PHENAZINE DERIVATIVE AND USE THEREOF FOR THE TREATMENT OF CANCER

A compound of formula (I),   wherein R1, R2, R3 and R4 are selected from a saturated or unsaturated, branched or unbranched, cyclic or non-cyclic alkyl, or an amide, or a functional group, or a salt or a solvate thereof, or a protonated form thereof, and to the use thereof for the treatment of cancer.

The invention relates to phenazine derivatives and uses thereof, in particular therapeutic uses thereof.

In the context of the treatment of pathologies and in particular tumors, new molecules and new approaches are constantly being developed and tested.

In particular, in recent years, photodynamic therapy has experienced a boom in particular for the treatment of skin pathologies. However, the sensitizing compounds known to date do not exhibit the best effects, and the failure rates are quite high. In addition, the wavelengths of the lasers used may generate adverse effects and induce deep lesions of the exposed tissues.

There is therefore a need to provide new compounds that would be effective for this type of non-invasive therapy.

One approach is to use fluorescent compounds capable of specifically targeting the cells of interest and exhibiting therapeutic properties under activation.

However, such compounds are rare.

Some phenazine derivatives are already known. Various compounds having such a phenazine structure have already been described in Laursen et al (chem Rev, 2004, 104:1663), Beifuss et al (above. Curr. Chem, 2005, 77), Terech et al (J Of Coll. Et entre. Sc, 2006:633), Llusar et al (Zeit. Fuer Anorg. Und Allge, chem, 2005, 631: 2215; J Of Mat. Chem, 2003, 13: 2505), Pozzo et al (Mol. Les cristaux et les Cristaux liquids Sc Et Tech, 2000, 344: 101; J Of Mat. Chem, 1998, 8: 2575) and US 2004/065227. However, to the knowledge of the inventors, these compounds have never been proposed as chemotherapeutic agents In addition, patent application WO2011117830 describes compounds derived from phenazine, but here again, to the knowledge of the inventors, such compounds do not have properties known to be used in photodynamic therapy.

There is therefore still a need for new compounds, and the invention aims to overcome this lack.

One of the aims of the invention is to provide compounds that can be used in photodynamic therapy that are effective, inexpensive and easy to produce.

Another object of the invention is to provide various therapeutic and diagnostic uses of these new compounds.

The invention relates to a compound of the following formula I:

independently of each other, R1 and R2 areeither a linear or branched, saturated or unsaturated, cyclic or non-cyclic C1-C18 alkyl, optionally substituted by one or more groups chosen from a hydroxyl group, an amino group, an aminoalkyl group, a C1-C5 alkoxy group, a C1-C5 alkyl, a peptide, a pyridine group, a phosphine group, a thiol, a C2 alkene, a C2 alkyne group and a halogen, or a benzyl radical optionally substituted by one or more radicals chosen from a hydroxyl group, an amino group, an aminoalkyl group, a C1-C5 alkoxy group, a C1-C5 alkyl group, and a halogen atom,or (hetero)aryl groups, optionally substituted by one or more groups chosen from a hydroxyl group, an amino group, an aminoalkyl group, a C1-C5 alkoxy group, a C1-C5 alkyl, a peptide, a pyridine group, a phosphine group, a thiol, a C2 alkene, a C2 alkyne group and a halogen, or a benzyl radical optionally substituted by one or more radicals chosen from a hydroxyl group, an amino group, an aminoalkyl group, a C1-C5 alkoxy group, a C1-C5 alkyl group, and a halogen,

in particular R2 possibly being a hydrogen atom, and

independently of each other, R3 and R4 are hydrogen H or correspond to R1 or R2 mentioned above, or R3 and R4 are either or both a carbonyl functional group forming amide functions including peptides or not,

or a salt or solvate thereof, or a protonated form thereof.

The invention is based on the surprising observation by the inventors that the aforementioned phenazine derivatives have variable fluorescent properties depending on the degree of protonation for in vitro and in vivo studies with an imaging objective. These molecules may also be used in the context of photodynamic therapy (PDT), with one and two photons.

Thus, the present invention describes a family of original compounds, low in toxicity and particularly promising for one- and two-photon imaging depending on the desired target. Their synthesis is carried out in few steps and at low cost. The intensity of the markings is remarkable, which objectively allows a more precise identification of the cytoplasmic targets to be envisaged. The intense fluorescence foci obtained in perinuclear regions could in particular correspond to the localization of the probe at the endoplasmic reticulum. These compounds therefore allow a crucial advance to be envisaged in the field of selective imaging and photodynamic therapy.

As will be demonstrated hereinafter in the examples, these compounds have allowed in vitro studies to be carried out on human cancer cells known to be a good model of xenograft, and one- and two-photon photon therapy have been shown to be effective in destroying such cells.

According to the present invention, the terms below have the following meanings, and the terms mentioned here having characteristics such as C1-C18, for example, can also be used with lower numbers of carbon atoms such as C1-C3 or C1-C5. If for example the term C1-C5 is used, it means that the corresponding hydrocarbon chain can comprise from 1 to 5 carbon atoms. If for example the term C3-C8 is used, it means that the corresponding hydrocarbon chain or ring can comprise from 3 to 8 carbon atoms.

The term “hydroxyl” corresponds to an alkyl-OH group, the alkyl group being as defined above.

The term “amino” corresponds to an amine which can be secondary, tertiary or quaternary. By extension, an “aminoalkyl” corresponds to an alkyl substituted by an amine.

The term “halogen atom” corresponds to a fluorine, chlorine, bromine or iodine atom. Chlorine is a preferred halogen atom within the scope of the present invention.

The term “halogen atom” corresponds to a fluorine, chlorine, bromine or iodine atom. Chlorine and fluorine are preferred halogen atoms within the scope of the present invention.

The term “aryl” used herein means a mono- or poly-cyclic aromatic group. An example of a monocyclic group can be phenyl.

The compounds of formula (I) possess unsaturations and can thus be in their tautomeric form. The present invention therefore also relates to the compounds of formula (I) in their tautomeric form.

The compounds of formula (I) may be in the form of a free base or in the form of addition salts with acids, which also form part of the invention.

These salts can be prepared with pharmaceutically acceptable acids, but additionally salts with other acids, useful for example to purify or isolate the compounds of formula (I), also form part of the invention.

Advantageously, the invention relates to the aforementioned compounds where R1 and R2 are, in particular independently of one another, linear or branched, saturated or unsaturated, cyclic or non-cyclic C4-C10 alkyls.

More advantageously, the invention relates to the aforementioned compound, where the protonated form of the compound of formula I is chosen from the following compounds:the compound of formula Ia:

the compound of formula Ib:

andthe compound of formula Ic:

The compounds of formula Ia are the most advantageous compounds according to the invention.

Thus, advantageously the invention relates to a compound of the following formula Ia:

independently of each other, R1 and R2 areeither a linear or branched, saturated or unsaturated, cyclic or non-cyclic C1-C18 alkyl, optionally substituted by one or more groups chosen from a hydroxyl group, an amino group, an aminoalkyl group, a C1-C5 alkoxy group, a C1-C5 alkyl, a peptide, a pyridine group, a phosphine group, a thiol, a C2 alkene, a C2 alkyne group and a halogen, or a benzyl radical optionally substituted by one or more radicals chosen from a hydroxyl group, an amino group, an aminoalkyl group, a C1-C5 alkoxy group, a C1-C5 alkyl group, and a halogen atom,or (hetero)aryl groups, optionally substituted by one or more groups chosen from a hydroxyl group, an amino group, an aminoalkyl group, a C1-C5 alkoxy group, a C1-C5 alkyl, a peptide, a pyridine group, a phosphine group, a thiol, a C2 alkene, a C2 alkyne group and a halogen, or a benzyl radical optionally substituted by one or more radicals chosen from a hydroxyl group, an amino group, an aminoalkyl group, a C1-C5 alkoxy group, a C1-C5 alkyl group, and a halogen,

optionally R2 is a hydrogen atom, and

independently of each other, R3 and R4 are hydrogen H or correspond to R1 or R2 mentioned above, or R3 and R4 are either or both a carbonyl functional group forming amide functions including peptides or not,

or a salt or solvate thereof, or a protonated form thereof.

Compounds according to the invention that are advantageous are the following:

More advantageously, the invention relates to the aforementioned compound, said compound having the following formula II, III or IV:

where X represents Cl, Br, OH, F or I.

The invention further relates to a pharmaceutical composition comprising, as active substance, a compound as defined above, in association with a pharmaceutically acceptable vehicle.

The invention relates to a compound as mentioned above, for use thereof as a drug.

According to the present invention, a pharmaceutically acceptable derivative comprises, but is not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or any other adduct or derivative that, upon administration to a patient in need, is able to provide, directly or indirectly, a compound as described above, or a metabolite or residue thereof, e.g. a prodrug.

The compounds according to the invention may also be vectorized, in particular via R4, by grafting natural ligands specifically recognized by cancer cells. These biomolecules may be steroids, sugars (glucose and derivatives), amines, amino acids or peptides.

As mentioned above, “pharmaceutically acceptable vehicle” means one or more solvents, diluents, or another liquid vehicle, dispersing or suspending auxiliaries, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, depending on the particular dosage form desired. Except to the extent that a conventional carrier is incompatible with the compounds of the invention, for example by producing any adverse biological effect or otherwise interacting in a detrimental manner with any other component(s) of the aforementioned pharmaceutical composition, the use of any known carrier is envisaged within the scope of the present invention.

Some examples of materials that can serve as pharmaceutically acceptable carriers are, without limitation, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethyl alcohol and phosphate buffer solutions, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweeteners, flavoring and perfuming agents.

Preservatives and antioxidants may also be present in the composition, depending on the formulator's judgment.

A compound according to the invention is preferably formulated in unit dosage form for ease of administration and uniformity of dosage. It is understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician as part of the medical evaluation. The specific therapeutically effective dose level for any particular patient or organism will depend on a variety of factors comprising the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition used; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration and rate of excretion of the specific compound employed; the duration of treatment; drugs used in combination or coinciding with the specific compound employed; and similar factors well known in the medical arts.

Furthermore, after formulation with an appropriate pharmaceutically acceptable excipient or vehicle in a desired dosage, the pharmaceutical compositions according to the invention may be administered to humans and other animals by the oral, rectal, parenteral, intracismal, intravaginal, intraperitoneal, dermal (e.g. by powders, ointments or drops) or buccal routes, as an oral or nasal spray, or the like depending on the severity of the disease or cancer being treated.

The active compounds according to the invention may also be in microencapsulated form, optionally with one or more excipients as noted above.

It is also appreciated that the compounds and pharmaceutical compositions of the present invention may be used in combination therapies, i.e., the compounds and pharmaceutical compositions may be administered simultaneously with, before, or after one or more other desired medical therapeutic agents or procedures.

Advantageously, the compound according to the invention has coloring properties, and may be used as a dye. Such dyes are in particular red in color, and are capable of coloring (or pigmenting) plant or animal fibers.

Due to their fluorescent properties, the compounds according to the invention may also be used as fluorescent “dyes,” in particular for labeling biological molecules or cellular organelles.

In another aspect, the invention relates to a compound as defined previously, for use thereof in the context of the treatment of pathologies by photodynamic therapy. In particular, the pathologies that may be treated with the compound according to the invention, using photodynamic therapy, are tumors (solid or hematopoietic) and keratoses.

Once a cancer is diagnosed, different methods are available to practitioners to treat the disease. These different techniques may be combined and the choice of treatment depends on the type of cancer and the stage at which it was discovered:surgery is traditionally used to remove the primary tumor and allows a large number of early cancers to be cured. It is now the most effective method for small tumor foci without metastasis. However, eliminating all cancer cells and preventing their spread during surgery can be difficult.radiotherapy, based on the action of ionizing radiation (X, α, β- or γ), is used to treat tumors, but poses the problem of the toxicity of ionizing radiation on surrounding healthy tissue.chemotherapy is the treatment of cancer with drugs that destroy cancer cells and prevent them from multiplying. There are many different drugs, with the choice of treatment depending on the type of cancer. However, they are not yet specific enough, since they do not yet differentiate between healthy cells and cancerous cells, thus causing many side effects.photodynamic therapy (PDT) consists in bringing pathological tissue into contact with a photo-activatable molecule (called photo-sensitizer), then photo-activating the molecule with light in order to produce singlet oxygen, which is very toxic, which will locally destroy the cancerous lesion. The major advantage of PDT is its selectivity. Indeed, the light used alone is not harmful, and the photosensitizer without light is not toxic. To induce the reaction, a joint action of light, photosensitizer and oxygen is required. Thus, by optimizing the concentration of the photosensitizer and the dose of light (power of the laser), it is possible to selectively destroy cells.

On the therapeutic level, conventional treatments have an imperfect selectivity with respect to tumor cells. One of the reasons is that scientists have long favored the race for the IC50(concentration of product necessary to kill 50% of a population of cells) rather than the search for specificity. They cause side effects, sometimes severe, which limit the doses at which they can be administered.

Clinical oncology therefore calls for the joint development of new, more sensitive and more effective diagnostic methods, as well as new, more effective, better tolerated, but also better understood therapies.

Based on this need, the inventors have taken advantage of the low toxicity properties of the compounds according to the invention and their ability to produce singlet oxygen under light excitation in order to propose a photodynamic therapy.

The inventors thus propose a photodynamic therapy using the aforementioned compounds, or:With one photon (λirrad.=514 nm), where the desired targets are surface tumors (melanoma, bladder, esophagus and bronchi) because the excitation wavelength is in the green. Although far-red (652 nm) or near-infrared (760 nm) excitation is preferred, studies have shown that for specific cases, excitation with green light is less toxic and more effective than excitation by red light. For example, in a study performed with Photofrin, on human mesothelioma xenografts in nude mice, photodynamic therapy performed with 514 nm light was shown to induce tumor-level effects, similar to those obtained with 630 nm excitation, with a decrease in normal tissue damage. Green light prevents deep tissue damage, thus reducing the risk of perforation. Since then, many studies on cells in culture or on laboratory animals have confirmed this.

With two photons (λirrad.=810 nm), the desired targets are deeper tumors (breast cancer, prostate cancer, retinoblastoma) because the excitation of the molecule with a laser in the far red or the near infrared allows deeper tissue penetration (zone of biological transparency).

Advantageously, the invention relates to a method for treating pathologies, in particular for treating tumors, by photodynamic therapy, comprising a step of administering an effective dose of compound as defined above to individuals in need, and exposure to a light beam having a wavelength varying from 450 to 850 nm.

Advantageously, the above-mentioned method is a method for treating pathologies, in particular for treating tumors, by one-photon photodynamic therapy, comprising a step of administering an effective dose of compound as defined above to individuals in need, and exposure to a light beam having a wavelength varying from 450 to 550 nm.

Advantageously, the above-mentioned method is a method for treating pathologies, in particular for treating tumors, by two-photon photodynamic therapy, comprising a step of administering an effective dose of compound as defined above to individuals in need, and exposure to a light beam having a wavelength varying from 750 to 850 nm.

In any of the aforementioned methods, it is advantageous to administer a compound according to the invention at a dose varying from 2 nmol·L−1(or nM) to 1 μmol·L−1(or 1000 nmol·L−1), in particular from 2 nmol·L−1(or nM) to 1 μmol·L−1per kg for oral administration. Thus, for an individual weighing 70 kg on average, the dose administered will be from 140 nM to 70 μM. In the context of targeted administration (by injection directly into the tumor for example), the dose will be directly that described above without the mass multiplier coefficient in kg.

Photodynamic therapy (PDT) has been used for many years in dermatology. Its theoretical principle is based on the use of a harmless molecule, accumulating preferentially in the cells to be treated. This molecule is transformed into a cytotoxic molecule after light excitation. The specificity of the treatment comes on the one hand from the pharmacokinetics of the molecule (diffusion, absorption and cellular metabolism), and on the other hand from the physics of the luminous flux.

Antitumor photodynamic therapy is therefore based on the combination of photosensitizing (Ps) molecules capable of concentrating in tumor cells, and focused light of the appropriate wavelength (Ps dependent). The combination of these two factors will allow tumor tissues to be specifically targeted and destroyed. This method still has a major drawback: only cancers accessible to light can be treated (red light, for example, only penetrates about 1 cm into living tissue).

The action of light (at a carefully selected wavelength) on the sensitizer will lead to the formation of singlet oxygen1O2(short-lived of about 0.01 to 0.004 μs), a molecule that is very reactive toward cellular components and therefore very toxic. The photosensitizer is injected intravenously; this will concentrate more or less selectively in the tumor tissue, the irradiation of which by laser light with a wavelength appropriate to the dye used leads to necrosis or apoptosis of the cancer cells.

The inventors have made the surprising observation that the compounds according to the invention, once activated at specific wavelengths, are capable of producing reactive oxygenated species in the form of singlet oxygen with a quantum yield ϕΔof about 0.1. This low efficiency is expected, since the vast majority of absorbed photons (76%) are converted into light.

Surprisingly, however, the inventors have noticed that this yield is largely sufficient to lead in vitro to the destruction of 98% of the tumor cells at very low concentrations of about 100 nM.

The invention also relates to a compound as defined previously, for use thereof in the context of diagnosing pathologies, in particular cancers.

Fluorescence imaging is one of the most powerful techniques for observing dynamic intracellular processes in living cells. Access to new adaptable fluorescent probes is of major importance because only very few biomolecules can currently be visualized due to inherent limitations in the structure of the probes used to date.

The imaging proposed, or the use of the compounds according to the invention, is based on conventional one-photon or two-photon fluorescence, as described above.

The invention further relates to a method of in vivo fluorescence imaging, comprising a step of administering an effective dose of compound as defined above to individuals in need, and exposure to a light beam having a wavelength varying from 450 to 850 nm, and a step of detection by appropriate means of fluorescent cells, tissues or cell organelles.

The invention further relates to a method of in vivo fluorescence imaging, comprising a step of administering an effective dose of compound as defined above to individuals in need, and exposure to a light beam having a wavelength varying from 450 to 550 nm, and a step of detection by appropriate one-photon fluorescence detection means of fluorescent cells, tissues or cell organelles.

The invention further relates to a method of in vivo fluorescence imaging, comprising a step of administering an effective dose of compound as defined above to individuals in need, and exposure to a light beam having a wavelength varying from 750 to 850 nm, and a step of detection by appropriate two-photon fluorescence detection means of fluorescent cells, tissues or cell organelles.

The invention also relates to the use of an aforementioned compound, for the in vitro or ex vivo visualization of living cells, of cytoplasmic organelles (mitochondria, endoplasmic reticulum, Golgi apparatus, vesicles, nucleus, etc.) or of tissues, by one- or two-photon fluorescence microscopy, said compound being used in particular at a concentration varying from 2 to 500 nmol·L−1.

The invention also relates to a method, in particular in vitro, for visualizing living cells, cytoplasmic organelles (mitochondria, endoplasmic reticulum, Golgi apparatus, vesicles, nucleus, etc.) or tissues, by fluorescence microscopy, comprising:a step of bringing living cells, cytoplasmic organelles or tissues, previously taken from an individual or an animal, into contact with a compound mentioned above, at a concentration of 2 to 500 nmol·L−1.a step of exposure to a light beam having a wavelength varying from 450 to 850 nm, anda step of detection by appropriate one- or two-photon fluorescence detection means of the fluorescent cells, tissues or organelles.

The invention also relates to a method, in particular in vitro, for eradicating cells comprising a step of using a light source emitting one or two photons to expose cells treated with a compound according to one of claims1to4, said compound being used at a concentration varying from 1 to 1000 nmol·L−1.

The invention also relates to the use of a compound mentioned above for the eradication, in particular in vitro, of cells.

Another aspect of the invention relates to a method of diagnosis, in particular in vitro, of a pathology involving a deregulation of the expression or the activity of one or more peptidases, amidases or both, from a biological sample from individuals affected by said pathology, comprising:a step of bringing said biological sample into contact with a compound as defined in one of claims1to3, where R4 is a functional group inhibiting the fluorescent properties of said compound,a step of fluorescence detection after exposure to a light beam having a wavelength varying from 450 to 850 nm.

The inventors have made the surprising observation that certain R4 groups, in particular (Carbonyl of the C(O-alkyl or C(O)-aryl type), drastically modify the fluorescence properties of the compounds according to the invention. However, once these groups are cleaved, by breaking the amide function, the compounds recover their initial fluorescence capacities.

Thus, the compounds of the invention where R4 is a group strongly impacting fluorescence may serve as a diagnostic probe for detecting abnormal peptidase or amidase activity within cells, a deregulation that is correlated with a pathology.

Therefore, if the cell is healthy, no fluorescence will be emitted after excitation, or fluorescence at a certain level FO will be measurable. If, however, the cell is a cell with increased peptidade/amidase activity compared to the healthy cell, or simply begins to express the peptidade/amidase that is not expressed in the healthy cell, the R4 group will be cleaved, or will be more cleaved, and a difference in fluorescence will be observed.

In the same way, if the pathological cell has a decrease in peptidade/amidase activity compared to the healthy cell, the fluorescence in the pathological cells will be weaker, or even disappear.

The invention will be better understood in the light of the following examples and drawings.

EXAMPLES

Commercial analytical-grade reagents were obtained from suppliers and used directly without further purification. The1H and13C NMR spectra are recorded in CDCl3, CD2Cl2, CD3CN, acetone-d6and DMSO-d6, determined with a Brucker AC250 spectrometer operating at 250 MHz or with a Jeol ECS400 spectrometer operating at 400 MHz. Chemical shifts are expressed in ppm and coupling constants (J) are in hertz. Separation patterns are designed in the form of s, singlet; br s, broad singlet; d, doublet; dd, doublet of doublets; t, triplet; td, triplet of doublets; qt, quintet.

The elemental and MS (mass spectrometry) analyses were carried out by the Spectropole de Marseille. ESI mass spectral analyses are recorded with a mass spectrometer 3200 QIRAP (Applied Biosystems SCIEX). High-resolution mass spectral analyses are recorded with a SYNAPT G2 HDMS mass spectrometer (Waters)

The preparative flash column chromatographies were carried out using silica gel G60 230-240 mesh (Merck).

Example 1—Syntheses of Compounds According to the Invention

Compound A: 1-Octylamine (v=830 μL, 2.05 equiv.) and N,N-diisopropylethylamine (DIPEA) (v=874 μL, 2.05 equiv.) was added to a solution of 1,5-difluoro-2,4-dinitrobenzene (DFNB) (m=500 mg, 1.00 equiv.) in ethanol (v=50 mL). The solution was heated to reflux for 1.5 h. After cooling to room temperature, the resulting solid in suspension was isolated by filtration, rinsed with EtOH and dried under vacuum to obtain compound A (m=1.04 g, quantitative yield) in an orange crystalline form.

Compound B: Boc2O (m=1.94 g, 8.89 mmol, 3.7 equiv.) and 4-dimethylaminopyridine (DMAP) (m=50 mg, 0.41 mmol, 17 mol %) were added to a solution of compound A (m=1.03 g, 2.43 mmol, 1.0 equiv.) in THF (10 mL). The solution was refluxed for 4 h. The solvent was removed under vacuum. The crude product was purified by flash chromatography (silica F60, DCM 100) to obtain compound B (m=1.52 g, 2.44 mmol, quantitative yield) in the form of a yellow solid.

Compound 8b: Compound B (m=5.15 g, 8.28 mmol, 1.0 equiv.) and hydrazine monohydrate (2.3 mL, 47.1 mmol, 5.7 equiv.) were added to a suspension of Pd on carbon carbon 5% (m=180 mg, 0.085 mmol, 1% mol) in EtOH (80 mL). The mixture was heated to reflux for 1.5 h. The Pd/C was removed by filtration through Celite 545, and the solid phase was rinsed with dichloromethane (3×100 mL). The combined organic phase was washed with water (3×150 mL) and brine (100 mL), dried with anhydrous MgSO4, filtered, concentrated and dried under vacuum to obtain compound 8b (m=4.43 g, 7.87 mmol, 96% yield) as a yellow solid.

Compound 12b: TFA at 0° C. was added to a solution of compound 8b (m=303 mg, 0.538 mmol) in dichloromethane (v=5 mL), HCl (12N, v=2 mL). This mixture was stirred under argon overnight. The resulting solid in suspension was collected by filtration, rinsed with CH2Cl2(v=20 mL), and dried under vacuum to obtain compound 12b (m=196 mg, 0.449 mmol, 84% yield) in the form of a light pink solid. This raw product was used directly without further purification.

Compound 13b: Compound 12b (m=306 mg, 0.703 mmol, 1.0 equiv.) was added to a solution of DFDNB (m=258 mg, 1.27 mmol, 1.8 equiv.) in MeCN (v=25 mL). The flask was closed with a septum and the solution was cooled in an ice water bath and degassed. N(iPr)2Et (v=735 μL, 4.22 mmol, 6.0 equiv.) was then added dropwise using a syringe under argon. The solution was stirred at 0° C. for 2 hours, then at room temperature for an additional two hours. The solution was concentrated in vacuo, and the residue taken up with EtOH (v=30 mL) and MeCN (v=10 mL). The solid obtained in suspension was recovered by filtration, rinsed with EtOH (v=100 mL) and Et2O (v=20 mL), and dried under vacuum to obtain compound 13b in the form of an orange powder (m=365 mg, 0.499 mmol, 79% yield).

Compound 3: Compound 12b (m=115 mg, 0.263 mmol, 1.2 equiv.) was added to a solution of compound 13b (m=160 mg, 0.219 mmol, 1.0 equiv.) in anhydrous MeCN (v=30 mL). The flask was closed, degassed and N(iPr)2Et was added dropwise (m=370 μL, 2.12 mmol, 9.6 equiv.) using a syringe under argon. The mixture was stirred at room temperature for 2 h with stirring, then refluxed overnight. After concentration of the solvent under vacuum, the residue was taken up with a mixture of acetone (v=5 mL) and ethanol (v=5 mL). The resulting solid product was isolated by filtration, washed with EtOH and dried under vacuum to obtain Compound 3 (m=155.5 mg, 0.148 mmol, 68% yield) as an orange powder.

Compound 1a: SnCl2.2H2O (343 mg, 1.52 mmol, 32 equiv.) and HCl (12M, 0.13 mL) were added to a solution of macrocycle 3 (50 mg, 0.05 mmol, 1 equiv.) in absolute ethanol (50 mL). The mixture was stirred at reflux overnight and neutralized with NaHCO3before adding ethanol (30 mL) and water (20 mL). After evaporation of the solvent under reduced pressure, the residue was extracted with a dichloromethane/ethanol mixture (3/1, v/v). The red organic layer was washed with an aqueous solution of HPF6(1 wt. % in water, 4×150 mL) and brine (100 mL), dried with MgSO4and concentrated in vacuo to obtain compound 1a [PF6] as a dark red solid (35 mg, 62% yield).

Compound 12h: a solution of compound 4 (628 mg, 1.66 mmol, 1 equiv.) in THF (25 mL) was hydrogenated (40 bars) overnight in the presence of Pd/C (5 wt. %, 36 mg, 0.02 mmol, 1 mol %). After reducing the pressure, the solution was degassed under sonication for 5 min. 1,5-difluoro-2,4-dinitrobenzene (320 mg, 1.58 mmol, 0.95 equiv.) was added to the solution with stirring at 0° C. The solution was left at this temperature for a further 10 min and the completion of the reaction was monitored by TLC. Then DIPEA (301 μL, 1.66 mmol, 1 equiv.) was added to neutralize the solution. The Pd/C was removed by filtration through celite. The crude product was purified by flash chromatography on silica gel using a dichloromethane/cyclohexane mixture (1/1) as eluent to obtain compound 12h in the form of a red solid (620 mg, 75% yield).

Compound 13d: a solution of compound 4 (628 mg, 1.66 mmol, 1 equiv.) in THF (25 mL) was hydrogenated (40 bars) overnight in the presence of Pd/C (5 wt. %, 36 mg, 1 mol %). After decreasing the pressure, the solution was degassed by sonication for 5 min, then cooled to 0° C. in an ice tray, and 1,5-difluoro-2,4-dinitrobenzene (320 mg, 1.58 mmol, 0.95 equiv.) was added to the solution with stirring. The reaction was maintained at 0° C. for 10 min. Then 1-octylamine (286 μL, 1.93 mmol, 1.1 equiv.) and DIPEA (289 μL, 1.66 mmol, 1 equiv.) were added. The mixture was stirred at room temperature for 3 days. After filtration through celite and concentration, the raw product was purified by flash chromatography on silica gel using a dichloromethane/cyclohexane mixture (50/50 to 55/45) as eluent to obtain compound 13d in the form of a red solid (498 mg, 49% yield).

Compound 1c: A solution of compound 13d (200 mg, 0.31 mmol, 1 equiv.) in methanol (40 mL) was hydrogenated (40 bars) overnight in the presence of Pd/C (5 wt. %) and HCl (12M, 0.1 mL). Then the mixture was stirred in air for 24 hours. The Pd/C was removed by filtration through celite. After removal of the solvent under reduced pressure, the resulting solid was taken up with dichloromethane (80 mL), washed with a solution of aqueous HPF6(1 wt. % in water, 2×50 mL) then distilled water (50 mL) and in fine concentrate. The residue was purified by flash chromatography on standard alumina 90 using a dichloromethane/cyclohexane mixture (100/0 to 99/1) as eluent to obtain compound 1c [PF6−] as a red solid (192 mg, 87% yield).

Synthesis of Compounds where R2 is Hydrogen

Compound 12 (808 mg, 5.18 mmol, 1.0 eq) and 1-octylamine (3.0 mL, 18.2 mmol, 3.5 eq) were introduced into a pressure bomb. The bomb was closed with a Teflon cap. The mixture was stirred at 140° C. for 1 hour. After cooling to room temperature, heptane (10 mL) was added. The resulting solid in suspension was isolated by filtration, purified by chromatography (60F silica, DCM, 100) to obtain compound 13 (1.23 g, 4.64 mmol, 90% yield) as a yellow solid.

Compound PR4 According to the Invention: N2,N7-dioctylphenazine-2,3,7-triamine

A solution of compound 20 (302 mg, 0.571 mmol) in MeOH (30 mL) was hydrogenated (20 bars) in the presence of Pd/C (5%) and HCl (12M, 0.5 mL) overnight. After addition of MeOH (30 mL), the solution was stirred under air for 24 h. The Pd/C was removed by filtration under Celite, and the solid phase was rinsed with

Compound 13c: A solution of compound 4 (625 mg, 1.66 mmol, 1 equiv.) in THF (35 mL) was hydrogenated (40 bars) overnight in the presence of Pd/C (5 wt. %, 36 mg, 1 mol %). After decreasing the pressure, the solution was degassed by sonication for 5 min, then cooled to 0° C. in an ice tray, and 1,5-difluoro-2,4-dinitrobenzene (320 mg, 1.57 mmol, 0.95 equiv.) was added to the solution with stirring. Then tert-butylamine (736 μL, 6.98 mmol, 4.2 equiv.) and DIPEA (602 μL, 3.46 mmol, 2.1 equiv.) were added. The mixture was stirred at room temperature for 4 days. After filtration through celite via dichloromethane and evaporation of the solution, the raw product was purified by flash chromatography on silica gel using dichloromethane/cyclohexane (1/1 to 6/4) as eluent to obtain the compound 13c as a red solid (575 mg, 63% yield).

Compound 1b: A solution of compound 13c (1 g, 1.71 mmol, 1 equiv.) in methanol (60 mL) was hydrogenated (20 bars) in the presence of Pd/C (5% by mass) and HCl (12M, 0.5 mL) for 6 hours. Then the mixture was stirred under air for 16 h. The Pd/C was removed by filtration through celite (AW) that had been rinsed several times with methanol and dichloromethane. After removal of the solvent under reduced pressure, the resulting solid was taken up in dichloromethane and washed with an aqueous solution of HPF6(5% by mass in water, 2×60 mL) and distilled water (60 mL). The organic layer was dried with Na2SO4, filtered and evaporated under reduced pressure. The residue finally obtained was precipitated in pentane and filtered to obtain product 1 b [PF6−] in the form of a red solid (1.05 g, 95% yield).

Example 2—Physicochemical Properties of the Compounds According to the Invention

The inventors have identified the following different properties of the compounds according to the invention:

The versatility of the synthesis method allows the introduction of various substituents ad infinitum to modulate the solubility properties. Depending on the hydrophilic/hydrophobic nature of R1, R2, R3 and R4, it is thus possible to solubilize type II compounds in polar, apolar, protic or aprotic solvents.

It is noted that although the compounds are more soluble in organic solvents, these exhibit the property of solubility in water, a property of major interest for use in vitro and in vivo on biological and cellular samples.

a. Characterization of Protonated Forms

The inventors have characterized the different protonated forms of compounds of formula I where R1and R2are C8H17, R3is tert-butyl and R4is H, of the following formula:

as well as the mono, di and tri protonated forms of the following respective formulas

The results obtained are shown inFIG. 1.

The1H NMR spectra of the compound

confirmed this hypothesis in particular with the presence of three protons, Ha, Hb and Hc, in the aromatic region with coupling constants traditionally encountered within a 1,2,4-trisubstituted benzene.1H NMR data also showed the presence of two magnetically non-equivalent octyl chains. All of these data suggest the formation of a phenazinium derivative

In addition, the proton signals Haand He—respectively at 5=7.17 and 7.00 ppm—experience a very strong shielding effect (δHd=6.25 ppm and (δHe=5.91 ppm) after addition of NaOD (40% w/w in D2O), an effect that is unusually observed within an aromatic system but already seen within certain triaminophenazines (δ=6.5-6.1 ppm and 5.9-5.4 ppm) described by Roy. This observation can be explained by a deprotonation reaction inducing a break in aromaticity in favor of a quinoidal-type structure

a. Optical Properties

In addition to characterizing the protonated forms, the inventors also tested the absorption and emission properties of the various compounds according to the invention.

i) Absorption Properties

The inventors tested the absorption of the four more or less protonated compounds mentioned above and evaluated ε (M−1cm−1) as a function of the wavelength λ (in nm).

FIG. 2shows the absorption spectrum of the type I molecule. The absorption spectrum reflects the presence of an uncharged type I species for which the degree of delocalization/conjugation is lower (hypsochrome effect) than for the type II cationic species.

FIG. 3shows the absorption spectrum of the type II molecule. The absorption spectrum reflects the presence of a mono-charged type II species for which the degree of delocalization/conjugation is greater (batochromic effect) than for the neutral type I species.

FIG. 4shows the absorption spectrum of the C-type molecule. A “super” acid, triflic acid (HOTf) (pKa(MeCN)=0.70), was used to protonate II in the UV cell (C≈1.44×10−5M, in MeCN). When acid is added (from 0.1 to 16 equiv.), the intensity of the initial bands located at 267, 308, 465 and 552 nm decreases in favor of the appearance of new bands at 274, 322, 507 and 686 nm. This spectral evolution reflects the disappearance of the starting compound II in favor of the formation of a single type C species whose absorption bands are of higher energy. The first protonation step is complete after adding 16 equiv. of HOTf

FIG. 5shows the absorption spectrum of the D-type molecule. Beyond the addition of 16 equiv. of triflic acid, a new species appears (new bands at 268, 287, 370, 475 and 506 nm) at the same time as the C cation disappears. Double protonation of II is complete after adding plus 2600 equiv. of HOTf. The C band at 686 nm has completely disappeared in favor of the spectrum of a D trication having a narrow band at 506 nm and equipped with a shoulder characteristic of a cyanine-type structure.

ii) Emission Properties

The inventors tested the fluorescence emission as a function of the wavelength λ (in nm) for the following compound taken up in acetonitrile

The results obtained are shown inFIG. 6.

The UV-Vis absorption spectrum of II showed the presence of two main bands located at λmax=265 nm and 549 nm and with respective shoulders located at λ=295 nm and 578 nm. Molecule II is also fluorescent and emits at λem=642 nm (excitation at λ=550 nm). This emission is comparable to that produced by the “neutral red” cationic analogue compound reported in the literature (λabs=534 nm and λem=616 nm)

iii) Fluorescence Yield and Emission

The inventors then set out to measure the value ϕf of the quantum yield of fluorescence of the following compound taken up in acetonitrile

To do this, the inventors measured the absorbance (relative intensity) as a function of the wavelength (in nm) in the presence of increasing doses of 1,8-DiazaBicyclo[4.3.0]Undec-7-ene (DBU—0 to 2 equivalents).

The results are shown inFIG. 7.

Phenazinium II and its II-H conjugate base fluoresce in MeCN at neutral or basic pH. Conversely, no luminescence property was observed in an acid medium.FIG. 7shows the spectral evolution of phenazinium II during the addition of DBU (excitation at 483 nm). This evolution clearly reflects the disappearance of the starting compound (decrease in the band at 637 nm) in favor of the formation of a single II-H species possessing an emission at higher energy much lower than that of II (increase in the band at 550 nm). Calculations of quantum yields indeed show that the deprotonated form [II-H] produces less fluorescence (ϕf=0.08 at 550 nm) than the starting form II (ϕf=0.76 at 637 nm). The reference used for the calculation of the fluorescence quantum yield is tetraphenylporphyrin in acetonitrile (ϕf=0.15).

The emission maximum is at a localized wavelength in the far red at 645 nm.

The coefficient ϕf found is 0.76, which attests to a very high fluorescence yield and a high brightness of about 50000.

The brightness (B) is proportional to the amount of light emitted by fluorescence at a given excitation light according to the relationship B=ε×ϕ (with ε=molar extinction coefficient and ϕ the emission quantum yield).

The calculation of ε is carried out using a spectrophotometer measuring the absorbance A (quantity without unit) of a dilute solution of known concentration C in a tank of thickness I.

The fundamental relation used in spectrophotometry is presented in the form: A=ε·I·c (A being the absorbance or optical density)

Calculation of ϕ: It is determined by measuring the emission intensity of a solution of known concentration; the reference used to calculate the fluorescence quantum yield here is tetraphenylporphyrin in acetonitrile (ϕf=0.15).

The quantum yield is defined by:

ϕ=number of photons emitted/number of photons absorbed

iv) Fluorescence In Vivo

Prior to fluorescent labeling tests on cell lines, the inventors tested the toxicity of the compounds according to the invention.

MCF-7 cancer lines were seeded in 96-well plates at a concentration of approximately 5000 cells/well in 200 μl of culture medium and left in culture for 24 hours. Then, the cells were incubated for 72 h, with or without the compound to be tested (from 1 nM to 1 μM). After incubation with the compounds, an MTT test was carried out in order to test the cytotoxicity of the compounds. The cells were briefly incubated in the presence of 0.5 mg mL−1of MTT for 4 h in order to measure mitochondrial activity. Then, the MTT precipitates were dissolved in 150 μL of an ethanol/DMSO (1:1) mixture solution and the absorbance was read at 540 nm.

The inventors came to the conclusion that the 100 nM dose did not significantly affect cell survival, as shown inFIG. 14. It is observed very clearly that the cytotoxicity in the dark is low at a concentration of 100 nM (FIG. 14)

The inventors tested the fluorescence of the compound of the following formula

The MCF-7 human breast cancer cells were seeded in petri dishes (World Precision Instrument, Stevenage, UK) having a glass plate at the bottom, in 2 mL of culture medium. The cells were then incubated for 16 h with the compound according to the invention at a concentration of 0.1 μM or 0.5 μM. 15 minutes before the end of the incubation, the cells were incubated with Hoechst 33342 (Invitrogen, Cergy Pontoise, France) at a final concentration of 5 μg·mL−1in order to label the cell nuclei. Then the cells were washed twice with culture medium.

One-photon fluorescence imaging was performed on live cells at a wavelength of 514 nm using a Carl Zeiss Confocal Microscope (LSM780). Two-photon fluorescence imaging was performed at wavelengths of 790 nm or 810 using the Chameleon laser available on the same microscope. All images were taken with the same objective, at the same magnification (63×/1.4 OIL DIC Plan-Apo).

The results obtained in single-photon microscopy are presented inFIG. 8, and in two-photon microscopy inFIG. 9.

These compounds showed remarkable one- and two-photon imaging properties (FIGS. 3 and 4). It clearly appears that the compounds are easily identifiable and that their localization is only cytoplasmic (the co-localizations are perfectly conclusive). It is interesting to note that the clearest and most intense markings are obtained with the lowest concentrations. Indeed, the markings are clearly finer and reveal intense cytoplasmic granules, which tends to show that a more precise identification of cytoplasmic targets is possible under these conditions. In particular, intense fluorescence foci obtained in perinuclear regions may correspond to the endoplasmic reticulum.

The inventors also tested the internalization of the compounds according to the invention over time.

The internalization kinetics were carried out with a CLARIOstar reader in order to quantify the internalization of the compound in the MCF-7 tumor cells. The values, corresponding to the ratio of the residual fluorescence/the total fluorescence, are presented in the form of means of three experiments, ±the standard deviation.

It is clearly seen (FIG. 10) that 10% of the fluorescent compound (II) penetrates the cell in less than 24 hours, allowing observation of very high-quality images.

The kinetics of incorporation by MCF-7 cells is shown inFIG. 10.

The absorption spectra were measured with a Perkin-Elmer double beam UV-visible spectrophotometer (Lambda EZ 210). The fluorescence spectrum was measured with a Fluorolog FL3-222 spectrofluorimeter (Horiba Jobin Yvon, Longjumeau, France) equipped with a 450 W xenon arc lamp, a thermostated compartment (25° C.), a photomultiplier UV-visible R928 (HAMAMATSU Japan) and an InGaAs infrared detector cooled with liquid nitrogen (DSS-16A020L Electro-Optical System Inc, Phoenixville, Pa., USA). The excitation beam is separated by an SPEX dual grating monochromator (1200 lines/mm blazed at 330 nm). The fluorescence was measured by the UV-Visible detector via the SPEX double grating emission monochromator (1200 lines/mm blazed at 500 nm). The production of singlet oxygen was measured by the infrared detector via the SPEX double grating emission monochromator (600 lines/mm blazed at 1 μm). All spectra were measured using 4-sided quartz cuvettes. The absorbance values at the excitation wavelength of the references and samples have been adjusted to approximately 0.2.

By this method, the inventors were able to measure the quantum yield ϕΔ, which is 0.11 for the compound

This low efficiency is expected, since the vast majority of absorbed photons are converted into light, about 76% of photons.

Example 3—Use of the Compounds According to the Invention in Photodynamic Therapy In Vitro

The inventors tested the effect of the compounds according to the invention in photodynamic therapy. MCF7 cells were incubated with the compound of formula

for 5 h and irradiated (or not) at 530 nm for 20 minutes. Two days later, the cells were subjected to a colorimetric cell viability assay (MTT).

The results are presented inFIGS. 12 and 13.

The results obtained under one-photon irradiation (λirrad.=514 nm) are exceptional, since 98% of tumor cells are killed at very low concentrations (C=100 nM).

As can be seen inFIG. 13, at a concentration of 100 nM, two zones are easily distinguished owing to the violet crystals of MTT, which only stain living cells (not irradiated).

Two-photon photodynamic therapy (λirrad.=810 nm) has shown very encouraging results, since nearly 50% of tumor cells are killed without optimization (irradiation for only 5 seconds at a very low concentration of 100 nM of compound according to the invention).

The inventors also tested the effect of the compounds according to the invention in photodynamic therapy on other models. Keratosis treatment tests were carried out at different concentrations of a photosensitizing compound (PS): either that used on the MCF-7 cells, or the compound of formula

on cultured keratinocytes. PS was added to the cells for 20 minutes at doses of 0 nM (A), 10 nM (B), 25 nM (C) or 50 nM (D) and the latter were irradiated (black columns) or not (gray columns) at 530 nm (FIGS. 15 and 17). Two days later, the cells are subjected to a colorimetric cell viability assay (MTT) (FIGS. 16 and 18).

Example 4—Internalization of the Compounds According to the Invention

The inventors evaluated the capacity of the cells to internalize the compounds according to the invention.

To do this, MCF-7 cells or healthy donor fibroblasts have been treated or not with 0.5 nM of compound of formula

for 1, 3, 6 or 24 h, and cell fluorescence was assessed by flow cytometry by detecting the number of cells with red fluorescence.

The results can be seen inFIGS. 20 and 21.

At an equal concentration (0.5 nM) after 6 h of incubation with the compound according to the invention, 46% of the cancerous cells (MCF-7) have internalized the compound, but only 18% of the healthy cells (fibroblasts) have. Similarly, 90% of cancer cells have internalized the compound according to the invention after 24 h compared with only 24% for healthy cells.

These results show that the compound according to the invention enters cancerous cells more rapidly than it enters healthy cells.

Example 5—Comparison Absorption of Phenazinium According to the Invention and Phenazine Described in the Prior Art

The cationic phenaziniums ([12]+; [23]+) are much more soluble than the neutral phenazines (24 and 25). The latter are indeed insoluble in alcohols and poorly soluble (C<10−4M) in MeCN, or acetone, whereas cationic phenaziniums ([12]+; [23]+) are soluble in all common solvents (i.e. toluene, Et2O, CH2CL2, CHCl3, acetone, MeCN, MeOH, EtOH DMF, DMSO) due to their amphiphilic character (the charged part being hydrophilic and the alkylated part being hydrophobic).

The absorption spectra of the compounds ([12]+; [23]+) are almost identical and show a band located in the region visible at λmax=553 nm (ε543=43400 M−1cm−1and ε543=41200 M−1cm−1) with a shoulder around 465 nm. Two absorption bands located in the ultraviolet at 265 nm and 320 nm complete these absorption spectra.

The absorption characteristics of the phenazine compounds 24 and 25 are similar to those of the cationic phenazinium compounds with, however, a 100 nm blue shift, their absorption appearing respectively at λmax=472 nm (ε472=16100 M−1cm−1) and λmax=472 nm (ε472=14800 M−1cm−1). The corresponding intensities are conversely much weaker (35 to 45% of the intensity of the main peak of the cationic phenazinium compounds). Furthermore, compounds 24 and 25 show several additional absorption bands located between 220 and 300 nm.

The data is shown inFIG. 19.

This demonstrates that phenazinium compounds are more suitable for photon therapy than neutral phenazines because they are more soluble and can be irradiated with a lower-energy laser (longer excitation wavelengths).