The present invention relates improved methods which allow the change of the Ca ²⁺ ion concentration of cells and tissues even by non-linear laser scanning microscopy, which may thus be suitable for the histological detection of viral infections, such as SARS-COV-2 viral infection. For the detection, it is essential to develop suitable dyes for the detection of Ca ²⁺ ions, which also give visible and evaluable signals to the scanning laser microscope. Thus, the present invention relates to a new fluorescent dye of the general formula

(I) which shows the amount of calcium present in both the extra-cellular and intracellular space because, in contrast to the prior art dyes, it is capable to enter the cell through the cell wall in an active state, so the intracellular and extracellular Ca ion activity can be measured parallelly.

Background of the invention

Calcium ions (Ca ²⁺ ) contribute to the physiology and biochemistry of cells. They play an important role in signaling pathways, as described by Brini et al. (Interrelations between Essential Metal Ions and Human Diseases. Metal Ions in Life Sciences. IB. Springer pp. 81- 137.), Brini at al. ("Chapter 5 Intracellular Calcium Homeostasis and Signaling". In Band, Lucia (ed.). Metallomics and the Cell. Metal Ions in Life Sciences. 12. Springer pp. 119-68.), M. J. Berridge, es mtsai., (Nat. Rev. Mol. Cell Biol. 2000, 1, 11-21.) where, as a secondary messenger, they play a role in neurotransmitter release of neurons (e.g., in the visual, auditory, and olfactory centers), contraction of all muscle cell types, and fertilization. Many enzymes require calcium ions as cofactors, including many coagulation factors such as troponin-C or calmodulin (Dougherty et al. Yew, AC (2005)) "Computational model of the cAMP-mediated sensory response and calcium-dependent adaptation in vertebrate olfactory receptor neurons". Proceedings of the National Academy of Sciences. 102 (30): 10415-20).. Proceedings of the National Academy of Sciences. 102 (30): 10415-20). Extracellular calcium is also important in regulating the potential difference between excitable cell membranes (during membrane depolarization caused by a Ca ²⁺ selective ion channel) and in maintaining proper bone formation (Rousset, M. et al. P. FEBS; 2004, 576, 41-45).

Several fluorescent molecules can be used to measure calcium concentration or spatial distribution in the cytoplasm. The following prior art fluorescent compounds are described in detail below, using the abbreviations used in the table below:

Ionic calcium concentration within a typical cell is around 100 nM, but it can increase up to a hundredfold during various cellular activities. This is relatively low compared to the extracellular concentration, where it can be up to 12,000 times. This is maintained by ATP- driven calcium ion pumps in cell walls. In electrically stimulable cells, such as skeletal and cardiac muscles and neurons, depolarization of the membrane leads to a calcium transient, in which case the concentration of calcium ion in the cytosol can reach up to 1 mM. (David E. Clapham; Review | Volume 131, ISSUE 6, P1047-1058, December 14, 2007; https://www.cell.com/abstract/S0092-8674(07)01531-0). In addition to the endoplasmic reticulum, mitochondria are also able to isolate and store some of the calcium ions. (Wilson, C.H. at al. Biochem J. 466(2): 379-390.) It is estimated that the free calcium concentration of the mitochondrial matrix increases to tens of micromolar levels in situ during neuronal activity. (Ivannikov, M.; et al. Biophys. J. 104 (11): 2353-2361.) [8] Various fluorescent calcium sensing dyes can be used to monitorthese processes, to map the distribution of calcium within the cell, and to monitor changes in calcium signaling and channel function in the host cell even in the case of viral infection. (Jacob L.Perrya at al. Methods Volume 90, 15 November 2015, Pages 28-38. https://doi.Org/10.1016/j.ymeth.2015.09.004).

Among the dyes, the advantage of using the BAPTA scaffold is that it is insensitive to intracellular pH changes, "releases" Ca ²⁺ ion much faster than EDTA and EGTA, it is difficult to metabolize, and its complex form of Ca ²⁺ ion is also it is highly soluble in water and its dissociation constant with Ca ²⁺ ion is in the range of biologically significant concentrations. Due to the rapidity of Ca ²⁺ ion binding, BAPTA has become a useful tool for studying local, rapid changes in Ca ²⁺ ion concentrations, such as the opening of Ca ²⁺ ion specific channels (Rousset, M. es mtsai. P. FEBS; 2004, 576, 41-45). Its advantage is that a fluorescent dye can be attached to one of its rings, which can indicate the binding of Ca ²⁺ ion by means of the PET effect. Their most common representative is Thermo Fischer Scientific Oregon Green ™ 488 BAPTA-AM. Its biological application is also made possible by its non-toxicity, so it can also be used in in vivo experiments. For this reason, BAPTA has many uses.

Due to the large role that calcium plays in cell function, it has many physiological effects, many diseases are associated with its absence or changes in the processes associated with it. Calcium intake is thought to reduce the risk of colon and rectal cancer, according to the U.S. Food and Drug Administration (FDA) (https://wayback.archive- it.org/7993/20171114023216/https:/www.fda.gov/Food/lngredien tsPackagingLabeling/Labe lingNutrition/ucm072771.htm). Research has shown that calcium also plays a major role in viral infections. In viruses, the use of host cell calcium signals is a general strategy to aid proliferation (Jacob L.Perry et al .; MethodsVolume 90, 15 November 2015, Pages 28-38). In the case of hepatitis B virus (HBV), it has also been found that its growth requires an increase in the amount of cytosolic calcium, which is achieved by influencing the mitochondrial calcium regulatory system (Michael J. Bouchard at al. Science 14 Dec 2001: Vol. 294, Issue 5550, pp. 2376-2378 DOI: 10.1126/science.294.5550.2376.). Similarly, the Herpes virus also activates calcium signaling pathways (Natalia Cheshenko et al., The Journal of Cell Biology, Volume 163, Number 2, October 27, 2003283-293, https://doi.org/10.1083/jcb. 200301084) and Influenza A virus affects human neutrophil calcium metabolism (KL Hartshorn et al. J Immunol August 15, 1988, 141 (4) 1295-1301).

An association with calcium has also been found for SARS-CoV-2 virus (COVID-19). Some antihypertensive drugs that block calcium channels (CCB drugs) have been found to be effective in inhibiting the proliferation of SARS-CoV-2 virus after infection (Lei-Ke Zhang at al. Cell Discovery volume 6, Article number: 96 (2020)). In addition, low levels of phosphorus and calcium in the blood have been observed in COVID - 19 infected patients in severe cases than in moderate ones. Based on this, low phosphorus and calcium levels may predict the severity of the infection and may be useful clinical biomarker for discriminatory diagnoses. (Caiting Yang at al. J. Med. Virol. 2021;93:1639-1651, DOI: 10.1002/jmv.26515).

Small molecule calcium sensors have many different uses. These include the study of neuronal activity by two-photon microscopy (Katona, G. et al .; Nature methods; 2012, 9 (2) 201-208; Rozsa, M. et al., Brain Struct Funct; 2017 222 (1) 651-659, Turi, GF et al., Neurochem Res; 2010 35 2086-2095) and testing the role of Ca ²⁺ ion in various cellular functions (Saoudi, Y. es mtsai: Eur. J. Biochem; 2004, 3264, 3255-3264). Recent research has shown the possibility of using synthetic calcium sensors as a contrast agent for magnetic resonance imaging (MRI), A. Barandov et al., Nat. Commun. 2019, 10, 1-9; 26: K. Dhingra et al. .: J. Biol. Inorg. Chem. 2008, 13, 35-46) or as a promising therapeutic agents (Z. Fu, es mtsai.: ACS Appl. Mater. Interfaces 2019, 11, 39574-39585). Furthermore, by combining these discovered techniques with a microendoscope (28: M. J. Levene et al., Neurophysiol. 2004, 91, 1908- 1912), fluorescent calcium chelators may have a bright future in human diagnostics and therapy. The BAPTA ionophore can be coupled with many fluorophores to provide a variety of dyes suitable for different purposes for fluorescent calcium imaging. (Takahashi, es mtsai.; Physiol. Rev. 1999, 79, 1089-1125). In analytical measurements, the increase in the fluorescence intensity of the dye molecule cased by complex formation is exploited and measured. This effect makes it suitable for use in the increasingly widespread two-photon (2P) laser scanning microscopic measurements, which has led to the major breakthrough in recent years, particularly in the field of neuroscience. (Chiovini, B., es mtsai.: Neuron, 2014. 82, 908- 924). Deliverability into cells and low toxicity are very important for biological applicability of such chemosensors. In the case of the fluorescent moiety, high two-photon absorption (2PA) and good quantum exploitation are important, in addition to which it is expected that the fluorescence emission will shift as much as possible in the direction of the red wavelength range (l> 600nm) (Tsien, R.Y., Biochemistry, 1980, 19, 2396-2404). In the case of the sensor part of the molecule, in order to having suitable fluorescent labeling and to be able to significantly influence the photoactivity of the labeling molecule by the photoinduced electron transfer (PET) effect, it is important the Calcium ion selectivity.

Fluorescent sensor compounds have been used in biological imaging for a decade, but they are also of great importance in analytical studies and are now widely used due to their ease of use and high sensitivity. Both chemical and optical properties of fluorescent chemosensors can be changed accurately and relatively easily (32: Li, X., et al., Chemical reviews, 2013. 114, 590-659; You, L. et al .: Chemical reviews, 2015 , 115, 7840-7892, Busschaert, N., et al., Chemical reviews, 2015, 115, 8038-8155, Wu, J., et al., Chemical reviews, 2015, 115, 7893- 7943). As a result, several chromophores described in the literature may be suitable for the preparation of an efficient sensor (see Tsien, RY, Biochemistry, 1980, 19, 2396-2404, Hamilton, GR, et al., Chemical Society Reviews, 2015, 44, 4415- 4432, Yin, J. et al., Chemical Society Reviews, 2015, 44, 4619-4644, Ashton, TD et al., Chemical Society Reviews, 2015, 44, 4547- 4595). During chelation, organic molecules bind to metal ions. Coordinated bonds are formed between the central atom and the ligands using the non-binding electron pairs of the ligands. In the case of chelation, the ligand has an increased affinity for that metal ion compared to non-chelating ligands.

The efficacy of chelating compounds can be characterized by an equilibrium constant called the dissociation constant (K _D ). This refers to the reaction with a particular metal ion and depends on the ionic strength, pH (Lindsay, WL et al., Soil Sci. Soc. Amer. Proc., 1969 vol. 33, 1969 62-68, Holloway, JH et al. : Analitycal Chemistry; 1960, 32 (2), 249-256 (Mirti, P .; Journal of Inorganic and Nuclear Chemistry, 1979, 41 323-330) and from the temperature (Arena, G. et al., Thermochimica Acta; 61, 129-138). The pH dependence is caused by the differently protonated forms of the chelating agents, since their equilibrium constants are different, so the equilibrium constant of the reaction changes as their amount changes.

Ca ²⁺ ions play a major role in biological processes. During membrane depolarization caused directly by a Ca ²⁺ selective ion channel, or indirectly as a secondary messenger that mediates the excitation of many Ca ²⁺ sensitive proteins (Rousset, M. et al. P. FEBS; 2004, 576, 41-45). Selectivity is important for reagents using for detection Ca ²⁺ , as Na ⁺ , K ⁺ , Mg ²⁺ , Ca ²⁺ ions provide majority of inorganic ions in living organisms.

Some common chelating reagents for complexing metal ions are shown in Figure 1. These are EDTA (ethylenediamine-N,N,N',N'-tetraacetic acid, 5a), EGTA (ethylene glycol bis (b- aminoethyl ether)-N, N, N', N'-tetraacetic acid, 4a), BAPTA (1,2-bis (o-aminophenoxy) ethane- N, N, N', N'-tetraacetic acid, 3a) molecules. The importance of the listed molecules lies in their ability to form a stable complex with up to several metal ions, so they are often used for analytical purposes. The most important complexing molecules are those in which complexes with similar metal ions have a large difference in stability, i.e. they selectively bind certain metal ions. (Boyd, S. es mtsai.: Chem. Soc, 1965, 7353-7358).

Figure 1. 3a: BAPTA, 4a: EGTA. 5a: EDTA and their calcium complexes EDTA is a widely used chelating reagent. It is very effective against almost all metal ions. It is characteristic of its ability to chelate even with alkali metal ions with relatively high stability. Because of these properties, it is ideal, for example, for determining water hardness by titration using an indicator ( Yappert, M. C. es mtsai.: Journal of Chemical Education ; 1997, 74 (12), 1422-1423). Its disadvantage is that due to the lack of selectivity it cannot be used for physiological purposes when the goal is to remove toxic metals that have entered the body, bacause it removes all metal ions from the blood plasma indiscriminately. (Arena, G. es mtsai.: Thermochimica Acta; 1983, 61, 129-138). The Ca ²⁺ ion selectivity of the chelating reagent over Mg ²⁺ ion is important for research, so non-selective EDTA would not be ideal.

EGTA prefers the Ca ²⁺ ion, so it is commonly used to make Ca ²⁺ buffers for biological purposes. The advantage of EGTA over other Ca ²⁺ ion binding reagents of similar strength is that while its binding to Ca ²⁺ , Sr ²⁺ , Ba ²⁺ ions is similar to EDTA's binding, it binds Mg ²⁺ ion five orders of magnitude less.

The dissociation constants of the complexing agents are summarized in the table below for the Ca ²⁺ and Mg ²⁺ ions in the deprotonated state and for the Ca ²⁺ ions at physiological pH. The data described in the literature varied over a large range and we did not find adequate data in all cases.

Table 1: Dissociation constants of known Ca ²⁺ ion complexing agents according to the literature

Deprotonated Ca Deprotonated Mg ^: physiological pH Ca ²⁺ , ,

BAPTA 6,15 nines adat 6,15

BAPTA is an analogue of EGTA. BAPTA also shows adequate selectivity and can be fluorescently labeled, although its dissociation constant for Ca ²⁺ ion is lower (see Table 1 above). The disadvantages of EGTA were slow buffering and high pH sensitivity, which were corrected with the development of BAPTA. BAPTA' nitrogen atoms do not protonate at physiological pH, as they are already aniline derivatives, which make them insensitive to pH change and faster Ca ²⁺ ion dissociation. The advantage of BAPTA is that it is insensitive to changes in intracellular pH, it releases Ca ²⁺ ion much faster than EDTA and EGTA, it is difficult to metabolize, and the complex form with Ca ²⁺ ion is also highly soluble in water and its dissociation constant with the Ca ²⁺ ion is in the biologically significant concentration range. Due to the rapidity of Ca ²⁺ ion binding, BAPTA has become a useful tool to study the local rapid changes in Ca ²⁺ ion concentrations, such as the opening of Ca ²⁺ ion specific channels (Lei-Ke Zhang et al. Cell Discovery volume 6, Article number: 96 (2020), Rousset, M. et al., P. FEBS; 2004, 576, 41-45).

Affinity for Ca ²⁺ ion is affected by substituents of ring. Electron withdrawing groups decrease, while electron donating groups increase the affinity for Ca ²⁺ ions (Pethig, R. et al., Cell calcium; 1989, 10491-498; Harrison, S. M et al., Biochimica et Biophysica Acta ; 1987, 925, 133-143.). Its advantage is that a fluorescent dye can be attached to one of its rings, which can indicate the binding of Ca ²⁺ ion by means of the PET effect. For this reason, BAPTA has several uses: testing the role of Ca ²⁺ ion in the cases of different cellular functions (Saoudi, Y. et al., Eur. J. Biochem; 2004, 3264, 3255-3264), testing the activity of neurons by two photon microscopy (Katona, G. et al .; Nature methods; 2012, 9 (2) 201-208; Rozsa, M. et al., Brain Struct Funct; 2017 222 (1) 651-659; Turi, GF et al .: Neurochem Res; 2010 35 2086-2095.). Developments have been going on for a long time in which the so-called BAPTA, or BAPTA-like, but compounds containing only one aromatic ring, the so-called half-BAPTA compounds are bound to a fluorescent dye so that the change during complex formation can also be measured optically. Such half-BABTA based dyes are described in US9103791B1 and US2019308991A1. There is no data in the patent to suggest that it would be more advantageous than known BAPTA derivatives.

According to the U.S. Pat. No. 5,049,673, BAPTA is attached directly to a fluorescent moiety, such as a dihydroxy-xanthine group. According to the application, such compounds are much more advantageous than previously used quin-2 or fluo-2, which have a significantly different structure. However, the BAPTA derivatives with direct coupling in switched of state have an intensity of 0, while during switching on this it is approx grow to 10-20%. Thus, there is no basal fluorescence that depicts the entire cell culture and thus gives an excellent preliminary image of the sample. U.S. Pat. No. 5,455,517 discloses BAPTA derivatives which are acylated on one or both amino groups in the 5-positions on the phenyl groups, for example, a fluorescein derivative in which the phenyl group in the fluorescein is a carboxyl group in the para position relative to the xanthine moiety. Thus, the BAPTA ester is attached to the fluorescent dye component via an arylamide group, e.g. for the dihydroxy-xanthinyl group. According to the patent, the compounds which are the subject of the patent are more preferred according to the patent than the similar compounds described previously. The structure of these or fluorescent indicators binds the aromatic BAPTA ring to a conjugated heterocyclic system via a trans ethylene bond that is either fixed (e.g., for Fura-2 and FURA RED) or rotating (e.g., for Fluo-3 and indo-1). Additional Ca ²⁺ selective fluorescence indicators have been described by Tsien (Intracellular Measurements of Ian Activities, ANN. REV. BIOPHYS. BIOENG. 12, 91 (1983)), but they have a limited fluorescence response or have other disadvantageous properties. Furthermore, in the examples described in the patent, the fluorescent moiety is not modified, so that they cannot enter the cells in the active state, so that they can be used only to a limited extent for the intended purpose.

The most common BAPTA representative is the BAPTA-AM product of Thermo Fischer Scientific Oregon Green ™ 488. Its biological application is also made possible by its non toxicity, so it can also be used in in vivo experiments. That compound was first disclosed in PCT Patent Application WO1997/039064. The application relates to compounds containing fluorinated xanthine derivatives which contain at least one fluorine atom. BAPTA-AM 488 is compound 104 which contains both protected BAPTA and two hydroxy groups of the xanthine group which are also protected by acetyl groups. Compound 103 is the active compound in which BAPTA is already present in acidic form, and the acetyl groups protecting the hydroxy groups on the xanthine backbone have also been cleaved. However, the activated form does not pass through the cell wall, so this can only be used delivered into the cells by using micropipettes. In contrast, protected BAPTAs enter cells from the extracellular space, and they are hydrolyzed by enzymes and converted to the active form within the cell. However, this is only suitable forthe study of living cells, since dead cells, even if they enter, are not hydrolyzed in them, so the amount of Ca ²⁺ released in dead cells cannot be measured with them. Furthermore, enzymes that enter the extracellular space after cell death also activate the compound outside the cell by hydrolysis of protected BAPTAs in the extracellular space. For example, if we try to map the proportion and location of healthy and dead cells in a cell culture, as it is the case for histological studies of viral infections, these compounds are not ideal for this purpose. Thus, there is a need to use BAPTA derivatives that are active but still allow the differentiation of cells in different states. This makes it possible to detect the consequences of a viral infection, for example, even in a living organism.

Brief description of the invention

It has been found that the compound of general formula

(I) wherein

Ri and R ₂ represent independently hydrogen or alkyl group with the proviso that one of Ri and R ₂ is at least non-hydrogen and one of R ₃ and R ₄ is a group of the general formula (II) wherein R ₅ and R ₆ represent independently a hydrogen atom, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy group, or R ₅ and R ₆ together form a -O-CH ₂ -O- group and R ₇ is hydrogen atom, C ₁ -C ₄ alkyl or C ₁ -C ₄ alkoxy group, these compounds are able to penetrate the cell wall in active form, excellent fluorescent dyes that selectively indicate Ca ²⁺ ion concentration based on the use of appropriate imaging techniques such as single or multiphoton, preferably during two-photon laser excitation microscopic procedures. The fact that the active form enters the extracellular space and enters the cell in the active state by penetrating the cell wall, thus not requiring the enzyme system of the living cell for activation, allows to use the imaging method study Ca ²⁺ ion concentration simultaneously in the extracellular space, in cells and in dead cells, so that the change in Ca ²⁺ ion concentration in a tissue sample can be examined in time frame, which is high importance e.g. in the investigation of viral infections.

DETAILED DESCRIPTION OF THE INVENTION

The solution to this problem has been found that if a BAPTA derivative is used in which neither the BAPTA nor the xanthine group hydroxy groups are protected, they may still be suitable for the purpose of simultaneously mapping cells in different physiological phases if the fluorescent dye on the xanthine backbone contains a C1-C6 alkyl or C1-C6 alkoxy group in at least one or more of the 2 'or 7' positions, according to the following numbering:

Thus, the present invention relates to fluorescent dyes which can be described by a compound of the general formula (I), in which

Ri and R ₂ represent independently a hydrogen atom, straight-chain or branched, unsubstituted C ₁ -C ₁₀ alkyl or C ₁ -C ₁₀ alkoxy group or substituted by one or more halogen atoms, a substituted or unsubstituted aryl or aralkyl, with the proviso that at least one of Ri and R ₂ is not hydrogen, one of R ₃ and R ₄ is a hydrogen atom, and the other represents a group of general formula

(II), wherein

R ₅ and R ₆ represent independently a hydrogen atom, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy group, or R ₅ and R ₆ together form a -0-CH ₂ -0- group,

R ₇ represent a hydrogen, Ci-C ₄ alkyl or Ci-C ₄ alkoxy group, and tautomeric forms and salts thereof. As it is known, the structure of the fluorescent dyes of the present invention can take various tautomeric forms:

In the present invention, for the sake of simplicity, only one form is indicated in the description. Taking into consideration that depending on the circumstances - e.g. under crystallization conditions - the equilibrium of the tautomeric forms may vary, even to the extent that the form indicated herein is not detectable, but the present invention relates to these tautomeric forms also.

According to the state of the art, there were two possibilities to determine the intracellular Ca ²⁺ ion concentration by imaging methods. One was that an active complexing agent consisting of an otherwise excellent fluorescent moiety and a BAPTA, which had the disadvantage of not being able to enter the cell wall, had to be introduced into living cells with a micropipette, even into many cells when a tissue sample was examined. This disadvantage has been eliminated by the developers by "protecting" the active complexing agent and the hydroxyl group of xanthine so that the structure, which is thus less polar but even more non ionic, enters the cells. After entry into the cell, they are activated using the enzyme system of the living cell. Thus, although it is possible to simply examine several cells and tissue samples, but the extracellular space cannot be monitored. Surprisingly, we have found that it is not necessary to protect the carboxy or hydroxy groups of the complexing or fluorescent dye molecule, to deactivate the compound to allow the dye molecule to enter the living cell, it is sufficient if in the xanthine backbone side chains are incorporated, which change the polarity (lipid solubility?) of the compound so that it easily penetrates easily trough the cell wall even in the active, ionic state and shows well the operation of Ca ²⁺ channels, the distribution of Ca ²⁺ in living cells, tissues and extracellular space. Additionally, they have green fluorescence, which requires a simpler laser and optical architecture, which significantly widens the range of applications and reduces costs.

Particularly, according to a highly preferred embodiment of the present invention is a compound of formula (I) wherein Ri and R ₂ represent independently C ₁ -C ₁₀ alkyl, preferably C ₁ -C ₆ alkyl, more preferably C ₁ -C ₄ alkyl group, most preferably hexyl, n-butyl, 2-butyl, tert- butyl, n-propyl, 2-propyl, ethyl or methyl group, as alkoxy group a C ₁ -C ₁₀ alkoxy group, preferably a C ₁ -C ₆ alkoxy group, more preferably a Ci- C ₄ alkoxy group, most preferably n-butoxy, 2-methylpropoxy, 2,2-dimethylethoxy, n-propoxy, 2-methylethoxy, ethoxy or methoxy group, and wherein the alkyl and alkoxy groups have 0 to n halogen substituents, wherein n is the total number of substitutable hydrogen atoms of the alkyl or alkoxy group, and wherein the halogen atoms are independently chlorine, bromine, iodine orfluorine atoms, most preferably fluorine atoms; as aryl group may be substituted or unsubstituted phenyl or naphthyl group, aralkyl group may be phenethyl, benzyl or naphthylmethyl group having 0 to m substituents independently selected from C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy group, halogen, which may be independently chlorine, bromine, iodine or fluorine, where m is the total number of substitutable hydrogen atoms of the aryl or aralkyl group;

R ₅ and R ₆ represent independently a hydrogen atom, ethyl, methyl, methoxy group, or R ₅ and R ₆ together form a -O-CH ₂ -O- group,

R ₇ represents a hydrogen, ethyl, methyl or methoxy.

More particularly, in highly preferred forms of the compounds of the present invention, Ri and R ₂ independently represent as alkyl group, hexyl, n-butyl, ethyl or methyl, as alkoxy group methoxy groups, and alkyl or alkoxy groups comprise 0-n halogen substituents, where n is the number of total hydrogen atoms of the alkyl or alkoxy groups, and wherein the halogen atoms are fluorine atoms, R ₅ and R ₆ represent independently a hydrogen atom, ethyl, methyl, methoxy groups, or R ₅ and R ₆ together form an -O-CH ₂ -O- group, R ₇ represents hydrogen atom, ethyl, methyl or methoxy group.

In the most preferred embodiments of the present invention, Ri and R ₂ independently stand for as alkyl group a hexyl, n-butyl, ethyl, or methyl group, R ₅ , R6 and R ₇ stand for hydrogen atoms.

In the compounds of the present invention, Ri and R ₂ may be identical. In some cases, their production may also be simplified.

Another embodiment of the present invention is a two-component isomeric mixture of compounds of formula (I) in any ratio, wherein in both isomers the meaning of Ri, R ₂ , R ₅ , R6 and R ₇ are the same as to the corresponding substituents of the other isomer, except that in the one of the isomeric component R ₃ is a hydrogen atom and R represents a group of general formula (II), while in the other isomeric component R ₃ represents a group of general formula (II) and R ₄ is a hydrogen atom, and a mixture of salts thereof. As it is clear from the general formula (I), the fact that the carboxyl-amino group linking to the BAPTA derivative is in the R ₃ or R position does not affect the formation of a similar conjugated system which is present in both cases which and changes the fluorescent properties of the dye in the case of complexation. Thus, even if there may be a small difference between the effects of the two compounds, this does not significantly affect the practical use. Furthermore, the isomer ratio formed in the synthesis depends on the reagents used and the reaction conditions, which are constant during stable synthesis, so that the proportion of isomeric mixtures in each product is essentially constant and Ri and R ₂ , which allow the penetration into the cell, are also the same in the isomers, the synthesis is shorter with two reaction steps, so it is much more efficient and cheaper.

The most preferred embodiments of the invention can be characterized by the following formulas:

Particularly preferred isomeric mixtures are: The compounds of the present invention contain an easily removable protons, thus they can obviously also be prepared in salt form. Such salts also form part of the present invention. The compounds of the present invention may also form salts of various compositions with organic or inorganic bases. According to a preferred embodiment of the invention, inorganic salts, alkali metal and alkaline earth metal salts can be used for salt formation. Of the alkali metal salts, the sodium, potassium and lithium salts are particularly suitable. Most preferably the potassium and sodium salts with 6 sodium ions or 6 potassium ions are particularly preferred. The compounds of the present invention may be crystalline or amorphous, and in some cases even liquids. Since the two nitrogen atoms of BAPTA are also capable of forming salts, the present invention also relates to salts of the compounds of formula (I) according to the invention with strong acids, such as sulfuric acid, para-toluenesulphonic acids.

In all forms, whether free acid or salt, or in any state, the corresponding tautomeric forms are in equilibrium, so that it is possible that some forms other than the tautomeric form used in the application, even more it may possible that the tautomeric from used in the description is undetectable. (In the present representation, the oxygen adjacent to R2 is represented as "quinone" oxygen.) However, these are essentially the same compounds and therefore all are within the scope of this invention. Another embodiment of the present invention is a process for the preparation of compounds of the general formula (I), a highly preferred embodiment of which is illustrated by the following route of synthesis: Thus, the object of the present invention to provide a compound of general formula (I) by reacting a compound of formula (III) wherein Ri, R ₂ are identical as Ri, R ₂ of the compound of general formula (I), Pri and Pr ₂ are identical or different hydroxy protecting groups, preferably acyl group, more preferably C ₁ -C ₄ substituted or unsubstituted aliphatic acyl group, most preferably acetyl group, one of R ₃ and R ₄ represents a hydrogen atom, and the other represents a group of general formula (IV) wherein R ₅ , R ₆ and R ₇ are as defined for R ₅ , R ₆ and R ₇ in the group of general formula (II), are as defined above Zi, Z ₂ , Z ₃ and Z ₄ are identical or different protected carboxyl groups, preferably alkyloxy carbonyl, more preferably C ₁ -C ₄ alkoxycarbonyl, most preferably ethoxy- or methoxycarbonyl, are deprotected and optionally transformed to a salt form.

The present invention is based on the above-mentioned recognition, that the compounds of the present invention able to penetrate cells not only in the form of protected compounds of formula (III) but also without protecting groups, in contrast to the fluorescent dyes used so far. Thus, by removing the protecting groups of compounds for which were developed for similar use, unexpectedly, even more advantageous fluorescent dyes are obtained. Particularly, the essence of our process is that the compounds of the general formula (III) are preferably deprotected by hydrolysis. Preferably, the compound of general formula (III) is hydrolyzed in water or in the presence of water in a protic or dipolar aprotic solvent, or in a mixture thereof, in the presence of a strong base, preferably an alkali metal hydroxide, more preferably potassium or sodium hydroxide. Particularly, as the polar solvent water, more preferably C1-C4 alcohols, methanol, ethanol, propanol, isopropanol, and normal butanol can be used. As suitable dipolar aprotic solvents include ether type solvents, preferably di isopropyl ether, methyl ether, butyl ether, dioxane, tetrahydrofuran, 2-methyl tetrahydrofuran, preferably dioxane, tetrahydrofuran, more preferably dioxane, as nitril type solvent acetonitrile, propionitrile, as amid solvent dimethylformamide or diethylformamide, as sulfur-containing solvent dimethyl sulfoxide or sulfolane can be used. According to the present invention, the hydrolysis is more preferably carried out in mixture of a water-miscible ether type solvent, in dioxane or tetrahydrofuran and water, more preferably in a mixture in which besides water a dipolar aprotic solvent, preferably ether type solvent and a C1-C4 alcohol as protic solvent are present. Most preferably, the hydrolysis is performed in a mixture of water-dioxane-ethanol or water-dioxane-methanol. As base an aqueous solution of KOH or NaOH is used. The preferred base for the hydrolysis is preferably used in aqueous solution. The used concentration KOH or NaOH solution is between 10 weight % by and the concentration of the saturated KOH or NaOH solution, preferably 20-40 weight%, most preferably 40 weight% of KOH or NaOH. The hydrolysis is preferably carried out at a temperature between 0° C and the boiling point of the solvent-water mixtures used, preferably between 10°C and 50°C, more preferably between 20°C and 30°C, most preferably at 25°C. The hydrolysis is carried out for 6 to 14 hours, preferably 8 to 12 hours, most preferably for 10 hours. Thus, we can proceed in such a way that the compound of general formula (III) is stirred or dissolved in one of the dipolar aprotic solvents listed above, or mixtures thereof, and then, optionally, ethanol or methanol is added to the mixture, followed by addition of 10 molar equivalents of KOH or NaOH solution and the resulting mixture stirred at room temperature for 8-12 hours. Most preferably, the compound of general formula (III) is dissolved in dioxane, one equivalent of methanol is added to the reaction mixture, and then 10 molar equivalents of aqueous KOH solution are added to the reaction mixture and stirred for 10 hours. From another aspect, the present invention also relates to the preparation of a key intermediate of general formula (III). The compound of general formula (III) according to the invention is prepared by using the compound of the general formula as starting material in which Ri stands for a hydrogen atom, straight-chain or branched Ci-Cio alkyl or Ci-Cio alkoxy, unsubstituted or substituted by one or more halogen atoms, substituted or unsubstituted aryl, or aralkyl group, Rs and Rg are hydrogen or carboxyl group, provided that one of Rs and Rg is hydrogen, and the other is carboxyl group.

The compound of the general formula (VIII) according to the invention is reacted with a compound of general the general formula in which R ₂ represents a hydrogen atom, straight-chain or branched, Ci-Cio alkyl or Ci-Cio alkoxy group unsubstituted or substituted by one or more halogen atoms, substituted or unsubstituted aryl or aralkyl group with the proviso that at least one of the group Ri of the compound of general formula (VIII) used and the group R ₂ of the compound of the general formula (IX) is not hydrogen. The reaction is carried out in the presence of sulfonic acids with or without a solvent in the sulfonic acid. When a solid sulfonic acid such as p-toluenesulfonic acid is used, the reaction is carried out in a solvent. In the case of liquid sulfonic acids, such as methanesulfonic acid, ethanesulfonic acid or propanesulfonic acid, the reaction can be carried out in the sulfonic acid itself without a solvent, because they also act as a solvent and a reagent in this case. As solvents, inert solvents such as aromatic hydrocarbons such as toluene, xylene, or aliphatic acyclic saturated or unsaturated C6-C12 hydrocarbons, preferably hexane, heptane, octane, decane, or saturated or unsaturated cyclic C6-C10 hydrocarbons such as cyclohexane, cyclohexane, cyclohexane halogenated solvents such as carbon tetrachloride, chloroform, dichloroethane, dibromethane, organic acids, preferably acetic acid, propionic acid can be used. The reaction mixture as a mixture of the two components in the reaction mixture, which also contains an organic sulfonic acid and optionally an additional solvent where appropriate is kept, preferably between 60 and 160°C, preferably between 80 and 120°C, more preferably between 90 and 110°C, most preferably at 100°C for 0,5-2 hours, preferably 1-1.5 hours, more preferably 1 hour. The used molar ratio of the diol of the general formula (IX) to the compound of the general formula (VIII) in the process according to the present invention is 0.8 to 1.2: 1, preferably 1: 1. In a most preferred embodiment of this process step, the diol of the general formula (IX) and the compound of general formula (VIII) are dissolved in a liquid sulfonic acid, preferably methane-, ethane-or propanesulphonic acid, most preferably methanesulfonic acid, and then the resulting mixture is kept at 90-110°C, most preferably at 100°C for 1-1.5 hours, so that the molar ratio of the diol of the general formula (IX) used to the compound of the general formula (VIII) is 1: 1. The hydroxyl groups of the thus given compound of the general formula are applied with protecting groups resulting the compound of the general formula (V) wherein the meaning of Ri, R ₂ , Rs and R _g are unchanged, Pri and Pr ₂ are identical or different hydroxy protecting groups, preferably an acyl group, more preferably a C1-C4 substituted or unsubstituted aliphatic acyl group is most preferably acetyl group. Most preferably, the acyl groups can be prepared by using acid anhydrides or acid halides in the presence of tertiary amines or inorganic acid scavengers. As the acid anhydride or acid chloride, acid chlorides or acid anhydrides formed from substituted or unsubstituted C1-C4 aliphatic carboxylic acids can be used. Most preferably, acetic anhydride, acetyl chloride, acetyl bromide, acetyl iodide, or propionic anhydride, propionyl halides such as chloride, bromide or iodide, or butyric anhydride or chloride can be used. As the solvent, dipolar aprotic solvents such as acetonitrile, propionitrile, dimethylformamide or pyridine or, in the case of use of acid anhydrides, the anhydride itself can be used. As the acid scavenger in the reaction, organic bases, or inorganic acid scavengers such as alkali or alkaline earth metal carbonates and hydrocarbons are used. As the organic base, tertiary amines such as triethylamine, diisopropylethylamine and the like, or pyridine can be used. Potassium or sodium carbonate is most preferably used as the inorganic acid scavenger. The acylation is carried out at a temperature between 60°C and the boiling point of the reaction mixture, preferably between 60°C and 110°C, more preferably between 80°C and 100°C, most preferably at 100°C, for 1 to 3 hours, preferably 1.5 to 2.5 hours, most preferably 2 hours. The acylating agent is preferably used in an excess of at least 1.5 to 2 times on the number of hydroxyl groups to be acylated.

By using the free carboxyl group of the thus given protected compound of the general formula (V) the amino group of a compound of the general formula (VI) wherein R ₅ , R ₆ , R7, Zi, Z ₂ , Z ₃ and Z are as defined for the group of formula (IV) is acylated.

The acylation reaction of the compound (VI) is preferably carried out in nonpolar or dipolar aprotic solvents, the nonpolar solvent being aromatic solvents, preferably toluene, xylene, saturated aliphatic solvents, preferably C6-C10 aliphatic solvents such as hexane, heptane octane, cycloparaxane, cyclohexane, e.g. cyclohexane. As the dipolar aprotic solvent, ether- type solvents such as methyl tert-butyl ether, diethyl ether, cyclic ethers such as dioxane, tetrahydrofuran or 2-methylfuran can be used. Suitable dipolar aprotic solvents include halogenated solvents such as dichloroethane, dichloromethane, chloroform, bromoform, dibromomethane, and acetonitrile or propionitrile can be used. As amide type solvent, formamide, dimethylformamide or diethylformamide can be used. Dimethyl sulfoxide, diethyl sulfoxide or sulfolane can be used as the sulfur-containing dipolar solvent. Organic or inorganic bases can be used as acid scavengers. As the organic base, tertiary amines such as triethylamine, diisopropylethylamine and the like, or pyridine can be used. Potassium or sodium carbonate is most preferably used as the inorganic acid scavenger. In the acylation reaction, the carboxyl group is first formed an anhydride with isobutyl chloroformate or a similar compound capable of forming an active anhydride (such as ethyl chloroformate, Dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIC), carbonyldiimidazole, tris (benzyltriazolylmethyl) amine (TBTA), O- (7-azabenzotriazol-l-yl) -N, N, N ', N'- tetramethyluronium tetrafluoroborate (TATU), (Benzotriazole- 1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate (BOP), (Benzotriazol-l-yloxy) tripyrrolidine phosphonium hexafluorophosphate (PyBOP), N, N, N',N'-Tetramethyl-0-(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU), followed by the addition of the amine component of the general formula (VI). Thus, according to the present invention, the reaction is carried out by dissolving the compound of formula (V) in one of the above solvents and then adding to the reaction mixture preferably 1 to 1.5 mol, preferably 1.1 mol, of organic compound per compound of formula (V) then isobutyl chloroformate is added in an amount of 1-1.5 mol, preferably 1.1 mol, based on the compound of formula (V). The reaction mixture is stirred for 2 to 4 hours, preferably 3 hours at -50 ° C to 100 ° C, preferably 0 ° C to 50 ° C, most preferably at 0 ° C, and then to the solution containing the mixed anhydride thus obtained is added the compound of general formula (VI). The product of general formula (III) is isolated.

The present invention relates to the preparation of compounds of general formula (VIII) for the preparation of compound of general formula (III). This is because the preparation of this makes it possible to introduce two different groups in two steps into the compound as substituents Ri, R ₂ . by reacting compound of general formula (IX) with a compound of general formula (VIII) having a different R group.

The compound of general formula (VIII) can be prepared in two steps according to the present invention by simple reaction of the compound of the general formula

(X) and 1,2,4-benzyltricarboxylic anhydride of the formula as starting materials. According to the present invention, the substituent Ri of the compound of the general formula (X) stands for a hydrogen atom, a straight-chain or branched, unsubstituted Ci-Cio alkyl or Ci-Cio alkoxy group or substituted by one or more halogen atoms, a substituted or unsubstituted aryl or aralkyl group, and Ri - as alkyl group comprises preferably a Ci-Cio alkyl group, more preferably a C ₁ -C ₆ alkyl group, even more preferably a C ₁ -C ₄ alkyl group, most preferably hexyl, n-butyl, 2-butyl, tert-butyl, n- propyl, 2-propyl, ethyl, or methyl group,

- as alkoxy group comprises preferably Ci-C ₆ alkoxy group, more preferably C ₁ -C ₄ alkoxy group, most preferably n-butoxy, 2-methylpropoxy, 2,2-dimethylethoxy, n-propoxy, 2-methyl-etoxy, ethoxy or methoxy group, and in which the alkyl and alkoxy groups contain 0 to n halogen substituents, where n is the total number of substitutable hydrogen atoms of the alkyl or alkoxy group, and in which the halogen atoms are independently chlorine, bromine, iodine or fluorine atoms, most preferably fluorine atoms;

- as aryl group comprises substituted or unsubstituted phenyl or naphthyl group, as aralkyl group comprises phenethyl, benzyl, or naphthylmethyl group, which aryl or aralkyl groups have 0 to m substituents which are independently C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy group, halogen atom, which may be independently chlorine, bromine, iodine, or fluorine atom, where m is the total number of substitutable hydrogen atoms of the aryl or aralkyl group.

The reaction results an isomeric mixture of the general formulas (Vll-A) and (Vll-B) in which mixture components Ri and R ₂ have the same meanings and may be as described for the general formulas Ri given in the general formula (X). Only the position of the two carboxyl groups is different between the two isomers. The preparation of the isomeric mixture is preferably carried out by carrying out the reaction in the presence or absence of a solvent in the sulfonic acid. When solid sulfonic acids such as p-toluenesulfonic acid are used, the reaction is carried out in a solvent. In the case of liquid sulfonic acids, such as methanesulfonic acid, ethanesulfonic acid or propanesulfonic acid, the reaction can be carried out in the sulfonic acid itself without a solvent, because they also act as a solvent and a reagent in this case. As solvents, inert solvents such as aromatic hydrocarbons such as toluene, xylene, or aliphatic acyclic saturated or unsaturated C6-C12 hydrocarbons, preferably hexane, heptane, octane, decan, or saturated or unsaturated cyclic C6-C10 hydrocarbons such as cyclohexane, cyclohexane, cyclohexane halogenated solvents such as carbon tetrachloride, chloroform, dichloroethane, dibromethane, organic acids, preferably acetic acid, propionic acid can be used. The reaction mixture is kept in a reaction mixture comprising an organic sulfonic acid and optionally an additional solvent, preferably between 60-160°C, preferably between 80- 120°C, more preferably between 90-110°C, most preferably at 100°C for 2-6 hours, preferably for 3-5 hours, more preferably for 4 hours. The molar ratio of diol of formula (X) to 1,2,4- benzyltricarboxylic anhydride of formula (XI) used in the process of the present invention is 1.2-2.5 : 1, preferably 2 : 1. According to a most preferred embodiment of this process step, the diol of the general formula (X) and the compound of formula general formula (XI) are dissolved in a liquid sulfonic acid, preferably methane-, ethane- or propanesulfonic acid, most preferably in methanesulfonic acid, and then the resulting mixture is heated to between 60- 160°C, preferably between 80 to 120°C, most preferably to 100°C for 2 to 6 hours, preferably 3 to 5 hours, most preferably 4 hours, so that the molar ratio of the diol of the general formula (X) to the compound of general formula (XI) used is 1.5-2, 5 : 1, preferably 2:1. The thus obtained isomers having general formulas (Vll-A) and (Vll-B) are optionally separated and the synthesis can be continued with the selected isomer. If the isomers are not separated, the mixture of isomers represented by the general formula (Vll-A) and (Vll-B) is hydrolyzed to give the compounds of general formulas (Vlll-A) and (Vlll-B) wherein Rs and Rg stand for a carboxyl group. The hydrolysis according to the present invention is carried out by reacting a mixture of the isomers represented by the general formulas (Vll-A) and (Vll-B) or, if the isomers have been separated, by reacting the isomers represented by the general formula (Vll-A) or (Vll-B) in water, or in a mixture of a protic solvent and water, with an aqueous solution of 10 to 50 weight %, preferably 40 weight% of NaOH, KOH or LiOH. As protic solvent one or more water- miscible alcohols, preferably methanol, ethanol, isopropanol, ethylene glycol, propylene glycol and glycerol are used. The reaction is preferably carried out at a temperature of 50 to 100°C oratthe boiling point of the given solvent-water mixture by stirring the reaction mixture at this temperature for 3 to 10 days, preferably 7 days. A mixture of the compounds of the general formula (Vlll-A) and (Vlll-B) is recovered from the resulting reaction mixture, and the mixture is separated by chromatography if necessary. From the thus obtained pure isomeric compounds of the general formula (VIII), the protected pure isomeric product of general formula (III) is obtained as described above, followed by hydrolysis to give the pure isomers of the compounds of the general formula (I).

According to another preferred embodiment of the present invention, the isomers (Vlll-A) and (Vlll-B) are not separated, but the further steps of the above synthesis are carried out as a mixture of the isomers, as shown in the following figure:

In this case, a mixture of isomers containing the compounds of the general formula (I) is obtained. However, as described above, it is clear from formula (I) that the fact that the carboxyl-amino group linking to the BAPTA derivative is in the R3 or R4 position does not affect the formation of the carboxyl-amino group in both cases, which not affects the similar formation of conjugated system that changes the fluorescent properties of the dye in the case of complex formation. Thus, even if there may be a small difference between the effects of the two compounds, this does not significantly affect the practical use.

However, if necessary, isomers of the compounds of the present invention may be prepared by either (Vll-A) + (Vll-B), (VA) + (VB), (lll-A) + (lll-B) intermediate mixtures or mixtures of the final product (IA) + (IB). Most preferably, a preparative HPLC method is used to perform the separation. The selection of methods for purification and isolation, in particular the development of chromatographic methods, is within the general knowledge of a person skilled in the art. Separation of the intermediate mixture (Vll-A) + (Vll-B) by, for example, formation of a diastereomeric salt and the resulting salts e.g. can also be performed by fractional crystallization.

Another embodiment of the present invention is a process for the preparation of compounds of the general formula (I) wherein Ri and R2 are the same. In this case, if it is not necessary to separate the isomers, the synthetic route can be reduced by two further steps. Namely, when, as described above, the resorcinol derivative of the general formula (X) having the Ri substituents listed above with the 1,2,4-benzyltricarboxylic anhydride of the formula (XI), the obtained (Vll-A) and (Vll-B ) in which the desired RI substituent is present at both appropriate positions on the xanthine group. The matching of the substituents Ri and R2 is a special case of the compounds of the formula (VII), so it is clear that in this case steps 2 and 3 of the synthesis, i.e. the compound of the formula (X) and the reaction of the compound of the formula (XI) does not require hydrolysis and subsequent reconstitution, which makes the process more efficient, shorter and less expensive.

Furthermore, the isomer ratio formed in the synthesis depends on the reagents used and the reaction conditions, which are constant during stable synthesis, so that the proportion of isomeric mixtures produced in each production is essentially constant. Thus, from the point of view of usability, the use of the mixture is not a disadvantage, but the production, especially in those cases where Ri, R2 - which allow penetration into the cells - are the same in the isomers, the production is shorter with two reaction steps, so it is much more efficient and cheaper:

Another object of the present invention is the use of compounds of the general formula

(I), their tautomeric forms and their salts as fluorescent dyes for the detection of Ca ²⁺ ions, wherein in the compounds of formula (I), as described above, Ri and R ₂ are independently hydrogen, straight or branched, unsubstituted or substituted by one or more halogen atoms C ₁ -C ₁₀ alkyl or C ₁ -C ₁₀ alkoxy, substituted or unsubstituted aryl or aralkyl, provided that one of Ri and R ₂ is at least one of hydrogen, R ₃ and R ₄ hydrogen atom and the other is a group of the general formula

(II) wherein R5 and R ₆ are independently hydrogen atom, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy group, or R ₅ and R ₆ together form an -O-CH ₂ -O- group, R ₇ is hydrogen atom, C ₁ -C ₄ alkyl or C ₁ -C ₄ alkoxy group. As described above, the compounds of formula (I) according to the invention may exist in the three tautomeric forms of the various Ri and R ₂ substituents, provided that Ri and R ₂ are the same, in two tautomeric forms which are in equilibrium with each other in solution under the conditions of use. Whichever tautomeric form is responsible for the superior effect, after complexation, the active tautomer is reconstituted from the remaining unreacted compounds of formula (I) and according to the rules of physical chemistry, the full amount of the compound is utilized if sufficient Ca ²⁺ ions are available. As mentioned above, the compounds of formula (I) are capable of simultaneously detecting the amount of Ca ²⁺ ion in the intra- and extracellular space.

Another, even more preferred embodiment of the invention is the use of compounds of the general formula (I), their tautomeric forms and their salts for the detection of Ca ²⁺ ions as a fluorescent dye, wherein Ri and R2 in the compounds of general formula (I) substituents are independently as C ₁ -C ₁₀ alkyl groups, preferably C ₁ -C ₆ alkyl groups, more preferably C ₁ -C ₄ alkyl groups, most preferably hexyl, n-butyl, 2-butyl, tert-butyl, n-propyl, 2-propyl, ethyl, or a methyl group, as alkoxy group comprise C ₁ -C ₁₀ alkoxy groups, preferably a C ₁ -C ₆ alkoxy groups, more preferably a C ₁ -C ₄ alkoxy groups, most preferably n-butoxy, 2-methylpropoxy, 2,2- dimethylethoxy, n -propoxy, 2-methylethoxy, ethoxy or methoxy groups, and which alkyl and alkoxy groups contain Oto n halogen substituents, where n is the total number of substitutable hydrogen atoms of the alkyl or alkoxy group, and in which the halogen atoms are independently chlorine, bromine, iodine or fluorine atoms, most preferably fluorine atoms; as aryl group comprises substituted or unsubstituted phenyl or naphthyl groups as aralkyl group comprises, phenethyl, benzyl or naphthylmethyl groups which aryl and aralkyl groups having 0 to m substituents independently selected from C1-C4 alkyl, C1-C4 alkoxy groups, halogen atoms, which may be independently chlorine, bromine, iodine or fluorine, where m is the total number of hydrogen atoms of the aryl or aralkyl group;

R5 and R6 are independently hydrogen, ethyl, methyl, methoxy groups, or R5 and R6 together form -O-CH2-O- group, R7 is hydrogen atom, ethyl, methyl or methoxy group.

An even more preferred embodiment of the present invention in the compound of the general formula (I), tautomer or salts used for the detection of Ca ²⁺ ion as a fluorescent dye Ri and R2 stand for as an alkyl group hexyl, n-butyl, ethyl or methyl group, as alkoxy group methoxy and which alkyl or alkoxy groups contain 0 to n halogen substituents, where n is the total hydrogen atom of the alkyl or alkoxy group, and in which the halogen atoms are fluorine atoms, R ₅ and R6 independently stand for hydrogen atom, ethyl, methyl, methoxy groups, or R5 and R6 together form an -O-CH ₂ -O- group and R ₇ stands for a hydrogen atom, ethyl, methyl, or methoxy group.

A further, even more preferred embodiment of the present invention the use as dye the compound of the general formula (I), tautomer or salts thereof used for the detection of Ca ²⁺ ion wherein Ri and R ₂ as alkyl groups stand for hexyl, n-butyl, ethyl or methyl groups and R ₅ , R ₆ and R ₇ stand for hydrogen atom.

In a further, more preferred embodiment of the invention, the dye used for the detection of Ca ²⁺ ion wherein the dye is a two-component mixture of the isomers of the compounds of the general formula (I) in any ratio or any tautomeric forms of those isomers and their salts, and in which mixture the meanings of Ri, R ₂ , R ₅ , R6 and R ₇ are the same as those of the corresponding substituents of the other isomer and in one isomeric component R ₃ stands for a hydrogen and R ₄ stands for a group of general formula (II) and in the other isomeric component R ₃ stands for a group of general formula (II) and R stands for a hydrogen atom and a mixture of salts of these isomers.

In another preferred embodiment of the present invention, the dye used to detect Ca ²⁺ ion is the following compounds or mixtures of isomers of the compounds of general formula (I) or mixtures of two-component isomers in any ratio, or tautomeric forms and salts of the compounds: As described above, the compounds of general formula (I) of the present invention are used as a fluorescent dye for the detection of Ca ²⁺ ions most preferably in living cells or tissues. These fluorescent dyes are particularly suitable for single or multiphoton irradiation, preferably 2 photon irradiation microscopy imaging.

According to the present invention, the amount of Ca ²⁺ ions in physiological solutions was measured using the compound MZ137 of Example 5 of formula (l/E-A), which proved that the compound is excellent for the detection of Ca ²⁺ ions (Example 6). The results show that the compounds of formula (I) according to the invention, such as MZ137A dye, have a low basic fluorescence in the absence of calcium, which makes them suitable for imaging. Studies have shown that with increasing Ca-ion concentration, the fluorescence intensity increased significantly (x5), allowing Ca-ion detection. The cell cultures used in the in vitro studies were VeroE6 (atcc crl-1586) and VeroE4 monkey cell line and HEK293 human cell line, respectively. The light absorption of the compound does not change substantially with the formation of the complex (Fig. 2/a), its fluorescence changes as a function of the Ca ²⁺ ion concentration (Fig. 2/b). The titration curve is shown in Figure 1/a. The compounds are excellent for up to 2- photon laser excitation studies. The two-photon cross section is shown in Fig. 1/b. Furthermore, Example 7 illustrates the use of compounds of formula (I) in cell cultures. The results show that e.g. using the compound MZ137, the distribution of Ca ²⁺ ion concentration in the intra and extracellular parts can be examined in one assay using one dye. Healthy and apoptotic and dead cells are well distinguished. During a viral infection, the presence of different amounts of virus and the tissue damaging effect can also be monitored, as shown in this example.

Example 7 also shows that the compounds (I) of the invention are non-toxic and can be used in in vivo mouse experiments, e.g. to examine brain activities.

It has surprisingly been found that the dyes according to the invention are also suitable for the detection of COVID infection (see Example 8). It was found that when cell cultures were stained with the MZ137 dye of the present invention as described in Example 7, which contained a sample from suspected Covid-infected individuals, fluorescence increased abruptly in some samples, while only a very slow linear increase of fluorescence was detected in others. Samples in which there was a sudden increase in fluorescence in parallel PCR assays were found to give a COVID positive result, while those in which fluorescence was slowly and steadily increased were PCR negative samples. The increase in positive samples is believed to be due to the ability of the compounds of formula (I) according to the invention to intercalate in the presence of active viruses due to their molecular structure, size, and polarity, while significantly increasing the fluorescence of the incorporated compound. Thus, the PCR test can be replaced by a simpler method.

Thus, in a further preferred embodiment of the invention, the dyes of formula (I) according to the invention are suitable for the detection of SARS-COV-2 virus infection.

Thus, the present invention relates to the use of compounds of the general formula (I) and tautomeric forms and mixtures and salts thereof for the detection of SARS-COV-2 virus infection in which Ri and R2 are independently hydrogen, straight or branched, unsubstituted C1-C10 alkyl or C1-C10 alkoxy group or substituted by one or more halogen atoms, a substituted or unsubstituted aryl or aralkyl group, provided that one of Ri and R2 is at least not hydrogen, one of R ₃ and R ₄ stands for a hydrogen and the other is a group of general the formula (II) wherein R ₅ and R ₆ are independently hydrogen atom, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy group, or R ₅ and R ₆ together form a -O-CH ₂ -O- group, R ₇ stands for a hydrogen atom, a C ₁ -C ₄ alkyl group or a C ₁ -C ₄ alkoxy group.

More particularly, a compound of the general formula (I) is preferably used to detect SARS- COV-2 virus infection, wherein the Ri and R ₂ substituents of the compound, tautomer or salt thereof stand for a C ₁ -C ₁₀ alkyl groups, more preferably a C ₁ -C ₆ alkyl groups, more preferably C ₁ -C ₄ alkyl groups, most preferably hexyl, n-butyl, 2-butyl, tert-butyl, n-propyl, 2-propyl, ethyl or methyl groups, as alkoxy groups comprises C ₁ -C ₁₀ alkoxy groups, preferably C ₁ -C ₆ alkoxy groups, more preferably C ₁ -C ₄ alkoxy groups, most preferably n-butoxy, 2-methylpropoxy, 2,2- dimethylethoxy, n-propoxy, 2-methyl-ethoxy, ethoxy or methoxy groups, and which alkyl and alkoxy groups comprise 0 to n halogen substituents, where n is the total number of substitutable hydrogen atoms of the alkyl or alkoxy groups, and in which the halogen atoms are independently chlorine, bromine, iodine or fluorine atoms, most preferably fluorine atoms; as aryl group comprise substituted unsubstituted phenyl or naphthyl groups as aralkyl groups comprise, phenethyl, benzyl or naphthylmethyl groups having 0 to m substituents which are independently C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy groups, halogen atoms, which may be independently chlorine, bromine, iodine or fluorine atoms, where m is the total number of substitutable hydrogen atoms of the aryl or aralkyl groups; R ₅ and R ₆ stand for independently hydrogen atom, ethyl, methyl, methoxy groups, or R ₅ and R ₆ together form -0-CH2-0- group, R ₇ stands for a hydrogen, ethyl, methyl, or methoxy group.

More preferably, the use of compounds of the general formula (I) or its tautomer or salts for the detection of SARS-COV-2 virus infection wherein, Ri and R ₂ independently represent a hexyl, n-butyl, ethyl or methyl group as alkyl group and methoxy as alkoxy group as alkoxy group, and which alkyl or alkoxy groups contain 0 to n halogen substituents, where n is the total number of hydrogen atoms of the alkyl or alkoxy group, and wherein the halogen atoms are fluorine atoms, R ₅ and R ₆ stand independently hydrogen atom, ethyl, methyl group or methoxy group, or R ₅ and R ₆ together form -O-CH ₂ -O- group, R ₇ stands for a hydrogen atom, ethyl, methyl, or methoxy group. In another preferred embodiment, a compound of the general formula (I) is used wherein Ri and R ₂ represent independently alkyl, hexyl, n-butyl, ethyl or methyl groups and R ₅ , R6 and R ₇ represent hydrogen atoms.

According to the present invention, a mixture of isomers of a compound of the general formula (I) in any ratio ora mixture of any tautomeric forms of those isomers and salts thereof for detection of SARS-COV-2 virus infection in which the meaning of Ri, R2, R5, R ₆ and R7 substituents are the same in the corresponding substituents of another isomer and in the one isomeric component R ₃ represents a hydrogen atom and R represents a group of general formula (II), while in the another isomeric component R ₃ represents a group of general formula (II) and R4 represents a hydrogen atom.

Most preferably, the SARS-COV-2 virus infection can be detected by using compounds is selected from the compounds formula (l/EA), (l/EB), (l/BA), (l/BB), (l/HA), (l/HB) or from a mixture of the compounds of formula (l/EA) + (l/EB), (l/BA) + (l/BB), (l/HA) + (l/HB) in any proportion.

When using the dyes of the present invention, either the detection of Ca ²⁺ ions or the examination of COVID test samples, it is performed by single or multiphoton irradiation, preferably 2 photon irradiation microscopy imaging preferably.

Advantages of the present invention can therefore be summarized as follows:

Fluorescent dyes of general formula (I) according to the present invention capable to enter the cells from the extracellular space in the active state, in contrast to the prior art dyes, which can only be introduced into the cells one by one by micropipette in the active state, while the protected compounds enter the cells and there they are activated. Thus, it is not possible to measure extra- and intracellular Ca ²⁺ ion activity simultaneously. OGB-AM cannot lose its protecting groups in the extracellular part, and the acetyl protecting groups on the xanthine ring prevent fluorescence. The novel dye molecules of the present invention are easier to prepare. Alkyl groups, such as ethyl, butyl, hexyl groups are nonpolar groups, which make the molecule so nonpolar that it can surprisingly cross the cell membrane. Thus, with the compounds of formula (I) according to the present invention, functional imaging is possible, a complex image can be obtained with one dye.

The compounds of the general formula (I) according to the present invention are non-toxic and therefore they can be used in vitro and in vivo environments.

It can also be used in cell viability tests: it distinguishes the different phases of cells: 1) living (healthy), 2) infected, 3) apoptotic and 4) dead cells, as we show in the examples and figures.

The fluorescent dyes of the present invention show a change in calcium concentration through calcium ion channels. Furthermore, another practical advantage of these compounds is that they have green fluorescence, which requires a simpler laser and optical structure.

Since the use of the dyes of the general formula (I) according to the present invention allows functional imaging, it allows to examine viral infections, e.g. cellular examination of covid infection. As described in Example 7, cell cultures were stained with the MZ137 dye of the present invention that contained a sample from alleged Covid infected individuals. Samples were also tested in parallel on PCR assays. The resulting fluorescence curves were arranged in two rows. In the samples resulting in the positive PCR test, the fluorescence of the samples increased sharply after a certain number of cycles. The increase in positive samples is due to the fact that the compounds of general formula (I) according to the invention are able to intercalate in the presence of active viruses due to their molecular structure, size and polarity, i.e. to significantly increase the fluorescence of the incorporated compound. Thus, the PCR test can be replaced by a simpler method.

A 4/d. As shown in Fig. 2, curve 1, which contains measurements of PCR-positive samples, increases significantly, while PCR-negative samples, the data of which are shown in Fig. 2, show a significant increase give a series of curves, showing only slow linear growth. This method thus allows the MZ137 dye to be used as an alternative dye to the PCR test, e.g. for detecting of COVID infection.

The preparation of the compounds according to the invention requires the customary organic chemical means available at the pharmaceutical and fine chemical production sites. The preparation of the subject matter of the invention is within the knowledge of the person skilled in the art based on the present description. Among the starting materials, alkyl and and alkoxy resorcinol derivatives (formulas IX and X), e.g. methyl, ethyl, butyl, methoxy, ethoxy resorcinol are commercially available and the preparation of analogs thereof is well known to those skilled in the art. 5-Amino-BAPTA tetramethyl ester was synthesized according to Cell Calcium, 10, 491-498 (1989), but both 5-amino-Bapta methyl (Synchem UG & Co.) and ethyl esters (e.g., Tianjin heowns Biochemical Technology Co., Ltd.) are also commercially available.

List of figures:

Figure 1/a. The two-photon Ca-ion titration curve of MZ137 (compound (l/E-A)) was recorded by excitation at 506 nm and detection at 526 nm. I = intensity (detected at 526 nm)

Figure 1/b. Determination of the two-photon cross section of MZ137 as a function of excitation wavelength using a Chameleon (Discovery) laser.

Figure 2/a. The absorption spectrum of MZ137 without Ca-ion (in the presence of EGTA) and in the presence of 0.588 mM Ca-ion concentration (Ca-ion buffer was used).

Figure 2/b. The fluorescence spectra of MZ137 at different Ca-ion concentrations (Ca-ion buffer) excited at 506 nm.

Figure 3 / a. cell culture

Figure 3/b. staining of cell culture with MZ137 (l/E-A) dye Figure 3/c. automated scanning with non-linear optics Figure 3/d. image of samples in cell culture

Figure 3/e. cell culture (a. nucleus, b. cell membrane, c. intracellular fluid, d. extracellular fluid) microscopic images analyzed in VeroE6 cell lines (x20 Olympus objective, two-photon microscope).

Figure 3/f. cell culture (a. apoptotic cell, b. cell membrane, c. intracellular fluid, d. extracellular fluid) Microscopic images analyzed in VeroE6 cell lines (x20 Olympus objective, two-photon microscope).

Figures 4/a., 4/b. and 4 / c. These figures show, in order, how distributed the injected dye into the mouse brain over time. The sharp white spots are staining marks of the glia marker sulforhodamine 101, which indicate the location of astrocytes. Images are in vivo recordings. Figure 4/d. Curves recorded during PCR (LAMP) evaluation detected at 520 nm. The curves show both positive (ascending, 1) and negative samples (linear, 2). Figure 5/a. 2',7'-dietil fluorescein-5 carbonyl-amino-BAPTA /(l/E-A) / ¹³ C NMR spectra.

Figure 5/b. Figure: ¹ HNMR spectrum of 2',7'-diethyl-fluorescein-5-carbonylamino-BAPTA /(l/E-A)/

The present invention is illustrated by the following examples without limiting the scope of the invention to the following examples:

Used devices:

1. HPLC-MS

This method and equipment was used to monitor the progress of the reaction during the syntheses based on UV and mass data, and to check the purity of the product after purification of the products.

Device details: Device type: Shimadzu 20AD + SIL20A, binary pump

Thermostat: CTO20A: 40 ° C

PDA: SPDM20A 190-800 nm

Column: Supelco 5pm, 50 x 4.6 mm, C18

Eluent A: 0.1% TFA / distilled water

Eluent B: 0.1% TFA/ AcN

Flow rate: 1 ml / min

Gradient: Eluent B: 0% - 100% (10 minutes)

Shimadzu LCMS2020; ESI ionizer: 10,000 V;

Nitrogen flow: 250 ° C, 14 I / min Splitter: 0.5

2. Preparative HPLC purification

A process for separating the main products of each step.

Device details: Device type: Armen

Column type, parameters: Phenomenex, Gemini 250x50,00 mm; 10 pm, C18, with 110 A charge

Eluent A: 10 ml TFA / 5 dm ³ in deionized water

Eluent B: AcN Elution: gradient, optimized for each material, based on HPLC analysis.

3. Flash chromatographic purification

It was used to separate the main product for larger quantities.

Device: puriFlash xs 520 plus, Interchim Column: 120 g, normal silica gel

4. NMR spectroscopy

Varian 400 MHz and Varian 600 MHz. 10-15 mg of sample dissolved in 0.7 ml of solvent (CDCI3 or DMSO-d6).

5. Single photon spectroscopy

The UV / Visible spectra of the samples were recorded on a Thermo Scientific Evolution 220 spectrophotometer in the wavelength range 220 to 700 nm in a quartz cuvette with a light path of 1.0 cm and closed with a Teflon cup. For a stock solution of the samples, about 1-3 mg was dissolved in 25 mL of the chosen solvent (MeOH). These were then diluted with the same solvent to a concentration (10-80 mM) suitable for recording the absorption spectrum and determining the wavelength corresponding to the absorption maximum ( max) and the corresponding molar extinction coefficient (e).

Emission data were collected on a Hitachi F-4500 spectrophotometer at room temperature. A gap of 5 and 10 nm was applied to the excitation and emitted rays, with background correction (subtraction of the spectrum measured at the solvent). Sample solutions were diluted from stock solutions used in UV/visible spectroscopy. The final concentration used for fluorescence measurements is approx. It ranged from 1 to 5 mM. Samples were excited at (or near) the absorption maximum ( max), which was determined from the previously measured UV / Visible spectrum. Examples

Example 1: Preparation of a mixture of isomers of 2 7'-diethylfluorescein-5-carbonyl-5-amino- BAPTA (preparation of a mixture of isomers of formula (l/E-A) + (I / E-B))

Step 1.1 Preparation of a mixture of 2',7'-diethyl-6-hydroxy-3-oxofluorescein dicarboxylic acid isomers ((Vll-A) + (Vll-B) (Ri and R2 = ethyl))

4-Ethylbenzene-l,3-diol (5.5 g, 2 eq., 40 mmol) was dissolved in methanesulfonic acid (18 mL) and 1,2,4-benzyltricarboxylic anhydride (3.83 g, 1 eq., 20 mmol) was added and stirred at 100°C for 4 hours. The reaction was monitored by HPLC-MS. After completion of the reaction, the product mixture was poured into a mixture of ice and water (20 ml), the precipitated orange material was filtered off and washed with water, and finally dried in a vacuum oven. The obtained mixture of isomers contaminated with methanesulfonic acid (12.8 g) was used in the second step without further purification. LCMS (ESI) t[ _¾ : 7.049 min and 7.141 (purity: 92%), m / z: 433 [M+H ⁺ ], UV _max = 450 nm

Step 1.2. Preparation of a mixture of carboxy-2',7'-diethylfluorescein diacetate isomers /(V- A) + (V-B) where (Ri and R2 = ethyl)/

A mixture of isomers of 2',7'-cliethyl-6-hyclroxy-3-oxo-fluorescein dicarboxylic acid (1.2 g, 2.8 mmol) was dissolved in acetic anhydride (4 mL) and pyridine (0.72 mL) was added and stirred for 2 hours. The reaction was monitored by HPLC-MS. After completion of the reaction, the product mixture was poured into water and extracted with ethyl acetate (2 x 20 mL). The combined organic layers were washed with water (2 x 20 mL), 10% NaHCC>3 (1 x 20 mL), 1 M HCI (1 x 20 mL), then water (1 x 20 mL) and saturated NaCI (1 x 20 mL), then dried over MgSC , filtered off and the solution is evaporated. The thus obtained product as a yellow powder (240 mg, 18%). LCMS (ESI) t _R : 9.417 min and 9.502 min, (purity: 92%), m/z: 517 [M+H ⁺ ], UV _max = 294 nm

Step 1.3. Preparation of a mixture of 2',7'-diethyl fluorescein diacetate 5-amino BAPTA tetramethyl ester isomer mixture /(lll-A) + (lll-B) (Ri and R2 = ethyl)/

A mixture of the isomeric mixture of carboxy-2',7'-diethyl fluorescein diacetate (240 mg, 1 equivalent, 0.46 mmol) was dissolved in dichloromethane (7 mL) and placed in an ice bath. 125 ul of DIPEA (2 equivalents, 0.97 mmol) was added, followed by 60 ul of isobutyl chloroformate (1 equivalent, 0.46 mmol) and stirred for 1 hour. Then 321 mg of amino-BAPTA (1.1 equivalents, 0.53 mmol) are added. The reaction was monitored by HPLC-MS. After completion of the reaction, the product mixture was poured into water (20 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with 10% NaHCC>3 and then saturated NaCI solution. It was dried over MgSC , filtered off and the solution is evaporated. The obtained product was 80 mg (15%) of a yellow powder. LCMS (ESI) t[ _¾ : 11.178 min and 11.192 min, (purity: 65%), m/z: 552 [M+2H ⁺ ], UV _ma x = 298 nm.

^X H NMR (400 MHz, cdcl ₃ ) d 8.72 - 8.64 (m, 1H), 8.56 (s, 1H), 8.48 (s, 1H), 8.30 (dd, J = 8.1, 1.6 Hz, 1H), 8.25 - 8.15 (m, 2H), 8.12 - 7.97 (m, 6H), 7.5 (m, 2H), 7.36 - 7.28 (m, 4H), 7.02 (s, 2H), 6.88 (dd, J = 18.0, 8.0 Hz, 4H), 6.62 (s, 2H), 4.67 (s, 8H), 4.40 - 4,25 (m, 4H), 4.36 - 4.20 (m, 2H), 2,45 - 2.30 (m, 12H), 1.16 (dt, J = 9.7, 7.1 Hz, 12H), 1.01 (t, J = 7.5 Hz, 6H).

¹³ C NMR (101 MHz, cdcl ₃ ) d 171.83, 171.58, 168.96, 168.61, 165.46, 163.72, 155.56, 150.41, 150.36, 149.65, 137.24, 134.88, 134.13, 133.68, 132.26, 130.99, 130.24, 129.73, 129.64, 128.81, 128.05, 126.52, 124.74, 123.61, 121.51, 119.29, 119.16, 115.76, 113.68, 113.06, 111.10, 107.22, 77.22, 69.00, 67.56, 66.95, 64.27, 63.55, 63.01, 62.92, 61.03, 60.94, 60.85, 53.76, 29.69, 23.01, 20.90, 14.13, 14.06, 14.03.

Step 1.4. Preparation of 2',7'-diethyl fluorescein carbonylamino BAPTA (5 and 6 carbonylamino) isomeric mixture. / (l/E-A) + (l/E-B)

In 2 ml of acetonitrile 80 mg of the isomer mixture 2',7'-diethyl fluorescein diacetate BAPTA tetramethyl ester was dissolved, then 321 mg of NaOH dissolved in 0.5 ml of water was added. The mixture was stirred at room temperature for 1 hour. The reaction was monitored by HPLC- MS. After completion of the reaction, the crude product was purified by preparative HPLC and lyophilized. 49 mg of product were obtained. LCMS (ESI) t[ _¾ : 7.5S2 min and 7.548 min, (purity: 96%), m/z: 454 [M+2H ⁺ ] UVmax = 451 nm.

Example 2: Preparation of a mixture of isomers of 2',7'-dibutylfluorescein-5-amino BAPTA (preparation of a mixture of isomers of formula (l/B-A) + (l/B-B))

Step 2.1 Preparation of a mixture of 2',7'-dibutyl-6-hydroxy-3-oxofluorescein dicarboxylic acid isomers ((Vll-A) + (Vll-B) (Ri and R2 = butyl))

4-Butylbenzene-l,3-diol (2 g, 12.03 mmol) was dissolved in methanesulfonic acid (3 mL), 1,2,4- benzyltricarboxylic anhydride (1.15 g, 6.015 mmol) was added, and the mixture was stirred at 100 C for 4 hours. The reaction was monitored by HPLC-MS. After completion of the reaction, the product mixture was poured into a mixture of ice and water (20 ml), the precipitated orange material was filtered off and washed with water, and finally dried in a vacuum oven. A mixture of isomers contaminated with methanesulfonic acid (3.11 g) was obtained and the product was used in the next step. LCMS (ESI) t[ _¾ : 6.604 min, m/z: 489 [M+H ⁺ ] UVmax = 452 nm

Step 2.2 Preparation of a mixture of carboxy-2',7'-dibuyl fluorescein diacetate isomers /(V- A) + (V-B) where (Ri and R2 = butyl)/ A mixture of the isomeric mixture of 2',7'-clibutyl-6-hyclroxy-3-oxofluorescein dicarboxylic acid (3.1 g, 6.3 mmol) was dissolved in acetic anhydride (12 mL) and pyridine (1.9 mL) was added, then the mixture was stirred for 2 hours. The reaction was monitored by HPLC-MS. After completion of the reaction, the product mixture was poured into water and extracted with ethyl acetate (2 x 30 mL). The combined organic layers were washed with water (2 x 30 mL), 10% NaHCC>3 (1 x 30 mL), 1 M HCI (1 x 30 mL), again with water (1 x 30 mL), and saturated NaCI (1 x 30 mL), then it was dried over MgSC , filtered and the solution is evaporated. The crude product (3.44 g) was dissolved in 40 mL of a solvent mixture (ACN: EtOH: water = 7: 2: 1) and purified by preparative HPLC. The pure fractions were collected, the acetonitrile was evaporated from the mixture and the aqueous solution was lyophilized after freezing. The product was obtained as a yellow powder (1.38 g, 38%). LCMS (ESI) t[ _¾ : 10.298 min (purity: 100%), m/z: 573 [M+H ⁺ ] UV _max = 294 nm

NMR

^X H NMR (400 MHz, dmso) d 13.64 (s, 2H), 8.29 (dd, J = 14.1, 8.0, 1.5 Hz, 2H), 7.24 (d, J = 1.4 Hz, 2H), 6.77 (d, J = 12.9 Hz, 3H), 2.31 (s, 4H), 1.27 (p, J = 7.5, 7.1 Hz, 4H), 1.21 - 1.05 (m, 4H), 0.75 (q, J = 7.0 Hz, 6H).

¹³ C NMR (101 MHz, dmso) d 168.91, 167.74, 166.05, 166.01, 155.73, 152.48, 150.36, 150.32, 148.98, 148.89, 137.49, 136.33, 133.09, 131.03, 130.88, 129.04, 128.91, 126.25, 125.70, 124.57, 124.45, 115.64, 115.58, 111.16, 81.54, 31.77, 31.62, 28.50, 28.45, 21.63, 21.55, 20.65, 13.54, 13.51.

Step 2.3. Preparation of a mixture of 2',7'-dibutyl fluorescein diacetate 5-amino BAPTA tetraethyl ester isomer /(lll-A) + (lll-B) (Ri and R2 = butyl)/

The mixture of acylated isomers (150 mg, 0.26 mmol) was dissolved in dichloromethane (6 mL) and placed in an ice bath. 50 ul of DIPEA (0.29 mmol) was added followed by 37.5 ul of isobutyl chloroformate (0.29 mmol) and stirred for 6 hours. Amino-BAPTA (237 mg, 0.39 mmol) was added and stirred at room temperature for 3 hours. The reaction was monitored by HPLC-MS. After completion of the reaction, the mixture was extracted between with water and the organic layer was dried over MgSC , filtered and the solution is evaporated. The concentrated mixture was dissolved in 10 mL of a solvent mixture (ACN: EtOH: water = 7: 2: 1) and purified by preparative HPLC. The pure fractions were collected, the acetonitrile was evaporated off the mixture and the aqueous solution was lyophilized after freezing. The product obtained as 105.9 mg (35%) of a yellow powder. LCMS (ESI) t[ _¾ : 9.994 min (purity: 99%), m/z: 580 [M+2H ⁺ ] UV _max = 298 nm

Step 2.4. Preparation of 2 ^, ,7'-dibutylfluorescein-5-amino-BAPTA isomeric mixture /(l/B-A) + (l/B-B)/ To 2',7'-dialkyl (diaryl) fluorescein diacetate BAPTA tetraethyl ester (60 mg, 0.05 mmol) dissolved in 4 mL of ethanol and NaOH (20.7 mg, 0.5 mmol) dissolved in water (0.4 mL) was added to the mixture. It was stirred at room temperature overnight. The reaction was monitored by HPLC-MS. After completion of the reaction, the mixture was evaporated. The concentrated mixture was dissolved in 10 mL of a solvent mixture (ACN: EtOH: water = 3: 4: 3) and purified by preparative HPLC. The pure fractions were collected, the acetonitrile was evaporated off the mixture and the aqueous solution was lyophilized after freezing. Thus obtained 37.8 mg (76%) of an orange powder. LCMS (ESI) t[ _¾ : 6.356 min and 6.465 min (purity: 99%), m/z: 482 [M+2H ⁺ ] UV _max = 452 nm.

Based on ¹ H-NMR, the ratio of isomer (A) to isomer (B) is 0.65 / 0.35.

^X H NMR (600 MHz, DMSO -d ₆ ) d 12.33 (bs, 2H), 10.41 (s, 0.65H), 10.05 (bs, 1.35H), 8.59 (d, J = 1.7 Hz, 0.65H), 8.31 (ddd, J = 21.0, 8.1, 1.5 Hz, 1H), 8.14 (d, J = 8.1 Hz, 0.35H), 7.48 (s, 0.35H), 7.50-7.20 (m, 3H), 6.99 (m, 1H), 6.90 - 6.73 (m, 4H), 6.73 - 6.70 (m, 2H), 6.40 (d, J = 9.4 Hz, 2H), 4.34 - 4.17 (m, 4H), 4.06 (s, 5H), 4.02 (d, J = 4.5 Hz, 3H), 2.35 (m, 4H), 1.31 (p, J = 7.6 Hz, 4H), 1.21 - 1.09 (m, 4H), 0.77 (dt, J = 9.6, 7.3 Hz, 6H).

Example 3. Preparation of a mixture of isomers of 2',7'-dihexyl fluorescein-5-amino-BAPTA (preparation of a mixture of isomers of formula (l/H-A) + (l/H-B))

Step 3.1. Preparation of a mixture of 2',7'-dihexyl-6-hydroxy-3-oxofluorescein dicarboxylic acid isomers /(Vll-A) + (Vll-B) (Ri and R2 = hexyl)/ 4-Hexylbenzene-l,3-diol (1 g, 5.15 mmol) was dissolved in methanesulfonic acid (3 mL), and 1,2,4-benzyltricarboxylic anhydride (0.5 g, 2.575 mmol) was added and stirred at 100°C for 4 hours. The reaction was monitored by HPLC-MS. After completion of the reaction, the product mixture was poured into ice water (20 ml), the precipitated orange material was filtered off and washed with water, and finally dried in a vacuum oven. A thus given mixture of isomers contaminated with methanesulfonic acid (1.61 g) was used in the next step. LCMS (ESI) t[ _¾ : 7.477 min and 7.619 min, m/z: 545 [M+H ⁺ ] UV _max = 452 nm

Step 3.2. Preparation of a mixture of carboxy-2',7'-dihexyl fluorescein diacetate isomers /(V-A) + (V-B) where (Ri and R2 = hexyl)/

A mixture of the isomeric mixture of 2',7'-clihexyl-6-hyclroxy-3-oxo-fluorescein dicarboxylic acid (1.6 g, 2.9 mmol) was dissolved in acetic anhydride (6.2 ml) and pyridine (1 ml) was added and the mixture was stirred for 2 hours. The reaction was monitored by HPLC-MS. After completion of the reaction, the product mixture was poured into water and extracted with ethyl acetate (2 x 20 mL). The combined organic layers were washed with water (2 x 20 mL), 10% NaHCC>3 (1 x 20 mL), 1 M HCI (1 x 20 mL), water (1 x 20 mL), and saturated NaCI (1 x 20 mL). It was dried over MgSC>4, filtered off and the solution is evaporated. The crude product (1.64 g) was dissolved in 20 ml of a solvent mixture (ACN: EtOH: water = 7: 2: 1) and purified by preparative HPLC. The pure fractions were collected, the acetonitrile was evaporated from the solution and the aqueous solution was lyophilized after freezing. The product was obtained as a yellow powder (0.78 g, 42%). LCMS (ESI) t[ _¾ : 9.606 min (Purity: 98%), m/z: 629 [M+H ⁺ ] UV _max = 294 nm.

NMR:

^X H NMR (400 MHz, dmso) d 8.28 (bs, J = 10.9, 8.0 Hz, 2H), 7.24 (m, 4H), 6.75 (d, J = 12.6 Hz, 3H), 2.32 (d, J = 9.7 Hz, 10H), 1.27 (hept, J = 7.2, 6.8 Hz, 4H), 1.15 - 1.03 (m, 14H), 0.75 (dd, J = 8.1, 4.0 Hz, 6H).

¹³ C NMR (101 MHz, dmso) d 168.89, 167.69, 165.94, 155.84, 152.54, 150.37, 150.34, 148.95, 148.86, 137.53, 133.08, 131.23, 130.89, 130.75, 129.09, 128.99, 128.86, 126.17, 115.52, 115.46, 111.15, 81.50, 30.77, 30.75, 29.28, 29.15, 28.57, 27.96, 27.88, 22.01, 21.97, 20.63, 13.82.

Step 3.3. Preparation of isomeric mixture of 2',7'-dihexyl fluorescein diacetate-5-amino- BAPTA tetraethyl ester /(lll-A) + (lll-B) (Ri and R2 = hexyl)/ The mixture of acylated isomers (150 mg, 0.24 mmol) was dissolved in dichloromethane (6 mL) and placed in an ice bath. 46 ul of DIPEA (0.26 mmol) was added followed by 34 ul of isobutyl chloroformate (0.26 mmol) and the mixture was stirred for 6 hours. Then amino- BAPTA (173 mg, 0.29 mmol) was added and stirred at room temperature. The reaction was monitored by HPLC-MS. After completion of the reaction, the mixture was extracted with water and the organic layer was dried over MgSC , filtered off and the solvent and evaporated. The concentrated mixture was dissolved in 10 mL of a solvent mixture (ACN: EtOH: water = 7: 2: 1) and purified by preparative HPLC. The pure fractions were collected, the acetonitrile was evaporated off the solution and the aqueous solution was lyophilized after freezing. As product 71.4 mg (24.6%) of an off-white solid is obtained. LCMS (ESI) t[ _¾ : 10.602 min (purity: 98%), m/z: 608 [M+2H ⁺ ] UV _max = 294 nm

Step 3.4. Preparation of Z'^'-dihexylfluorescein-S-amino-BAPTA isomer mixture /(l/H-A) + (l/H-B)/

To 2',7'-dialkyl (diaryl) fluorescein diacetate BAPTA tetraethyl ester (130 mg, 0.2 mmol) dissolved in dioxane (5 mL) was added ethanol (5 mL) then KOH (112 mg, 2 mmol) dissolved in water (1 mL) is added to the mixture. It was stirred at room temperature overnight. The reaction was monitored by HPLC-MS. After completion of the reaction, the mixture was evaporated. The concentrated mixture was dissolved in 10 mL of a solvent mixture (ACN: EtOH: water = 3: 4: 3) and purified by preparative HPLC. The pure fractions were collected, the acetonitrile was evaporated off and the aqueous solution was lyophilized after freezing. The formed product was 38 mg (16%) of an orange powder. LCMS (ESI) t _R : 7.160 min and 7.221 min (purity: 99%), m/z: 509 [M+2H ⁺ ] UV _max = 453 nm.

Example 4: Preparation of 2',7'-diethyl fluorescein-5-carboxylamino-BAPTA (preparation of isomer of formula (l/E-A))

Step 4.1. Preparation of a mixture of 2',7'-diethyl-6-hydroxy-3-oxofluorescein dicarboxylic acid isomers ((Vll-A) + (Vll-B) (Ri es R2 = ethyl))

This step is described in step 1.1 of Example 1. The isomeric mixture prepared by that method.

Step 4.2. Preparation and separation of a mixture of 2-(2,4-dihydroxy-5-ethylbenzoyl) terephthalic acid (Vlll-A) and (Vlll-B) isomers /(Vlll-A) and (Vlll-B) (Ri = ethyl)/

The isomeric mixture prepared according to Example 4.1 (12.6 g, ca. 0.04 mol, overweight, contaminated with methanesulfonic acid) was dissolved in 28 ml of 40% aqueous sodium hydroxide and stirred at 100°C for 7 days. The reaction was monitored by HPLC-MS. After completion of the reaction, the product mixture was poured into water and, after cooling, acidified to pH 2 with using 4M hydrochloric acid. The aqueous layer was extracted with EtOAc and THF. The organic layer was washed with NaCI, then concentrated and dried in a vacuum oven. The product contained the pure component VII I/A, the other isomer (Vlll/B) was hydrolyzed, not recovered. The crude product recovered was 7.57 g (overweight).

LCMS (ESI) t _R : 6,714 min, (purity: 99 %), m/z: 433 [M+EG], UV _max = 443 nm Step 4.3. Preparation of 5-carboxy-2',7'-diethylfluorescein diacetate ((V-A) where (Ri and R2 = ethyl))

The procedure is the same as described in step 2 of Example 1, except that instead of the use of mixture of the 2',7'-diethyl-6-hydroxy-3-oxofluorescein dicarboxylic acid isomer, only one of the isomers, the 2', 7'-Diethyl-6-hydroxy-3-oxofluorescein-3,5-dicarboxylic acid was used.

LCMS (ESI) t _R : 9,417 min, (purity: 91 %), m/z: 517 [M+EG], UV _max = 294 nm

Step 4.4. Preparation of 2',7'-diethyl fluorescein diacetate-5-carbonylamino-BAPTA- tetraethyl ester / (lll-A) (Ri and R2 = ethyl) /

The procedure is the same as described in Step 3 of Example 1, except that 5-carboxy-2',7'- diethylfluorescein diacetate was used as the starting carboxylic acid component.

LCMS (ESI) t _R : 9,994 min (purity: 97 %), m/z: 580 [M+2EG] UV _max = 298 nm

Step 4.5. Preparation of 2',7'-diethyl fluorescein-5-carbonylamino-BAPTA /(l/E-A)/

The procedure of step 4 of Example 1 was repeated except that 2',7'-diethylfluorescein diacetate-5-carbonylamino-BAPTA-tetraethyl ester was used as starting material.

LCMS (ESI) t _R : 7,532 min es 7.548 min, (purity: 98 %), m/z: 454 [M+2EG] UV _max = 451 nm

^X H NMR (600 MHz, DMSO -d ₆ ) d 12.38 (bs, 3H), 10.41 (s, 1H), 10.12 (bs, 2H), 8.58 (s, 1H), 8.32 (dd, J = 8.0, 1.7 Hz, OH), 7.46 (d, J = 2.3 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.36 (dd, J = 8.7, 2.3 Hz, 1H), 7.03 - 6.99 (m, 1H), 6.90 - 6.84 (m, 2H), 6.80 (d, J = 8.8 Hz, 1H), 6.78 - 6.75 (m, 1H), 6.72 (s, 2H), 6.40 (s, 2H), 4.40 - 4.17 (m, 4H), 4.06 (s, 8H), 2.37 (ddp, J = 21.7, 14.7, 7.4 Hz, 4H), 0.94 (t, J = 7.5 Hz, 6H). (Figure 5/b)

¹³ C NMR (151 MHz, dmso) d 172.42, 168.22, 163.44, 158.20, 157.96, 149.98, 149.55, 149.47, 139.36, 136.64, 135.61, 132.85, 127.32, 121.53, 121.16, 118.52, 118.43, 115.05, 113.52, 108.57, 107.50, 101.81, 67.25, 67.11, 53.50, 22.36, 14.15. (Figure 5/a)

Example 5: Preparation of 2',7'-diethyl fluorescein-6-carboxylamino-BAPTA (preparation of isomer (l/E-B)) Step 5.1. 2 ^, ,7 ^, -Diethyl-6-hydroxy-3-oxofluorescein-3,6-dicarboxylic acid ((Vll-B) (Ri and R2 = ethyl))

4-Ethylbenzene-l,3-diol (1 equivalent) and 2-(2,4,-dihydroxy-5-ethylbenzoyl) terephthalic acid which was prepared similarly as described in Step 2 of Example 4 and separated by preparative HPLC during work-up (1 equivalent) was dissolved in 10 ml/g of methanesulfonic acid and stirred at 100°C for 1 hour. The reaction was monitored by HPLC-MS. After completion of the reaction, the crude product was dissolved in 10 ml of a solvent mixture (ACN: TEA: water = 8: 1: 1) and purified by preparative HPLC. The pure fractions were collected, the acetonitrile was evaporated off and the aqueous solution was lyophilized after freezing. The product thus obtained was used in the next step.

Step 5.2. Preparation of 6-carboxy-2 ', 7'-diethylfluorescein diacetate ((V-B) where (Ri and R ₂ = ethyl))

The procedure was carried out in same way as it is described in Step 3 of Example 4, except that instead of 2',7'-diethyl-6-hydroxy-3-oxofluorescein-3,5-dicarboxylic acid, 2',7'-diethyl-6- hydroxy-3-oxofluorescein-3,6-dicarboxylic acid was used which was prepared according to Step 5.1.

Step 5.3. Preparation of 2',7'-diethyl fluorescein diacetate-6-carbonylamino-BAPTA- tetraethyl ester /(lll-B) (Ri and R ₂ = ethyl) /

The procedure of Step 4 of Example 4 was repeated except that 6-carboxy-2',7'- diethylfluorescein diacetate was used as the starting carboxylic acid component.

Step 5.4. Preparation of 2',7'-diethyl fluorescein-6-carbonyl-5-amino-BAPTA /(l/E-B)/

The procedure of Step 5 of Example 4 was repeated except that as starting material 2', 7'- diethylfluorescein diacetate-6-carbonylamino-BAPTA-tetraethyl ester was used.

Example 6

Measurement of Ca-ion at physiological pH A fresh solution of the compound of formula (l/EA) according to Example 4 (MZ137) was prepared by accurately weighing about 1 mg of the substance dissolved in MOPS buffer (10 cm3) pH 7.4 (physiological) and with 10% of EtOH, that the thus given stock solutions have a concentration of ~100 mM. Then two Ca ²⁺ ion buffer solutions were prepared, one (Solution A) containing 1 mM EGTA and the other containing 1 mM Ca ²⁺ ion in addition to 1 mM EGTA (Solution B). Buffers were also prepared in MOPS. Then 0.05 cm ³ of dye stock solution is added to 10 cm ³ of the prepared solution A and 0.10 cm ³ is added to 20 cm ³ of solution B, so that in both cases the concentration of the sensor molecule was the same (1 pM). After that, we changed the small parts of solution A to solution B, so we practically increased the concentration of free Ca ²⁺ in very small steps, which was determined by the given buffer composition. The calculation of the current log [Ca ²⁺ ] _free values are calculated automatically by an algorithm written in a spreadsheet, giving only the replaced quantity. The exchange is always carried out in a cuvette, the contents of which are homogenized with a small magnetic stir bar. Thus, the fluorescence was measured at the maximum fluorescence emission wavelength in solutions containing several concentrations of buffered Ca ²⁺ ion. The work was performed according to the SOP used by Thermo Fischer Scientific. In all cases, the exchange of solution parts is done empirically, depending on the result of the last measurement. The results of the measurements are shown in Figure 1. The data show that MZ137A significantly increases the fluorescence response (by about 5-fold) with increasing Ca ion concentration. MZ 137A gives a weak basic fluorescence even without Ca ion, which is very useful because the contours of the cell and cell culture can be seen even without Ca ion. In this case, no special staining procedure is required during imaging.

Example 7 Use of compounds of formula (I) in in vivo and in vitro assays

Cell lines used: VeroE6 (atcc crl-1586) and VeroE4 monkey cell line and HEK293 human cell line, respectively.

Preparation of cell cultures on plates were performed using the standard procedure.

Optical scanner unit specifications

Working distance: 2-8 mm

Lens: water-immersion 16x lens (Olympus x20)

Field of view: 900 x 900 pm.

Resolution: <500 nm Scanning speed: 16 s / FOV = 25 s / sample holder = 2500 s / plate (40 min / plate)

Laser: Pulse laser 690-1040 nm, eg Chameleon (Discovery)

Excitation at 700 or 740 nm

Detector: multi-channel tunable detection

Sample preparation, and steps of measurement:

1. Cell culture 1 (Figure 3/a)

2. Cell staining with MZ137 (1/ E-A) dye (Figure 3/b)

3. Automated scanning with non-linear optics (Figure 3/c)

4. Image of the samples in the cell culture (Figure 3/d)

Presentation of measurement results:

Microscopic images analyzed in VeroE6 cell lines (x20 Olympus objective, two-photon microscope).

3 / e. Figure: Microscopic image of the cell culture: a. nucleus b. cell membrane c. intracellular fluid d. extracellular fluid

3 / f. Microscopic image of a cell culture of a culture containing apoptotic cells: a. apoptotic cells b. cell membrane c. intracellular fluid d. extracellular fluid

Demonstration of the effect of viral infection

Microscopic image of VeroE6 cell culture in a sample well on a 96-well plate and the process of viral infection stained with MZ137 dye (Olympus x20):

3/g. Healthy cell culture. (Healthy cells are circled in the image.)

3/h. Low virus concentration. (Healthy and apoptotic cells are circled in the image.) 3/i. Medium virus concentration. (Apoptotic cells are circled in the image.)

3/h. High virus concentration. (The picture shows large dead areas.)

In vivo testing of MZ137 dye:

Images show the effect when injected into mouse brain tissue. It is clear from the image on the left that the neurons appear as dark spots (Fig. 4a), while in the middle (Fig. 4b) and right images (Fig. 4c) a small amount of dye has already entered the neurons. Astrocytes also took up the dye and the specific glia marker sulforhodamine 101 (white, near-white spots).

8. Histological detection of SARS-COV-2 virus infection (intercalation of MZ137 dye in the presence of SARS-COV-2 virus)

The intercalating efficacy of the MZ137 dye was also verified in parallel PCR assays on samples taken from allegedly Covid-infected patients.

The sample tested according to the present invention was taken by using a sample taken from the nasal or pharyngeal mucosa for PCR analysis according to the prior art by the standard method (SWAB). Following RNA isolation, the solution was stained with MZ137 before PCR or LAMP and then amplified by standard PCR in the presence of the appropriate primer. The resulting fluorescence curves are arranged in two rows. In the samples resulting in the positive PCR test, the fluorescence of the samples increased sharply after a certain number of cycles.

A 4/d. As shown in Fig. 2, curve 1, which contains measurements of PCR-positive samples, increases significantly, while PCR-negative samples, the data of which are shown in Fig. 2, show only slow linear growth. Thus, this method allows the detection of COVID infection as an alternative to the PCR test.