EP1517903A2

EP1517903A2 - Marked maleimide compounds, method for preparing same and use thereof for marking macromolecules

Info

Publication number: EP1517903A2
Application number: EP03761670A
Authority: EP
Inventors: Frédéric DOLLE
Original assignee: Commissariat a lEnergie Atomique CEA
Current assignee: Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2002-07-01
Filing date: 2003-06-30
Publication date: 2005-03-30
Also published as: WO2004002984A3; FR2841554B1; AU2003259312A1; WO2004002984A2; US20050249662A1; JP2006504640A; US7456202B2; FR2841554A1; CA2491193A1; CA2491193C

Abstract

The invention concerns maleimide compounds marked with Fluor 18 of general formula (I), wherein: m represents an integer from 0 to 10; n represents an integer from 1 to 10; Y represents a group selected among optionally substituted heterocyclic, monocyclic or bicyclic groups; X represents a radical of formula (U) a -((CR1R2) b -(V) c) d -((CR3R4) e- (W) f) g-, wherein: a, b, c, d, e, f, g represent each independently an integer from 1 to 10; 0 A 10; U, V and W represent each independently -NR1-, -O-, -S-,formula (II), ethynyl, -CR1=CR2-, -(C=0)-, -(C=S)-, -C=NR1)-, -C(=0) 0-, -(C=S) S-, -C(=NR1)NR2-, -CR1R2-, -CR1OR2-, -CR1NR2R3. The invention also concerns a method for preparing said compounds, their use for marking macromolecules and complexes of said compounds with a macromolecule. The invention further concerns a kit for analysis, detection, or diagnosis using said complexes. The invention finally concerns the use of the complexes in a medical imaging method such as position emission tomography (PET).<>

Description

MALEIMIDE BRANDS,

THEIR PREPARATION PROCESS

AND THEIR USE FOR MARKING MACROMOLECULES

DESCRIPTION

The present invention relates to maleimide compounds labeled with fluorine-18.

The invention also relates to a process for the preparation of these compounds.

The invention finally relates to the use of these maleimide compounds, in particular labeled with fluorine-18, for the labeling of. macromolecules, such as oligonucleotides, proteins, antibodies and peptides.

The technical field of the invention can be defined, in general, as that of radioactive labeling of macromolecules and, in particular, of proteins and peptides. Indeed, for use in research or diagnosis, macromolecules, such as proteins or even peptides can be coupled to a labeling molecule allowing their detection, this labeling molecule can be, for example, a fluorescent molecule, gold particles, a paramagnetic compound or a molecule carrying a radioelement.

Proteins have been radioactively labeled with radioisotopes, iodine and various metal radioisotopes, such as technetium, indium and gallium. More recently, proteins have been labeled with fluorine-18.

For example, peptides coupled to radioelements, such as fluorine, allow "in vivo" detection of the localization of thrombotic zones during vascular accidents of all kinds, in particular apoptotic and inflammatory foci, using systems of imaging.

Thus, radioactive atoms emitting positrons with a short lifespan and in particular ¹⁸ F can, in particular, be detected by positron emission tomography (PET) devices (PET or “Positon Emission Tomography”).

Radioactive labeling with fluorine-18 poses, in particular due to the very short period of fluorine-18 (close to 109.8 minutes), specific problems which make labeling with fluorine-18 fundamentally different from that with other halogens, such as iodine. The above coupling can be carried out by all the conventional techniques of organic chemistry known to those skilled in the art, and by the synthesis of protein and peptide markers carrying one or more radioactive atoms with a short lifespan, in particular ¹⁸ F. This marker generally consists, on the one hand, of a part capable of receiving, for example, an ¹⁸ F atom and, on the other hand, of a part comprising any conventional function of binding to the macromolecule, by for example, protein. These markers must meet the requirement for rapid and easy synthesis, because due to the short lifespan of radioisotopes such as ¹⁸ F, the duration of synthesis should not generally exceed a few hours.

In addition, this synthesis, due to the high radioactivity of the compounds used, must be able to be carried out by robotic means.

Thus, the methods for labeling proteins or peptides with fluorine-18 make use of markers also called “conjugated” or “synthon” labeled, which are classified into three main families, depending on whether they react with the amino groups, sulfhydryl groups, or carbohydrate groups of macromolecules, such as proteins and peptides.

Among the compounds or conjugates reacting with amino groups, there may be mentioned imidates, such as 3- [ ¹⁸ F] fluoro-5-nitrobenzoimidate, which react, for example, with the ε-NH _{2 group} of lysine to bind to a protein; activated esters, such as

N-succinimidyl- [ ¹⁸ F] fluorobenzoate; carboxylic acids, such as N- (4- [ ¹⁸ F] fluorobenzoic acid); aldehydes, such as 4- [ ¹⁸ F] pentafluorobenzaldehyde and isothiocyanates, such as 4- ([ ¹⁸ F] fluoromethyl phenylisothiocyanate).

Activated halides, such as (4- [ ¹⁸ F] fluorophenacyl bromide), react with groups amino, such as the ε-NH _{2 group} of lysine or the -SH group of cysteine.

Amines, such as 1- (4- ([ ¹⁸ F] fluoromethyl) benzoyl) -aminobutane-4-amine react with the CO ₂ H groups, for example glutamic acid or aspartic acid or with CHO groups of glycoproteins.

Nitrenes with photochemical active centers, such as azidophenacyl fluoride [ ¹⁸ F] also react with amino groups, for example the ε-NH _{2 group} of lysine.

The most efficient and best described method for labeling proteins and peptides is that which uses activated acids, but it is also the method which has the greatest non-specificity since all the nucleophilic sites of amino acids of proteins or peptides will react with the marker, co judued, or labeled synthon.

Two more specific methods for labeling peptides and nucleotides have good specificity with respect to sulfur atoms, for example cysteine for the peptides and a phosphoro-thioate function for the nucleotides.

These are, first of all, processes using haloacetamide "synthons", which, although satisfactory, have the drawback of being very slow and therefore not very suitable for ¹⁸ F, due to the period of this one.

These are then processes involving activated maleimides, which can bind to SH groups with very good specificity because the reaction is very slow vis-à-vis, for example ε-NH ₂ sites of lysine.

The scheme of the reaction involving the maleimido group is as follows, in the case of a protein:

X-PROTEIN

in which X represents -S-.

For any labeling, whatever the type, the molecules comprising a maleimide radical are, currently considered to be the best, as regards their reactivity with macromolecules, such as peptides or proteins. The document by SHIUE C.-Y. et al., J. Label

Compounds Radiopharm 26: 287-289 (1989), describes the compounds:

The first of these compounds is not easy to label with fluorine-18 with high specific activity.

Indeed, only fluorine F _{2 would} allow easy labeling of (as with iodine) and it is precisely that F ₂ is generally a product with low specific activity.

In particular, F _{2 is} not suitable for the manufacture of so-called “radiotracers” compounds which are preferably targeted according to the invention, simply because the injected mass of labeled molecule becomes large and that, then, the basic principle governing this “tracer”, ie the extremely low occupation (for example, less than 5%) of the receiving sites, is not respected.

In addition, the synthesis of the first of these compounds is difficult, it is, in fact, carried out in four stages requiring a long period of time with very low yields, and relatively complex chemical transformations. This process is therefore not likely to be easily automated.

The second of the compounds cited in the document by SHIUE et al. has an amide chain which is not chemically very solid and which is easily cleaved, broken, in vivo.

Its implementation for diagnostic applications is therefore not possible. In addition, the synthesis of this second compound comprises three stages and the final yield is low, close, for example, to 10% (EOB: "End Of Bombardment" in English, that is to say end of irradiation) . The document US-A-4,735,792 relates to molecules of formula:

in which X is a radioactive halogen chosen from bromine-75, bromine-76, bromine -82, iodine-123, iodine-125, iodine-131 and fluorine-18.

However, only the molecule labeled with iodine-125 is actually prepared.

The preparation of a molecule labeled with fluorine-18 is neither mentioned nor mentioned, and the remarks already made above, with regard to the first compound of the document of SHIUE et al. , also apply within the framework of document US-A-4,735,792.

A person skilled in the art, on reading this document, does not have any information allowing him to specifically prepare a fluorine-18 labeled compound and if he plans to do so, he would use F ₂ and thus lead to a compound of low specific activity, unusable in “PET” imaging.

It can also be considered that the chemistry used to manufacture the fluorinated compound of document US Pat. No. 4,735,792 is a complex and long-term chemistry. There is therefore a need for maleimide compounds labeled with fluorine-18, which exhibit high reactivity, high selectivity as well as good specific activity. There still exists a need for maleimide compounds labeled with fluorine-18, which can be manufactured at high yield by a simple, reliable, easily automated, robotizable, rapid and short-lived process. The object of the present invention is to provide a fluorine-18 labeled maleimide compound which meets, among other things, these needs.

The aim of the present invention is also to provide a fluorine-18 labeled maleimide compound which does not have the drawbacks, defects, limitations and disadvantages of the compounds of the prior art and which solves the problems of the prior art.

This object, and others still are achieved, in accordance with the invention, by providing a compound of general formula (I):

in which: m represents an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n represents an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

Y represents a group chosen from monocyclic or bicyclic heterocyclic groups chosen from imidazolyl, pyrazolyl, benzimidazolyl, pyridinyl, piridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, possibly quinoxalinyl substituted by one or more substituents each of said substituents being independently selected from hydrogen, halogen (non-radioactive), phenyl, alkyl Cι- ₆ alkoxy Cι- _6, aryloxy, amino, mono- or di (alkyl Cι_ ₆₎ alkylamino, mono- or di (aryl) amino, thio, alkyl Cχ- ₆ alkylthio, arylthio, formyl, alkyl Cι- ₆ alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, -C ₆ alkoxy carbonyl , aryloxycarbonyl, Cι- ₆ -aminocarbonyl, arylaminocarbonyl, trifluoromethyl; - X represents a radical of formula:

(U) _a - ((CR _! R ₂ ) _b - (V) _c ) _d - ((CR ₃ R ₄ ) _θ - (W) _f ) _g -

in which: - a, b, c, d, e, f, g each independently represent an integer of 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9;

- U, V and W each independently represent o -NRi-, -O-, -S-, ^• -, ethynyl, -CRi≈CRs-, - (C = 0) -, - (C = S) -, -C (= 0) 0-, - (C≈S) S-, -C (= NRι) NR ₂ -, -CRιR ₂ -, -CRιOR ₂ -, -CRιNR ₂ R ₃ -, where Ri R ₂ R-3 and R ₄ are each independently chosen among hydrogen, halogens, phenyl, Cι_ ₆ alkyl, C _1-6 alkoxy _/ aryloxy, amino, mono- or di (Cι- ₆ alkyl) amino, mono- or di (aryl) amino, thio , Cι- ₆ -thio alkyl, arylthio, formyl, Cι- ₆ -carbonyl alkyl, arylcarbonyl, carbonyl Cι- ₆ -carbonyl, aryloxycarbonyl, Ci-β-aminocarbonyl, arylaminocarbonyl, trifluoromethyl.

Generally, in the present description, halogen means fluorine, chlorine, bromine or iodine. Cι_ ₆ alkyl corresponds to saturated hydrocarbon radicals with linear and branched chains having from 1 to 6 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl and hexyl.

The attachment and substitution of heterocycles, aryl group, etc., can be done at any position. Similarly, the attachment of ¹⁸ F to Y or X can be done at any position, in particular at any position on a heterocycle.

Advantageously, in the compound of formula (I) above, n = 1, and Y is a 3-pyridinyl group.

The compounds of formula (I) can belong to various families, a first family can be defined as that of "alkyl ethers", which correspond to the following formula (II):

in which p is an integer from 1 to 10, such as 2, 3, 4, 5, 6, 7, 8 or 9. The preferred compounds of formula (II) are chosen from the following compounds:

[(2- [ ¹⁸ F] f luoro-pyridin-3-yloxy) -methyl) -pyrrole-2, 5-dione

[2- (2- [F] f luoro-pyridin-3-yloxy) -ethyl] -pyrrole-2, 5-dione

1- [3- (2- [F] fluoro-pyridin-3-yloxy) -propyl] -pyrrole-2, 5-dione

[1- [4- (2- [F] fluoro-pyridin-3-yloxy) -butyl] -pyrrole-2, 5-dione

1- [5- (2- [ ¹⁸ F] f luoro-pyridin-3-yloxy) -pentyl] -pyrrole-2, 5-dione

1- [6- (2 - [ ¹⁸ F] fluoro-pyridin-3 -yloxy) -hexyl] -pyrrole-2, 5-dione

A second family of compounds of formula (I) can be defined as those of “phenylalkyl ethers”, which correspond to the following formula (III):

wherein q and r independently represent an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9.

The preferred compounds of formula (III) are chosen from the following compounds:

uoro-pyridin-3-yloxy) -ethyl] -phenyl} -

1- [4- (2- [ ¹⁸ F] Fluoro-pyridin-3-yloxymethyl) -phenyl] pyrrole-2, 5-dione

1- [4- (2- [ ¹⁸ F] Fluoro-pyridin-3-yloxymethyl) -benzyl] - pyrrole-2, 5-dione

A third family is that of the compounds which correspond to the following formula (IV):

in which s is an integer from 1 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9. A preferred compound of formula (IV) is the following compound:

1- [3- (6- [ ¹⁸ F] Fluoropyridin-3-yl) -propyl] -pyrrole-2, 5-dione

A fourth family is that of the compounds which correspond to the following formula (V):

in which t is an integer from 0 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 and T is a group -CH = CH- or -C≡C-. Preferred compounds of formula (V) are the following compounds:

(6- [ ¹⁸ F] Fluoro-pyridin-3-yl) -prop-2-ynyl] -pyrrole-2, S-dione

The compounds according to the invention have never been described, nor mentioned, in the prior art.

The compounds according to the invention differ fundamentally from the compounds of the prior art, because of their specific structure in which the part carrying the atom of fluorine-18 is constituted, according to the invention, by a specific group Y which is in particular a pyridinyl group, the linking part, of coupling to a macromolecule, such as a protein or a peptide, is constituted, according to the invention by a specific function, namely a maleimido function, and, finally, the part of bond to a macromolecule and the part carrying the fluorine-18 atom are linked according to the invention by a new specific chain or spacer arm, for example of the alkyl type (generally from 2 to 6C), alkyl ether, ethers phenylalkyl, alkenyl, which are not fragile and are not susceptible to ruptures "in vivo". The invention relates to the use of a compound, as described above for labeling macromolecules.

This macromolecule can be any macromolecule, in particular known biological, but it can be chosen, for example, from the oligonucleotides, proteins, antibodies and peptides. Said macromolecule is advantageously a macromolecule for recognizing a specific site chosen, preferably, from sites presenting target molecules specific for a pathology, such as sites of apoptosis, necrosis or tumor area.

The invention also relates to a complex comprising a macromolecule coupled to a compound according to the invention, as described above.

Said macromolecule is preferably chosen from oligonucleotides, proteins, antibodies and peptides.

Said coupling is carried out by reaction of the double bond of the maleimido group of the compound according to the invention with specifically an -SH (thiol) function of cysteine in the case of a peptide, or a phosphoro-thioate function in the case of an oligonucleotide. This is one of the advantages linked to the specific structure of the compounds according to the invention than allowing specific, or even exclusive, labeling of cysteines, while most of the other “synthons” only allow non-specific labeling of lysines and cysteines. The selective or even exclusive labeling of cysteines is due to the presence in the molecule of the invention of a “dedicated” function, namely the maleimido function, which is a dedicated function for chemoselectivity towards the thiols of cysteines , or in a similar manner towards the phosphoro-thioate functions.

The labeling or coupling, via cysteine, can be a direct labeling or coupling, that is to say that the cysteine already exists in the macromolecule which it is desired to couple to the compound according to the invention, either cysteine or a molecule (peptide) comprising it, can be introduced (coupled beforehand or not with the compounds of the invention) in the macromolecule which did not contain cysteine and coupling is then carried out if this has not been carried out beforehand cysteine or the molecule comprising it.

The cysteine or molecule comprising it can, for example, be introduced "on order" into the macromolecule by engineering proteins / peptides in a position which does not compete with, or does not disturb the biological function.

Said macromolecule is advantageously a macromolecule for recognition of a specific site, as described above. The coupling, that is to say the labeling, is preferably such that it does not affect the recognition activity of the target, of the site, by the macromolecule. The invention also relates to an analysis and detection kit, for example for imaging medical comprising a compound according to the invention and a macromolecule.

The invention also relates to an analysis and detection kit, for example for medical imaging, comprising a compound according to the invention coupled to a macromolecule, that is to say a complex according to the invention.

The invention also relates to a diagnostic kit comprising a compound according to the invention and a macromolecule.

The invention further relates to a diagnostic kit comprising a complex, as described above.

Finally, the invention relates to the use of the complex or the compound, as described above, in a medical imaging method, such as positron emission tomography (PET) and the use of a complex or a compound according to the invention for manufacturing a product intended for medical imaging, for example for positron emission tomography (PET).

Finally, the invention relates to a product for medical imaging, in particular positron emission tomography (PET) comprising a complex or a compound as described above and a pharmaceutically acceptable vehicle.

In their application, in the context of “PET”, the compounds and complexes, according to the invention, comprising an atom of fluorine-18 show numerous advantages over the compounds with another radioactive halogen, for example iodine. Indeed, the only isotope of the positron emitting iodine is iodine-124, which could allow PET.

However, it remains produced in small quantities (some mCi against curies for F-18). It is also difficult to produce. Finally, iodine-124 is not a pure positron emitter (unlike fluorine -18.97%) and decreases by beta + emission at only 25% and by electronic capture at 75%; it has a large number of gamma lines ranging from 0.603 MeV (62%) to 2.75 MeV (1%).

The invention also relates to a process for the preparation of a compound of formula (I), as described above, in which: a) a precursor compound of formula (la) is brought into contact:

PR>

in which PRi and PR ₂ independently represent a hydrogen atom or a group protecting the amino function, provided that PRi and PR ₂ are not both (simultaneously) a hydrogen atom, or PRi and PR ₂ together with the nitrogen atom form a cyclic protecting group for the amino function, Gp represents a leaving group capable of being replaced by a fluorine atom, 18 and X, Y, m and n have the meaning already given more high ; with a source of fluoride ions F ^" marked with [ ¹⁸ F], to give a compound of formula (Ib):

PR

_{PRl N} (x ^ _{Çf) n F (} ib ₎

b) eliminating in compound (Ib), the protective group (s) PR _X and / or PR ₂ of the amino function to give a compound of formula (Ie):

H, N- 18, -

(X) n (Y) n- (Ic)

c) reacting the compound (le) with a reagent capable of giving a maleimido group from an amino group, to obtain the final compound of formula (I).

The method according to the invention is simple, reliable, easy to implement and can be easily robotized. It only has three steps, one of which is an extremely simple deprotection step. The overall duration of the process is short: for example, it is generally from 60 to 120 minutes, preferably from 75 to 85 minutes.

The incorporation of the halogen fluor-18 is carried out extremely efficiently with a high yield, for example 70 to 100%, because, in particular, it is carried out on a heterocyclic group, such as pyridine. The final yield of the whole process for a purified product is extremely high, for example from 15% to 25% and the potential quantities of “synthon” compound, at the end of the synthesis, are also very large.

In the compound (la), the groups PR ₁ and PR ₂ when they are protective groups can be any protective group known in organic chemistry. They are preferably chosen from the tert-butoxycarbonyl (BOC) and fluorenylmethoxy carbonyl (FMOC) groups.

When PR _X and PR ₂ form together with the nitrogen atom of the amino function, a protective group thereof, this protective group can be, for example, a pthalimido group.

In compound (la), the group Gp may be any leaving group capable of being replaced by a fluorine-18 atom; Gp is preferably chosen from halogens, such as F, Cl, Br, I, mesyl, tosyl and triflate groups, when Y is an alkyl group; and Gp is preferably chosen from halogens, ammonium salts, such as trimethylammonium-trifluoromethane sulfonate, and the nitro group, when Y is an aromatic or heterocyclic group.

In step a), the source of fluoride ions marked with ¹⁸ F comprises said fluoride ions and a counterion, chosen from large cations, such as rubidium, and tetrabutylammonium, and small cations , such as potassium, sodium and lithium, said small cations being trapped, stabilized, for example, by a cryptand or a crown ether, etc., said cryptand or crown ether being adapted to the small cation used. An example of cryptand is the product

KRYPTOFIX ^® K ₂₂₂ : (4, 7, 13, 16, 21, 24-hexaoxa-1,10-diazabicyclo [8.8.8] hexacosane) which traps, for example, potassium ion.

The counterion or cation can be brought in the form of any salt, for example, it can be K ₂ C0 ₃ , in the case of potassium.

Step a) is generally carried out in a solvent, which can be any suitable solvent, such as DMSO. Step a) can be carried out under conditions known to a person skilled in the art, with heating generally at a temperature of 50 to 200 ° C., for example, 145 ° C., for a duration generally of 1 to 30 minutes, for example from 4 to 6 minutes. Step b) of removal of the protective group from the amino function, of deprotection, to give the compound of formula (Ic), where the amino group is free, can be carried out by any known deprotection method. It is possible, for example, to put the compound (Ib) in contact with TFA in CH ₂ C1 ₂ for a duration generally of 1 to 5, for example of 2 minutes. It should be noted that TFA is generally used only if the protective group is removed in an acid medium, for example when PR _X = BOC and PR ₂ = H. In step c), the reagent capable of giving a maleimido group from an amido group can be any known compound. It can thus be chosen from N-methoxycarbonylmaleimide and succinimide.

Step c) can be carried out under conditions known to a person skilled in the art, for example in a solvent, such as xylene, THF, with heating generally at a temperature of 100 to 200 ° C., for example 190 ° C, for a period of 1 to 20 minutes, for example 5 minutes.

Step c) can in another embodiment also be carried out in a two-phase mixture for example of dioxane and aqueous sodium bicarbonate, at room temperature for a period of 3 to 15 minutes, for example 10 minutes; This ^mode of implementation of step c) offers the advantage of better performance and be implemented at room temperature, without the need to heat the mixture.

The compound of formula (la) can correspond to the following formula (IIa):

The compound (Ha) preferably corresponds to the following formula (11b):

The compound of formula (la) can, in another embodiment, correspond to the following formula (Illa):

The compound (Illa) preferably corresponds to the following formula (Illb):

The compound of formula (la) can, in yet another embodiment, correspond to the following formula (IVa):

The compound (IVa) preferably corresponds to the following formula (IVb):

In another embodiment, the compound of formula (la) can correspond to the following formula (Va):

The compound (Va) preferably corresponds to the following formula (Vb):

The invention also relates to the precursor compounds of formulas (la), (lia), (Ilb), (Illa),

(Illb), (IVa), (IVb), (Va), (Vb), as described above, as synthesis intermediates for the compounds of formulas (I) to (V) according to the invention.

The precursor compounds can be chosen, in particular, from the final compounds defined, listed above, in which the [ ¹⁸ F] is replaced by a non-radioactive halogen, such as ¹⁹ F, Cl, Br, I, a salt d ammonium, such as trimethylammonium-trifluoromethanesulfonate or an NO ₂ group and the group l-pyrrole-2,5-dione is replaced by a tert-butoxycarbonylamino group.

Preferred precursor compounds are, for example [3- (3-tert-butoxycarbonylamino-propoxy) - pyridin-2-yl] -trimethyl-ammonium, trifluoromethane sulfonate; and [3- (2-nitro-pyridin-3-yloxy) -propyl] -carbamic acid tert-butyl ester.

The invention will now be explained in more detail in the following description, given by way of illustration and not limitation, in relation to examples of preparation of compounds according to the invention, as well as complexes according to the invention. Experiment conditions

Chemicals, thin layer chromatography (TLC ") and high pressure liquid chromatography (" HPLC ").

The chemicals were obtained from various suppliers (ALDRICH, FLUKA or SIGMA France) and they were used without further purification, except when mentioned. The TLCs are carried out on plates coated, beforehand, with silica gel 60F ₂ 5 from MERCK. The compounds were located (1) if possible at 254 nm, using a UV lamp and / or (2) by iodine staining and / or (3) by soaking the TLC plates in an ethanolic solution at 1 % ninhydrin (or a 1% aqueous solution of KMn0 ₄ ) and by heating on a hot plate. Radioactive points, spots or "spots" are detected using a BERTHOLD TRACE MASTER 20 automatic linear analysis device.

spectroscopy

The NMR spectra are recorded on a BRUKER AMX device (300 MHz), using the hydrogenated residue of deuterated solvents (DMS0-d ₆ , δ: 2.50 ppm; CD ₂ C1 ₂ , δ: 5.32 ppm; CD ₃ OD , δ: 4.78; CD ₃ CN, Ô: 1.93 ppm) and / or TMS as internal standards for ¹ H NMR, and deuterated solvents (DMS0-d _6; δ: 39.5ppm; CD ₂ C1 ₂ , δ: 53.8 ppm; CD ₃ OD, δ: 49.3 ppm) and / or TMS, as internal standards for ¹³ C NMR.

The chemical shifts are given in ppm with TMS (tetramethylsilane) as a reference, the chemical shift of which is fixed at 0. s, d, t, dd, q, q5, m, b represent singlet, doublet, triplet, doublet of doublet , quadruplet, quintuplet, multiplet, and broad respectively). Mass spectra

("MS") are measured on a Quadripolair Finnigan 4 600 (DCI / NH ₄ ⁺ ).

Production of the radioactive isotope

Aqueous fluoride [ ¹⁸ F] ions were prepared in a CGR-MeV 520 cyclotron by irradiation of a 2 mL water target, using a 20 MeV proton beam on water enriched in [ ¹⁸ 0 ] to 95% by the nuclear reaction [ ¹⁸ 0 (p, n) ¹⁸ F]. The fluoride ions were transferred to the appropriate shielded cell. Typical production: 550-650 mCi (20.3 to 24.0 GBq) of [18F] F ^" at the end of the bombardment for an irradiation of 20 μA, 30 minutes (36,000 μC).

Various

The radiosyntheses using fluor-18, including the purifications by semi-preparative HPLC were carried out in a 7.5 cm cell shielded with lead, using a ZYMATE robot system controlled by computer (from ZYMACK CORP., USA) . activation in the microwave is carried out with a MICRO ELL 10 oven (2.45 GHz), supplied by LAB ELL AB, Sweden.

The specific radioactivity is determined as follows: the area of the UV absorbance peaks, corresponding to the radiolabelled product, is measured on the HPLC chromatogram and compared to a standard curve giving the mass, as a function of the UV absorbance.

Example 1

General procedure for coupling MITSUNOBU of 3 - (N-tert-butylcarbonylamino) -1-propanol with various 3-hydroxypyridine derivatives substituted in 2.

To a solution of 3.0 g of triphenylphosphine (molecular weight: 262.69; 11.4 mmol) in THF (60 mL), add 1.8 mL of diethyl azocarboxylate (DEAD, molecular weight: 174, 16; d: 1.106; 11.4 mmol; 1 eq). After stirring at 0 ° C for 10 to 15 minutes, 1.95 ml of 3- (N-tert-butoxycarbonylamino) -1-propanol (molecular mass: 175.23; d: 1.025; 11.4 mmol; 1) are added. eq) and the 3-hydroxypyridine derivative substituted in 2 (11.4 mmol _/ 1 eq). The mixture is stirred at room temperature overnight, then concentrated to dryness. The residue is taken up with CH ₂ C1 ₂ and the solution obtained is washed with a 10% aqueous solution of NaHC0 ₃ , water, brine, and dried with Na ₂ S0 ₄ before concentration until dryness. The residue is chromatographed on silica gel to give the desired derivative: 3- [3- (N-tertbutyloxycarbonylamino) - l-propoxy] pyridine (or tert-butyl ester of [3- (pyridin-3-yloxy) -propyl] acid] carbamic).

Example 2

General procedure for deprotection by TFA of N-tertbutoxycarbonylamino functions.

To 3 to 7 mmol of the terbiobutyl ester of [3- (pyridin-3-yloxy) -propyl] -carbamic acid suitable in 5 ml of CH ₂ Cl ₂ , 2 ml of TFA are added. The solution is stirred for 45 minutes at room temperature and concentrated to dryness.

The residue is redissolved in 2 mL of CH ₂ C1 ₂ and concentrated again to dryness (2 times) to give 3- (pyridine-3-yloxy) -propylamine, in the form of an oily residue.

Example 3

General procedure for the formation of maleimido.

A from 2 to 3 mmoles of the

3- (adequate pyridin-3-yloxy) -propylamine in 5 ml of

THF, 500 mg of maleic anhydride are successively added (molecular weight: 98.06; 5.1 mmol) and

200 mg hydrated p-toluenesulfonic acid (mass molecular: 190.22; 1.0 mmol). The solution is refluxed for 24 hours and concentrated to dryness. The residue is chromatographed on silica gel to give the desired 1- [3- (pyridin-3-yloxy) -propyl] -pyrrole-2,5-dione derivative.

Example 4

Preparation of 2-fluoro-3-hydroxypyridine

At 100 mL of pyridine hydrogen fluoride (Py. (HF) _X , from FLUKA, 70% of the sample consisting of hydrogen fluoride, 30% of the sample consisting of pyridine) cooled to 0 ° C. successively and carefully add 3.7 g of 2-amino-3-hydroxypyridine (molecular weight: 110.12; 33.6 mmol) and 3 g of NaN0 ₂ (molecular weight: 69.00; 43.5 mmol). The mixture is stirred at 0 ° C for 1 hour, then slowly made basic with an aqueous solution of NaOH, 10 N, transferred to a decanter and extracted with EtOAc. The organic phases are combined, washed with water, brine, dried with Na ₂ S0 ₄ and concentrated to dryness. The residue is purified by passage through a column of silica gel (eluent: heptane / EtOAc: 50/50) to give 2.5 g (65%) of 2-fluoro-3-hydroxypyridine in the form of a solid which is used without further purification.

Rf (EtOAc / heptane: 80/20): 0.65 mp:

131 ° C. ² H NMR (DMSO-d ₆ , 298K): δ: 10.41 (s, 1H); 7.64 (td, J: 1.7 & 4.7 Hz, 1H); 7.42 (dd, J: 1.7, 1.7 & 10.8 Hz, 1H); 7.17 (ddd, J: 1.3, 4.7 & 7.8 Hz, 1H). ¹³ C NMR (DMSO-298K): δ: 152.8 (d, jc: 233 Hz, C); 140.2 (d, J ² _F _c: 27 Hz, C); 135.6 (d, J ³ _FC ): 13 Hz, CH); 126.2 (d, J ³ _F. _C : 5 Hz, CH); 122.6 (CH) .MS (DCI / NH ₄ ⁺ ): C ₅ H ₄ FNO: 131 [M + NH ₄ ⁺ ]; 114 [M + H ⁺ ]. Anal. (C ₅ H ₄ FNO) C, H, N.

Example 5

In this example, we describe the preparation of a reference molecule which is 1- [3- (2-fluoro-pyridin-3-yloxy) -propyl] -pyrrole-2, 5-dione, in which fluorine n is not radioactive fluorine, but ¹⁹ F.

a) Preparation of the tert-butyl ester of [3- (2-fluoro-pyridin-3-yloxy) -propyl] -carbamic acid.

The procedure described above in Example 1 is used with the 2-fluoro-3-hydroxypyridine prepared in Example 4 (1.29 g; 11.4 mmol) to give 1.9 g (62%) of tertiary butyl ester of [3- (2-fluoro-pyridin-3-yloxy) -propyl] -carbamic acid in the form of a yellow oil, after “flash” chromatography (eluent: CH ₂ C1 ₂ pure, heptane / EtOAc: 70/30 to 50/50). Rf (CH ₂ CL ₂ / EtOAc: 95/5): 0.45. ^X H NMR (CD ₂ C1 ₂ , 298K): δ: 7.68 (dt, J: 4.8 & 1.8 Hz, 1H); 7.28 (td, J: 7.8 & 1.5 Hz, 1H); 7.10 (dd, J: 5.1 & 0.9 Hz, 1H); 4.96 (b, ι ₂ : 20 Hz, 1H); 4.07 (t, J 6.0 Hz, 2H); 3.28 (q, J: 6.0 Hz, 2H); 1.98 (q ⁵ , J 6.0 Hz, 2H); 1.39 (s, 9H). ¹³ C NMR (CD ₂ C1 ₂ , 298K): δ 156.3 (C); 154.1 (d, J1F-C: 235 Hz, C); 142.5 (d, J ² _F -c: 25 Hz, C); 137.5 (d, J ³ _FC : 13 Hz, CH); 123.0 (CH); 122.2 (CH); 79.2 (C); 67.3 (CH ₂ ); 38.0 (CH ₂ ); 29.8 (CH ₂ ); 28.5 (CH ₃ ).

b) Preparation of the

3- (2-fluoro-pyridin-3-yloxy) -propylamine.

The procedure described above in Example 2 is used with the tert-butyl ester of [3- (2-fluoro-pyridin-3-yloxy) -propyl] - carbamic acid prepared in a) (1, 0 g; molecular weight: 270.30; 3.7 mmol) to give 1.4 g / 95%) of 3- (2-fluoro-pyridin-3-yloxy) - propylamine. 2 TFA, in the form of a yellow oil.

^ NMR (CD ₃ 0D, 298K): O: 7.51 (bt, J <2.0 Hz, 1H); 7.36 (bt, J <3.0 Hz, 1H); 7.04 (bq, J <3.0 Hz, 1H); 4.03 (t, J: 6.0 Hz, 2H); 2.99 g (Q, J: 6.0 Hz, 2H); 2.01 (q ⁵ , J: 6.0 Hz, 2H). ¹³ C NMR (CD ₃ 0D, 298K): Ô: 159.3 (q, J ² _F - _C : 41 Hz, C, CF ₃ C0 ₂ H); 155.3 (d, J ^{1F "C} : 237 Hz, C); 143.5 (d, J ² _F. _C : 24 Hz, C); 138.5 (d, J ³ _FC : 13 Hz, CH) ; 125.1 (CH); 123.8 (CH); 116.4 (q, J _C • '284 Hz, C, CF ₃ , C0 ₂ H); 67.9 (CH ₂ ); 38.6 ( CH ₂ ); 29.4 (CH ₂ ). c) 1- [3- (2-fluoro-pyridin-3-yloxy) -propyl] -pyrrole-2, -5 dione.

The procedure, described above in Example 3, is used with 3- (2-fluoro-pyridin-3-yloxy) - propylamine .2TFA (1.0 g; molecular weight: 398.23; 2, 5 mmol) to give 310 mg (48%) of 1- [3- (2-fluoro-pyridin-3-yloxy) - propyl] -pyrrole-2,5-dione, in the form of a yellow powder, after "flash" chromatography (eluent: heptane / EtOAc: 50: 50 to 30/70). For analysis purposes, an aliquot (100 mg) was further purified by preparative or semi-preparative HPLC.

Rf (EtOAc): 0.7 Rf (EtOAc / heptane:

80/20): 0.5. ^X H NMR (CD ₂ C1 ₂ , 298K): δ: 7.69 (bd, J: 3.0 Hz, 1H); 7.27 (t, J: 6.0 Hz, 1H); 7.11 (dd, J: 3.0 & 6.0 Hz, 1H); 6.69 (s, 2H); 4.05 (t, J: 6.0 Hz, 2H); 3.82 (t, J: 6.0 Hz, 2H); 2.11 (q ⁵ , J: 6.0 Hz, 2H). ¹³ C NMR (CD ₂ Cl ₂ , 298K): δ: 171.2 (2xC); 154.0 (d, J ^{1F "C} : 235 Hz, C); 142.4 (d, J ² _F. _C : 25 Hz, C); 137.7 (d, J ³ _F -c: 13 Hz, CH) _/ 134.5 (2xCH); 123.2 (CH); 122.2 (CH); 67.5 (CH ₂ ); 35.4 (CH ₂ ); 28.5 (CH ₂ ). MS ( DCI / NHR ^+): Cι HnFN ₂ ₂ 0 _3: 251 [M + H ^+].

Example 6

Preparation of the tert-butyl ester of [3- (2-nitro-pyridin-3-yloxy) -propyl] - carbamic acid. The procedure described above in Example 1 is used with 2-nitro-3-hydroxy pyridine (1.6 g; molecular weight: 140.10; 11.4 mmol) to give 2.2 g (65 %) of the tert-butyl ester of [3- (2-nitro-pyridin-3-yloxy) -propyl-] carbamic acid in the form of a yellow oil, after “flash” chromatography (eluent: heptane / EtOAc: from 60/40 to 40/60). For analysis purposes, an aliquot (100 mg) is further purified on a preparative or semi-preparative HPLC apparatus.

Rf (EtOAc / heptane: 50/50): 0.35. ^X H NMR (CD ₂ C1 ₂ , 298K): δ: 8.04 (t, J: 3.0 Hz, 1H); 7.53 (d,

J: 3.0 Hz, 2H); 4.95 (b, w _1/2 : 15. Hz, 1H); 4.18 (t,

J: 6.0 Hz, 2H); 3.26 (q, J: 6.0 Hz, 2H); 1.99 (q ⁵ ,

J: 6.0 Hz, 2H); 1.40 (s, 9H). ¹³ C NMR (CD ₂ C1 ₂ , 298K): δ: 156.3 (C); 149.2 (C); 147.3 (C); 139.5 (CH); 129.2 (CH); 124.0 (CH); 79.2 (C); 68.3 (CH ₂ ); 37.9

(CH ₂ ); 29.5 (CH ₂ ); 28.4 (CH ₃ ).

Example 6 bis:

Preparation of trifluoromethanesulfonate

[3- (3- fcerfc-butoxycarbonylamino-propoxy) -pyridin-2-yl] - trimethyl-ammonium

The procedure described above in Example 1 is used with 2-dimethylamino-3-hydroxypyridine (0.250 g; molecular weight: 138.17; 1.8 mmol) to give 0.290 g of the tert-butyl ester of [3- (2-dimethylamino-pyridin-3-yloxy) -propyl] -carbamic acid (58%) as a yellow oil after “flash” chromatography (eluent: heptane / EtOAc: from 70/30 to 50/50).

Rf (CH ₂ Cl ₂ / EtOAc: 50/50): 0.30. ^ Η NMR (CD ₂ C1 ₂ , 298K): δ: 7.79 (dd, J: 4.0 & 1.5 Hz, 1H); 7.00 (dd, J: 7.8 & 1.2 Hz, 1H); 6.73 (dd, J: 7.8 &. 4.8 Hz, 1H); 5.15 (b, vr _1/2 : 15 Hz, 1H); 4.02 (t, J: 6.0 Hz, 2H); 3.30 (q, J: 6.0 Hz, 2H); 2.95 (s, 6H); 1.99 (q ⁵ , J: 6.0 Hz, 2H); 1.42 (s, 9H). ¹³ C NMR (CD ₂ C1 ₂ , 298K): δ: 156.2 (C); 153.7 (C); 146.2 (C); 139.0 (CH); 118.8 (CH); 115.9 (CH); 79.1 (C); 67.4 (CH ₂ ); 41.1 (CH ₃ ); 38.8 (CH ₂ ); 32.3 (CH ₂ ); 28.5 (CH ₃ ).

The tertiary butyl ester of [3- (2-dimethylamino-pyridin-3-yloxy) -propyl] -carbamic acid is then diluted in toluene (2 ml per 100 mg of ester) and the solution is cooled to 0 ° C (ice bath). To this solution is added methanetrifluoromethanesulfonate (50 microliters for 100 mg of ester) and the reaction medium is stirred for 1 hour at 0 ° C. The precipitate of trifluoromethanesulfonate of [3- (3-tert-butoxycarbonylamino-propoxy) -pyridin-2-yl] -trimethyl- ammonium is filtered, washed with small portions of ethyl ether and dried under vacuum to yield a fine powder. white (145 mg per 100 mg of ester).

^X H NMR (CD ₂ C1 ₂ , 298K): δ: 8.09 (dd, J: 3.3 & 1.0 Hz, 1H); 7.67 (bd, J: 8.0 Hz, 1H); 7.61 (dd, J: 7.0 & 4.2 Hz, 1H); 5.20 (b, v _{1 2} : 15 Hz, 1H); 4.31 (t, J: 6.3 Hz, 2H); 3.71 (s, 9H) 3 .: 31

(q, J: 6.3 Hz, 2H); 2.12 (q ⁵ , J: 6.3 Hz, 2H) / 1 .: 38 (S, 9H). ¹³ C NMR (CD ₂ C1 ₂ , 298K): δ: 156.6 (C) 147 .7

(VS) ; 142.6 (C); 139.0 (CH); 129.0 (CH) 124 .6

(CH); 121.2 (q, J: 319 Hz, CF ₃ ); 79.3 (C) 68.1

(CH ₂ ); 54.8 (CH ₃ ); 37.5 (CH ₂ ); 30.0 (CH ₂ ) 7 28 .4 (CH ₃ ).

Example 7

In this example, the preparation of a compound according to the invention is described, which is 1- [3- (2- [ ¹⁸ F] fluoro-pyridin-3-yloxy) -propyl] -pyrrole-2,5 - dione.

a) Complex K [ ¹⁸ F] FK ₂₂₂ .

In order to recover and recycle the water target [ ¹⁸ 0], it is made to pass through an anion exchange resin (AGlx8, from Bio-Rad, 100-200 mesh). The fluoride ion [ ¹⁸ F] is then eluted from the resin, using 1.0 ml of an aqueous solution of KCO ₃ at 4.5 mg / ml.

After addition of 11.0 to 15.0 mg of KRYPTOFIX ^® K ₂₂₂ (4, 7, 13, 16, 21, 24-hexaoxa-1,10-diazobicyclo [8.8.8] hexacosane), the resulting solution is then gently concentrated to dryness at 145-150 ° C, under a stream of nitrogen for 10 minutes to give a pure K [ ¹⁸ F] FK ₂₂₂ complex, in the form of a white semi-solid residue. b) 1- [3- (2- [ ¹⁸ F] fluoro-pyridin-3-yloxy) - propyl] -pyrrole-2, 5-dione.

DMSO, freshly distilled (600 μL), containing 4.0 to 6.0 mg of the marker precursor “nitro” (tert-butyl ester of [3- (2-nitro-pyridin-3-yloxy) -propyl acid) ] -carbamic) is added directly to the tube containing the dried K [ ¹⁸ F] -K ₂₂₂ complex. The tube (unsealed) is then placed in a heating block (at 145 ° C for 4 minutes). The tube is then cooled using an ice / water bath and the remaining radioactivity is measured.

85% to 95% of the initial activity placed in the container is still present. The reaction mixture obtained, dark in color, is then analyzed by radiochromatography. The incorporation yields are calculated from the TLC radiochromatogram and are defined by the area ratio of the tert-butyl ester derivative of [3- (2- [ ¹⁸ F] fluoro-pyridin-3-yloxy) -propyl acid. ] - carbamic on the total activity of ¹⁸ F fluor-18 (Si0 ₂ -CCM; eluent: EtOAc; Rf:: 0.75 and Rf: fluoride ion [ ¹⁸ F]: 0.0). The reaction mixture is diluted with 1 ml of water and transferred to a C18 Sep-pak (waters) cartridge. The tube is rinsed twice with 1 mL of water, which is also transferred and added to the diluted reaction mixture on the cartridge.

The assembly is then passed through the cartridge. The cartridge is washed with 3 ml of water and partly dried for 0.5 minutes, sending a stream of nitrogen. The tert-butyl ester derivative of • [3- (2- [ ¹⁸ F] fluoro-pyridin-3-yloxy) -propyl] - carbamic acid is eluted from the cartridge with 3 mL of dichloromethane in a reaction flask containing 0.1 mL of TFA. 2 ml of dichloromethane are used twice to wash the cartridge and to completely transfer the derivative labeled with [ ¹⁸ F] mentioned above (5% of the amount of total radioactivity, involved in the fluorination process, remains on the cartridge) . The incorporation yield is also confirmed after the elution of Sep-pak by the ratio of the count values of CH ₂ C1 ₂ to total eluted radioactivity (DMSO / H ₂ 0 + CH ₂ C1 ₂ ). The resulting CH ₂ C1 ₂ / TFA solution (50/1, V / V) is concentrated to dryness (at 65-75 ° C) under a moderate stream of nitrogen for 4 to 6 minutes). The deprotection yield is quantitative: No molecule, described above, protected by BOC can be detected by radiochromatography. The residue, above, is redissolved in 2 mL of CH ₂ C1 and concentrated again to dryness to minimize the presence of TFA (at 65-75 ° C under a moderate stream of nitrogen for 4 to 6 minutes). The residue is then diluted with 0.5 mL of xylene containing 25 mg of N-methoxycarbonylmaleimide. The container is then hermetically closed, heated for 5 minutes at 190 ° C (strong reflux), then cooled for 2 minutes, using an ice / water bath. The reaction mixture is then injected onto a column of semi-preparative HPLC. Isocratic elution [eluent: heptane / EtOAc: 50/50; flow rate: 6.0 mL / minute] which gives 1- (3- (2- [ ¹⁸ F] fluor-pyridin-3-yloxy) -propyl] -pyrrole-2, 5- labeled dione, pure, retention time: 7.5 to 8.0 minutes.

Typically, 60 to 70 mCi of 1- [3- (2- [ ¹⁸ F] fluor-pyridin-3-yloxy) -propyl] -pyrrole-2, 5-dione labeled, pure, can be obtained in 75 to 85 minutes , from 550-650 mCi of a production batch

[ ¹⁸ F] F ^" of a cyclotron.

Example 7a:

The compound labeled with fluorine-18, 1- [3- (2- [ ¹⁸ F] fluoro-pyridin-3-yloxy) -propyl] -pyrrole-2, 5-dione can also be prepared by repeating steps in) and b) of the process described in Example 7, still using as labeling precursor, the compound “nitro” (tert-butyl ester of [3- (2-nitro-pyridin-3-yloxy) -propyl] acid] -carbamic), but by modifying the final part of the preparation (step c)) in the following way (variant according to which step c) is carried out in a biphasic mixture of dioxane and aqueous sodium bicarbonate).

After deprotection of the amine function (TFA / CH ₂ C1 ₂ ), the residue obtained after concentration to dryness is taken up in 0.250 ml of dioxane containing 25 mg of N-methoxycarbonylmaleimide. To this solution, 0.750 L of a saturated aqueous sodium bicarbonate solution are added and the preparation is vortexed at room temperature for 10 minutes. The reaction mixture is then diluted with 1 ml of water and transferred to a C18 Sep-pak cartridge (Waters). The flask is rinsed 2 times with 1 mL of water, which is also transferred and added to the diluted reaction mixture on the cartridge. Finally 8 ml of water are further added to the reaction mixture diluted on the cartridge. The assembly is then passed over the cartridge. The cartridge is washed with 3 mL of water and partially dried for 0.5 minutes, sending a stream of nitrogen. The derivative labeled with fluorine-18 (1- [3- (2- [ ¹⁸ F] fluoro-pyridin-3-yloxy) -propyl] -pyrrole-2, 5-dione) is eluted from the cartridge with 3 mL of dichloromethane in a new empty vial. 1 ml of dichloromethane is used twice to wash the cartridge and to completely transfer the derivative labeled with [ ¹⁸ F] mentioned above. The solution containing the derivative labeled with [ ¹⁸ F] mentioned above is concentrated (at 65-75 ° C, under a moderate stream of nitrogen for 3 to 5 minutes) to a volume of approximately 1 mL and injected on a column of semi-preparative HPLC. The purification is identical to that described in Example 7.

Example 7b:

The fluorine-18 labeled compound, 1- [3- (2- [ ¹⁸ F] fluoro-pyridin-3-yloxy) -propyl] -pyrrole-2,5-dione can also be prepared by repeating steps a) and b) of the process described in Example 7 or 7a, but using as labeling precursor, the compound "trimethylammonium-trifluoromethane sulfonate" (trifluoromethanesulfonate of [3- (3-tert- butoxycarbonylamino-propoxy) -pyridin-2-yl] -trimethyl- ammonium).

Example 8

In this example, the labeling of a peptide, namely the peptide N-acetyl-Lys-Ala-Ala-Ala-Ala-Cys-amide, is described by a compound according to the invention, which is 1- [3 - (2- [ ¹⁸ F] fluor-pyridin-3-yloxy) -propyl] -pyrrole-2, 5-dione.

The procedure is as follows:

To an equivalent of peptide (2 mg / mL; 200 nmol; 36.2 μL) in solution in 50 mM Tris buffer, 150 mM NaCl, pH = 7.4, 1 equivalent of TCEP in Tris buffer is added (7.9 mg / mL; 7.3 μL; 200 nmole).

The sample is left 5 min at room temperature and is then diluted in 1 ml of Tris buffer. Dry synthon

1- [3- (2- [ ¹⁸ F] fluor-pyridin-3- ^ yloxy) -propyl] -pyrrole-2, 5- dione is taken up in lOOμL of a 50/50 heptane / ethyl acetate mixture, and the reduced peptide solution is added. The sample is left for 10 minutes at ambient temperature, shaking from time to time.

The labeled peptide is purified by HPLC on column C18 with a gradient of 0 to 34% acetonitrile / 0.1% TFA in

H ₂ 0 / 0.1% TFA in 30 min (DeltaPak C18 column,

R _t - _pept i _d e = 28mn).

Claims

1. Compound of general formula (I)

in which: m represents an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; - n represents an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

Y represents a group chosen from monocyclic or bicyclic heterocyclic groups chosen from imidazolyl, pyrazolyl, benzimidazolyl, pyridinyl, piridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, possibly quinoxalinyl substituted by one or more substituents each of said substituents being independently selected from hydrogen, halogen, phenyl, alkyl Cι_ ₆ alkoxy Cι_ _6, aryloxy, amino, mono- or di (alkyl Cι_ ₆₎ amino, mono- or di (aryl) amino, thio, Cι_ ₆ -thio alkyl, arylthio, formyl, alkyl. Cι_ ₆ -carbonyle, arylcarbonyle, carbonyle, alkoxy Cι_ ₆ -carbonyle, aryloxycarbonyle, 04/002984

44

Cι- alkyl. ₆ - aminocarbonyl, arylaminocarbonyl, trifluoromethyl;

X represents a radical of formula:

(U) _a - ((CRιR ₂ ) _b - (V) _c ) _d - ((CR ₃ R ₄ ) _e - (W) _f ) _g -

in which: a, b, c, d, e, f, g each independently represent an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 _/

U, V and W each independently represent o -NRi-, -0-, -S-, —N—, ethynyl, -CRι = CR ₂ -, - (C = 0) -, - (C = S) -, -C (= NR _ι ) -, -C (= 0) 0-, - (C = S) S-, -C (= NRι) NR ₂ -, -CR _X R ₂ -, -CRιOR ₂ -, - CRιNR ₂ R ₃ -, where i, R ₂ , R ₃ and R ₄ are each independently chosen from hydrogen, halogens, phenyl groups, C _α - ₆ alkyl, Cι- ₆ alkoxy, aryloxy, amino, mono- or di (Cι_ _ë ) amino, mono- or di (aryl) amino, thio, Cι- ₆ -thio alkyl, arylthio, formyl, Cι-6-carbonyl alkyl, arylcarbonyl, Cι alkoxy carbonyl _{- 6} -carbonyl, aryloxycarbonyl, C1-s-aminocarbonyl, arylaminocarbonyl, trifluoromethyl.

2. Compound of formula (I) according to claim 1, in which n = l, and Y is a group

3-pyridyl.

3. Compound according to claim 2, which corresponds to the following formula (II): 04/002984

45

where p is an integer from 1 to 10, such as 2, 3, 4, 5, 6, 7, 8 or 9.

4. Compound of formula (II) according to claim 3, which is chosen from: 1- [2- (2- [ ¹⁸ F] fluoro-pyridin-3 -yloxy) - ethyl] -pyrrole-2, 5-dione ; 1- [4- (2- [ ¹⁸ F] fluoro-pyridin-3 -yloxy) -butyl] -pyrrole-2,5-dione;

- 1- [5- (2- [ ¹⁸ F] fluoro-pyridin-3 -yloxy) - pentyl] -pyrrole-2, 5-dione;

- 1- [6- (2- [ ¹⁸ F] fluoro-pyridin-3 -yloxy) - hexyl] -pyrrole-2, 5-dione; - l - [(2- [ ¹⁸ F] fluoro-pyridin-3-yloxy) - methyl] -pyrrole-2, 5-dione; 1- [3- (2- [ ¹⁸ F] fluoro-pyridin-3 -yloxy) - propyl] -pyrrole-2, 5-dione.

5. Compound according to claim 2, which corresponds to the following formula (III):

4/002984

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in which q and r independently represent an integer of 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9. 6. Compound of formula (III) according to claim 5, who is chosen from:

- 1- {4- [2- (2- [ ¹⁸ F] fluoro-pyridin-3-yloxy) -ethyl] -phenyl} -pyrrole-2, 5-dione;

- 1- [4- (2- [ ¹⁸ F] fluoro-pyridin-3-yloxymethyl) -phenyl] -pyrrole-2, 5-dione;

- 1- [4- (2- [ ¹⁸ F] fluoro-pyrridin-3-yloxymethyl) -benzyl] -pyrrole-2, 5-dione.

7. Compound according to claim 2, which corresponds to the following formula (IV):

where s is an integer from 1 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9.

8. Compound of formula (IV) according to claim 7, which is: 1- [3- (6- [ ¹⁸ F] fluoro-pyridin-3-yl) - propyl] -pyrrole-2, 5-dione.

9. Compound according to claim 2, which corresponds to the following formula (V): 04/002984

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in which t is an integer from 0 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 and T is a group -CH≈CH- or -C≡C-.

10. Compound according to claim 9, which is chosen from:

- 1- [3- (6- [ ¹⁸ F] fluoro-pyridin-3-yl) - allyl] -pyrrole-2, 5-dione; - 1- [3- (6- [ ¹⁸ F] fluoro-pyridin-3-yl) - prop-2-ynyl] -pyrrole-2, 5-dione.

11. Use of a compound according to any one of claims 1 to 10, for labeling a macromolecule.

12. Use according to claim 11, wherein said macromolecule is chosen from oligonucleotides, proteins, antibodies and peptides.

13. Use according to any one of claims 11 and 12, in which the macromolecule is a macromolecule for recognition of a specific site.

14. Use according to claim 13, in which said specific site is chosen from sites presenting specific target molecules 4/002984

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pathology, such as sites of apoptosis, necrosis, tumor area.

15. Complex comprising a macromolecule coupled to a compound according to any one of claims 1 to 10.

16. The complex according to claim 15, wherein said macromolecule is chosen from oligonucleotides, proteins, antibodies and peptides.

17. The complex as claimed in claim 16, in which said coupling is carried out by reaction of the double bond of the maleimido group with, specifically, an -SH function of cysteine in the case of a peptide, or a phosphoro-thioate function in the case of an oligonucleotide.

18. Complex according to any one of claims 15 to 17, in which the macromolecule is a macromolecule for recognition of a specific site.

19. The complex as claimed in claim 18, in which said specific site is chosen from sites presenting target molecules specific to a pathology, such as sites of apoptosis, necrosis, tumor area.

20. Analysis and detection kit, for example for medical imaging comprising a compound according to any one of claims 1 to 10 and a macromolecule.

21. Analysis and detection kit, for example for medical imaging comprising a complex 4/002984

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according to any one of claims 15 to 19 coupled to a macromolecule.

22. Diagnostic kit comprising a compound according to any one of claims 1 to 10 and a macromolecule.

23. A diagnostic kit comprising a complex according to any one of claims 15 to 19.

24. Use of a complex according to any one of claims 15 to 19 or a compound according to any one of claims 1 to 10 in a medical imaging method, such as positron emission tomography (PET) ).

25. Use of a complex according to any one of claims 15 to 19 or of a compound according to any one of claims 1 to 10 for manufacturing a product intended for medical imaging, for example by emission tomography of positrons (PET).

26. A product for medical imaging, in particular positron emission tomography comprising a complex according to any one of claims 15 to 19 or a compound according to any one of claims 1 to 10 with a pharmaceutically acceptable vehicle.

27. A process for the preparation of a compound of formula (I) according to any one of claims 1 to 10, in which: a) a precursor compound of formula (la) is brought into contact: 4/002984

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PR ₂

in which PRi and PR ₂ independently represent a hydrogen atom or a group protecting the amino function, provided that PR _X and PR ₂ are not both a hydrogen atom, or PR _X and PR ₂ form together with the nitrogen atom a cyclic protecting group for the amino function, Gp represents a leaving group capable of being replaced by a fluorine-18 atom, and X, Y, m and n have the meaning already given in the claim 1, with a source of fluoride ions F ^" labeled with [ ¹⁸ F] to give a compound of formula (Ib):

b) eliminating in compound (Ib), the protective group or groups PRi and / or PR ₂ of the amino function, to give a compound of formula (Ie):

H ₂ N-_ ₍ χ _{) π} 18r

(Y) n- (le)

c) the compound (le) is reacted with a reagent capable of giving a maleimido group to 04/002984

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from an amino group, to obtain the final compound of formula (I).

28. The method of claim 27, wherein the PRi and PR ₂ groups when they are protecting groups are chosen from tert-butoxycarbonyl (BOC) and fluorenylmethoxy carbonyl (FMOC) groups, or else they form together with the atom amino function nitrogen a phthalamido group.

29. A method according to any one of claims 27 and 28, wherein the group Gp is chosen from halogens, such as F, Cl, Br, I, mesyl, tosyl and triflate groups when Y is an alkyl group; and among the halogens, ammonium salts such as trimethylammonium-trifluoromethane sulfonate, and the nitro group, when Y is an aromatic or heterocyclic group.

30. Method according to any one of claims 27 to 29, in which, in step a), the source of fluoride ions labeled with ¹⁸ F comprises said fluoride ions and a counterion chosen from large cations , such as rubidium, and tetrabutylammonium, and small cations, such as potassium, sodium and lithium, said small cations being stabilized by a cryptand or a crown ether suitable for said small cation .

31. Method according to any one of claims 27 to 30, in which step b) is 04/002984

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carried out by bringing the compound (Ib) into contact with TFA in CH ₂ C1 ₂ for a period of 1 to 5 minutes.

32. Process according to any one of claims 27 to 31, in which the reagent capable of giving a maleimido group from an amino group, in step c), is chosen from N-methoxycarbonylmaleimide and succinimide.

33. Process according to any one of claims 27 to 32, in which step c) is carried out in a solvent such as xylene, THF with heating at a temperature of 100 to 200 ° C, for example 190 ° C for a period of 1 to 20 minutes, for example 5 minutes.

34. Process according to any one of claims 27 to 32, in which step c) is carried out in a biphasic mixture, for example of dioxane and aqueous sodium bicarbonate, at room temperature for a period of 3 to 15 minutes , for example 10 minutes.

35. Process according to any one of claims 25 to 34, in which the compound of formula (la) corresponds to the following formula (IIa):

in which pa the meaning already given in claim 3. 4/002984

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36. The method of claim 35, in which the compound of formula (IIa) corresponds to the following formula (Ilb):

37. Method according to any one of claims 27 to 34, in which the compound of formula (la) corresponds to the following formula (Illa):

in which q and r have the meaning already given in claim 5.

38. The method of claim 37, in which the compound of formula (Illa) corresponds to the following formula (Illb): 04/002984

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39. Process according to any one of claims 27 to 34, in which the compound of formula (la) corresponds to the following formula (IVa):

in which s has the meaning already given in claim 7.

40. Method according to claim 39, in which the compound of formula (IVa) corresponds to the following formula (IVb):

41. Method according to any one of claims 27 to 34, in which the compound of formula (la) corresponds to the following formula (Va): 4/002984

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in which t and T have the meaning already given in claim 9.

42. Process according to claim 41, in which the compound of formula (Va) corresponds to the following formula (Vb):

43. A precursor compound of formula (la), as defined in any one of claims 27 to 29.

44. Compound precursor of formula (IIa),

(Ilb), (Illa), (Illb), (IVa), (IVb), (Va), (Vb) as defined, respectively, in claims 35, 36, 37, 38, 39, 40, 41 and 42.

45. Compound, precursor of a compound of general formula (I), which is chosen from the compounds of claims 4, 6, 8, and 10, in which the

[ ¹⁸ F] is replaced by a non-radioactive halogen, such as ¹⁹ F, Cl, Br, I, an ammonium salt, such as trimethylammonium-trifluoromethanesulfonate or a 4/002984

56

group -N0 ₂ , and the group l-pyrrole-2, 5-dione is replaced by a tert-butoxycarbonylamino group.

46. A precursor compound according to claim 45, which is the

[3- (3-tert-butoxycarbonylamino-propoxy) -pyridin-2-yl] - trimethyl-ammonium trifluoromethanesulfonate; the tert-butyl ester of the acid

[3- (2-nitro-pyridin-3-yloxy) -propyl] -carbamic.