CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from Italian Patent Application No. 102017000117226 filed on 17/10/2017, the disclosure of which is incorporated by reference.

TECHNICAL FIELD

The present invention relates to a new class of fluorescent probes for detection in biological systems.

BACKGROUND ART

A fluorescent probe, in the area of biology, is a molecular system that allows selective detection of specific components of complex biological systems, such as tissues and cells, both fixed and in culture. The fluorescent probes allow marking, within a live cell, of subcellular elements of interest (organelles, proteins etc.) or measurement of important cellular parameters.

In recent years there has been a marked increase in the use of fluorescence microscopy as an experimental approach for the direct observation of biological phenomena due, in particular, to two factors: the technological progress of detection instruments, with the development of confocal microscopy and the mathematical analysis of traditional fluorescence images (digital imaging) , and the development of specific probes to mark and dynamically follow cell structures or physiological parameters.

Although in recent years important progress has been made in the development of new fluorescent probes, today there is still a need for fluorescent probes that have greater chemical stability to allow their preservation and a simpler utilization protocol. The inventors of the present invention have produced a new class of compounds that has demonstrated its ability to meet both the above needs and to guarantee, at the same time, a more effective biological detection.

DISCLOSURE OF INVENTION

The subject of the present invention is a class of molecules

(I)

wherein :

X and Y, equal to or different from each other, are chosen from 0, S and NR, where R is H or an aliphatic group Ci-Cis; n, r, m and s, equal to or different from one other, can assume the values 1 or 2.

Preferably, R is an aliphatic group C1-C6

Preferably, X and Y are both NC¾ and n = r = s = m = l

A further subject of the present invention is a fluorescent probe for detection in biological systems characterized in that it is composed of a molecule comprised in the general formula ( I ) .

BEST MODE FOR CARRYING OUT THE INVENTION

Examples are given below for purely illustrative non-limiting purposes .

- preparation of the molecules with general formula (I) -

AJ2NBD

The molecule 1 , 7-bis ( 7-nitrobenzo [ 1 , 2 , 5 ] oxadiazol-4-yl ) -4 , 10- dimethyl-1, 4, 7, 1 O-tetraazacyclododecane was produced (here and in the following referred to by the name AJ2NBD) as tetrahydrochloride salt, comprised in the general formula (I) . In particular, with reference to the above general formula (I), the synthesized molecule is characterized in that X and Y are NCH ₃ .

By way of example the synthesis procedure used is reported, without this constituting a limiting aspect of the present invention.

1 , 4 , 7 , 1 O-tetraazacyclododecane (1) (1 g, 5 mmol) and K2CO3 (10.2 g, 74 mmol) were suspended in acetonitrile (100 cm ³ ); a solution of 4-chloro-7-nitrobenzo [ 1 , 2 , 5 ] oxadiazol (2) (2.2 g, 11 mmol) in acetonitrile (80 cm ³ ) was added drop-wise in 1 hour; at this point the suspension was maintained under stirring at room temperature for 48 hours and then filtered. The resulting solution was evaporated under a vacuum at reduced pressure, obtaining an orange-coloured crude product. The product was recrystallized from hot ethanol/aqueous HCl at 37% obtaining AJ2NBD-4HC1 as a microcrystalline solid (2.7 g, 80%) .

To confirm its identity, the elemental analysis and the NMR analysis of the product obtained is given below.

Elemental analysis: C, 39.5; H, 4.7; N, 20.6.

¾ NMR analysis (DMSO-d ₆ , 30°C, δ in ppm) : 2.92 (s, 6H) , 3.14 (m, 4H) , 3.35 (m, 4H) , 4.41 (m, 8H) , 6.42 (d, J=9.2 Hz, 2H) , 8.52 (d, J=9.2 Hz, 2H) , 13.46 (br s, 1H) .

¹³ C NMR analysis (DMSO-d ₆ , 30°C, δ in ppm) : 41.0, 51.7, 58.7, 103.7. 121.8, 136.5, 145.1, 145.5, 145.9.

N202NBD

The molecule 4 , 10-bis ( 7-nitrobenzo [ 1 , 2 , 5 ] oxadiazol-4-yl ) -1 , 7- dioxa-4 , 10-diazacyclododecane was prepared (here and in the following it will be referred to by the name N202NBD) comprised in the general formula (I) . In particular, with reference to the above general formula (I), the synthesized molecule is characterized in that X and Y are 0.

By way of example the synthesis procedure used is reported, without this constituting a limiting aspect of the present invention . 1 , 7-dioxa-4 , 1 O-diazacyclododecane (3) (87 mg, 0.5 mmol) and K2CO3 (1.0 g, 7.4 mmol) were suspended in acetonitrile (10 cm ³ ) . 4-chloro-7-nitrobenzo [ 1 , 2 , 5 ] oxadiazole (2) (0.22 g, 1.1 mmol) in acetonitrile (8.0 cm ³ ) was added drop-wise over 1 h, after which the suspension was stirred at room temperature for 48 h and then filtered. The solution was evaporated under vacuum to yield the crude product, and the orange solid obtained was chromatographed on neutral alumina (70-230 mesh), eluting with chloroform. The eluted fractions were collected and evaporated to dryness to obtain an orange solid (0.18 g, 73%) . C2oH2oN ₈ 0 ₈ : C, 48.00; H, 4.03; N, 22.39. Found: C, 48.2; H, 4.1; N, 22.2. ¾ NMR (DMSO-cte, 30°C, δ in ppm) : 3.63 (m, 8H) , 3.70 (m, 8H) , 6.55 (d, J=9.2 Hz, 2H) , 8.55 (d, J=9.2 Hz, 2H) . ¹³ C NMR (DMSO-cte, 30°C, δ in ppm) : 49.8, 70.0, 97.2, 120.4, 131.7, 134.8, 141.3, 156.9.

- fluorescence tests -

The fluorescence tests were performed using the molecule AJ2NBD.

AJ2NBD was tested as a fluorescent probe on adherent cell lines (HT22, HeLa) and on a line that grows in suspension (Jurkat) . AJ2NBD · 4HC1 was solubilized in DMSO to the final concentration 15mM. The solution thus obtained was kept at - 20°C showing a high stability. In conclusion, AJ2NBD has a good solubility and is stable at the storage temperature. AJ2NBD was then used in the above-mentioned lines in a concentration range of 100-200 uM in flow cytometry and at a concentration of 500 μΜ in confocal microscopy for an incubation time of 10-15 min. The concentrations used are compatible with the cell viability of all the lines used.

The procedure with which AJ2NBD was used as a fluorescent probe is reported below for illustrative non-limiting purposes.

- Preparation of the adherent cells (HT22, HeLa) and marking

The adherent cells were brought to the desired confluence, the culture medium was removed and AJ2NBD was added in a concentration interval of 100-200 μΜ, after being appropriately diluted from the master solution (concentration of 15mM in DMSO) . The cells were subsequently incubated at 37°C, 5% CO2, for 10-15 min and then processed by flow cytometry also without washing in PBS.

- Preparation of cells in suspension (Jurkat) and marking -

The cells in suspension were centrifuged in order to obtain a pellet so that they could then be re-suspended at the desired concentration in a medium pre-heated to 37 °C where AJ2NBD was added in a concentration interval of 100-200 μΜ, after being appropriately diluted from the master solution (concentration of 15mM in DMSO) . The cells were subsequently incubated at 37°C, 5% CO2, for 10-15 min and then processed by flow cytometry also without PBS washing.

The decision to use several models (adherent cells and cells in suspension) was dictated by the need to evaluate in a differentiated and consequently more detailed manner the intracellular compartments highlighted by AJ2NBD as fluorescent probe: these compartments/organelles are variable in terms of extent and intracellular location both in relation to the metabolic moment of the cell and in relation to the cell type.

Relative to the spectral characteristics of AJ2NBD, it has been found that it has an emission band which is fairly broad but which at the same time does not prevent it from being used together with other fluorescent probes.

In this regard, AJ2NBD was used simultaneously with other probes on the market: Lysotracker Deep RED, Mitotracker Red, Dil, Bodipy, each able to mark specific cell organelles, such as mitochondria and lysosomes. This type of evaluation was very important as it allowed highlighting of any superimpositions with the fluorescence of the new marker; it is also useful since in cell biology it is often necessary to mark several organelles and identify a colocalization. It was also very important to ascertain that AJ2NBD does not interfere with the other probes used. In fact, if the fluorescent probes interfere with one another, this would necessarily result in incorrect interpretation of the biological phenomenon.

Having ascertained the fluorescent probe capacity on the above cell cultures, AJ2NBD was tested to establish whether it was also useful as a probe for study of the vesicular traffic. With the object of further verifying the potential of the fluorescent probes subject of the present invention, the HeLa and HT22 cells were cultivated in the absence of serum for 24h (a fundamental component for the normal growth of cells in vitro) and in the presence of rapamycin (50nM for 24h) as a positive control of autophagy, a process in which the formation of a large number of new vesicles is observed. In this regard it should be remembered that in literature it is known that the deprivation of serum is able to induce an oxidative stress with consequent activation of the autophagy pathway. The autophagy pathway is involved in the cell turnover of macromolecules and cell organelles. The first step of autophagy entails the development of isolated membranes able to delimit portions of the cytoplasm and/or organelles. These membranes then give rise to vacuoles surrounded by double membrane containing material to be eliminated. Recent studies have shown the presence of Rab30, a typical protein of the Golgi apparatus, on the autophagic vacuoles, thus demonstrating the involvement of this organelle in the genesis of autophagic vacuoles.

Colouring with the probe in question and

Mitotracker/Lysotracker highlighted compartments clearly distinct from the mitochondria, and a partial colocalization with the lysosomal compartment was detected. The best defined and most brilliant marking was in the perinuclear area, normally occupied by the Golgi apparatus. During the autophagic process, induced by deprivation of serum (SD) , the Golgi apparatus provides the membranes for construction of the autophagic vacuoles.

The presence of fluorescent vacuoles or particles suggested the physiological process of gemmation between the Golgi cisternae. A result of this type qualified AJ2NBD as a probe for study of the vesicular traffic, suggesting its use for commercial purposes in cell biology studies.

From the above description, it is evident that the fluorescent probes subject of the present invention, in addition to having a high chemical stability and an extremely simple utilization protocol, offer the significant advantage of representing a class of vesicular traffic markers. Lastly, the fluorescent probes subject of the present invention have the further advantage of penetrating inside the cells without the need to permeabilize them and alter their viability and functionality. This advantage allows the use of said fluorescent probes to study some cell processes as they take place.