WO2014202814A1 - Flourescent compounds - Google Patents

Abstract

The invention relates to the compounds of formula I, a kit comprising said compounds, and to the use of said compounds as selective markers for mitochondria. The compounds are designed such that they can be internalised by the living cell, selectively accumulate in the mitochondria, and stain said mitochrondria in preference to other cell organelles. The compounds also have an increased photostability. In addition, the compounds according to the invention can be used in cell cultures, both in primary cultures and cell lines, in living cells or paraformaldehyde-fixed living cells, with the selectivity and staining efficacy thereof being maintained, allowing same be used in real-time visualisation experiments.

Description

 Fluorescent compounds

Technical sector

 The present invention is directed to the compounds of formula I, a kit comprising said compounds and their application as selective markers for mitochondria.

Background of the invention

 Mitochondria are responsible for the production of more than 90% of the cellular energy of mammals and play a fundamental role in the cellular function and of certain tissues. Due to its critical involvement in the cells it is important to detect them and identify their behavior, which depends on the metabolic state of the cell. In this sense, selective fluorescent staining agents for mitochondria have been designed and several are commercial, for example Mitotracker ® or Rodaminal23.

The publication Cancer Research, 2002, 62, 7219-7229, describes diphenylfuran diamidines and how the incorporation of aromatic rings as end groups of furamidine side chains can influence the accumulation of said compounds between the nucleus and the cytoplasm. Specifically, it is described that furamidine derivatives that possess an aromatic or heteroaromatic ring in the terminal position of a side chain accumulate more easily in the cytoplasm, mostly in the mitochondria, unlike the corresponding alkyl derivatives that accumulate selectively in the nucleus.

 The recent Journal of the American Chemical Society, 2012, 135, 62-65, describes a compound that combines an aggregation-induced light-emitting group and two triphenylphosphonium groups, which is known to direct the entry of probes to mitochondria. The resulting compound showed specificity for mitochondria and higher photostability than other reagents.

However, commercial staining agents have problems and the industry is constantly looking for fluorescent agents with improved properties. For example, in many cases these agents have low photostability, poor selectivity and / or insufficient internalization in mitochondria. Thus, it is still necessary to design compounds that accumulate selectively in the mitochondria, are able to label them fluorescently and have high photostability.

Brief Description of the Invention

 The authors of the present invention have developed new compounds with properties that make them suitable for use in research applied to cells. These compounds are designed to be able to internalize in the living cell, selectively accumulate in the mitochondria. More specifically, the authors found that bis-amidinium-derived compounds with extended aromatic systems are able to internalize in the cell with surprising effectiveness and selectively accumulate in the mitochondria. Additionally, the compounds of the invention can be applied in cell cultures, both primary cultures and cell lines, in living cells that can also be subsequently fixed, for example, with paraformaldehyde.

In addition, it has been found that some compounds of the invention are fluorescent and stain mitochondria selectively. In addition, they have surprisingly high photostability, so that they can be irradiated for a long time without their photodegradation taking place, which allows a longer observation time during the experiment. The photostability of these compounds allows a wide variety of experiments to be designed, for example irradiating the sample at different times during the experiment or the cell study, irradiating the sample for a long time, or combining these compounds with other reagents during the experiment. They also allow the realization of real-time visualization experiments (time-resolved fluoride escence). It has also been proven that they are not cytotoxic, and that they allow monitoring the mitochondrial recycling process, which gives it added value. Thus, in one aspect the invention is directed to compounds of formula I

 I

 where A and B are each independently selected from optionally substituted aryl and heteroaryl,

 each of n, m, or, q is independently selected from 1, 2, or 3,

 W is selected from single bond, optionally substituted alkyl, aryl and heteroaryl,

 Ri is selected from hydrogen, hydroxyl, -OR ', -N (R') (R ") -, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, azido, tetrazino and an acceptor fluorophore group,

 K is selected from single bond, alkyl, -O-alkyl-, -O-alkyl-N (R ') -, heterocycle and heteroaryl,

each of R _a and R _b is independently selected from hydrogen and a photolabile protecting group,

each of Pi and P ₂ are independently selected from hydrogen, Ci-alkyl

C12, C ₆ -Ci5 aryl, C3-C15 heteroaryl and optionally substituted C3-C15 heterocycle, N0 ₂ , CN, halogen, -OR ', -SR', -S (0) R ', -S (0) ₂ R ', -OS (0) ₂ R', -N (R ') (R "), - C (0) R', -C (0) OR ', -C (0) N (R') (R "), -OC (0) R 'and - N (R') C (0) R",

each R 'and R "is independently selected from hydrogen, Ci-C ₆ alkyl, C3-C7 cycloalkyl, C ₆ -Ci5 ri, C3-C15 heteroaryl and optionally substituted C3-C15 heterocycle,

 and comprises at least one organic or inorganic anion to maintain neutrality.

In another aspect the invention is directed to a kit for cell study comprising a compound of formula I as described above.

In another aspect the invention is directed to the use of a compound of formula I as described above, as a selective reagent for mitochondria. In a particular embodiment the reagent is a marker. In a particular embodiment, the invention is refers to the use of a compound of formula I as described above as a staining agent.

In another aspect the invention is directed to a method for labeling mitochondria or a mitochondrial component comprising incubating a sample, wherein said sample comprises mitochondria or a mitochondrial component, with an aqueous solution of a compound of formula I as defined above.

 In a particular embodiment, the invention is directed to a method for staining mitochondria or a mitochondrial component, which comprises incubating a sample, wherein said sample comprises mitochondria or a mitochondrial component, with an aqueous solution of a compound of formula I as defined. above, where said compound is in an amount sufficient to stain mitochondria or a mitochondrial component and incubation takes place for a time sufficient to detect fluorescence.

A further aspect of the invention is a process for the preparation of a compound of formula I

 I

 which comprises the reduction of a compound of formula IX

IX

and optionally coupling with a photolabile protective group, where A, B, Pi, P ₂ , m, n, or, q, W, K, Ri, Ra, Rb are as defined above, and comprises at least one organic or inorganic anion to maintain neutrality. A further aspect of the invention are the compounds of formula IX and X used as intermediates in the synthesis of the compounds of formula I.

Description of the figures

Figure 1. Above: Structure of the Compound (3). Bottom: Water fluorescence emission spectrum of Compound (3). Excitation wavelength 329 nm, the spectrum is collected from 385 to 600 nm.

 Figure 2. Top row: Left: CEF cells incubated with Compound (3) 5μΜ and observed in the blue channel at 1000X. Center: CEF cells incubated with MitoTracker 500 nM and observed in the red channel at 1000X. Right: Overlap of both images demonstrating the mitochondrial location of Compound (3) in a primary culture. Bottom row: Same protocol performed on VERO cells fixed with para-formaldehyde.

 Figure 3. Left column: Different cell lines incubated with Compound (3) 5μΜ, observed in the blue channel at 1000X. Central column: Same cell lines incubated with MitoTracker 500 nM, observed in the red channel at 1000X. Right column: Superposition of both images that demonstrates the mitochondrial location of Compound (3) in different immortal lines.

 Figure 4. Above: Vero cells incubated with Rodaminal23 2μΜ for 30 min and observed in the green channel at 1000X at time 0, 30 and 60 seconds. Medium: Vero cells incubated with MitoTracker 500 nM for 30 min and observed in the red channel or at 1000X at time 0, 30 and 60 seconds. Below: Vero cells incubated with Compound (3) 5 μΜ for 30 min and observed in the red channel at 1000X at time 0, 2 and 8 minutes.

Figure 5. Left: Vero cells incubated with Compound (3) 5μΜ observed in the blue channel at 1000X after 24 hours. Center: Vero cells incubated with Rodaminal23 2μΜ before being observed in the green channel at 1000X. Right: Overlay both images demonstrating the mitochondrial non-localization of Compound (3) after 24 hours.

 Figure 6. Left column: Vero cells incubated with LysoTracker 30 minutes before being displayed, at different incubation times with Compound (3), in the red channel at 1000X. Central column: Vero cells incubated with Compound (3) 5μΜ for 30 minutes and, after washing, the culture is allowed to evolve at time 0, 3 and 5 hours displayed in the blue channel at 1000X. Right column: Overlap of both images demonstrating the mitochondrial recycling of Compound (3) and how it is found in Lysosomes.

Figure 7: Electron microscopy photographs of untreated Vero cells (7A and 7B), or incubated with Compound (3) 5μΜ under the conditions previously described (7C and 7D). Figure 8: Photographs of the cell culture stained with trypan blue at 48 and 72 hours after being incubated with Compound (3) 5 M for 30 minutes under the conditions previously described. Dead cells are stained blue and are indicated in a photo with an arrow.

 Figure 9. A r r i b a: E s t ru rure of 6- {3 - [(6-carbamimidoylnaphthalen-2- yl) oxy] propoxy} naphthalen-2-carboximidamide. Center: Vero cells incubated at 5 μΜ under the conditions described. Below: Water fluorescence emission spectrum of this compound. Excitation wavelength 329 nm, the spectrum is collected from 355 to 550 nm

 Figure 10. Above: Structure of the 6 - ((3-hydroxypropyl) amino) -2-naphthimidamide. Center: Vero cells incubated at 5 μΜ under the conditions described. Below: Water fluorescence emission spectrum of this compound. Excitation wavelength 329 nm, the spectrum is collected from 385 to 600 nm.

Figure 11. Above: Structure of the 6 - ({3 - [(naphthalen-2-yl) amino] propyl} amino) -naphthalen-2-carboximidamide midamide (Compound (18)). Bottom left (HA): Vero cells incubated with Compound (3) Ι μΜ under the described conditions observed in the blue channel at 1000X. Bottom center (1 1B) Vero cells incubated with Lysotracker® under the described conditions observed in the red channel at 1000X. Bottom right (1 1C): Overlay of both images demonstrating the mitochondrial non-location of the Compound (18).

Figure 12. a) Left column: Double staining in live cell of Vero and CEF cells with compound 3 and Mitotracker after 3 hours. Central column: Double staining in freshly seeded CEF cells (above) and aged CEF cells (below) with compound 3 and Mitotracker after 3 h. Right column: Staining of the same CEF cells that show a high activity of B-galactosidase in aged CEF cells, typical of senescence, b) Double staining of newly seeded (control) CEF cells and others incubated for 24 h, with peroxide of hydrogen (100 μΜ) after incubation with compound 3 and red Mitotracker (after 3 h of compound (3)). Cells subjected to oxidative stress show low colocalization of both markers. Figure 13. Mitochondrial trapping of compound (23) 20 μΜ. Above: CEF cells treated with compound 3 and compound 23 using stvAF594 as a secondary marker. A) blue channel; b) red channel; c) overlay of both images. Bottom: CEF cells treated with compound 23 20 μΜ, d) stvAF405 as secondary marker, blue channel; e) Qdot565 as secondary marker and compound (3) 5 μΜ (green channel); f) absence of secondary marker (blue channel).

 Figure 14. CEF cells treated with the protocol of example 11, were treated with compound (23) 20 μΜ and compound (3) 5 μΜ and subsequently incubated with st-

QDot565. a) Fluorescent photography in the green channel, b) Fluorescent photography in the blue channel, c) Superposition of the same field of vision. The images were taken at 1000X, ISO 400 and an exposure time for the image a) of ls and b) 200 ms.

 Figurel5. CEF cells treated with the protocol of example 1 and incubated with st-AF405. a) Fluorescent photography in the blue channel, b) Visible photography of the same mink field. The images were taken at l OOOx, ISO 400 and an exposure time for image a) of 1 s.

 Figurelo. CEF cells treated with the protocol of example 1 and incubated with st-AF594. a) Fluorescent photography in the red channel, b) Visible photography of the same mink field. The images were taken at lOOOx, ISO 400 and an exposure time for image a) of 1 s.

 Figurel7. CEF cells treated with the protocol of example 1 and incubated with st-QDot565. a) Fluorescent photography in the red channel, b) Visible photography of the same mink field. The images were taken at lOOOx, ISO 400 and an exposure time for image a) of 1 s.

Detailed description of the invention

Definitions "Alkyl" refers to an acyclic linear or branched hydrocarbon chain, consisting of carbon and hydrogen atoms, without unsaturations, of, unless otherwise indicated, 1 to 12, preferably 1 to 8, more preferably 1 to 8 6, and even more preferably 1 to 4 carbon atoms, and which is attached to the rest of the molecule by a single bond. The alkyl radicals may be optionally substituted by one or more substituents selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, an alkoxy group, a cyano group, a nitro group, a thioalkoxy group, an amino group , a heteroalkyl or CF _{3 group} , for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, etc. "Alkenyl" refers to an acyclic linear or branched hydrocarbon chain, consisting of carbon and hydrogen atoms, which has, unless otherwise indicated, between 1 and 3 alkene groups, 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms. Alkenyl radicals may be optionally substituted by one or more substituents selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, an alkoxy group, a cyano group, a nitro group, a thioalkoxy group, an amino group , a heteroalkyl or CF _{3 group} , for example, ethenyl, n-propenyl, n-butenyl, n-pentenyl, etc.

"Alkynyl" refers to an acyclic linear or branched hydrocarbon chain, consisting of carbon and hydrogen atoms, which has, unless otherwise indicated, between 1 and 2 alkyne groups, 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms. Alkynyl radicals may be optionally substituted by one or more substituents selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, an alkoxy group, a cyano group, a nitro group, a thioalkoxy group, an amino group , a heteroalkyl or CF _{3 group} , for example, propynyl, n-butynyl, n-pentinyl, etc.

"Cycloalkyl", "cycloalkenyl" and "cycloalkynyl" refer to a non-aromatic monocyclic or polycyclic ring comprising carbon and hydrogen atoms, preferably of, unless otherwise indicated, between 3 and 10 carbon atoms and respectively is saturated (cycloalkyl), it has at least one alkene (cycloalkenyl) group, it has at least one alkyne (cycloaquinyl) group. Examples of these groups, although not limited to this list, are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl, cyclohexenyl, cyclooctenyl, cyclohexinyl. "Halogen" is fluorine, chlorine, bromine or iodine.

"Aryl" refers to an aromatic hydrocarbon of, unless otherwise indicated, 6 to 15, preferably 6 to 10 carbon atoms, such as phenyl or naphthyl. The aryl radicals may be optionally substituted by one or more substituents selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, an alkoxy group, a cyano group, a nitro group, a thioalkoxy group, an alkyl group or CF ₃ .

"Heterocycle" refers to a stable ring of, unless otherwise indicated, 3 to 15 members consisting of carbon and hydrogen atoms and from 1 to 5 heteroatoms, where heteroatoms are selected from nitrogen, oxygen and sulfur, preferably a 3 to 6 member ring formed, and more preferably a 5 to 6 member ring and 1 to 3 heteroatoms. For the purposes of this invention, the heterocyclic groups may be monocyclic, bicyclic or tricyclic systems, which may include fused rings; and the nitrogen or sulfur atom in the heterocyclic ring may be optionally oxidized; the nitrogen atom may be optionally quartearized; and the heterocyclic radical may be partially or fully saturated. The heterocyclic radicals s may be aromatic (for example, they may have one or more aromatic rings) in which case they are considered as "heteroaryls" for the purposes of the present invention. The heterocyclic ring may be substituted by one or more substituents selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, an alkoxy group, an alkyl group, a thioalkoxy group, a cyano group, a nitro group or CF ₃ . Examples of such heterocycles include, for example, furan, thiophene, pyrrole, imidazole, triazole, alkyltrizaol, tetrazol, isothiazole, benzothiophene, benzofuran, indole, benzoimidazole, tetrahydrofuran, cyclooctan [d] pyridazine, pyridine, bipyridine and terpiridine.

"Good leaving group" is an atom or molecular fragment that efficiently detaches itself from a molecule in a heterolytic bond breakage, carrying with it the electronic pair of the bond. Normally the outgoing groups are detached in the form of anions and a good leaving group is considered to be a weak base. Examples of a good outgoing group are generally known by the average expert and the choice of the most appropriate in each situation is part of their daily work. Non-limiting examples are OTs (tosyl), OMs (mesyl) or halogen. The compounds of the present invention are positively charged so they are always accompanied by an organic or inorganic anion, which constitutes a salt of the compound of formula I. Said anion may be, although the following list is not limiting, formate, acetate, trifluoroacetate, oxaloacetate, succinate, phosphate, chloride, bromide, iodide, sulfate, hydrogen sulfate, etc.

Compounds

 The compounds of formula I are able to internalize in the cells and once inside they accumulate in the mitochondria with excellent selectivity. An additional advantage of the compounds of formula I is that they can selectively direct towards the mitochondria other molecular charges to which they are conjugated.

In an articular embodiment, the invention relates to compounds of formula la

 the

where A, B, Ra, Rb, K, Ri, Pi, P ₂ , m, n, or, q are as defined above, and comprise at least one organic or inorganic anion to maintain neutrality.

In a particular embodiment of the compounds of formula I the group K-Ri is -OR 'or - N (R') (R ") -, wherein R 'and R" are as defined above.

In another particular embodiment, the invention relates to compounds of formula Ib

compuesto de fórmula Ic

compound of formula Ic

 Ic

wherein A, B, Ra, Rb, Pi, P ₂ , m, n, or, q are as defined above, and comprise at least one organic or inorganic anion to maintain neutrality, and where the rings C and D are heterocycles.

 These derivatives are especially suitable for obtaining coordination compounds with transition metals that allow modulating the properties of the compounds of the invention. In a particular embodiment, the compounds of the invention of formula Ib or Ic are coordinated with transition metals, for example those selected from the group consisting of Ru, Ir, Os, Rh and Re.

In a particular embodiment, rings C and D of the compounds of formula Ib or Ic are a pyridine, forming the following bis-pyridinium group:

 In another particular embodiment the invention relates to compounds of formula Id

 Id

where Z is an acceptor fluorophore group and A, B, Ra, Rb, W, K, Pi, P ₂ , m, n, or, q are as defined above, and comprise at least one organic or inorganic anion to maintain neutrality

Another particular embodiment of the present invention relates to compounds of formula le

 Ie

where A, B, Ra, Rb Pi, P ₂ , m, n, or, q are as defined above, and comprise at least one organic or inorganic anion to maintain neutrality. In addition, it has been found that when irradiating the cells treated with the compounds of formula If, a fluorescence emission is observed that signals the mitochondria and / or mitochondrial components. In a particular embodiment, the compounds of formula If emit fluorescence at a wavelength between 380 nm and 750 nm. In a more particular embodiment, the compounds of formula If emit fluorescence at a wavelength between 420 nm and 570 nm.

 In addition, it was found that the compounds of formula If are useful in real-time visualization experiments (time-resolved fluorescence) since they allow to maintain the efficiency of the staining and its selectivity by mitochondria in living cells. Thus these compounds have a special relevance when studying mitochondrial behavior.

 An additional advantage of these compounds of formula If is that they allow monitoring material transfer processes between the mitochondria and lysosomes, their speed and the differences that may exist when subjecting cells to different conditions such as oxidative stress.

 Thus, another particular embodiment of the present invention relates to compounds of formula If

 If

where o and q are as defined above, and comprise at least one organic or inorganic anion to maintain neutrality. Another particular embodiment of the present invention relates to compounds of formula Ig

 Ig

where Pi, P ₂ , m, n, or, q are as defined above, and comprise at least one organic or inorganic anion to maintain neutrality.

 Another particular embodiment of the present invention relates to compounds of formula Ih

 Ih

where Ra, Rb, A, B, Pi, P ₂ , m, n, o, q, K and Ri are as defined above, and comprise at least one organic or inorganic anion to maintain neutrality. In a particular embodiment, in a compound of formula I, la, Id or Ih, together K-Ri and KZ is selected from the following groups

IVh IVi ivj

IVh IVi ivj

where Z is an acceptor fluorophore group, p is selected from 1, 2 and 3, and X is oxygen or nitrogen, and R 'is as defined above.

 In a particular embodiment R 'in the fragments of formula IVa-n is an alkyl group of 1 to 4 carbon atoms.

 In a particular embodiment, K is a single bond and Ri is hydrogen.

In a particular embodiment or and q are the same and are selected from 1 to 2.

In a particular embodiment A and B are the same and are selected from optionally substituted phenyl and pyridine.

In a particular embodiment Pi and P ₂ are hydrogen.

In a particular embodiment, W is -CH ₂ -.

In a particular embodiment the sum of o and q is 2, 3 or 4.

In the present invention, the term "acceptor fluorophore" refers to a species that participates in a resonance energy transmission (FRET) process accepting the energy transferred by another species that is in an excited state (this transfer can take place at through any photophysical process, including Forster or Dexter type mechanisms). A key parameter for the transfer of energy between two fluorophores is the combination of species with nearby energy levels. This proximity is related to the transfer theory so that the higher the value of the overlap integral between the donor and acceptor (IDA), between the emission spectrum of the donor and the absorption spectrum of the acceptor, the better the FRET . In the present invention, the theory of energy transfer is incorporated, as well as the calculation of said integral, the interpretation of its values and the detection of transfer energy by means of the following article: "Resonant Energy Transfer: Methods and Applications", Analytical Biochemistry, 1994, 218, 1-13. In addition, these energy requirements can be easily visualized by the overlapping of the emission spectrum of the donor fluorophore and the excitation spectrum of the acceptor fluorophore.

 When an acceptor fluorophore group is present in a compound of the invention, they have the additional advantage that they emit light at an even higher wavelength than in compounds in which these groups are not present, due to the transfer process of intramolecular energy Thanks to this transfer process, the range of emission wavelengths at which mitochondria or mitochondrial components can be monitored is effectively extended. In addition, by modifying the acceptor fluorophore group it is possible to design derivatives that emit light at different wavelengths that are of interest for application as luminescent biosensors. Another additional advantage is that the intensity of the emission band increases when an acceptor fluorophore group is present in a compound of formula I, which leads to greater detection sensitivity.

In a particular embodiment, the acceptor fluorophore group is selected from a lanthanide ion complexed with a bonding agent or a coumarin of general formula III

where R ₂ is selected from hydrogen, halogen and C ₁ -C ₁₂ alkyl, optionally substituted, and R ₃ is selected from - R ₄ R ₅ , -OH, -OR 4, -OCOR 4, R 4 and R 5 being the same or different selected from hydrogen, alkyl C ₁ -C ₁₂ , optionally substituted.

In a particular embodiment, the lanthanide ion is selected from Eu ⁺³ and Tb ⁺³ .

In a particular embodiment, the chelating agent is selected from 1,4,7, 10-tetraazacyclodecane-1, 4,7, 10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTP A), 4,7, 10- Tetraazacyclododecane-l, 4,7, 10-tetra (methylene phosphonic acid) (DOTP), 1,4,8, 11-tetraazacyclo-dodecane-1,4,8, 11-tetraacetic acid (TETA).

In the present invention, the term "photolabile protecting group" is defined as a protecting group whose binding to a molecule is broken or released by exposure to light of an appropriate wavelength. Photolabile protecting groups, as well as the conditions for their preparation and subsequent deprotection, are known in the state of the art (eg GCR Ellis-Davies, Nature Methods, 2007, 4, 619-628, SR Adams and RY Tsien, Annu. Rev. Physiol., 1993, 55, 755-784, Mayer G, Heckel A., Angew Chem Int. Ed. Engl, 2006, ¥ 5 (30), 4900-21) and include, for example, derivatives of o- nitrobenzyl, benzoin derivatives, phenacil derivatives, etc.

Preferably, in the present invention the photolabile protecting group is deprotected by irradiation with UV light (preferably of a wavelength between 200 and 400 nm, more preferably> 365 nm), preferably with a power of between 5 and 10 W, more preferably 8 W.

 In a particular embodiment, the photolabile protecting group is selected from the following structures:

 Ha Ilb

where each of P ₃ and P4 are independently selected from hydrogen, Ci- C12 alkyl, aryl C ₆ -Ci5, heteroaryl Ci ₃ C ₅ and C ₃ Ci ₅ heterocycle optionally substituted,

N0 ₂ , CN, halogen, -OR ', -SR', -S (0) R ', -S (0) ₂ R', -OS (0) ₂ R ', -N (R') (R " ), -C (0) R ', -C (0) OR', -C (0) N (R ') (R "), -OC (0) R' and - N (R ') C (0 ) R ", wherein each R 'and R" are independently selected from hydrogen, alkyl Ci-C _6, C ₃ -C chyle cicloal _7, C ₆ aryl -Ci5, heteroaryl Ci ₃ C ₅ heterocycle C ₃ Ci ₅ optionally substituted; or two P ₃ groups form, together with the phenyl ring to which they are attached, a heterocycle group, and r is selected from 1, 2, 3 and 4.

In a preferred embodiment the invention relates to the compound 6,6 '- [1, 3-propanedi-yl-di (imino)] dinaphthalene carboximidamide (Compound (3)). This compound is of special interest since it emits fluorescence in a range between 450 and 520 nm and allows it to be distinguished from most known mitochondrial markers.

Synthesis of the compounds of the invention

The compounds of the present invention can be performed following a special sequence of reactions following conditions known in the state of the art. Thus, for example, the amidinium group of the compounds of formula I of the invention can be obtained by reducing a group -C- (H (OH)) -H2 (amidoximes, Synthetic Communications 1996, 26 (23) 4351-4367) in a compound of formula IX, which in turn is obtained by treating a nitrile group (compounds of formula X) with hydroxylamine chloride:

 compounds of the invention

[net]

 IX

[NH ₂ OH]

 X

 There are many conditions to effect the reduction that leads to the compounds of formula I, only limited by compatibility with other functional groups present in the molecule. The person skilled in the art knows the appropriate conditions compatible with different functional groups. Thus, for example, in case there are alkene or alkyne groups in the compounds of formula I sensitive to certain reduction conditions, other compatible ones can be sought. Non-limiting examples of such conditions are hydrogenation in AcOH / EtOH in the presence of Pd / C (Ismail MA, Brun R., Wenzler T., Tanious FA, Wilson WD, Boykin DW Bioorg. Med. Chem 2004, 72 (20) , 5405-5413).

Appropriate conditions for the introduction of the fluorophore group are known to the person skilled in the art and there are many in the literature. Some non-limiting examples are: Chem. Commun., 2010, 46, 5518-5520, Chem. Sci., 2012, 3, 2383, J. Am. Chem. Soc. 2012, 134, 9199-9208, Angew Chem Int Ed Engl. 2012, 51, 2443-2447, Org Lett. 2011, 13, 5937-5939, J Am Chem Soc. 2012, 134, 3720-3728, or J Am Chem Soc. 2008, 130 (41): 13518-13519.

The compounds of formula X are obtained by coupling the aromatic compounds with the incorporated nitrile group of formula (XI) with a compound of formula (XII) (linker). The reaction may be of the nucleophilic type wherein the leaving group (L) may be in the compound of formula XI as shown in the scheme below, or it may be in the compound of formula XII, the amino group being in the compound of formula XI. Alternatively, L can be an aldehyde group (- C (= 0) H) by coupling using a reductive amination (March, j. "Advanced Organic Chemistry; Reactions Mechanism and Structure", Wiley-Interscience, 4th Edition, reaction 6- 15, page 898; Klyuev and Khidekel, Russ. Chem. Rev 1980, 49, 14-27, Rylander, Catalytic Hydrogenation over Platinum Metals pp 291-303, Academic Press New York, 1967).

 XII

Non-limiting examples for coupling compounds of formula XI and XII, for example to prepare compounds of formula Ib or Ic, can be found in the Journal of Organic Chemistry 76 (5) 1333-1341, 2011. Conditions and compounds suitable for This transformation can also be found in Inorganic Chemistry 43 (13) 3965-3975, 2004 and European Journal of Organic Chemistry 28, 4777-4792, 2009.

The compound of formula XI can be obtained commercially with the nitrile group or as an acid derivative, in which case it is usually convenient to transform it into nitrile before the reaction with XII. This can be done by methods known in the state of the art. For example, an acid ester of formula XIII can be transformed into acid of formula XIV by methods known in the state of the art (March, J. "Advanced Organic Chemistry; Reactions Mechanism and Structure", Wiley-Interscience, Fourth Edition, page 378, reaction 0-10), such as treatment in aqueous basic medium. In turn, the acid of formula XIV can be transformed into an amide of formula XV through acyl chlorides or acid anhydrides, according to the methods described in March, J. "Advanced Organic Chemistry; Reactions Mechanism and Structure", Wiley -Interscience, fourth edition., Page 417, reaction 0-52 and page 418, rection 0-53, which is transformed into nitrile (March, J. "Advanced Organic Chemistry; Reactions Mechanism and Structure", Wiley-Interscience, fourth Edition., Page 1041, reaction 7-39, Yamato, Sugasawa, Tetrahedron Lett., 1970, 4383; Appel, Kleinstuck and Ziehn, Chem. Ber., 1971, 104, 1030; Harrison, Hodge and Rogers, Synthesis 1977, 41 ).

Strategies similar to those described above can be found in Chem. Sci., 2012, 3, 2383, Angew. Chem. Int. Ed. 2012, 51, 7541-7544 or Chem. Commun., 2010, 46, 5518-5520. Compounds of formula XII are commercially available and are known to the person skilled in the art, for example some are described in the references cited here. The compounds of the invention can carry photolabile groups coupled to the amidinium group (groups Ra and Rb, for example groups of formula Ha or Ilb), which can be coupled following methods known to those skilled in the art, such as those described in Examples 1 to 4 of ES 2 396 076 Al.

From a compound in which K-Ri is IVb-h, another can be obtained in K-Ri is IVi-n, by the click reaction between an azide and an alkene or alkyne following the usual conditions known to the person skilled in the art. matter (see for example the conditions described in Kolb HC, Finn MG, Sharpless KB., Angew Chem Int. Ed. Eng., 1 2001, 40 (\ \), 2004-2021; VV Rostovtsev, LG Green, VV Fokin , KB Sharpless, Angew. Chem. Int. Ed., 2002, 41, 2596-2599 or in F. Himo, T. Lovell, R. Hilgraf, VV Rostovtsev, L. Noodleman, KB Sharpless, VV Fokin, J. Am Chem. Soc, 2005, 727, 210-216). For example, a compound of formula I with a fragment of formula IVk can be obtained from the corresponding compound with a fragment of formula IVg by reaction with an azide of formula R'-N ₃ .

In a particular embodiment, the imidinium group is first prepared from a compound of formula XI, and the photolabile group of formula Ra and / or Rb is protected or incorporated before coupling it with a compound of formula XII, and directly obtaining the compounds of the invention.

In addition, from a compound of formula I it is possible to covalently bind other compounds, markers or molecules of interest by reactions known to those skilled in the art. Thus, for example, from a compound of formula I where the K-R1 assembly is a hydroxyl group (formula IVb) or amino (formula IVc), a nucleophilic substitution reaction can be carried out on a compound of formula XVI, where L is a good outgoing group; alternatively L may be an aldehyde group and the reaction that would be carried out by the person skilled in the art would be a reductive amination; or L is an ester, amido or a carboxylic group and the person skilled in the art would obtain compound 1-2 by trans-esterification.

XVI

 1-2

where A, B, Pi, P ₂ , m, n, or, q, W, K, Ri, Ra, Rb are as defined above,

each of t, u are independently selected from 4, 5 or 6, and

each of Yi and Y ₂ is independently selected from -O-, - H-, - H (C = 0) -, - C (= 0) 0-, and comprises at least one organic or inorganic anion to maintain neutrality . Mitochondrial staining

 In another aspect the invention is directed to a method for labeling mitochondria or a mitochondrial component comprising incubating a sample, wherein said sample comprises mitochondria or a mitochondrial component, with an aqueous solution of a compound of formula I as defined above.

The present invention is also directed to a method for staining mitochondria or a mitochondrial component, which comprises incubating a sample, wherein said sample comprises mitochondria or a mitochondrial component, with an aqueous solution of a compound of formula I as defined above. , wherein said compound of formula I is in an amount sufficient to stain mitochondria or a mitochondrial component and incubation takes place for a time sufficient to detect fluorescence.

In a particular embodiment, the compound of formula I is a compound of formula If. In a particular embodiment, said method further comprises a fixing step. In another particular embodiment, said method further comprises a permeabilization step. Fluorescence detection can be carried out after the incubation stage, and / or after the fixation stage and / or after the fluorescence stage. The amount of the compound of formula I or If required for staining mitochondria in living cells is generally equal to or greater than ImicroM; typically between 1 and 5 microM. The incubation time is generally between 20 and 60 minutes, typically between 30 and 40 minutes.

 The method described above also allows additional reagents to be incorporated. In a particular embodiment, said method further comprises adding an additional reagent to the sample, wherein said additional reagent is a reagent that produces a detectable response due to a specific cellular component, intracellular substance, or a cellular condition. The additional reagent may comprise a protein, an amino acid sequence, a labeled antibody and / or a labeled oligonucleotide. The invention is further directed to a kit comprising a compound of formula I. The compounds of formula I may be in the form of a solution, for example in water and / or DMSO, or as a solid in powder, pellets, granules etc. It should be noted that the compounds of formula I are water soluble and stable in said solution.

The invention is illustrated below with some examples, although these should not be construed as a limitation thereof.

Examples

 Example 1A. Preparation of 6,6 '- [1, 3-propanedi-yl-di (imino)] dinaphthalene carb damida.

 1.1. Preparation of 6-bromonaphthalen-2-carboxylic acid.

A suspension of methyl 6-bromonaphthalene-2-carboxylate (4) (500 mg, 1.88 mmol) in dioxane (9 raL) and MeOH (9 mL) at room temperature was stirred until all the solid dissolved. KOH (318 mg, 5.66 mmol, 3 e) was added slowly over the solution and the mixture was heated at 50 ° C overnight, cooled to room temperature and treated with 2M HC1 to pH 3 in order to precipitate the product, which was filtered, washed with water and dried under vacuum to provide 6-bromonaphthalene-2- acid carboxylic (5) as a white powder (427 mg, 1.70 mmol, 90%). 1 H NMR δ (400 MHz, DMSO-de): 7.71 (dd, J = 8.8, 1.9 Hz, 1H), 7.97-8.93 (m, 2H) 8.08 (d, J = 8.8 Hz, 1H), 8.28 (s, 1H), 8.61 (s, 1H). ¹³ C NMR δ (DMSO-d ₆ ): 121.7 (C), 126.3 (CH), 127.4 (CH), 128.6 (C), 129.6 (CH), 129.8 (CH), 130.4 (CH), 130.7 (C) , 131.4 (CH), 135.9 (C), 167.1 (C). ESI-MS: [M + H] ⁺ caled, for CnH ₆ Br0 ₂ 248.9546 found 248.9542. CnH ₇ Br0 ₂ (MW 251.0761).

 1.2. Preparation of 6-bromonaphthalen-2-carboxamide.

A suspension of 6-bromonaphthalene-2-carboxylic acid (5, 251 mg, 1 mmol) in toluene (10 mL) was treated with SOCl ₂ (1189.7 mg, 10 mmol) and DMAP (23 mg, 0.188 mmol), and was heated to reflux 2 h. The excess solvent and SOCl ₂ was removed by distillation under reduced pressure. The precipitate was dissolved in CH ₂ C1 ₂ (25 mL) at room temperature and bubbled with dry ammonia gas for 30 min. The mixture was concentrated to provide a white solid, which was purified by flash chromatography on silica gel (1% MeOH / CH ₂ Cl ₂ ) to provide 6 as a white solid (114 mg, 0.46 mmol, 46%). 1H NMR δ (400 MHz, DMSO-d ₆ ): 7.51 (s, 1H), 7.70 (dd, J = 8.8, 1.9 Hz, 1H), 7.95-8.00 (m, 3H), 8.15 (s 1H), 8.27 (d, J = 1.4 Hz, 1H), 8.49 (s, 1H). ¹³ C NMR δ (DMSO-d ₆ ): 121.4 (C), 126.0 (CH), 127.5 (CH), 128.3 (CH), 130.0 (CH), 130.2 (CH), 131.2 (C), 131.6 (CH) , 132.7 (C), 135.7 (C), 168.2 (C). ESI-MS: [M + H] ⁺ caled, for C _n H ₉ BrNO 249.9862 found 249.9857. Cn¾BrNO (MW 250.0913).

 1.3. Preparation of 6-bromonaphthalen-2-carbonitrile.

On a solution of 6-bromonaphthalen-2-carboxamide (6, 84 mg, 0.336 mmol) and pyridine (53.4 mg, 0.675 mmol) in dioxane at 0 ° C TFAA was added dropwise (77.5 mg, 0.369 mmol) and the The reaction was stirred at room temperature for 5 h. The crude was poured into water and extracted with EtOAc (3 ^χ 50 ml). The combined aqueous phases were washed with water, dried with Na ₂ S0 ₄ , concentrated and purified by flash chromatography on silica gel (30% AcOEt / hexanes) to provide 7 as a white powder (50 mg, 0.215 mmol, 64%) 1H NMR δ (400 MHz, DCC1 ₃ ): 7.64 (dd, J = 8.5, 1.3 Hz, 1H), 7.69 (dd, J = 8.8, 1.8 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H) , 7.84 (d, J = 8.5 Hz, 1H), 8.07 (s, 1H), 8.20 (s, 1H). ¹³ C MR δ (DCC1 ₃ ): 109.9 (C), 118.8 (C), 123.6 (C), 127.5 (CH), 128.3 (CH), 129.9 (CH), 130.2 (CH), 130.7 (C), 131.2 (CH), 134.0 (CH), 135.5 (C). ESI-MS: [M + H] ⁺ caled, for C _n H ₇ BrN 231.9756 found 231.9761. CnH ₆ BrN (MW 232.0760).

1.4. Preparation of 6 - ({3 - [(6-cyanonaphthalen-2-yl) amino] propyl} amino) -naphthalen-2-carbonitrile.

A mixture of 6-bromonaphthalen-2-carbonitrile (7, 373 mg, 1.60 mmol), propan-1,3-diamine (52.9 mg, 0.714 mmol), K ₃ P0 ₄ (700 mg, 5 mmol), L-Proline (68 mg, 0.50 mmol) and Cul (68 mg, 0.5 mmol) was dissolved in 3.6 mL of DMSO and heated at 90 ° C for 20 h. The cold mixture was partitioned between water and AcOEt, and the aqueous phase was extracted with AcOEt (3 ^χ 40 mL). The combined organic phases were washed with a saturated NaCl solution, dried over Na ₂ S0 ₄ and concentrated under reduced pressure. The residual oil was purified by flash chromatography on silica gel (60% AcOEt / hexanes) to provide the desired dinitrile (10) as a brown solid (236.5 mg, 0.63 mmol, 88%). 1 H NMR δ (400 MHz, DMSO-d ₆ ): 1.99 (p, J = 6.8 Hz, 2H), 3.30 (dd, J = 12.3, 6.6 Hz, 4H), 6.63 (t, J = 5.2 Hz, 2H) , 6.78 (d, J = 1.8 Hz, 2H), 7.12 (dd, J = 8.9, 2.2 Hz, 2H), 7.47 (dd, J = 8.5, 1.6 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 7.71 (d, J = 8.9 Hz, 2H), 8.18 (s, 2H). ¹³ C NMR δ (DMSO-d ₆ ): 27.2 (CH ₂ ), 40.3 (CH ₂ ), 101.5 (CH), 101.8 (C), 119.5 (CH), 120.0 (C), 124.8 (C), 126.1 ( CH), 126.4 (CH), 129.1 (CH), 133.5 (CH), 137.0 (C), 149.2 (C). ESI-MS: [M + H] ⁺ caled, for C ₂₅ H ₂ iN ₄ 377.1761 found 377.1761. C ₂₅ H ₂₀ N ₄ (MW 376.4531).

1.5. Preparation of 6,6 '- [1, 3-propanedi-yl-di (imino)] dinaphthalene carboximidamide (Compound (3)).

 Hydroxylamine hydrochloride (279 mg, 4 mmol) was dissolved in anhydrous DMSO (2 mL) under argon. DIEA (646 mg, 5 mmol) was added slowly at room temperature. After stirring the mixture 30 min, dinitrile 10 (75 mg, 0.2 mmol) was added over the reaction mixture. The resulting mixture was heated overnight at 55 ° C. After verifying by analytical RP-HPLC that all the starting material had been transformed into the desired product, the mixture was purified by preparative reverse phase chromatography (gradient: 5% B 5 min, 5% to 50% B 30 min). The appropriate fractions were collected, concentrated and lyophilized. The isolated solid was identified as the desired intermediate.

The intermediate Bis- (benzamidoxime), ammonium formate (22.7 mg, 0.36 mmol) and Pd / C (10%, 22 mg) were mixed with glacial AcOH (1.5 mL) and heated at 110 ° C under an argon atmosphere. The starting material was consumed after 4h (analytical RP-HPLC) and the sample was cooled and filtered on celite. 2 volumes of H ₂ 0 were added on the filtrate to carry out purification by preparative reverse phase chromatography (gradient: 5% B 5 min, 5% to 65% B 30 min). The appropriate fractions were collected, concentrated and lyophilized to provide 3 as a yellow solid (0.072 mmol, 46 mg, 36%). 1 H NMR δ (400 MHz, DMSO-d ₆ ): 2.01 (p, J = 6.7 Hz, 2H), 3.32 (t, J = 6.8 Hz, 4H), 7.14 (dd, J = 8.9, 2.0 Hz, 1H) , 7.62 (dd, J = 8.8, 1.5 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H), 6.81 (d, J = 1.5 Hz, 1H) , 8.94 (s, 4H), 9.16 (s, 4H). ¹³ C MR δ (DMSO-d ₆ ): 27.8 (CH ₂ ), 40.8 (CH ₂ ), 101.8 (CH), 119.4 (C), 120.1 (CH), 124.2 (CH), 125.0 (C), 126.1 ( CH), 129.8 (CH), 130.4 (CH), 138.5 (C), 149.8 (C), 165.7 (C). ESI-MS: [M + H] ⁺ caled, for C ₂₅ H ₂₇ N6 411.2292 found 411.2295. C ₂₉ H ₂₈ F ₆ N ₆ 0 ₄ (MW: 638.5608).

Example IB Preparation of 6 - ({2-amino-3 - [(6-carbamimidoylnaphthalen-2-yl) amino] -propyl} amino) naphthalen-2-carboximidamide. 1 .6. Preparation of 6 - ({3 - [(6-Cyanonaphthalen-2-yl) amino] -2-hydroxypropyl} amino) -naphthalen-2-carbonitrile.

 L-Proline (20)

 DMSO 90 ° C

A mixture of 6-bromonaphthalene-2-carbonitrile (500 mg, 2.15 mmol), serinol (92 mg, 1.0 mmol), K3PO4 (871 mg, 4.1 mmol), L-Proline (118 mg, 1.0 mmol) and Cul (97 mg, 0.5 mmol) in 5.1 mL of dimethylsulfoxide heated at 90 ° C for 20 h under argon. After cooling to room temperature, the mixture was partitioned between water and ethyl acetate and the aqueous phase extracted with ethyl acetate (3 ^χ 40 mL). The combination of organic phases was washed with a saturated NaCl solution, dried over Na ₂ S0 ₄ and concentrated under reduced pressure. The residual oil was purified by flash chromatography on silica gel (60% AcOEt / hexanes) to provide the compound (20) as a brown solid (111 mg, 0.49 mmol, 91%). 1H NMR δ (250 MHz, DMSO - </ _é ): 3.16-3.24 (m, 4H), 4.04 (m, 1H), 5.21 (d, J = ¥ .5 Hz, 1H, OH), 6.61 (t, J = 4.8 Hz, 2H, NH), 6.79 (s, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 8.9 Hz, 2H), 8.17 (s, 2H). ¹³ C NMR δ (DMSO-d ₆ ): 46.9 (CH2), 66.8 (CH), 101.7 (CH), 101.8 (C), 119.6 (CH), 120.0 (C), 124.8 (C), 126.1 (CH) , 126.4 (CH), 129.1 (CH), 133.5 (CH), 136.9 (C), 149.3 (C). ESI-MS:

[M + H] ⁺ cale, for C ₂₅ H ₂₁ N ₄ O 393.1710 found 393.1697 Ci ₄ Hi ₄ N ₂ 0 (MW 392.4525). one . 7. Preparation of 6 - ({2-Azido-3 - [(6-cyanonaphthalen-2-yl) amino] propyl} amino) -naphthalen-2-carbonitrile.

A suspension of compound (20) (178 mg, 0.45 mmol) in CH ₂ C1 ₂ (4.5 mL) was cooled with a water-ice bath. Methanesulfonyl chloride (60 mg, 40 μΕ, 0.52 mmol) and triethylamine (69 mg, 94 μΕ, 0.68 mmol) were added, and the mixture was stirred under argon for 2 h. The crude was partitioned between a solution of HC1 and aqueous 10% ethyl acetate, and the aqueous phase was extracted with ethyl acetate (3 ^χ 40 mL). The combined organic phases were washed with a saturated NaCl solution, dried over Na ₂ SÜ4 and they were concentrated under reduced pressure. The residual oil was dissolved in dimethylformamide and sodium azide (87 mg, 1.35 mmol) was added; The mixture was allowed to react overnight at 80 ° C, cooled to room temperature and partitioned between water and ethyl acetate. The aqueous phase was extracted with ethyl acetate (3 ^χ 40 mL) and the combined organic phases were washed with saturated NaCl solution, dried over Na ₂ S0 ₄ and concentrated under reduced pressure to give an oil which was purified by Flash chromatography on silica gel (40% AcOEt / hexanes) to provide the compound (21) as a brown solid (125 mg, 0.30 mmol, 67%). 1H NMR δ (300 MHz, CDC1 ₃ ): 3.44-3.66 (m, 4H), 4.16 (m, H), 4.52-4.55 (m, 1H). 6.81 (d, J = 2.3 Hz, 2H), 7.01 (dd, J = 8.8, 2.4 Hz, 2H), 7.42 (dd, J = 8.6, 1.6 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H ), 7.65 (d, J = 8.8 Hz, 2H), 7.99 (s, 2H). ¹³ C NMR δ (CDC1 ₃ ): 45.1 (CH ₂ ), 59.7 (CH), 103.9 (CH), 104.8 (C), 119.2 (CH), 119.9 (C), 126.2 (C), 126.7 (CH), 127.2 (CH), 129.9 (CH), 133.7 (CH), 136.7 (C), 147.3 (C). ESI-MS: [M + H] ⁺ cale, for C ₂₅ H _{2 or} N7 418.1775 found 418.1773 C ₂₅ Hi ₉ N ₇ (MW 417.4653).

 1 .8. Preparation of 6 - ({2-amino-3 - [(6-carbamimidoylnaphthalen-2-yl) amino] propyl} -amino) naphthalen-2-carboximidamide.

Hydroxylamine hydrochloride was dissolved in anhydrous dimethylsulfoxide (3.8 mL) under argon, diisopropylethylamine (496 mg, 680 μΕ, 3.8 mmol) was added portionwise at room temperature. After stirring the mixture for 30 min, the compound (21) was added to the reaction mixture (100 mg, 0.19 mmol). The resulting mixture was heated overnight at 55 ° C. The crude was purified by preparative chromatographic column in reverse phase (gradient: 5% B 5 min, 5% to 50% B 30 min). The fractions of interest were collected, concentrated and dried. The resulting benzamidoxime (125 mg, 0.17 mmol) was dissolved in acetic acid (1.7 mL) and added acetic anhydride (36 mg, 0.35 mmol); The reaction suddenly changed color to a dark brown. After checking that all the starting material had been acetylated by RP-HPLC, the mixture was diluted 10 times in methanol and hydrogenated over Pd / C (10%, 12 mg) at room temperature for 4 h. Catalyst was removed by filtration. The filtrate was concentrated under reduced pressure and purified by reverse phase semi-preparative chromatographic column (gradient: 5% B 5 min, 5% to 50% B 30 min). The fractions of interest were collected, concentrated and dried to obtain the compound (22) as a pale yellow solid (42 mg, 0.05 mmol, 29%). 1H NMR δ (500 MHz, MeOO-d ₄ ): 3.63-3.76 (m, 4H), 3.90 (m 1H), 4.33 (m, H), 6.89 (s, H), 6.97 (d, J = 2.2 Hz , 2H), 7.20 (dd, J = 8.9, 2.3 Hz, 2H), 7.57 (bs, 4H), 7.83 (d, J = 8.9 Hz, 2H), 8.23 (s, 2H). ¹³ C NMR δ (MeOD- <¾): 43.0 (CH ₂ ), 49.2 (CH), 102.8 (CH), 1 19.6 (CH), 120.1 (C), 123.0 (CH), 126.1 (C), 126.5 ( CH), 129.0 (CH), 130.2 (CH), 138.3 (C), 148.7 (C), 166.8 (C). ESI-MS: [M + H] ⁺ cale, for C ₂₅ H ₂₈ N ₇ 426.2401 found 426.2386. C ₃₁ H ₃₀ F ₉ N ₇ O ₆ (MW 767.5988).

Example 1C. Preparation of (6- {5 - [(3aR, 4R, 6aS) -2-oxo-hexahydro-lH-thieno [3,4- d] imidazolidin-4-yl] pentanamide} -N- {l, 3-bis [(6-carbamimidoylnaphthalen-2- yl) amino] propan-2-yl} hexanamide).

To a solution of compound (22) (15 mg, 19 μιηοΐ) in dimethylformamide (325 μΐ.), Diisopropylethylamine (10 mg, 14 μΐ., 78 μmol) and biotin succinimidyl ester (7.4 mg, 16 μιηοΐ) were added and the resulting mixture was stirred at room temperature for 2 h. The disappearance of the starting material and the appearance of the desired product was confirmed by RP-HPLC. The residue was purified by HPLC (gradient: 15% B, 5 min; \ 5% → 95% B, 30 min.). The fractions of interest were collected, concentrated and dried to obtain the compound (23) as a pale yellow solid (7.6 mg, 7.62 μιηοΐ, 39%). 1H NMR δ (500 MHz, MeOO-d ₄ ): 1.26-1.30 (m, 2H), 1.38-1.43 (m, 4H), 1.53-1.64 (m, 4H), 1.65-1.72 (m, 2H), 2.16 (t, J = 7.4 Hz, 2H), 2.18-2.24 (m, 2H), 2.69 (d, J = 12.7 Hz, 1H), 2.91 (dd, J = 12.7, 4.9 Hz, 1H), 3.05 (t, J = 6.9 Hz, 2H), 3.10 (t, J = 6.9 Hz, NH), 3.14-3.18 (m, 2H), 3.40-3.49 (m, 2H), 3.53- 3.63 (m, 2H), 4.15 (dd , J = 6.5, 5.8 Hz, 1H), 4.26 (dd, J = 7.7, 4.6 Hz, 1H), 4.47 (dd, J = 7.8, 4.7 Hz, 1H), 6.92 (s, NH), 6.98 * (s , 1H), 7.05 * (s, 1H), 7.12 * (dd, J = 8.8, 2.1 Hz, 1H), 7.16 * (dd, J = 8.8, 2.3 Hz, 1H), 7.57-7.65 (m, 2H) , 7.68 * (d, J = 3.2 Hz, 1H), 7.70 * (d, J = 3.3 Hz, 1H), 7.77 (t, J = 8.4 Hz, 2H), 8.21 (s, 2H). (* ') ¹³ C NMR δ (MeOD- <¾): 26.6 (CH ₂ ), 26.9 (CH ₂ ), 27.5 (CH ₂ ), 29.5 (CH ₂ ), 29.7 (CH ₂ ), 30.1 (CH ₂ ) , 36.8 (CH ₂ ), 37.0 (CH ₂ ), 40.1 (CH ₂ ), 41.0 (CH ₂ ), 42.5 (CH ₂ ), 45.9 (CH ₂ ) *, 46.1 (CH ₂ ) *, 53.1 (CH), 57.0 (CH), 61.6 (CH), 63.4 (CH), 103.7 (CH) *, 104.3 (CH) *, 120.7 (C) *, 120.9 (C) *, 121.0 (CH) *, 121.1 (CH) *, 124.3 (CH) *, 124.4 (CH) * 127.1 (C) *, 127.1 (C) *, 127.7 (CH) *, 127.8 (CH) *, 130.5 (CH), 131.5 (CH) *, 13 1.5 (CH ) *, 140.1 (C), 150.6 (C) *, 151.0 (C) *, 166.1 (C), 168.2 (C), 175.9 (C), 176.9 (C). ESI-MS: [M + H] ⁺ cale, for C ₄₁ H ₅₃ N ₁₀ O ₃ S 765.4017 found 765.4015. C ₄₅ H ₅₄ F ₆ N ₁₀ O ₇ S (MW 993.0285).

Example ID. Preparation of compounds with a photolabile protecting group.

1.9. Preparation of the compound (24).

Compound (3) (10 mg, 15.7 μιηοΐ) was dissolved in 750 μΐ _^ DMF and DIEA (19.4 mg, 150 μιηοΐ) and nitro veratril succinimide (22.3 mg, 63.0 μιηοΐ) were added, the mixture was allowed to react for overnight and the reaction crude was purified directly by preparative reverse phase chromatography (gradient: 5% B 5 min, 5% to 75% B 30 min). The appropriate fractions were collected, concentrated and lyophilized to provide the compound (24) as a yellow trifluoroacetic acid salt (2 mg, 2.6 μιηοΐ, 17%). 1H NMR (400 MHz, DMSO-d ₆ δ): 2.01 (p, J = 6.6 Hz, 2H), 3.32 (dd, J = 10.6, 6.4 Hz, 4H) 3.90 (s, 3H), 3.95 (s, 3H ), 5.66 (s, 2H), 6.81 (s, 2H), 7.14 (d, J = 9.0 Hz, 2H), 7.38 (s, 1H), 7.61-7.79 (m, 7H), 8.23 (s, 1H) , 8.27 (s, 1H), 8.81 (s, 2H), 9.19 (s, 2H), 10.4 (s, 1H). ESI-MS: [M + H] ⁺ caled, for C ₃₅ H ₃₆ N ₇ O ₆ 650.2722 found 650.2710. C ₃₇ H ₃₆ F ₃ N ₇ O ₈ (MW 763.7190).

1.10. Preparation of the compound (25).

Compound (3) (10 mg, 15.7 μιηοΐ) was dissolved in 750 μL · of DMF and DIEA (19.4 mg, 150 μιηοΐ) and succinimide nitra veratril (22.3 mg, 63.0 μιηοΐ) were added, the mixture was allowed to react for overnight and the reaction crude was purified directly by preparative reverse phase chromatography (gradient: 5% B 5 min, 5% to 75% B 30 min). Appropriate fractions were collected, concentrated and lyophilized to provide compound (25) as a yellow solid (3.3 mg, 3.7 μπιοΐ, 24%). 1H NMR (400 MHz, DMSO-d ₆ δ): 2.01 (p, J = 6.7 Hz, 2H), 3.32 (t, J = 6.8 Hz, 4H), 3.90 (s, 6H), 3.94 (s, 6H) , 5.66 (s, 4H), 6.82 (d, J = 1.4 Hz, 2H), 7.14 (dd, J = 9.0, 1.9 Hz, 2H), 7.37 (s, 2H), 7.62-7.69 (4H), 7.76 ( s, 4H), 8.27 (s, 2H), 10.4 (s, 2H).

Example 2. Preparation of oxygenated derivative (comparative).

2.1. Preparation of l, 3-Bis (6-amidinonaphthalen-2-yloxy) propane

 (13)

To an stirred solution of 4-cyano phenol (169 mmol) in DMF was added anhydrous K _{2 C 3} (414 mg, 3 mmol). After 30 min, 1,3-dibromopropane (80.8 mg, 0.4 mmol) was added and the reaction was stirred at 50 ° C for 24 hours. The mixture was partitioned between water and AcOEt, and the aqueous phase was extracted with AcOEt (3 ^χ 50 mL). The combined organic phases were washed with a saturated NaCl solution, dried over Na ₂ S0 ₄ and concentrated under reduced pressure to provide an identified white solid. as the desired compound 12 (321mg, 1.9 mmol) that was used in the next reaction without further purification.

(M.W. 640.5304). To a solution of said bisnitrilo (12), 67.3 mg, 0.178 mmol) in THF under argon at 0 ° C was added 1 mL of LiN (TMS) ₂ 1M in THF (149 mg, 0.89 mmol) and the resulting solution It was stirred at room temperature for 12 hours. It was then cooled to 0 ° C and acidified with ethanolic HC1 (1.25 M). After stirring 4h, the solvent was removed by reduced pressure. The residue was dissolved in DMF purified by preparative reverse phase chromatography (gradient: 5% B 10 min, 5% to 55% B 30 min.) The appropriate fractions were collected, concentrated and lyophilized to provide 13 as a white solid (54 mg, 0.084 mmol, 50%). 1H MR δ (400 MHz, DMSO-de): 2.37 (p, J = 6.1 Hz, 2H), 4.38 (t, J = 6.2 Hz, 4H), 7.36 (dd, J = 9.0, 2.5 Hz, 2H), 7.54 (d, J = 2.4 Hz, 2H), 7.79 (dd, J = 8.7, 1.9 Hz, 2H), 8.00 (s, 2H), 8.02 (s, 2H) 8.43 (d, J = 1.6 Hz, 2H) , 9.27 (s, 4H), 9.36 (s, 4H). ¹³ C NMR δ (400 MHz, DMSO-de): 28.3 (CH ₂ ), 64.6 (CH ₂ ), 106.7 (CH), 120.3 (CH), 122.8 (C), 124.1 (CH), 127.0 (C), 127.3 (CH), 129.1 (CH), 130.7 (CH), 136.8 (C), 158.6 (C), 165.6 (C). ESI-MS: [M + H] ⁺ caled, for C ₂ 5H ₂₅ N ₄ 0 ₂ 413.1972 found 413.1793.

(MW 640.5304).

Example 3. Preparation of nitrogenous derivative (Comparative)

In order to carry out comparative studies, the compound 6 - ((3-hydroxypropyl) amino) -2-naphthimidamide (14) was prepared according to the sequence indicated below in Scheme 1:

 L-Proline (16)

 (15) DMSO 90 ° C

1- NH ₂ OHxHCI DIEA, DMSO

 2- A (¾0, AcOH

3- H ₂ , Pd / C

 MeOH

 Scheme 1

By the reaction of 6-bromonaphthalene-2-carbonitrile (15) with excess of 3- aminopropan-l-ol (approximately 9 equivalents) in the presence of K ₃ PO ₄ , L-Proline and Cul, and DMSO as solvent was prepared 6 - (3-hydroxypropylamino) naphthalen-2-carbonitrile (16). For conditions for this reaction see Journal of Organic Chemistry 2005, 70, 5164-5173, Organic Letters 2003, Vol 5, No. 6 793-796, Organic Letters 2002, Vol 4, No 4. 581-584 or Chem. Comm ., 2009, 1715-1717. In three stages from the latter, 6 - ((3-hydroxypropyl) amino) -2-naphthimidamide (14) was obtained: (i) treatment with hydroxylamine and base; (ii) acylation with acetic anhydride and (iii) catalytic hydrogenation with palladium. 1H NMR δ (400 MHz, DMSO-d ₆ ): 1.78 (p, J = 6.5 Hz, 2H), 3.20 (t, J = 7.0 Hz, 2H), 3.55 (t, J = 6.2 Hz, 2H), 6.77 (d, J = 2.1 Hz, 1H), 7.10 (dd, J = 8.9, 2.3 Hz, 1H), 7.63 (dd, J = 8.7, 2.0 Hz, 1H), 7.70 (d, J = 5.6 Hz, 1H) , 7.73 (d, J = 5.8 Hz, 1H), 8.23 (d, J = 1.8 Hz, 1H), 9.02 (s, 2H), 9.16 (s, 2H). ¹³ C NMR δ (400 MHz, DMSO-de): 31.5 (CH ₂ ), 39.5 (CH ₂ ), 58.4 (CH ₂ ), 101.2 (CH), 118.8 (C), 119.6 (CH), 123.6 (CH) , 124.4 (C), 125.6 (CH), 129.3 (CH), 129.9 (CH), 138.1 (C), 149.5 (C), 165.3 (C). ESI-MS: [M + H] ⁺ caled, for Ci ₄ Hi ₈ N ₃ 0i 244.1444 found 244.1438. Ci ₆ Hi ₈ F ₃ N ₃ 0 ₃ (MW 357.3276).

Example 4. Preparation of monofunctionalized nitrogenous derivative (comparative) Also for comparative purposes, 6 - ({3 - [(naphthalen-2-yl) amino] propyl} amino) naphthalen-2-carboximidamide (18) was prepared following the scheme described in Scheme 2:

 Scheme 2

First 6 - ({3 - [(naphthalen-2-yl) amino] propyl} amino) naphthalen-2-carbonitrile (19) was prepared by reacting 6-bromonaphthalene-2-carbonitrile with the compound of formula (20) in presence of K ₃ PO ₄ , L-Prolina and Cul, and using DMSO as solvent. Following a sequence analogous to that already described in the previous example, it was prepared in three stages from 6 - ({3 - [(naphthalen-2-yl) amino] propyl} amino) naphthalen-2-carbonitrile (19) 6- ({3 - [(naphthalen-2-yl) amino] propyl} amino) naphthalen-2-carboximidamide (18): (i) hydroxylamine and base treatment; (ii) acylation with acetic anhydride and (iii) catalytic hydrogenation with palladium. 1H NMR (400 MHz, MeOD-d ₄ á): 2.20-2.09 (m, 2H), 3.41 (t, J = 6.7 Hz, 2H), 3.63-3.54 (m, 2H), 6.83 (s, H ), 7.08 (d, J = 8.9 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 7.43-7.52 (m, 4H), 7.58-7.66 (m, 2H), 7.73 (dd, J = 8.9, 4.3 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 8.04 (s, NH), 8.18 (d, J = 1.7 Hz, 1H). ¹³ C NMR (63 MHz, MeOD-d ₄ to): 27.02 (CH _2), 41.4 (CH _2), 48.7 (CH _2), 120.3 (CH), 120.7 (C), 120.9 (CH), 121.0 (C), 124.1 (CH), 124.3 (CH), 126.9 (CH), 127.6 (CH), 127.9 (C), 128.3 (CH), 128.4 (CH), 128.9 (CH), 129.7 (CH), 130.4 (CH), 131.1 (C), 131.3 (CH), 131.4 (CH), 135.1 (C), 140.0 (C), 150.6 (C), 168.2 (C). ESI-MS: [M + H] ⁺ caled, for C ₂₄ H ₂₅ N ₄ 369.2074, found: 369.2077. C ₂ 6H ₂₅ F ₃ N ₄ 0 ₂ (MW 482.4975). Example 5. Internalization, fluorescence and cell localization

 The day before the cell internalization experiments, the cells were seeded in twelve-well plates containing glass coverslips (15 mm). The images were obtained with a DP-71 digital camera mounted on an Olympus BX51 fluorescence microscope. The images were processed (cropped, resized, global contrast and brightness adjustment) with Adobe Photoshop (Adobe Systems). The spectroscopic parameters of the fluorescent channels are as follows:

 • Blue channel: U-MWU2 ultraviolet excitation: 360 to 370 nm excitation filter, 420 nm emission filter and 400 nm dichroic mirror.

· Green channel: blue excitation U-MWB2: excitation filter 460 at 490 nm, emission filter at 520 nm and dichroic mirror at 500 nm.

 • Red channel: green excitation U-MNG2: excitation filter 530 at 550 nm, emission filter at 590 nm and dichroic mirror at 570 nm.

 5 μΜ of compound (3) was incubated in chicken embryo fibroblast monolayers (Chicken Embryo Fibroblasts, CEF) in Dulbecco's Modified Eagle Medium medium (DMEM) for 45 minutes at 37 ° C. The cells were then washed three times with phosphate buffered saline (PB S), and observed directly under the fluorescence microscope without further fixation (blue channel). The treated cells showed fluorescence emission in the expected range for Compound (3), demonstrating that the compound can penetrate the membrane of living cells.

 The fluorescence of Compound (3) shows a cytosolic filamentous pattern consistent with mitochondrial localization (Figure 2A, image on the left). To confirm this possibility, the cells were co-labeled with the commercial mitochondrial marker MitoTracker®, Invitrogen (Figure 2B, central image) and it was found that both markers show completely superimposable labeling patterns, demonstrating that Compound (3) is directed the mitochondria and constitutes a novel fluorescent marker for this organelle (figure 2C, right panel)

To confirm that Compound (3) can be used as a universal mitochondrial marker, we decided to carry out the same double staining experiment described in Figure 1 with additional cell lines of different origins. Figure 2 shows representative results obtained with the cell lines: Vero, BHK, DF1 and HeLa. All of them showed staining patterns similar to those obtained with CEF. Conclusion: Compound (3) has a satisfactory capacity for cellular internalization and selective accumulation in mitochondria. Its spectroscopic properties make it a blue fluorescent marker for this cell organelle. This mitochondrial fluorescent marking has been demonstrated in different cell lines of different origin, such as mammal (human, hamster or monkey) or bird (chicken). This pattern is reproducible both in immortalized cell lines and in primary cultures. Compound (3) is constituted as a universal fluorescent mitochondrial marker. Example 6. Photoestability and discoloration

 Previous experiments show that Compound (3) can be used in living cells as a universal mitochondrial marker, which in turn suggests that it could also be used in time-lapse experiments. As a condition, the probe or marker must not only be able to enter the cell, but must also be photostable enough to tolerate repeated irradiation (or long exposure) so that its resistance to fading (in English "photobleaching ") must be minimal.

Having demonstrated its ability to cross the cell membrane, we decided to check and compare the stability of Compound (3) with specific and commercial mitochondrial markers. To do this, Vero cells were incubated with 5 μΜ Compound (3), 2 μΜ Rhodamine 123, or 500 nM MitoTracker under the conditions previously described. Each sample was continuously irradiated under the fluorescence microscope light source emitted through the 100X objective, and the images were collected after specific irradiation times. The Rodaminal23 green dye (figure 4, top row) shows a very poor photostability, it appears almost completely photolised after 30 seconds of irradiation under these conditions. The MitoTraker® (figure 4, center row) showed greater resistance to fading, but again photobleaching was completed after 60 sec. In contrast to the poor photostability of Rhodamine 123 and MitoTracker, Compound (3) hardly shows degradation in its emission after 2 minutes under the same conditions, and was still clearly visible after 8 minutes of irradiation (Figure 4, row lower). Conclusion: Compound (3) has a high resistance to discoloration and great photostability at the same exposure and observation conditions, being far superior to commercial mitochondrial markers such as Mitotracker® and Rodaminal23. Example 7. Viability and cytotoxicity

After checking the excellent photostability and a selective marking towards mitochondria, Compound (3) could be considered perfectly as an in vivo marker reagent for "time-lapse" experiments, as long as it does not affect cell viability. To check its cytotoxicity, Compound (3) 5 μΜ was incubated in Vero cells as described above. After incubation, the medium was replaced with fresh DMEM with 10% FBS (Fetal Bovine Serum) and cells were incubated at 37 ⁰ C for several days.

 Interestingly, 24 hours after incubation with the dye, the blue fluorescence attributed to Compound (3) did not show the typical filamentous pattern of mitochondria, but instead, all the fluorescence was concentrated inside spherical vesicles, (Figure 5 A, left image). To confirm that the mitochondria were not corrupted by the presence of Compound (3), we stained these cells with Rodaminal23, which exclusively marks the functional mitochondria. The green fluorescence associated with Rodaminal23 demonstrated the mitochondrial presence with a filamentous morphology (Figure 5B, central image) typical of this cell line. We checked how the fluorescence of Rodaminal23 and that of Compound (3) did not collocate. (Figure 5C, central image).

 When investigating the nature of these spherical vesicles possessing the fluorescence derived from Compound (3), Compound (3) 5 µΜ was incubated in Vero cells as described above. 30 minutes before the cells are visualized at different times, they are incubated with a commercial lysosomal marker, LysoTracker Red ® (Invitrogen). Colocalization of the fluorescent signal demonstrates how Compound (3) has accumulated in the lysosome. This form of recycling is perfectly known and justifies the non-toxicity of Compound (3) in addition to reflecting the dynamics and mitochondrial recycling.

In a parallel experiment, cells where Compound (3) 5μΜ was incubated for 30 min and subsequently washed with PBS, allowed to evolve for 24 hours only with Compound (3) were studied by electron microscopy. The images demonstrate how Compound (3) does not cause any increase in the number of lysosomes present inside the cell (Figure 7, photos 7C and D), since analyzing the morphology and amount of the organelles it is impossible to differentiate a cell exposed to the Compound ( 3) and another one that does not (figure 7, photos 7A and 7B).

These results suggest that all internalized Compound (3) is recycled to lysosomes from the mitochondria without affecting the viability of any of the exposed cells. The same pattern was present at 48 and 72 hours after the initial incubation with the dye, but the fluorescence intensity was reduced over time and was undetectable on the fifth day. As irrefutable evidence of the null cell cytotoxicity, cells that had been treated with Compound (3) 48 and 72 hours before were incubated with trypan blue (it is a dye that is used in histological stains for viability tests, which allow differentiating cells alive of dead cells), and very few cells in the cultures (indicated by arrows) were stained by the dye, which demonstrates the viability of the monolayers.

Conclusion: After a certain time Compound (3) is recycled from the mitochondria to the lysosome without affecting mitochondrial function and without encouraging the increase in the presence of lysosomes in the cytoplasm. Staining with a vital dye to a cell culture exposed to Compound (3) 48 and 72 hours earlier demonstrates the low number of dead cells. Both data demonstrate the zero cellular toxicity of Compound (3).

Example 8. Structure-Activity Relationship

 In the search for a structure-activity relationship we have synthesized several similar compounds that try to mimic their structure with some variations. Once synthesized we have studied its cellular internalization and luminescent properties.

Sintetiz or 6- {3 - [(6-carbamimido-yl-naphthalen-2-yl) oxy] propoxy} naphthalene-2-carboximidamide (Figure 9, above) consisting of the oxygenated derivative of compound 3, where Exocyclic nitrogen from the naphthalene ring is replaced by oxygen. The 6- {3 - [(6-carbamimido-yl-naphthalen-2- yl) oxy] propoxy} naphthalene-2-carboximidamide was incubated at a concentration of 5 μΜ in Vero cells in Dulbecco's Modified Eagle Medium medium (DMEM) during 45 minutes at 37 ^° C. The cells were then washed three times with phosphate buffered saline (PBS), and observed directly under the microscope of fluorescence (blue channel). The treated cells showed no fluorescence emission in the expected range (Figure 9B), which demonstrates that the compound must not efficiently internalize through the membrane of living cells.

To try to find out the need for the presence of the two naphthalene units, 6 - ((3-hydroxypropyl) amino) -2-naphthimidamide was synthesized. 6 - ((3-hydroxypropyl) amino) - 2-naftimidamida incubated at a concentration 5 μΜ in Vero cells in Dulbecco's Modified Eagle Medium (DMEM) for 45 minutes at 37 ⁰ C. The cells were then washed three times with phosphate buffered saline (PBS), and were observed directly under the fluorescence microscope (blue channel). The treated cells showed a faint emission of fluorescence in the expected range, of less intensity compared to Compound (3) under the same observation conditions (Figure 10B), which shows that the compound seems to present no difficulties in its internalization, but yes in its accumulation inside the cell and in the mitochondria.

Finally we synthesize the 6 - ({3 - [(naphthalen-2-yl) amino] propyl} amino) naphthalene-2-carboximidamide (compound 18). In it we evaluate the influence of the two amidinium groups. Compound (18) at a concentration of 1 μΜ was incubated in Vero cells in Dulbecco's Modified Eagle Medium medium (DMEM) for 45 minutes at 37 ^° C. The cells were then washed three times with phosphate buffered saline (PBS), and were observed directly under the fluorescence microscope (blue channel). The treated cells showed a high intensity in fluorescence emission in the expected range under the same observation conditions as Compound (3) (Figure 11 A). A co-appointment with the Mitotracker ® (figure 11B) showed that its intracellular marking profile does not correspond to the mitochondria as evidenced by the superposition of both photographs (figure 11C).

 Conclusion: The presence of the second unit of naphthalene provides the molecules of the invention with a surprising capacity for cell accumulation, while the amidinium groups confer excellent selectivity in mitochondrial marking.

Example 9. Monitoring mitochondrial recycling processes in different cell lines. Freshly seeded CEF and Vero cells were incubated with compound (3) 5 μΜ for 45 min at 37 ° C as described above. Both cultures were labeled with Mitotracker at different times after they were incubated with the compound (3). The comparison of staining patterns of compound (3) and Mitotracker allowed us to verify that in the case of compound (3) there is a fluorescence transfer, probably associated with material recycling processes between the mitochondria and lysosomes, which resulted slower in the Vero line than in CEF. For example, after three hours after initial marking with compound (3), Vero cells still show considerable overlap between the tides of compound (3) and Mitotracker while CEF cells showed a different staining pattern, (Figure 12a). Similarly, in the case of CEF cells, the distribution of staining between mitochondria and lysosome is different depending on cell age. Freshly seeded cells appear to have a slow recycling rate, since they exhibit considerable overlap between the compound (3) and the Mitotracker after 3 h, unlike the senescent CEF cells, which showed a high B-galactosidase activity ( Figure 12a, right column), these have a completely remodeled tidal pattern of the compound (3) after 3h (a redistribution equivalent to that observed in a fresh culture after 7 h). Example 10. Study of the oxidative process.

 An experiment was carried out on the behavior of the marking with the compound (3) in cells subjected to different states of oxidative stress by incubation with hydrogen peroxide.

As a control, CEF cells were incubated with compound (3) as described above and with Mitotracker after 3 hours. As expected, the Mitotracker stained a filamentous mitochondrial network and the compound (3) revealed a high degree of overlap with the Mitotracker emission profile (Figure 12b, control); however, when CEF cells were incubated with hydrogen peroxide (100 μΜ) 24 h before being labeled with compound (3) and Mitotracker (3 h later), a high mitochondrial fragmentation (Mitotracker) was observed and there was hardly any overlap with the compound (3), a sign that could indicate a high mitochondrial recycling. If the damage from mitochondrial stress is severe, you practically do not know observe mitotracker dizziness while compound (3) still stained the damaged mitochondria (Figure 12b, H ₂ 0 ₂ , cells marked with arrows).

Example 11. Internalization and fluorescent marking with the biotin derivative (23). CEF cells were washed 3 times with 1 mL of PBS, treated with compound (23) 20 µΜ and kept in an incubator for 2 hours. The supernatant was suctioned, the cells were coated with 1 mL of DMEM and kept in the incubator for another 5 h. Compound (3) 5 μΜ or Mitotracker 500 nM was added in DMEN for 45 min prior fixation for subsequent mitochondrial localization, if needed. The cells were washed 3 times with PBS buffer and fixed with 4% PFA in PBS for 45 min. After fixation, the cells were washed again in the same manner and permeabilized with 0.5% Triton 10% PBS for 3 min and washed once more. The cells were coated with a 2% BSA solution in PBS for 30 min and the supernatant was sucked, and the streptavidin conjugates (st-AF594, st-AF405 or st-QDot565) were added in the same buffer for 90 min. Finally the cells were washed and deposited on slides, to be immobilized as mowiol before being observed under the fluorescence microscope.

As expected, the emission in the blue channel, the channel corresponding to the emission of the compound (3), showed the typical mitochondrial pattern (Figure 13 a; more importantly, the st-AF594 conjugate distribution is consistent with the mitochondrial marking ( red channel, Figure 13b) and fully coincides with the emission of compound (3) (superposition, Figure 13c) .This result is reproducible with other streptavidin conjugates, such as that of AlexaFluor 405 (stvAF405), or quantum conjugate dot Qdot565, which are found in the mitochondria in the presence of the compound (23). (Figure 13d and, respectively.) It should be noted that control experiments demonstrated that st-AF405 does not concentrate on the mitochondria in the absence of the compound ( 23), exhibiting poor marking and emission under the microscope using the same observation parameters.As expected, no emission of the compound (23) was observed if streptavidin conjugated with cotton is not added n fluorescent marker (Figure 13 f). Taking these results into account, we conclude that compound (23) is an efficient and versatile mitochondrial locator.

Figure 14 shows the CEF cells that have been incubated with compound 3 and 23, and subsequently have been fixed, permeabilized and incubated under the conditions described above with a streptavidin conjugate covalently bound to a quantum dot (st-Qdot 565). As you can see, compound 3 (seen through the blue channel) marks a typical mitochondrial pattern (Figure 14a); It is important to note that the st-Qdot 565 also shows exactly the same staining profile (green channel), which is confirmed by the superposition of both images (Figure 14b and 14c respectively). And in Figure 15 the CEF cells that have been subjected to the fixation, permeabilization and incubation protocol with the secondary marker st-AF594 in the absence of compound 23 are observed. As it can be verified, the emission of this is negligible under the same conditions of observation and there is no defined location pattern (figure 15a).

Claims

Claims 1. A compound of formula I

 I  where A and B are each independently selected from optionally substituted aryl and heteroaryl,  each of n, m, or, q is independently selected from 1, 2, or 3,  W is selected from single bond, optionally substituted alkyl, aryl and heteroaryl,  Ri is selected from hydrogen, hydroxyl, -OR ', -N (R') (R ") -, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, azido, tetrazino and an acceptor fluorophore group,  K is selected from single bond, alkyl, -O-alkyl-, -O-alkyl-N (R ') -, heterocycle and heteroaryl, each of R _a and R _b is independently selected from hydrogen and a photolabile protecting group, each of Pi and P ₂ are independently selected from hydrogen, C 1 -C 12 alkyl, C ₆ -C ₅ aryl, C ₃ -C 15 heteroaryl and optionally substituted C ₃ -C ₁₅ heterocycle, N ₂ , CN, halogen, -OR ', -SR' , -S (0) R ', -S (0) ₂ R', -OS (0) ₂ R ', -N (R') (R "), -C (0) R ', - C (0 ) OR ', -C (0) N (R') (R "), -OC (0) R 'and - N (R') C (0) R", each R 'and R "are independently selected from hydrogen, _Ci-C6, C3-C7cycloalkyl, aryl C ₆ -Ci5, heteroaryl and heterocycle-C15 C3 C3-C15 optionally substituted, and comprises at least one organic or inorganic anion to maintain neutrality. 2. The compound of formula according to claim 1,

 the wherein A, B, Ra, Rb, K, Ri, Pi, P ₂ , m, n, or, q are as defined in claim 1, and comprises at least one organic or inorganic anion to maintain neutrality. 3. The compound of formula Ib according to claim 1,

 Ib  or the compound of formula I-lc according to claim 1,

 I-lc wherein A, B, Ra, Rb, Pi, P ₂ , m, n, o and q are as defined in claim 1, and comprises at least one organic or inorganic anion to maintain neutrality, and wherein the C rings and D are heterocycles. 4. The compound of formula I-d according to claim 1, z  /  K HIH where Z is a fluorophore acceptor group and A, B, Ra, Rb, W, K, Pi, P _2, m, n, o, q are as defined in claim 1, and comprising at least one organic anion or inorganic to maintain neutrality.  The compound of formula according to claim 1,

 you wherein A, B, Ra, Rb, Pi, P ₂ , m, n, or, q are as defined in claim 1, and comprises at least one organic or inorganic anion to maintain neutrality. 6. The compound of formula If according to claim 1,

 If  wherein o and q are as defined in claim 1, and comprise at least one organic or inorganic anion to maintain neutrality.  The compound of formula Ig according to claim 1 or claim 6,

 Ig wherein Pi, P ₂ , m, n, or, q are as defined in claim 1, and comprise at least one organic or inorganic anion to maintain neutrality. 8. The compound of formula Ih according to claim 1,

 Ih wherein Ra, Rb, A, B, Pi, P ₂ , m, n, o, q, K and Ri are as defined in claim 1, and comprise at least one organic or inorganic anion to maintain neutrality.  9. Trifluoroacetic salt of 6,6 '- [1, 3-propanedi-yl-di (imino)] dinaphthalene carboximidamide. 10. Procedure for the repair of a compound of formula I,

 I  which comprises the reduction of a compound of formula IX

 , IX  and optionally coupling with a photolabile protective group, wherein A, B, Pi, P ₂ , m, n, or, q, W, K, Ri, Ra, Rb are as defined in claim 1, and comprise at least one organic or inorganic anion to maintain neutrality . 11. Kit for cell study comprising a compound of formula I as defined in any of claims 1 to 9. 12. Use of a compound of formula I as defined in any one of claims 1 to 9, or of a kit as defined in claim 11, as a selective reagent for mitochondria.  13. Use of a compound of formula I as defined in any one of claims 1 to 9, or of a kit as defined in claim 11, as a staining agent.  14. Method for marking mitochondria or a mitochondrial component comprising incubating a sample, wherein said sample comprises mitochondria or a mitochondrial component, with an aqueous solution of a compound of formula I as defined in any one of claims 1 to 9.  15. Method for staining mitochondria or a mitochondrial component, comprising incubating a sample, wherein said sample comprises mitochondria or a mitochondrial component, with an aqueous solution of a compound of formula I as defined in any one of claims 1 to 9, wherein said compound of formula I is in an amount sufficient to stain mitochondria or a mitochondrial component and incubation takes place for a time sufficient to detect fluorescence.  16. A method according to claim 14 or 15, further comprising a fixing step. 17. Method according to any of claims 14 to 16, further comprising a permeabilization step.  18. Method according to any of claims 14 to 17, wherein the incubation time is between 20 and 60 minutes.  19. A method according to any of claims 14 to 18, further comprising adding an additional reagent to the sample, wherein said additional reagent is a reagent that produces a detectable response due to a specific cellular component, intracellular substance, or a cellular condition.  20. The method of claim 19, wherein the additional reagent comprises a protein, an amino acid sequence, a labeled antibody and / or a labeled oligonucleotide.  21. Compound of formula IX as defined in claim 10. 22. Compound of formula X

 X wherein W, K, R¡, A, B, P¡, P ₂ , m, n, or, q are as defined in any of claims 1 to 8.