 21 , wherein R3 is a group of formula (c), and in this formula R' is hydrogen, alkyl group, alkyl-O-CO- group or dihalo-substituted 1 ,3,5-triazin-2-yl group.
32
23. A compound as claimed in any of claims 20 to 22, wherein R4 is halo, preferably chloro.
24. A compound as claimed in claim 20, which is a compound of formula (12), (13), (14), (15), (16) or (17)
( 15 )
33
16 ) ( 17 )
25. A process for the preparation of a compound as claimed in claim 1 or a salt thereof, characterised in that
(i) to prepare a compound of formula (I) wherein R* is a group of formula (d) or (e), a compound of formula (II),
( ll )
wherein Rx is a group of formula (d) or (e), L is a suitable leaving atom or group, and Ry and R4 are as defined in claim 1 , is reacted with a compound of formula R3-H, wherein R3 is as defined in claim 1 ; or
(ii) to prepare a compound of formula (I) wherein Rx is a group of formula (f), a compound of formula (III),
( til )
wherein R9 and R10 are as defined in claim 1 , and L is a suitable leaving atom or group, is reacted with a compound of formula (IV),
( IV ) wherein Rx, Ra and R4 are as defined in claim 1 ,
and in these reactions one or both of the reactants are optionally used in protected form, and the optionally present protecting group is removed from the product,
and/or, if desired and possible, a resulting compound is converted into its sa!t.
26. A compound as claimed in claim 1 for use in detecting sodium ions and optionally potassium ions.
27. A reagent composition or kit for detecting sodium ions and optionally potassium ions, which comprises/beside the conventional components of such com-
positions and kits, a compound as claimed in claim 1 as ion-sensitive fluorescent dye.
28. Use of a compound as claimed in claim 1 for detecting sodium ions and optionally potassium ions.
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|---|---|---|---|
| HU1000630A HU1000630D0 (en) | 2010-11-23 | 2010-11-23 | Sodiumion sensitive fluorescent dyes, method for their preparation and their use |
| HUP1000630 | 2010-11-23 |
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| Publication Number | Publication Date |
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| WO2012069859A1 true WO2012069859A1 (en) | 2012-05-31 |
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| EP2752464A4 (en) * | 2011-08-30 | 2015-06-17 | Fujifilm Corp | Novel compound having xanthene derivative multimeric structure, colored composition, ink for inkjet recording, inkjet recording method, color filter, and color toner |
| JP2015193800A (en) * | 2014-03-27 | 2015-11-05 | 富士フイルム株式会社 | Compound, coloring composition, ink for inkjet recording, inkjet recording method, inkjet printer cartridge, and inkjet recorded matter |
| WO2016042171A3 (en) * | 2015-07-16 | 2016-05-19 | Iridos Ltd | A xanthene compound, and a colorant comprising the same |
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| RU2247117C1 (en) | 2003-06-17 | 2005-02-27 | Общество с ограниченной ответственностью Фирма "ОЛБО" | Yellow-luminescence colorless organic luminophor and a method for preparation thereof |
| WO2007012905A1 (en) | 2005-07-28 | 2007-02-01 | Állami Nyomda Nyrt | Colourless water-soluble organic luminophores and intermediates thereof |
| DE102007053664A1 (en) * | 2007-11-08 | 2009-05-14 | Friedrich-Schiller-Universität Jena | Optical sensor for the detection of ions, gases and biomolecules, comprises a matrix with indicator dyes consisting of thin layer of ionic fluids, which is absorbed with the indicator dyes in inert or reactive thin carrier material |
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| EP2752464A4 (en) * | 2011-08-30 | 2015-06-17 | Fujifilm Corp | Novel compound having xanthene derivative multimeric structure, colored composition, ink for inkjet recording, inkjet recording method, color filter, and color toner |
| JP2015193800A (en) * | 2014-03-27 | 2015-11-05 | 富士フイルム株式会社 | Compound, coloring composition, ink for inkjet recording, inkjet recording method, inkjet printer cartridge, and inkjet recorded matter |
| WO2016042171A3 (en) * | 2015-07-16 | 2016-05-19 | Iridos Ltd | A xanthene compound, and a colorant comprising the same |
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| HU1000630D0 (en) | 2011-02-28 |
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