WO2013011242A1

WO2013011242A1 - Novel water-soluble chromophore

Info

Publication number: WO2013011242A1
Application number: PCT/FR2012/051711
Authority: WO
Inventors: Cyrille MONNEREAU; Chantal Andraud; Olivier Maury
Original assignee: Ecole Normale Superieure De Lyon; Centre National De La Recherche Scientifique
Priority date: 2011-07-21
Filing date: 2012-07-19
Publication date: 2013-01-24

Abstract

The present invention concerns a water-soluble chromophoric entity comprising at least one lipophilic chromophore bonded covalently to at least two water-soluble polymer chains, characterised in that each water-soluble polymer chain is only covalently bonded to one single lipophilic chromophore, and each polymer chain bears several hydroxy functions and is obtained by polymerisation of one or more monomers, at least one of which is chosen from those defined in claim 1; it also concerns a method for preparing such chromophoric entities.

Description

NEW CHROMOPHORE WATER SOLUBLE

The invention relates to the technical field of biophotonics. More specifically, the present invention provides an original strategy, simple to implement, effective and adaptable to the main families of compounds of interest in this context, allowing the solubilization of these compounds while ensuring good biocompatibility of the latter in a cellular environment .

Biophotonics is defined as "the use of visible rays, ultraviolet rays, infrared rays, or even X-rays for the analysis or modification of biological objects that are inherently complex. This science is currently in full development, with in particular the emergence of new microscopies, such as bi-photonic fluorescence microscopies, which make it possible to obtain very high resolution images compared to conventional fluorescence techniques. Moreover, biophotonics makes it possible to envisage a large number of other applications, of which a non-exhaustive list covers: dynamic phototherapy (at one or two photons), fluorescent probes for the detection and recognition of molecules of biological interest , fluorescent pH probes, oxygen partial pressure probes ...

In this context, the use of molecules that can interact with visible, UV, IR ... to produce the expected response, is necessary and essential. These molecules can be molecules of the living, modified by genetic way (case of the "Protein green fluorescent" approach, whose discovery gave rise to the Nobel prize of Shimomura, Chalfie and Tien in 2008) or synthetic molecules, called chromophores (from the Latin "chromos phorus", "which brings color").

The use of synthetic chromophores requires to provide the latter with chemical modifications making them compatible with the physiological medium, namely:

i / Water-soluble: most chromophores, and particularly two-photon chromophores, are intrinsically hydrophobic, it is necessary to incorporate one or more solubilizing functions into the system. While being water-soluble, it is necessary that the object obtained incorporating the chromophore, once solubilized, retains the spectroscopic properties necessary for the target application, such as its fluorescence quantum yield, for example.

ii / Bioavailable; For in vivo applications in particular, it is necessary for the chromophore to have the capacity to cross the different biological barriers leading to the target organ / tissue. Bioavailability will depend on several factors, the main ones being the lipophilicity index (logP) and the size of the object. In-vitro, the problem is simplified, and will be limited to the ability of the object to diffuse into the studied tissue or to enter the target cell compartment.

iii / Bioselectives; it is important that the object incorporating the chromophore be routed to a given target organ, preferably selectively, ideally exclusively.

iv / non-toxic: The object must also have the lowest toxicity, or, in the case of objects intended for therapy, the lowest toxicity outside the target cell / organ, or in the absence of stimulus necessary for its activation.

Among these constraints, the problem of hydrosolubilization of chromophores, and particularly of fluorophores, is undoubtedly the one that mobilized the greatest number of research works. This theme has also been expanding rapidly over the past fifteen years: a keyword search on the ISI Web Of Knowledge engine, using the "water-soluble" and "fluorescent" entries, indicates that the number of publications on the subject has been steadily increasing year-on-year since 1995, from 37 publications (1995) to over 240 for 2010. Many strategies have been suggested to achieve the desired water solubility properties. If one is interested in the chromophores with nonlinear optical properties, which on the one hand are the most interesting in term of properties, and on the other hand present a structure with a carbon skeleton of a great length of conjugation in general , the making it particularly lipophilic and difficult to hydrosolubilize, the hydrosolubilization strategies can be divided into several categories:

those involving the introduction of solubilizing functional groups on the fluorescent molecule, such as anionic or cationic groups of the alcohol functions, or oligo- or polyethylene glycol derivatives (OEG or PEG). An example of cationic entities is described by Prasad et al. (J. Phys Chem., 1996, 100, 4521-4525), with two-photon fluorophores bearing a pyridinium group. Bazan et al. (X Am Chem Soc 2005, 127, 820-821) have developed a similar strategy with quaternary ammoniums as solubilizing groups. In these two examples, a sharp drop in the fluorescence quantum yield of the objects in an aqueous medium is observed. Nicoud et al. (Photochemical and Photobiological Sciences, 2006, 5, 102-106), have described the synthesis of two symmetrical fluorescent chromophore structures with two photons, one having a bi-phenyl core and the other a biphenetic heart. butylfluorene, each of these chromophores having a substitution on the 1,2,3-benzene triol end groups with methyl ether groups of oligoethylene glycol having 1,2 or 3 ethylene glycol units. The synthesized structures have variable water solubilities which decrease very strongly when the number of ethylene glycol units is reduced. Of the five structures synthesized, only three have acceptable water solubilities, and this water solubility is excellent for two of the compounds. The fluorescence properties in water are relatively well conserved, relative to the same chromophore in dichloromethane: it is approximately divided by a factor of two for water-soluble biphenyl core compounds, to be about 39-45%, and is about 72% for the fluorenyl core compound. However, the compounds studied have two photon absorption properties in the relatively small window of biological interest (between 750 and 950 nm), due to the absence of a strong electron donor group. These chromophores absorb in the near UV and the blue to a photon, and not enough in the red to two photons and are therefore not adapted to work in a biological environment. This lack of charge transfer may explain why fluorescence quantum yield is relatively better preserved. This last point is corroborated by the work of Ogilby et al. (1 Org Chem 2005, 70, 7065-7079) implementing a similar strategy on charge transfer chromophores, which made it possible to demonstrate a drop in the quantum yield of the object in water ( Q = 0.04) relative to its analogue in toluene (Q = 0.17). In addition, work by the inventors performed on chromophores for two-photon imaging described by Andraud et al. (Org Biomol Chem, 2010, 8, 142-150) has shown that the use of short PEG chains, if it allows a relatively efficient dissolution of the object in a biological medium and its vectorization in a cell, leads to a drastic decrease in the fluorescence quantum yield of the object in water relative to dichloromethane, which can range from a factor of 5 to a factor of 10. Moreover, in all the examples cited, despite the modest size of the objects, the synthesis of water-solubilized objects is relatively complex, with, for the simplest objects, a minimum of 6 steps from commercial products, and at least 15 steps for the chromophores of Andraud et al.

- those involving the dispersion of the chromophore within a micellar system. This strategy generally requires the synthesis of an amphiphilic block copolymer, and a micelle preparation step, in an aqueous and organic solvent mixture allowing the incorporation of the chromophore into the micellar system in formation. The work of Jen et al. (Advanced Functional Materials 2007, 17, 1691-1697), for example, describe two-photon fluorescent chromophores that are incorporated into a polystyrene / poly (acrylic acid) block copolymer, synthesized by ATRP methods in protected form from the (tert-butyl ester) acid. The objects obtained have a high gloss, due to the large number of fluorophores present in each object. Nevertheless, the results show that, according to the method of preparation used for the preparation of the micelles, the quantum yields obtained can be severely affected, this decrease being up to a factor of 14 compared with the chromophore alone in toluene. The authors logically attribute this decrease to the aggregation of the chromophore. This effect is almost systematically encountered in chromophores of biological interest (mainly non-linear properties), except in rare paradoxical examples of aggregation-induced emission (Prasad et al., Advanced Materials, 2007, 19, 371-3795). , and is therefore an almost systematic limitation of micellar systems, as recently shown (Andraud et al., Photochem, Photobiol, Sci., 2011, 10, 1216), with a charge transfer chromophore.

- those involving the use of polymers. The polymers may be obtained by copolymerization of two monomers, one providing the water-solubility and the other incorporating the chromophore (where a function that will allow the subsequent grafting of the chromophore), or obtained by modifying a polymer synthesized by controlled methods. on the alpha or omega ends. A recent review Charreyre et al. (Prog Polym Sci, 2011, 36, 568-602) lists the examples of the literature resulting from these two approaches, by anionic polymerization techniques, RAFT, ATRP, or NMP, in the context of partially related applications. to fluorescent polymers with biomedical aims. Among the monomers used in the few water-soluble systems described, the quasi-exclusivity concerns species carrying anionic (sulfonate) or cationic (quaternary ammonium) groups, or alternatively on monomers bearing groups of the PEG or OEG type. The authors of this review also point out that the amphiphilic character of the formed species, in which the terminal chromophore constitutes a lipophilic head, very often results in the formation of micellar systems, with extinction of the emission or appearance of excimer bands.

those in which the chromophore is incorporated covalently within a dendritic system, either at the periphery of the dendrimer or at the center of the dendrimer. In a 2008 article, Frechet et al. (J.Am.Chem.Soc, 2008, 130, 664-645) have shown that fluorophores peripherally interact to form J-aggregates, which modifies their fluorescence signal. This property is used in the context of the realization of pH sensors, the ester bond linking the fluorophore to the polymer can be hydrolysed by immersion in an acid medium (pH = 4) for a few hours, which allows to restore the fluorescence of the individual object in solution. In a 2007 article, Jang et al. (Chem Mater, 2007, 19, 5557) measured the kinetics of fluorescence decline of porphyrins located in the heart of dendrimers of different generations and showed that this kinetics decreased with the increase of dendrimer generations. In other words, the dendrimer forms a protective "shell" around the fluorophore, which allows it to maintain good emission properties.

In general, the deleterious effect of protic polar solvents, particularly of aqueous solvents, on the quantum yields of many fluorophores, and mainly of fluorophores emitting from an excited state of CT ("charge-transfer") type, either through aggregation of the fluorophore species (generally lipophilic, see De Schyver et al., Chem Rev. 1993, 93, 189-221), or by dipolar interactions with the OH functions of the solvent (see Zachariasse et al. J. Phys Chem 1992, 96, 10809-10819), leading in both cases to deactivation of the fluorophore by non-radiative route. Andraud et al. (Photochem, Photobiol, Sci., 2011, 10, 1216), in the past, faced similar difficulties, with fluorophores of the same type as those exemplified in our invention, dispersed either in micellar systems or attached "in clusters" in side chains of water-solubilising polymers.

The points, ii / (bioavailability), iii / (bioselectivity) and iv / (lack of toxicity) were less specifically studied in the context of chromophores / fluorophores than in the more general context of the vectorization of molecules of therapeutic interest. For dendrimers, Fréchet et al. supra have listed the various parameters of dendritic systems that can influence the characteristics pharmacological ii, iii / and iv / of the object mentioned above, and whose control ultimately allows to obtain an ideal vector.

The low polydispersity of the object, that is to say its homogeneity of size, allowing a reproducible pharmacokinetics,

-The absence of ionic groups, known to accumulate in the liver and have high toxicity,

-The size of the object, which can, particularly in the case of tumor treatment, promote vectorization of the product by passive transport to the organ to be treated. However, the study by Jang et al. supra, indicates that the size of the object may also be an obstacle to its vectorization, probably in cases where active transport is involved.

The possibility of including on the periphery of the structure, "active targeting units", making it possible to induce a selectivity of the object for cells carrying specific receptors.

-The possibility for the object to be removed from the body after treatment, by natural routes (with or without biodegradation), which can be promoted by the presence of water-solubilizing groups at the periphery.

At present, according to the authors, only dendrimers have characteristics that make it possible to envisage such a control. However, the synthesis of dendrimers, particularly high-generation dendrimers, is a process that can be long, difficult and impractical to implement when targeting high generations.

In this context, the present invention provides novel water-soluble chromophoric entities which may be at least as useful as the solutions of the prior art, but which may have one and / or the other of the following characteristics;

- An ease of preparation,

- Good preservation of luminescence properties in water, - A satisfactory bioavailability, allowing them a good internalization in living cells, at least in vitro.

The present invention relates to chromophoric entities, and in particular water-soluble fluorescent entities comprising at least one lipophilic chromophore covalently bound to at least two water-soluble polymer chains, characterized in that:

each water-soluble polymer chain is only covalently bound to a single lipophilic chromophore,

and each polymer chain carries several hydroxyl functions.

Advantageously, the polymer according to the invention consists of at least one iterative monomer unit carrying a hydroxy function.

In the context of the invention, the polymer chains preferably belong to the family of polyacrylamides, polymethacrylamides, polymethacrylates, polyacrylates or polyallyl esters, in homopolymer or copolymer form.

In the context of the invention, the chromophore is solubilized by covalent bonding to at least 2 chains of water-soluble polymers bearing OH bonds. In particular, the chromophore may, for example, be linked to a number ranging from 2 to 10 and for example to 2, 3, 4, 5 or 6 polymer chains defined within the scope of the invention.

The term "polymer" means a species having at least one iterative unit (also called monomeric unit) within its structure, and whose synthesis (or growth) is carried out by a sequence of identical reactions. This sequence of identical reactions occurs during the same synthesis step, that is to say without intermediate treatment during the growth reaction, which therefore excludes the fact that the polymers include dendrimers. In the context of the invention, the polymer chains comprise, for example, from 4 to 100 monomeric units per polymer chain. The concept of polymer thus encompasses that of oligomer. The term "homopolymer" means a polymer consisting of a single monomeric unit repeated and whose growth therefore involves only one monomer.

The term "copolymer" means a polymer formed by at least two different monomeric entities that are repeated and whose growth therefore involves at least two monomers. These two monomeric entities can either be organized in successive segments ("block copolymer"), or distributed randomly within the polymer chain ("random or random copolymers").

The polymer chains may be obtained from at least one monomer below having a hydroxy pendant function present on a side chain or a function which after treatment leads to a hydroxy function, which allows the repeated introduction of OH functions. .

The polymer chains can be obtained from one or more monomers, at least one or all of the monomers used being chosen from:

In the case of the last monomer, basic hydrolysis after polymerization makes it possible to obtain the desired -OH functions. The polymers according to the invention thus obtained are homopolymers or copolymers comprising at least one iterative monomeric unit bearing at least one OH function which corresponds to one of the above monomers, or in the case of the last monomer, to said monomer having underwent basic hydrolysis. More generally, and in a similar manner, the chromophore-linked polymer chains can be obtained from at least one monomer having a pendant hydroxy function present on a side chain. The pendant hydroxyl groups will preferably be separated from the main chain of the polymer by 0 to 10 atoms, and preferably by 2 to 7 atoms, preferably chosen from carbon, oxygen or nitrogen atoms.

Advantageously, each polymer chain present on the chromophoric entity will comprise from 4 to 50 monomeric units, or even from 4 to 30 units, and preferably from 10 to 15 monomeric units, which makes it possible to obtain a better preservation of the luminescence in an aqueous medium. The reaction time and the polymerization conditions will be adapted by those skilled in the art to obtain such a chain length.

By "chromophore" is meant any chemical species that can transform a light influx (Ultra-Violet, Visible, Infrared, X). This transformation can be detected by spectroscopic method, or produce an identifiable modification of the medium in which the chromophore is located. The chromophore entities according to the invention are capable of absorbing at least one photon, and of reaching, by an internal energy conversion process, a metastable excited state from which the desired physicochemical process can take place (for example a photon by fluorescence or phosphorescence, singlet oxygen generation ...).

Preferably, the lipophilic chromophores used in the context of the invention are charge-transfer chromophores, that is to say conjugated chromophores having an electronic delocalization between the ground state and the excited state. Such chromophores exhibit photoinduced excited states of charge transfer type, that is to say giving rise to a delocalization of the electron cloud resulting from the absorption of one or more photons. In the context of the invention, chromophores with non-linear optical properties are preferred, the latter being of interest for applications related to two-photon fluorescence, membrane process imaging, fluorescent pH probes, anionic probes . Such chromophores are, in particular, in the form of dipoles, quadrupoles or octupoles and may be represented

with D which is an electron-donating group, for example of the N-substituted aniline type, phenol ether or thiophenol ether, and A which is an acceptor group, for example of the nitro or cyano type,

and the connecting arm symbolized by the solid line is an uninterrupted conjugated sequence,

parenthesis meaning that the presence of the group in these parentheses is incidental to the definition.

The uninterrupted conjugated chain is, in particular, a hydrocarbon chain which may be substituted or unsubstituted, in which saturated bonds and unsaturated bonds chosen from double and triple bonds alternate. The unsaturated bonds, and in particular the double bonds, may be incorporated into a ring, preferably a hydrocarbon ring, for example chosen from:

The two chromophores used in the examples correspond to this definition, the N-substituted aniline groups acting as electron donor groups and the groups:

electron acceptor group.

The chromophores used in the context of the invention may also belong to the family of cyanines or merocyanines, which may be considered as special cases of charge transfer compounds. Such chromophores are described in particular by Maury et al. in 1 Am. Chem. Soc. 2010, 132, 4328, or by Bouit et al. in Chem. Mater. 2007, 19 (22), 5325-5335. We can also refer to Chem. Rev. 2008, 108, 1245-1330 and 1994, 94, 195-242 which list of lipophilic chromophores which can be water-solubilized in the chromophoric entities according to the invention.

Luminescence is the phenomenon by which a chromophore, after absorption of a photon, will emit from an excited state, a lower energy photon (possibly equal) to the incident photon by an internal energy conversion process. If the emissive excited state has the same spin multiplicity as the ground state, we speak of fluorescence, if this multiplicity is different, we speak of phosphorescence.

The term "water-soluble entity" means an entity soluble in pure water at 25 ° C. at a concentration of at least 10 ^-5 mol.L ^-1 , preferably at a concentration of 10 ^-3 mol.L ^-1 , and ideally at a concentration of ¹ mol.L ^-1 . "Soluble" means that the resulting solution must be clear, homogeneous, and without visible precipitation by the naked eye.

The term "lipophilic" in the context of the invention means that a molecule is soluble in organic solvents exclusively, preferably in solvents other than protic polar solvents, and preferentially soluble mainly in solvents of solvents. hydrocarbon type. The solubility in organic solvents and the non-solubility can in particular be observed at 25 ° C. at a concentration of at least 10 ^-5 mol.L ^-1 , preferably at a concentration of 10 ^-3 mol.L ^-1 , and ideally at a concentration of 10 ^"1 mol.L ^{" 1} .

In the chromophoric entities according to the invention, each water-soluble polymer chain may be obtained by controlled radical polymerization.

Radical polymerization has three stages: initiation (creation of free radicals and reaction with the first monomer unit), propagation (successive additions of monomer units on the growing ((macro) radical chain) and termination ( stopping the chain) by coupling or disproportionation between two growing chains or by transfer of a proton on a growing chain. Termination and transfer reactions affecting (macro) radicals are responsible loss of control of polymerization (polymeric chains of unpredictable mass, high polymolecularity).

"Controlled polymerization" means a process in which the polymerization reaction proceeds without parasitic reactions (terminations, inter-chain coupling reactions, transfer reactions), and with constant and homogeneous kinetics throughout the polymerization reaction. , giving rise to the formation of chains of uniform and controlled length. This homogeneity is characterized by the polymolecularity index, ratio of the weight-average molecular weight to the number-average molecular weight of polymer chains obtained;

With the terms M _w and M _n defined by:

With M _x the molar mass considered and n _Xr the number of chains having this molar mass M _x .

The polymolecularity index is necessarily greater than 1. The lower the polymolecularity index, the more the object is uniform.

The subject of the invention is also a method for preparing a chromophoric entity as defined in the context of the invention in which each water-soluble polymer chain is obtained by controlled polymerization of at least one of the previously defined monomers.

By using such a controlled radical polymerization strategy based on a controlled growth of water-soluble polymer chains, it is possible both to induce good water solubility, while exercising control over the factors mentioned in points ii /, iii / and iv / previously. This strategy offers efficiency and versatility similar to those obtained in dendrimer systems, but with a much simpler implementation. It can be considered that in the chromophoric entities according to the invention, the chromophore is located at the heart of polymer chains of controlled size. Preferably, the polymolecularity index of the polymer chains is less than 1.5, preferably less than 1.3, ideally less than 1.1.

In particular, each polymer chain can be obtained by ATRP, RAFT or NMP polymerization. The ATRP and NMP techniques use the reversible termination, either by coupling with an initiating agent comprising a nitroxide in the case of NMP (Nitroxide Mediated Polymerization), or by coupling of an initiating agent comprising a halogen atom in the case of ATRP (Atom Transfer Radical Polymerization) (Wang JS et al., 1995, Macromol., 28, 791).

The RAFT technique uses reversible chain transfer, with an initiator agent having the following pattern;

... - C-S- ...

He

s

In the context of the invention, it is possible to use any type of initiator agents used in ATRP, RAFT or NMP techniques. By way of example, mention may be made of initiating agents, for example chosen from:

The last initiator agent is occasionally used as an ATRP initiator, but the presence of a chlorine at the end of the chain of the polymer then obtained, is more difficult to substitute by an N ₃ type group in particular.

2 / RAFT

with Y which represents, for example, a functionalization allowing the binding to the chromophore, of the activated ester or acetylene type for coupling to the chromophore by click-chemistry. The concept of activated ester is well known to those skilled in the art. An activated ester function can be defined as an ester whose "alcohol" part is a good leaving group with respect to nucleophilic substitution reactions. Such activated esters have, for example, been described by W. Anderson et al, in American Society, 1964, 46, 1839-1842 and by R. Arshady in Advances in Polymer Science, 1994, 111, 1-41.

3 / N

These different modes of controlled polymerization are characterized by the fact that, after polymerization reaction, at one or the other end of the polymer chain, the polymerization transfer agent: a bromine for the ATRP is found. , a thioester or thiocarbonate for RAFT, a nitroxide for NMP. Also, in the context of the invention, according to certain embodiments, the chromophoric entity comprises, at the Ω end of at least one polymeric chain, or all of the polymer chains present, a bromine, a dithioester (- C (S) -S-) or a nitroxide (-O-N-). For exemple dithioester linking, there may be mentioned -CH ₂ -C (S) -S- or trithiocarbonates (-SC (S) -S-).

In the case of the RAFT and NMP processes, it is conceivable, in theory, to attach the chromophore to one side or the other of the initiating agent (transferring side or transferred side). It is preferable that this attachment be transfering side as illustrated above, because this allows to cleave the transfer function: dithioester or trithiocarbonate for RAFT, which becomes a thiol function after cleavage, and nitroxide for NMP, which becomes a hydroxy function after hydrolysis, without removing the chromophore from the polymer chain. It should be noted that, for the initiator agents used in the RAFT and NMP processes, there is a multitude of possibilities for connecting the nitroxide or dithioester or trithiocarbonate groups to the chromophore, but the techniques are more complex than those used for the ATRP. As an example of functionalization technique that can be implemented, reference may be made in particular to Liangliang Qiang et al. in Macromol. Rapid. Common. 2006, 27, 1779-1786, to Charreyre et al. in Prog. Polym. Sci, 2011, 36, 568-602 and the publications cited in this latter publication. Those skilled in the art will be able to make the appropriate modifications to the chromophore used to allow its covalent bond with the envisaged initiating agent.

In some embodiments, each polymer chain is covalently bound to the lipophile chromophore via a -OC (O) - or -O- or -NH-C (O) - linkage (in the chromophore sense -polymer).

For more details on the conditions to be implemented for each of the controlled polymerization techniques to be envisaged, reference may be made to the very detailed reviews available listing the main operating techniques used in carrying out controlled polymerization as a function of the reagents available. : K. Matyjaszewski in Controlled Living Radical Polymerization, Progress in ATRP, NMP and RAFT, Vol. 768 (Ed K. Matyjaszewski), ACS Symposium Series, 2000, p2; for ATRP, see Matyjaszewski et al. Adv. Mater. 1998, 10, 901-915 and Chem. Rev 2001, 101 (9), 2921-2990 and the thesis of the University of Maine de David Fournier, defended on December 6, 2005 "Atomic controlled radical polymerization by atom transfer (ATRP) of dimethylvinylazlactone - Application to the development of reactive supports" and in particular the table on page 17; for RAFT see Charreyre et al. Macromol. Rapid Common. 2006, 27, 653-692, for the NMP see Detrembleur et al. Chem. Rev. 2008, 108, 1104-1126.

According to preferred embodiments of the chromophoric entities and the method according to the invention, each water-soluble polymer chain is obtained by controlled radical polymerization by reaction of the functionalized lipophilic chromophore with a polymerization initiator and at least one monomer. It is also possible to carry out the controlled radical polymerization with an initiating agent and finally to graft the chromophore onto a function present at the end of the polymer originating from the initiator agent. The chromophores used for coupling to the initiating agent or the polymer will comprise, for example, a minimum of two, ideally four, primary hydroxy or amine functions not involved in the conjugation of the system. Such hydroxy functions may then be esterified with 2-bromoalkyl bromide groups.

The above initiating agents may therefore be grafted or, in a preferred embodiment, on the chromophore to carry out the polymerization reaction, or in free form, optionally protected. In the latter case, the polymer obtained will then be coupled to a chromophore carrying at least two reactive functions for grafting the chromophore at the alpha end of at least two polymer chains.

A simple way to envisage the grafting of the polymerization initiator function on the chromophore consists of an esterification reaction [- (CO) -O-] or of amide formation (peptide coupling - (CO) -NH-) between an initiator functionalized with a carboxylic acid group or an activated carboxylate (N-hydroxysuccinimide carboxylate, or acyl halide), on a chromophore, functionalized in as many positions as the desired number of chains, by a function of alcohol type or amine (optionally aniline or phenol). The initiator may therefore include carboxylic acid or acyl halide function, and may in particular be chosen from commercially available products, with regard to the examples ATRP and RAFT, synthesizable in 3 steps for the NMP example (Studer et al., Org Biomol Chem. ., 2011, 9, 2403-2412),

In order to disturb as little as possible the spectroscopic properties of the chromophore, it is preferable that the alcohol or amine function used for the coupling is not involved in the photoinduced CT-type electron transition, and thus is not located on the direct sequence. conjugate of the chromophore. It may therefore in particular be alcohol or aliphatic primary amines. For example, in the case of chromophores of the dipole, quadrupole or octupole type as described in the figure on page 9, if the electron donor group D is an aniline derivative, functionalization of the Ν, Ν- type can be envisaged. dihydroxyethyl. Similarly, if the group D is a phenol derivative, benzenediol or benzenetriol (O-hydroxyethyl) or thiophenol (S-hydroxyethyl). N, N-dihydroxyethylaniline, as well as its para aldehyde equivalent, are commercial, and the reactions making it possible to obtain from these structures a great diversity of Potential target chromophores by various reactions (halogenation, Sonogashira, Sille, Heck, Suzuki, Negishi ..., Knoevenagel condensations) are well known to those skilled in the art. From the terminal hydroxy functions, the amino functions are accessible in three simple steps clearly described by Jung et al. (Chem Mater 2006, 18, 4713-4715).

For cyanine chromophores, the functionalisation by N-alkylation of indolinium rings is well known. Processes for the preparation of N-substituted cyanine on indolinium have been described in various patent documents, including DE 3721850.

In general, such derivatives can be obtained by reaction of 2-bromohydroxyethyl with 2,3,3-trimethylindoline at reflux of toluene for 15 hours, the resulting indolium salt being obtained by filtration of the precipitate formed, and reacted. in a Knoevenagel condensation reaction with a derivative of 5-hydroxypenta-2,4-dienal or 3- (hydroxymethylene) cyclohex-1-enecarbaldehyde (Andraud et al., Chem.

-5335). The synthetic route is described below.

The different chromophores can be involved in esterification or peptide coupling reactions. Detailed and critical reviews are available concerning the different methodologies for activation of the carbonyl function developed for the realization of peptide couplings (Montalbeni et al., Tetrahedron 61 (2005) 10827-10852) or the formation of esters (Haslam, Tetrahedron 36 (1980) 2409-2433). As examples illustrating the variety of chromophores functionalized by an initiator group that can be envisaged, the following three cases are detailed:

According to a preferred embodiment of the chromophoric entities and the method according to the invention can be combined with the above, each water-soluble polymer chain is uncharged.

According to preferred embodiments of the chromophoric entities and the process according to the invention which can be combined with the preceding ones, the chromophoric entity comprises at the Ω end of at least one polymeric chain, or even of all the polymeric chains present:

a functionalization allowing the subsequent grafting of biological ligands or other molecules of interest, in particular by "click chemistry". In particular, such functionalization can correspond to a function N ₃ or -G≡X 'with X' = N or CH. The function -N ₃ , for example, can be easily obtained from a bromine present at the Ω end of the polymer and originating from the initiating agent, by the action of NaN ₃ (for example, as described in J. Am. Chem Soc., 129, 13, Apr. 4, 2007, 3979-3988).

or a biological ligand covalently bonded to the end of at least one polymer chain. In this case the biological ligands can be grafted by reaction of "click chemistry" on a function of the type -N ₃ or -C≡X 'with X' = N or CH. The mechanism of chemical bonding referred to as "click chemistry" or Huisgen reaction was first described by Huisgen and Szeimies in Chem. Ber. 1967, 100, 2494 and Ber. 1965, 98, 4014. These publications describe a 1,3-dipolar cycloaddition reaction of an azido derivative on an alkyne derivative at high temperature, according to the scheme below:

Such reactions will be carried out with copper (I) catalysis to control the regioselectivity of the cycloaddition between an alkyne derivative and an azide derivative as described in the prior art (H. Kolb,

Mr. Finn, K. Sharpless. Angew. Chem. Int. Ed. (2001), 40, 2004 in particular) or else activated thermally, or activated by release of cycle voltage. For more details, we can also refer to Chem. Soc. Rev., 2007, 36, 1249-1262 and Macromol. Rapid. Common. 2007, 28, 15-54.

In the context of the invention, the term "biological ligand" means a compound which has at least one recognition site enabling it to react with a target molecule of biological interest, for example of the protein, membrane protein, enzyme or polypeptide type. , polynucleotides, polysaccharides, DNAs, RNAs, etc.). By way of example of biological ligands, mention may be made of mono or polysaccharides, polynucleotides of the DNA or RNA type, polypeptides, antibodies, antigens, proteins, etc.

As monosaccharide, there may be mentioned, for example, glucose, galactose, mannose, fructose, and as a polysaccharide, for example, sucrose, lactose, maltose.

The term "polynucleotide" means a sequence of at least 2 deoxyribonucleotides or ribonucleotides optionally comprising at least one modified nucleotide. The polynucleotide can be an oligonucleotide, a natural nucleic acid or its fragment such as a DNA, an RNA ribosomal, a messenger RNA, a transfer RNA, a nucleic acid obtained by an enzymatic amplification technique.

By "polypeptide" is meant a sequence of at least two amino acids. By amino acids, we mean the primary amino acids that code for proteins, amino acids derived after enzymatic action such as fra / 75-4-hydroxyproline and natural amino acids but not present in proteins such as norvaline, V- methyl-L-leucine, Stalin (Hunt S. in Chemistry and Biochemistry of amino acids, Barett GC, ed., Chapman and Hall, London, 1985), amino acids protected by chemical functions for use in solid support synthesis or in the liquid phase and non-natural amino acids.

The term "antibodies" includes polyclonal or monoclonal antibodies, antibodies obtained by genetic recombination and antibody fragments. The term "antigen" refers to a compound capable of being recognized by an antibody for which it has induced synthesis by an immune response. The term "protein" includes holoproteins and heteroproteins such as nucleoproteins, lipoproteins, phosphoproteins, metalloproteins and both fibrous and globular glycoproteins.

In the context of the invention, and as is apparent from the examples below, it has been demonstrated that it is possible to hydrosolubilize a lipophilic chromophore, while preserving its luminescence in an aqueous medium, with a drop in the lower luminescence 40% in particular, while maintaining sufficient lipophilicity to maintain the internalisation of the chromophoric entities obtained. The term "conservation of luminescence in an aqueous medium" means that a water-soluble chromophoric entity according to the invention has a quantum yield in water close to that in a reference organic solvent, in particular dichloromethane, of the chromophore parry, that is to say the chromophore used to conduct the polymerization. By "close" is meant at least 50%, preferably 75%, ideally 100% of the quantum yield of the parent chromophore in dichloromethane. "Quantum luminescence yield" means the ratio between the number of photons emitted by a given quantity of chromophores and the number of photons absorbed by this same quantity of chromophores. This ratio is less than or equal to 1, the higher it is, the more luminescent the molecule.

The proposed solution relies on the choice of the polymers used which comprise hydroxyl functions, in particular the pendant hydroxy functional groups present on the side chains of the monomers used. The process according to the invention is simple and makes it possible to obtain chromophoric entities which do not precipitate during preparation. It leads to very homogeneous chromophoric entities.

The examples below illustrate the invention but are not limiting in nature.

Ant-In synthesis of formula (1):

To a solution deoxygenated by argon bubbling of 1,8-dibromoanthracene (100 mg, 0.3 mmol) and 4-ethynyl-N, N (bishydroxyethyl) aniline (125 mg, 0.6 mmol) in THF (3 ml) and triethylamine (1mL) are added Pd (PPh ₃ ) 2Cl 2 (10mg, 0.014mmol) and Cul (5mg, 0.027mmol). The mixture is stirred for 16H at 70 ° C, giving rise to the precipitation of a dark red solid. The solvents are evaporated and the residue is suspended in 10 ml of dichloromethane and then filtered. The solid obtained is again suspended in 10 ml of dichloromethane and then filtered. The collected solid is dissolved in about 10 mL of pyridine, filtered on paper, and the filter paper is washed with a large volume of THF until only a brown residue remains on the paper. The filtrate is isolated, and the solvents are removed under reduced pressure, and the solid is dried under high vacuum. The solid is then dissolved in a mixture of THF (5mL) and pyridine (80μί). Bromoisobutyryl bromide (340 mg, 5eq) is then added to the dark red solution. The brilliant orange solution thus obtained is stirred at ambient temperature for one hour and then poured into 50 ml of a 1/1 ice / aq HCl mixture. 0.5M. Dichloromethane is added, the organic phase is extracted and then washed again with aq HCl. 0.5M. The organic phase is collected, dried over sodium sulfate, and the solvents are evaporated under reduced pressure. The product is chromatographed on a silica column, eluting with dichloromethane. The orange band is collected, and 215 mg of a dark orange oily solid are obtained after evaporation (0.18 mmol, 60% in two steps). ^X H NMR (200 MHz, CDCl ₃ , ppm): δ 8.68 (dd, J = 3.2 Hz, J = 6.6 Hz, 4H), 7.65 (m, 8H), 6.85 (d, J = 9 Hz, 4H), 4.41 (t, 7 = 6Hz, 8H), 3.80 (t, J = 6Hz, 8H), 1.92 (s, 24H). ¹³ C NMR (50 MHz, CDCl ₃ , ppm): δ 171.2, 147.7, 133.2, 131.9, 127.4, 126.5, 118.5, 112.2, 111.7, 103.2, 85.5, 63.0, 55.5, 49.3, 30.8. HRMS calc for [C54H57Br4N2O8 + H] ⁺ 1177.0843, found 1177.0812. UV-vis (CH ₂ CI ₂ ): _max (nm)

= 474 [4.38]

- Synthesis of DBB-In of formula (2):

To a deaerated solution of 1,4-diiodo-2,5-dibromobenzene (120 mg,

0.245 mmol) (200mg; 0.6 mmol) and 4-ethynyl-N, N (bishydroxyéthyl) aniline (lOOmg, 0.49 mmol) in THF (3mL) and triethylamine (lml) were added Pd (PPh ₃ ₂ CI ₂ (7 mg, 0.010 mmol) and Cul (3.8 mg, 0.02 mmol). The mixture is stirred for 16H at 70 ° C, resulting in the precipitation of an ocher orange solid. The solvents are evaporated and the residue is suspended in 10 ml of dichloromethane and then filtered. The collected solid is dissolved in about 10 mL of pyridine, filtered on paper, and the filter paper is washed with a large volume of THF until only a brown residue remains on the paper. The filtrate is isolated, and the solvents are removed under reduced pressure, and the solid is dried under high vacuum. The solid is then dissolved in a mixture of THF (5mL) and pyridine (150μί). Bromoisobutyryl bromide (285 mg, 1.25 mmol, 5eq) is then added to the orange solution. The brilliant yellow solution thus obtained is stirred at ambient temperature for one hour and then poured into 50 ml of a 1/1 ice / aq HCl mixture. 0.5M. Dichloromethane is added, the organic phase is extracted and then washed again with aq HCl. 0.5M. The organic phase is collected, dried on sodium sulfate, and the solvents are evaporated under reduced pressure. The product is chromatographed on a silica column, eluting with dichloromethane. The orange band is collected, and 140 mg of a dark yellow solid are obtained after evaporation (0.12 mmol, 45% in two steps). ^X H NMR (200 MHz, CDCl3, ppm): δ 7.71 (s, 2H), 7.43 (d, J = 8.6 Hz, 4H), 6.75 (d, J = 8.6 Hz, 4H), 4.36 (t, J = 5.6 Hz, 8H), 3.75 (t, J = 5.6 Hz, 8H), 1.90 (s, 24H). ¹³ C NMR (50 MHz, CDCl3, ppm): δ 171.7, 147.6, 135.5, 133.4, 126.3, 123.3, 111.9, 110.4, 97.6, 85.9, 62.9, 55.5, 49.1, 30.8. HRMS calc for [C54H57Br4N2O8 + H] ⁺ 1234.8720, found 1234.8665 UV-vis (CH ₂ Cl ₂ ):

General polymerization procedure

0.02 mmol of previously prepared DBB-In gold or Ant-In initiator are dissolved in a mixture of THF (1 mL) and 2- (2-hydroxyethoxy) ethyl acrylate (HEA, lmL) in a tube. The mixture is then intensely deoxygenated by bubbling nitrogen for 15 minutes, then CuBr (12 mg, 0.085 mmol) and 2,2'-bipyridine (25 mg, 0.016 mmol) and added and the nitrogen bubbling maintained for 1 hour. minute. The tube is then sealed and heated at 85 ° C for 20 minutes. The tube is opened and the mixture is poured on distilled water (10 mL). The resulting solution is placed in a dialysis bag and dialyzed against water (the dialysis bag is stirred for 48 hours in a 10 L beaker filled with distilled water, the contents of which are regularly replaced by fresh water). . The light yellow solution is transferred from the dialysis bag to a 100 mL flask, and lyophilized for 24 h, giving 100 mg of a fibrous powder.

According to such a procedure, different objects are prepared by carrying out the polymerization with hydroxyethyl acrylate (HEA);

with the Ant-In initiator of formula (1):

the fluorescent entity Ant-PHEA of formula (3) is obtained:

the fluorescent entity DBB-PHEA of formula (4) is obtained:

* Η NMR (500 MHz, CD ₃ OD, ppm): δ 8.7 (b, 4H), 7.7 (m, 8H), 7.03 (b, 4H), 4.2 (b, "110H), 3.80 (b," 110H) ), 2.45 (b, * 50H), 1.8 (b, "100H), 1.22 (b, 24H) ¹³ C NMR (50 MHz, CD ₃ OD, ppm): δ 177.1, 175.7, 169.6, 142.3, 132.8, 131.1 , 127.5, 126.6, 118.2, 112.0, 110.3, 106.2, 86.1, 71.8, 67.2, 66.8, 62.1, 60.1, 59.8, 41.2, 34.7, 24.4.

DBB-PHEA:

XH NMR (500 MHz, CD ₃ OD, ppm): δ 7.79 (b, 2H), 7.46 (b, 4H), 6.93 (b,

4H), 4.6 (b, "50H), 4.2 (b, * 110H), 3.80 (b," 110H), 2.45 (b, * 50H), 1.8 (b, "100H), 1.18 (b, 24H). δ 177.6, 175.7, 169.6, 148.5, 135.1, 133.1, 131.1, 126.3, 122.9, 112.1, 109.2, 86.4, 73.2, 66.1, 62.9, 60.1, 59.8, 41.2, 34.7, 24.4.

Their fluorescence characteristics are given in the TABLE

1 below. TABLE 1

The visible UV spectra were recorded with a JASCO 670 UV-Visible spectrophotometer. The luminescence spectra were measured with a spectrophotometer -Jobin Yvon Fluorolog-3. Luminescence is induced on samples in dilute solutions placed in 10mm-thick, polished, four-sided quartz cuvettes, by non-polarized light from a 450W xenon CW lamp, and detected at an angle of 90% by a Hamamatsu R928 photomultiplier. Spectra are corrected to a reference signal to compensate for fluctuations in incident light intensity (due to lamp and monochromator) and spectral response to emission wavelength (due to detector and monochromator ). The quantum fluorescence yields Qx _f are measured in dilute solution (with an optical density less than 0.1, corresponding to a concentration of less than about 2 μΜ), using the Stern-Volmer method based on the following equation

QQ = [Λ (λ) / Λ (λ)] [η _χ ² / η _Γ ² ] [ζνα] With A the absorbance of the solution at the excitation wavelength (λ), n the index of refraction and D the integral of the measured luminescence signal, "r" and "x" respectively meaning "of the reference" and "of the sample". Here, the references used are coumarin 153 in methanol (Qr = 0.45) at 417 nm for DBB-In and DBB-PHEA and fluorescein in 1M aqueous sodium hydroxide solution (Qr = 0.92) at 470 nm for Ant- In and Ant-PHEA.

The excitation of the reference and of the sample is carried out at the same wavelength.

In the case of DBB-PHEA, the conservation of fluorescence is 92% and 62% in the case of Ant-PHEA.

Flow cytometry incorporation experiments indicate that DBB-PHEA is better incorporated in the tested cell lines (baf-3 and B16-F10) than their non-oligomerized analogs solubilized in DMSO and at least equivalent to DBB in a micellar system of Pluronic type (c). The possibility of using these compounds for one- and two-photon imaging applications, and for photodynamic therapy applications, has also been established by means of microscopy and cell death measurements after irradiation, for Ant-PHEA and DBB-PHEA respectively.

Claims

1 - Water-soluble chromophoric entity comprising at least one lipophilic chromophore covalently bound to at least two water-soluble polymer chains, characterized in that:

each chain of water-soluble polymer is covalently bound to only one lipophilic chromophore,

and each polymer chain bears several hydroxyl functions and is obtained by polymerization of one or more monomers, at least one of the monomers used being chosen from;

in the case of the last monomer, a basic hydrolysis being carried out before polymerization to obtain the desired hydroxyl functions.

2 - water-soluble chromophoric entity according to claim 1 characterized in that each chain of water-soluble polymer is obtained by controlled radical polymerization. 3 - water-soluble chromophoric entity according to one of the preceding claims characterized in that each polymer chain is obtained by ATRP, RAF or NMP polymerization.

4 - water-soluble chromophoric entity according to one of the preceding claims characterized in that each water-soluble polymer chain is obtained by controlled radical polymerization by reaction of at least one monomer with the lipophilic chromophore functionalized with a polymerization initiator.

5 - water-soluble chromophoric entity according to one of the preceding claims characterized in that the polymolecularity index of the polymer chains is less than 1.5, preferably less than 1.3, ideally less than 1.1.

6 - Water-soluble chromophoric entity according to one of the preceding claims, characterized in that each polymer chain is linked to the lipophilic chromophore covalently via a bond - OC (O) - or -0- or -NH- CO)-.

7 - water-soluble chromophoric entity according to one of the preceding claims characterized in that each water-soluble polymer chain is uncharged.

8 - water-soluble chromophoric entity according to one of the preceding claims characterized in that it comprises, at the end Ω of at least one polymeric chain, or all of the polymer chains present, a bromine, a thioester (-C ( S) -S-) or a nitroxide (-O-N-).

9 - water-soluble chromophoric entity according to one of claims 1 to 7 characterized in that it comprises, at the end Q to 'at least one polymeric chain, or all of the polymeric chains present, a functionalization allowing the subsequent grafting of biological ligands or other molecules of interest, in particular by "click chemistry".

10 - chromophoric entity according to claim 9 characterized in that the functionalization corresponds to a function N ₃ or -C≡X with X = N or CH. 11 - water-soluble chromophoric entity according to one of claims 1 to 7 characterized in that a biological ligand is covalently bound to the end Ω of at least one polymeric chain, or all of the polymer chains present.

12 - water-soluble chromophoric entity according to one of the preceding claims characterized in that the polymer chains are polyacrylamides, polymethaacrylamides, polymetacrylates, polyacrylates or polyallyl esters, in homopolymer or copolymer form.

13 - water-soluble chromophoric entity according to one of the preceding claims characterized in that each polymer chain comprises from 4 to 30 monomeric units.

14 - water-soluble chromophoric entity according to one of the preceding claims characterized in that the lipophilic chromophores are chromophores with nonlinear optical properties.

15 - water-soluble chromophoric entity according to one of the preceding claims characterized in that the lipophilic chromophores are fluorophores.

16 - water-soluble chromophoric entity according to one of the preceding claims characterized in that the lipophilic chromophores are chosen from dipoles, quadrupoles or octupoles.

17 - water-soluble chromophoric entity according to one of the preceding claims characterized in that the lipophilic chromophores are chosen from dipoles, quadrupoles or octupoles schematically represented by:

dipole

18 - Water-soluble chromophoric entity according to claim 17, characterized in that the uninterrupted conjugated chain is a hydrocarbon chain which may be substituted or unsubstituted, in which saturated bonds and unsaturated bonds chosen from double and triple bonds alternate, the bonds unsaturated, and in particular double bonds, which can be incorporated into a cycle, preferably a cycle

19 - water-soluble chromophoric entity according to one of the preceding claims characterized in that the lipophilic chromophores belong to the family of cyanines or merocyanines.

20 - Process for preparing a chromophoric entity according to one of the preceding claims characterized in that each water-soluble polymer chain is obtained by controlled radical polymerization.

21 - Process according to claim 20 characterized in that each polymer chain is obtained by ATRP, RAFT or NMP polymerization.

22 - Process according to claim 20 or 21 characterized in that each polymer chain is obtained by controlled radical polymerization by reaction of the functionalized lipophilic chromophore with a polymerization initiator and at least one monomer.