EP3870558A1

EP3870558A1 - Novel compounds and uses of same for near-infrared cherenkov luminescence imaging and/or for deep tissue treatment by cherenkov dynamic phototherapy

Info

Publication number: EP3870558A1
Application number: EP19813116.1A
Authority: EP
Inventors: Richard Decreau; Vivian LIORET; Yann BERNHARD; Bertrand COLLIN
Original assignee: Universite de Bourgogne
Current assignee: Universite de Bourgogne
Priority date: 2018-10-26
Filing date: 2019-10-25
Publication date: 2021-09-01
Also published as: US20220001012A1; FR3087772A1; FR3087772B1; WO2020084267A1

Abstract

The present invention concerns compounds of the following general structure (I): (I) in which A, B, C, D, L1, L2, L3, L4, n and m are as defined in claim 1. The invention also concerns the use of these compounds for an application for near-infrared Cherenkov luminescence imaging and/or for the treatment of deep biological tissues by Cherenkov dynamic phototherapy.

Description

New compounds and their uses for near infrared Cherenkov luminescence imaging and / or for the treatment of deep tissue by dynamic Cherenkov phototherapy

The present invention relates to new compounds which can in particular be used for near infrared Cherenkov luminescence imaging and / or for the treatment of deep biological tissues by dynamic Cherenkov phototherapy.

Imaging-guided surgery is a practice used to refine the surgical procedure, aiming to achieve complete tumor resection and minimizing excision of healthy tissue. Several preclinical studies have shown that Cherenkov Luminescence Imaging (hereinafter referred to as CLI imaging which is the acronym for "Cherenkov Luminescence Imaging"), which is optical imaging, can be successfully used to guide resection surgical treatment of tumors and lymph nodes but also for the detection of cancerous lesions using Cherenkov luminescence endoscopy (CLE, English acronym for "Cherenkov Luminescence Endoscopy") "(Several clinical studies are underway on preoperative and intraoperative use CLI imagery for different cancers, such as prostate cancer, breast cancer, gastrointestinal cancer and metastatic lymph nodes "(By improving the accuracy of surgical resection, CLI imagery has the potential to become a breakthrough technology in cancer surgery.

However, surgery guided by CLI imagery harbors potential challenges inherently linked to the source used for CLI imagery, namely Cherenkov radiation (hereinafter referred to as CR radiation, “CR” being the acronym). "Cherenkov radiation"), which has the disadvantage of being a relatively weak signal

The compounds of the invention will in particular make it possible to improve the signal in the zone of transparency of the tissues, for an equivalent quantity of doses of radiopharmaceuticals. Radiopharmaceuticals are substances which, due to their physico-chemical and nuclear characteristics, can be used for the diagnosis and treatment of cancer. One class of radiopharmaceuticals contains beta energy emitters that can emit CR radiation usable for CLI imaging, which is a fairly recent technique (2009) ^ and which arouses great interest. CR radiation emits in the ultraviolet (UV) and blue, namely in the field of the electromagnetic spectrum having a wavelength (l) ranging from 300 to 500 nm (nanometers), where the biological tissues are the most opaque and therefore not very transparent.

In order to be able to fully exploit CLI imagery, it is therefore necessary to transfer part of the CR radiation to the area of the electromagnetic spectrum where the tissues are much more transparent, namely in the near infrared (IR) area having a wavelength. (l) ranging from 650 to 900 nm.

The transfer of part of the CR radiation in the near IR has already been described in the literature.

Some authors ^{(2, 4 '>} have thus proposed the use of nanoparticles of the “Quantum Dots” type (“QDs”) which represent effective platforms for carrying out such a transfer, but which nevertheless have the drawback of being toxic due to the presence of cadmium for some of them.

The inventors have proposed the activation of fluorophores by CR radiation in order to transfer part of this CR radiation in the near IR. A fluorophore is a chemical capable of emitting fluorescent light after excitation. More particularly, in the document Bemhard et al. 2014 ^<5) , “radiochelate fluorophore” conjugates are described in which the radiochelate is linked to the fluorophore by a bond. The radiochelate is an energy beta emitter which produces CR radiation and which allows a transfer of energy of the CRET type (“CRET” being the acronym of “Cherenkov Radiation Energy Transfer”) towards the fluorophore which, after having received the radiation CR, emits fluorescence radiation. These fluorophore radiochelate conjugates however only allow modest Stokes displacements, of the order of about 20 nm; the transfer to the near IR region is therefore not achieved. The Stokes displacement represents the difference, in wavelength, between the position of the peak of the absorption spectrum and that of the peak of the emission spectrum.

In the document Bemhard et al. of 2017 ^<6) , the Inventors described multi-molecular systems comprising several fluorophores. More particularly, these systems include a radiometal and two or three fluorophores (fluorophore-1, fluorophore-2 and possibly fluorophore-3) which are not linked to each other by a bond. The radiometal is an energy beta emitter which produces CR radiation and which allows a transfer of CRET type energy to the fluorophore-1, which in turn will transfer the energy received to the fluorophore-2 by an energy transfer. of FRET type (“FRET” being the acronym of “Fôrtser Résonnance Energy Transfer”) then fluorophore-2, after having received the energy of fluorophore-1, then emits a fluorescence radiation. In the case where three fluorophores are present, the fluorophore-2 transmits the energy received towards the fluorophore-3 by an energy transfer of the FRET type, then the fluorophore-3 then emits a fluorescence radiation.

The disadvantage of this multimolecular system is in particular a / that the energy transfer is done with significant losses and that the fluorescence emission is low (there is therefore a poor energy transfer efficiency and a low gloss) b / that it is not unimolecular therefore one cannot imagine an identical bio-distribution from one component to the other of such a multimolecular system and c / that it is not biovectorized, therefore non-specific for a biological target.

In the document by Bizet et al. from 2018 ⁽⁷⁾ , “fluorophore- 1 fluorophore- 2” conjugates, activated by an external source of radiation, are described, the fluorophore- 1 absorbing in the UV region of the spectrum and the fluorophore-2 emitting in the near IR region. , for application in cell imaging, and more particularly for visualizing B16F10 melanoma cells. However, the conjugates described in this document are not soluble in water and are moreover very slightly soluble in organic solvents.

Conjugates dyads type fluorophores, tryades etc. which correspond to the excitation criterion in the low wavelengths and re-emitting in the wavelengths have been described in the literature ^{(10, No.} ^{12 , 13)} . However, these systems are not suitable for CLI imagery and have neither a conjugation site nor a site for introducing a radioactive entity.

A first aim of the invention is to obtain new compounds which can advantageously be used for near IR CLI imaging.

By near IR CLI imagery is meant the transfer of CR radiation to the near IR. The properties which are sought by the inventors for such compounds are in particular those of exhibiting a high gloss, large Stokes displacements, namely approximately 300 to 400 nm, with emissions around 800 nm, and / or good energy transfer yields, namely yields greater than 40%, preferably greater than 50%.

The "Brillance" is the product of the quantum fluorescence yield "c | F "and the molar extinction coefficient" e "(or probability of absorption): Brightness = c | F x e.

Thus, a gloss considered as "correct" or "good" may be the result of a good quantum fluorescence yield while the molar extinction coefficient is less good or vice versa. Ideally, when the quantum fluorescence yield and the molar extinction coefficient are both good then the brightness is high.

To meet the aim of the invention described above, it would ideally be necessary for all of these parameters to be as high as possible for the compounds of the invention in order to “transport” a maximum of Cherenkov photons with the greatest efficiency since the UV up to near IR, so that the radiance in the near IR area resulting from CR radiation alone is increased once the compound of the invention is brought into contact with CR radiation.

However, if one of these parameters decreases (for example the brightness) but the other increases (for example large Stoke displacements), then the compounds of the invention remain interesting for their envisaged application.

The term "high" gloss means a gloss greater than 10,000 M 'cm ¹ , and preferably greater than 100,000 IVT'crrf ¹ .

A "good" gloss is a gloss ranging from 1000 M ^_1 cm l to 10 000 IVT'crrf ¹ .

Another property sought by the inventors for the compounds of the invention is their good solubility in water and / or solvents, in particular organic solvents.

After intense research by the inventors, the latter have developed compounds which meet the needs described above, these compounds comprising one or more radioactive entities producing CR radiation, several fluorophores and possibly a vector molecule, all linked together. to others so as to form a unimolecular structure.

A second object of the invention is to obtain new compounds which can advantageously be used for the treatment of deep biological tissues by dynamic Cherenkov phototherapy.

Dynamic phototherapy, hereinafter referred to as PDT (“PDT” being the acronym for “PhotoDynamic Therapy”), is a technique used in the clinic for the adjunctive treatment of cancers in superficial areas which are accessible by a source light (skin, melanoma, esophagus, neck and head, bladder, prostate), but also for the treatment of acne or in ophthalmology

However, due to the limit of penetration of light into tissues ^<2, conventional PDT only allows to reach “non-deep” or superficial tissues, which are defined as fabrics where light from an exogenous source can penetrate, ie from a few millimeters up to a maximum of 10 mm. PDT therefore does not allow reaching deep tissue areas (beyond 10 mm). PDT, like optical imaging, therefore suffers from the fact that light is absorbed by the tissues and can only be used for surface treatments.

PDT more particularly involves the concomitant use of a photosensitizer and light at a certain wavelength. A photosensitizer is a compound that, under irradiation, has the ability to transfer its electronic excitation energy to another compound. In PDT, the role of the photosensitizer is to absorb light and transfer the energy thus captured to the oxygen present in the organism to transform it into reactive oxygenated species, hereinafter referred to as ROS species ("ROS" being the English acronym for "Reactive Oxygen Species"). The ROS species will react with and destroy the tumor cells. The photosensitizer, previously injected or applied topically, is intended to concentrate as selectively as possible in the tissues to be treated. The wavelength of the excitation light is adapted to the absorption spectrum of the photosensitizer. The irradiation then triggers a cascade of chemical reactions which will produce the ROS species. PDT, like CLI imaging, is therefore an optical technique since it relies on the activation of photosensitizers (anticancer drugs) by light.

However, many photosensitizers absorb in the UV / blue region of the electromagnetic spectrum, where biological tissues are not transparent. Other research has been done on photosensitizers that absorb in the near IR region of the spectrum where tissue transparency is most pronounced.

The use of CR radiation to serve as a light source for PDT has already been described in the literature. The authors Anyanee KamKaew et al. ^<> thus proposed chlorines (photosensitizers) immobilized on silica nanoparticles which are radiolabelled. These immobilized chlorines, which intrinsically capture CR radiation, are however not optimized to absorb CR radiation optimally, and therefore they do not generate a significant amount of ROS. In addition, they are not covalently conjugated to a biomolecule, so there is no possibility of selectively reaching one biological target over another. Finally, the molecular structures proposed by these authors are nanoparticulate systems and not molecular. After intense research by the inventors, the latter imagined compounds in which the light source is an integral part of the compounds of the invention. The compounds of the invention include a radioactive entity which is conjugated to a photosensitizer. The radioactive entity which emits light (CR radiation) can thus be conceived as an on-board light source, since it is entrained with the photosensitizer towards the tumor cells. There is therefore no longer a limit on the depth of use of the photosensitizer.

PDT can thus also be used in deep tissue, this is Cherenkov PDT. The invention will thus advantageously allow PDT access to any type of tissue depth, greater than 10 mm and more. By deep biological tissue is meant according to the invention, the tissues lying at a depth ranging from 1 cm to 30 cm below the horny layer of the skin.

In addition, when the photosensitizer thus radiolabelled according to the invention is combined with a vector entity targeting the receptors of cancerous tissues, this will make it possible to be selective of the cancerous tissues which it is desired to treat in depth.

According to a first subject, the invention relates to new compounds comprising one or more radioactive entities A, one or more fluorophores B, a fluorophore and / or photosensitizer C, and optionally a vector entity D, all linked together so as to form a unimolecular structure.

According to a second object, the invention relates to the use of these compounds:

- for near IR CLI imaging when C is only a fluorophore,

- for the treatment of deep biological tissues by PDT Cherenkov when C is only a photosensitizer,

- for near IR CLI imaging and Cherenkov PDT when C is both a fluorophore and a photosensitizer.

According to a third subject, the invention relates to the process for the preparation of the compounds of the invention.

A more particular subject of the present invention is a compound having the following general structure (I):

in which :

* A is a radioactive entity which is a beta energy emitter which produces Cherenkov radiation, said radioactive entity preferably being a radiochelate, namely a radiometal surrounded by a chelate or a non-metallic radioelement;

* B is a fluorophore which absorbs electromagnetic radiation with a wavelength l ranging from 300 nm to 500 nm;

* It is a fluorophore which emits electromagnetic radiation with a wavelength l ranging from 650 nm to 950 nm,

and or

* It is a photosensitizer which produces reactive oxygenated species ROS;

* D can be present or absent, and represents, when it is present, a vector entity, said vector entity preferably being a biomolecule or a nanoparticulate vector,

* Ll, L2, L3 are each independently of the other a group linking at least divalent

1.3 iA) ,,

or a covalent bond, with the condition that are not present simultaneously, which means that if is present then <A} „is absent and vice versa,

* L4 is present if D is present and represents, when it is present, an at least divalent linking group or a covalent bond;

* n is an integer equal to 1, 2, 3, 4 or 5;

* m is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8;

and in which:

* A activates B by a CRET type energy transfer,

* B transfers the energy received from A to C, by an energy transfer of the intramolecular FRET type or by an energy transfer of the TBET type.

"TBET" is the acronym for "Through-Bond-Energy-Transfer".

FRET transfer is the transfer of energy between two fluorophores, the emission spectrum of the first of which corresponds to the absorption spectrum of the second. It is a transfer that takes place in space.

TBET type transfer means that the transfer does not take place in space but through the links. The radioactive entity A is designated in the following by A. It can consist of a radioelement alone (non-metallic) or of a radiometal type radioelement surrounded by a chelating agent: the radiometal + chelating agent is called radiochelate.

Fluorophore B is designated in the following by B. B can also be called “antenna”.

The fluorophore and / or photosensitizer C is designated in the following by C. C can also be called “platform”.

The vector entity D is designated in the following by D.

The compounds of the invention can therefore have a variable number of A (which can range from 1 to 5) and / or B (which can range from 1 to 8).

The A (s) is (are) represented by (A) _n .

The (or) B is (are) represented by (B) _m .

(A) _n is linked to C or (A) _n is linked to (B) _m .

(B) _m is always linked to C and vice versa (C is always linked to (B) _m ).

D, if present, is still linked to C.

When it is said in the present application that L1, which binds (A) _n to C, is a covalent bond, this means that (A) _n is directly linked to C without the intermediary of a linking group (in other words a electron of A forms a bond with an electron of C).

In the same way when L2, which links C to (B) _m , is a covalent bond, this means that (B) _m is directly linked to C without the intermediary of a linking group.

The same is true for L3, which links (B) _m to (A) _n , and L4, which links C to D.

When L1, which links (A) _n to C, is a linking group, the latter will be an at least divalent radical (namely divalent, trivalent, tetravalent etc.) depending on the meaning of n.

Similarly, when L2, which links C to (B) _m , is a linking group, the latter will be an at least divalent radical (namely divalent, trivalent, tetravalent, etc.) depending on the meaning of m.

It is the same for L3, which links (B) _m to (A) _n .

When L4, which links C to D, is a linking group, the latter will be a divalent radical.

Due to the conformational structure of the compounds of the invention, it is always A which activates B then B which activates C, even in the case where A is linked to C.

According to one embodiment of the invention, C is a photosensitizer which produces reactive oxygenated species ROS, and in particular singlet oxygen with a quantum yield (f _D ) greater than 5%, preferably greater than 30%. When in the compound of formula (I) described above, ^Lî · ^{! Rt)} 'is absent, then it has the following structure (1-1):

(1-1),

wherein A, B, C, D, Ll, L2, L4, n and m are as defined above.

When L1, L2 and / or L4 are simple covalent bonds, then (A) _n is directly linked to C, C is directly linked to (B) _m and / or C is directly linked to D.

By way of examples, if L1, L2 and L4 each represent a single covalent bond, the compound (1-1) is then simply represented by:

(AT), . VS . (BS, ..

D

When D and L4 are absent, then the compound of formula (1-1) is simply represented by:

When in the compound of formula (I) described above, (A) „ is absent, then it has the following structure (1-2): (1-2), in which A is a non-metallic radioelement and B, C, D, L2, L3, L4, n and m are as defined above.

When L3, L2 and / or L4 are simple covalent bonds, then (A) _n is directly linked to (B) _m , (B) _m is directly linked to C and / or C is directly linked to D.

By way of examples, if L3, L2 and L4 are each a single covalent bond, the compound of the invention (1-2) is then simply represented by:

₀ - _c - ( _B ) _m - (A) „

When D and L4 are absent, then the compound of formula (1-2) above is simply represented by: By way of examples, when n = m = 1, D is absent and L1 and L2 are each a covalent bond, then the compound (I) of the invention, and more particularly (1-1) can simply

A - C - B

to be represented by:

If there is an L1 between A and C which is a linking group, then the above compound is represented by:

If D is present and Ll, L2 and L4 each represent a covalent bond, then the compound (1-1) is represented by:

If there are linking groups L1 and L4, then the compound is represented by:

When n = m = 1, D is absent and L3 and L2 are each a covalent bond, then the compound (I) of the invention, and more particularly (1-2) can simply be represented

A - B - C

by :

If there is an L3 between A and B which is a linking group, then the above compound is represented by:

When n = m 1, D is present and L4, L2 and L3 each represent a single bond

covalent, then compound (1-1) is represented by:

If there are linking groups L3, L2 and L4 then the compound is represented by:

Still by way of examples, if n = 5, m = 1, L1 is a linking group, L2 a covalent bond and D is absent, the compound (I) of the invention and more particularly (1-1) is represented. by : Still by way of examples, if m = 4, n = 1, L2 is a linking group, L1 is a covalent bond and D is absent, the compound of the invention (1-1) can be represented by:

If D is present and L4 is a linking group, then the above compound is represented by:

If m = 4, n = 1, L2 and L1 are linking groups, D is absent, compound (1-1) can also be represented by:

or by :

if L2 is a single covalent bond.

The examples given above are not understood to be exhaustive.

Furthermore, the formulas exemplified are only diagrams intended to illustrate and understand the structure of the compounds of the invention. These diagrams are not representative of the conformational structures of the compounds of the invention

Within the meaning of the invention, the term "radioactive entity" without further specification can denote both a radiochelate and a non-metallic radioelement.

When A is a radiochelate then A is always linked to C.

When A is a non-metallic radioelement then A can either be linked to C or to

B.

In the compounds of formula (1-1) A is always linked to C and A can indifferently represent a radiochelate or a non-metallic radioelement. In the compounds of formula (1-2) A is always linked to B and A then represents a non-metallic radioelement.

According to an advantageous embodiment of the invention, A is:

* a radiochelate whose radiometal is chosen from the group comprising ⁹⁰ Y, ¹⁷⁷ Lu, ⁶⁸ Ga, ⁸⁹ Zr, ⁶⁴ Cu, ⁸⁹ Sr, ²¹² Bi, ²¹³ Bi, ⁴⁴ Sc, ²²⁵ Ac and ⁴⁴ Sc and whose chelating agent is chosen from the group comprising DOTAGA, DOTA, NOTA, NODAGA and DFO.

* a non-metallic radioelement chosen from the group comprising F, I, I and P.

DOTAGA means “2,2 ', 2” - (10- (2,6-dioxotctrahydro-2H-pyran-3-yl) - 1, 4,7, 10- tetraazacyclododecane-1,4,7-triyl) triacetic acid "

DOTA means "1,4,7,10- tetraazacyclododecane tetraacetic acid".

NOTE means "4,7-triazacyclononane-N, N’, N "-triacetic acid".

NODAGA means "2- (4,7-bis (carboxymethyl) -l, 4,7-triazonan-l-yl) pentanedioic acid". DFO stands for "A ¹ - (5-aminopentyl) -A ¹ -hydroxy- / V ⁴ - (5- (4 - ((5 - (/ V-hydroxyacetamido) pentyl) amino) -4-oxobutanamido) pentyl) succinamide" .

An example of a radiochelate is the radiometal ⁹⁰ Y chelated with the chelating agent DOTAGA or DOTA. They are respectively represented by "[ ⁹⁰ Y] -DOTAGA" and "[ ⁹⁰ Y] -DOTA". The radioactive entity [ ⁹⁰ Y] -DOTA within the compound of formula (I) can be represented by the following radical:

The radioactive entity [ ⁹⁰ Y] -DOTAGA within the compound of formula (I) can be represented by the following radical:

As already indicated, the radioactive entity A is an energy beta emitter which produces CR radiation. The compounds of the invention therefore do not need to be activated by an external source of radiation, since they themselves produce CR radiation, continuously, until the decrease of A.

The half-lives of radioactive entities A are very variable and can range from 68 minutes to 6.6 days.

As examples, the life time:

- radiochelate [ ⁹⁰ Y] -DOTA or [ ⁹⁰ Y] -DOTAGA is 64.1 hours,

- ¹⁸ F is 109.7 minutes,

- [ ⁶⁸ Ga] -NOTA or [ ⁶⁸ Ga] -NODAGA radiochelate is 68 minutes,

- of [ ⁸⁹ Zr] -DFO is 78.4 hours,

- from [ ¹⁷⁷ LU] -DOTA OR [ ¹⁷⁷ LU] -DOTAGA is 6.64 days.

According to one embodiment of the invention, B is chosen from the group comprising a coumarin-type nucleus; coumarin substituted, in particular by one or more hydroxy and / or by a pyridinium, itself optionally substituted; pyranine; pyrene; BODIPY; Substituted BODIPY, in particular phenyl-BODIPY, hydroxyphenyl-BODIPY, aza-BODIPY; fluorescein; rhodamine, in particular rhodamine 6G, rhodamine 101, rhodamine B, rhodamine 123; eosin, especially eosin B, eosin Y; tryptophan and mixtures thereof. BODIPY is the abbreviation for boron-dipyromethene.

By way of example of coumarin substituted by a hydroxy, mention may be made of hydroxycoumarin, substituted by two hydroxy, there may be mentioned dihydroxy coumarin.

By way of example of hydroxycoumarin substituted with a pyridinium, mention may be made of 4-methylpyridinium 7-hydroxycoumarin or 4-propylsulfonatepyridinium 7-hy droxycoumarin.

According to the invention B can also comprise at least one solubilizing group, in particular chosen from the group comprising a sulfonate (SO3); a carboxylate (COO); ammonium (NR4) with R = H, alkyl or aryl; a phosphonate (PO3 ² ); a pyridinium, preferably substituted; an imidazolium and their mixtures.

By way of example, B can be a pyrene-type nucleus comprising at least one solubilizing group of formula SO ^ Na, and preferably three SO 3 Na groups.

B can be a coumarin or hydroxycoumarin nucleus substituted with a methylpyridinium or a propidylsulfonate pyridinium. According to an advantageous embodiment of the invention, when B is greater than 1, then the B can independently be identical or different within the compound (I) of the invention.

Having different B's in the same compound covers a wider absorption window.

As an example of such an embodiment, when n = 2, then one B can represent a nucleus of the coumarin or substituted coumarin type and the other B a nucleus of the BODIPY or substituted BODIPY type.

According to yet another advantageous embodiment of the invention, in the compounds of formula (1-2) where A represents a non-metallic radioelement, the latter can replace a part of B. In other words a part of B is replaceable by A which means that A is then an integral part of B.

In this case L3, which binds A to B, never represents a linking group but always a covalent bond (A replaces part of B).

As an example of such an embodiment, when B is a substituted BODIPY or BODIPY type nucleus, one of the two fluorines naturally present in BODIPY can be replaced by the non-metallic radioelement ¹⁸ F.

In the invention is meant by a “type” X nucleus, a nucleus “originating from a compound

X ".

More specifically, once the compound X is engaged in a bond with one or more other compounds it will no longer be the compound X as such but a compound originating from X or a compound of type X.

This compound of type X is compound X as it appears once it is engaged in one or more bonds.

By way of example, in a compound of formula (1-1) where D is absent, when B is linked to C, and when B is a hydroxycoumarin type nucleus, for example of the 7-hydroxycoumarin type, it may be represented by the radical:

while 7-hydroxycoumarin as such is represented by: Still by way of example, in a compound of formula (1-2) where D is absent, when B is linked to C but also to A, and when B is a dihydroxycoumarin type nucleus, for example of type 4, 7 -hydroxy coumarin, it can be represented by the divalent radical:

Still by way of example, in a compound of formula (1-1) where D is absent, when B is linked to C, and when B is a pyranine type nucleus, it may be represented by the monovalent radical:

In a compound of formula (1-2) where D is absent, when B is linked to C but also to A, and when B is a ring of 8-phenyl-BODIPY type, it may be represented by the divalent radical:

As more specific examples of B, mention may be made of a nucleus of the coumarin, hydroxycoumarin, dihydroxy coumarin, methylpyridinium hydroxycoumarin, propylsulfonatepyridinium hydroxycoumarin, pyranin, pyrene, BODIPY, phenyl- BODIPY, hydroxyphenyl-BODIPY type.

According to another advantageous embodiment of the invention, C is chosen from the group comprising a cyanine-type nucleus, in particular cyanine-7, cyanine-5, cyanine-3; phthalocyanine, especially phthalocyanine of silicon, zinc, magnesium, phosphorus, aluminum, indium; naphthalocyanine, in particular naphthalocyanine of zinc, magnesium, phosphorus, aluminum, silicon, indium; chlorine, especially chlorine of zinc, magnesium, phosphorus, aluminum, silicon, indium; bacteriochlorine, in particular bacteriochlorine of zinc, magnesium, phosphorus, aluminum, silicon, indium.

According to the invention C can also comprise at least one solubilizing group, in particular chosen from the group comprising a sulfonate (SO3); a carboxylate (COO); ammonium (NR ₄ ) with R = H, alkyl or aryl; a phosphonate (PO3 ² ); a pyridinium, preferably substituted; an immidazolium and their mixtures.

For example, C may be a silicon phthalocyanine substituted by a pyridinium itself substituted (for example by a methyl or a propylsulfonate at the nitrogen atom of the pyridinium).

According to another advantageous embodiment of the invention, C is a cyanine-type nucleus. Cyanines are the name of a family belonging to the group of polymethines. They have many applications as fluorescent markers. The cyanines used in the context of the invention absorb mainly above 600 nm.

By way of example, in a compound of formula (1-1) where D is absent, n = 1 (only one A), the ring C of cyanine type may be represented by the radical of the following general formula:

in which :

p is an integer ranging from 0 to 4,

R 'represents CH ₂ , NH, N (alkyl), CO, NHCO, NHCOO, NHCOO (CH ₂ ) _p , p = 0 to 4,

R represents N ₃ , COOH, CH ₃ , NHCOO (alkyl),

More specific examples of such cyanine-type nuclei are:

Still by way of example, in a compound of formula (1-1) where D is absent or present, n = 1 (a single A) or greater than 1, the nucleus C of cyanine type may be represented by the radical of following general structure:

in which :

p is an integer ranging from 0 to 4,

each R 'represents independently of the other CH ₂ , NH, N (alkyl), CO, NHCO, NHCOO, NHCOO (CH ₂ ) _p .

More specific examples of such cyanine-type nuclei are:

In a compound of formula (1-2) where D is absent, the nucleus C of cyanine type may be represented by the radical of the following general structure:

in which :

p is an integer ranging from 0 to 4,

each R independently of one another represents N ₃ , COOH, CH ₃ , NHCOO (alkyl),

More specific examples of such cyanine-type nuclei are: H

According to another advantageous embodiment of the invention, C is a phthalocyanine type nucleus.

In a compound of formula (1-1), the nucleus C of phthalocyanine type can be represented by one of the multivalent radicals of the following formula:

in which M represents Zn (zinc), Mg (magnesium), P (phosphorus), Al (aluminum), In (indium).

In addition to the solubilizing group or groups which can be carried by C, it is also possible, according to an advantageous embodiment of the invention, for C to carry one or more functional groups. These functional groupings can in particular act as a “hanging function” for D.

Thus, when C is for example a phthalocyanine type nucleus with a metal M at the center of the cycle, then it will be possible to link a functional group to metal M, the latter being intended to react with another functional group which is an integral part a precursor of D (the precursor of D designating D before its bond with C) or else which is specially grafted on the precursor of D. The reaction between the two functional groups of C and the precursor of D will form the linking group L4, which links C with D.

Examples of functional groups which can be linked with the metal M of phthalocyanine are: -0- (CH ₂ ) _q -N ₃ with q an integer ranging from 1 to 4,

According to an advantageous embodiment of the invention, in the compounds of formula (1-1) or (1-2), when m = 1 (only one B), the linking group L2, which binds C and B, is :

* a divalent radical -O-, -S-, -NH-, -N (alkyl);

* a divalent saturated or unsaturated hydrocarbon radical having from 1 to 4 carbon atoms, preferably 2 carbon atoms, in particular chosen from -CºC-, -CH ₂ -CºC-CH ₂ -,

-CH = CH-, -CH ₂ -CH = CH-CH ₂ - OR - (CH ₂ ) _q -, q is an integer ranging from 1 to 4, preferably from 1 to 2.

O is oxygen, S is sulfur and N is nitrogen.

Still according to the invention, in the compounds of formula (1-1) or (1-2), when m = 2, 3 or 4, L2 is a multivalent linking group which plays the role of a platform making it possible to collect the B and link them with C.

Examples of L2 multivalent binder radicals are:

According to another embodiment of the invention, in compounds (1-1) and / or (1-2), L2 is a covalent bond.

According to yet another advantageous embodiment of the invention, in a compound of formula (ii), the linking group L1, which links A to C, can comprise a function of amide, carbonyl, amine, triazole, pyridazine, peptide type , urea, thiourea, thioether, maleimide.

Examples of multivalent bonding radicals L1 are:

_* (CH ₂ ) _p -CO- (CH ₂ ) _{p *} , _* NH- (CH ₂ ) _q -NH _* , _* NH-C ₆ H ₄ -0- (CH ₂ ) _{p *} ,

_* (CH ₂ ) _q -CO-NH- (CH ₂ ) _{q *} , with q = 1 to 4 and p = 0 to 4.

According to the invention in a compound of formula (1-1), when n = 2, 3, 4 or 5, A = radiochelate, L1 is a multivalent linking group which plays the role of a platform making it possible to collect all the A then make a connection with C.

By way of example of such a multivalent compound, mention may be made of the radical “1, 2, 3, 4,5,6- benzenehexamethanamine” which makes it possible to carry up to five radiochelates A:

When he wears the five A, he is represented by the radical of formula

According to another embodiment of the invention, in the compounds (1-1), L1 is a covalent bond.

According to yet another advantageous embodiment of the invention, in the compounds of formula (1-2), when n = m = 1, A is a non-metallic radioelement, the linking group L3, which binds A and B, is :

- (CH ₂ ) _q -0- (CH ₂ ) _q -; - (CH ₂ ) _q -0- (CH ₂ ) _q -0-; - (CH ₂ ) _q -S- (CH ₂ ) _q -; - (CH ₂ ) _q -S- (CH ₂ ) _q -S-; - (CH ₂ ) _q -NH- (CH ₂ ) _q -; - (CH ₂ ) _q -NH- (CH ₂ ) _q -NH-; - (CH ₂ ) _q -; q is an integer ranging from 1 to 4.

According to another embodiment of the invention, in compounds (1-2), L3 is a single covalent bond. According to yet another advantageous embodiment, the compound of the invention is of formula (1-1) in which A is a radiochelate linked to C via a linking group L1, and C is linked to B by l intermediate of L2 which is a covalent bond.

According to another particularly advantageous embodiment, the compound (I) of the invention comprises a vector entity D.

This vector entity can be a biomolecule such as a peptide; a protein; an antibody-like protein; an antibody fragment-like protein, such as "Fab", "Fab'2", "Fab '", "ScFv", "nanobody", affïbody, diabody; an aptamer.

According to one embodiment of the invention, the group linking L4, namely the group linking D to C or vice versa, can comprise a function of amide, carbonyl, amine, triazole, pyridazine, peptide, urea, thiourea, thioether type, maleimide.

As examples, the group linking L4 can be represented by one of the following radicals:

According to another embodiment of the invention, L4 is a single covalent bond in the compounds (I) of the invention.

By way of example, in a compound of formula (1-1) of the invention, when n = m = 1, A = radiochelate and that C is a cyanine-type nucleus linked to D, then C can be represented by the trivalent radical of the following formula:

in which :

p is an integer ranging from 0 to 4, each R 'independently of the other represents CH ₂ , NH, N (alkyl), CO, NHCO, NHCOO, NHCOO (CH ₂ ) _p .

Another object of the present invention lies in the process for preparing the compounds of the invention.

First a first unimolecular structure is prepared which includes C and from one to four B.

Then, up to five A can be grafted onto the conjugate thus formed, so as to form a new unimolecular structure.

Finally D can be grafted on this unimolecular structure, to reform a new unimolecular structure.

Those skilled in the art will know which method to use to attach a specific molecule to a selected substrate.

By way of example, the cyanines which will be used to prepare a compound of the invention of formula (I) in which C is a cyanine-type nucleus, will be symmetrical and asymmetrical cyanines having one of the following structures:

(Tf: Triflate, Ms: Mesylate, Ts: Tosylate)

B can be grafted to cyanine by replacing the halogen (for example chlorine as shown above) with cyanine.

In a compound of formula (1-1), A will be grafted to cyanine via the functional group NH ₂ , N ₃ , COOH, NHCOO (alkyl) etc.

The group linking L4, namely the group linking D to C or vice versa, results from the reaction between (1 /) a functional group of a precursor of D and (2 /) a functional group carried by C before it either engaged in an affair with D.

By "precursor" of D is meant the entity D before it is engaged in a bond with C.

C therefore advantageously comprises at least one functional group in order to be able to react with a functional group of the precursor molecule of D.

When the compound of the invention comprises a ring C of cyanine type, the functional group of the ring of cyanine type may for example be an azide (N ₃ ), tetrazine, activated ester group (which is an activated form of an acid function carboxylic) or triazine.

Thus L4, which is the group linking D and C, can be a radical comprising a function:

- triazole, which results from the reaction of an azide function of C with an alkyne function of the precursor of D,

- pyridazine, which results from the reaction of a tetrazine function of C with a bicyclononyne function of the precursor of D,

- amide which results from the reaction of the activated form of a carboxylic acid function of C with an amine function of the precursor of D.

When the compound of the invention comprises a nucleus C of phthalocyanine type, phthalocyanine is linked to D via a functional group, comprising an azide function N ₃ , linked to the metal M of phthalocyanine. In general, the methods used in the context of the invention are the general methods of organic synthesis, purification by chromatography and LC-MS ("Liquid chromatography-mass spectrometry").

The syntheses are convergent (synthesis of the C platform, or even of certain B antennas) and each compound is characterized by a range of spectroscopic methods: proton and carbon NMR, high and low resolution mass spectrometries, UV / Vis spectrometries (Visible ) and infrared.

The purity of the synthons and of the targets is determined by HPLC. At the end, the BC compounds carrying the entity suitable for radiolabelling are radiolabelled using the techniques of radiolabelling chemistry with dedicated protections on a dedicated site. The radiochemical purity is controlled by radio-TLC and / or by radio-HPLC.

The compounds are then conjugated to a biomolecule, for example an antibody labeled with a bio-orthogonal chemical function, the techniques for analysis and purification of the bio-conjugates include MALDI-TOF mass spectrometry, UV / Vis and purification is carried out on Sephadex and FPLC steric exclusion columns.

Table 1 below illustrates the compounds (I) of the invention.

The new compounds of the invention can advantageously be used:

- for near IR Cherenkov luminescence imaging when C is a fluorophore, - for the treatment of deep biological tissue by Cherenkov dynamic phototherapy when C is a photosensitizer,

- for both when C is a fluorophore and a photosensitizer.

The invention therefore also relates to the use of a compound of formula (I) as defined above, for an application for imaging by near infrared Cherenkov luminescence.

Compounds numbers 1, 3, 5-7, 11-18, 21, 22 in Table 1 are particularly advantageous for an application for near IR CLI imaging. The invention also relates to a method of diagnosis by near IR Cherenkov luminescence imaging, said method being characterized in that it comprises the administration to a subject of a compound of formula (I), said compound preferably comprising D . The invention also relates to a compound of formula (I) as defined above, for use for the treatment of deep biological tissues by dynamic Cherenkov phototherapy.

Another object of the invention resides in a compound of formula (I) as defined above, for use for the treatment of deep biological tissues by dynamic Cherenkov phototherapy, said dynamic Cherenkov phototherapy being used in combination with at least one other anti-cancer treatment.

Indeed, the stress induced by the PDT-Cherenkov effect on the deep tumor can induce cell mortality by direct PDT therapeutic effect. When small amounts of the compounds of the invention are used, the stress induced by the PDT-Cherenkov effect can have a non-lethal effect which nevertheless makes it possible to weaken the tumor tissues and thus makes them more sensitive to other therapies. Thus, a first stage of treatment of the tumor by the action of PDT-Cherenkov using the compounds of the invention makes it possible to potentiate the action of one or more other therapies to be carried out in a second stage.

Compounds 2, 4, 8-10, 19, 20 of Table 1 are particularly advantageous for use in the treatment of deep biological tissue by PDT Cherenkov.

In addition, these compounds 2, 4, 8-10, 19, 20 are also advantageous for an application for CLI near IR imaging.

The invention also relates to a method of treating cancer by dynamic Cherenkov phototherapy, and in particular a method of treating deep biological tissue, said method being characterized in that it comprises the administration to a subject of a compound of formula ( I) as defined above, said compound preferably comprising D.

The method of treatment of cancer by dynamic Cherenkov phototherapy as described above can also be combined with at least one other method of anti-cancer treatment.

The optical luminescence properties of the compounds of the invention and of the BC intermediates are examined by conventional spectrofluorimetry (laser source) and in the case of radiolabelled compounds, by spectrofluorimetry in the presence of radioelements in bio luminescence mode, and optical imager. The photosensitizing properties of the compounds dedicated to PDT-Cherenkov are examined by UV / Vis spectrometry by monitoring the decrease in the absorption band of DPBF (diphenylbenzofuran), but also by cellular tests and study of cytotoxicity.

Finally, the in vivo studies take place on xenografted mice carrying cancer models, which are chosen as being superficial cancers in the case of CLI imaging studies, or else as being deep cancers in the case of PDT-Cherenkov studies .

FIG. 1 is a diagram of synthesis of asymmetric cyanines (35) and (36) which will be used to prepare compounds (I) of the invention in which C is a nucleus of asymmetric cyanine type.

Figure 2 is a synthetic diagram of compound 1 of the invention.

Figure 3 is a synthetic diagram of the compounds 5 (Figure 3a) and 6 (Figure 3b) of the invention.

Figure 4 is a synthetic diagram of the compounds 21 (Figure 4a) and 11 (Figure 4b) of the invention.

FIG. 5 is a synthetic diagram of the compounds 4 and 19 of the invention.

The following examples illustrate the invention, they do not limit it in any way.

Example 1

Preparation of compounds f1) of the invention

The methods for preparing several compounds which are the subject of the invention are described in detail in this example.

Separations and analyzes by HPLC

System A: HPLC-MS (Hypersil C18 column, 2.6pm, 2.1 x 50 mm) with H ₂ 0 0.1% Lormic acid (LA) as eluent A and CFL.CN 0.1% LA as eluent B [gradient linear from 5 to 100% B (5 min) and 100% B (1.5 min)] at a flow rate of 0.5 mL / min. UV detection is carried out at 650 and 750 nm.

System B: HPLC (Hypersil Cl 8 column, 5mhi, 10 x 250 mm) with H ₂ 0 0.1% LA as eluent A and CFLCN 0.1% LA as eluent B [linear gradient from 20 to 60% of B in 40 min] at a flow rate of 3.5 mL / min. UV detection is carried out at 700 and 780 nm.

1 / Synthesis of asymmetric cyanines of formulas (see Figure 1)

In compounds 1, 3, 5-7 and 12-17 of the invention, C is an asymmetric cyanine type nucleus.

The method of synthesis of asymmetric cyanines (35) and (36) used to prepare the compounds of the invention has no precedent and has been developed by the inventors. It is therefore described in detail below (see also Figure 1).

Compound synthesis (30)

Phosphorus oxychloride (5.6 mL, 60 mmol) is added dropwise at 0 ° C to anhydrous DMF (6.5 mL, 84 mmol). After 30 min, the cyclohexanone (2.75 mL, 27 mmol) is then added leading to a change in the color of the reaction mixture which becomes orange and which is brought to reflux for 1 h in a water bath. After the mixture has cooled to room temperature, an aniline / ethanol mixture [1: 1 (v / v), 90 mL] is added dropwise. This is followed by an exothermic reaction, generation of HCl and solidification. After adding aniline, the reaction mixture, deep purple in color, is poured into a mixture of ice water / concentrated HCl [10: 1 (v / v) 80 ml]. The solution stored at 4 ° C. for 12 h sees the formation of crystals. After filtration, the crystals are washed with cold water and then with diethyl ether and dried to yield 7.19 g (75%) of the product (30).

Synthesis of (31)

To a solution of 1-bromo-3chloropropane (1.57 g, 10 mmol) in 15 mL of DMF (N, N-dimethylformamide) is added sodium azide (650 mg, 10 mmol). After stirring for 5 h at room temperature, the reaction mixture is poured into 80 ml of water and extracted with ether (3 x 50 ml). The organic phases are combined and washed with water (2 x 50 mL), brine (100 mL), then dried over MgSO ₄ and finally concentrated under reduced pressure. To the residue obtained (0.98 g, 8.23 mmol) which is resolubilized in acetone (50 mL) is added sodium iodide (2.47 g, 16.47 mmol). The resulting mixture is brought to reflux with stirring for 16 h, then is poured into 50 ml of water and extracted with ethyl acetate (3 x 50 ml). The organic phases are washed with water (2 x 50 mL), dried over MgSO ₄ and concentrated under reduced pressure to yield 1.27 g (60%) of the product (31), which is in the form of a yellow oil. No purification is necessary. 1H NMR (500 MHz, CDCfi, 300 K): d (ppm) = 2.03 (m, 2H); 3.25 (t, J = 6.6 Hz, 2H); 3.43 (t, J = 6.6 Hz, 2H). ¹³ C NMR (125 MHz, CDCfi, 300 K): d (ppm) = 2.42; 32.46; 51.59. Synthesis of (32)

A solution of 2,3,3-trimethylindolenine (377 mg, 2.37 mmol) and azido-3-iodopropane (31) (lg, 4.74 mmol) in racetonitrile is brought to reflux for 2 days. The color of the solution changes from light orange to dark green. The solvent is evaporated under reduced pressure and 5 ml of dichloromethane are then added. This solution was added dropwise to diethyl ether (50 mL) resulting in the precipitation of a dark green compound. The solid is recovered by filtration, then 5 ml of dichloromethane are added and the process is repeated twice. The solid is dried under vacuum to give 596 mg (68%) of the product (32) which is in the form of a dark green to brown solid.

1H NMR (500 MHz, CDCl ₃ , 300 K): d (ppm) = 1.65 (s, 6H); 2.32 (m, 2H); 3.19 (s, 3H), 3.70 - 3.77 (m, 2H); 4.91 (t, J = 7.2 Hz, 2H), 7.52 - 7.64 (m, 3H); 7.84 - 7.89 (m, 1H).

¹³ C NMR (125 MHz, CDCl ₃ , 300 K): d (ppm) = 196.67; 141.62; 141.08; 130.30; 129.80; 123.40; 115.87; 54.85; 49.04; 47.87; 27.44; 23.26; 17.50.

Synthesis of

A mixture of 2,3,3-trimethylindolenine (33 mg, 2.08 mmol) and 3-bromopropylamine hydrobromide (456 mg, 2.08 mmol) is heated to 120 ° C. in a sealed tube for 10 h. The solid residue formed is cooled and washed thoroughly with diethyl ether and then a mixture of Et ₂ O: CHCl ₃ (1: 1) to give 574 mg (74%) of the product (33).

¹³ C NMR (125 MHz, MeOD, 300 K): d (ppm) = 199.28; 143.38; 142.32; 131.36; 130.60; 124.80; 116.40; 56.19; 46.46; 37.86; 29.79; 16.81; 22.85.

Synthesis of

Compound (32) (300 mg, 0.81 mmol) and sodium acetate (70 mg, 0.85 mmol) are dissolved in 30 ml of dry ethanol leading to a green solution. Compound (30) (313 mg, 0.97 mmol) is then added with 10 ml of dry ethanol leading to a purple / blue solution. The reaction mixture is brought to reflux for 2 h and the progress of the reaction is monitored by LC-MS (Liquid Chromatography-Mass Spectrometry). Half the volume of solvent is distilled under reduced pressure and the more concentrated reaction mixture is poured into 70 ml of Et ₂ 0. The solid is washed with Et ₂ 0 (3 x 50 ml) and dried under vacuum. The solid is then purified with a chromatographic column on silica gel (DCM / MeOH 98/2 vol.) To yield 175 mg (36%) of the pure product (34). Note that the color of the compound depends on its protonation state, it appears blue in TLC due to the acidity of the silica.

1H NMR (500 MHz, CDCl ₃ , 300 K): d (ppm) = 1.66 (s, 6H); 1.87 (p, J = 6.2 Hz, 2H); 1.93 - 2.08 (m, 2H); 2.61 - 2.67 (m, 2H); 2.79 (t, J = 6.1 Hz, 2H); 3.42 (t, J = 6.2 Hz, 2H); 3.79 (m, 2H); 5.57 (d, J = 12.6 Hz, 1H); 6.70 (d, J = 7.8 Hz, 1H); 6.92 (t, J = 7.4 Hz, 1H); 7.14 - 7.23 (m, 5H); 7.37 (m, 3H); 7.62 (d, J = 12.6 Hz, 1H); 8.84 (s, 1H). ¹³ C NMR (125 MHz, CDCl ₃ , 300 K): d (ppm) = 159.86; 158.13; 129.50; 129.40; 129.30; 128.17; 125.77; 122.12; 121.36; 121.23; 121.19; 120.82; 106.69; 93.40; 77.51; 77.26; 77.01; 66.10; 49.03; 46.49; 39.67; 36.09; 29.95; 28.61; 26.94; 26.20; 21.60; 15.52.

Cyanine synthesis (35) (fluorophore)

Compounds (34) (105 mg, 0.175 mmol), (33) (119 mg, 0.315 mmol) and sodium acetate (26 mg, 0.315 mmol) are dissolved in 10 ml of dry ethanol to yield a solution green. The mixture is brought to reflux with stirring for 7 hours and its color turns to dark blue. The progress of the reaction is monitored by UV-Visible and LCMS. The reaction mixture is concentrated under reduced pressure and then poured into 30 ml of Et ₂ 0. The resulting solution is filtered to give a brownish solid which is washed with Et ₂ 0 and purified on a steric exclusion column using chloroform as eluent for lead to 300 mg (40%) of a pure product (35) which is in the form of a green solid. 1H NMR (600 MHz, DMSO, 323 K): d (ppm) = 1.70 (s, 6H); 1.70 (s, 6H); 1.86 - 1.91 (m, 2H); 2.01-2.07 (m, 4H); 2.74 - 2.77 (m, 4H); 2.99 (m, 2H); 3.53 (t, J = 6.5 Hz, 2H); 4.32 (q, J = 7.0 Hz, 4H); 6.32 (d, J = 13.9 Hz, 1H); 6.41 (d, J = 14.3 Hz, 1H); 7.27 - 7.67 (m, 8H); 7.87 (s, 2H); 8.27 (d, J = 14.0 Hz, 1H); 8.32 (d, J = 14.2 Hz, 1H). Mass spectrum: m / z = 595.3331 [M-2Br] - (calculated for C ₃₆ H ₄₅ Br ₂ ClN ₆ : 754.1751).

Cyanine synthesis (fluorophore)

Compound (35) (300 mg, 0.396 mmol), di-tert-butyl dicarbonate (130 mg, 0.594 mmol) and DIPEA (N, N-diisopropylethylamine) (255 mg, 1.98 mmol) are dissolved in 15 mL of chloroform to give a green solution. The mixture is brought to reflux with stirring, and the progress of the reaction is monitored by LCMS. After cooling to room temperature, the crude mixture is washed with water (2 x 40 mL) and with a 0.2 M solution of hydrochloric acid (30 mL). The organic phases are combined, concentrated under reduced pressure, then the compound is isolated and purified by steric exclusion column using chloroform as eluent to yield the pure product (36), which is found under form of a green solid. Mass spectrum: m / z = 695.5 [M-Br] (calculated for C _4i H ₅₂ BrClN ₆ 0 ₂ : 774.30). HPLC, retention time: 5.95 min. UV-Vis: 777 nm.

2 / Synthesis of compound 1 (see Figure 2)

Synthesis of the coumarin-cyanine conjugate (37)

7-hydroxycoumarin (4.1 mg, 0.026 mmol) and sodium hydride (2.0 mg, 0.0515 mmol) are dissolved in 1 ml of DMF and the mixture is stirred at room temperature for 10 min. Cyanine (36) (10 mg, 0.0128 mmol) is then added and the progress of the reaction is followed by LCMS. After approximately 20 minutes, the DMF is distilled under reduced pressure, the product is taken up in CHCf, then washed with water and purified on a small plug of silica gel (solvent: DCM). Compound (37) is obtained in the form of a green solid (10 mg, 90%). Mass spectrum: m / z = 821.4 [M-Br] (calculated for CsoFLvBrN _ô Os: 900.35). HPLC, retention time: 5.6 min. UV-Vis: 307, 777 nm.

Compound synthesis

Compound (37) (10 mg, 0.013 mmol) is dissolved in 2 ml of a TFA / DCM mixture (1/9 vol.) And the resulting solution is stirred for 1 h at room temperature. To this mixture are added 10 mL of DCM, the organic phase is then washed with a saturated solution of NaHC ^' CF (2 x 25 mL), then dried with MgSO ₄ and then concentrated under reduced pressure to obtain 9 mg (90%) of product (38). Mass spectrum: m / z = 721.4 [M-CF ₃ CO ₂ ] (calculated for C ₄ 5H ₄ 9N ₆ 03: 721.92). HPLC, retention time: 4.4 min. UV-Vis: 307, 777 nm.

Compound synthesis

To a solution of compound (38) (9 mg, 0.0107 mmol) in 1.5 mL of DMF are added DOTA-GA anhydride (2.2 ', 2 ”- (10- (2,6-dioxotetrahydro- 2H-pyran-3-yl) - 1,4,7,10- tetraazacyclododecane-1,4,7-triyl) triacetic acid) (9.1 mg, 0.0198 mmol) and triethylamine (11 mg, 0.105 mmol), then the resulting mixture is stirred for 24 h at 50 ° C. After evaporation of the DMF, the product is taken up in CHCl3 and washed with water. The product is then purified by steric exclusion column with CHCl 3 to obtain 7 mg (50%) of pure product (39). Mass spectrum: m / 2z = 590.2 (calculated for C6 ₄ H ₇₉ NioOi2: 1180.39). HPLC, retention time: 4.3 min. UV-Vis: 307, 777 nm.

Synthesis of compound 1 of the invention The precursor (39) is placed in a buffer of ammonium acetate pH 4.5 and placed in the presence of a source of radioactivity (source ⁹⁰ YCl3), so as to obtain the specific activity desired. The mixture is heated at 80 ° C for 2 hours and the reaction is monitored by radio-ITLC. Compound 1 of the invention is obtained.

3 / Synthesis of the compounds 7, 12 and 13 of the invention

Compounds 7, 12 and 13 are synthesized according to a synthesis method comparable to that described for compound 1 but with the following differences: the steps corresponding to the introduction of radiochelate type ⁹⁰ Y- [DOTAGA] are not carried out.

Instead, the ¹⁸ F radiolabelling steps for compounds 7 and 13 are carried out using the corresponding alcohol which is transformed into a triflic ester which is then placed in the presence of an Na ¹⁸ F type salt to lead to the substitution of the triflate by ¹⁸ F.

The synthesis of compound 12 of the invention is carried out according to a process comparable to compound 1 (introduction of a BODIPY derivative carrying a 4-hydroxy-phenyl group in meso). The BODIPY compound carrying two non-radioactive fluorine atoms ¹⁹ F will react with DMAP (dimethylaminopyridine) by substitution of one of the two fluorine atoms, thus leading to a BODIPY-DMAP adduct where DMAP now in quaternized form is a good group. leaving. In the presence of an Na ¹⁸ F type salt, ¹⁸ F will replace the quaternized DMAP, leading to the species radiolabelled at ¹⁸ F.

4 / Synthesis of the compounds 5 and 6 of the invention (see FIG. 3)

Compounds 5 and 6 can be obtained from an asymmetric cyanine (40) leading to compound (42) or (41), which are respectively the precursors of compounds 5 (Figure 3a) and 6 (Figure 3b) of invention.

Compound synthesis

Compound (34) (170 mg, 0.28 mmol), compound (31) (88 mg, 0.28 mmol) and sodium acetate (23 mg, 0.28 mmol) are dissolved in 12 mL d dry ethanol leading to a brown solution. The mixture is stirred and brought to reflux for 2 hours where it becomes dark green. After UV-Visible and LCMS controls, the reaction mixture is concentrated under reduced pressure and poured into 75 ml of Et ₂ 0. The solution is filtered to give a brownish solid which is washed with Et ₂ 0 and purified by chromatography on silica gel (using a solvent gradient DCM / MeOH 98/2 vol. to 90/10 vol) to provide 75 mg of pure product (40) in the form of a dark green solid. 1H NMR (500 MHz, CDCl ₃ , 300 K): d (ppm) = 1.9 (m, 12H); 1.90 (m, 2H); 2.07 (t, J = 6.5 Hz, 2H); 2.56 - 3.08 (m, 6H); 3.53 (t, J = 6.0 Hz, 2H); 4.14 (t, J = 7.1 Hz, 2H); 4.60 (m, 2H); 6.07 (d, J = 13.7 Hz, 1H); 6.58 (d, J = 14.4 Hz, 1H); 7.11 - 7.54 (m, 8H); 8.21 (d, J = 13.4 Hz, 1H); 8.42 (d, J = 14.2 Hz, 1H).

Synthesis of compound 5 (Figure 3a)

The coumarin group is reacted with 2-benzyloxy-1,3-dichloropropane, the adduct is deprotected to lead to bis-coumarin alcohol (called multi-B amplifier head) which then reacts with chloro-cyanine ( 40). The DOTAGA-ethylenediamine group reacts with the acid function of the intermediate (42) to give the compound (43). The radiolabelling step makes it possible to obtain compound 5.

Synthesis of compound 6 (Figure 3b)

Hydro coumarin (21 mg, 0.13 mmol), sodium hydride (6.3 mg, 0.26 mmol) is dissolved in 2 mL of DMF. After 10 min, the compound (40) (50 mg, 0.07 mmol) is added and the course of the reaction is followed by LCMS. As soon as the reaction no longer progresses, 20 ml of diethyl ether are added. The solid is filtered, then washed with diethyl ether, acetone (the purity is followed by HPLC) to yield 11 mg of pure product (41) in the form of a green solid.

One of the six amine functions of 1,2,3,4,5,6-Benzenehexamethanamine is protected while the other five are involved in the reaction with DOTAGA (tBu) ₄ . The adduct formed (also called “multi-A” amplifying head) is engaged in the reaction with cyanine. The system obtained is saponified and then radiolabelled with Yttrium to yield compound 6.

5 / Synthesis of the compounds 21 and 11 of the invention (see FIG. 4)

Compounds 21 and 11 are obtained from a symmetrical cyanine (44) carrying two azide groups, said cyanine leading to intermediate (45), which itself leads to compound (46) which is a precursor of compound 21 (Figure 4a) of the invention or to the compound (48) which is a precursor of the compound 11 (Figure 4b) of the invention.

Synthesis of the precursor

Pyranin (74 mg, 0.14 mmol) and triethylamine (43 mg, 0.42 mmol) are dissolved in 2 mL of dry DMSO. The mixture is stirred at 50 ° C for 4 h then a solution of cyanine (44) (50 mg, 0.07 mmol) in 1 mL of DMSO is added. After 4 h at 45 ° C., 20 ml of DCM are added to the reaction mixture. After filtration which eliminates the excess of pyranine then concentration under reduced pressure, the reaction crude is diluted in water (20 mL) and extracted with diethyl ether (2 x 30 mL) to yield, after lyophilization at 41 mg (54%) of pure product (45) in the form of a green solid.

1H NMR (500 MHz, Methano / ₄ , 300 K): d (ppm) = 0.53 (s, 6H); 1.41 (s, 6H); 1.99 (p, J = 6.7 Hz, 4H); 2.10 - 2.25 (m, 2H); 2.82 (m, 2H); 2.95 (m, 2H); 3.46 (td, j = 5.8; 4.0 Hz, 4H); 4.11 - 4.20 (m, 4H); 6.27 (d, j = 14.1 Hz, 2H); 7.06 (t, j = 7.5 Hz, 2H); 7.12 - 7.17 (m, 2H); 7.18 (d, J = 8.0 Hz, 2H); 7.24 - 7.32 (m, 2H); 8.09 (d, j = 14.0 Hz, 2H); 8.28 (s, 1H); 9.01 (d, j = 9.6 Hz, 1H); 9.23 (s, 2H); 9.50 - 9.56 (m, 2H).

HRMS ESI: m / z = 1041.2740 [M-2Na ⁺ H] (calculated for C ₅₂ H ₄₉ N ₈ O ₁₀ S ₃ : 1041.2739). UV-Vis (water): 241.9; 287.4; 360.0; 394.8; 770.4 nm.

Synthesis of compound 21 (FIG. 4a)

Compound synthesis (46)

Compound (45) reacts with DOTA-GA-ethylenediamine-BCN under the following conditions. 6.3 mg of DOTA-GA-ethylenediamine-BCN (0.0129 mmol) are dissolved in 1 ml of phosphate buffer at pH = 7.4. Then 0.48 mL of a solution of cyanine (45) (1.97 mg; 0.00181 mmol) in water is added. The mixture is then stirred at room temperature for 3 h then lyophilized and the crude product obtained is purified by HPLC to give after lyophilization 3.55 mg (78%) of target compound (46).

UV-Vis (PBS buffer): 242.0; 288.1; 360.5; 395.2; 770.8 nm. HPLC analysis with system A: 3.9 min, 77% MeCN 0.1% FA. HPLC with system B: 25.0 min, 45% MeCN 0.1% FA.

Preparation of compound 21 of the invention

A conjugation step (pH control, temperature, antibody concentration) gives access to the "compound 46-antibody" system, that is compound (47). The bioconjugate is radiolabeled at 1.90 to give the compound 21 of the invention.

Synthesis of compound 11 (Figure 4b)

Compound synthesis

Fa cyanine (45) is dissolved in 2 mL of dry DMSO then DOTA-GA-ethylenediamine-BCN is added. The mixture is stirred at 50 ° C for 16 h. Compound (48) is obtained. 1H NMR (500 MHz, Methano / ₄ , 300 K): d (ppm) = 0.53 (s, 6H); 1.41 (s, 6H); 1.99 (p, J = 6.7 Hz, 4H); 2.10 - 2.25 (m, 2H); 2.82 (m, 2H); 2.95 (m, 2H); 3.46 (td, j = 5.8; 4.0 Hz, 4H); 4.11 - 4.20 (m, 4H); 6.27 (d, j = 14.1 Hz, 2H); 7.06 (t, j = 7.5 Hz, 2H); 7.12 - 7.17 (m, 2H); 7.18 (d, J = 8.0 Hz, 2H); 7.24 - 7.32 (m, 2H); 8.09 (d, j = 14.0 Hz, 2H); 8.28 (s, 1H); 9.01 (d, J = 9.6 Hz, 1H); 9.23 (s, 2H); 9.50 - 9.56 (m, 2H). HRMS ESI: m / z = 1041.2740 [M-2Na + H] (calculated for C52H49N8O10S3: 1041.2739). UV-Vis (water): 241.9; 287.4; 360.0; 394.8; 770.4 nm.

Compound 11 synthesis

The cyclic bismacro compound (48) in a buffer is incubated for several hours in the presence of a defined quantity (MBq) of Yttrium-90 ⁹⁰ UO ₃ trichloride in order to achieve the desired specific activity. The radiochemical purity is checked by RI-TLC.

Compound 11 of the invention is obtained.

6 / Synthesis of the compounds 4 and 19 of the invention (see FIG. 5)

Compound synthesis (49)

Coumarin (646 mg, 4 mmol), 4,5-dichlorophthalonitrile (788 mg, 4 mmol) are dissolved in 10 ml of DML (dimethylformamide) in the presence of K2CO3 (2.21 g, 16 mmol). The resulting mixture is stirred at 45 ° C for 16 h and is then recovered by filtration. The filtrate is concentrated under reduced pressure and purified by column chromatography using as eluent a mixture of DCM / MeOH 9 solvents (5/5 vol.) To give 920 mg (70%) of the desired compound (49) in powder form .

1H NMR (500 MHz, CDCl ₃ , 300 K): d (ppm) = 6.45 (d, d = 9.6 Hz, 1H); 6.97 (dd, J = 8.5; 2.4 Hz, 1H); 7.02 (d, j = 2.4 Hz, 1H); 7.26 (s, 1H); 7.59 (d, j = 8.4 Hz, 1H); 7.73 (d, j = 9.6 Hz, 1H); 7.94 (s, 1H).

¹³ C NMR (125 MHz, CDCl ₃ , 300 K): d (ppm) = 108.17; 111.80; 114.06; 114.16; 115.77; 115.97; 116.68; 117.02; 122.79; 130.16; 131.19; 136.13; 142.55; 155.74; 156.37; 156.60; 159.82.

MALDI-TOL (Calculated for C17H7CIN2O3: 322.0145, found 323).

Synthesis of the phthalonitrile-coumarin-pyridine system

The phthalonitrile-coumarin conjugate (49) (400 mg, 1.24 mmol), 4-hydroxypyridine (176 mg, 1.85 mmol) and K2CO3 (512 mg,, 71 mmol) are dissolved in 20 mL of DML. The resulting mixture is stirred at 45 ° C for 16h. Then, K ₂ CO ₃ is separated by filtration on a frit, then the filtrate is concentrated under reduced pressure and purified by chromatography using as eluent the mixture of solvents (DCM / MeOH 90/10) to give 200 mg (%) of the desired product. (50).

Synthesis of the diiminoisoindoline-coumarin-pyridine system

A solution of dicyanobenzene (50) in methanol is brought to reflux under bubbling of ammonia for several hours. After distilling off the solvent, the compound obtained (51) is immediately used in the following synthesis step.

Steps to transform (51) into compounds 4 and 19 of the invention.

Diiminoisoindoline (51) is reacted with a silicon salt. The reaction mixture is brought to reflux then the solvent is distilled under reduced pressure. The residue obtained is washed with a series of solvents, then dried and immediately used in the next step. Intermediate (52) reacts with 3-azidoethanol in the presence of base is brought to reflux. After purification, a suspension of phthalocyanine (53) in methyl iodide is brought to reflux. At the end, the excess methyl iodide is distilled and the phthalocyanine (54) is purified by semi-preparative HPLC. Intermediate (54) and DOTAGA-ethylenediamine-BCN are reacted, after distillation of the solvents, the target conjugate (55) is separated from the compound (54) and the by-products by HPLC.

Example 2

Use of the compounds of the invention for PDT-Cherenkov

An in vitro tube and in vitro cell study shows the intramolecular CRET / TBET or CRET / FRET transfer properties of the compounds of the invention.

The measurement of activated oxygen species (ROS) is carried out by UV / Visiblc spectrometry by following the disappearance of the absorbance band of DPBF (diphenylbenzofuran) following the reaction with the ROS generated by the dynamic photo process Cherenkov.

In vitro cell study The cells are plated on 96-well microplates, incubated with the solution of a compound of the invention in the total absence of a source of extraneous stray light capable of exciting the photosensitizing compound.

A control plate is prepared in the presence of the non-radiolabelled compound to prove that the toxicity measured does not come from:

- a parasitic cytotoxic effect resulting,

- the intrinsic cytotoxicity of the compound,

- photocytotoxicity resulting from excitation by an exogenous light source.

Furthermore, a control study using the non-radiolabelled parent compound - and in the absence of an exogenous light source - confirms the origin of the cytotoxicity.

PDT-Cherenkov in vivo protocol

It begins with the injection of a compound of the invention intravenously into xenografted mice carrying a deep cancer model of cancer cells.

A batch of control mice injected with a radiolabelled bioconjugate, of structure AD, that is to say containing no BC, is prepared. The tumor volume is monitored by performing PET imaging of the tumor. This imaging is carried out as follows: the mice are anesthetized and then injected with 18F-Fluorodeoxyglucose (18F-FDG) and are then imaged on an mTER imager. Throughout the experiment, every precaution was taken so that no stray light, that is to say other than Cherenkov radiation, could reach the tumor marked by the compound of the invention.

The following checks are carried out:

- the tumor is deep (> 1 cm deep),

- the mice have hair, and possibly a screen can be affixed on the study area.

This batch of control mice studied makes it possible to show the evolution of the volume of the tumor in the event of prolonged exposure to an exogenous and potentially parasitic light source. At a sufficient depth, the evolution of the tumor volume of control mice versus treated mice makes it possible to demonstrate the effectiveness of the compounds of the invention.

Example 3

Use of the Compounds of the Invention for Near IR CLI Imaging

CLI imaging protocol A study on an optical imager makes it possible to measure the transfer properties by CRET / TBET or CRET / FRET intramolecular of compound 1 of the invention.

Furthermore, an in vitro study has shown that the non-radiolabelled parent compound does not exhibit cytotoxicity.

The CLI imaging protocol begins with the injection of compound 1 of the invention intravenously into xenografted mice carrying a tumor.

A batch of control mice injected with an AD type radiolabelled bioconjugate, that is to say one containing no BC, is also prepared.

When the AD bioconjugate has reached the tumor, after several hours (the number of hours will depend on the nature of the biomolecule), the mice are anesthetized and are then placed in the optical imager.

The mice are imaged in Cherenkov mode and in Bio luminescence mode, first by examining the radiance over the entire spectral window of the optical imager (500-850 nm) then using filters to examine the radiance over the near-infrared zone only.

After the experiments, the mice are sacrificed.

The measurement of the radiance is the step making it possible to demonstrate the transfer to the near infrared and the effectiveness of the compounds of the invention. This implies a direct comparison of the radiance between the batch of control mice injected with AD (the radiolabelled biomolecule, ie the Cherenkov radiation alone, not amplified by the BC antenna), and the batch of mice injected with the compound 1 of the invention.

The comparison of the result obtained with compound 1 of the invention and the result obtained by the authors Bemhard et al. ° makes it possible to demonstrate the relevance of a unimolecular probe rather than a multi-molecular probe, and the interest of the compounds of the invention.

Bibliographic references

(1) Grootendorst, M. R., Cariati, M., Kothari, A., Tuch, D. S., Purushotam, A. Cerenkov luminescence imaging (CLI) for image-guided cancer surgery, Clin. Transi. Imaging (2016), 4, 353-366.

(2) Demian van Straten, Vida Mashayekhi, Henriette S. de Bruijn, Sabrina Oliveira and

Dominic J. Robinson, Oncologie Photodynamic Therapy: Basic Principles, Current Clinical Status and Future Directions, Cancers, 2017, 9, 19, 1-54, doi: l 0.3390 / cancers 9020019.

(3) Robertson, R. et al. Optical imaging of Cerenkov light generation from positron- emitting radiotracers. Physics Medicine Biology 54, N355-N365 (2009).

(4) Dothager, R. S., Goiffon, R. J., Jackson, E., Harpstrite, S. & Piwnica-Worms, D.

Cerenkov Radiation Energy Transfer (CRET) Imaging: A Novel Method for Optical Imaging of PET Isotopes in Biological Systems. PLoSone 5, el3300 (2010).

(5) Boschi, F. & Spinelli, A. E. Quantum dots excitation using pure beta minus radioisotopes emitting Cerenkov radiation. RSC Advance 2, 11049-11052 (2012).

(6) Bemhard et al., Chemical Communications, 2014, 50, pp. 6711-6713.

(7) Bemhard et al., Scientifïc Reports, 2017, 7, 45063.

(8) Bizet et al., Bioorganic & Medicinal Chemistry 26 (2018), pp. 413-420.

(9) Anyanee KamKaew et al., Applied Materials & Interfaces, 2016, 8 (40), pp 26630-26 637.

(10) C. Gôl, M. Malkoç, S. Yeçilot, M. Durmuç, Dyes Pigm, 111 (2014), pp. 81-90.

(11) S. Osati, H. Ali, J.E. van Lier, Tetrahedron Lett, 56 (2015), pp. 2049-2053.

(12) H. Yamk, M. Gôksel, S. Yesilot, M. Durmus, Tetrahedron Lett, 57 (2016), pp. 2922-2926.

(13) A. Loudet, C. Thivierge, K. Burgess, Dojin News (2011), p. 137, http: //www.doi indo. co .ip / letteri / l 37 / review / 01.html.

(14) Wen-Hai Zhan, Jian-Li Hua, Ying-Hua Jin, Xin Teng, and He Tian, Res. Chem.

Intermed., Vol. 34, No. 2-3, pp. 229-239 (2008).

Claims

1. Compound characterized in that it has the following general structure (I):

in which :

and or

* It is a photosensitizer which produces reactive oxygenated species ROS;

* L1, L2, L3 are each, independently of one another, an at least divalent linking group or a covalent bond, with the condition that

are not present simultaneously, which means that if is present then

^! '"is absent and vice versa,

* n is an integer equal to 1, 2, 3, 4 or 5;

* m is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8;

and in which:

* A activates B by a CRET type energy transfer,

* B transfers the energy received from A to C, by an energy transfer of intramolecular FRET type or by an energy transfer of TBET type.

2. Compound according to claim 1, characterized in that it has the following structure (1-1):

(i-i),

wherein A, B, C, D, Ll, L2, L4, n and m as defined in claim 1.

3. Compound according to claim 1, characterized in that it has the following structure (1-2): wherein A is a non-metallic radioelement, and B, C, D, L2, L3, L4, n and m are as defined in claim 1.

4. Compound according to any one of claims 1 to 3, characterized in that A is:

* a radiochelate whose radiometal is chosen from the group comprising ⁹⁰ Y, ¹⁷⁷ Lu, 6 ⁸ Ga, ⁸⁹ Zr, ⁶⁴ Cu, ⁸⁹ Sr, ²¹² Bi, ²¹³ Bi, ⁴⁴ Sc, ²²⁵ Ac and ⁴⁴ Sc and whose agent chelating agent is chosen from the group comprising DOTAGA, DOTA, NOTA, NODAGA and DFO,

* a non-metallic radioelement chosen from the group comprising 18 F, 131 I, 124 I and 32 P.

5. Compound according to any one of claims 1 to 4, characterized in that B is chosen from the group comprising a nucleus of the coumarin type; coumarin substituted, in particular by one or more hydroxy and / or by a pyridinium, itself optionally substituted; pyranine; pyrene; BODIPY; Substituted BODIPY, in particular phenyl-BODIPY, hydroxyphenyl-BODIPY, aza-BODIPY; fluorescein; rhodamine, in particular rhodamine 6G, rhodamine 101, rhodamine B, rhodamine 123; eosin, especially eosin B, eosin Y; tryptophan and mixtures thereof.

6. Compound according to any one of claims 1 to 5, characterized in that C is chosen from the group comprising a cyanine-type nucleus, in particular cyanine-7, cyanine-5, cyanine-3; phthalocyanine, in particular silicon phthalocyanine, zinc phthalocyanine, magnesium, phosphorus, aluminum, indium; naphthalocyanine, in particular naphthalocyanine of zinc, magnesium, phosphorus, aluminum, silicon, indium; chlorine, especially chlorine of zinc, magnesium, phosphorus, aluminum, silicon, indium; bacteriochlorine, in particular bacteriochlorine of zinc, magnesium, phosphorus, aluminum, silicon, indium.

7. Compound according to claim 5 or claim 6, characterized in that B and / or C comprises (s) at least one solubilizing group chosen from the group comprising a sulfonate (SO3); a carboxylate (COO); ammonium (NR ₄ ) with R = H, alkyl or aryl; a phosphonate (PO3 ² ); a pyridinium, preferably substituted; an immidazolium and their mixtures.

8. Compound according to any one of claims 1 to 7, characterized in that D is a biomolecule, in particular a peptide; a protein; an antibody-like protein; an antibody fragment type protein, such as "Fab", "Fab'2",

"Fab '", "ScFv", "nanobody", affïbody, diabody; an aptamer.

9. Use of a compound according to any one of claims 1 to 8, for an application for near infrared Cherenkov luminescence imaging.

10. A compound according to any one of claims 1 to 8, for use in the treatment of deep biological tissue by Cherenkov dynamic phototherapy.