WO2020065200A1

WO2020065200A1 - Cycloheptyne-cobalt carbonyl complexes and their applications in bioconjugation

Info

Publication number: WO2020065200A1
Application number: PCT/FR2019/052235
Authority: WO
Inventors: Philippe Hermange; Morgan CORMIER; Eric FOUQUET
Original assignee: Universite de Bordeaux; Institut Polytechnique De Bordeaux; Centre National De La Recherche Scientifique
Priority date: 2018-09-25
Filing date: 2019-09-24
Publication date: 2020-04-02
Also published as: FR3086290B1; FR3086290A1

Abstract

The present invention relates to a cycloheptyne-cobalt carbonyl complex of formula (I). The invention also relates to a method for preparing such a compound of formula (I) and to the use thereof for labeling, imaging and/or orthogonal modification of a target molecule.

Description

CYCLOHEPTYNE-COBALT CARBONYLES COMPLEXES AND THEIR APPLICATIONS IN BIOCONJUGATION

OBJECT OF THE INVENTION

The present invention relates to the field of “click” chemistry and, in particular, to new cyclic alkynes protected in the form of cobalt complexes for applications in bioconjugation.

BACKGROUND OF THE INVENTION

In the early 2000s, conjugation reactions compatible with the study of living organisms were developed. These so-called bio-orthogonal reactions make it possible to study, and sometimes even to visualize, biological phenomena in real time, within or on the surface of a living system, such as a cell or even an organism.

Among the various conjugation reactions existing at present, click chemistry reactions, in particular the cycloaddition between an alkyne and an azide, are among the most exploited, in particular for imaging and labeling of biomolecules. The coupling between a molecule comprising an azide, for example a protein modified with an azide obtained by chemical conversion or by expression in auxotrophic bacteria, with an alkyne, linked in turn to a diagnostic agent, makes it possible to quickly and selectively create a link between the two entities, in the form of a triazole motif. The advantages of this reaction have also made it possible to extend the applications to the conjugation of biomolecules with a therapeutic agent. In particular, the development of antibody-drug conjugates or ADC (for "Antibody-Drug Conjugate") has recently been the focus of attention of researchers. Indeed, this approach, which aims to target the action of an active principle through a cell recognition process mediated by antibodies, makes it possible to limit the doses injected for the same effectiveness, to reduce side effects and / or to consider use of molecules known to be effective but not very selective. More particularly in the case of cancer treatment, the advantage of ADC is to eliminate cancer cells without affecting healthy cells. The ADC market is therefore a promising emerging market. Advances in this area are therefore closely linked to the development of effective compounds ensuring the conjugation of the biomolecule with the therapeutic or diagnostic agent. It has been particularly found that 1 ^'' use cyclic alkynes forced (or tensioned) was used to achieve the cycloaddition reaction with an azide, but also with a nitrone, or a nitrile oxide, in the absence of catalyst.

The bicyclo [6.l.0] nonyne (BCN), described in application WO 2011/136645, is one of the constrained cyclic alkynes most used for bioconjugation. However, its reactivity is not optimal and its synthesis is relatively complex, since it requires in particular a separation of the diastereoisomers.

BCN

3,3,6,6-tetramethyl-thiacycloheptyne (TMTH) and its derivatives are cycloheptynes which have been described by King et al. (Chem. Commun. 2012, 48, 9308) for applications in bioconjugation. However, despite their good reactivity, the synthesis of such compounds is long and complex, which limits their use and their dissemination.

TMTH

Compounds having a cycloheptyne-hexacarbonyl dicobalt motif, in particular compound (A), represented by the formula below, have been described by Green (Synlett. 2001, 2, 243 and Eur. J. Org. Chem. 2008, 36, 6053). These compounds were prepared by a double Nicholas reaction between a bissilylated nucleophile and a butyn-1,4-diether derivative protected by a hexacarbonyl dicobalt group. The absence of substituents on the formed cycle and of a second functionality does not, however, allow this type of compound to be used in bioconjugation.

Brâse (RSC Adv. 2014, 4, 15493) and Tomooka (Angew. Chem. Int. Ed. 2015, 54, 1190) described the preparation of the 8 and 9-membered cyclic compounds, as represented by the formula (B) :

in which X and Y each independently represent O, NH, NTs (Ts = Tosyle), NNs (Ns = Nosyle), NBz (Bz = Benzoyl) or S. The synthesis strategy for these compounds is similar to that described for the compound (AT). However, the reactivity of these compounds is relatively low.

There therefore remains a real need to develop new cyclic alkynes, having improved reactivity and which can be obtained by a simple and inexpensive process, for the efficient and selective conjugation of a target molecule with an agent, in particular a therapeutic agent or diagnostic.

SUMMARY OF THE INVENTION In this context, the inventors have developed new cyclic alkynes of the cycloheptyne type, substituted by alkyl groups, and whose alkyne function is protected by a dicobalt-carbonyl group. The use of these cycloheptynes complexed with dicobalt makes it possible both to obtain reactive cycles for “click” reactions of the cycloaddition type, while being precursors stable over the long term in their protected form. The cycloheptyne motif of these compounds is also substituted by a chain carrying a second functionality, thus offering the possibility of coupling two different molecules having a suitable reactive group such as an azide, a thiol or another equivalent group, so orthogonal. These compounds can be obtained by a simple, rapid, inexpensive synthesis process and offering very good yields.

The subject of the invention is a compound of formula (I):

in which

Ri, R _2, R ₃ and R ₄ each independently represent an alkyl -CY ,, cycloalkyl, C ₃ -C ₁₂ heterocycloalkyl or C ₃ -C _12, and / or Ri and R ₂ together with the carbon to which they are attached, a ring selected from cycloalkyl and C ₃ -C ₁₂ heterocycloalkyl, C ₃ -C ₁₂ and / or R ₃ and R ₄ together with the carbon to which they are attached a ring selected from C ₃ -C ₁₂ cycloalkyl and a C ₃ -C12 heterocycloalkyl;

Q represents a chain comprising at its end a functionality allowing conjugation to a molecule; and

L and L ’each independently represent a donor ligand of two electrons chosen from:

^■ a CO ligand,

^■ a nitrogenous ligand, such as dinitrogen, pyridine, amine N (Ra) (Rb) (Rc), or a nitrile R-CN,

^■ a phosphorus ligand such as a phosphine P (R _e) (R _f) (R _g) or a phosphite P (OR _h) (OR) (OR _j),

Ra, Rb, Rc, Rd, Re, Rf, Rg, Rh, Ri, and Rj, being independently selected from hydrogen, C ₁ -C ₁₂ alkyl, C ₃ -C ₁₂ cycloalkyl, aryl and heteroaryl, and,

^■ a carbene ligand C (Rk) (Ri), R _k and Ri being independently chosen from C1-C4 alkyl, C ₃ -C ₁₂ cycloalkyl, aryl and heteroaryl, or forming together with the carbon to which they are attached a C ₃ -C12 cycloalkyl or a C ₃ -C12 heterocycloalkyl. According to a particular embodiment, Ri, R ₂ , R ₃ and R ₄ represent a methyl. According to another particular embodiment, L and L ′ represent a CO ligand.

According to another particular mode, Q is a chain -X-Y, in which:

X represents a divalent unit chosen from - (C ₁ -C ₁₂ alkyl) -, - (C ₃ - C ₁₂ cycloalkyl) -, - (aryl) -, - (heteroaryl) -, - (aryl) - (alkyl in CY-CY) -, - (alkyl-CY) - (arylC) -, - (heteroaryl) - (alkyl CY-CY,) -, - (alkyl-C ₆₎ - (heteroaryl) - , - (CY-CY alkyl,) -

(aryl) - (alkyl CY-CY,) -, - (alkyl-C ₆₎ - (heteroaryl) - (alkyl CY-CY,) -, and - (alkyl-C ₆₎ - ( cycloalkyl C ₃ -C ₂₎ - (alkyl-C ₆₎ -; and

Y is a functionality selected from an alcohol, an amine, a carboxylic acid, an ester, an acid chloride, an anhydride, a true alkyne, and an alkene.

According to a preferred embodiment, X is a C ₁ -C ₁₂ alkyl, in particular a C ₃ alkyl, and Y is a true alkyne.

Preferably, a compound of formula (I) has the following structure:

Another subject of the invention relates to a process for the preparation of a compound of formula (I), comprising the following successive steps:

(a) a step of reacting the compound of formula (II):

in which Ri, R _2, R 3 and R ₄ each independently represent an alkyl -CY ,, cycloalkyl, C ₃ -C ₁₂ heterocycloalkyl or C ₃ -C _12, and / or Ri and R ₂ together with the carbon to which they are attached, a ring selected from cycloalkyl and C ₃ -C ₁₂ heterocycloalkyl, C ₃ -C ₁₂ and / or R ₃ and R ₄ together with the carbon to which they are attached, a ring selected from cycloalkyl C ₃ -C ₁₂ and a C ₃ -C12 heterocycloalkyl, and

R ₅ represents a group chosen from:

^■ R _Ô C (O) - with R ₆ being a CY-CY alkyl, a C ₃ -C ₁₂ cycloalkyl, a CY-CY alkoxy, or an aryl, and

^■ R ₇ S (0) 2- with R ₇ being a C ₁ -C ₆ alkyl, a C ₃ -C ₁₂ cycloalkyl or an aryl,

with a dicobalt complex Co2 (CO) 6 (L) (L ’), in which L and L’ each independently represent a donor ligand of two electrons chosen from:

^■ a CO ligand,

Ra, Rb, Rc, Rd, Re, Rf, Rg, Rh, Ri, and Rj, being independently selected from hydrogen, C ₁ -C ₁₂ alkyl, C ₃ -C ₁₂ cycloalkyl, aryl and heteroaryl , and,

^■ a carbene ligand C (Rk) (Ri),

R _k and R being independently selected from alkyl CY-CY ,, cycloalkyl, C ₃ -C _12, aryl and heteroaryl, or together with the carbon to which they are attached a cycloalkyl C ₃ -C 12 or a C ₃ -C12 heterocycloalkyl,

preferably L and L ’represent a CO ligand;

(b) a step of reacting the compound obtained in step (a) with a compound of formula (III):

(III),

wherein Rs, R ₉ , and Rio each independently represent a C 1 -CY alkyl, or an aryl, preferably methyl;

(c) a step of reacting the compound obtained in step (b) with a borane, preferably BH ₃ ; (d) a step of reacting the compound obtained in step (c) with an oxidant, preferably sodium perborate; and

(e) a step of reacting the compound obtained in step (d) with a compound of formula (IV):

in which Q represents a chain comprising at its end a functionality allowing conjugation to a molecule, preferably a -X-Y chain, where:

X represents a divalent unit chosen from - (C1-C12 alkyl) -, - (C3-C12 cycloalkyl) -, - (aryl) -, - (heteroaryl) -, - (aryl) - (C1-Calkyl ₆₎ -, - (alkyl-C ₆₎ - (aryl) -, - (heteroaryl) - (alkyl-C ₆₎ -, - (alkyl-C ₆₎ - (heteroaryl) -, - (alkyl-CV,) - (aryl) - (alkyl-C ₆₎ -, - (alkyl-C ₆₎ - (heteroaryl) - (alkyl-C ₆₎ -, and - ( alkyl-C ₆₎ - (C3-Ci2) - (alkyl-C ₆₎ -; and

Y is a functionality chosen from an alcohol, amine, carboxylic acid, ester, acid chloride, anhydride, true alkyne, and alkene;

or an ester, carboxylic acid, or anhydride derivative thereof.

According to a particular mode, Ri, R ₂ , R ₃ and R ₄ of the compound of formula (II) represent a methyl. According to another particular mode, R5 of the compound of formula (II) represents a group R _Ô C (O) - in which Rr, is methyl.

According to a preferred embodiment, Q of the compound of formula (IV) is a chain -XY where X is a C ₁ -C ₁₂ alkyl, preferably a C ₃ alkyl, and Y is a true alkyne.

A further object of the invention relates to the use of a compound of formula (I), for labeling, imaging and / or orthogonal modification of a target molecule.

FIGURES

Figure 1: Diagram illustrating the process for the synthesis of a compound of formula (I).

Figure 2: Diagram illustrating the preparation of a bioconjugate molecule from a compound of formula (I) according to the invention. DETAILED DESCRIPTION

The expression "between" is used to describe a range of values in which the mentioned lower and upper limit values are included.

By "alkyl" is meant a saturated, linear or branched aliphatic group. The term "alkyl-C _6" may particularly denote a methyl, ethyl, propyl, isopropyl, butyl, /.v -butylc, tert-butyl, pentyl, or hexyl.

By “cycloalkyl” is meant a mono-, bi- or tri-cyclic alkyl group, bridged or not. The expression "C3-C12 cycloalkyl" can more particularly designate a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, or cyclododecyl.

By “heterocycloalkyl” is meant a hydrocarbon group, mono-, bi- or tri-cyclic, bridged or not, saturated or unsaturated, and comprising at least one heteroatom, such as N (azacycloalkyle), O (oxacycloalkyle) or S ( thiacycloalkyle). The expression “C3-C12 heterocycloalkyl” can more particularly designate an oxirane, oxetane, 3-dioxolane, benzo- [1,3] -dioxolyl, pyrazolinyl, pyranyl, thiomorpholinyl, pyrazolidinyl, piperidyl, piperazinyl, 1,4-dioxanyl , imidazolinyl, pyrrolinyl, pyrrolidinyl, piperidinyl, imidazolidinyl, morpholinyl, 1,4-dithianyl, pyrrolidinyl, quinolizinyl, oxozolinyl, oxazolidinyl, isoxazolinyl, isoxazolidinyl, thiazolinyl, thiazolidinyl, isiaziazinyl, isothiazolinyl. Preferably, a C3-C12 heterocycloalkyl is an oxetane.

By "alkyloxy" or "alkoxy" is meant an -O-alkyl group where the alkyl group is as defined above. The term "alkoxy-C _6" may particularly denote a methoxy, ethoxy, propyl, isopropoxy, butoxy, /.v -butoxy, tert-butoxy, pentoxy, or hexyloxy.

By "aryl" is meant a monocyclic or polycyclic aromatic hydrocarbon group. In particular, an aryl denotes a phenyl, a biphenyl, or a naphthyl, and preferably a phenyl. By “heteroaryl” is meant a monocyclic or polycyclic aromatic group, comprising at least one heteroatom such as nitrogen, oxygen or sulfur. Particularly, a heteroaryl designates a pyridinyl, thiazolyl, thiophenyl, furanyl, pyrrolyl, imidazolyl, triazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzimidazolyl, triazinyl, triazinyl, pyridazinyl, indolizinyl, isoindolyl, indazolyl, purinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, b-carbolinyl, phenanthridinyl, acridinyl, pyrididinyl, pheninrolinazinyl, pheninolazinyl, pheninolazinyl, pheninolazinyl, pheninolazinyl, phenidrolyl phenyl, phenyl) oxindolyl, benzothienyl, benzothiazolyl, s-triazinyl, oxazolyl, or thiofuranyl.

The groups as defined above also include the alkyl, cycloalkyl, alkoxy, heterocycloalkyl, aryl and heteroaryl groups mono or poly substituted by groups including in particular alkyls, cycloalkyls, aryls, perfluoroalkyls, alkoxy, alkylthio, alkylamino, halogens, cyano, nitro, etc.

By "alkylthio" is meant a group -S- (alkyl), where the alkyl group is as defined above. An example of alkylthio is notably methylthio.

By "alkylamino" is meant a group -NH- (alkyl) or -N (alkyl) ₂ where the alkyl group is as defined above. Examples of alkylamino are in particular methylamino, ethylamino, or dimethylamino.

By "perfluoroalkyl" is meant an alkyl group in which the hydrogens have been replaced by a fluorine. An example of perfluoroalkyl is in particular CU.

By "halogen" is meant a fluorine, chlorine, bromine or iodine atom.

Compound

The subject of the invention is a compound of formula (I):

(I),

in which

Q represents a chain comprising at its end a functionality allowing conjugation to a molecule; and,

^■ a CO ligand,

^■ a carbene ligand C (Rk) (Ri), R _k and Ri being independently selected from C1-C6 alkyl, C ₃ -C ₁₂ cycloalkyl, aryl and heteroaryl, or forming together with the carbon to which they are attached a C3-C12 cycloalkyl or heterocycloalkyl in C3-C12.

According to a particular mode:

Ri, R ₂ , R ₃ and R ₄ each independently represent:

^■ a CY-CY alkyl, or

^■ heterocycloalkyl, C ₃ -C _12, preferably C ₃ oxacycloalkyl - C12; and or

Ri and R ₂ together with the carbon to which they are attached form a C ₃ -C ₁₂ heterocycloalkyl, preferably a C ₃ -C ₁₂ oxacycloalkyl and / or, R ₃ and R ₄ together form with the carbon to which they are attached a C ₃ -C ₁₂ heterocycloalkyl, preferably a C3-C12 oxacycloalkyl.

According to a particular embodiment, the groups Ri, R ₂ , R ₃ and R ₄ are identical.

According to a particular mode Ri and R ₂ , and R ₃ and R ₄ respectively form a cycle "(Ri, R ₂ )" and a cycle "(R ₃ , R ₄ )" as defined above. In this context the cycle “(Ri, R ₂ )” designates the cycle formed by Ri and R ₂ taken together with the carbon to which they are attached, and the cycle “(R ₃ , R ₄ )” designates the cycle formed by R ₃ and R ₄ taken together with the carbon to which they are attached. Preferably, the cycles “(Ri, R ₂ )” and “(R ₃ , R ₄ )” are identical.

In these particular embodiments, the compound of formula (I) according to the invention is symmetrical, which makes it possible to avoid the formation of multiple isomers thus simplifying its purification.

According to a preferred embodiment, Ri, R ₂ , R ₃ and R ₄ are identical and represent a methyl or an oxetane, or the rings (Ri, R ₂ ) and (R ₃ , R ₄ ) are identical and form an oxetane . More preferably, R 1, R ₂ , R ₃ and R ₄ represent a methyl.

By “donor ligand of two electrons” is meant any electronically neutral molecule capable of coordinating with a metal, in particular cobalt, via two electrons, in the form of an electronic pair paired or not, of one of the atoms constituting said molecule.

By “nitrogenous ligand” is meant a donor ligand of two electrons comprising a nitrogen atom and capable of coordinating with a metal, in particular cobalt, via the non-binding doublet of the nitrogen atom. Examples of nitrogen ligand are in particular dinitrogen, a pyridine, an amine, or a nitrile. Preferably, the amine is of formula N (R _a ) (R _b ) (R _c ) in which R _a , R _b and R _c are independently chosen from hydrogen, C ₁ -C ₆ alkyl, C ₃ -C ₁₂ cycloalkyl, aryl and heteroaryl. Preferably, the nitrile has the formula Rj-CN in which R _d is chosen from hydrogen, C ₁ -C ₁₂ alkyl, C ₃ -C ₁₂ cycloalkyl, aryl and heteroaryl.

By “phosphorus ligand” is meant a donor ligand of two electrons comprising a phosphorus atom and capable of coordinating with a metal, in particular cobalt, via the non-binding doublet of the phosphorus atom. Examples of a phosphorus ligand are in particular a phosphine or a phosphite. Preferably, the phosphine has the formula P (R _e ) (R _f ) (R _g ), in which R _e , R _f , and R _g , are independently chosen from hydrogen, C 1 -C 6 alkyl, a C ₃ -C ₁₂ cycloalkyl, aryl and heteroaryl. Preferably, the phosphite is of the formula P (OR _h) (OR) (OR _j), wherein R _h, R i and R _j are independently selected from hydrogen, alkyl Ci-C ₆ cycloalkyl C ₃ -C ₁₂ , aryl and heteroaryl.

By "carbene ligand" is meant a ligand donor of two electrons comprising a carbene function and capable of coordinating with a metal, in particular cobalt, via the two electrons of the carbene function. Preferably, the carbene ligand is of formula C (R _k ) (Ri), in which R _k and Ri:

- are independently selected from C ₁ -C ₆ alkyl, C ₃ -C ₁₂ cycloalkyl, aryl and heteroaryl, or

- Together with the carbon to which they are attached, form a C ₃ -C ₁₂ cycloalkyl or a C ₃ -C ₁₂ heterocycloalkyl, such as an imidazolidinyl or an imidazolinyl.

According to a preferred embodiment, L and L ’represent a CO ligand.

According to the invention, Q represents a chain comprising at its end a functionality allowing conjugation to a molecule, such as a biomolecule, a therapeutic or diagnostic agent. By “functionality” is meant a chemical group reactive with respect to another chemical group carried by said molecule, and thus making it possible to create a bond between the compound of formula (I) and said molecule. Examples of functionality include:

- an alcohol (-OH);

- an amine (-NH ₂ );

- a carboxylic acid (-COOH); - an ester, in particular -COOR ', in which R' represents a C ₁ -C ₁₂ alkyl, C ₃ -C ₁₂ cycloalkyl, aryl, or heteroaryl;

- an acid chloride (-COC1);

- an anhydride, in particular - (CO) -0- (CO) -R ”, in which R” represents a C1-C12 alkyl, C3-C12 cycloalkyl, aryl, or heteroaryl;

- a true alkyne (-CºCH), or

- an alkene (-CLUCH ₂ ).

Preferably, the functionality at the end of the Q chain is a true alkyne.

According to a particular embodiment, Q is a chain -X-Y, in which:

X represents a divalent unit chosen from - (C ₁ -C ₁₂ alkyl) -, - (C ₃ - C ₁₂ cycloalkyl) -, - (aryl) -, - (heteroaryl) -, - (aryl) - (alkyl Ci-CY,) -, - (C1-CY alkyl) - (arylc) -, - (heteroaryl) - (CY-CY alkyl) -, - (C1-C6 alkyl) - (heteroaryl) -, - (alkyl in CY-CY,) - (aryl) - (alkyl in CY-CY,) -, - (alkyl in C1-C6) - (heteroaryl) - (alkyl in CY-CY,) -, and - ( alkyl-C6) - cycloalkyl (C ₃ -C ₂₎ - (alkyl-C ₆₎ -; and

The divalent unit X as defined above must be read from left to right, and relates the carbon of the carbonyl (ester function) and the functionality Y in the compound of formula (I). The order of the groups constituting X is given so that the left-most group is linked to the carbon of the carbonyl and the right-most group is linked to Y. When X consists only of a group, this ci is connected to the carbon of carbonyl and to Y.

For example, when X is - (aryl) - (alkyl in CY-CY,) -, the aryl is connected to the carbon of carbonyl and the alkyl in C ₁ -CY, is connected to Y, the aryl and l 'C ₁ -CY alkyl, being interconnected. When X is a CY-CY alkyl, the C ^' ₁ -CY alkyl is linked to the carbon of the carbonyl and to Y.

According to a particular embodiment, X is a C ₁ -C ₁₂ alkyl, in particular a C ₃ alkyl. According to another particular embodiment, Y is a true alkyne.

A compound of formula (I) preferred according to the invention has the formula:

Synthesis process The invention also relates to a process for the preparation of a compound of formula (I). The process according to the invention is a simple, rapid synthesis process, and allows access to the compound of formula (I) with very good yields, and from easily accessible commercial products. The method according to the invention comprises the following successive steps:

(a) a step of reacting the compound of formula (II):

(P),

in which

Ri, R _2, R ₃ and R ₄ each independently represent an alkyl -CY ,, cycloalkyl, C ₃ -C ₁₂ heterocycloalkyl or C ₃ -C _12, and / or Ri and R ₂ together with the carbon to which they are attached, a ring selected from cycloalkyl and C ₃ -C ₁₂ heterocycloalkyl, C ₃ -C ₁₂ and / or R ₃ and R ₄ together with the carbon to which they are attached a ring selected from C ₃ -C ₁₂ cycloalkyl and a C ₃ -C12 heterocycloalkyl, and

R ₅ represents a group chosen from:

^■ R _<5 C (0) - with Re is alkyl Ci-C _6, cycloalkyl C ₃ -C ₁₂ alkoxy CY-CY, or aryl, and

^■ R ₇ S (0) 2- with R ₇ being a C ₁ -C ₆ alkyl, a C ₃ -C ₁₂ cycloalkyl or an aryl, with a dicobalt Co ₂ (CO) ₆ (L) (L ') complex, in which L and L' each independently represent a donor ligand of two electrons chosen from:

^■ a CO ligand,

Ra, Rb, Rc, Rd, Re, Rf, Rg, Rh, Ri, and Rj, are independently selected from hydrogen, alkyl Ci-C _6, cycloalkyl C ₃ -C _12, aryl and heteroaryl , and,

^■ a carbene ligand C (Rk) (Ri),

R _k and Ri being independently chosen from C1-C4 alkyl, C ₃ -C ₁₂ cycloalkyl, aryl and heteroaryl, or forming together with the carbon to which they are attached a C ₃ -C12 cycloalkyl or a C ₃ -C ₁₂ heterocycloalkyl;

wherein Rs, R ₉ , and Rio each independently represent C ₁ -C alkyl or aryl;

(c) a step of reacting the compound obtained in step (b) with a borane;

(d) a step of reacting the compound obtained in step (c) with an oxidant; and

in which Q represents a chain comprising at its end a functionality allowing conjugation to a molecule,

or an ester, carboxylic acid, or anhydride derivative thereof.

The conditions (comprising in particular the parameters such as the temperature, the concentration, the number of equivalents of reagents and the solvent) for each stage of the process according to the invention are described below for particular and / or preferred embodiments, and can be modified or adjusted by those skilled in the art from his knowledge, without departing from the invention. Each intermediate obtained at the end of each step or sub-step of the process according to the invention can be isolated, and optionally purified, for example by chromatography or recrystallization, and reacted in the next step. Several steps or sub-steps of the method according to the invention can be carried out successively without isolating an intermediary.

By solvent is meant both an organic solvent and an inorganic solvent, or a mixture of these. Examples of organic solvents are in particular, aliphatic, cyclic or acyclic hydrocarbons, such as cyclohexane or pentane, aromatic hydrocarbons such as benzene, toluene or xylene, halogenated hydrocarbons such as dichloromethane, chloroform or chlorobenzene, nitrogenous solvents such as acetonitrile or triethylamine, oxygenated solvents, in particular ketones such as acetone, ethers such as diethyl ether, tert-butyl methyl ether (TBME), tetrahydrofuran (THF), alcohols such as methanol or ethanol, esters such as ethyl acetate, or amides such as dimethylformamide (DMF), and a mixture thereof. An example of an inorganic solvent is, in particular, water.

Stage (a) of the process according to the invention aims to protect the alkyne function of the compound represented by formula (II):

(P),

in which

Ri, R _2, R 3 and R ₄ each independently represent an alkyl -CY ,, cycloalkyl, C ₃ -C ₁₂ heterocycloalkyl or C ₃ -C _12, and / or Ri and R ₂ together with the carbon to which they are attached, a ring selected from cycloalkyl and C ₃ -C ₁₂ heterocycloalkyl, C ₃ -C ₁₂ and / or R ₃ and R ₄ together with the carbon to which they are attached, a ring selected from cycloalkyl in C ₃ -C ₁₂ and a heterocycloalkyl in C3-C12, and

R ₅ represents a group chosen from:

^■ R ₇ S (0) 2- with R ₇ being an alkyl-C ₆ cycloalkyl or C 3 -C 12 aryl, with a dicobalt complex Co2 (CO) 6 (L) (L '), in which L and L' each independently represent a donor electron ligand chosen from:

^■ a CO ligand,

^■ a carbene ligand C (Rk) (Ri),

R _k and Ri being independently chosen from C 1 -CV _> alkyl, C ₃ -C ₁₂ cycloalkyl, aryl and heteroaryl, or forming together with the carbon to which they are attached a C3-C12 cycloalkyl or a C3-C12 heterocycloalkyl.

According to a particular embodiment, step (a) of the process according to the invention comprises the reaction of the compound of formula (II) with a dicobalt complex Co2 (CO) 6 (L) (L ') where L and L' are as defined above. Step (a) can be carried out at a temperature between 0 ° C and 50 ° C, preferably between 10 ° C and 35 ° C. Step (a) can be carried out in an organic solvent such as dichloromethane. Preferably, step (a) is carried out in the absence of water and oxygen. The amount of dicobalt complex Co2 (CO) ₆ (L) (L ') used in step (a) is preferably between 1 and 3 equivalents, advantageously, between 1 and 1.5 equivalents, relative to the compound of formula (II). The compound obtained in step (a) is preferably isolated and purified, in particular by chromatography, before being used in step (b) of the process according to the invention.

According to a particular embodiment, the groups Ri, R ₂ , R ₃ and R ₄ of the compound of formula (II) are identical or the rings (Ri, R ₂ ) and (R ₃ , R ₄ ) of the compound of formula ( II) are identical. According to a preferred embodiment, Ri, R ₂ , R ₃ and R ₄ of the compound of formula (II) are identical and represent a methyl or an oxetane, or the rings (Ri, R ₂ ) and (R ₃ , R ₄ ) of the compound of formula (II) are identical and form an oxetane. More preferably, R 1, R ₂ , R ₃ and R ₄ represent a methyl.

According to a preferred embodiment, L and L 'of the Co2 (CO) ₆ (L) (L') complex represent a CO ligand. According to a particular embodiment, the compound of formula (II) used in step (a) is such that R ₅ represents R ₆ C (O) - in which R ₆ is a C1-C6 alkyl, preferably methyl.

The compound of formula (II) can be obtained via an initial step (i) allowing the activation of the alcohol functions of the compound of formula (Ili):

in which Ri, R ₂ , R ₃ and R ₄ each independently represent a CY-CY alkyl, a C ₃ -C ₁₂ cycloalkyl, or a C ₃ -C ₁₂ heterocycloalkyl, and / or Ri and R ₂ together form with the carbon to which they are attached, a ring selected from cycloalkyl and C ₃ -C ₁₂ heterocycloalkyl, C ₃ -C ₁₂ and / or R ₃ and R ₄ together with the carbon to which they are attached, a ring selected from cycloalkyl and C ₃ -C ₁₂ heterocycloalkyl, C ₃ -C _12. The activation of these alcohol functions can be obtained by any conventional reaction known to those skilled in the art, for example an acetylation or esterification reaction, or a sulfonylation reaction.

Step (i) can comprise the reaction of the compound of formula (Ili):

wherein Ri, R _2, R ₃ and R ₄ each independently represents an alkyl CY-CY ,, cycloalkyl, C ₃ -C ₁₂ heterocycloalkyl or C ₃ -C _12, and / or Ri and R ₂ form together with the carbon to which they are attached, a ring selected from cycloalkyl and C ₃ -C ₁₂ heterocycloalkyl, C ₃ -C ₁₂ and / or R ₃ and R ₄ together with the carbon to which they are attached a ring selected from cycloalkyl, C ₃ -C ₁₂ heterocycloalkyl and C ₃ -C _12, with a compound:

- R ₅ -Z, where R ₅ represents a group chosen from:

^■ R _e O) - with Re is alkyl Ci-C _6, cycloalkyl C ₃ -C ₁₂ alkoxy, Ci-C _6, or an aryl, and

^■ R ₇ S (0) 2- with R ₇ being an alkyl-C ₆ cycloalkyl or C 3 -C 12 aryl,

and Z is a group selected from chlorine, bromine and OR ₁₁ wherein R ₁₁ is hydrogen, alkyl Ci-C _6, or aryl; or - R5-O-R5 where R5 represents a group chosen from:

^■ R < ₅ C (0) - with Re being a C ₁ -C ₆ alkyl, a C ₃ -C ₁₂ cycloalkyl, a C ₁ -C ₆ alkoxy, or an aryl, and

^■ R ₇ S (0) ₂ - with R ₇ being an alkyl-C ₆ cycloalkyl or C 3 -C 12 aryl.

According to a preferred embodiment, step (i) comprises the reaction of the compound of formula (Ili) as defined above with a compound of type R5-O-R5, where R5 represents RoCfO) - with R ₆ being methyl .

Step (i) can be carried out at a temperature between 0 ° C and 50 ° C, preferably between 10 ° C and 35 ° C. Step (i) is preferably carried out in an organic solvent such as dichloromethane. The amount of the compound of type R ₅ -Z or R ₅ -OR ₅ used in step (i) is advantageously between 2 and 10 equivalents, preferably between 3 and 7 equivalents, relative to the compound of formula (Ili) . Step (i) is advantageously carried out in the presence of one or more Bronsted bases. Examples of Bronsted bases are in particular hydroxides such as sodium or potassium hydroxide, carbonates or hydrogen carbonates such as sodium carbonate, alcoholates such as sodium methanolate, nitrogen bases in particular, dimethylaminopyridine (DMAP) , pyridine or amines such as triethylamine. Preferably, the base of Bronsted is chosen from triethylamine, DMAP, and a mixture of these. The compound obtained in step (i) is preferably isolated and purified, in particular by chromatography.

Step (b) of the process according to the invention allows the formation of a motif of the 5-methylene-cyclohept-1-yne type by a double Nicholas reaction, starting from the compound obtained in step (a) and a bissilylated precursor.

According to a particular mode, step (b) comprises the reaction of the compound obtained in step (a) with a compound of formula (III):

wherein Rs, R _9, and Rio are each independently an alkyl-C ₆ or aryl. According to a particular embodiment, Rs, R _9, and Rio are each independently an alkyl C -C _6, in particular methyl or tert-butyl, preferably methyl.

Step (b) can be carried out at a temperature between -80 ° C and 0 ° C, preferably between -55 and -25 ° C. Step (b) is preferably carried out in an organic solvent such as dichloromethane. Step (b) is preferably carried out in the presence of a Lewis acid such as BF3, AICI3 as well as the solvates thereof. According to a preferred embodiment, the Lewis acid is BF3 diethyl etherate (denoted BF3.0Et ₂ ). The amount of the compound of formula (III) used in step (b) is advantageously between 1 and 3 equivalents, preferably between 1 and 1.5 equivalents, relative to the compound obtained in step (a) and used in step (b). The amount of Lewis acid used in step (b) is advantageously between 1.5 and 5 equivalents, preferably between 1.8 and 3 equivalents, relative to the compound obtained in step (a) and used in step (b). The compound obtained in step (b) is preferably isolated and purified, in particular by chromatography, before being used in step (c) of the process according to the invention.

The compound of formula (III) can be prepared by reacting 3-chloro-2-chloromethylprop-1-ene with an electrophilic source of the group (R ₈ ) (R ₉ ) (Rio) Si, in which Rs, R ₉ , and Rio each independently represent a C1-C6 alkyl, preferably a methyl, or an aryl, in the presence of metallic lithium and naphthalene. In particular, said electrophilic source is (R ₈ ) (R ₉ ) (Rio) Si-Cl. Said reaction is preferably carried out in an organic solvent such as THF. The electrophilic source is advantageously added at a temperature between -95 ° C and -50 ° C, preferably between -80 ° C and -70 ° C. The amount of said electrophilic source used is advantageously between 2 and 4 equivalents, preferably between 2 and 2.5 equivalents, relative to 3-chloro-2-chloromethylprop-1-ene. The amount of metallic lithium used is advantageously between 4 and 12 equivalents, preferably between 5 and 10 equivalents, relative to 3-chloro-2-chloromethylprop-1-ene. The amount of naphthalene used is advantageously between 0.01 and 2 equivalents, preferably between 0.05 and 0.5 equivalents, relative to 3-chloro-2-chloromethylprop-1-ene.

The compound obtained in step (b) of the process of the invention is subjected to formal hydration of its alkene, of anti-Markovnikov type, converting said alkene into primary alcohol.

According to a particular embodiment, the conversion of G alkene into primary alcohol is carried out in two successive stages: stage (c) then stage (d). Step (c) comprises reacting the compound obtained in step (b) with a borane.

By “borane” is meant an organic or inorganic compound comprising at least one BH bond. Examples of borane are in particular BH3, B ₂ H ₆ , 9-borabicyclo [3.3.l] nonane (9-BBN), monoisopinocamphéylborane, dicyclohexylborane, dimesitylborane, disiamylborane, catecholborane, pinacolborane and a mixture of these. Preferably, the borane is BH3.

Borane can be used in the form of a solvate, such as a THF solvate (e.g. BH3.THF). According to a particular embodiment, step (c) is carried out at a temperature between -15 ° C and 50 ° C, preferably between -5 ° C and 30 ° C. Step (c) is preferably carried out in an organic solvent such as THF. The amount of borane used in step (c) is advantageously between 1 and 4 equivalents, preferably between 1.2 and 2 equivalents, relative to the compound obtained in step (b) and used in step ( vs). The compound obtained in step (c) is advantageously subjected to step (d) without being isolated and purified.

Step (d) of the process of the invention comprises the reaction of the compound obtained in step (c) with an oxidant, such as sodium or potassium perborate, dioxygen, hydrogen peroxide, or a mixture of these. Preferably, the oxidant used in step (d) is sodium perborate. The oxidant is advantageously used in the form of a suspension in water. The amount of oxidant used in step (d) is advantageously between 2 and 10 equivalents, preferably between 4 and 8 equivalents, relative to the compound obtained in step (c) and used in step (d ). Step (d) can be carried out at a temperature between 0 ° C and 50 ° C, preferably between 10 ° C and 35 ° C.

The compound obtained in step (d) is a compound of the cycloheptyne type and contains a primary alcohol function. The compound obtained in step (d) is preferably isolated and purified, in particular by chromatography, before being used in step (e) of the process according to the invention.

Step (e) of the process of the invention makes it possible to functionalize the primary alcohol with a chain comprising at its end a functionality allowing conjugation to a molecule. According to a particular embodiment, step (e) comprises the reaction of the compound obtained in step (d) with a compound of formula (IV):

in which Q represents a chain comprising at its end a functionality allowing conjugation to a molecule.

Examples of functionality at the end of the Q chain of the compound of formula (IV) are in particular an alcohol (-OH), an amine (-NH ₂ ), a carboxylic acid (-COOH), an ester, in particular - COOR ', in which R' represents a C ₁ -C ₁₂ alkyl, C ₃ -C ₁₂ cycloalkyl, aryl, or heteroaryl, an acid chloride (-COC1), an anhydride, in particular - (CO) - 0- (CO) -R ”, wherein R” represents C1-C12 alkyl, C3-C12 cycloalkyl, aryl, or heteroaryl, true alkyne (-CºCH), or alkene (-CH = CH ₂ ) .

Preferably, the functionality at the end of the Q chain is a true alkyne.

According to a particular mode, Q in the compound of formula (IV) is a chain -X-Y, where:

X represents a divalent unit chosen from - (C ₁ -C ₁₂ alkyl) -, - (C ₃ - C ₁₂ cycloalkyl) -, - (aryl) -, - (heteroaryl) -, - (aryl) - (alkyl in C1-CY,) -, - (alkyl in CY-CY) - (arylc) -, - (heteroaryl) - (alkyl in CY-CY) -, - (C1-C6 alkyl) - (heteroaryl) -, - (alkyl in CY-CY) - (aryl) - (alkyl in CY-CY,) -, - (alkyl in C1-C6) - (heteroaryl) - (alkyl in CY-CY,) -, and - (alkyl Cl-C6) - cycloalkyl (C ₃ -C ₂₎ - (alkyl-C ₆₎ -; and

Y is a functionality chosen from an alcohol, amine, carboxylic acid, ester, acid chloride, anhydride, true alkyne, and alkene.

The divalent unit X as defined above must be read from left to right, and relates the carbon of the carbonyl and the functionality Y in the compound of formula (IV). The order of the groups constituting X is given so that the left-most group is linked to the carbon of the carbonyl and the right-most group is linked to Y. When X consists only of a group, this ci is connected to the carbon of carbonyl and to Y.

For example, when X is - (aryl) - (alkyl CY-CY) -, the aryl is attached to the carbonyl carbon and the alkyl-C ₆ is connected to Y, aryl and C ₁ -CY alkyl, being interconnected. When X is a CY-CY alkyl, the C ^' ₁ -CY alkyl is linked to the carbon of the carbonyl and to Y.

According to a particular embodiment, X is a C ₁ -C ₁₂ alkyl, in particular a C ₃ alkyl.

According to another particular embodiment, Y is a true alkyne.

According to a preferred embodiment, X is a C ₁ -C ₁₂ alkyl, in particular a C ₃ alkyl, and Y is a true alkyne. According to a particular embodiment, step (e) is carried out in the presence of a Bronsted base. Basic examples of Bronsted include those described above. Preferably, the base of Bronsted is triethylamine. The amount of base used in step (e) is advantageously between 1 and 5 equivalents, preferably between 1 and 2 equivalents, relative to the compound obtained in step (d) and used in step (e) . Step (e) can be carried out at a temperature between 0 ° C and 50 ° C, preferably between 10 ° C and 35 ° C. Step (e) is preferably carried out in an organic solvent such as dichloromethane.

Alternatively, step (e) may comprise the reaction of the compound obtained in step (d) and comprising a primary alcohol, with the compound of formula (IV) as defined above in the form of a derivative ester, such as the methyl or ethyl ester, carboxylic acid or anhydride, and / or the functionality of which at the end of the Q chain is in a protected form. By "protected form" is meant a chemical form rendered inert with respect to the reaction implemented in step (e) by means of a protective group. The choice of a suitable protective group can be made by those skilled in the art using their general knowledge of synthetic chemistry. A deprotection step, restoring functionality in its original form by eliminating the protective group, can be carried out at the end of step (e), under suitable conditions and determined by a person skilled in the art.

The compound obtained in step (e) is preferably isolated and purified, in particular by chromatography.

According to a preferred embodiment of the invention, the process comprises the following successive stages: (i) a stage of reaction of the compound of formula (Ili):

in which Ri, R ₂ , R3 and R ₄ each independently represent a C 1 -CY alkyl, a C ₃ -C ₁₂ cycloalkyl, or a C ₃ -C ₁₂ heterocycloalkyl, and / or R 1 and R ₂ form together with the carbon to which they are attached, a ring selected from cycloalkyl and C ₃ -C ₁₂ heterocycloalkyl, C ₃ -C ₁₂ and / or R ₃ and R ₄ together with the carbon to which they are attached, a ring selected from cycloalkyl and C ₃ -C ₁₂ heterocycloalkyl, C ₃ -C _12, with a compound:

- R ₅ -Z, where R ₅ represents a group chosen from: ^■ R _<5 C (0) - with Re is alkyl Ci-C _6, cycloalkyl C ₃ -C ₁₂ alkoxy, Ci-C _6, or an aryl, and

^■ R ₇ S (0) ₂ - with R ₇ being an alkyl-C ₆ cycloalkyl or C 3 -C 12 aryl,

and Z is a group selected from chlorine, bromine and OR ₁₁ wherein R ₁₁ is hydrogen, alkyl Ci-C _6, or aryl; or

- R ₅ -OR ₅ where R ₅ represents a group chosen from:

^■ REC (O) - with Re is alkyl Ci-C _6, a C3-C12 alkoxy, Ci-C _6, or an aryl, and

^■ R ₇ S (0) 2- with R ₇ being an alkyl-C ₆ cycloalkyl or C 3 -C 12 aryl;

and steps (a) (b), (c), (d), and (e) as defined above.

use

The compound of formula (I) according to the invention contains an alkyne function protected by a dicobalt group, and a second reactive functionality at the end of its Q chain, preferably a true alkyne, and is particularly suitable for the bioconjugation of a target molecule to an agent, in particular a diagnostic or therapeutic agent.

In the context of the present invention, labeling, imaging and / or modification of the target molecule is carried out by conjugation of the target molecule with an agent suitable for the desired application (diagnostic or therapeutic) via a compound of formula (I) which ensures the link between said target molecule and said agent.

By "conjugate" is thus meant a target molecule / compound of formula (I) / agent complex. This conjugation can in particular be achieved by means of “click” reactions or coupling reactions. Examples of “click” reactions are in particular the cycloaddition reactions between an alkyne and a 1,3-dipole or a 1,3- (hetero) diene, alkene hydrothiolation reactions (“thiol-ene” reaction between a thiol and an alkene) or alkyne (“thiol-yne” reaction between a thiol and an alkyne). Examples of coupling reactions include esterification or amide formation reactions. The choice of reaction used for conjugation depends on the reactive functions involved.

The term “orthogonal” or “bioorthogonal” is understood to mean any reaction or process which may occur in a living system, such as a living cell or organism, without interfering with the natural biochemical reactions or processes of said living system. The invention relates to a use of a compound of formula (I) for labeling, imaging and / or orthogonal modification of a target molecule.

By “target molecule” is meant any molecule, in particular a biomolecule, comprising (or modified with) at least one reactive function with respect to an alkyne and / or of the functionality at the end of the Q chain of the compound of formula (I), in particular a 1,3-dipole, a 1,3- (hetero) diene, a thiol, amine, alcohol, carboxylic acid, ester, anhydride, or acid chloride function, and preferably a 1,3-dipole.

Examples of the 1,3-dipole are in particular azides, nitrones, diazomethanes, nitrile oxides, or nitrilamines. Examples of 1,3-diene are in particular 1,3-butadiene, 1,3-cyclohexadiene or furan. Examples of 1,3-heterodiene are in particular l-oxa-1,3-butadiene or 3-aza-1,3-butadiene. Preferably, said target molecule comprises an azide function.

By "biomolecule" is meant any molecule naturally present in a living organism. Examples of biomolecules include antibodies, proteins, glycans, proteoglycans, lipids, enzymes, hormones, acids or nucleic bases. Preferably, the biomolecule is an antibody. Said biomolecule comprising (or modified with) said at least one reactive function can in particular be obtained by expression of autotrophic bacteria, genetic engineering or chemical conversion.

According to the invention, an agent, in particular a therapeutic or diagnostic agent, includes any molecule comprising (or modified with) at least one reactive function with respect to an alkyne and / or of the functionality at the end of the Q chain of the compound of formula (I), in particular a 1,3-dipole, a 1,3- (hetero) diene, a thiol, amine, alcohol, carboxylic acid, ester, anhydride, or acid chloride function , and adapted to the desired application. Preferably, said agent comprises an azide function.

Examples of agents used for labeling, imaging and / or modification of a target molecule are, for example, fluorophores such as Alexa Fluor 555, fluorescein, rhodamine, chromomycin, or cyanine 3, biotin, polyethylene glycol (PEG) or polypropylene glycol (PPG) chains, radioactive isotopes, steroids, pharmaceutical compounds, oligo- or polysaccharides, nucleotides, oligonucleotides or polynucleotides, or peptide labels such as FLAG. According to a particular embodiment, a compound of formula (I) is used for labeling, imaging and / or orthogonal modification of an antibody. According to this mode, the compound of formula (I) conjugates an antibody with a therapeutic agent.

The conjugates (target molecule / compound of formula (I) / agent complexes) can be prepared by two methods which differ according to the order of the steps implemented, in particular the step of deprotection of the cyclic alkyne of the compound of formula (I).

According to a first embodiment, a method for preparing a conjugate as defined above comprises the following successive steps:

(1) a step of deprotection of the cyclic alkyne of the compound of formula (I);

(2) a step of reacting the compound obtained in step (1) with a target molecule as defined above; and,

(3) a step of reacting the compound obtained in step (2) with an agent as defined above.

This method is illustrated in particular in Figure 2 ("Method 2A").

Deprotection step (1) comprises eliminating the dicobalt group (Co ₂ (CO) ₄ (L) (L ')) from the compound of formula (I) to restore the cyclic alkyne function. This deprotection can be carried out by reacting a compound of formula (I) with a suitable deprotection agent, in particular a tertiary amine N-oxide, such as trimethylamine N-oxide (TMANO) or N-N-oxide -methylmorpholine (NMO). Preferably, the deprotective agent is TMANO.

Step (1) is advantageously carried out at a temperature between -10 ° C and 50 ° C. Step (1) is advantageously carried out in an organic solvent such as acetonitrile, or in water. The amount of deprotection agent is advantageously between 2 and 10 equivalents, preferably between 3 and 8 equivalents, relative to the compound of formula (I).

Step (2) comprises reacting the deprotected compound obtained in step (1) with said target molecule. In step (2), a reaction occurs between the deprotected cyclic alkyne of the compound obtained in step (1) and the reactive function of said target molecule, preferably an azide. Step (2) is advantageously carried out at a temperature between -10 ° C and 50 ° C. Step (2) is advantageously carried out in an organic solvent such as acetonitrile, or in water. The quantity of target molecule is advantageously between

I and 5 equivalents relative to the deprotected compound obtained in step (1).

Steps (1) and (2) are advantageously carried out successively in a "one pot", that is to say without isolating the compound obtained in step (1). According to a preferred embodiment, steps (1) and (2) are carried out simultaneously, in other words the compound of formula (I) and said target molecule react in the presence of a tertiary amine N-oxide.

Step (3) comprises the reaction between the functionality at the end of the Q chain of the compound obtained in step (2), preferably a true alkyne, with the reactive function of said agent, preferably an azide.

According to a particular embodiment, step (3) is implemented in the presence of a catalyst such as CuS0 ₄ , and optionally of sodium ascorbate.

Step (3) is advantageously carried out at a temperature between -10 ° C and 50 ° C. Step (3) is advantageously carried out in an organic solvent such as tert-butanol, in water or in a mixture of these. The amount of agent is advantageously between 1 and 5 equivalents relative to the compound obtained in step (2). The amount of catalyst is advantageously between 0.01 and 0.5 equivalents, preferably between 0.05 and 0.2 equivalents, relative to the compound obtained in step (2).

It is understood that steps (2) and (3) are interchangeable by conjugating, for example, the agent firstly with the deprotected compound obtained in step (1), then then the target molecule.

According to a second embodiment, a method for preparing a conjugate as defined above comprises the following successive steps:

(1 ’) a step of reacting the compound of formula (I) with a target molecule;

(2 ’) a step of deprotecting the cyclic alkyne of the compound obtained in step (G); and,

(3 ’) a reaction step of the compound obtained in step (2’) with an agent.

This method is illustrated in particular in Figure 2 ("Method 2B"). Step (L) comprises the reaction between the functionality at the end of the Q chain of the compound of formula (I), preferably a true alkyne, with said target molecule comprising at least one reactive function, preferably an azide. The experimental conditions of step (G) can be similar to the experimental conditions described above for step (3) of method 2A.

The deprotection step (2 ′) comprises the elimination of the dicobalt group (Co ₂ (CO) ₄ (L) (L ′)) from the compound obtained in step (G) to restore the cyclic alkyne function. The experimental conditions of step (2 ') can be similar to the experimental conditions described above for step (1) of method 2 A.

Step (3 ’) comprises the reaction between the cyclic alkyne of the deprotected compound obtained in step (2’) with an agent, which comprises at least one reactive function, preferably an azide. The experimental conditions of step (3 ’) can be similar to the experimental conditions described above for step (2) of method 2 A.

It is understood that steps (1 ’) and (3’) are interchangeable by example combining the agent first with the compound of formula (I) and then the target molecule.

The methods for preparing a conjugate of said target molecule and said agent from the compound of formula (I) can be carried out in vitro or in vivo.

According to a particular embodiment for the preparation of conjugates, the compound of formula (I) is grafted onto a solid support. The use of a solid support makes it possible to facilitate the stages of purification during bioconjugation, or even to get rid of it. More particularly, the use of a solid support makes it possible to effectively remove traces of cobalt which can contaminate the bioconjugation product.

A solid support can in particular be silica, glass or a polystyrene resin optionally onto which is grafted another polymer such as polyethylene glycol. This solid support is advantageously functionalized with at least one linking group. By “linking group” is meant any chemical group capable of forming a bond with a compound of formula (I), in particular with at least one of the cobalt (Co) atoms of the compound of formula (I), so that said compound of formula (I) is grafted to the solid support. The linking group may especially be a phosphino group of the formula -PR ₂ wherein R is an alkyl-C ₆ cycloalkyl CVC1 2, or aryl, preferably phenyl.

EXAMPLES

The invention will be better understood in the light of the following examples, which are given purely by way of illustration and are not intended to limit the scope of the invention, defined by the appended claims.

Example 1:

Unless otherwise stated, all commercial products were used without prior purification. 1 H and ¹³ C NMR spectra were recorded on a Bruker Avance I 300 MHz spectrometer ('H: 300 MHz, ¹³ C: 75.3 MHz). The chemical shifts d for the NMR spectra are expressed in ppm relative to the residual peak of the solvent. The coupling constants J are expressed in hertz (Hz). The following abbreviations are used for the multiplicity of peaks: s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of doublet; dt, triplet doublet. The yields are given for the products isolated after purification. TLC analyzes (Thin Layer Chromatography) were performed with Lluka Silica Gel 60 L254 plates. The high resolution mass spectra were obtained using a Qq-TOL tandem mass spectrometer (API Q-STAR Pulsari, Applied Biosystems). The infrared spectra were recorded on a Perkin Elmer Spectrum 100 LT-IR spectrometer.

1) Preparation of 2,5-dimethylhex-3-yne-2,5-diyle diacetate 1

1

To a solution of 2,5-dimethylhex-3-yne-2,5-diol (5 mmol, 710 mg, 1 eq.) In anhydrous dichloromethane under nitrogen atmosphere (C = 0.1 M), were added triethylamine (50 mmol, 6.9 mL, 10 eq.), DMAP (5 mmol, 610 mg, 1 eq.) then acetic anhydride (25 mmol, 2.5 mL, 5 eq.) at temperature ambient. The resulting solution was stirred at room temperature for 16 hours. After evaporation of the solvent under vacuum, the crude residue was purified directly by chromatography on silica gel (petroleum ether / Et ₂ 0 7: 3). Compound 1 was obtained in the form of a colorless liquid (m = 848 mg, yield = 75%).

NMR (300 MHz, CDCh): d [ppm] = 2.00 (s, 6H); 1.62 (s, 12H). 2) Preparation of compound 2a

2a

To a solution of compound 1 (1.2 mmol, 270 mg, 1 eq.) In anhydrous dichloromethane under nitrogen atmosphere (C = 0.1 M) under nitrogen atmosphere, was added Co ₂ C0 ₈ ( 1.2 mmol, 450 mg, 1 eq.) At room temperature. The resulting solution was stirred for 4 hours at room temperature, then the solvent was evaporated in vacuo. The complex was then purified by chromatography on silica gel (petroleum ether / Et ₂ 0 8: 2). Compound 2a was obtained in the form of a red solid (m = 660 mg, yield = 99%). Rf: 0.62 (8: 2 cyclohexaneÆUO).

A solution of 3-chloro-2-chloromethylprop-1-ene (8 mmol, 1 mL, 1 eq.) And trimethylsilyl chloride (16 mmol, 2 mL, 2 eq.) In THF (5 mL) was added to a suspension of pieces of freshly cut lithium (56 mmol, 400 mg, 7 eq.) and naphthalene (0.8 mmol, 100 mg, 0.1 eq.) in THF (10 mL) at -78 ° C under atmosphere nitrogen. The resulting mixture was then brought back to room temperature, while stirring for 24 hours, until a reddish solution appeared. The mixture was then treated with water (10 mL), extracted with diethyl ether (3 x 20 mL), and the organic phase was dried over sodium sulfate. The crude of compound 2b was obtained and used thus without purification (m = 1.1 g, crude yield = 68%).

[ppm] = 4.39 (s, 2H); 1.49 (s, 4H); 0.05 (s, 18H). 4) Preparation of compound 3

To the solution of compound 2a (1.2 mmol, 660 mg, 1 eq.) In anhydrous dichloromethane (C = 0.1 M) under nitrogen atmosphere, the 1,3-bis (trimethylsilyl) was added - 2-methylenepropane 2b (1.2 mmol, 240 mg, 1 eq.). The resulting solution was cooled to -40 ° C then BF ₃ .0 Et ₂ (2.4 mmol, 0.72 mL, 2 eq., C = 46.5%) was added. The resulting solution was stirred for 1 hour at -40 ° C, then the solvent was evaporated in vacuo. The compound was purified by chromatography on silica gel (100% petroleum ether). Compound 3 was obtained in the form of a red solid (m = 245 mg, yield = 45%). Rf: 0.71 (100% petroleum ether).

2H).

To a solution of compound 3 (0.5 mmol, 227 mg, 1 eq.) In anhydrous THF (C = 0.1 M) under nitrogen atmosphere, was added BH ₃ .THF (0.75 mmol, 0.75 mL, 1.5 eq., C = 1 M in THF) at 0 ° C. Fa resulting solution was stirred at room temperature, until total consumption of the substrate (typically 16 hours - TLC control) then NaB0 ₃ .4H ₂ 0 (2.5 mmol, 382 mg, 5 eq.) Suspended in the water (2 mL) was added to the mixture at room temperature. The resulting mixture was vigorously stirred for 4 hours at room temperature (two-phase mixture). The mixture was then treated with water (10 mL), extracted with ethyl acetate (3 x 10 mL), and the organic phase was dried over anhydrous sodium sulfate. The compound was purified by chromatography on silica gel (petroleum ether / Et ₂ 0 8: 2). Compound 4 was obtained in the form of a red solid (m = 142 mg, yield = 60%). Rf: 0.25 (7: 3 cyclohexane / ethyl acetate).

NMR (300 MHz, CDCh): d [ppm] = 3.45 (t, J = 6.1 Hz, 2H); 2.13-1.96 (m, 1H); 1.79 (d, J = 13.5 Hz, 2H); 1.34 (d, J = 13.3 Hz, 2H); 1.29 (s, 12H).

¹³ C NMR (75 MHz, CDCh): d [ppm] = 69.1; 46.9; 37.9; 35.6; 33.8; 31.9.

FT-IR (u / crn- ¹ , KBr): 3371, 2994, 2929, 2086, 2043, 2019, 1591, 1469, 1379, 1362, 1248, 858, 837. 6) Preparation of compound 5

To a solution of compound 4 (0.3 mmol, 141 mg, 1 eq.) In anhydrous dichloromethane (C = 0.1 M) under nitrogen atmosphere, were added 5-hexynoyl chloride (0.45 mmol, 59 mg, 1.5 eq.) and triethylamine (0.3 mmol, 41 mL, 1 eq.) at room temperature. The resulting solution was stirred at room temperature, until total consumption of the substrate (typically 1 hour, TLC control). After evaporation of the solvent under vacuum, the crude residue was purified directly by chromatography on silica gel (petroleum ether / Et ₂ 0 8: 2). Compound 5 was obtained in the form of a red solid (m = 86 mg, yield = 51%). Rf: 0.44 (8: 2 petroleum ether / Et ₂ 0).

NMR (300 MHz, CDCh): d [ppm] = 3.94 (d, J = 6.4 Hz, 2H); 2.50 (t, J = 7.4 Hz, 2H); 2.30 (td, J = 6.9 and 2.6 Hz, 2H); 2.00 (t, J = 2.6 Hz, 1H); 1.94 - 1.83 (m, 2H); 1.74 (d, J = 13.0 Hz, 2H); 1.47 - 1.39 (m, 2H); 1.32 (s, 12H).

¹³ C NMR (75 MHz, CDCh): d [ppm] = 173.0; 109.9; 70.1; 69.2; 46.8; 37.7; 33.6; 32.9; 32.2; 31.7; 23.6; 17.9.

FT-IR (u / cm ¹ , KBr): 3313, 2966, 2937, 2086, 2044, 2019, 1737, 1591, 1469, 1451, 1381, 1363, 1157, 858.

Claims

1. Compound of formula (I):

in which

Ri, R _2, R 3 and R ₄ each independently represent an alkyl -CY ,, cycloalkyl, C ₃ -C ₁₂ heterocycloalkyl or C ₃ -C _12, and / or Ri and R ₂ together with the carbon to which they are attached, a ring selected from cycloalkyl and C ₃ -C ₁₂ heterocycloalkyl, C ₃ -C ₁₂ and / or R ₃ and R ₄ together with the carbon to which they are attached, a ring selected from cycloalkyl C ₃ -C ₁₂ heterocycloalkyl and C ₃ -C 12;

^■ a CO ligand,

Ra, Rb, Rc, Rd, Re, Rf, Rg, Rh, Ri, and Rj, being independently selected from hydrogen, C ₁ -C ₁₂ alkyl, C ₃ -C ₁₂ cycloalkyl, aryl and heteroaryl, and

^■ a carbene ligand C (Rk) (Ri),

R _k and R being independently selected from alkyl CY-CY ,, cycloalkyl, C ₃ -C _12, aryl and heteroaryl, or together with the carbon to which they are attached a cycloalkyl C ₃ -C 12 or a C ₃ -C12 heterocycloalkyl.

2. Compound according to claim 1, in which Ri, R ₂ , R3 and R ₄ represent a methyl.

3. A compound according to claim 1 or 2, wherein L and L ’represent a CO ligand.

4. A compound according to any one of claims 1 to 3, in which Q is a chain -X-

Y, in which:

X represents a divalent unit chosen from - (C1-C12 alkyl) -, - (C3-C12 cycloalkyl) -, - (aryl) -, - (heteroaryl) -, - (aryl) - (C1 -CY alkyl ,) -, - (alkyl-CY) - (arylC) -, - (heteroaryl) - (alkyl CY-CY,) -, - (alkyl-C ₆₎ - (heteroaryl) -, - ( alkyl in CY-CY,) -

(aryl) - (alkyl CY-CY,) -, - (alkyl-C ₆₎ - (heteroaryl) - (alkyl CY-CY,) -, and - (alkyl-C ₆₎ - ( C3-Ci2) - (alkyl-C ₆₎ -; and

5. The compound of claim 4, wherein X is C1-C12 alkyl, preferably C3-alkyl, and Y is true alkyne.

6. Compound according to any one of claims 1 to 5, in which the compound has the formula

7. Process for the preparation of a compound as defined according to any one of claims 1 to 6, comprising the following successive steps:

(a) a step of reacting the compound of formula (II):

(P),

in which

R ₅ represents a group chosen from:

^■ R _b C (O) - where R _b is an alkyl-C ₆ cycloalkyl, C ₃ -C ₁₂ alkoxy CY-CY, or aryl, and

^■ a CO ligand,

^■ a carbene ligand C (Rk) (Ri),

preferably L and L ’represent a CO ligand;

(III),

(c) a step of reacting the compound obtained in step (b) with a borane, preferably BH ₃ ;

(d) a step of reacting the compound obtained in step (c) with an oxidant, preferably sodium perborate; and

(IV),

X represents a divalent unit chosen from - (C1-C12 alkyl) -, - (C3-C12 cycloalkyl) -, - (aryl) -, - (heteroaryl) -, - (aryl) - (C1 -CY alkyl ,) -, - (alkyl-CY) - (arylC) -, - (heteroaryl) - (alkyl CY-CY) -, - (alkyl-C ₆₎ - (heteroaryl) -, - (C in CY-CY,) - (aryl) - (alkyl CY-CY,) -, - (alkyl-C ₆₎ - (heteroaryl) - (alkyl CY-CY,) -, and - (C Ci-C ₆₎ - (C3-Ci2) - (alkyl-C ₆₎ -; and

Y is a functionality selected from an alcohol, an amine, a carboxylic acid, an ester, an acid chloride, an anhydride, a true alkyne, and an alkene;

or an ester, carboxylic acid, or anhydride derivative thereof.

8. The method of claim 7, wherein Ri, R ₂ , R ₃ and R ₄ represent methyl.

9. The method of claim 7 or 8, wherein R ₅ represents a group R ₆ C (O) - in which Re is methyl.

10. Method according to any one of claims 7 to 9, in which Q is a chain -X- Y where X is a C ₁ -C ₁₂ alkyl, preferably a C ₃ alkyl, and Y is a true alkyne .

11. Use of a compound according to any one of claims 1 to 6, for labeling, imaging and / or orthogonal modification of a target molecule.