WATER-SOLUBLE ACTIVABLE MOLECULAR PROBES, INTERMEDIATES FOR THEIR SYNTHESIS AND DETECTION METHODS TH

专利摘要:
The invention relates to probes of formula (I): in which: X1 = NH, O or S; X2 = O or S; SE is a leaving group, which may be eliminated under the action of a stimulus which may notably be the presence of an enzyme, a chemical compound or a physicochemical characteristic of the medium in which the probe is located; A is an aromatic group, which after cleavage in aqueous solution of the C (X2) -O bond, leads to the release of a chromophore or a fluorophore, R represents a hydrogen atom or - (L) n- GP, where n is 0 or 1, L is a linker, and GP is a water-solubilizing group, and their physiologically acceptable salts, solvates or hydrates. The invention also relates to the intermediates for their synthesis and detection methods implementing them.
公开号:FR3022784A1
申请号:FR1456014
申请日:2014-06-26
公开日:2016-01-01
发明作者:Jens Hasserodt；Maxime Prost
申请人:ECOLE NORM SUPERIEURE LYON；Centre National de la Recherche Scientifique CNRS；Universite Claude Bernard Lyon 1 UCBL；Ecole Normale Superieure de Lyon；
IPC主号:

专利说明:

[0001] The invention relates to s. Monomers useful in the diagnosis, incorporating a DrI - E Ir, which in response to a si rulus which may include a particular enzyme, a cyclic compound or a characteristically pHlysicochemical in itself the Sundcii will self-strive for a viable entity of the Pacific Rim type c. cre. bblogic or chemical, the detection of an enzymatic activity can be very useful. The organisms er11 {72.77 '. These cells or cell extracts, biological fluids or examples of biological or chemical samples in the enzymatic enzyme can be detected. Leensymes constitucini: ne: enix biomarqueus various pathologies also in many cellular processes and are therefore the subject of innumerable studies by cell biologists. Thus, their detection of information relating to a particular metabolic or morbid state, for example, therefore, probes capable of detecting a highly useful enzymatic activity and which have been subject to work in particular can be detected. the work of one of the inventors, the present invention, which is based on the corresponding patent application, and the applications WO 201, substrates, eptidases and WO 2014/020285, which describes Those which use a substance which is a substrate of the enzyme of interest to a gro, which, after the aging of the substrate, causes the cytoplasty of the sp ... The lemming of a rophorite (fluorophore leading to a molecular proton transfer in an excited nitrate, named ESIPT for Excited State Intramolecular Proton Ti Gold, a good solution for enzymatic activity. reliability of the results that they orgi to dissolve a single molecule rnle in order to ensure the Ffet, the aiM.Et of o-solvent obes generally decreases the activity of 7.7mtante enzymes. In addition, while low solubility and some in vitro testing, the passage of the in vivo systems requires the use of high solubility solutions. they are going to be the same way as in the case of Uri CO-5, in vivo is exc. The molecular molecule of hydrom L, of ad X tissues, not the ti7s, u_. This is the case, perhaps, of a Sideri, as experienced by Anal. Biochem. 1991, a preoccupation for 10 The first was, so many times. substrates, ::.: rizymatic5 ries for ph) sphatase caspasc (Liu et al., J. Am. (Jem Soc., 2012, 134, 1 ',' example, functi ,,, very well but it limits the The choice of the hepatic enzymes, since they can not be targeted, can not be targeted, and the negatively negatively charged cells have difficulty in membrane binding of extracellular enzymes. the grafting of the water-solubilizing groups on the signalophore used, often suironate grottoes in a one-ehetrahedron 2006, 62, 578, ShabEtt al.Am.Chem.Soc, 2011, 133, 10960. Still another hydrosolysis k The result is that it is unattractive to reduce the performance of the signal released in this way because of the enzymatic activity. we want to use ophores in the solid state, possibilities are not generalised at the highest level. with molecular spores, no enzymatic activity. There are, however, very few systems which allow the association of a hydrosolubilized resin with an enzymatic substrate for the purpose of releasing an active molecule. These are generally based on a spacer 770 eliminating 3022 784 (Sha t I. I am Chem, 372-7.1, DU on a combination of a cyclizing pacifier and min: .habat et al., Bioorg. Che,), 2e 15, 7318). The problem is that the substitute spacer is used to produce toxic hauterbent intermediates (Rokita, Quinone Methides, 1st ed., 2009, Uhn Wiley and Sons Inc., Bolton et al., Chem Res Toxicol. 1995, 8, 323, Thatcher et al., J. Org Chem 7, 62, 1820. On the other hand, the cyclization / elimination period is far from it because it is so complex that it is very small. 2LP in series) in kinetics which are to be satisfied by Bioorg, Mec: hem., 2012, 20, 3465), or a hydrostatic flessability compared with Negligible (Romieu et al Ora, Lett 200 $ 1 in, 1517) .These constructions deliver their ro: -Ecule tens of hours (Shabat et al., Bioconjugate Chem 201: 7, 1432), which 15 makes them difficult to use to quickly detect the absence or absence of certain analytes in a given sample.There is a desire to develop a new generation of cyclizing spacers for the versatile use of The enzyme substrate gateway, whether or not, is non-polar and meets the solubility requirements. Néanrions. this spacer will have to keep Lflc, effective collapse ra. In the generation t devn .: p.7,: - put unc. Recognition of the invention, the inventors propose bridges yes, on the one hand, offers a new solubility satil isante and, on the other hand, are easily synthesizable and motu I of the presence of a piperazine group, one of the atoms, orter various water-solubilising groups. The I have chosen to propose a solub.::in that incorporates a water-solubilizing portion st-comoosanteibe the probe, namely in a rele e 30 and substrate which allows choibr import any -nzym and any molée. The probes according to the invention correspond to formula (I): ## STR1 ## wherein X 1 = NH, O or, X 2 = O or S, SE is a leaving group, and in particle A is an aromatic group which after polishing the aqueous C (X 2) -O bond leads to the erection of a chromophore or a fluorophore, R represents a hydrogen atom or - (L n-GP, where n is 0 or 1, L is a linker, GP is a water-solubilizing group, and their salts, solvates or hydrates are physiologically acceptable. The present invention makes it possible to detect a chemical or enzymatic analyte capable of cleaving the X1-SE bond, by detecting the chromophorophorophore which will be released following the cyclization of the piperazinyl type spacer of the formula: The substrate according to the invention acts as a molecular probe capable of revealing the presence of a stimulus LI that an enzyme, which is iniquitous - a physicochemical property of the medium in which a change in pH is found. When the sea is cured by said ie is fragmented via a C77.:7'-I interaction to release a chrompphore or fluorophore is possible that the grouping. A also behaves chromophore or a fluorophore, when bound to the probes of this case of spectral properties probes, and er. The intensity of the absorbance of fluorescent fluorescence will be different: (a) under the action of an enzyrT-7, a chemical composition or a physicochemical characteristic of the medium in which it is located. In an advantageous manner, but with the proviso that the probe may be selected, it is possible for the probe of the formula (I) to be undetectable by mbon or emission. of light, before encountering the stimulus, especially form a chemical compound or physicochemical character of the (which is the probe, 15 (that is to say that is called "iurtive") The leaving group SE is such that its elimination, for example by enzym, or following a purely chemical action (such as a pH variation or the presence of a reducing agent), ase, by reiterating Preferably, the ir-molecular molecule of self-collapsing bi-release a fluoroonore or chrr-ophore, especially in the form of A-OH or A-O-, or A '= 0 (A' = 0 being t for I obtained after rearrangement of .4-0H, a tautomerism or polymerized form of A-OH J. The cleavage of the SE-X1 bonding nération of the fi or chromophore, especially sou A-DH or A-0-, or A1 = (A '= 0 and a form obtained after rearranging A-OH t A-O-, a tautomeric or polymerized form of A-C'1) by cyclizing the spacer as shown in Scheme 1 ci below: Scheme 1 A-OH S -H SE chemical / enzymatic reaction cyclization R. - composed of d. catr cc,,.,.,)) its an intelligent e., which carries, at one extreme, up to the other, iii an organic group A is a carbamate or thiocarbamate moiety which, when released by fragmentation of the spacer-AO, to give the corresponding product, leads to the formation of a chromophor or fluorophore detethle, iv) a water-solubilising group of the invention, the presence of the nitrogen atom carrying R group makes it possible to modulate the proprd of the probe and introduces a third functionalization, beside the SE leaving group and group A leading, after removal of the SE group and the cyclization of space, to the release of a detectable chore, dd-1 (,) phore or fluorophore.
[0002] Indeed, the forrnd probes (I) according to the invention comprise n multifunctional piperazine-type spacer which allows Vina a wide range of enzyme substrates, and CL ', the annex group R cu perncdcc ,,;. N function of its nature, to make (nolecule still pi '' s of the nitrogen atom, allows starting from a'eme z, r .. (1 - :, (dr-dediaire of synthesis to lead to Furthermore, the probes according to the invention have one, with respect to their homo-types comprising a piperidinyl type spacer, exemplified in US Pat. Nos. 2013/045854 and WO 2014/020285 and to the essential reagents, but not all of the spacers, such as polyvinyl acetate and the like. C) coumarin or - impure 4-methyl-7- (phenylacetamido) wumirine 4-n lumbelliferyl acetate Furthermore, as dn: 7 cases -; 7,77names WO 2013,15854 and WO 5 2014/020285, the sonci.E: has pre-organized spacer t for its cyclization after cleavage Ue, 'its SL-Xi, which accelerates the procer-- collapse of the spacer, while ensuring a good recon enzyme t on. However, it is by no means obvious that, with the exception of the spacer or in the context of the invention, in which groups 10 introduced to increase the water-solubility, there is no impact. report, probes. Now, in contrast, the introduction of a cerebral solution was slightly sterile at the level of the R group, and therefore, a p: UOR; close to the site of action of the stimulus (ie, an enzyme, a chemical compound or a variation of a physicochemical characteristic of millet, 3ns is found in 7, in f7: 7y7or the nature of the SE group) did not modify the naked reaction under the action of the corresponding Umulus. The invention therefore relates to the compounds of formula (I), whatever their implementation variant described in the present patent application, for the detection in vivo, in humans or animals, in vitri 7ulo or A stimulus such as an enzyme, a compound, has a physicochemical characteristic of the medium in a probe, the presence of which causes cleavage of the SE-X1 bond. another ace. The invention relates to a method according to the invention in which a stimulus such as an enzyme, a compound or a physicochemical characteristic of the medium in CQ can be detected. the presence of which leads to the cleavage of the SE-X1 bond, ie to detect the presence of a chemical compound or a characteristic, is found in the sorrlo. The medium in which the SE-X1 bond cleaves is cleaved, comprising contacting a suspect sample with a probe. (a) according to the invention, - the applicab, of covalent cleavage conditions between X1 and SE, which e5 10 of a Mye of the liai! C (X.2) the OA roup following a cyclization of the spacer prese - -onde of formula (I), and - quantitative analysis o .. chromopho.'e .4.) ore The échan, lion can be all é appropriate biological change. He may 15 notam; 3 of a biological sample, a sample of blood, I, serum! : .q, asma, urine, from a specimen or from isolated cells, especially in the case of an enriched type of preparation, may be used in combination with one another. slow, well known to those skilled in the art. Advantageously, the compounds of formula (I) behave as probes operating in a dye / dye mode than before cleavage by stimulation. The enzyme, a pure compound, is one of a chemical characterization of the medium in the probe, depending on the nature of the probe, the probe is susceptible. It is also possible to choose (- A, in order to obtain different spacers the group released after) C (X2) and the gr -, :: OO, and ii) the formula scrd ( I). In particular, the salt detection method (for example, it is carried out under physiological conditions, is aqueous at a pH of from 1 to 4, and in particular: The pH of the order of 7.4 In one embodiment of the invention, a fluorophore is released and its analysis comprises the steps of exposing the crematorium to a bright light capable of producing light at room temperature. The length of the fluorine ion, and the ionization of the ions in the method of the invention, are released and its analysis made by the source of the light produced by the light source. at a wrinkle absorption of the chromophore, and - detection of light absorbed in cases where a chromophore is lib, a color change will be detected following the enzyme, a chemical compound or a variatior. physicochemical characterisitics of the m in which is the concern, according to the nature of the group SE). Preferably, but not necessary, the fluorophore and chromophore, in particular in the form of A-OH or A-O-, or -D 20 (A '= 0 eta: a form obtained by rearrangement of A -Ori or A- (e-tiy ... ib .-. 11-1-ic or polym A-OH), forms an aqueous precipitate The invention will now be described in more detail. SE is a labile, water-soluble, aqueous group under the action of a stimulus, the presence of an enzyme, a chemical compound of a physicochemical cell, and a medium which is (-) uvea probe.The SE elir can be notoriously ::: To be carried out as an example As an example, it can be referred to as pH, or as a redox potential, lerrh,: n In the case where X = S. in particular, where X 1 = NH or O, the abile SE is susceptible to a compound, it is cleaved. chemical (target) or enzyme. An enzyme is defined as a protein which catalyzes e group 3E ensures the reconr of a target ompos' chemical or enzymatic physicochemical metmeter digue di and especially of enzyme, _ctivernelt localized in a tissue ....- 1 in a cell type The It is characterized by the ability of the molecule to act under the action of a molecule which is less susceptible to the presence of an enzyme, to the presence or physiochemical environment of the medium in which the probe is located. In the case where SE is an enzymatic substrate, the enzyme then acts as a catalyst for the between SE and the X1 atom to which it is attached. Such a cleavage is the consequence of hydrolysis in an aqueous medium for which lzyme will act as a catalyst. Although in this case even cleavage involves hydrolysis, there will be talk of cleavage under the actior which is catalytically active for the reactant, olyse. The choice of X1 will be adapted according to the size SE, and notamnrv selected enzyme substrate, so as to o, .., Hold the desired cleavage. In particular, X1 will be oxygen in the case where SE will be an esterase, glycosidase, phosphatase, sulphatase, or uronidase substrate, or NH in the case where SE will be a protease substrate, in particular ariclase, or transferase and X1 will be sulfur in the case where X1-SE will be a disulfide bridge nui will be cleaved by a reducing agent. In particular, the homology, depending on the a for the probes according to the invention, may choose a substrate for H. an enzyme selected from esterases such as carboxylesterases or lipases; alkaline phosphatases; glucuronidases such as 13-gluc monidase; y, rcosidases; proteases such as proteases; the peptidases and the SE group will preferably be specific to an enzyme of interest. On the other hand, these enzymes have the capacity to cleave a set of grotipemertits 5. among which we may mention hexosaminich.377, and esterases. Such F-enzyme substrates have been chosen from among the carbon-linked ecosyl yams, which have the remainder of the fatty acid molecule bound by their structure of the molecule. The remainder of the equilibrium is determined by a terminal acyl function or by a lat-link, or a group is defined as follows. with X1 which then represents NH. SE can be a substrate for a glycoside enzyme, and is a group containing a carboxylic acid, which is anomerically identical to the remainder of the molecule. "Glycosidase" is understood to mean a glycoside hydrolase enzyme. It has the capacity to catalyze the hydrolysis of glycolic bonds, to release at least one saccharide compound: "Glycosyl" skirt means any sugar, mono or ps linked to the rest of the molecule by a glycosyl linkage, ie via SV II anomeritic, the anomeric carbon adopt alpha or beta configuration.As an example of group SE, mono glycosyl groups are that is to say formed of saccharide units that are identical or different, the saccharide units can be selected from a hexose or pentose type, and selected by -tose, tmose, gulose, allose, altrose, Vidas (talose, fuc, ose, lyxose, ribose and: xylose, ru example The saccharidit.y..v units. can be used for staining L or qlycosyl groups possi. Substituting substrates for glycidylase can be used; as SE. The 7-alkyl groups can be used without, in particular, with the aid of acetyl or 10-amylol, LL 3 -3-acetylhexosamines, are preferably glycoside derivatives. Most often, the compounds of the invention are in the case of a glyosyl, hepar, or a copolymer with a block alea structure.
[0003] Examples of such compounds include the following glycidase substrates: mono-glycosylated esters selected from: osyl, glucosyl, mannosyl, gulosyl, allosyl, osyl, lido, talosyl and the like. fucosyl, fructos arabinosyl, lyxosyl, idyl, glucuronyl. and -hexosaminyl and 3-yl groups formed, especially 2 to 20, of prAferEni. 3 and especially from 4 to 6) of these monoglycosyl moieties ide read or c`` Examples of enzymes which may be targeted by the probes according to the invention include N-acetyl-β-galactosar, N-acetyl-β-glucosaminidase (γ-amylase; -arabinofuranosidase, α-JUR% β-chitobiosidase, α-galactosidase, lactosidase, α-glucosidase, β-glucosidase, β-glucuronidase, e-methylaltase, α-mannosidase, -mannosidase, p-xylosidase osidase, α-Fucosidase, 13-L-fucosidase, L-iduronidase and cellulase (Orenga, S., James A., Manafi, M., Perry, J. I , & Pincus, DH (2009) .Enzymatic substrates: rol. "- Logy Journal of Microbiological Methods, 79 (2), 139-155) SE can also be a 20-galactosid ubstrate. β-galactosidase, induronidase, glucosidase, N-acetyl D-glucosaminidase, N-acetyl-D-galactosaminidase, mannosidase, fucosidase or glucuron, especially P-glucosidase, Examples of such snout: s SE behave Galactosidase substrates are hereinafter adorned: mono-glycosyl groups selected from L-iduronyl, D-glucopyranosyl, D-galactopyranosinyl, N-acetyl-D galactosaminyl: D-mar. Fucopyranosyl and glycosyl-containing groups, preferably 2 to 20, of 1 to 10, and more particularly 6) of these identical or different groups. can also be an esterase substrate, especially lipase. In this case, an acyl group bonded by its C EU function remains in the molecule. In particular, SE is slightly lower than R 1, which represents, for example, an alkyl group of 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, and aryl SE. be a . a protease or drr tidase, in particular. a catechol or metalloprotease substrate, or PSMI Speciece Memt., which is rare, is characterized by a Dmino acid skirt bound by an acyl function, a side link remains of a molecule. 10 By group &quot; Neptidyl &quot; is understood to mean at least one amino acid linkage between them. In c. the invention, the oc., ir amines preser, L., in. S, ie, naturally occurring amino acids: no, but will preferably be selected from the crude amino acids (= proteinogenic), optionally in salified or protected form. The end time of the terminal amino acid may eventually be salified. As an example of salified form, there is little: citu, f3 hydrochloride, tosylate or trifluoroacetate. All peptidyl groups and peptidase substrates can be used as the SE group. Whether or not the amino acids are ionalised, particularly at the end of the particular embodiment, the peptidyl residue SE has amino acids to be the same or those selected from the group consisting of amino acids. Is. However, the N-terminal of the amino acid or of the epoxide group may be acyl-C 7, .alpha., Where R 1 is a (C 1 -C 6) alkyl or a group - 0- (Cr-C6) alkyl. The possible function of the N-terminus of a given probe with u, a (D-, CO-) is slow because the endoproteases interfere with the substrate. Tho grot free at its end. N-terminal functionalization will be preferred in the case where SE. In this case, an N-terminal acid is preferably salified under ammonium. Solid synttp (SPPS) by means of it. air protection groups which also contain groups Ps this context) often allows ntb. We simply have a probe with a carbameter at the end of the end. In the case of amino-p-libriase, it is preferred to use one amino acid in the endopoies; it is preferable to use a gro1. For example, the following peptidyl groups may be PitA-: Leu (for leucine aminopeptidase), Ser-Gln-Asn-Tyr (N-ter part of li-D perice). preferred cleavage of VIN-1), Asp-Glu-vA1- (,: His-Ser-SEr-Lys-Leu-C1 aspase, the prostate-specific antigen 'PSA xtrery N-terminal may be or be selected from a group consisting of a (C 1 -C 6) alkyl group or a -O- (CH 2) group (for example, group COMe). The amino acid selected for its suitability for DDRI-targeted activity, which is: 1: 1-peptidyl-1-floac, may have been for A-20 only a small molecule involved. This will be seen in particular in the following: Examples of peptides may be used as biomarkers for biologic phenomena of diseases during repeated imaging. -11nn ria pi H i .--- n-nutra) "--- - Maturation poePP, Zincma peptides I HIV abon cancer AIDS As [furv-- If: Serir Trypta 's, p', Ser of Angiotensin I bloodseed) Tl Mnlaria té.oporc Hypertension Alzheimer EC ".. ', r of Cat.hepsine 1Pqaprnepsines I and II The invention also relates to bone mineralization, but also to corticosteroids, which can be used to inhibit the binding of amide linkages. bstrat -penicillin amidases, amide hydrolase a. fat, 01 fi: '3malonarrlidas, 7p, p a substiPat of frflSfLc- --- 00yseur tiansfert. of a moiety of a moiety, a moi, ubp,: a substrate of the trs, p, pjr3ses, of A, ai: None glutamyl / In this case, SE corresponds to amino acid by an acyl function carried by its carbo terminus by a chain. In particular, sulfonyl sulphonyl sulfonyl sulphonyl sulphonyl sulfonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulfonyl sulphonyl sulphonyl sulfonyl sulphonyl sulfonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl sulphonyl bromide acid res 42 D 503), n ', (, Bater and - refer SE is also a suu reactant with a: hW-- and in particular a, pe forming a disulfide bridge with X, uu is a sulfur atom which will be cleaved by a reducing agent, in particular by the dithiothreitol or by the analyte to be analyzed, or a reagent reacting by cleavage acid labile lonon upon incation sample sample and in particular a forma ° ac binding group with X1 which is an oxygen atom, whereas SE groups corresponding to methyloxymethyl (-CH2-0-013), methyloxyethyl (-CFL: H2C1-1 Ph-hyloxyallyl (-012); -C umdque cui shelter : 'age 17 He den Cr allows the nFhor Li'un cht-omoph-we o' IUC This chomoououc or fluorophr, re can: -,: pprespondre directly to e ',, q, dared released A-Q; What form of aqueous solution can be used , whether at a post-cleave position, or at a com- munity or at a comple! - post storage cflv compos -0F or p> Di pi pc-_ pt - ',. k ..... jre a tori-re. tautomèri this Lus ip er rl Mille A '= 0. For example, the term "aromatics" is understood to include one or more members of the group, whether or not they are selected from one or more of their own or from a single member, and / or ## EQU1 ##, preferably from 4 to 4.0, with from 10 to 10 carbon atoms selected from among the atoms of In the case of sulfur, an aromatic group has a carbocyclic ring containing one carbomer of poly with more than two cycou, and may have the form of a carbon containing, for example, from 40 to 40 carbon atoms, and preferably from 6 to 15 carbon atoms, and comprising at least one aromatic hydrocarbon ring, optionally 1: 1, in a heteroatom with oxygen , within the carbocyclic ring and / or several carbocyclic carbon atoms in the carbonyl form C = O, such a carbocycle may be unsubstituted by one or more substituents. arorne omprennent norarrrnent ary groups heterc__ / le, non substitut n substril. xample such car bocycles, under their non-subshtuated form, phenyl, naphthyl, 2-, 3- or 4-pedanyl, 2- or 3-furoyl, 10 or 3-iophenyl, pyrrolyl, imidazolyl, pyrazolyic, U. ), oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyrimidinyl, tetrazolyl, thiadiazolyl, oxadiazolyl, triazo yridazinyl, indolyl, pyronyl. Ut) mophor st a molecule able to absorb a spectrum part of the visible lum. As chromophore (1-xEr.mp), paranitrophenol and s ivés, dyes of the type indigokles obtained after dimerization rivé enol corresponding (and corresponding unc to A '= 0) which form a precipitate in medium aqueous p) bic, or compounds such as cyclohexenoesculetine (CHE), alizarin or hydroxyflavone which form a colored precipitate in the presence of certain metals: x3 H 3 -hydroxyindcly O -1- hydroxyflavone NO2 paranitrophenol indigo 0 0 OH OH Alizarin CHE 3022 784 18 iivent: be substituted pllLI7's: X3 iden its chles atoms lorer brome, iodine or fluor el. positioned on any position of the benzene ring. A fluorophore is an e of fluorescence, when it is subjected to an excitation, a luminous wave length. Adar - - The fluorescence is the result of the fact that the egoect is excessed: by the light of a given wave length, it wanders at a great distance. Opacity is an iomene that is of the int (fludroph (a -., / /) An incident photon.This process is the absorption of photon causes an electron in fluoro to pass from its melting state to a J In the upper energy, it returns to its original level by emitting a photon, called -: reser Uti -, .. urophor,: n -'-, and then clE: A greater This is simply because the energy photon is less than that of the absorbed photon, because of the energy dye, during the lifetime of the photon absorbed. This application is described in patent application WO 2.0C 4/058 787. Examples of enhancements for the release of such fluorophores are given below: ferone For example, the OA 1 group containing the fluorine liberator after rearrangement of the released R-α-OH 0 can be cited as the following fluorophores (the second is described below). by: Shabat et al J. Am. Soc., 2012, 134, 20412): fluorescent HO non-fluorescent fluorescent fluorescent non-fluorescent modes of separation, the OA group is selected; in a manner which corresponds to a fluorophore ESIPT, which may or may not be cipitated. Aqueous solution. Examples of such OA moieties are AA). X4 X5 dan 'Inn is x, is t. Oxygen and X4 is -NH2, -OH, -SH, alkyl, aryl, -O-alk, -O-phenyl, -NH-a- .--, H-phenyl, -S -alkyl or -S-aryl, said alkyl and phenyl groups may be substituted or unsubstituted, or X5 represents a nitrogen atom and is bonded to X4 which then CH, O, S, N or Ni "to form a substituted heteroaryl, or rosyl, 3022 784 C and represents an aryl or a heteroArv, for example cholo non o said groups i-) to be substituted or unsubstituted, with X6 which represents n " NR ", and R" which represents a hydrogen atom or a group Ci The ESIPT fluorophores show a StokE delta, which> exceeds 100 nm and often reaches 200 nm. All the E3IPT fluorophores lose this fluorescence emission corresponding to a Stokes shift greater than 100 nm, if the phenolic OH group is alkylated, acylated or otherwise unsaturated, which prevents the transfer of a hydrolene atom. ... X5 meronoma on the illustratior (1) with the formula (AA), during excitation by irradiation, and so E) èC -nission fluorescence characteristic. a process of trt of prot7.,. Most preferably 7.11 such C7 group A corresponds to a phenyl group which is nonsulted and / or which is fused with one or more urbocyclyl, wherein the nitrogen atom is optionally substituted. This oxy-OA derivative, when it is not a substrate, is in the form of phenol HO-A, which belongs to the fluorophores ESIPT. OA of the phenoxy type, correspond, for example, to the following pioneered structures (BB) or (CC): Rb (BB) in which - T is -NH-C 0 -S-, -O-, -NH, N -alkyl or N-Ra is hydrogen or a carbon substituent (of electrc as -CN or -COORd, with Rd n;.: represents a g.:J) ;, -C4) a kyle well Ra is - CONReRf, with Re and Rf, identical or different, which represent hydrogen or a (C1-C4) alkyl group, or Ra is -CF3 or a 2-oxazolyl group, 5H 5H zolyl, 2-imidazolyl, 2-bel 4-pyrimidinon-2-yl or quinazolinon-2-yl, b is hydrogen chloride, bromine, e or fluorine, -OH, -NH2, -NRgRh, -Rg and Rh which Each of them is independently reactive or alternatively R a and R b is hydrogenated to form saturated or unsaturated, substituted or unsubstituted hydrocarbon-comprising or chain-substituted hydrocarbons. optionally interrupted by one or more heteroatoms selected from N, S and O, - Rc is hydrogen, Br, Cl, I or F, R'a R'b (CC) wherein: ## STR2 ## wherein: is NH 2, OH., aryl -in gro (C 1 -C 4) alkyl, SH, NHR'c, OR'c, NR'cR'd or - or different, which represent a group. ryle, - R'a is the hydrocyanate or ur _ Jar: racteu, 'f-tr. as -CN, or -COOR'e, with ._ _ .represent,; roup (ou'c -CONR'fRig, with R'f and R'g, identiqus or different, which reprénten ', hydrogen or a g- (C1-C4) alkyl, Lian R '-CF3 or a 2-axazolyie, i-ben2oh171-7zolyl, 4-pyrim-dn-2-yl or quin-1inon-2 Rb is the hydroo ... 2, a bromine atom, iodine or -OH, -NH2, -NR'N. R'h and RI, identical or different, represent a group - or R'7, and R'hccnt them between them to form a 4 or 5-membered cocorenar carbon, saturated or unsaturated, 15 '.i'stadituted or unsubstituted optionally interrupted by one or more heteroatoms chosen prcL We can notably E applications WO 2013/045854 and WO 2014 / 020285A which give examples of such fluoropines) ESIPT res for more details. By way of example of the group OA, corresponding to a fluorophore of the ESIPT type, the following may be particularly: ## EQU1 ## The very large Stokes of fluoropolymers (approximately 70 nm for the HPQ shown in FIG. 25 ana of tz will contribute to the ex sensit ya renara probe liberated easily distinguishable from the nuores of the biological sample which the a, led led 3022 784 23 Defirutior es gn _ R can represent I atorr or - (L), with equal to 0 or 1. Often, for reasons c nthk n = is a lo liaiçon, and LL., T-nrnment an arm 2 'years the cn -> s GP', - lentiqt j, which are selected 3m -0-, -NH- -N (C 1-6 alkyl) -, -N (phen-C (O) -, -C (OO) -OC (O) -O-, - NHC (O) -O-, -OC (O) - '-S-, -NHC (O) -10 and -C:' Slow: C1-20 aIlL C1-20 alcéu, -20 alcyny -24 Jryl, C7-44 alkenylaryl, Cr44alkynylaryl, C7-C14cycloalkyl, alkenylcycloalkyl, C7-alkynylcycloalkyl, C7-H, alkenylheterocycloalkyl, C7-44alkynylhetyl erocyeivalkyl; H drcii. It is preferable to terminate with a triazo group, which may be substituted without substitution, including one or more of the following groups: ## STR5 ## Substituents selected from among, alkoxy, C110 alkyl, C6 1, 1, 2, 4, 6, 6, 8, 9, 9, 9, 10, 10, 10, 10, 10 or 10, respectively, which are equal to 0 or 1. The L arm is present to remove the wildebeest from the opium. or for only 5% / rItilACP '1'; PInn a real mode L presents' 1 with Li. = - C (0,, rra. = m2 = 1 Oet and above, and, -, fent -C (O) - (CH 2) p-L- with p which is 1, 2, 3 or 4 and the triazole is a group of 111-1,2,3-triazole. Hydrosolubilizer By "hydrosolubilized group, it is meant ur hrrin-Inhile which) allows to improve the solubility of a probe which differs naked by the REVERSE of 111 :, by a CH2, to obtain a 1iperidine as described d715ins 3022 784 24 applications WO 2013 045854, n.10 2014/020285, as an example hydro group solubilizer, ocltef .: cable groups of espen form aqueous c. As an alternative to GP, F1 functions selected from the group consisting of 5 (seconal primers or F 2 C 8 functions, such as sulfonate or phosphate, include those peptide polysaccharide functions of aromatic excipients in which - arnidine, or tetrazole; .35 or N-carboxylic acid, including methyl esters, sulfurylose lactose; the groups, -, ', n7, es TAT-peFandes ... have been quoted -NH2, -NHC1-4alkyl, and In) esalk-1. ") are identical or different and comprise from 1 to 4 As used herein, the term "alkyl group" is used to mean an acyl group. It is understood that the term "alkyl group" is intended to mean a hydrocarbon chain, e, linear or branched. The alkyl groups have 1 to 6 carbon atoms and are preferably used. Examples of alkyl groups having 1 to 4 carbon atoms, such as alkyl or (C 1 -C 6) alkyl, include methyl, 7-propyl, iso-propyl, n-butyl and n-butyl groups. , sec, -, yl, n-pentyl, n-hexyl. For example, a caub (mono-, bi- or polycyclic, includes at least one aryn group, for example, phenyl, c. Groups comprising from 6 to 12 carbon atoms, wherein the group means that a substituted group is substituted for one or one of the following groups: Suitable substituents are selected from chlorine, bromine, iodine, cyano, trifluoromethyl, trifluoromethyl, alkenyl, acynyl, alkyl, heterocyl, amino, alkylamino, alkylamino, hydroxy, and the like. a coxy '. Aromatic groups (notarvit and said groups, .alpha., Enyl, alkynyl, thioalkyl, isocyanate, acyl groups and aromatic groups may be substituted for the terms used in these 77, ..bstituart5 are recognized by the man of the same way. <i'v> design._ a string. ... carbon, linear branched qq.i.Compound: At least one (131 Hemn, ,, cs (having a preference of 2 to 20 carbon atoms preferably 2 to 6 carbon atoms; "Aicy denotes a hydrocarbon-based, linear or branched chain with 15 mnl- a triple liaishn, and preferably present from 2 to 1 of cad, - e, and preferably from 2 to 6 carbon atoms; term _ Da I a chain A saturated branched hydrocarbon in which at least one hydrogen atom has been replaced by a fluorine atom, preferably having from 2 to 12 atoms, comprises a saturated, saturated amine ring. The term "bridged alkyl groups" is defined by the term "heterocycloalkyl" in which a cyclocycloalkyl group is defined in which at least one aboly It is preferred that the carbon, preferably at least 6 carbon atoms, be carbonyl, alkoxy, alkoxy, aryl, phenyl or trifluoroalkyl, preferably from 1 to 3 carbon atoms.Examples of these are the groups, cyclopentyl, cyclohexyl, cyclohexyl, naphthalene, cyclohexyl, cyclohexyl, cyclohexyl, cyclohexyl, cyclohexyl, cyclohexyl, cyclohexyl, cyclohexyl, and the like. The term "clay" is understood to mean a carbocene; mono-, bi- or C07- -7-UY-, preferably from 6 to 12. Examples of these are carbon monoxide, and, for example, phenyl cinnarine and naphthyl groups, which are group-free. The aryl group is particularly preferred. Pz - "heteroryl" group means a monocycle, or polycarbonyl, which is preferably 5 to 12 carbon atoms, and, at least one aromatic and at least one nitrogen-containing compound. or sulfur, used in the case of the heteroaryl group, for example: or 3-pyrrolyl, aminothiazolyl, OXE-hidridyl, pyrazinoyl, tetrazolyl, thiadiazolyl, oxadiazolyl, triazol pyridazinyl, inaolyl, etc. The hetero groups also include such groups in which one or more atoms are present. The carbon atom is (are) in the form of an aryl carbon C = O, acyl, a carbon-bonded group of an acyl function, C = O and especially a 4: 17.001-1, -C., l-, (C 1 -C) alkyl, R 1, R 4, R 4, R 4, R 4, R 4, R 4, R 4, R 4, R 4, R 4, R 4, R 4, R 4, R 4, R 4, R 4, C 1 -C 4 alkyl or aryl. - C7-4.4 al ("n" means respectively a rum nest island, or alkynyles interrupted or terrrirkr n: year / le, It is preferred to -l, 25 alkyl aryl and aicyr .ary from 7 to 22 and ci .: The invention is hereby incorporated by reference to the following: The same applies to the carbon atom or to the water-like processes used in the Examples. us according to) 1, .T1er '.. 30flueS of formula (I) and P1 and P2 are temporary protective groups of the amine functions in particular under the reaction conditions used, Y is a leaving group of the type Cl, 7-nitrophenol, imidazole or N-methylimidazolium, or N-hydroxysuccinimide, and Rp represents R or a temporary protective group of the amine functions, in particular under the reaction conditions used, or Rp is a precursor group of the R group. FIG. 2 OR: P2 P2, SE, SE (V) (VI) A 6,, x 2, SE 3022 784 28 A protective group of amines, such as those in Protective Gr; 2s in Orgfd. -3is, Greene T.W. and Wuts rjM ,, cd John V`, / ley and Sons 5 -t in Proteciing Groups, Koc. L 1994, Georg Thieme, creY title, example of tek nrouinPrrents, o can cite the Brome hEn -.- - the group a / the "Joe 123R" i with which C groups _yloxy g- 1 to 12 ': 21.0nees Carbon represents an aryl group: ## STR2 ## and in particular ycarbanyl (Boc) and Dxycarbonyl (Alloc). The compounds (V) for commercial use according to a prcK..a. d: it describes in t, 2010, renylmethyluAy, orbonyi Jbln.n -, us to narb I1 / 41 III 15 (V) apart- ": the bis-protected piperazine,: ut Ueb grOUr-- Group, butyloxycarbonyl (Bac), thanks to a gold reaction su ive. reaction avc. uu pa; uforrrnr1. The compound wherein X 1 = The amine derivative NH is then obtained, for example, by synthetic synthesis, while the compound (V) = S fear + by n nuclear substitute. It is also possible, in a manner, to illustrate them in Schemes 3 and 4 which are necessary when X1 = or NH p01 is prepared in accordance with the present invention. 3 and 4, A, X 1, X 2, and SE are as for the probes of formula (I), and R2 is a gram of F carbon atoms, R group is (n. In particular, the following reactions are employed, and Y is a partan or para group, or itrophenol, imidazole or N-n-triindazolium, and N-hydrofluoric, Fip is R or a particular ternary protective agent. Reaction R3-NH R3-NH R3-NH3 R3-NH R3-NH3 R3-NH R3-NH3 In the procedure described in Scheme 3, cy: perazine is obtained by a double oleophilic substitute of ur ipose and ethylene diamine (X) on d: ## STR2 ## In the Scheme 4, piperazinone ring (PIII), piperazinone ring reductant, also from a malonium bromide, and a compc: pe etily diamine (X). The groups R different from H, which correspond to a group 1-GP, can be introduced by means of the reactions of the reductive amination, the linkage between the linkages and the Ion synthesis pathways. For example, it can be found on the link. L. ligands such as nucleopedes, peptides, cycles of Hu or Diels-Alder cyclization, clysyls such as Sc'-4; Dshra or Heck. It is possible for an intermediate compound of the formula (I ') to be a precursor group of a group R or Rp which is protected in the group. 1 2 2- (LI) mil.-2 and A Xç (I "p) 10 llJ R'p 15 so nue their or; 'ECH, N3, a roncoon a group p; rotectepr de, ^ nt such as dér for (I), - and m - Io - good - preferably uSioai - ini the groupos ber es groups - C.) o'in which, rarp:.: sente an alkyl or nyle group of there 12 atoms of C: 71- or a CH 2 R 3 R 3 with R 11 is an aryl, cycloalkyl or aromatic group and M 3 is R 3. For example, from R 1 to R 2, R 3 is preferred. Examples are n-tert-butyloxycarbonyl, 9-, ..., phenylmethyloxycarbonyl, and allyloxycarbonyl carbons. in some cases ,. arnelt, a fblrnb, to serve as an interest in the pr ~ reon of a fa .., 4dn, (.: oripose of formula (I). This is particularly the case ciie.s compoundJ what = = or - which can be used: especially pov sJs compc sian- which 1,1 = -CONH- or -NHCC- tr'CI = 1. The starting reagents are commercially or fe ... The molecules of Formula (J) are only accessible to me as simple, and can not be used in the same way. In the case of the U-domain which is considered to be low-cost, 10 μl of the compounds selected according to the invention are prepared in accordance with standards of the art. The salts of the compounds of the formula ## STR1 ## are present in those with acids or bases, depending on: ## STR1 ## These acids may be chosen from inorganic and organic acids and bases which 15 porrne's --- it a seppa oj a crystallization LOilVe: 1: d: The rles srnposés F's e (I), as well as salts pfd, iologiquement acceph ,, ble, ie Ipatib, es with applatiuns vii, A1 and in vitro, as long as it is possible to use acyl or a specifically free acid, tartaric acid, alpha-ethyl acid, methacrylate, acid, or those which form: Hydrogen sulphate, hydrochloride, bromide, hydrate, (Snonhosphate, maleate, Escherite, 2-naphthalenesulfonate, pard-VA-sulphonyl mesylate, isotionate Examples of suitable bases are arginine, benzyl alcohol, benzyl alcohol, and the like: those containing physiological salts, such as: of potassium, calcium. Examples of hydrated compounds include, for example, serni-hyclrat. hydrates and polyhydrates By solvate. use: elm of the compound associated with one or more molecules of, t, especially used during its synthesis or during its purification, without being in solution in the latter. , ..,!, composed rits to find s..Ofyou, the forms o in rnebnge according to all proprbons Selo: mode c._ the rac e inve rnmr cu r es R and 5 equals H_Tut're mode of - - the invention is to be saved under the condition that it does not, in particular, the zf -,: posés te be - a for._ in proportions se7-s, rolment composed of formuHF. a one or diastereoisomeric form e, with an enantiomeric excess at J%, or even greater than, 95%, is a pure isomeric form, that is to say with a dimeroisor-1. higher than 99%. The poses (I) may be isolated in a form enriched in a stereoisor or enantior by the conventional techniques of curing. For example, racemic fractional recrystallizations with an optically active acid or base, the principle of which is the well known or the technical. In the context of the invert, all the preferred and preferred embodiments for A, R, X, and X may be combined. If the ion is present or has the following characteristics, the values = 0, ie X1 = 0 and SE is one of glycosidase or gH. -idase and 25 corresponds to a carbon number of 77 at the time of the test. and is, for example, preferably 1 to 20 carbon atoms, or 20 carbon atoms, ie, X 1 = NH and SE is a radical. Proteasease = peptide epitope I to peptides by a function 7: Crenal by a lateral, 3022 784 33 following 2.0: ## EQU1 ## -C (0) -. = rin2 = 1 nor 1 = 1 or 0 as defaults z mn enmmmtion 14, and notar sente - p which is equal to 1, and. L3 which is a triazole group and in particular a 13-triazole group, is it. JREFR, MONIN, carnoxy-aeff7, te and pa and PC A is one, npkifiquement described in the prc patent application., probes according to the invention are attractive plus sensitivity a Scjenc- and to reduce the activity, particularly of the enzyme, expressed by bacterial colonies on a colon (colony); (2) the detection of an in vitro drug. ivite, 2J-rp - 3tique, in biological fluids atol. detection of an activity, in particular a uizymatic activity of a unicellular cell; In particular, the activity of a cytoplasm, particularly of an active activity, is intracellular, in which the fibrinoscopy is (5) the tissue. Also, (6) in vivo, the following are examples of the following: The following are examples of potential applications, examples of which are included in the scope of the assays. on mn down.t,, alnes. These are currently grown on the Petri base or up to 300C (71.7) Orlie. can not say without actively separating them. He is, for example, able to conceive of them, say on a function The samples were taken from a bacterial sample to a bacterial sample. The phosphors can be fluorescently imaged in an innoscopic manner, that is, in an entire organism. In this (L, BS probe will penetrate the cellular r to achieve eret (a stimulus one in 77, y - a chemical compound or a '-'7..This physicochlonal metrology mWeu -: 15 The following examples illustrate the invention as follows: ## STR1 ## Abbreviate 3-methylfluoroacetylmethyl-DCV: - -loromethane HOBt: 1-hydru., nybenzotria; Boc: te, n -butyloxycarboxy Ac: acetyl HATU hexaluon ... i., phosphates of 1-sBu sec-butyl Bis (dimethylamino) methyltrik71-n "t 1,2,3-triazolo {4,5-ujyridiniun yde TA: Tem nature Ambient DIAD: izodicarboxylate of: N, N-methyl-coumarin diisopropyl THF: Tetrahydrofuran DCC: Dicyclohexyl carbodiimide DMF: N-dimethylformamide iPr: O-6: Chemical (pp. RFU), Fluorescence R: AU: L "Arbitrator absorbance of on (exc (nm) Aem: Length of goldP ---- e magr L que (nm) re LC: C liquid: Mass spectroscopy ESI: lectrospray _7: Cor range HH (Hz) d: t: trip 3022 784 35 dd: double doublet The NMR spectra have been increased to 297K thanks to a speci fi c: --- ADVANCE 300 (300 MHz & 75 MHz for Flet 13C, respectively one Bruker AVANCE 500 (500 MHz & 125 MHz for 1H and 13C, respectively). Low resolution mass spectra were obtained by means of a spectrometer coordinated with the chromatography of AGILENT 1100. Chromatographic analyzes were carried out on C-1 gel plates. :: 75: ee 60Â filed (Aldrich). The high-resolution measurements were obtained by means of a mass spectrometer. The absorbance measurements were obtained using a fluorometer. LA - Syntheses: Al (4-benzylpiperazin-2-yl) methyl) -2-trifluoroacetic acid: I - Preparation of the intermediate 8 phenylacetamide, sulfur 20 a) Compound preparation Boc.20 D t MI To a solution of P4: (5N) 1 (10Molmol, 2.0eq) in DCM (30Cr) is added dropwise as a solution, Boc2O (8.07g, 50mmol, 25%). in (150mL) u: neprock from 3h to 7: 3022 784 36 7. Most importantly, the addition of the reaction mixture to the reaction mixture is then concentrated by This solution is then washed with an appropriate aqueous solution of NJEd-1 (2 x 100 mL) and a solution of 100 ml of water.
[0004] Dve. :: the brine (100mL). It is dried with N 504, fired, and dredged to obtain the 4th of a lull lore qt: crystallizes with time (7,571 yield: 81%). MHz, CDCl 3): δ = 3.33 (t,, 2.75 (t, 1 1 = 5Hz, 4 17 (s, 11 1.41 (s, 9H) ppm.
[0005] 13C NMR (75 MHz, CDCl3: 5 = 154.9, 79.5, 56.3, 46.0, 44.8, 30.4, 28.5 ppm) MS: ESI: [M + H] +4 mz 187.3 c I am "Drim rmrfise.trt Prepared 3: N-Boc-W-benzyl-p Benzene Bacon: Iden 'N molecular sieve 4A H DCM, 0 ° C to T 3h 2 86% A solution, daring 2 (10.0g, 5.7rrmol, 1.0eq.), Benzalic acid, m.p., mol. Sieve 4), DCM (200 -.) - DidiP 9 ° C several portions of NaBii (uAc) 3 (17 8U, The reaction is stirred at 0 ° C. for 1 hour at RT at the end of the reaction, the mixture is filtered and the filtrate is washed three times with a saturated aqueous solution of NaHCO 3 (x 150 ml). The organic phase is dried with Na 2 SO 4, filtered and evaporated and the crude oil is purified by silica gel chromatography on pure DCM edge and then DCM: Me (H / 95: 5 / v: v) to obtain C. (3), with a colorless oil crystallized with 6 mmol, 6 mmol, 86%) .MN (300 MHz, = 7.29 (m, 2 (s, 2H), 3.45 (t, J). = 51-1z, L40 (t, J = 5Hz), 1.48 (s, 41-1) pp m.
[0006] ## EQU1 ## where: ## EQU1 ##, ## STR1 ## 137, 79, 128, 128, 128, 127, 79.63, 63.18, 43.61, 28.54. MS: ESI: F L11 + -uvée 277.3, cF, 7ul .. 77.3 Rf = d ethyl / 8: 2 I v: v) Preparation of (hydroxynyl) -4-tert-butyl-4-benzyl-2-oxoic acid, -30 ° C, half-OH 2, paraformaldehyde -30 ° C to RT, 2. ## EQU1 ## In a balloon dried and placed under an inert atmosphere of 10 °, dissolves the compound (1.0 g, 3.6 mol, 1.0 eq) in THF (CH 3 H). This solution is cooled to -30 ° C. the sBuLi (1.3M in C exarie, 5eq.) Is added dropwise. The reaction mixture was stirred at -30 ° C for 6 minutes with 3-aldehyde (350mg, 11.6mmol, 3.2eq) and introduced rapidly. This suspension is stirred at -30 ° C. for 30 minutes, and 1:30 at RT before being quenched thanks to the saturated aqueous solution of NH4Cl (20 ml). The organic phase is the aqueous phase is washed twice with Et 2 O (2 × 20 mL). The combined phases are combined, dried with Na 2 SO 4, filtered and evaporated.
[0007] The crude reaction product is purified by chromatography column (petroleum ether eluent: ethyl acetate / 7: 3 / v: v). PCl 4 is treated as a yellow oil (t325 mg, 2.0 mmol of water: 57%). 1 H-NMR (500 MI-I CDCl3): δ = 7.2 (m, 5H), 4.12 (br s, 1H), 3.92 (m, 3H), 3.54 (s, 2H), 3.40 rs, 1H), ( d, J = 11.6Hz, 1H), 2.86 (d, J = 8 2.33 (dd, J = 11.61-t 3.9Hz, 2.13 (td, 1 = 11.6Hz, 3.9Hz, 1H), 1 ppm.
[0008] 13C-NMR (127 NLz, CDCl3): δ = 155.3, 137.3, 129.0, 128.6, 127.5, 80.66.5, 63.1, 57. 72.6, 51.4, 41.7, 28.5 ppm. MS: EST: [M + H] found: -307.2, ceP-. Rf = 0.28 (n-2-trole ether: acetate) iperazine-2-yl (1,3-phthalimide) PPh 3 DIAD, AT 18h A cooled to 0 ° C. Triph, ν, p) (1.07 g, nil, 1.2e, ec (oummy, 4.03 mmol, 1.2 eq) in dt: anhydrous (L) is added dropwise the DIA!) (845 μL). The reaction mixture is stirred at 0 ° C. for 30 min at room temperature and the resulting oil is purified by silica gel chromatography. : ethyl acetate / 8: 2 / v: v) petroleum ether to obtain Compound 5 in the form of a white solid (1.1 H NMR (500 MHz, CDCl 3): δ = 7.79 (4.45 (m, 0.5H), 3.98 ./. '1 = 13Hz, 4H), 2.> 1 2.05 2.76 mmol, yield: 82%). i), 7.30 (m, 51-0, 4.55 (m, 1.5H), 75 (d, J = 12Hz, 0.4H), 3.48 (), 1.08 + 1.03 (2xs, 9H) ppm.
[0009] 13C-NMR (125 MHz, = 168.5, 168.2, 167.9, 155.1, 154.4, 138.2, 134.5, 134.1, 133.7, 132.8, 1: 2.4, 129.0, 128.7, 128.5, 127.4, 123.7, 123.4, 123.3, 79.7, 62.9 , 54.5, 53.3, 50.3, 48.8,, 38.6, 37.8, 28.4, 27.9 ppm, MS: E5 + H1, found 436.3, c: Rf Rf = 0.32 (petroleum ether: ethyl acetate, 8%) (2) The compound 6: tert-butyl 2- (aminomethyl) -4-benzylpioe-carboxylate 302 2,784 39 (NI-12 EION 5 6 A-1,71 con7ose 5 ( 2.76 mg, in EtO 2 (nonohydrophilic acid), 12.0 mmol, 4.0eq.) And the malfunction is refluxed at 18 hrs., In which the suspension is cooled. The filtrate is filtered and evaporated to give a solid which is resuspended in (50 ml), filtered once more, the filtrate is washed twice with NaClCO 3-saturated softening solution. (2x50mL) and once with brine (50% organic phase is dried with Na2SO4 and evaporated to give 6 (610mq) as an oil. it is next step without further purification. 11.1 q l (500 MHz, THIS is δ = 7. 3.97 (bru, 2H), 3.57 (d, J = 13.2 11-1), 3.43 (d, J = 13.1 H ::, (dd, J = 13.1 , 7.0 Hz, 1.94 J = 13.1, 7.0 Hz, H, (m, 2H), 2.10 (m, 2H), 1.50 ppm.
[0010] 13 C-NMR (125 MHz, CDCl 3): δ = 155.33, 138.36, 128.76, 12.328.35 128.13, 127.19, 79.74, 62.82, 53.55, 53.30, 53.13, 48.55, E4, 28.48, 21.99 ppm. MS: ESI: [M-306.3, calc. 305.2. P Primer 7 hrthutv4-: phenyl-5-phenyl-5-phenyl ether, iPrEt2N Ph DCM, 0 ° C to RT, 2h 43% over 2 steps Bn Compound 6 (600 mg, 1.96 mmol, 25 (DIPEA) (530 μL, 3.0 mmol, 1.5 eq) in DCM 3022 was added at 0 ° C to the solution of 318 chloride solution. L. 2, mmol, 1.2eq.) 1.111K11.1v1 1., 5mL). The mixture was stirred at 0 ° C for 3.5 hours. At the end of the reaction the mixture is saturated with a saturated aqueous solution (3 × 20 ml) .The crude phase is dried from Na 2 SO 4, filtered and evaporated at room temperature. 1-ions are chromatographed on silica gel (eluent: Ethei Dle: acel / 55:45 to obtain compound 7 under solid form, 1.16 mm em. 6% over 2 steps) .
[0011] 10 I, 500 MHz, 3): 5 =. 7.06 (m, 10H), 6.12 (s, 1H), 4.08 (s, 1H), 3.43-3.3H, 3.21 (d, J = 13.0Hz, 1H), 2.61- = 11.6Hz, 1H), 2.57 ( d, 1, 1Hz, 1H), 1.99 (dd, 1 = 11.6, 3.8Hz, 1H, 11.6, 3.8Hz, 1H), (DG) 1-1) ppm. 3 C NMR (125 MHz: 471.0, 137.8, 135.1, 129.4, 128.9, 128.9, 128.8, 128.4, 127. 32.8, 54.2, 53.0, 49.7, 43.9, 41.0, 40.1, 2-. 1 ppm I: [M + Hr m / z, alculated 424.3 Rf = 0.23 (ether of V: v) g) Preparation of Compound 8: ((4-b) Erazin-2-y) Mette 2 -1. Ethanol, in the form of a solution of compound 7 (OOmg, 1.18 mmol, 1.0eq.) in DM. (4m) is trifluoroacetic acid (FA) (4mL) and the meta-reactant 1.10, which is stirred at RT during the reaction, the reaction mixture is evaporated under pH1. This gives two times, and the resultant oil is dissolved in the equilibrium, and the product is p.
[0012] 4 Jans 3022 784 41 of the Et20. The suspension is dried at room temperature. To obtain the compound in the form of a powder (401 mg, 31, yield: 78%).
[0013] 1H-NMR (500 MHz, CD-Or ': 5 = 7.42 - 7.22 (m, 9H), 3.70 - 3.59 (m, 2H), 3.57 (s, 2H), 3.53 - (m, 1H), 3.41 -, 36 (m, 3H), 3.15 (td, 1 = 11.3, 3.5 Hz, 1H;, 8 (d,, = 11.3 Hz, 2H), 2.z, t, J = 11.3 Hz, 1H), 2.24 (t , J = 11.3 I-1H) ppm (125 MHz, CD3OD): ν max, 136 130.39, 130.26, 129.70, 129.x, 2, 128.85, 128.13, 63.15, 56, 49.54, 44.80, 43.69, 40.74 for 10 MS: ESL: L] z found 324.3, calculated 324.3 II - Preparation of intcary 10,: 4-methyl-2-oxo-2H-c-7-yl carbonochloridate H triphosgene, NaOH (1M aq) To a cooled solution at 0 ° C. of triphosgene (420 mg, 1.4 mmol, 0.7 eq) M (10 ml) is: 4-methylumbelliferone 9 (360 g, 1 g); This suspension is added to the suspension by adding 3% NaOH (2M, 1.1 mL, 2.2 mmol, 1.1 mL) and the reaction mixture is stirred at 0.degree. TA for 18h, the 20s pension is then filtered and the resulting solid is washed twice with d (DCM and dried to give about half of the The expected mass of compound (202 mg, 0.85 mm) was then washed with water and the organic phase was dried from Na 2 SO 4 -PtO 4 to give a H 2 O, comontion. D 10 (240 mg, 1.0 as a powder che. The rendem_ I this is%. NMR (300 MHz, CD: -), 7.33-7.19 (m, 1H), 2.46 (s, Rf = 0.47 (cyclohexyl, ethyl / 6: 4 Cl in 2.0 EL). 12, hydroCoxo-21-1-2- ((2-phenylethanol) -, 75-carboxylate iposulphonylpiperoxide iP7Et2N DC, 0 ° C to RT, 4h30 33% 10 A cooled at 0 ° C. (237 mmol, 1.05 eq) anhydrous (10 ml) under argon inert atnips is added at room temperature. (400mg, 0.91mmol, and c, 1.2EA (48 (, 3.0eq) in DCM at 0 ° C, 301 ° C. After 4 hours at the end of the reaction, the mixture is reacted with a saturated aqueous solution of aHCO 3 (3 × 2 O 2, the organic phase with Na 2 SO 4, a filtrate, and a reaction mixture). The column is purified by column d) matogra, He on silica gel using DCM MeO0-1-1 9q: 1, q7: 1, V) to obtain compound I, in the form of a 16 U huH, 31 mmol, yield: 33%) 11-1-NMR (500 lx 7. '6 (2xd, 5Hz, 1H), 7.26-7.06 (m, 10H),, = 9 / 1H) , 6.97 (t, J = 7.4 Hz, 1H), 6.25, 0.5H), 6.13 (br s, 1H 4 (s, 0.5H), 4.33 (s, 0.5H), 4.16 (s, 0.5H) , 4.99 (m, 0.5H), 3.9a. t, J = 13.2 Hz, 1H), 3.70 - 3.61 (m, 0.5H), 3.50 - (m, 41-1), 3.28 (t, J = 12.6 .4 1H), 3.20 - 3.05 (m, 1H); ), 2.72 (d, J = 11.9 H 2.65 (d, J = 2.31 (d, 3 = 7.7 Hz, 3H), 2.13 (dd, 3 = 11.9, Hz, 11-1: 2.06 (t, 3 = 11.8 Hz, 1H) - 160.68, 154.08, 13C-RIvb 7, CDCl3): 5 = 11.23, 152.76, 152, 2, 137.53, 134.76, 129.38, 129.31, 128.90, 127.40, 127.1 125.22, 118.11, 117.96, 117.37, 117.24 , 153.82, 153.40, 128.88, 128.44, 114.20, 114.12, 302, 784 43 MS: EAST: Rf = 110.07, 62.63, 54.00, 53.85, 53.51, 52.74, 51.14, 43.79, 40.97, 37, 47, 72 ppm. 1 / z found 526.3, calculated 526.3 10H / 97: 3 rv) ketamiu azi cart (10% w) MeC1.-1Bh, 78% HCl HCl A flask containing a solution of compound 11 (120mg, 0.23m 1.0eq.) And 1,1,2-trichloroethane (25μl, 0.25mmol, 1.1eq.) In ME (5mL) is purged pervint 5min with H2. The Pd / C (10%, 24mg)) was removed and the flask was again purged with H2 for 5min. The reaction mixture is then stirred at RT for 18 h under a H 2 - atmosphere. At the end of the reaction, the mixture is filtered through Celite® and the filtrate is evaporated under reduced pressure to an approximate volume of 2 ml. Et20 is then added to precipitate the product. The resulting slurry was dried in air to give compound 12 as a white pudra (85mg, 0.18mmol, yield: 78%). 11-1-NMR (500 MHz, CD30D): = 7.80 (d, J 8.1 7.35 - 7.07 (m, 7H), 6.33 (s, 1H), 4.63 (2xs, 1H), 4.33 (2xs, 3H) 3.45 - 3.32 (m, 3H), 3.21 (s, 1H), 2.49 (3.78 - 3.66 (m, 1H), 3.53 3H) ppm, C-NMR (125 MHz, CD30D): δ = 175.07. 162. 155.25, 155.00, 136.46, 130.19, 129.71, 128.O), 127.15, 119.38, 119.02, 114.83, 111.09, 50.92, 50.42, 44.39, 44.04, 43.75, 38.6-38.32, 37.44, 37.39, 18.72 ppm. HRMS: C24H26N3O5 [M + H] m / zt 436.1858, calculated 436.1867 IV - Preparation of Intermediate 14, Yl-N- (ylmethyl) tetrahydrofuride DTP, DCM 0 C at RT To a suspension of pheretic acid (1 g, 11.2 mmol HOBt Oleq (1.66 g, 12.3 mmol), a solution of dicyclohexide DCM (10 mL). 2- (Aminomethyl) peridine in DIF 30 mL: 0.1 mmol (2.56 g, 12.3 mmol, 1.1 eq), ns of the isant is stirred at RT for 30 min. The reaction mixture is then stirred at RT until the resulting solution is filtered and the filtrate is washed twice with an aq. It is saturated with NaHCO 3 (e: x 50 mL), washed several times with 10% KH 2 PO 4, and the pH of the phase is then added to the recycle phase. The aqueous solution of NaOH M) is added to the aqueous phase as follows: oi tr mol, yield: bu H), 3.57 (s, 2H), .56 (m, 2H), 1..5 .H 12. The second organ is withdrawn D .x100 mL), combined, dried SVac Compound 14 as salt 34, filtered c (2.0H ddO MHz, CDCl3): δ = (m, 5H) (m, 1H), 3.U4 (2.59 (nl, 2H), 1.75 (m 4H) ), 1.03 (m, 1H) MS: ESI: [M + H + I found 233 3, calculated 233.3 3030 784 45 V - Preparation of the compound e 4-methyl-2-oxo-2 / - -chromen-2- ( (2-phenylacetamido) methyl) piperidine-1-carboxylate (Comparative 11) To a suspension cooled to 0 ° C. of Compound 10 (39 mmol), 1.05 eq.) In anhydrous DCM (5 ml) under an inert atmosphere. Argon was added at 14 (25 mg, 0.001 m, 1.0 eq) followed by DIPFe (55 μL, 0.30 mmol, 2.0 eq). The -actionnal mixture is stirred at 0 ° C. for 30 minutes and at RT for 4 hours. At the end of the reaction, the reaction mixture is washed three times with saturated aqueous NaHLO 3 solution (3 × 20 mL) and the organic phase is dried with Na 2 SO 4, filtered and evaporated. The crude reaction product is purified by chromatography column on a gel (eluent: gradient of DCM: MeOH / 99: 1, 98: 2, 97: 3 / v: v) to obtain compound 15 in the form of a colorless oil (3 g / l). n.31mmol,, ..., ent: 15 54%). 11-I-NMR (500 MHz, CDCl ): Δ = 7.49 (s, 1H), 7.30 - 7.04 (m, 5H), 7.04 - 6.88 (m, 2 -1), 6.16 L, .90 (s, 1H), 4.48 - 4.23 (m, 1H), 4.03 (d, J 13.5 Hz, 1H), 3.82 (s', 64 - 3.27 (m, 3H), 3.12 (s, 1H), 2.35 (s, 3H); , 1.77 - 1.36 (m, 6H) ppm.
[0014] 13 C-NMR (125 MHz, CDCl 3): δ = 171.42, 160.73, 154.14, 153.99, 153.83, 152.12, 134.62, 129.75, 129.36, 129.00, 128.38, 127.35, 126.76, 125.19, 118.10, 117.32, 114.20, 110.34, 53.51. , 51.09, 50.93, 46.72, 43.69, 41.28, 40.83, 40.64, 39.53, 38.94, 31.69, 26.53, 25.28, 24.39, 23.04, 19.09, 18.76 ppm.
[0015] HRMS: C2 2 Na05 [M + Na] m / z found 457.1734 calc. 457.1734 Rf = 0.27 DCM: MeOH / 98: 2 / v: v) o 10 14 0 o DOM, 0 ° C to RT, 4h30 33% 3022 784 (4-Benzyl-zin-2-yl) methyl VI - Preparation of the Intermediate: Ethanoate Under the Salt with the Same Value thionyl chloride HO 2h CO 17 16 e octanoyl :: 16 10.0 g, 69 mmol, 1.0 eq is added to the chloride of t, L, nyl (30 ml, 410 mol, 6.0e - dionne continued for 2i, then re volatiles, ± - .: evaporated under reduced pressure. The resulting oil is taken up again and the solution is again evaporated, this procedure being repeated for a further time, the compound 17 is prepared in the form of a very fragrant yellow liquid (11, C 66, mol). , yield (300 MHz, CDCl 2, 2.87, J 7.3 Hz, 2H), 1.70 i = 7.3 Hz, 2H), 1.42 - 1.22 (m, 8H), 0.88 (t, J = H) ppm. 1: 1 '(- 2, -PNM (75 MHz, CL 22.65, 14.11 ppm, b) Preparative ((octanoyloxy) ... h 5 = 173.89, .., 31.64, 28.85, .20, 4 , e _zirtz e 28.51, 25.20, 4- Jen zy1-2- Bac Bac RI OH 17, Et3N DCM, 0 ° C to RT, 3h 44% 20 Bn 4 To a cooled dilution of thamine (78C 'additio-drop at 1 I in 25 obi (RnOmg 3.72mmol, - Oeq.) And anhydrous DCM, and a solution of compound 17 (667m; (5mL). The racial mixture is attached to OC pendulum. l is then diluted with DCM once with saturated aqueous NaHCO 3 solution (50mL), water (50mL), once with aqueous solution and once with water. brine (50 mL) HCl phase (1M organic) is dried with Na2SC and rinsed out to give the crude oil. This silica gel (pure DCM of ethyl is 18% by weight of a C4mmol, 4% sieve) (CD30D): 5 = 7.34 - 7.16 0 4.18 (rn, (d, J 13.1 Hz, 1H), 3.37 (d , Hz, 1H), 3.0'2; = 1: i Hz, 1H), 2.77 (d, = 11.5 Hz, 2H), 2.20 7.6 Hz, 2H), 2.14 - 1.95 2H), 1.60 -. , 2H), 1.44 (s, 9H), 1.25 (s, 8H), 0.87 (t, = 6.4 Hz, 3H) ppm.
[0016] 13C-RM (.25MHz, CD30D): S = 173.72, 154.91, 138.16, 128.93, 128.42, 127.19, 79.97, 62.83, 61.56, 53.10, 52.82, 34.36, 31.80, 29.25, 29.04, 24.95, 22.73, 14.20 ppm. .
[0017] Hi miz found 433.3. Calculated 433.3 J COI É (elme: ide trifluoroz .TFA est, TLC yellow 1-11-NMR (500 3.88 (s, 1H ,, DCM, TA, 2h quantitative FJ 13n 20 The vr-cédure used is the same as This is described for the following: Rr. of 8, but using the compound C7Cb mmol,.) in (5mL) and TFA for compound white mudre form (775mg, rement: qua IN (500MHz, CD30D): 5 = 11 - 7.27 (n-, 51-i), 4.30 (dd, J = 12.5, 3.9, 25 "), 4.22 (dd, J = mz, 1H), 3.70 - 3.63 (m, 1H), 3.46 1 = 13.2 Hz, 1H), 3.28 - 3.12 I), 2.71 (td, 3 = 12.2, 12.2, 2.6 Hz, 1H), 2.61 (t, J = 11.8 Hz, 1H), 2.38 (t, 3 = 7.5 Hz, 2H). ), 1.66 - 1.52 (m, 2H), 1.30 (s, 8H), 0.89 (t, 3 = 6.7 Hz, 3H) ppm.
[0018] 3022 784 48 13C-NMR (125 MHz, CD30D): at = 174.57, 162.06, 161.77, 135.40, 13095 129.86, 129.62, 118.70, 116.40, 62.75, 62.55, 54.98, 52.20, 49.8 (- 34.47, 32.84, 30.13, 30.07 , 25.72, 23.65, 14.38 ppm, MS: ESI: [M + H] m / z found 333.3, C, J 333.2 V I-) ari of 4-methyl-2-oxo-2H-c-iromethyl 7-eylamine hydrochloride compound -carboxylate (EXAMPLE 4), which is identical to that described for the compound of compound 11, with the proviso that L-Lcmg, 1,. Compound 19 (750mg, 1.67mmd), EA (8L, 5.02mmol, 3.0eqd anhydrous DCM (+/- 17%). silica (acetate): acetate as colorless solid (3mg, 22%), 20H-NMR (500MHz, CD30D): m.p. ,, J = 8.6 Hz, 1H), 7.24, 1H), 7.11 - 7.00 (m, 2H), 6.18 11), 4.57 - 4.3 m, 2H 4.27 1H), 4.09 - 3.95 54 (d, J = 13.1 Hz, 1H ', (d, J = 13.0 Hz, 1H), 3.23 (t, J = 1Hz Hz, 0.5H), 2.82 (d, J =.) I'L, LH), 2.36 (s,, 2.21 - 2.10). (m, 3H), 1.55 1.43 (m, 2H), 1.26 - 1 12 y.LH, .87 - m 3H) 25 ppm.
[0019] 13C-NMR (125 MHz, CD30D): 173.46, 160.56, 154.1 3.82, 152.88, 152.00, 137.65, 11.76, 128.35, 127.29, 125.17, 118.11, 117.90, 117.29, 114.14, 110.25, L05, 62.57, 61.57, 61.28, 52.75, 52.62, 51.16, 50.57, 41.02, 40.21, 34.13, 31.58 28.86, 24.76, 22.54, 18.65, 14.03 3022 784 49 MS: ESI: [M + H] m / z found 535.3, calculated 535.3 Rf = 0.26 (ether of Petroleum: ethyl acetate: Preparation of compound 21: 4-methyl-2-oxo-chromen-7 12 - ((octanoyloxy) meth) piperazine-1-carboxylate hydrocure 20 HCI H2 (1311-1.). Pd / C (1,1,2-tr .: ticirc: ethane MeOH, TA, 15h 51% The procedure used is the same as that for the synthesis of crr-12, with compound 20 (180 mg, 0.3: 1.11 hr, 1, Oeq.), 1,1,2-ethane (37 μL, 0.39 mmol, 1.1 eq.), Pd / C (38 mg, 20% w). 10 MeOH (5mL) for di-11-Pnir 21 compound in the form of a pot (95mg, 0.20mmol, - ant: 61%).
[0020] 1H-NMR (500 MHz, CD30D): δ = 7, = 8.6 Hz, 1H), 7.32 - 7.13 (m, 1H), 4.67 (s, 1H), 4.4 ..) (m, 2H), 3.53 (d. , J = 13.4 Hz, 2H), 3.45 = 11.7 Hz, 3H), 3.26 1H), 2.49 (3H), 2.35 (t, J = 7.5 Hz, 2H), 1.57), 1.26 (dd, J = 28.7). , 21.9 H, 0.84 (t, J = 6.8 Hz, 3H) ppm.
[0021] 13C-NMR (125 MHz, CD30D): at = 174.84, 162.51, 155.27, 154.96, 154.90, 154.05, 127.20, 119.25, 119.06, 114.87, 111.01, C.14, 43.94, 43.50, 34.83, 32.80, 30.17, 30.07, 25.89, 23.63, 18.67, 14.36 prq 20 HRMS: C24H33N2O6 [M + Hr m / z found 445.2318 calculated 445.2333 3022 784 50 VTIT - Preparation of the compound, 4- (4 - (((4-methyl-2-gold) 2H-cbron; yl) carl, yloxy) methyl) piperazin-1-yl) buta-sulphonic acid. MPLE31 A suspension of the compound (0.52mmol, 1.0eq) (10mL) is added. NaHCO (1M, 10% organic phase is taken up in water and is washed three times with combined ethyl acetate (3x10mL), dried with Na2SO4, filtered and evaporated to give an inc. This oil is dissolved in THF (2.5 mL), and butane is added (2 drops) and the mixture is heated to reflux. <48 h At the end of the reaction, with THF (2.5 mL) and EL, 15 (5 mL) and separate water is separated. lyophilized to give the compound 22 in the form of a very hygroscopic white solid (g, 0.022 mmol, yield: 43%).
[0022] ¹H-NMR δ (2H), 6.23 (d, J = 1. (m, 4H), 3.12 - 2.8 D): = 7.72 (d, J = 8.6 Hz, 1H), 7.21-7.03 (m, (s , 1H), 4.38 - 4.12 (m, 2H), 3.62 - 3.24 80 (t, J = 6.9 Hz, 2H), 2.42 - 2.35 (m, 3H), 2.29 - 2.17 (m, - 1.73 (m, 4H)) , 1.47 (s, 2H), 1.25-10H), 0.74 (t, J = 6.9 Hz, 3H) ppm.
[0023] 13 C NMR (CD3OD): δ = 162.54, 155.30, 155.02, 154.93, 127.23, 119.30, 118.88, 114.88, 34.i = 2.83, 30.21, 30.10, 25.92, 23.67, 23.24, 18.71 ppm. HRMS. , 40N2NaO9S [M + Na] mp [13.2339 calculated 603.2347 3022 784 51 IX - Preparation com -.,: anoylonyr ré.'at on ethyl-2-1xo- Li-hromen-7-yl 2e (comp 3tif 2) il-Boc. ## STR5 ## to the solution r - oid is DCM 24; MOI, Meg.) LrS from a solution of Boc2O '-, 6g, mel7rige "and at 0 ° C for AT nci, -Int 3h. Then diluted DCM (30ml) and washed once with saturated aqueous solution of 10 (3x60mL). The stain is dried with Na2Sal, evaporated in vacuo. uutenir (6, g 28mmol, endurance: 65%) u yellow form. NMR (300 MHz, CDCl3) δ = 4.36 - 4.22 (m, 1H), 3.94 (d, J = 12.3 Hz, 73 (m, 1H), 2.87 (t, J = 12.3 Hz, 1H), 1.38 (m, 111-1) ppm ..) 5.43, 79.92, 6. 6, 52.68, 40.14, 28.57, 1H), 3.88-3.75 (m, 1H), 2.01 (br s, 1H), 1.73-1.52. (1'.ntl.
[0024] 13 C-NMR (75 MHz 2539, 25.35, 19.7) (1-Octanoloyloxy-Hyl) piperacline Boc 1: 1 20 24 OH 17, Et, N DCM, 0 ° C -A, 2H quenched 25: 25 at 0.degree. C. (CdT.L 8mmo Oeq.) And triethylamine, 42mmol, 1.5eq, of the DO, c (40mL) is added dropwise soluticn of the compound 24 (5.0g, 30.6 mmol, 1.1 g of dry DCM, the reaction mixture is stirred at 0.degree. C. for 25 minutes and with DCM (40 ml) was washed once with water (50 nm with 1M NaOH) ( The organic phase is dried with Na 2 SCl 3, and evaporated to dryness to dry. The organic phase is dried with Na 2 Cl 2 and evaporated to dryness. (9.82 g, yield C, lti as u 1, e used without further purification 1 H-NMR (300 MHz, CDCl3) 6 = 4.40 (s, 4.26 - 4.11 (m, 0.9H), 4.10 - 3.99 (m, 1.3H), 3.99 - 3.80 (m, 0.8H), 3 - 3.64 (m, 0.2H), 3.59 - 3.47 (m, 0.2H), 2.86 - 2.65 Cm, 0.8H), 2.48 (s). , 0.2H), 2.37 (J = 7.5 1-1z, 0.2H), 2 J = 7.5 Hz, 2H), 1.68-1.6H), 1.43-1.30-1 .1, 101-I) 0, R0 ( t, 3 = 5.9 F IL, mr.
[0025] 13 C NMR (75 CDCl3, c5 = 173.77, 155.15, 79.54, 61.71, 34.38, 31.75, 29.21, 28.99, 13.52, 24.98, 22.67, 19.42, 14.13 ppm). The procedure was as described for the synthesis of compound 8, but using compound 25 (700 mg, 1.62 mm -1, 1.0eq) in DCM (3mL) and ## STR3 ## to give compound 26 as a white powder (9.56 mmol, 88% yield) .RMN (300 MHz, CDCl 3) δ = 8.93 (s, 1H), 8.25 (s) , 1H), 4.54 (d, J = 5.4 Hz, 2, .1H), 4.32 (d, J = 3.4 Hz, 0.3H), 4.28 (d, J = 3.4 hr, 0.6H), 4.23 - 4.07 (m, 1H), 3.50 (d, J = 12.7 Hz, 1H), 3.43 - 3.26 (m,) 3.03 - 2.83 (n 2.30 (t 3 = 7 Hz, 1H), 2.06 - 1.46 (m, 8H) , 1.39 - 1.17 (m, 9H), 0.87 L, = 173.81, 161.67, 117.60, 63.67, 45.11, 33.69, 31.71, 29.06, 28.95, 25.16, 24.67, 22.66, 22.06, 21.97, 14.1 ppm. ESI: [M + Hr m / z found 241.2, calculated 241.2 3022 784 53 d, repaired, posed 27: 4-r, '... y..2-oxo-2H-chromen-7-y 2- ((octanoyloxy) methyl) piperidine The procedure used is the same as that for the syrtse of compound 11, with the compound 1.0eq, the compound 0, and the compound of formula (I). 79mmol, 1.0eq.) (700pL, 3.95mn, anhydrous (10mL). Chromatography on a gel of petroleum ether of Ethanol (15:25) gives the product (27) in the form of a crude oil, 0.34 mol, 43%) 1-1-1-NMR (500 MHz, CDCL 7.57 (d, J = 8.6 1H), 7.16 - 7.05 (n 6.24 (s, 1H), 4.68 (s, 1H), 4.41 (t, J) = 9.9 Hz, 1H), 4.22-4.10 (m, 2H), 3.13 (brs, 0.5H), 2.98 (brs, 0.5H), 2.42 (s, 3H), 2.28 (t, J = 6.8 Hz, 2H), 1.85 1.66 (m, 4H), 1.65-4H), 1.24 (brs, 0.84 (t, J = 6.6 Hz, 3H) 15 ppm.
[0026] 13C-NMR (125 MHz, CDCl3) at = 173.77, 160.82, 154.33, 154.20, 153.29, 152.12, 125.22, 118.25, 117.39, 114.31, 110.39, 61.67, 50.35, 50.00, 40.09, 34.37, 31.75, 29.22, 29.03, 25.81 , 25.12, 24.99, 22.70, 19.32, 1 14.17 ppm. 20 ° C: Na06 [M + Na: i-466.2179 calculated 466.200 s, = 0.34 (petroleum ether: Etl of etk / 75:25 / v: v) X - Preoarat c. 4-methyl-2-oxo-2.11-chromen-7-3- (1-methoxyethoxy) ethoxy] ethyl) -1H-1: 2,3-triazole-1 - [(2-acetamido)] 1-Methypiperazine-1-carboxylate (E- -: L4) Preparation of compound 29, 2-azido-2-methoxyethoxy) ethoxy) ethane NaN3 N3 DMF, H2O, 70 ° C, 18h 29 69% 3022 784 54 A -ne solution (5mL) in the form of a mixture of 1.0% and 1.0% of a mixture. ## STR3 ## (2.04 g, the reaction is carried out in a solvent, the resulting product is acetal (50mL)). The susp, anion c and the filtrate and: times with a saturated aqueous solution (NaKCO3 2x50mL), puff, one with umure (50mL). The organic phase is dried with filtered and evaporated to give the compound 29 as end-color (820 mg, 4.33 mmol, yield 69%).
[0027] 1H-R.
[0028] 300 MHz. CDCl3) = 3.613337 (m, 8H), 3.55-3.48 (m, 2H), 3.38 1, 0.71, 70.68, 70.63, 70.05, 50.6; PERP. Rf = 0.56 (p-ethyl acetate: tyl, v / v) b) Pre-ionization of OSE (pentynoyl) -2 - ((z-3o) m-) piperazin-1-carboxy pentynoic acid, HATU, DIPEA DCM, TA, 2 h 30, quantitative 20 At one instance, 11.1 compound 12 (50mg 0.106mmol, 1.0eq): 2mg, 0.17mmol, 1, leq) and HATU, , 0,117mrtioD, 1 aq.) In anhydrous DCM (5m1 ...) under DIPEA additive (4 2,2eq.) And the actual mixture is ag at the end of the reaction. the reaction mixture is. DC (20mL) was washed twice with NaHCO3 (2x20mL) and once with sodium. Phase L H is dried, L E. The reaction crude purified by chromatography column. ply on neutral alumina 3022 784 55 (Petroleum Ether: 21 "oe.e / 1: 9 / v: v) to obtain the compound 30 in the form of a homogenous emulsion. 500 MHz, CDCl3) δ = 7.59 (dd, 3 = 14.8, 8.6 Hz, 1: 1), 7.36 - 7.27 (m, 2H), 7.27 - 7.21 (m, 2H), 7.21 - 7.02 ( m, 2H), 6.73 - 6.45 (br, 1H), 6.32 - 6.04 (m, -I), 4.67 - 4.25 (m -.17 - 3.69 (m, 3H), .47 (m, 3H), 3.45; - 3..9 -I), 3.18-2.89 on, 2H), 2.81 (s, 3H), L4 - 2.63 (m, 1H), 2.60 - 2.47 3H), 2.44 (s, TH), 2.20 (s, 3. - 1.96 (m, 1H) ppm.
[0029] 13C-1e (125 MHz, CDCl3) = 171.52, 171.36, 170.69, 170.37, 165.73, 160.62, 154.03, 153.52, 153.23, 152.78, 152.16, 134.78, 134.65, 129.23, 128.97, 128.88, 127, 33, 127.21, 125.35, 117.9 /, 117.58, 117.45, 114.33, 114.22, 110.28, 110.16, 83.32, 83.05, 69.34, 69.03, 53.52, 51.33, 45.59, 45.20, 45.03, 43.62, 42.37, 42.19, 41.21, 40.48, 40.19. , 39.98, 38.62, 38.05, 37.50, 31.77, 18.74, 14.66, 14.36 ppm.
[0030] MS: ESI: [M + H] m / z found 515.3, calculated 515.2 Rf = 0.20 (petroleum ether: ethyl acetate / 1: 9 / v: v) c) Prepared: omposed 31, 4-meth - oxo-2H-chromen-7-yl 4- (3- (1- (2- (2- (2-methoxyethoxy) ethoxy) ethyl) -1H-1,2,3-triazol-4-yl) propanoyl) -2- ((2-phenylacetamido) methyl-1-carboxylate 28. CuSO4.5 ascorbate from a partition of the compound btrnmol, 1.0eq.) And the compound 28 2.1 0.128mmol, 2.2eq. mixed. DCM (1.mL) and water (1mL) were acidified CuSO4.5H2O (9mg, 6 O4mmor,,) and sodium ascorbate (10mg, 0.08mmol, 1.2eq.) To Uri2 portion. This reaction mixture is then VA prrirn 2T, At the end of the onnel is with DC71, L) and water CiOr 3022 784 56 orna ue is withdrawn and the phas2 -'i.se is washed three times with DM The organic phases are dried with Nt 7; and evaporated. The Krlit r4, jinel is purified by DCM gradient killing gel column: MeOH / 100: 50, 98: 2, 97: 3, 96: 4, 93: v / v: v). compound 31 in an oven, for example, 11-V 500 MHz,) δ = 1-1,), 7.47 (m 1. 32 - 6.94 (m,, I), 3.61 (s, 0.2H), 6.21 (m, 0.2H), 4.73 3.25H) , 4.55 - 4.17H), 4.01 - 3.69 (ri, 3.6! - 7; (m, 1.5H), 3.59 - 3.50 (m,, 1 = .0 - 3.38 (m, 3H), 3.31 2.5H), 3.26 - 2.55 (6.51-1), 2.36 (ppm) 3C-NMR (125 MHz, CDCl2 172.66, 54, 170.71, 60.77, 154.23, 153.64, 152.93, 152.10, 134.96, -r.70, 129.42, 129.03, 128.85, 12 '.35, 127.14, 125.36, ..96, 118.17, 117.60, 114.42, 110.45, 72.04, 7 69.56, 67.21, 59.18, 5: .., 50.21, 45.48, 45.21 44.77, 14.02, 43.86. , 42.43, 42.22, 41.15, 40.55, 40.24, 40.00, 39.36, 38.12, 37.72, 37.42, 32.04, 31.66, 29.81, 28.81, 213, 0.57, 18.86 ppm MS: ES, I + m / z tr (p 704.5 , calcd 704.3 Rf = 0.30 (DCM: MeOH, 27: 7 / v: v) XI - Preparation of 4-valentyl (4-ylmethyl) -2-phenylacetamide 9. The salt form with the Trifluoroacetyl Acid Prepared Late Utyl (A): Methyl Ester D: Male 1-12 Atrn, (10% w 1, 2, i) Methane MeOH 18h, 76% The procedure used is the same asthat described for the synthesis of compound 12, with compound 7 (605 mg, 1.53 mmol, 1.0 eq), 1,1,2-trichloroethane (161 μL, 1.70 mmol, 1.1 eq.), Pd / C (12 r. 2u) in MeOH (30mL) to obtain the compound: ## STR2 ## a white (41mg, 1.16mmol, yield: 76%). - MHz, CDCl3) C = 7.43 - 7.15 (-1.5I-1), 6.25 (s, 1H), 4.47 - 4.15 (m, 2H '- 3.57 (n, 2.H) 3.52 (d, J = 2.9) z, 2H), 3.33 (m, 0.7H), 3.28 (rn, 2.8H), 2.68-2.29 (m, 4.5H), 2.00 (d, J = 8.4Hz, 1H), 1.47n (7sMHz, CDCl 3) at = 171.53, 171.33, 170.51, 170.26, 165.70, 34.84, 1, 12.11, 128.78, 127.26, 127.09, 83.36, 83.06, 80.81, 68.90, 7, 45.37, 43.60, 42.10, 41.32, 31.77. , 31.69, 28.29, 14.55, 333.2 MS: ESI: [M4 found 3312, cp 17 ° 'Preparation of compound 33, tert-butyl 4- (pel -ynoyl) -2 - ((2-nylacetamido) r) piperazine 1-Carboxy: H HCl 32 To a suspension at 0 ° C. CH 2, 32 (410 mg, 1, 1.0eq), of pentyndic acid) and '= -IgAlCl', 1,22mmol 1,1eq.) In DCIA argon oil, addi - tional DIPEA Immol 2; and rearranging is rIll74 at 0 ° C hung then at RT for 7h15. At the end of the reaction, the reaction ruddle is diluted with DC L) and washed with an aqueous solution of 7% NaHCO 3 (2 × 5 C and a brine (7.). After drying with or without evaporation, the reaction is purified by 25 hours, on alumina ne-fre (petroleum ether: ac 15:85 / v: v) to obtain the residue 33 under nitrogen. Yellow (552 mg, yield: 302, 784, 581, 1H NMR (300 MHz, CDCl3) = 7.43 - 7.15 (m, 5H), 6.25 (s, 1H), 4.47 - 415 (m, ( m, 2H), 9 - 3.57 (m, 2. 52 (d, J = 2.9 Hz, 3.28 - 2.8 2.8H), 2.68 - 2.29 (m, 4.5H), 2.00 (d, J = 8.4 Hz, 1H) , 17 (s, 9H) ppm.
[0031] 13 C-NMR (75 MHz, CDCl 3) = 171.53, 171.33, .170.26, 165.70, 154.91, 1M. 0, 129.22, 128.92, 128.78, 127.26, 12709, 83.36, 83.06, 80.81, 69.0, 100, 53.49, 49.97, 45.37, 43.60, 42. 28.29, 14.3, 1.29 ppm. MS: ESI: [1v1-1] m, z, 413.2 Rf = 0.29, -iey pet. (c) Preparation of compound 34, 11 - ((4- (pent-4-, 1-piperoyl) methyl) -2-phenylacetamide under fora with trifluoroacetic acid. TFA DCM, TA, 3h 88% The procedure used is the method described in the synthesis of compound 8, using: ## STR1 ## in DCM (1 Tn Ern) to give h7: 34 compound under (500rr.-1 mmol) :: 88%) (50 MHz, MeOH) = 7.33 - 7.22 (m) , 4H), 7.22 - 7.15 (m, 1H), 4.4E, J = 13.4 Hz, 1.H 3.97 (t, J = 13.4 Hz, 1H), 3.45 - 3.11 (m, 6.5H), 3.06 (t. , J = 10.6 Hz, 0.5H), 3.01 - 2.81 (m 2 67 - 2.46 (m, 2H), 2.42 (m, 2H), 2.24 (d, J = 1.6 Hz, 1H), 1.34 - 1.26 (m 1 1) ppm.
[0032] 13 C-NMR (125 MHz, MeO D) δ = 175.33, 172.22, 172.09, 163.50, 1023, 162.96, 162.68, 1-30, 130.27, 130.17, 129.70, 128.09, 128.03, 83.85, 70.32, 70. :, 56.37, 56.22, 55.75, 54.81, 46.53, 44.58, 44.48, 43.72, 43.66, 43.55, 43.26, 42.64, 40.34, 40.19, 40.08, 39.63, 39.42, 32.73, 32.59, 18.65, 15.11, 13.09 pp 302 and 2 784 59 MS: ESI: [M + H] z found 313.2, calculated 313.2 XII - Preparation of the compound 4- (3- (1- 2- (2- (2-methoxyethoxy) ethoxy) 1H-1,2,3-a 1- 4-yl) propanoyl) -2-phenylacetamido) metha, ylate (EXAMPLE 5) ~ arenium of 4- (pent-4-yl) -4-phenyl-2- (2-phenylacetamido) rn (ine-1-carboxylate) paranitrophenylchloroformate, 02N iPr2EtN IrT, C at RT, 2 h 30, Aantitative 10 at 0 ° C cool and 0.014 mmol, 1.0eq.), and at room temperature. 5e in anhydrous DCM (2mL) is the para-nitrofenolchloroformate (28mg, 0.130mmol, 1.1eq) once The reaction mixture is stirred at 0 ° C for 30min and then AT hangs at the end of the reaction the m The reaction mixture was diluted with DCM (20 mL) and washed twice with saturated aqueous NaHCO 3 solution (2 x 25 mL) once with brine (25 mL). The organic phase is dried with Na 2 SO 4, filtered and evaporated. The reaction product was purified by chromatography on silica gel (petrol-ol: acetate 2: 8/20: v) to give compound 35 in solid form (a) quantitative. 11-I-NMR (500 MHz, CDCl3) b = = 9.8 Hz, 2H), 7.33 -, 7H), 6.23 fc n 41-1), 6.08 - 5.82 (m, - 4.36 (m, 1.5H), 4.3 - 17 (m, - 3.88 (m, 1.4H) 3.77 - 3.62 (m, 0.8 3.'32 - 4.5H), 3.12 (t, J = 12. 2.96 - 2.65 (m 1.8I-1 2.65 - 2.26 (r 4.2H), 2.08 - 1.86 (, 1.29-1.13 (m, 0.5 1) 13C-R 1 CDCl 3) 6 = 171.59, 171.35, 170.80, 1 170.28, 40, 155.85, 152.84, 152.40, 145.05, 144.94, 134619, 134.48, 129.35, 129.25, 3022 784 60 129.08, 128.96, 127.50, 127.33, 125.17, 122.27, 122.13, 121.85, 83.28, 83.03, 69.37, 69.10, 53.54, 51.50, 51.25, 45.58, 45.23, 45.03, 43.68 , 42.43, 42.09, 41.20, 40.59, 40.18, 38.70, 38.10, 37.48, 31.86, 31.75, 14.71, 14.40 ppm MS: ESI: [M + H] + m / z found 478.3, calculated 478.3 Rf = 0.32 (ether of: ethyl acetate / 2- b) Preparation of compound 36, trc 2- (2- (2-methoxyethoxy) ethoxy) ethyl) -1H-1,2,3-tria2-dyl -2- (2- phenylacetamido) methyl) piperazine-1-carbox The procedure used is the same as that described for the synthesis of compound 31, with the co Mposé 35 (30mg, 0.063mmol, 1.0ec compound 28 (26mg, 0.138mmol, 2.2eq.), CuSO4.5H2O (9mg, 0.04mr), 6 (sodium ascorbate (16mg, 0.08mmc) 1.eq.) in a mixture C.sub.15 (1mL) and water (1mL). After purifying the compound 35 as a light oil (39mc 058mmol, yield: 93%). 1H-NMR (500 MHz): 8.29-8.01 (m, 2H), 7.54-7.01 (m, 7H), 4.56-4.24 (m, 5H), 3.66 (m, 5H), 3.66-3.37 (m.p. m, 12H), 3.31 (d, 3 = 4.7 Hz, L.55 (m, 7H), 2.18 - 1.84 (m, 1H) ppm.
[0033] 13C-RM MHz, CDCl3) b = 172: 71.62, 156.11, 155.95, 152.46, 145.10, 144.97, 135.45, 134.92, 29.1, 128.97, 128.80, 127.31, 127.10, 125.18, 122.21, 72.00, 70.64, 70.60, 69.43, 59.14, 51. 1.26, 50.69, 50.44, 50.18, 45.45, 45.20, 44.63, 44.03, 43.80, 42.40, 4; 11.06, 40.40.25, 40.10, 39.39, 38.05, 37.72, 37.48, 31.85, 31.39, 30.40, 29.77, 29.51, 28.59, 26.99, 21.36, 20.53 ppm. MS: ESI: [M + H] + m / z found 667.3, calculated 667.3 Rf = 0.31 (DCM: JH / 95: XIP Preparation of 9,4-chloro-2- ( 6-chloro-4-oxo-3,4-iminazolin-2-yl) phenyl-2-meth (etho) ethoxy) ethyl) -1,3-triazol-4-yl) propar. 1-methylacetar, Ho) methyl) piperazine-1-oxylate (EXAMPLE 6) of Dopo-chloro-2 (6-chloro-4-oxo-3,4-ihydroquazolin-2-yl) phen-1-yn-4-ynoyl) 2 - ((2-acetamido) n-lipérazine-1-carboxylate CI 0 CI 1. triphosgene, iPr2EtN DCM, 0 ° C to RT, 18h C 2. 34, iPr2EtN, DCM, 0 ° C to RT, 18h 69% CI To a slurry, cooled to 0 ° C and under an inert atmosphere of argon of 10 ....: cenpose 37 (-milling, 0.13mm 1.1eq) in anhydrous Ce-K1 (5mL) is added a solution of tryposgene (130mg, 0.43mmol, 3.3eq) years' 2mL) followed by the addition of DIPEA (80pL, 0.43mmol qeq.). This reaction mixture is stirred at 0 ° C. for 30 min. The results were determined by evaporation under a 15% wavelength, and was recorded in DCM anhyd, e (5mL). This solution was added to a solution of compound 34 (50 mg, 0.117 μm, eq) in dry DCM (mL), DIPEA (85 μL, 0.47 mol, 4 eq.). and this reaction mixture was reacted for 18 hours, the reaction mixture was d (20 mL) and 20 times with a saturated aqueous solution of NaHCO 3 (2 x 25 mL) and a salt with brine (25 mL). The organic phase is dried with Na 2 SO 4 and evaporated The crude reaction product is purified by two successive columns of silica gel on silica gel (petroleum ether: acetyl, ethyl acetate 2.8%). and DCM-MeOH / 99: 1, 98: 2, 97: 3 / v: v gradient). The compound 38 was pure as a white solid (52mg, 0.080, t: 59%). 1H CDCl3) δ = 11.12-10.16 (r 7_14 (s, 0.7H), 8.01-7. 7.64 (s, -L, 7.41 (s, 2) H), λu - (m, 5.3H), El 2 5 - 6.46 (m, 0.9H), 6.10 (s, 0.3I-fl 4.56 - 401 2H) - 2.99 (m, YiH 2.99 - 23 (m, 81-I), 2.60 - 2.15 (m, 3.6 W, 2.10-L59 (m, 1.6H), mp (ppm).
[0034] 13C-Rn (17) MH = 17305, 171.80, 170.88, 170.50, 170.31, 161.41, 152.46, 149.45, 147.43 /.20, 147.09, 135.64, 135.41, 135.19, 1: 34.72, 134.55, 133.55, 1, 39, 133.14, 132.11, 131.72, 130.88, 13ii.52, 130.29, 129.61, 129.43, 129.27, 12.20, 129.05, 128.85, 127.99, 127.66, 127.45, 127.20, 127.1G, 125.96 - 80, 124.81, 124.35, 124.16, 122.27, 122.06, 121.85, 83.37, 87.08, 82.95, 69.53, 69.39, 69.06, 6897, 51.86, 51.67, 45.26, 45.09, 44.96, 43.58, 43.38, 42.44, 41.78, 41.11, 40.58, 40.37, 39.74, 39.33, 38.86. , 38.41, 38.02, 31.76, 29.72, 14.68, 14.33 ppm MS: ESI: [M + H] m / z found 6452 ,, - peak 645.2 Rf = 0.19 (DCM: MeOH / 97: 3 / v: v) 302 2 784 63 b) Preparation compound 39, 4-chloro. -2,6-Chlok, -4-oxo-3,4-dihydroquinazolin-2-yl) enyl 443'11 .- (2- (2- (2-methoxyethoxy) ethoxy) ethyl) -1H-1,2,3 -tria; 1-propyloyl) -2- ((2-phenylacetamido) 111 + yipiperazine-1-carooxyiate CI The procedure used is that it is written for the compound 31, with the ammol compound, 1.0eq.), the Lohi daring 28 (31mg, 0.170mmol, 2.2ecP CuSO4.5H.0 LCnq, 0.05mmol, 0.6G3.) Sodium (20mg, 0.10mm, in a 2-liter mixture) and water (2mL), after purification, the compound 39 was obtained in the form of a slightly yellow oil (50 mg, 0.060 mmol, yield: 78%). 111-F / 500 MHz, CDCl3) δ = 11.1.2 (s, 0.4H), 10.89 (s, 0.2H), 10.14 - 8.33 - 7.94 (m, 21-7.71 (m, 2H), 7.70 - 7.48 (m, 7.4: - 7.32 (m, 2H), 7.30 - 7.1 (m, 3.5H), 4.54 (m, 4H), 3.93 (m, 4H), .77 - 3.45 - 2.72 (m, 2.39). C11C3 (s, 3H) ppm.
[0035] 13C-RM MHz, CDCh at = 174.00, 173 1, 72.11 171.24, 161.27, 152.45, 52.24, 149.84 71, 147.59, 1.41.54, 147.30, 147.19, 135.53, 135.30, 135.16, 135.11 133.08, 133.02, 132.14, 132.09, 131.98, 131..67, 131.54, 131.04, [29 79, 129.44, 1.29 ... 128.84, 128.78, 128.68, 20.64, 127.19, 126.97, 126.04, 125.83, 125, 124.32, 124Y, 122.54, 2.31, 70.64, 70.59, /0.43, 69.27, 59.15, 2.06,: ... b (,), 51.5t, bO90, 50.58, 45.52, 45.47, 45.3 45.17, 44.5: 3.88, 43, 72, 43.53, 41.87, 41.17, 28, CuSO4.5H2O sodium ascorbate DCM: H20 (1: 1), TA, 2h 78% Cl 39: 3022 784 64 40.84, 40.42, 40.00, 39.20, 39.11, 38.30, 38.28, 30.34, 29.70, 29.43, 21.39 , 20.64 Drr, MS: ESI: H, m / z found 834.5, calculated 834.3 = 0.13 (D (.M v: v) B - Evaluation of the rosolubility of corn, The hydrosolubli, is compcy function of the presence or c of these compounds, which are the following: (::, the known product quantity is dissolved in a volume of DM). to obtain a clear mother solution at the end of the DN in the best case. has found a troubled solution, the cond. ns and the 15 sc) 1u_. .1 mother in DMSO was lowered to 50 mM. THIS.; Mothers are then diluted in a PBS flask as follows to obtain a range of concentrates, including the following: HiM and A compound is declared insolubi if a precipitate appears and persists after U: ion.
[0036] The US presented the results of these solubility tests.
[0037] 3022 784 65 Concentrations Product 1mM 500pM 250pM 100pN 50pM 2-Ex soluble soluble soluble soluble soluble insoluble insoluble insoluble soluble soluble soluble soluble soluble soluble 9 Ex 6 soluble soluble soluble soluble soluble AMC- solubble soluble Dlub e insolt soluble insoluble insoluble insoluble insoluble insoluble 21 - Ex 2 insoluble insoluble soluble soluble solubility 22 - Ex 3 soluble soluble soluble soluble soluble soluble insoluble insoluble soluble soluble insoluble Dar, the cases presented, the co -elon 7. C..11UMCD7 rap Ân described in the dema ,,, subs. corporbii, a 2013/045854 and WO 2014f. Donationist Act: certain: 10 _Lee under F3 Cti .states for scence 3022 784 66 A. a srdid-ir, n of probe according to invervdonin PBS in a plate 9 (7, - puts rescence or ' A solution of GA (Penici A. melase Vaterstone Tech.) of PBS or an elution of Lipase (Lipase, Candida Rugosa, Sign-SOride, COM BS.) Concentration between 10 μM and 50 μM Rinse PGA Concentration : 5U Or concentration rriaqe: 4U iue and ene, ufte incubate 37 ° C and the fluopE :: s ,,: nude 10 the abs) e course of time with a ', cf: el ... 1- J E-nSpire rou, .. e). The resulting :: 50: nt ": e triplicate rests For the, e: thylunn1- 1.1: ert: omposed 12, 15, 31 Fluorescence: ex = 370 l- m nrn: 15 - Hard 530 nm, paranitrophenolene (Absorbance compound Aabs = 405) -exposed results on k to 1. Annexed, the evolution of the fluorescence supressed ULLIVGIUUI I UU 'posed 12 by the 20 PGA is presented FIGURE 'fAvc ° r: D: ence sui composed rir the resentee - The evolutior this follows,' aç.-...: b ., 7tion of compo_ -, ra PGA.is presented ri 25 La (1E7 ° * response of the ccuI and 3e the invention, and compo utj ~ .5e e, and their activation by the PGA) ésente 4.. The PGA is shown to be PGA based on rad: Tv = 7: by fluorescence evolution following activation of compound 21. The lipase is pre-set FIG. 7. The ratios of the compound, the invention, and the compound 27 'are as follows. As long as COTrip., following their activation by Lipase, n FIGURE 8. Conclusions the compounds of the invention, effectively erect, to detect the presence of a virus enzymato ev through c mesudv fluorescence or diabsorbance Lai '..' c ... :: :(,) enzymabq birth. It is a relatively small group of cells or a steric view of the spaced-apart arm.

权利要求:
Claims (5)
[0001]
REVENDICATIONS1. s I formula (I): wherein: = NH, O or S X2 = O or S, SE is aqueous under the action: UiTiULUS From 1 "- ::. be the pie.ence of one of a compound In a medium or medium it is found that the probe, A is an aromatic group, which after cleavage of the aqueous C (X2) -O bond, leads to a phosphorus. or a fluorophore R representing a hydrogen atom which is equal to 0 or 1, L is a bromine and a hydrolysing agent or hydrates, prysLo.
[0002]
2. Probes the UJon I carck -'-. In that group 20 labil 'ubstrat
[0003]
3. Probes according to claim 1, wherein E is an oxygen and E is an extender substrate. and where S is X1 is a reducing agent. However, this is a non-binding or transferase binding substrate, and one that is c by a
[0004]
4. Probes according to any one of Claims 1 to 3 in which the number is in the range of at least one of said esterases is greater than that of said carboxylesterases. Phosphatases such as glucuronidases, such as cosine, proteases such as meta-cetases, peptides, and aminophosphates.
[0005]
5. Probes s = cl = I === 7: = 7neque of veve4clication 4 cari rized in that IF groups one anc = rr = l-rique ec-. ; groups peptc, = / - es: 72 10 the rule by Ln n, Dr7 rbOrrcrrr dna, tate-at Scetles according to -7 -) =. 1 = that-denicatiorr 1 5 cari, == 7 > 3e in that gE is n by iple of N-acetyl-p-ctosamin = d, E, = icosa idase; α-Amylase a-1α, 1α-1) inofurarrsiclase .. arabin = sidase; p-cellobiosidase pchit (== '. = 7' -, == ida; galactosidase rilucosi; p-glt. ase tosidase; --- = annos -se; I mannos; d: Y ::: p-xylOSivac; pD-lucosidase; O.-L-fucosidase; L-fucosidase L-iduronidase or cell; and corresponds to 20 g; nuccrylation; The fluorocarbon carbon is a molecule, and SE is a group: the galactosyl, the glucosilane, the mistletoe, the methyl, the frucupsyl, the araoinos, the ribosyl, the xylosyl, yl and acetyl-hexosam ==== - == y - k: ^ lycosyl Cuu group d ~ - "rieurs, by eyernpl, n d- 2 to 20, prefére-ice 3 to and read -nt of 4 t = '., _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _. ) sidase, hc N-acetyl-D-cosatnoce, r-rni = -Tb ': 3.7; annanosidase, fucosidase or glucirrnidase .. especially the 0 -glu cwo; and cc Lià, .... upernent gyc. Bora are the rest of I tolecule, also mono-hydroxyalkyl selected by, D-glucuron nyl, atonyl-anosyl, β-glucosyl. D-rran nfe, leiUcopyranosyl or a g, e7., Ment 1Ssurs, for example 20, of ft: »p, 1H from 4 to 0 oa's monoglycosylated groups identiqui- 8 - Probes according to claims 1 to 5 In that SE is: idase, let N be a raw substrate 7,137: .MA te., J7; eE substrate (: ', e rater> rostate Spe nbran- gen); and SE is a mixture of the aptidyl and the remainder of the acylated molecule. As a result, a staphylatiline transterase, gamma-pyrraseferase, and PEEP-or amino acid bound to the by a fon-te-a-1 dortinated by its carbon or by a sporular side hawk to a sulfon group. a, ejnamide with, then represents NH. Probes according to the invention: Claims 1 to 8, wherein: A compound comprising one or more aromatic rings or not; -1 / 4-, rn take a "", L, the atom ((the o>, -: -: -) or of and / or or several _as carbon D i for-ne a carbonyl C = O. 10 - Probes according to one of the following: wherein R 1 is an aryl compound, R 1 resulting in the liberator selected from paranitr and its derivatives, the indigoids, the oxyflavone, and the probes according to one of claims 1 to 9, characterized in that A is a compound with -OA selected from: fluorescein resorufin DAO 12 - Probes according to one of the claims characterized in that A is an aromatic compound with -OA q in the formula (AA): wherein: X 5 is an oxygen atom and an aryl, -O-alkyl, 2-enyl group, wherein said phenyl groups may be substituted or n- is X 5 is nitrogen and is attached to X 4 is CH 3 , S, N or NH for substituted or unsubstituted heteroaryl, and: whether or not by exen,, e chosen pan ,. groups ph & e, aphthyl, and: o said groups may be substituted or unsubstituted, with X6 which represents 5, NR ", and R" which represents a hydrogen atom or a group (Cr kyle, preferably, DA is of the type phenoxy, and corresponds to the following preferred structures (BB) or (CC): Rb (BB) in which - T is -N - iC (O) -, -5-, -O-, - 4, Na alkyl or N-aryl, - Ra is hydrogen or carbon attractant, such as CN or --COOP, J, with a (C 1 -C 4) alkyl group, or R.sub.a is: ## STR2 ## of the different, which represents: hydrogen or a (C 1 -C 4) alkyl group, or R a is -CF 3 or a gr 2 -oxazolyie, 2-imidazolyl, 2-benzoimidazolyl, 4-pyrimidine, 2-yl or 1-yl, a chlorine atom, bromine, iodine or - Rb is hy-o fluorine, -OH, -NH2, -NRgRh, each independently one (Cr with Rg and Rh which represent well Ra and Rb are linked together to form a column comprising 4 or the optionally substituted or unsubstituted In one embodiment of the invention, Li or the group consisting of N, S and O, ydrogene, Br, Cl, I or F, R 'a (CC) in - T' is OH, L, R, NHR'c, O1, NR '. cR'd no SR'c - and R'd c.fférents, which represent a gr.) up, nu 15 R'a is the hyt) gene OUfl The cari locator carPcnr uttracteur d -CN, or -COOR'e, with OR P: e.St -CONR177.tg, with hydrogen or L is a 2-oxazoly, 2-rolmidinon-2 ttn or quinaz: u r47.1reSeH., a group (C1-, ck Nucleic acids or di-nents, which either R'a is -CF3 or imidan, 2-benzenamide. In one, bromine, or with R'h and Pt, cntiques (- 'erei which represent a group (C I, rote them to form a _ R'h 2, -NP2hR-i fluorine); ## STR5 ## wherein R is unsubstituted, optionally interrupted by one or more hetero atoms. Probes selc lueio :::: incp from 1 to 9 ..Orisées in this oi CC7: 0 aromatiquo -OA which answers to one of the following formulas: CI re --- idîciJons 1 to 13 14 - waves according to characters in this that R 1 is n bonding, and in particular a brE 1- (L2) m2- (L11) can7 the piper direction -> .gru'.1.7e GP av (Li L'l, the same or different , which are moldy among -O-, -NH, .alpha. -N (phenyl) -, -N (ar: C (O) -, -C (O) O, -C1 .., O) -, -OC (O ') -NHC (O) -O-, -OC (O) -Nh "NHC (O) -NH -SO2-, which is selected from C1-20 alkenyl, C1- C7-C7-44 alkynyl, C7-C17-alkyl, C7-C7-44alkenylcycloalkyl, -C7-alkylheterocycloal-C (1-) -C1- and -CONH-; C4-44 alkenylhdocycyno alcy: iterocycc or to terminate by a cell selected from C 1-10 alkyl (70XY, C1-10 alkyl, C6-10 aryl, amido, iruuo, phosprici my alkynyl E 25 ml, r1 and m2, identical .nts, which are equal to 0 or 1 and GP being as defined in claim 1. 15 - Eebn n resells pea 14, characterized in that L represents - (L1) m1 .- () 2 - (L '= C (O) - ml = m2 = 1, the; Rupees new in the subset, n-ipus are unsubstituted or -rn.1 = 1 or 0 and L2 and LU k as in claim 14, and in particular L is -C (O) - (CH) -L3- with p which is equal to 1, 3 or 4 and L3 which is a triazole group and in particular a group 1., L, 41 16 - Probes according to one, that revendiri, , TJen.s In this process, the hydrogen is charged with water, and the hydrogen is charged with an aqueous charge of 7 to 7 carbon atoms. 1 to 15% - one of the amines, ecu-guanidine, izole, and the use of ammonium, diisolate, or phosphonate, such functions F1 and / or [J, Sugar or lysacrid such as glucose, ictose and m3nnose, peptic group, etc., such as poly, ginine, and FAT-pe-Probes one. Claims 1 to 17, characterized in that X2 is a diatomic oxygen atom, in accordance with one of claims 1 to 18, characterized in that: For example, a glycosidase substrate or a glycosidase group, a glycosidase, a glycosidase, a glycosidase, a glycosidase, a glycosidase, a glycosidase, a glycosidase, a glycosidase, a glycosidase, a glycosidase, a glycosidase, or a glycosidase, or a glycosidase, or qu. for example, an alk erence group of 1 to 20 cc), a group of 1 to 2C r3 of 1: 1 Dne, a small protease substrate or p-ptidase 30 and the like. lyl bound to the rest of the molecule, by an acyl function portE-air its car-bai-the arra aiia or by ur l-L-GP with: represents - (L1) m1- (L2) m2- (The 1) m 1 with L1 = - C (0) -, 1 '= m_ = 1, m'l = 1 yes ti and L2 and L1 as defined at refurbishment. 14, and L is - C (O) - (CH2) p -Li, p aat equal to 1, 2, 3 is i. In addition, it is preferred to use a fatty acid salt, which is preferably used in the presence of hydrogen, phosphate, and polyethylene lycols. Probes according to the invention selected from: H-Aloriiire de 4-a-2airichrorriaa.J 2 - ((2-pheriilacetamido) n .: hyl) piperazine-1-aiiiraicaialaiiiaia (Compound I: 12 NH 21 HCl (N-octanoyloxy) methylol-N-1-carbox-) -2H-Crirornen-7-yl] -alkyl hydrochloride 21) 4- (4 - (((4-methyl-2-oxo-21-yl) -acetate) chromen-7-yl) (b) carbonyl) -3- ((octanoyloxy) methyl) piperazia-1 ') butane-1-sulfonic acid (Compound 22) SO3H 22 o 22 77 4-methyl-2-oxo-2H-chromen- 7-yl 4- (3- (1- 2- (2- (2-methoxyethoxy) ethoxy) ethyl) -1H-1,2,3-triazol-4-yl) propanoyl) -2 - ((2-phenylacetamido) Methylpiperazine-1-carboxylate (Compound 31) 4-Nitrophenyl 4- (3- (1- (2- (2- (2-methoxyethoxy) ethoxy) ethyl) -1H-1,2,3-triazole 4 1-yl) propanoyl) -2 - ((2-phenylacetam) -1-olpiperazine-1-carboxylate 10 (Compound 36) oo 364-chloro-2- (6- (2-methoxy) -4,4-rroquinazolin -2-yl) phenyl 4- (3- (1- (2- (2-Athyl) -1H-1,2,3-triazol-e-yl, phenylacetamido) -amine-1-carboxyla :: 39) 39 formulas ("p) (1.1) m1- (L) 2) m2- (1 and (I "p) R '10 in the following: X2 to L1, L1, L2, ml. are as defined for (I) in claims 1 to 15, where: EL 19, 3022 784 79 and Z is CECH, N3, a N-function; Inimid 3 or Rcp represents a protective group consisting of a group consisting of a group - (0-12) m 3 fluorine and the groups with R '1 which 1: at 12 tDn.7s of carbon or a corresponding urnyl, cycica or 0, 1, 2 or 3, 71u: carbo-sn darbPnyl groups, 9-fluorenylnthoxycarbonyl, and nor vioxycarbonyl, eurorries Pu hvdrF 10 22 - Are: -7: .. ElOn Only one of the claims I detecion in vivo, cne: Naked man nln: al, of a stimulus such a chemical compound or cerss ,,: t The invention relates to a catheter, a catheter, a catheter, a catheter, a catheter, a catheter, a catheter, a catheter, a catheter, a catheter, a catheter, a catheter, a catheter, a catheter, and a catheter. It is claimed that the presence of an enzyme is a compound (or a physical, chemical characteristic of the locus in which the probe is located, the presence of which is the presence of cleavage. The following steps are followed: - the setting in C: c) ncct of a sample uspec cLnir said stimuk with a probe lon h. Revolutions 1 to 20; Binding of the action of the stimulus, which is related to the cleavage of the 70L17, is one of a group, and the group, at a concentration of the present spacer. in the probe U (I) quantitative or qualitative, E, of the chroma or fluorophore released. 25 - 24 or 24, characterized in that it is subjected to drunkenness in lysiological conditions, in particular in an aqueous medium, preferably tanned to a pH belonging at pH 4 to 9, and pH at orc 7.4, 302, 784, 80 - Method according to one embodiment, in which a fluorophore is and its anal, e comprises the following: position of the fluorophrn a brninous source; the pro a era at a wavelength (i'b '.- iorption of fluorine () orna; and The method according to one of claims 23 to 25, wherein the analysis of the invention comprises the 10-compounds of the ch-c: Jmophore. to a luminous light capable of a light to a lorcp, the absorption wave of the chromophore of the absorbed light, corresponding to a tank, 3 - color 28 - method according to one or more claims 23 to 27 15 - in that the fluc e chro mc ore in 3 form of a compound 4 ,, - (117, H ... 0- or ,,. a fo ~ment, or of a terrique form or polym OE, 1 -ne a pr7: 7 -i in scutioror

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FR1456014A|FR3022784B1|2014-06-26|2014-06-26|WATER-SOLUBLE ACTIVABLE MOLECULAR PROBES, INTERMEDIATES FOR THEIR SYNTHESIS AND DETECTION METHODS THEREOF|FR1456014A| FR3022784B1|2014-06-26|2014-06-26|WATER-SOLUBLE ACTIVABLE MOLECULAR PROBES, INTERMEDIATES FOR THEIR SYNTHESIS AND DETECTION METHODS THEREOF|

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CN201580034492.9A| CN106470977B|2014-06-26|2015-06-24|Water-soluble activatable molecular probe, synthetic intermediate thereof and related detection method|

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US15/320,448| US10179929B2|2014-06-26|2015-06-24|Water-soluble activatable molecular probes, intermediates for the synthesis thereof and associated detection methods|

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PCT/FR2015/051705| WO2015197981A1|2014-06-26|2015-06-24|Water-soluble activatable molecular probes, intermediates for the synthesis thereof and associated detection methods|

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JP2016575183A| JP6793040B2|2014-06-26|2015-06-24|Activateable water-soluble molecular probes, intermediates for their synthesis, and related detection methods|

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CA2952332A| CA2952332A1|2014-06-26|2015-06-24|Water-soluble activatable molecular probes, intermediates for the synthesis thereof and associated detection methods|

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EP15742341.9A| EP3160517A1|2014-06-26|2015-06-24|Water-soluble activatable molecular probes, intermediates for the synthesis thereof and associated detection methods|