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

R ^x is

a group of formula (d)

wherein

X is -S-, -0- or -C(=0)0-, and

R ¹ and R ² 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 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, 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 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

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 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; and

R ⁴ 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 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.

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 R ³ 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) R ¹ and/or R ² stands for an optionally sub ^¬ stituted group, it is preferred that said group is unsubstituted. Where in the group of formula (d) 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. The more polar molecule is to be prepared, the lower should be the number of carbon atoms contained in these groups. When 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. When 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. Particularly preferably, 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.

When R ^x represents a group of formula (e), in this group both R and R ⁶ are preferably hydroxy groups, whereas both R ⁷ and R ⁸ 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 R ³ 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, 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. When R ⁴ is halogen, it is particularly preferably chloro.

The following group combinations proved to be preferred: - R ^x is a group of formula (d), R ^y is hydrogen, R ³ is a group of formula (a) or (b), and 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.

The following group combinations proved to be particularly preferred:

- R ^x is a group of formula (d) wherein both R ¹ and R ² are hydrogen or one of the?ry su!phonic acid group, R ^y Is hydrogen, ³ is a group of formula (a) wherein R is . hydrogen or alkyl-C(=0)- or HOOC-alkyl-, and R ⁴ is chloro;

- 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 ;

- R ^x 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 R ⁵ and R ⁶ are hydroxy and both R ⁷ and R ⁸ are hydrogen, R ^y is hydrogen, R ³ is a group of formula (a) wherein R is alkyi or HOOC-alkyl-, and R ⁴ is halo;

- R ^x 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 R ⁵ and R ⁶ are hydroxy and both R ⁷ and R ⁸ are hydrogen, R ^y is hydrogen, R ³ is a group of formula (c) wherein R' is alkyi, and R ⁴ 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 R ^x is a group of formula (d) or (e) are prepared by reacting a compound of formula (II),

( » ) wherein R ^x is a group of formula (d) or (e), L is a suitable leaving atom or group and R ^y and R ⁴ are as defined above, with a compound of formula R ³ -H, wherein R ³ is as defined above.

Those compounds of formula (I) wherein R ^x is a group of formula (f) are prepared by reacting a compound of formula (III),

wherein R ⁹ and R ¹⁰ are as defined above and L is. a suitable leaving atom or group, with a compound of formula (IV),

( IV ) wherein R ^y , R ³ and R ⁴ 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 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. 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 R ⁴ and R ^y are as defined above and L is a suitable leaving atom or group, with a compound of formula R ³ -H, wherein R ³ 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.

¹ H NMR analysis: The spectra were recorded with a Bruker AC-300 type apparatus at 300 MHz and 25°C; DMSO-d ₆ 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 R ^x 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 t _R 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 t _R 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 K _d values, i.e. are the most sensitive to sodium ions, wherein R ³ is a group of formula (a) and at the same time R ⁴ 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 R ^x 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: t _R = 2.95 min., 965 (M+H) ⁺ .

Similarly were prepared the following compounds: formula (10); LCMS: t _R = 3.34 min., formula (1 1 ): LCMS: t _R = 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 (F ₀ ). 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 R ^x 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]-diethyla mmonium chloride of formula (14) were obtained. LCMS: t _R = 2.95 min., 1111 (M+H) ^* .

Similarly were prepared the following compounds from the appropriate starting subsiances:

formula (12); LCMS: t _R = 3.10 min-. ,

formula (13); LCMS: t _R = 2.54 min.,

formula (15); LCMS: t _R = 4.32 min.,

formula (16); LCMS: t _R =? 3.33 min.,

formula (17); LCMS: t _R = 2.84 min.

For all of the above compounds the extinction maximum was 540, and the emission maximum was 625 nm.