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WO2006006196A2 - Synthese de molecules organometalliques pouvant etre utilisees en tant que marqueurs de substances organiques - Google Patents

Synthese de molecules organometalliques pouvant etre utilisees en tant que marqueurs de substances organiques Download PDF

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Publication number
WO2006006196A2
WO2006006196A2 PCT/IT2005/000406 IT2005000406W WO2006006196A2 WO 2006006196 A2 WO2006006196 A2 WO 2006006196A2 IT 2005000406 W IT2005000406 W IT 2005000406W WO 2006006196 A2 WO2006006196 A2 WO 2006006196A2
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compound
bis
formula
following groups
compound according
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PCT/IT2005/000406
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WO2006006196A3 (fr
Inventor
Stefano Maiorana
Clara Baldoli
Clara Rigamonti
Patrizia Romana Mussini
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Universita' Degli Studi Di Milano
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Publication of WO2006006196A2 publication Critical patent/WO2006006196A2/fr
Publication of WO2006006196A3 publication Critical patent/WO2006006196A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F17/00—Metallocenes
    • C07F17/02—Metallocenes of metals of Groups 8, 9 or 10 of the Periodic Table

Definitions

  • the present invention concerns the synthesis of organometallic molecules used to recognise biological substances by spectroscopy or electrochemically.
  • nucleic acids for example: nucleic acids, proteins, amino acids
  • PNAs peptide nucleic acids
  • nucleic acids For the analysis of nucleic acids, the biomolecules most often used are nucleic acids themselves or else synthesis oligomers normally having from 10 to 20 monomeric units both carrying known base sequences. For the same purpose more recently peptide nucleic acids
  • PNAs DNA peptido- mimetic, in the structure of which the sugar phosphate sequence, negatively charged, has been replaced by a neutral aminoethyl glycine chain to which the nucleobases are linked .
  • biomolecules are immobilised on supports, for example glass or silicon plates or polymeric membranes, etc., so as to form the so-called microarrays.
  • This type of sensor is very important both in gene diagnosis and for other purposes, and the recognition of a molecule can be detected through spectrometry, fluorimetric, electrochemical methods etc. (Wang J.
  • the detection of the signal is obtained by introducing onto the biosensor suitable markers, such as fluorescent compounds or organometallic complexes (Fischer-Durand, N. Sa ⁇ main ,M.; Bogna, R. ; Vessieres, A.; Zakrwewski, J.; Jaouen, G. Chem. BioChem.,5, 519-525, 2004; Metzler-Nolte, N.; Angew. Chem. Int. Ed., 2001, 40, 6, 1040-1043; Verheijen, J. C; Van der Marel, G. A.; Van Boom, J. H.; Metzler-Nolte, N.; Bioconjugate Chemistry, 11, 6, 2000, 741-743;) .
  • markers such as fluorescent compounds or organometallic complexes
  • the present invention concerns organometallic compounds of formula (I) , their salts and solvates:
  • n is from 0 to 5;
  • A is selected from: O, N, S;
  • R 5 is selected from H and -(CH 2 )0-5-;
  • X is selected from the following groups: -(CH 2 ) 0-5-/ -CO-, o 9 9 C-N , C-(CH 2 )L 5 , C-S ;
  • Y is selected from the following groups: -(CH 2 ) 0-5- , (CH 2 ) L5 -N-C ? ⁇ o , (CH 2 ) 1-5 —S—C ;
  • OM is selecteedd ffrroomm tthhee ffoollilcowing groups:
  • R 1 is selected from the following groups : H, CO , o
  • R 7 is selected from the following groups: H, - (CH 2 ) 1-
  • n is from 0 to 2, more preferably it is equal to 0.
  • A is preferably selected from 0 and N.
  • R 2 , R 3 , R 4 are preferably selected from H and
  • D is preferably selected from N and O.
  • R 5 is preferably equal to -(CH 2 ) 0-2-, more preferably it is -
  • D is N
  • p is equal to 2 and R 5 is a radical -CH 2 - •
  • X is selected from: -CO-, -(CH 2 ) 0-2- and -CO-NH-; more preferably it is selected from: -CO-, -CH 2 - and -CO-NH-.
  • OM is preferably selected from the following groups:
  • OM is selected from:
  • R 7 is preferably selected from: H, -CH 2 Ph, - (CH 2 ) x . 3 /
  • NO 2 Ph, -C 6 F 5 ; more preferably it is selected from: H, -CH 2 Ph, -C 6 F 5 , -S-S-
  • A can be equal to NH.
  • the most preferred organometallic compounds of formula (I) are: Tris(ferrocene methylene oxymethyl)aminomethane (20);
  • the compounds of formula (I) can be used as markers for the determination of biological molecules.
  • biological molecules we mean: nucleic acids, oligonucleotides, proteins, peptides, amino acids etc, preferably DNA and RNA.
  • the biomolecules are DNA, RNA and PNA.
  • PNA PNAs having up to 20 monomeric units. Thanks to the presence of a polyfunctional unit containing many organometallic groups (for example ferrocenyl) , which are determined with an electrochemical method (for example: cyclic voltammetry) , the analysis sensitivity is substantially increased with respect to the corresponding substrates in which there is just one organometallic complex.
  • the products of formula (I) can also be used as markers of substances of pharmaceutical interest, for example antibodies (Fischer-Durand, N. Salmain ,M. ; Bogna, R.; Vessieres, A.; Zakrwewski, J.; Jaouen, G. Chem. BioChem.,5, 519-525, 2004) or antiepileptics (Vessieres, A.; Salmain, M.; Brossier,P. ; Jaouen, G.; J. Pharm. Biomed.
  • antibodies Fischer-Durand, N. Salmain ,M. ; Bogna, R.; Vessieres, A.; Zakrwewski, J.; Jaouen, G. Chem. BioChem.,5, 519-525, 2004
  • antiepileptics Vessieres, A.; Salmain, M.; Brossier,P. ; Jaouen, G.; J. Pharm. Biomed.
  • analogues of Tamoxifen (anitumour drug) containing a ferrocene group are known in literature. (Top, S.-; Vessieres, A.; Leclercq, G. ; Tang, J.; Vasserman, J.; Hunche, M.; Jaouen, G. Chemistry: A Europ. J., 21, 5223-5236, 2003) .
  • organometallie groups can increase the lipophily of the substance or molecule to which they are linked and increase their cellular permeability.
  • the present invention concerns the possibility of detecting biomolecules marked with the compounds of the invention through an electrode, preferably a microelectrode.
  • Said electrode can be used by itself or else immobilised, together with other microelectrodes, on a support such as glass, silicon, gold or polymeric membranes, so as to construct the so- called microarrays. (Wang J. Nucleic Acids Research, 28, 3011-3016,2000) .
  • step (a) the compound (2) is reacted with the compound of general formula R x -E (3) , where R 1 is as described above and E is a leaving group, preferably halogen, more preferably Cl or Br, even more preferably Cl.
  • R 1 is as described above and E is a leaving group, preferably halogen, more preferably Cl or Br, even more preferably Cl.
  • the reaction conditions depend heavily upon the nature of the group R 1 and are known in the field. For example, when R 1 is equal to group -COOCH 2 Ph, a mixture of H 2 O and AcOEt at a temperature of about 20 0 C is used as solvent vigorously shaking for about 5 hours. The desired product is obtained through separation of the pahses and extraction with solvent.
  • step (a) is optional; in some cases it is possible to start from the compound (4) in which R 1 is equal to hydrogen and to carry out step (b) directly; or else the compound (4) , in which R 1 is as described above, can be commercially available.
  • the step (a) is preferably carried out when A is equal to N.
  • step b) the intermediate (4) is reacted with the reactant of general formula (5) , where R 5 , X,. Y and OM are as described above and Q is selected from OH and halogen, preferably it is selected from OH or Cl.
  • the step (b) is preferably carried out in a solvent selected from: toluene, CH 2 Cl 2 , DMF, THF, dioxane or mixtures thereof, more preferably in mixtures of toluene/CH 2 Cl 2 2:1, CH 2 Cl 2 or DMF.
  • solvents are preferably anhydrides and the reaction is preferably carried out in an atmosphere of nitrogen or argon.
  • the reaction temperature is generally between 15 0 C and 100 0 C, preferably between 20 0 C and 80 0 C, even more preferably between 23 0 C and 65 0 C.
  • the reaction can be conducted in the presence of acids or of bases which are preferably selected from, respectively, CF 3 COOH 7 HCl, H 2 SO 4 , CH 3 COOH; PTSA, preferably CF 3 COOH, or Et 3 N, pyridine, KOH, N,N- dimethylaminopyridine, piperidine, K 2 CO 3 , Na 2 CO 3 , diisopropylamine, preferably Et 3 N.
  • acids or of bases which are preferably selected from, respectively, CF 3 COOH 7 HCl, H 2 SO 4 , CH 3 COOH; PTSA, preferably CF 3 COOH, or Et 3 N, pyridine, KOH, N,N- dimethylaminopyridine, piperidine, K 2 CO 3 , Na 2 CO 3 , diisopropylamine, preferably Et 3 N.
  • bases preferably selected from, respectively, CF 3 COOH 7 HCl, H 2 SO 4 , CH 3 COOH; PTSA, preferably CF 3 COOH,
  • the reactant of formula (5) can be used in molar excess with respect to the intermediate (4) ; the amount used depends upon the number of lateral chains present in the reactant (4) (for example one from R 2 , R 3 and R 4 can be equal to H) and upon the type of end product that one intends to obtain, i.e. having one, two or three organometallic groups. For example, if one wishes to obtain a trisubstituted end product the reactant (5) can be used in molar excess of 4-5 times or more with respect to (4) .
  • the product (6) can be deprotected and acylated in situ, for example by reaction with a base and then with an acylating reactant.
  • two products are formed: one acylated product and one intramolecular transacylation product (see example 5) .
  • step (d) the compound (7) is reacted with the reactant (8) of formula R 8 -OH, where R 8 is a linear or branched alkyl group (Ci_ 8 ) , preferably it is a linear alkyl group (CV 3 ) , more preferably it is a methyl group.
  • the reaction takes place at a temperature generally between 15 0 C and 100 0 C, preferably between 25 0 C and 75 0 C, more preferably at the reflux temperature of the reactant (8) , if it is a liquid, or else at the reflux temperature of the solvent used.
  • step (e) the intermediate (9) is reacted with the reactant (10) of general formula G- (CH 2 ) 1 -5-G, where G is selected from a group NH 2 and SH, preferably it is a group NH 2 and the group (CH 2 ) 1 - 5 is an ethylene group.
  • the reaction temperature is between 15 0 C and 100 0 C, preferably between 20 0 C and 80 0 C, even more preferably between 23 0 C and 65 0 C.
  • the duration of the reaction depends upon the starting products used.
  • the reactant (10) is also used as solvent.
  • step (f) the intermediate (11) is reacted with (12) of general formula Q-CO-OM, where Q and OM are as defined above.
  • reaction conditions are the same as those foreseen for step (b) .
  • the molecules of formula (I) are anchored to the biomolecule (amino acids, peptides, nucleic acids, preferably PNA, DNA, RNA, more preferably PNA) suitably derivatized according to the known methods for peptide synthesis, for example, by reaction with activated esters of carboxyli ⁇ groups present in the biomolecule.
  • amino acids, peptides, nucleic acids, preferably PNA, DNA, RNA, more preferably PNA suitably derivatized according to the known methods for peptide synthesis, for example, by reaction with activated esters of carboxyli ⁇ groups present in the biomolecule.
  • PNA amino acids, peptides, nucleic acids, preferably PNA, DNA, RNA, more preferably PNA
  • the compounds of formula (I) can be transformed into the corresponding isocyanate (13) that can react with amino or alcohol groups present in most biological and organic molecules.
  • Example 8 shows the synthesis of glycine labelled with a compound of formula (I) . This example can be generalised for all amino acids.
  • Fc At room temperature and in an atmosphere of nitrogen, a solution of ferrocenyl methanol (0.6 mmol, 0.13 g) in CH 2 Cl 2 (12 ml) is added to with N-Cbz- tris (hydroxymethyl)aminomethane (0.2 mmol, 0.05 g) dissolved in CH 2 Cl 2 (4 ml) and CF 3 COOH (10 ⁇ l). It is heated to 40 0 C for 4 h, then ferrocenyl methanol (0.32 mmol, 0.07 g) is added again and it is heated for another 4h to 4O 0 C. It is left overnight at room temperature then 20 ml of an aqueous solution of 5% NaOH is added.
  • EXAMPLE 3 N,N'-diferrocencarbonyl-1,3-dia ⁇ un.ino-2- propanol (24) .
  • the benzyl chromotricarbonyl acid (0.6 g, 2.32 mmol) is dissolved in anhydrous DMF, obtaining an orange solution that is heated to 6O 0 C.
  • the solid carbonyldiimidazol (0,38 g, 2,32 mmol) is added in portions over 5 minutes, the solution becomes deep red. It is heated to 6O 0 C for 30 minutes, noting the development of CO 2 .
  • l,3-diamine-2-propanol 0.1 g, 1.16 mmol
  • the combined organic phases are treated in a separatory funnel with a solution of KHSO 4 0.3M (3x5ml) , the organic phases are dried over Na 2 SO 4 and evaporated.
  • the crude reaction product is purified on a chromatography column (eluent: CH 2 Cl 2 /AcOEt/MeOH in increasing MeOH gradient) to give the product (26) in a yield of 63% and (27) as by-product in a yield of 23%.
  • the aqueous phases are once again removed with CH 2 Cl 2 (2x25 ml) .
  • the combined organic phases are washed with H 2 O (30 ml) and dried over Na 2 SO 4 .
  • the solvent is evaporated and a pale yellow coloured oil is recovered that is used as such for the subsequent reactions.
  • the aqueous phase is extracted with CH 2 Cl 2 (3x20 ml), the combined organic phases are washed with a 0.3 M solution of KHSO 4 (3x10 ml) and then with H 2 O (20 ml) , dried over Na 2 SO 4 , filtered and evaporated.
  • the crude reaction product is purified through a chromatography column on silica gel (ETPrAcOEt,4/6) followed by CH 2 Cl 2 :MeOH, l/l to elute the product, which is precipitated from pentane. It is filtered recovering 135 mg of (37) as a yellow solid in a yield of 77%.
  • Pt radius 1 mm.
  • GC glassy carbon
  • SCE saturated calomelane reference electrode
  • the compounds were studied: through cyclic voltammetry on electrodes made from Pt or GC with a non-rotating disc, at concentrations between 10 "4 and ICT 3 M, at different scanning speeds of the potential (from 20 to 500 mV/s) ; through low-speed scanning voltammetry (5 mV/s) on rotating disc working electrodes made from Pt or GC, at different rotation speeds (from 500 to 3000 revolutions per minute) . Both of the electrodes before being used underwent washing with HNO 3 and H 2 O.
  • the compounds of the invention determine an increase in the analytical sensitivity with the same amount of biological substance to be analysed. This result is obtained thanks to the presence of many organometallic groups that provide a current intensity that increases as the number of groups increases.
  • Using the compounds of the invention there is also the advantage of being able to link many organometallic groups to the biological molecule through a single reaction, avoiding having to replicate many times the introduction reaction of the organometal; this saves time and money.
  • the compounds of the invention can also be used as labels of substances of. pharmaceutical interest (for example antibodies etc.) and of drugs, in order to easily determine their localisation; for example to check whether interaction of the drug with cells and receptors has or has not occurred.
  • organometallic groups linked to a drug can also substantially increase its lipophily and therefore its bioavailability.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Steroid Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

L'invention concerne des composés de formule générale (I). Ces produits sont de préférence utilisés en tant que marqueurs de biomolécules (par exemple, acides nucléiques, oligonucléotides, ANP, peptides, protéines, stéroïdes, etc.) principalement utilisés dans le domaine diagnostique.
PCT/IT2005/000406 2004-07-15 2005-07-15 Synthese de molecules organometalliques pouvant etre utilisees en tant que marqueurs de substances organiques WO2006006196A2 (fr)

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ITMI2004A001427 2004-07-15
ITMI20041427 ITMI20041427A1 (it) 2004-07-15 2004-07-15 Sintesi di molecole organometalliche utilizzabili come marcatori di sostanze organiche

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006095181A3 (fr) * 2005-03-09 2006-12-14 E2V Biosensors Ltd Groupes de marquage pour biocapteur
US9127308B2 (en) 2002-03-07 2015-09-08 Atlas Genetics Limited Nucleic acid probes, their synthesis and use
US9421025B2 (en) 2008-11-05 2016-08-23 Shockwave Medical, Inc. Shockwave valvuloplasty catheter system
WO2019236954A1 (fr) 2018-06-07 2019-12-12 Seattle Genetics, Inc. Conjugués de camptothécine
CN114262275A (zh) * 2021-12-15 2022-04-01 华中师范大学 一种高效低毒性dna及rna脂质递送载体
CN117342983A (zh) * 2023-12-05 2024-01-05 康羽生命科学技术(苏州)有限公司 过乙酰化GalNAc-L96合成方法
WO2024108053A1 (fr) 2022-11-17 2024-05-23 Sanofi Conjugués anticorps-médicaments de ceacam5 et leurs méthodes d'utilisation

Family Cites Families (2)

* Cited by examiner, † Cited by third party
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US5310687A (en) * 1984-10-31 1994-05-10 Igen, Inc. Luminescent metal chelate labels and means for detection
US20050059834A1 (en) * 2003-09-16 2005-03-17 Ming Zhou Dendritic supramolecular compound for electrochemiluminescent analysis

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9127308B2 (en) 2002-03-07 2015-09-08 Atlas Genetics Limited Nucleic acid probes, their synthesis and use
US10094800B2 (en) 2002-03-07 2018-10-09 Atlas Genetics Limited Assays and apparatus for detecting electrochemical active markers in an electric field
WO2006095181A3 (fr) * 2005-03-09 2006-12-14 E2V Biosensors Ltd Groupes de marquage pour biocapteur
US9421025B2 (en) 2008-11-05 2016-08-23 Shockwave Medical, Inc. Shockwave valvuloplasty catheter system
WO2019236954A1 (fr) 2018-06-07 2019-12-12 Seattle Genetics, Inc. Conjugués de camptothécine
US12194321B2 (en) 2018-06-07 2025-01-14 Seagen Inc. Camptothecin conjugates
WO2023109881A1 (fr) * 2021-12-15 2023-06-22 华中师范大学 Vecteur d'administration lipidique d'adn et d'arn à haute efficacité et à faible toxicité
CN114262275B (zh) * 2021-12-15 2023-09-29 华中师范大学 一种高效低毒性dna及rna脂质递送载体
CN118434711A (zh) * 2021-12-15 2024-08-02 瑞达信使(杭州)生物技术有限公司 一种高效低毒性dna及rna脂质递送载体
CN114262275A (zh) * 2021-12-15 2022-04-01 华中师范大学 一种高效低毒性dna及rna脂质递送载体
WO2024108053A1 (fr) 2022-11-17 2024-05-23 Sanofi Conjugués anticorps-médicaments de ceacam5 et leurs méthodes d'utilisation
CN117342983A (zh) * 2023-12-05 2024-01-05 康羽生命科学技术(苏州)有限公司 过乙酰化GalNAc-L96合成方法
CN117342983B (zh) * 2023-12-05 2024-02-06 康羽生命科学技术(苏州)有限公司 过乙酰化GalNAc-L96合成方法

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