PL228280B1 - New asymmetric substituted derivatives of 1,4,7- triazacyclononane, method of their manufacturing, their application and the protein chip and method of the protein immobilization - Google Patents

Abstract

Triazamacrocyclic compounds are characterized by the fact that they have three different substituents attached to nitrogen atoms and have the ability to complex metal ions (e.g. Ni, Cu). Compounds with an attached moiety of the alkyl-oligoethylene glycol type are preferred and their use for monolayer formation, especially for the attachment of a His-tag protein.

Description

Description of the invention

The subject of the invention is new asymmetrically substituted 1,4,7-triazacyclononan derivatives, in other words triazamacrocyclic compounds with the ability to complex metal ions, especially Ni, Cu, having the ability to specifically bind proteins with a histidine affinity label (Histag) and a method of their synthesis. Preferred according to the invention are compounds with an attached alkyl-oligoethylene glycol moiety (with an alkanoligo (oxyethylene) moiety) which are particularly suitable for the immobilization of proteins on the surfaces on which they form a selfassembled monolayer. These compounds have a group binding them to the support at one end and a metal complexing triazamacrocyclic moiety at the other end which binds to the protein. These compounds can form self-assembling monolayers on the following substrates: gold, silver, glass, silicon and polymer, and minimize the non-specific attachment of proteins to the surface during protein immobilization on the substrate. The invention also relates to a process for the preparation of 1,4,7-triazacyclononane derivatives suitable for in situ functionalisation of substrates. The compounds of this invention may have potential use in the construction of biochips, biosensors, microarrays and other substrates on which proteins are immobilized.

Azamacrocycles form stable complexes with a wide range of dip block metals (Hancock RD, Martell AE Chem. Rev. (1989) 89, 1875; Wainwright KP Coord. Chem. Rev. (1997) 166, 35-90; Reichenbach-Klinke R ., Konig BJ Chem. Soc., Dalton Trans. (2002) 121-130). However, the stability of the complexes depends on the size of the crown (macrocyclic backbone) and the influence of the attached substituents on the crown. Lanthanide chelates (III) based on the azacorona structure (e.g. NOTA, DOTA) are among the most stable compounds synthesized so far that chelate lanthanides (Craig AS et al. J. Chem. Soc., Chem. Commun. (1989) 1793; Duma TW and J. Coord. Chem. (1991) 23, 221; Clarke ET and Martell AE Inorg. Chim. Acta (1991) 181, 273-280; Clarke ET and Martell AE Inorg. Chim. Acta (1991) 186, 103 -111). The Ga (NOTA) complex remains intact in nitric acid for 6 months (Broan J.P. et al. J. Chem. Soc., Perkin Trans. 2 (1991) 87-99). Among the macrocyclic amines, 1,4,7-triazacyclononane is one of the most extensively researched in recent years. 1,4,7-Triazacyclononane (1,4,7-triazonane, tacn, [9] aneN3) and its derivatives form very stable complexes with metals (Yang R., Zompa LJ Inorg. Chem. (1976) 15, 1499- 1502).

One way to change the chelating properties of a macrocycle is to introduce various chemical moieties into its structure. New strategies for the synthesis of azamacrocyclic compounds are still being developed to allow the preparation of azacorone compounds with more than one type of substituent in high yield. Unfortunately, the synthesis of a polyazamacrocyclic compound with a fixed number of substituents, including even the synthesis of a compound with only one introduced substituent per azakorone amino group, is difficult even with the available, proven methods. The introduction of only one substituent into the triazacoron by conventional methods produces a statistical mixture of compounds (Fallis I.A. et al. Chem. Commun. (1998) 665-666; Kovacs Z. and Sherry D. Tetrahedron Lett. (1995) 36, 9269-9272). The critical point in the synthesis is that leading to the differentiation of the three equal nitrogen atoms (secondary amine groups) during the synthesis. Conventional synthetic methods result in a mixture of the expected product with other macrocyclic compounds with a different distribution of substituents and their variable amounts, forcing laborious and often unsuccessful separation and purification procedures. Purification and isolation from the compound mixture is also difficult due to the chemical nature of unprotected amine azamacrocycles, which are basic, highly polar and have no chromophore.

Introduction to the synthesis of 1,4,7-triazatri-cyclo [5.2.1.0 ⁴ & ^10] decane (ortoamid-TACN) (Atkins TJJ Am. Chem. Soc. (1980) 102, 6364-6365) allowed the selective attachment of various moieties to triazacorons. This synthon enables mono-, di- and tri-N-substituted azamacrocycles to be obtained in high yield in a three-step synthesis (Schulz D. et al. Inorg. Chim. Acta (1995) 240, 217-229; Blake AJ et al. J Chem. Soc., Dalton Trans. (1996) 4379-4387; Ellis D. et al. Chem. Commun. (1996) 1817-1818; Blake AJ et al. J. Chem. Soc., Dalton Trans. (1996 ) 31-43; Creaser SP et al. J. Chem. Soc., Perkin Trans. (1999) 1211-1213; McGowan PC et al. Inorg. Chem. (2001) 40, 1445-1453). A. Warden et al. In Org. Lett. (2001) 3 (18), 2855-2858 and MJ Belousoff et al. In Polyhedron (2007) 26, 344-355 reported the synthesis of di-N-substituted tacn derivatives with two different substituents, including one tacn derivative with three different substituents (1- (3-aminopropyl) -4-benzyl-7-isopropyl-1,4,7-triazacyclononane) synthesized

A method different from that of the present invention. This method fails to efficiently synthesize the compounds of this invention. They used a method involving tacn-orthoamide.

One of the key technologies used for the construction of biochips or biosensors is the technology of protein immobilization (Rusmini F. et al. Biomacromolecules (2007) 8, 1775-1789). Existing methods of protein immobilization on a solid support use the physical adsorption of proteins on surfaces without the possibility of controlling the spatial orientation or the formation of a covalent bond by a non-selective chemical reaction (Chatelier RC et al. J. Biomater. Sci., Polym. Ed. (1995) 7 (7) ), 601-22; Nakanishi K. et al. J. Biosci. Bioeng. (2001) 91, 233-244). Protein covalent immobilization methods do not only immobilize specific proteins, but rather nonspecifically immobilize all proteins without defining their spatial orientation. Additionally, it is necessary to use highly purified proteins to eliminate non-specific binding of non-target proteins. The reactive site of the protein can be blocked following an immobilization procedure, resulting in decreased activity of the protein; the chemical reagents used may have a degrading effect on the structure and function of the protein (Gershon PD, Khilko SJ Immunol. Meth. (1995) 183, 65-76; Johnsson B. et al. Anal. Biochem. (1991) 198, 268-277; Collioud A. et al. Bioconjugate Chem. (1993) 4, 528-536; Veilleux JK IVD Technol. (1996) 26-31; Sweeney RY et al. Anal. Biochem. (2000) 286, 312-314; Wilson DS and Nock S. Curr Opin. Chem. Biol. (2002) 6, 81-85). The methods of non-covalent attachment of proteins to the surface most often use tags inserted into proteins. The surfaces exposing molecules (ligands) that bind to proteins via these labels ensure stable, specific binding and allow the proteins to be repeatedly attached and given a specific orientation (Cha T. et al. Proteomics (2005) 5, 416-419 ).

The methods of targeted protein immobilization are mainly based on the use of antibodies for this purpose. Specific interactions between biotin and streptavidin or the hexahistidine affinity label and nickel chelators are also exploited (Keller T.A. et al. Supramol. Sci. (1995) 2, 155-160). The hexahistidine affinity label is the most common tag used for insertion into recombinant protein sequences. A wide variety of templates and detection methods based on the interaction of the oligohistidine fragment with the metal-chelating compound complex have also been described (Ueda E.K. et al. J Chromatogr. A (2003) 988 (1), 123).

Self-assembling monolayers (SAMs) (Ulman A. Chem. Rev. (1996) 96, 1533-1554), formed by chemisorption of substituted alkanothiols on the gold surface, are among the best-characterized synthetic organic monolayers (Dubois LH, Nuzzo RG Annu. Rev. Rev. Phys. Chem. (1992) 43, 437-463) and have been used as model surfaces in studies of the interaction of proteins with surfaces (Spinke J. et al. Langumuir (1993) 9, 1821-25; Willner I. et al. J. Am Chem Soc (1992) 114, 10965-66; Song S. et al. J. Phys, Chem. (1993) 97, 6564; Mrksich M. et al. J. Am Chem Soc. (1995) 117,12009-10). The most attention is paid to organothiol monolayers on the gold surface. Monolayers are usually formed on substrates such as: glass plates, silicon, silicone, microplates, nitrocellulose or PVDF membranes, magnetic or other microparticles (microspheres). Alkanothiols can also be adsorbed on gold nanoparticles. Nanoparticles called MPCs (monolayer-protected gold clusters) are nanometer-sized gold particles completely covered by a protective monolayer consisting of thiol ligands (Brust M. et al. J. Chem. Soc., Chem. Commun. (1994) 801. ).

Modification of surfaces based on self-assembled monolayers with oligo- or polyethylene glycol is used to minimize non-specific interactions between peptides, polypeptides, proteins, nucleotides, and cells and the surface (Love JC et al. Chem. Rev. (2005) 105, 1103). Derivatives of alkanothiols are used that have a proximal alkyl fragment (segment) that facilitates their self-assembly into a monolayer, and a distal segment that minimizes non-specific interactions between the protein and a monolayer formed from such components (Singhvi R. et al. Science (1994) 264, 696-698). Alkanothiol monolayers on gold (SAMs), which constitute the majority of systems studied so far, i.e. alkanothiol monolayers with a glycol end fragment, have a minimal denaturing effect on proteins, providing them with a friendly, bioinert surface. By means of ellipsometry, it was found that the irreversible adsorption of proteins to SAMs formed from alkanothiols terminated with ethylene glycol fragments was blocked (Prime KL et al. J. Am. Chem. Soc. (1993) 115, 10714; Prime KL et al. Science (1991) 252, 1164 ).

In the purification process of genetically recombinant proteins, the IMAC (immobilized metal affinity chromatography) method has been used for many years.

Biotechnol. (1994) 1 (2), 151-64; Zachariou M. Methods Mol. Biol. (2004). ) 251, 89-102). The bed is previously functionalized with a metal chelating compound, most often nitrilotriacetic acid (NTA). Or iminodiacetic acid (IDA). Binding of the oligo-histidine label to the metal-chelating compound complex occurs by coordinating bonding of the N-atoms of the imidazole residues in the oligo-histidine label to free coordination positions in the Ni2 + ion, which is complexed by the chelating compound and thus partially saturated with coordination (Arnold FH Bio / Technology (1991) 9, 151-156).

The affinity (Kd) of a protein with a single histidine label for Ni-NTA modified surfaces was estimated to be 1 μΜ by SPR (Nieba L. et al. Anal. Biochem. (1997) 252, 217-228), while Hochuli E. et al. determined it at about 10 ¹³ M ^-1 (Hochuli E. et al. Bio / Technology (1988) 6, 1321-25), while in an assay using fluorescent Ni-NTA conjugates, it was determined that a single Ni-NTA complex binds from His with a Kd constant of 10 µM (Lata S. et al. J. Am. Chem. Soc. (2005) 127, 10205-10215).

The Ni-NTA complexes were used to purify proteins that had a tag consisting of six histidine residues introduced by recombinant DNA technology (Hochuli E. et al. J. Chromatogr. (1987) 411, 177-184; Porath J. et al. Nature (1975) 258, 598-599; Ueda EK et al. J. Chromatogr. A (2003) 988, 1-23).

The IMAC method using the interaction between a protein with a histidine affinity label and a surface presenting a metal chelating ligand, e.g. Cu, Ni, has many advantages and is suitable for direct transfer and application in protein microarray technology (Cha T. et al. Proteomics (2004) 4, 1965-1976). His-tagged protein is released from the complex with the metal chelating compound in the presence of a competitor e.g. imidazole or chelating compounds e.g. EDTA. In the case of IMAC based on the use of NTA, metal loss and protein dissociation in the absence of competition compounds are often reported (Dorn IT et al. J. Am. Chem. Soc. (1998) 120, 2753-2763; Nieba L. et al. Anal Biochem. (1997) 252, 217-228; Mateo C. et al. Biotechnol. Bioeng. (2001) 76, 269-276; Chaga GSJ Biochem. Biophys. Method (2001) 49, 313-334; Jiang W. et al. Anal Biochem. (1998) 255, 47-58).

The affinity of the metal-chelator complex and the interaction between the components of the Ni-NTA-oligohistidine fragment complex are too weak to provide a stable and stoichiometrically defined bond. Partially stable binding in the complex can be achieved by a high density of NTA groups on a chromatographic support or planar surface, then several histidine residues from an oligohistidine label can be bound simultaneously to several chelator-metal units (Dorn IT et al. Biol. Chem. (1998) 379 (8-9), 1151-9; Frenzel A. et al., J Chromatogr. B Analyt. Technol. Biomed. Life Sci. (2003) 793 (2), 325-9; Paborsky LR et al. Anal Biochem. (1996) 234, 60-5; Lauer SA, Nolan JP Cytometry (2002) 48 (3), 136-45).

Several papers presented a method of immobilizing His-tag proteins through their complexes with an NTA-functionalised medium. The use of multiplet NTA for attachment of proteins to surfaces was described in 1997 (Paborsky L.R. et al., Anal. Biochem. (1996) 234, 60-65). Using this technique, Schmid et al. Demonstrated a method of immobilizing His-tagged 5HT3 receptor for studies using total internal reflection fluorescence microscopy (Schmid, E.L. et al. Anal. Chem. (1997) 69, 1979). An analytical detection method based on surface modification with metal chelating groups was also presented (Schmid E.L. et al. Anal. Chem. (1998) 70, 1331-1338).

Sigal G. B. et al. Reported a method of immobilizing His-tag proteins via a thiol group on a planar gold substrate after prior formation of a metal chelating complex between the histidine imidazole ring and the carboxyl groups of the chelating compound. For this purpose, they used glass plates with a layer of gold, which they placed in an ethanol solution containing tri (oxyethylene) alkanothiols and a derivative of these alkanothiols with an attached NTA (Sigal G.B. et al. Anal. Chem. (1996) 68, 490-7). The density of the NTA-displaying molecules on the surface was regulated by the presence of another triethylene glycol-terminated thiol in the solution. The resulting mixed monolayer on gold was used for testing by the SPR method. Unfortunately, the combination with NTA is of the urethane type, which makes this compound susceptible to decomposition in an acidic environment and under the influence of sunlight. Self-organizing monolayers (SAMs)

On the gold surface, showing the NTA that binds to the Fab fragment of the antibody was used for FTIR studies (Nieba L. et al. Anal. Biochem. (1997) 252, 217). Detection of metal ions by means of SAMs presenting NTA has been proposed (Liley M. et al. Langmuir (1997) 13, 4190).

The above-described solutions based on chelating compounds, ie IDA or NTA, have many limitations. First, the stability of the complex, i.e. the interaction between the metal chelator, metal and protein, is often too weak to immobilize the protein for a sufficiently long period of time for observation and testing to be performed. Second, metal chelators interact non-specifically with untagged proteins. Moreover, the complexes formed may decay in the presence of some interfering compounds.

One way to increase the potency of the interaction between the affinity label and the metal chelating ligand is to alter the affinity label. Accordingly, on the one hand, extended His-tag tags are described which consist of 10 histidine residues instead of the standard six (Guignet E.G. et al. Nat. Biotechnol. (2004) 22 (4), 440-4). On the other hand, proteins and protein complexes are described which have two histidine labels introduced and bind more stably to the surface of Ni-NTA-chips (Nieba L. et al. Anal Biochem. (1997) 252 (2), 217-28 ; Steinhauer C. et al. Proteomics (2006) 6 (15), 4227-34) and two His-tag labels separated by a peptide fragment (Khan F. et al. Analytical Chemistry (2006) 78, 3072-3079).

However, not all proteins can incorporate one or more affinity tags at their N or C-terminus without too much disruption of their biological function (Freydank A.-C. et al. Proteins: Struct. Funct. Bioinf. ( 2008) 72 (1), 173-83).

A second option to increase the strength of the interaction between the affinity label and the metal chelating ligand is to improve the metal chelating ligand. Bivalent chelating complexes (two chelating groups (NTA)) for in situ labeling of proteins with a fluorophore introduced for their detection are described (Kapanidis AN et al. J. Am. Chem. Soc. (2001) 123 (48), 12123-5) . These complexes show increased affinity for the histidine affinity labels as compared to the monovalent ones. Also shown are ligand-attached derivatives of alkanothiols, which were obtained by increasing the number of NTA molecules attached to a linker. Such multivalent chalators bind proteins more strongly to His-tag. Monolayers (SAMs) formed from such derivatives have also been proposed (Tinazli A. et al. Chem. Eur. J. (2005) 11, 5249-5259).

The azamacrocyclic derivatives have much greater metal bond constants than those achieved with nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA). The stability (Kd) of the various metal complexes with NTA is in the range 10 ⁹ -10 ¹⁴ (Anderegg G. Pure Appl. Chem. (1982) 54, 2693; Anderegg G. et al. Pure Appl. Chem. (2005) 77 ( 8), 1445-1495).

PCT International Patent Application Publication: WO03 / 042249 describes the use of immobilized 1,4,7-triazacyclononan "tacn" derivatives (metal complexes thereof) for purifying proteins having a histidine affinity tag (His-tag) by affinity chromatography (IMAC). Immobilized tacn derivatives provide significantly increased metal bond stability, even during prolonged experiment, compared to NTA.

Recently, D.L. Johnson and L.L. Martin in J. Am. Chem. Soc. (2005) 127, 2018-2019 presented the use of the "tacn" compound for the immobilization of proteins having a histidine affinity label on a gold electrode. First, they used an alkanothiol to form a monolayer on gold and then added the synthesized tacn substituted with 2 benzyl and carboxyl groups (after the authors name: 1-acetato-4-benzyl-1,4,7-triazacyclononane). The monolayer functionalization process took place in situ on the electrode. They confirmed that tacn derivatives bind nickel and enable the immobilization of proteins while controlling their spatial orientation after adsorption on the monolayer.

Thiol monolayers can be used to construct sensors, biosensors, biocatalytic surfaces, biological microarrays and the like. SAMs-mediated gold surface modifications have been successful in nanotechnological biosensor applications, e.g., commercially available chips for analyzing biomolecular interactions using surface plasmon resonance (SPR) (Lofoas S. et al. Colloids Surf. B (1993) 1, 83- 89). Also shown is a protein microarray on which proteins are immobilized to a surface-modified Ni-NTA (Zhu H. et al. Science (2001) 293, 2101-2105).

One of the most important elements that make up a protein microarray is a solid support covered with a monolayer on which a protein or a protein binding compound can be immobilized. Unlike DNA, proteins tend to bind nonspecifically to surfaces

PL 228 280 Β1 and losing its biological activity at the same time. Therefore, the conditions for the substrate to which proteins bind are different from those for DNA microarrays, which means that it is not only intended to provide functional groups on the surface of the substrate enabling the protein-binding compound to attach, but also to prevent non-specific binding. proteins on a surface where no protein binding compounds are exposed (Merkel JS et al. Curr Opin Biotechnol. (2005) 16 (4), 447-52).

Many applications, in particular the manipulation of proteins in solution or techniques involving the immobilization of proteins on the surface, such as SPR or microarray analyzes, require high molecular level stability of the chelator-metal-oligohistidine labeled protein.

The progress in the development of the technology of protein microarrays depends to a large extent on the availability of suitable surfaces and monolayers meeting the above-mentioned requirements.

Taking into account the state of science described above, there is a need to improve the existing solutions by proposing a new method that will solve most of the problems described above. Therefore, work was undertaken to improve the method of immobilizing proteins with the His-tag tag, so that the following criteria were simultaneously met: the immobilization resulted in a defined and non-random orientation of the protein on the surface and high selectivity and specificity for the target protein on bioinert surfaces, and highly limited non-specific interactions between this surface and proteins. Accordingly, compounds, triazamacrocyclic derivatives, having a linker have been designed that have the ability to form self-assembled monolayers (SAMs) on surfaces. By using the azamacrocyclic derivatives according to the invention, which have better metal complexing properties, stable / stable binding to the oligo-histidine label was ensured at the molecular level. The described methods of obtaining triazamacrocyclic derivatives make it possible to synthesize derivatives with three different substituents on nitrogen atoms of triazacorones. By introducing further functional units into the azamacrocyclic (chelating) compound, the chelating properties of the azacorones can be regulated, and proteins that contain an oligo-histidine affinity label can be stably bound. The new asymmetrically substituted triazamacrocyclic derivatives, object of the present invention, prepared by the process of the invention, are capable of complexing metal ions, preferably Ni and Cu. Their preferred structures according to the invention are shown and their preparation processes are illustrated.

The derivatives according to the invention have not yet been described anywhere.

The invention is described in detail below, also by means of appropriate examples and schemes.

New, asymmetrically substituted 1,4,7-triazacyclononane derivatives capable of complexing metal ions, especially Ni and Cu, of the general formula I, possibly in the form of salts, hydrates or complexes with metals, h ₂ n

wherein

R2 and R3 are the same or different and represent, independently of each other, hydrogen, an optionally substituted saturated or unsaturated alkyl group with a carbon number of 1 to 6, especially substituted with amino, amide, nitro, cyano, hydroxyl, halogen, carboxyl or ester of a carboxylic acid having 1 to 6 carbon atoms in the chain, and is also an optionally substituted heterocyclic group or an optionally substituted aromatic or heteroaromatic group with a carbon number of 6 to 10, or a protecting group, and Z is -CH2- p is an integer of 3, with R2 or R3 being the same only when they represent a hydrogen or a protecting group.

PL 228 280 Β1

Particularly preferred are the derivatives of the general formula I according to the invention in which:

R2 are protecting groups selected from formyl, tert-butyloxycarbonyl, formyl or benzyl.

Particularly preferred are derivatives of general formula I, selected from the group consisting of:

4- (3-Amino-propyl) -7-tert-butoxycarbonylmethyl- [1,4,7] triazonane-1-carboxylic acid tert-butyl ester,

(3-Amino-propyl) -7-benzyl- [1,4,7] triazonane-1-carboxylic acid tert-butyl ester, {4- (3-Amino-propyl) -7-formyl- acid tert-butyl ester [1,4,7] triazonan-1-yl} -acetic, 4- {3-amino-propyl) -7-benzyl- [1,4,7] triazonan-1-carbaldehyde,

[4- (3-Amino-propyl) -7-benzyl- [1,4,7] triazonan-1-yl] -acetic acid.

The invention also relates to new, asymmetrically substituted 1,4,7-triazacyclononan derivatives with an oligo (oxyethylene) fragment of the general formula 2:

R3 formula 2 optionally in the form of salts, hydrates and metal complexes in which:

R2 is as defined above;

R3 is hydrogen; p is an integer of 3; n is an integer 11; m is an integer of 3; k is an integer 1;

B is -OC (O) NH-, -C (O) NH-;

Z is as defined above;

Y is CH2 = CH-, -SH, CH3C (O) S- and R7 is C1 to C2 alkyl.

Particularly preferred are derivatives of general formula 2, selected from the group consisting of: N- [3- (4-benzyl-7-formyl- [1,4,7] triazonan-1-yl) propyl] -2- {2- [2- (2-undec-10-enyloxy-ethoxy) ethoxy] -ethoxy} -acetamide,

4-Benzyl-7- [3- (2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy] -ethoxy} -acetylamino) -propyl] - [1, 4,7] triazonane-1-carboxylic acid,

S- (11- {2- [2- (2 - {[3- (4-benzyl-7-formyl- [1,4,7] triazonan-1-yl) propylcarbamoyl] methoxy} ethoxy) - ester ethoxy] -ethoxy} -undecyl) thioacetic acid,

4- {3- [2- (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy] -ethoxy} ethoxy) -acetylamino] -propyl} -7-benzyl- [1 , 4,7] triazonane-1-carboxylic acid,

N- [3- (4-benzyl- [1,4,7] triazonan-1-yl) propyl] -2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] ethoxy} -ethoxy) -acetamide,

{4-Formyl-7- [3- (2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy] -ethoxy} -acetylamino) -propyl] - [1 , 4,7] triazonan-1-yl} -acetic,

4-tert-Butoxycarbonylmethyl-7- [3- (2- {2- [2- (2-undec-10-enyloxyethoxy) -ethoxy] -ethoxy} -acetylamino) -propyl] triazonan-1- acid tert-butyl ester carboxylic acid,

(4- {3- [2- (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy] -ethoxy] ethoxy) -acetylamino] -propyl} -7-formyl- [ 1,4,7] triazonan-1-yl) -acetic,

4- {3- [2- (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy] -ethoxy} ethoxy) -acetylamino] -propyl] -7-tert-butoxycarbonylmethyl- acid tert-butyl ester [1,4,7] triazonane-1-carboxylic acid, (4- {3- [2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -acetylamino ] -propyl} - [1,4,7] triazonan-1-yl) -acetic,

N- [3- (4,7-diformyl- [1,4,7] triazonan-1-yl) propyl] -2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy ] ethoxy-acetamide,

S- (11 - {2- [2- (2 - {[3- (4,7-diformyl- [1,4,7] triazonan-1-yl) propylcarbamoyl] methoxy} -ethoxy) ethoxy ester ] -ethoxy} -undecyl) thioacetic acid,

PL 228 280 Β1

2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -N- (3- [1,4,7] triazonan-1-yl-propyl) acetamide,

N- (3- [1,4,7] triazonan-1-yl-prc> pyl) -2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy] -ethoxy} -acetamide ,

N- [3- (4-benzyl- [1,4,7] triazonan-1-yl) -prc> pyl] -2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy ] -ethoxy} -acetamide, {4- [3- (2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy] -ethoxy} -acetylamino) -propyl] - [1, 4,7] triazonan-1-yl} -acetic.

Particularly preferred are the derivatives containing an amide bond of general formula 2a, which are a special case of general formula 2:

ABOUT

R3 formula 2a and their optional salts, hydrates, and metal complexes where

R2 and R3 are as defined above; p is an integer of 3; n is an integer 11; m is an integer of 3; k is an integer 1;

while R4 is independently selected from the group consisting of hydrogen, thiol protecting groups, preferably CH3C (O) -.

Particularly preferred are compounds of general formula 2a selected from the group consisting of:

S- (11- {2- [2- (2 - {[3- (4-benzyl-7-formyl- [1,4,7] triazonan-1-yl) propylcarbamoyl] methoxy} ethoxy) ester -ethoxy] -ethoxy} -undecyl) thioacetic acid,

4- {3- [2- (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy] -ethoxy} ethoxy) -acetylamino] -propyl} -7-benzyl- [1 , 4,7] triazonane-1-carboxylic acid,

N- [3- (4-benzyl- [1,4,7] triazonan-1-yl) propyl] -2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy } -ethoxy) -acetamide,

(4- {3- [2- (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy] -ethoxy} ethoxy) -acetylamino] -propyl} -7-formyl- [ 1,4,7] triazonan-1-yl) -acetic,

4- {3- [2- (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy] -ethoxy} ethoxy) -acetylamino] -propyl) -7-tert-butoxycarbonylmethyl- acid tert-butyl ester triazonane-1-carboxylic acid,

Acid (4- {3- [2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -acetylamino] -propyl} - [1,4,7] triazonane -1-yl) acetic,

S- (11 - {2- [2- (2 - {[3- (4,7-diformyl- [1,4,7] triazonan-1-yl) propylcarbamoyl] methoxy} ethoxy) ethoxy ester -ethoxy-undecyl) thioacetic acid,

2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -N- (3-triazonan-1-yl-propyl) -acetamide.

It is evident that the type of substituents and the pattern of the substituent distribution in the structures defined by the above general formulas can be determined in such a way as to produce new chemically stable compounds which can then be synthesized by techniques known in organic synthesis, like those preferred compounds described herein. invention.

Combinations of substituents described by the structures and compounds of this invention are preferred and result in stable and synthesized compounds. It is expected that compounds of the structure according to the invention with the remaining combinations of the substituents mentioned above will behave similarly.

PL 228 280 B1

Terms such as triazamacrocyclic, azamacrocyclic, triazacoronic, azacoronic, crown are used interchangeably in describing this invention.

The subject of this invention is also the process of synthesizing these new triazamacrocyclic derivatives having the ability to complex metal ions, especially Ni and Cu.

The subject of this invention is also the use of the compounds according to the invention that can be used to attach proteins to surfaces.

Moreover, nowhere have derivative monolayers according to the invention or proteins, especially recombinant proteins, specifically immobilized on a suitable surface, preferably gold, by means of the derivatives according to the invention been described.

By the term "functionalization", and more specifically "monolayer functionalization", is meant the process of attaching an azacorone compound of general formula 1 to a previously prepared gold monolayer consisting of compounds of the alkanothiol or oligo (ethoxyethylene) alkanothiols, some of which have a terminal carboxyl group; an amide bond is formed between the compound of general formula 1 and a compound that is one of the components of the already formed monolayer on gold and has a terminal carboxyl group, so the attachment of the azacorone takes place after the formation of the monolayer (otherwise it is the case of compounds of general formula 2, 2a, which have the azacorone fragment already attached and are ready to form a monolayer which presents the azacorone group).

By the term "ligand" is meant a molecule that binds specifically to another molecule.

By the term "immobilization" is meant the immobilization of a protein on a surface by its covalent or non-covalent bonding to the surface.

The term "self-assembly" refers to the phenomenon whereby specific nanostructures with new physical properties are formed spontaneously under the influence of interatomic covalent bonds or intermolecular attractive forces. Typically this phenomenon occurs in self-assembled monolayers, DNA-containing biomaterials, nano-micro-particles, and the like.

By the term "protecting group" or "protecting group" is meant a group that protects sensitive functional groups against adverse transformations, side reactions and is stable during the reaction during which it performs protective functions. The release of the functional group (deprotection), after carrying out the appropriate reaction or a whole series of reactions, should take place under conditions in which the product obtained does not undergo undesirable transformations.

Protecting groups can be used to facilitate synthesis; if necessary, other than those mentioned in the examples and according to the synthetic strategy presented in schemes I, II, III, IV and in the description of the invention. Protecting groups and reaction conditions can be found e.g. in T. W. Greene, Protective Groups in Organic Synthesis, (3rd, 1999, John Wiley & Sons, New York, N.Y.) and in Protective Groups in Organic Chemistry, Plenum Press, London, New York 1973.

For the purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, the general principles and terminology of organic chemistry are contained and described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, and March's Advanced Organic Chemistry, 5th Ed., J Wiley & Sons, NY: 2001, the content of which therefore serves as a reference material. reference.

The derivatives of this invention include those described above by general formulas 1, 2 and 2a, which are further illustrated in the descriptions and examples.

The new derivatives according to the invention may be intermediates for the synthesis of oligo (oxyethylene) alkanothiols, in particular:

2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -N- (3- [1,4,7] triazonan-1-yl-propyl) -acetamide , (4- {3- [2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -acetylamino] -propyl} - [1,4,7] -triazonan-1-yl) -acetic and N- [3- (4-benzyl- [1,4,7] triazonan-1-yl) -propyl] -2- (2- {2- [2- (11 -merkapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -acetamide, compounds with an end-linked azacorone moiety that are used to form self-assembled monolayers on a metal surface, most often on gold.

The new derivatives, according to the invention, have the ability to complex metal ions (e.g. Ni, Cu) through their fragment with an azacorone structure. Nickel (II) complexes with organic ligands immobilized on a protein-inert monolayer can bind proteins with a histidine affinity label.

PL 228 280 B1

The invention also presents a new strategy to obtain asymmetrically N-substituted 1,4,7-triazacyclononane derivatives as well as derivatives suitable for the synthesis of tacn derivatives with an attached terminal alkanolyxyethylene moiety. This strategy allows the synthesis of triazamacrocyclic derivatives with an amine-terminated linker. This linker enables the convenient attachment of azacorones to various chemical compounds / biomolecules via e.g. an amide bond and thus opens the way to new asymmetrically substituted metal coordinating azamacrocycles. The introduction of various substituents into the azacorone allows the properties of the complex to be regulated, e.g. it may increase the selectivity of the ligand for specific ions.

Chemical modifications of the amine groups of azamacrocyclic compounds change the physical properties and often lead to changes in the ability to form coordination bonds with metals. Effective and selective modification, however, presents many difficulties due to the presence of several, equal groups in the azakoron, as well as the reactivity of the already introduced groups and the use of several orthogonal protecting groups. The developed method for the synthesis of azamacrocyclic derivatives, as well as the method of selective protection of amino groups enables the synthesis of various azacorone compounds, which can have each amine group of the crown substituted with a different chemical group.

The new tacn derivatives have three different substituents, one or two of which may be a formyl or tert-butyloxycarbonyl protecting group. They find application in the functionalisation of oligo (oxyethylene) alkanothiols (including their precursors and thiol-protected derivatives) terminated with a carboxyl group. Alkanothiols of this type are used to form monolayers on gold. The oligo (oxyethylene) fragment (otherwise: oligoethylene glycol) reduces the occurrence of non-specific interactions between the protein and the synthesized ligand. The monolayer formed from such modified alkanothiols is biologically inert and shows a very low level of non-specific interaction with proteins. Such monolayers with a surface-present Nikel chelating azacorone fragment can selectively bind proteins with a histidine affinity label. The method using self assembled monolayer (SAM) using oligo (oxyethylene) alkanothiols according to the invention with an attached azacorone fragment is a convenient solution for the formation of biocompatibile and bioresistant SAMs and does not require the functionalisation of the monolayer in situ with the compound azamacrocyclic.

Preferred routes for synthesizing the compounds of the invention are set out below in general terms.

The method of producing asymmetrically substituted 1,4,7-triazacyclononane derivatives of the general formula I according to the invention consists in:

(a) the tacn-orthoamide derivative obtained from the tacn compound is introduced with an amino protected linker by reaction with a halogenated aminoalkyl (haloaminoalkyl) derivative, resulting in the corresponding quaternary ammonium derivative being the main reaction product;

(b) a tacn derivative of a quaternary ammonium salt having a protected linker is hydrolyzed to a tacn derivative with a formyl group on the azacoron;

(c) a tacn derivative having a protecting group protected linker and a formyl group protecting the azakorone secondary amino group, selectively introducing a further substituent, preferably from the group consisting of e.g. benzyl, -CH2COOH or a methyl, ethyl, tert-butyl ester or a group formyl or other protecting to afford a tacn derivative with three substituents on the azacoron;

(d) a tacn derivative having a protected linker and a formyl group protecting the azakorone secondary amino group and a third other substituent, preferably a formyl group, is selectively deprotected, preferably with hydrazine, to yield the tacn derivative with free the primary amino group of the linker represented by general formula 1; or (dl) a derivative of a tacn compound having a protective linker and a formyl group protecting the azacorone secondary amino group and a third other substituent, preferably from the group consisting of e.g. benzyl, -CH2COOH or a methyl, ethyl, tert-butyl formyl ester , deprotected to yield a tacn derivative with a free linker primary amine group and a free azakorone secondary amine group;

(e) the derivative of tacn having an amino terminated linker is treated with ethyl trifluoroacetate which selectively introduces a trifluoroacetyl protecting group on the primary amine of the linker;

(f) the derivative of the tacn compound with the selectively protected primary amino group of the linker is introduced with further substituents of the R 3 type, including a protective group or groups stable in a medium in which the trifluoroacetyl group is depleted from the amino group, preferably Boc;

(g) the trifluoroacetyl group is then removed from the derivative by selective deprotection, yielding a tacn derivative with a free primary amine linker (linker) and having two different substituents on the azacoron, one or two of which may be a protecting group, preferably Boc, or gives a tacna derivative with three different substituents without any protecting group among the substituents represented by the general formula 1.

Scheme A shows a synthetic route for a triazamacrocyclic derivative (chelating compound) having an amine terminated linker attached to one of the azacorone nitrogen atoms and having a further two substituents which may be protecting groups. In the synthesis described in this scheme, a linker is first selectively introduced into the azacorona, followed by further, preferably different, substituents. The protecting groups are removed selectively so as to ultimately obtain a triazamacrocyclic derivative with a linker terminated with a free primary amine group. The resulting chelating compound is then linked to the alkyl-oxyethylene segment (Scheme B) by forming a preferably amide bond between them.

Scheme A

HN NH

C _N .....>

H tacn

- N N

Y - Ń - Y tacn orthoamid (a) /

R1 LZJ; \ _iblem R1, LZ-L <'

N {and? N. , N < ^b > N l ΪΝ NH

Η / γ \ ~ H.

Y-L./ \ - N ~

R1 Γ, Ζ-J and \ R2 N t J? N N

H, /

\. NY! «/ _O .J

Z \ <d)

H, N N Ν '

N

H.

1. <e) \ 2. (f) f-ZJ and \ R2

Η, ΝΊ 1? NN ² / \

Y-N-Y (pattern!)

O ^F 'N' ^Z ! »'Ń N' ^{R2 F} F ^H <?

. N (g)

R3

PL 228 280 Β1

In Scheme A: R1 is a protecting group, R2 and Z, p are as defined above in the description of the derivative of general formula 1. The description of steps a, b, c, d, di, e, f, g in the text below.

The method of producing asymmetrically substituted 1,4,7-triazacyclononan (tacn) derivatives of general formula 2 and general formula 2a according to the invention consists in:

(a) performing a synthesis reaction between a derivative of general formula 1 and an alkyl chain derivative optionally with an oligo (oxyethylene) moiety, optionally in a preferred case, to prepare derivatives of formula 2a.

(ai)) an amide bond formation reaction is performed between an alkenyl-oligoethylene glycol or an acetylthioalkyl-oligoethylene glycol derivative terminated with one carboxyl group and an asymmetrically substituted 1,4,7-triazacyclononane derivative of general formula 1 having a linker terminated an amino group to give oligo (oxyethylene) alkanothiols derivatives with a triazamacrocyclic fragment having amino protecting groups on the azacoron and a thiol group (if present).

Scheme B

Η, Νί J? N ² /

R2 (formula 1) ν - .. / i

R3

(pattern 2)

The 1,4,7-triazacyclononane derivatives of general formula 2 and 2a according to the invention are prepared by the processes shown in general Scheme B, wherein R2, R3 and Y, A, Z, p, n, m, k are as defined above in the description of the compound of general formula 2 and 2a, X represents substituents selected from the group consisting of -OC (O) -, -C (O) OH-, active esters of the carboxyl group, carboxyl groups upon activation, e.g. with carbonyldiimidazole, acid chlorides of carboxylic acid, -CH2OH-, hydroxyl groups after activation, preferably with mesyl chloride, -CH2OS (O) 2CH3, -CHL-halogen, -NH2, epoxy, whereby when X is an active ester, said ester is preferably an N-hydroxysuccinimide ester, p -nitrophenol or pentafluorophenol.

Suitable methods for the preparation of amide compounds are carboxyl activation methods and condensation reactions known from peptide chemistry.

As activating reagents for the carboxyl group, substances known to those skilled in the art may be used, such as thionyl chloride, oxalyl chloride, pivaloyl chloride, chloroformic acid ester derivatives or Ν, Ν'-carbonyldiimidazole. After in situ preparation, active derivatives of compounds of formula 2 are reacted with amino derivatives of formula 1.

A suitable condensing agent may also be a mixture of couplers with additives, e.g. a mixture of Ν, Ν'-dicyclohexylcarbodiimide, 1-hydroxy-1H-benzotriazole, (benzotriazol-1-yloxy) tripyrrolidinophosphonium (PyBomorpholine) and N-ethylcarbodiimide hexafluorophosphate.

PL 228 280 Β1

A description of amide bond-mediated coupling methods and examples of coupling compounds can be found in Tetrahedron (2005) 61, 10827-10852 (Ch.A.G.N. Montalbett, V. Falque).

The precursor compounds for the preparation methods described in Schemes A and B are either commercially available or can be prepared according to procedures described in the literature, or by adaptation of these procedures (references are listed below in the text).

Preferably, individual compounds of the invention are prepared by the exemplary processes detailed in Schemes I, II, III, IV.

Scheme I

h ₂ n

ABOUT

AT

A preferred method of preparing compounds of general formula I according to the invention is that the following process steps, shown in Scheme I, are carried out sequentially:

(a) reaction of ^{1,4,7-triazatri-cyclo [5.2.1.0 41cl} ] decane (tacn-orthoamide) (3) obtained according to the recipe from the literature with N- (3-bromopropyl) phthalimide or N- (4-bromobutyl) an amino protected phthalimide to give the corresponding quaternary ammonium derivative as the major reaction product (4);

(b) hydrolyzing the resulting salt to obtain a tacn derivative with a formyl group (5);

(c) selectively introducing a further substituent, preferably from the group consisting of, for example, benzyl, -CH2COOH or methyl, ethyl, tert-butyl ester and providing compound (6) as the main reaction product;

(d) selective deprotection of the phthalimide protecting group with e.g. hydrazine in ethanol in the presence of intact formyl protecting group leads to (7).

The detailed preparation of compounds (6) and (7) from Scheme I is described in Example I. Following the description of Scheme I and replacing the -ChCOOtBu substituent with a benzyl substituent, 4- (3-amino-propyl) -7-benzyl- can also be easily obtained. [1,4,7] triazonane-1-carbaldehyde.

According to a first synthetic strategy, compounds of general formula I are obtained by the procedure described above consisting of the steps mentioned (scheme I).

A variation of the process for the preparation of compounds of general formula I according to the invention consists in carrying out the following process steps as shown in scheme II in sequence:

(a) introduces a formyl protecting group to compound (5), to obtain derivative (8),

PL 228 280 Β1 (b) the phthalimide protecting group is selectively removed, preferably with hydrazine in ethanol, in the presence of intact formyl protecting groups, yielding the tacn derivative (9) with two of the same protecting groups covered by general formula 1. In this way, it is possible to obtain 7- (3-amino-propyl) - [1,4,7] triazonane-1,4-dicarbaldehyde (9, scheme II).

Scheme

ABOUT

H _Z N

>

/

The detailed preparation of the compounds according to the procedure in Scheme II is described in Example II.

A further variant of the process for the preparation of the compounds of general formula I according to the invention consists in carrying out the following process steps as shown in scheme III in succession:

(a) Compound (10), a tacn derivative with a linker terminated with a free primary amino group, is reacted with ethyl trifluoroacetate at reduced temperature, preferably -5-0 ° C, to give compound (11) as the main product with a selectively protected primary amino group on a linker;

(b) selectively introducing a tert-butyloxycarbonyl (Boc) (or acid labile) protecting group to obtain compound (12);

(c) selective removal of the trifluoroacetyl protecting group over the intact Boc protecting group with e.g. NaOH, NH ₄ OH, NaBH ₄ or K 2 CO 3 χ H 2 O in methanol yields (13).

Scheme III

PL 228 280 B1

The detailed preparation of the compounds according to the procedure in Scheme III is described in Example III.

4- (3-Amino-propyl) -7-tert-butoxycarbonylmethyl- [1,4,7] triazonane-1-carboxylic acid tert-butyl ester can also be readily obtained by following the description of Scheme III and replacing the benzyl substituent with -CH2COOtBu. .

Precursors of compounds covered by this patent such as 1,4,7-triazacyclononane,

1,4,7-triazatricyclo [5.2.1.0 ⁴ & ^10] decane derivatives ftaloimidowe (4), (5), (6) and the derivative (8) can be easily prepared according to procedures described in the following publications. However, derivative (6) is a novel compound not yet described.

The synthesized precursor (5) is further converted into new derivatives according to the procedures of this patent as described and illustrated in Schemes I, II, III, IV and in the examples showing the synthesis of the new preferred compounds of this patent. The synthetic strategies presented in Schemes I, II, III, IV can be used to obtain a much wider group of compounds than described in the examples. The synthesis of these compounds is available to any technician with intermediate knowledge of organic synthesis. The structures of these compounds are represented by general formulas 1, 2 and 2a.

1.4.7-triazacyclononate (tacn) can be obtained according to a standard procedure described in the literature (Richman, JE, Atkins, TJJ Am. Chem. Soc. 96 (1974) 2268; Atkins, TJ, Richman, JE, Oettle, WF Org. Synth 58 (1978) 86; Koyama, H., Yoshino, T. Bull, Chem. Soc. Jpn. 45 (1972) 481-484; Wieghardt, K., Schmidt, W., Nuber, B., Weiss J. Chem. Ber. (1979) 112, 2220-2230).

1.4.7- Triazatri-cyclo [5.2.1.0 ⁴ & ^10] decane (ortoamid-TACN) (3) can be obtained preferably in a manner known from the literature (Atkins, TJJ Am. Chem. Soc. (1980) 102, 6364- 6365; Weisman, GR, Johnson, V., Fiala, RE Tetrahedron Lett. (1980) 21, 3635-3638.

The phthalimide derivatives (4), (5), (6) and the derivative (8) can be obtained in a manner known from the literature [A. Warden, B. Graham, M. T. W. Hearn, and L. Spiccia Org. Lett. (2001) 3 (18), 28552858].

A method for the preparation of asymmetrically substituted 1,4,7-triazacyclononane derivatives having an alkenyl function or a thiol group, of general formula 2 and formula 2a, with an alkyl-oligoethylene glycol fragment having the ability to complex metal ions, preferably Ni and Cu, according to the invention consists in carrying out the following process steps as shown in Scheme III in succession:

(a) the amide bond formation reaction is performed between the aminoalkyl derivative of tacn (in this case represented by a derivative with two formyl groups) (9) and a compound of formula (14) containing a carboxyl group, in the presence of coupling reagents, preferably dicyclohexylcarbodiimide (DCC) , (b) the obtained compound (15) is converted into the compound (16) in which the thiol group is protected with an acetyl group in a free radical reaction, (c) then the protective groups: acetyl and formyl (or other protecting group) are removed from the compound ( 16), preferably in an acidic environment, especially in 0.1 M HCl in methanol, to obtain compound (17).

Compound (14) with a carboxyl terminal group can be obtained according to standard procedures from J. Phys. Chem. B (2004) 108, 7665-73 (Lee J.K. et al.). However, the method of introducing a thioacetyl group (CH3C (O) S-) is described in: J. Am. Chem. Soc. (1991) 113, 12-20 (Pale-Grosdemange C.E.S. et al.).

It is evident that the compound (16) can also be obtained, preferably by an amide bond formation reaction between an oligo (oxyethylene) alkanothiol with a thiol protected group, e.g. with (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy) ] -ethoxy} -ethoxy) -acetic acid, and compound (9) in the presence of coupling reagents, especially DCC, EDC.

The detailed preparation of the compounds according to the procedure in Scheme III is described in Example III.

By following the description of Scheme III and replacing one formyl substituent with -CH2COOtBu, i.e. instead of compound (9) using compound (7), acid (4- {3- [2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy) -ethoxy) -acetylamino] -propyl} -triazonan-1-yl) -acetic.

Following the description of Scheme IV and replacing the formyl substituent with a benzyl substituent, i.e. 4- (3-Amino-propyl) acid tert-butyl ester 16

PL 228 280 Β1

-7-benzyl- [1,4,7] triazonan-1-carboxylic acid, also N- [3- (4-benzyl- [1,4,7] triazonan-1-yl) propyl] -2 can be obtained easily - (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -acetamide.

ABOUT

Scheme IV

Following the description of Scheme IV and replacing the formyl substituent with -ChCOOtBu, i.e. 4- (3-amino-propyl) 7-tert-butoxycarbonylmethyl- [1,4,7] triazonanoacid tert-butyl ester 1-carboxylic acid, the acid (4- {3- [2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -acetylamino] -propyl} - [1,4,7] triazonan-1-yl) acetic.

The synthesis consisted in such modification of the trays, which ensured easy introduction of the linker to only one amino group of the crown, without modifying the other two. A method was used that used the transformation of tacna into its orthoamide form. The 1,4,7-triazatricyclo [5.2.1.0 ⁴¹⁰ ] -decane thus obtained enables the selective introduction of a phthalimide protected linker, and then the easy introduction of a carboxyl or benzyl group after prior tacn-orthoamide hydrolysis. After removing the protective phthalimide group with e.g. hydrazine in ethanol, we obtain tacn with one crown amine protected by a formyl group with two other substituents attached to the azakorone, one of which is a linker terminated with a free primary amino group. One formyl group is revealed after the tacnorthoamide is hydrolyzed (i.e. it does not need to be introduced in a separate chemical reaction). It is also possible a simple method to selectively introduce other non-formyl protecting groups such as Boc or trityl. At the same time, this method allows for the differentiation of amino groups and is especially useful for the synthesis of tacn derivatives having a linker with a primary amino group. This primary group was protected selectively with a trifluoroacetyl (Tfa) group in the presence of 11-th. crown amino group, which can then be easily protected with a tert-butoxycarbonyl (Boc) group. The method is selective and efficient - only the primary amino group is protected with the Tfa group.

PL 228 280 Β1

The developed synthesis methods allow the synthesis of compounds of general formula 1, (scheme I, scheme II, scheme III) as well as those with an attached alkanoligo (oxyethylene) moiety of general formula 2 and general formula 2a (scheme IV).

The process for the preparation of 1,4,7-triazacyclononan (tacn) derivatives of the general formula I according to the invention is carried out in such a way that on a tacn derivative having a linker protected with a protecting group and a formyl group protecting the azacorone secondary amino group represented by the formula 1i, in which R1, Z, p are as defined in the description of general formula 1, selectively introducing a further substituent or protecting group to give a tacn derivative with three substituents on the azacoron of formula 1 ii, in which R1, Z and p have the meaning given above

R1

NH \

>

formula 1 i

The substituent for R2 is -ChbCOOtBu, which is preferably introduced in the alkylation reaction of the secondary amine group of azacorones with tert-butite bromoacetate. The introduced substituent R2 may also be a protecting group,

R1

-Z-.1 /

Jp _y N (

R2 • Preferably a formyl group, introduced by reaction of a tacn derivative with ammonium formate. The tacn derivative represented by the general formula 1 such that R2 is -ChbCOOtBu and R3 is a formyl group is {4- [3-N-phthalimidopropyl] -7-formyl- [1,4,7] triazonan-1- ylo} -acetic. The tacn derivative represented by the general formula 1, wherein R2 and R3 are formyl groups, is 7- [3-N-phthalimidopropyl] - [1,4,7] triazonane-1,4-dicarbaldehyde.

The preparation of the 1,4,7-triazacyclononan (tacn) derivatives of the general formula 1 according to the invention is preferably carried out in that a tacn derivative having a linker protected with a protecting group and a formyl group protecting the secondary amine group of azacorones and a third another substituent, which preferably may also be a formyl group, represented by formula 1 and i (where R 1 is a protecting group, Z, p are as defined in general formula 1) is selectively deprotected to yield a tacn derivative with a free primary the amino group of the linker described by the general formula 1.

In the method according to the invention, after selective deprotection, the derivative described by the general formula 1 is obtained.

The preparation of the 1,4,7-triazacyclononan (tacn) derivatives of the general formula 1 according to the invention is carried out in that the tacn derivative having an amine terminated linker and the R2 substituent introduced on the azacorone secondary amine group represented by the formula liii (wherein R2, Z, p are as defined in general formula 1) selectively insert a trifluoroacetyl protecting group onto the primary amino group of the linker at reduced temperature, preferably from -5 ° C to 5 ° C, with ethyl trifluoroacetate ( methyl),

PL 228 280 Β1 h ₂ n

/ \

R2 is the formula liii to give the tacn derivative of formula I iv (wherein R2, Z, p are as defined in the description of general formula 1).

R2 formula 1 iv

The method of the invention employs the selective protection of the primary amino group of the linker with a trifluoroacetyl group in the presence of 1,4,7-triazacyclononan secondary amino groups.

The preparation of the 1,4,7-triazacyclononan (tacn) derivatives of formula 1 according to the invention is carried out by derivatizing tacn with a selectively protected primary amino group of the linker by means of a trifluoroacetyl group represented by formula 1 and iv (wherein R2 , Z, p have the meaning given in the description of the general formula 1) a further substituent R3 is introduced, but it may be a stable protecting group in which the trifluoroacetyl group is descended from the amino group, preferably a tert-butyloxycarbonyl (Boc) group with to form a tacn derivative of formula 1v (wherein R2, R3, Z, p are as defined in the description of general formula 1).

ABOUT

N - J i

R3 formula 1v

The preparation of the 1,4,7-triazacyclononan (tacn) derivatives of the general formula I according to the invention is carried out in that a tacn derivative having a trifluoroacetyl protected linker and a Boc group protecting the azacorona secondary amino group and a third other substituent , represented by formula 1v (where R2, R3, Z, p are as defined in general formula 1) is selectively deprotected, yielding a tacn derivative with a free primary amino group of the linker represented by general formula 1, simultaneously having two different substituents on the azacoron.

The tacn derivative represented by the general formula 1 such that R2 is benzyl and R3 is a Boc group is 4- (3-amino-propyl) -7-benzyl- [1,4,7] triazonane-1-carboxylic acid tert-butyl ester.

The preparation of asymmetrically substituted 1,4,7-triazacyclononane (tacn) derivatives of general formula 2 and general formula 2a is carried out according to the invention using a synthesis reaction between a derivative of general formula 1 and an alkyl chain derivative optionally with an oligo (oxyethylene) moiety of formula 2i,

PL 228 280 Β1

formula 2i (where Y, A, n, m, k are as defined in the description of general formula 2, and X represents substituents selected from the group consisting of -OC (O) -, -C (O) OH-, active esters of the carboxyl group , carboxyl groups after activation with e.g. carbonyldiimidazole, acid chlorides of carboxylic acid, -CH2OH-, hydroxyl groups after activation, preferably with mesyl chloride, -CH2OS (O) 2CH3, -CH2- halogen, -NH2, epoxy group, where X is active ester, said ester is preferably N-hydroxysuccinimide (NHS ester), p-nitrophenol or pentafluorophenol ester) to give a derivative of general formula II (wherein R3 is a protecting group, Y, A, n, m, k are as defined above, and B, Z, R2, p are as defined in the description of general formula 2); and the compound of general formula 2 is released from this derivative upon selective deprotection.

In the method according to the invention, after selective deprotection, the derivative described by the general formula 2 is obtained.

The preparation of asymmetrically substituted 1,4,7-triazacyclononane (tacn) derivatives of general formula 2a is carried out according to the invention by the formation of an amide bond between an alkenyl-oligoethylene glycol or an acetylthioalkyl-oligoethylene glycol derivative terminated with one carboxyl group of formula 2i, wherein the substituents are as defined above and the asymmetrically substituted 1,4,7-triazacyclononane derivative of general formula 1 having an amine terminated linker to give the derivative of general formula 2 (wherein R3 is protecting, Y, A, n, m, k are as defined above, and B, Z, R2, p are as defined in the description of general formula 2); and then this derivative gives derivative 2a after removal of the protecting groups.

In the method according to the invention, after selective deprotection, the derivative described by the general formula 2a is obtained.

Compounds of general formula I, in particular those having an amino-terminal linker and having a protecting group, preferably formyl or Boc, are novel synthons that can be easily chemically converted into many new asymmetrically N-substituted tacn derivatives; they also allow easy association with other molecules preferably terminated with a carboxyl group.

According to the invention, the obtained derivatives of the tacn compound have the ability to chelate d and p block metals, preferably Ni, Cu, Co, Fe, Zn, and also lanthanides, preferably Gd, Eu, Sm.

The metals most commonly used to bind proteins to a histidine affinity label via their complex with a chelating ligand are Ni and Cu.

Self-assembled monolayers (SAMs) are the preferred substrate for the immobilization of biomolecules according to the invention. They are formed spontaneously by chemisorption and self-assembly of functionalized, long-chain organic molecules on the surfaces of appropriate carriers. SAMs are typically prepared by immersing a carrier in a solution containing a ligand that is surface reactive. There are many ways of producing SAMs known in the art. Forming ordered SAMs on gold from alkanothiols is a relatively quick process. Highly ordered SAMs from hexadecanethiols on gold can be obtained by immersing the gold coated substrate in a solution of hexadecanethiol in ethanol (about 2 mM) for a few minutes.

The process of forming an alkanothiol monolayer on gold takes place in two kinetically different stages. The initial adsorption of the thiols from their millimolar solution takes place within minutes. After this initial adsorption phase, the thiolates reorient to maximize the van der Waals and other stabilizing interactions that occur over the course of hours. The reorientation phenomenon manifests itself as a slow increase in monolayer thickness.

Oligoethylene glycol, like polyethylene glycol, is known to inhibit the non-specific interaction of proteins with the surface; also because of their properties which make particles or materials bioinert. An inert group is a group that prevents non-specific adsorption or non-specific binding of proteins. In particular, when the derivatives of this invention are used for the immobilization of proteins, it is preferable that they are mixed with

In order to provide the surface with properties that prevent nonspecific binding of biomolecules and also prevent changes in their spatial structure. These inert groups are often hydrophilic groups. A preferred inert group according to this invention is oligo (ethylene) glycol (OEG), especially triethylene glycol or hexaethylene glycol. It is also preferred that the ligand-bearing derivatives contain an inserted glycol fragment in their structure, since then the formed mixed monolayer does not have a disturbed, disordered structure.

Therefore, in the structure represented by the general formula 2, there is an oxyethylene fragment introduced to minimize non-specific binding of biomolecules. More specifically, it is introduced into a molecule in order to later impart bioinertial characteristics to a mixed monolayer composed of two compounds: oligoxyethylenealkanethiol and ligand-exposing oligoxyethylenealkanethiol (hereinafter an azamacrocyclic compound). Additionally, and importantly, a mixed monolayer formed of these two compounds will be a more ordered structure.

SAMs monolayers, formed from the compounds of the invention, should preferably be mixed monolayers. The mixed monolayer consists of two types of molecules - alkanothiols, i.e. alkanothiols with an oxyethylene fragment and oxyethylenealkanethiols linked to an azamacrocyclic segment (or alkanothiols whose oxyethylene fragment has been linked to a ligand). Alkanothiols with an oxyethylene fragment form the basic monolayer, the main property of which is the lack of non-specific interactions with proteins and giving the surface bioinert and anti-bioadhesive properties. This layer displays ligands presented by functionalized oxyethylenealkanethiols, whose task is to interact specifically with the target protein.

The use of alkanothiols with a terminal oligo (oxyethylene) fragment gives the monolayer bioinertia and eliminates the possibility of non-specific adsorption of proteins on the monolayer. As a result, it is possible to specifically attach specific biomolecules to the monolayer. The PEG or OEG chain may have a reactive or activated group that allows them to be attached to the surface of a substrate, e.g. a metal. This creates a system consisting of SAMs-OEG. A biological or non-biological receptor, ligand, reporter at each of the active or reactive ends of the SAM / OEG system can be attached to the SAMs-OEG thus formed.

Mixed monolayers tend to form microdomains predominantly containing one type of thiolate (Stranick, S. J .; Parikh, A. N .; Tao, Y. T .; Allara, D. L .; Weiss, P. S. J. Phys. Chem. (1994) 98, 7636). Microdomains reduce the homogeneity of the surface. They also introduce an uncontrolled factor, which results in the lack of repeatability during the formation of such monolayers, obtaining different characteristics of the monolayers after repeating the same procedure. Surface migration of thiolates appears to be a significant factor that enables the formation of microdomains. When we use two types of thiols, one of which has an inert-terminated chain and the other has the same structure extended with an attached ligand, we obtain a more uniform distribution of thiolates and the formation of microdomains should be limited, resulting in more uniform and reproducible surface characteristics.

Likewise, the derivatives of the invention can be used to form a mixed monolayer of the alkaneothiol derivatives of the invention on a gold surface by a standard monolayer formation process (SAMs). In this process, the metal surface is contacted with a solution of the thiol mixture forming the basic monolayer representing groups that are non-reactive, inert towards biomolecules, and with the thiol introducing ligands into this monolayer, more precisely with the same structure as the thiols forming the bioinert monolayer, but extended by an additional segment exposed outside this monolayer. monolayers. The ordered monolayer has a specific density of alkanothiols, and through the ratio of non-modifiable to ligand-presenting alkanothiols, the amount and density of these ligands in the monolayer can be controlled, and consequently the amount of protein that can be immobilized.

The mixture of thiols making up the mixed monolayer is in an inert solvent. Thus, the solution contains both thiols, undecyl thiol combined with triethylene glycol and undecyl thiol combined with triethylene glycol combined with azacorona. The solution of these alkanothiols contains monolayer-forming components in a predetermined proportion. When a substrate with a gold layer is placed in this solution, a mixed alkanothiol monolayer is spontaneously formed on the surface. The ratio of both types of thiols established in the starting solution determines the density of the azacoronium ligand on the metal surface.

PL 228 280 B1

The solvent is one of the factors influencing the SAMs formation process from thiol solutions when they are brought into contact with the metal surface. Ethanol and methanol are such solvents. Those familiar with the art will appreciate that other solvents can also be used. A solvent such as DMSO is not suitable as it does not allow formation of a monolayer.

Typically, according to the state of the art, the gold monolayer is prepared by immersing the gold surface in an ethanolic solution of appropriate thiols that are components of the later formed monolayer. If the alkanothiol derivative is poorly soluble in ethanol, DMSO is added, and then the surface covered with a thin gold layer, e.g. a SPR chip, is placed in this solution for 3 to 8 hours to form a self-assembled monolayer (SAMs).

The compounds of the invention may have a wide variety of uses.

In particular, the triazamacrocyclic derivatives according to the invention, taken as a whole, are a linker that allows proteins and other biomacromolecules to be linked to the surface. It is understandable that this is a requirement for many highly sensitive analytical systems, i.e. sensors, biochips.

A monolayer formed from the compounds according to the invention can be used as a diagnostic tool or any other tool using monolayers formed from derivatives according to the invention or obtained after functionalization of monolayers, coatings, films with the compounds according to the invention. Currently, polycarbonates and polyurethanes, apart from glass, are substrates that can be coated with gold according to methods known and described in the literature. These monolayers can also be formed on other substrates as long as the gold layer adsorbs thereon and thus makes the material useful for immobilizing functionalized organic molecules thereon.

According to the invention, the derivatives of general formula 2 or 2a are used to form a self-assembled monolayer on a metal surface, preferably on gold.

Alkanothiol monolayers from newly synthesized compounds described by general formula 2 and 2a, preferably monolayers obtained from 2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] ethoxy} -ethoxy) -N- (3 - [1,4,7] triazonan-1-yl-propyl) -acetamide, consisting of only one compound and mixed (together with 2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy } -ethanol) were formed by a process of their self-assembly from their 2 mM ethanol solution on gold.

The substrate for gold is most often glass, i.e. basic and cover microscope slides. The gold coating is vacuum sprayed onto a pre-formed adhesive titanium layer on a clean glass surface. Tooling for forming such a gold coating on a glass surface is commercially available (e.g., Pharmacia Biosensors).

Alkanothiol monolayers formed on gold (planar surface, e.g. glass plate) can also be formed on gold or gold-plated micro- or nanoparticles (microspheres), or on colloidal gold.

According to the invention, spherical media (beads, micro / nano-particles) and membranes can be examples of substrates for protein immobilization.

According to the invention, a preferred affinity tag that allows interactions between a protein and a metal chelating compound to occur is the histidine (His) n-tag tag, where n is an integer from 4 to 15.

Among the preferred target molecules that can be immobilized are peptides, polypeptides, proteins, peptide modified polymers or dendrimers, and peptide mimetics. Peptide mimetics and protein mimetics are molecules that contain functional groups similar to those found in peptides or proteins, but differ in the composition of the backbone. For example, peptoids differ from peptides or proteins in their side chain attachment site.

Preferred molecules are an immobilized protein or a target molecule complexed with a metal chelating compound represented by the general formula 1 or 2 or 2a.

A preferred molecule is the target molecule when immobilized via a compound represented by the general formula 1 or 2 or 2a on a planar surface.

A preferred molecule is the target molecule when immobilized via a compound represented by the general formula 1 or 2 or 2a on a membrane or spherical microparticle.

The compounds of the invention can be used for modification, immobilization, conjugation, purification, detection, monitoring, analysis or for the detection of a target molecule.

It is preferred to use the compounds of the invention to form self-assembled monolayers (SAMs) on noble metal surfaces.

PL 228 280 B1

A preferred application of the SAMs obtained from the compounds of the invention is in measuring methods, i.e. surface plasmon resonance (SPR). Moreover, the compounds according to the invention can be used in the production of micro- and / or nano-structural surfaces and protein microarrays.

According to the invention, derivatives of general formula 1 are used to functionalize a monolayer in situ with a compound of general formula 1.

According to the invention, derivatives of general formula 1, 2 or 2a are used for the formation of a monolayer in which the compounds of general formula 2, 2a are one of the components forming a monolayer on which a protein with a histidine affinity label is immobilized or the functionalization of the monolayer takes place in situ using a compound of general formula 2 for the construction of protein microarrays.

The method of forming a protein chip or microarray according to the invention consists of the following steps:

(a) modifying the surface by forming a self-assembled monolayer (SAMs) from the compounds of general formula 2 and 2a on the substrate;

(b) attaching the protein to the exposed binding site (metal chelating azamacrocyclic fragment).

The protein chip or microarray according to the invention is characterized in that the His-tagged protein is bound to the substrate via a compound described by general formula 2 and 2a.

According to the invention, a method of immobilizing proteins is preferred by immobilizing the protein on a support via a compound described by general formula 2 and 2a.

The protein chip or microarray according to the invention is characterized in that the His-tagged protein is bound to the substrate via a compound described by general formula 1.

According to the invention, a method of in situ functionalisation of the substrate with the compound of the general formula 1 is preferred.

According to the invention, a method of immobilizing proteins is preferred by immobilizing the protein on a support via a compound represented by the general formula 1, when the support has a chemical moiety capable of forming a covalent bond with the compound represented by the general formula 1.

The invention is illustrated in the following examples.

The examples show representative syntheses of 'tacn' derivatives according to the invention.

It is obvious to anyone familiar with organic synthesis that, while the precise reagents and reaction conditions are set forth in the following examples, modifications are possible while still remaining in accordance with the subject matter of this invention.

The guidelines provided in the description of the invention relating to the synthesis of the compounds described by the general formulas 1, 2, 2a according to the invention should be considered as non-restrictive. These guidelines and examples are merely illustrative of the preferred methods of preparing the compounds of this invention. The effectiveness of the presented methods of synthesis was tested experimentally by the syntheses presented in the examples below, and the structures of the derivatives obtained were confirmed by appropriate spectroscopic techniques.

All reactants, reagents, catalysts, and solvents used during the synthesis were purchased from commercial suppliers of purity part. or cz.d.a. and used without further purification unless otherwise stated.

The course of the chemical reactions was monitored by thin layer chromatography (TLC) on silica gel plates with a UV indicator. Detection of compounds on the plates was made against UV or under an atmosphere of sublimating iodine. Column chromatography was performed on silica-gel (Merck). The extraction solvents were treated with magnesium sulfate, unless otherwise stated. The solvents were evaporated on a rotary evaporator under reduced pressure at a temperature below 40 ° C. The structure of the compounds obtained was confirmed by ESI-TOF MS (and. Electrospray ionization time of flyight mass spectrometry) and NMR spectroscopy (nuclear magnetic resonance). NMR spectra were recorded on a Varian spectrometer operating at 400 MHz for ¹ H. The measurements were made in CDCl3 solution using the residual solvent signal as the chemical shift standard (7.26 ppm for ¹ H and 77.0 ppm for ¹³ C). The ¹ H and ¹³ C NMR spectra of the depicted tacn derivatives are complex, reflecting the presence of rotamers and isomers. Mass analyzes (HRMS) were recorded on a Micromass spectrometer.

PL 228 280 B1

The following are examples of syntheses of the compounds of the invention.

'Tacn' was prepared according to the procedure described by Richman, J.E. and Atkins, T.J. (J. Am. Chem. Soc. 96 (1974) 2268), yielding 2.45 g of product.

ESI-TOF MS: calcd for C6H15N3 130.21 (M + H) +, is 130.406.

From 700 mg (5.42 mmol) of 'tacn', 790 mg of 'tacn-orthoamide' (3) was obtained (from Weisman, G. R. et al. Tetrahedron Lett. (1980) 21.3635-3638).

ESI-TOF MS: calcd for C7H13N3 140.20 (M + H) +, is 140.086.

From 770 mg of (3), 656 mg (4) was obtained according to the procedure described in: Org. Lett. (2001) 3 (18), 2855-2858 (A. Warden et al.).

ESI-TOF MS: calcd for C18H23N4O2 327.41 (M + H) +, is 327.158.

985 mg of (5) was obtained from 1.215 g of (4) according to the procedure in: Org. Lett. (2001) 3 (18), 28552858 (A. Warden et al.).

ESI-TOF MS: calcd for C18H24N4O3 345.42 (M + H) +, is 345.134.

103 mg (10) was prepared according to the procedure described in: Org. Lett. (2001) 3 (18), 2855-2858 (A. Warden et al.).

ESI-TOF MS: calcd for C16H28N4 276.43 (M + H) +, is 277.247.

2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethanol was synthesized according to the procedure described in J. Am. Chem. Soc. (1991) 113, 12-20 (Pale-Grosdemange CES et al.). First thioacetate S- (2- {11- [2- (2-hydroxy-ethoxy) -ethoxy] -ethoxy} -undekylu) ^[1 H NMR (CDCl3) δ 1,221,38 (m, 12H), 1.52 -1.6 (m, 2H), 2.31 (s, 3H), 2.38 (br, OH), 2.85 (t, 2H), 3.44 (t, 2H), 3.56- 3.76 (m, 12H); ESI-TOF MS: calcd for C19H38O5 379.58 (M + H) +, is 379.248], from which the protecting group was removed to yield 22 mg of product as a pale yellow oil.

ESI-TOF MS: calcd for C17H36O4 337.54 (M + H) +, is 337.683.

P r z x l a d I.

This example illustrates the preparation of [4- (3-amino-propyl) -7-formyl- [1,4,7] triazonan-1-yl] -acetic acid tert-butyl ester (7) following the procedure in Scheme I.

1.

{4- [3-N-Phthalimidopropyl] -7-formyl- [1,4,7] triazonan-1-yl} -acetic acid tert-butyl ester (6)

To (5) (257 mg, 0.75 mmol) in 30 mL of acetonitrile was added 722 mg of potassium carbonate. Then tert-butyl bromoacetate (0.75 mmol) was added. The mixture was stirred at 60 ° C for 72 hours. At this time, the suspension was filtered to give an orange filtrate. The solvent was removed by rotary evaporation under reduced pressure. An orange oil was obtained, to which chloroform was added and washed with water (4 x 10 ml), yielding 196 mg of product (6) after evaporation of chloroform (6) as a light yellow viscous oil.

¹ H NMR (CDCl3) δ 1.45 (m, 9H), 2.2-2.4 (br, 2H), 2.7-3.3 (m, 14H, resonance forms), 3.5 (s , 2H), 3.8 (m, 2H), 7.7 and 7.8 (s, 4H, resonance), 8.0 and 8.1 (s, 1H, resonance);

gHSQC {1H / 13C} NMR (CDCl3) δ (rotamers) 1.45 / 28.14, 2.32 / 22.57, 2.83 / 55.05, 2.93 / 57.41, 3.08 / 51.82, 3.22 / 51.47, 3.47 / 61.06, 3.51 / 55.87, 3.58 / 60.49, 3.59 / 50.59, 3.67 / 59, 11, 3.79 / 53.26, 3.86 / 34.81, 3.90 / 58.73, 4.00 / 50.93, 4.00 / 60.77, 4.02 / 53.10, 4.02 / 34.84, 7.73 / 134.21, 7.83 / 123.16, 8.1 / na;

ESI-TOF MS: calcd for C24H34N4O5 459.56 (M + H) +, is 459.165.

2.

[4- (3-Amino-propyl) -7-formyl- [1,4,7] triazonan-1-yl] -acetic acid tert-butyl ester (7)

To a solution of (6) (68 mg, 0.15 mmol) in absolute ethanol (1 ml) was added hydrazine hydrate (0.74 mmol) at room temperature. The reaction mixture was refluxed for 1 hour. Upon cooling the reaction mixture, a white precipitate formed which was filtered off and washed with chloroform. The filtrate was rotary evaporated under reduced pressure to give compound (7) (49 mg) as a yellow oil.

¹ H NMR (CDCl3) δ 1.46 (m, 9H), 2,2-3,3 (m, 16H, rotamers), 3.48 (br, 2H), 3.76 (br, 2H), 8 , 2 and 8.4 (s, 1H, 2 resonances);

gHSQC {1H / 13C} NMR (CDCl3) δ (rotamers) 1.45 / 20.18, 1.46 / 37.26, 1.48 / 28.79, 1.55 / 31.71, 2.31 / 25.61, 2.81 / 56.42, 3.15 / 51.79, 3.48 / 60.81, 3.48 / 49.46, 3.57 / 60.61, 3.58 / 51, 39, 3.75 / 53.58, 3.97 / 51.53, 4.01 / 60.73, 4.46 / 57.44, 8.42 / 164.1;

ESI-TOF MS: calcd for C16H32N4O3 329.46 (M + H) +, is 329.181.

PL 228 280 B1

P r z x l a d II.

This example illustrates the preparation of 7- (3-amino-propyl) - [1,4,7] triazonane-1,4-dicarbaldehyde (9) according to the procedure in Scheme II.

1.

7- [3-N-phthalimidopropyl] - [1,4,7] triazonane-1,4-dicarbaldehyde (8)

Ammonium formate (1.71 mmol) was added to compound (5) (393 mg, 1.14 mmol) in acetonitrile (10 mL). It was heated under reflux for 12 hours. The solvent was evaporated under reduced pressure on a rotary evaporator. The residue was dissolved in ethyl acetate (20 ml) and washed with water (2 x 10 ml). (8) (321 mg) was obtained as a colorless liquid.

¹ H NMR (CDCl3) δ 1.83 and 1.99 (m, 2H, resonance forms), 2,60-3,99 (m, 16H, rotamers), 7.73

7.84 (s, 4H, resonance forms), 7.83 and 8.21 (s, 2H, resonance forms);

gHSQC {1H / 13C} NMR (CDCl3) δ (rotamers) 1.83 (27.34, 1.99 / 27.48, 2.60 / 54.96, 2.69 / 58.86, 2.73) 54.58, 2.84 / 51.85, 3.28 / 50.09, 3.36 / 49.83, 3.60 / 45.39, 3.66 / 48.214, 3.75 / 36.09, 3.77 / 50.22, 3.82 / 46.78, 3.991 / 36.06, 3.999 / 49.54, 4.05 / 47.36, 7.73 / 134.16, 7.84 / 123, 18, 7.83 / 164.32, 8.21 / 164.10;

ESI-TOF MS: calcd for C19H24N4O4 373.43 (M + H) +, is 373.709.

2.

7- (3-Amino-propyl) - [1,4,7] triazonane-1,4-dicarbaldehyde (9)

The synthesis was performed in a similar manner to compound (7) in Example 1 except that the starting material was compound (8) (159 mg, 0.43 mmol). Product (9) was obtained as light yellow oil (45 mg).

¹ H NMR (CDCl3) δ 1.67 (m, 2H), 2.80 (m, 2H), 2.71 and 2.81 (m, 2H, resonance forms), 2,59-2,74 (m , 4H, resonance forms), 3.23-4.00 (m, 2H, resonance forms), 8.16 and 8.20 and 8.33 (s, 2H, resonance forms);

gHSQC {1H / 13C} NMR (CDCl3) δ (rotamers) 1.67 / 28.32, 1.67 / 30.37, 2.59 (54.93, 2.66 / 57.96, 2.71) 54.62, 2.80 / 39.88, 2.81 / 51.67, 3.23 / 50.17, 3.31 / 49.99, 3.32 / 36.49, 3.40 / 46, 46, 3.59 / 45.81, 3.64 / 48.43, 3.67 / 50.74, 3.72 / 47.14, 3.96 / 50.21, 4.01 / 47.90, 8.20 / 160, 8.33 / 164;

ESI-TOF MS: calcd for C11H22N4O2 243.32 (M + H) +, is 243.134.

P r x l a d III.

This example illustrates the preparation of (3-amino-propyl) -7-benzyl- [1,4,7] triazonane-1-carboxylic acid tert-butyl ester (13) following the procedure in Scheme III.

1.

N- [3- (4-benzyl- [1,4,7] triazonan-1-yl) propyl] -2,2,2-trifluoroacetamide (11) Compound (10) (63 mg, 0.23 mmol) and triethylamine (0.23 mmol) was dissolved in methanol (5 mL) followed by the addition of ethyl trifluoroacetate (38.9 mg, 0.27 mmol). The temperature was kept between -5 ° C-0 ° C for 1.5 hours. It was then stirred for an additional 2 hours without cooling and the solvent was evaporated on a rotary evaporator under reduced pressure. G of compound (11) is obtained as a viscous clear light yellow oil.

¹ H NMR (DMSO-d6) δ 1.5-1.6 (m, 2H), 2.3 (m, 2H), 2.4-3.6 (m, 14H), 3.8 (s, 2H), 7.4 and 7.9 (s, 5H);

ESI-TOF MS: calcd for C18H27F3N4O 373.44 (M + H) +, is 373.273.

2.

4-Benzyl-7- [3- (2,2,2-trifluoro-acetylamino) -propyl] - [1,4,7] triazonane-1-carboxylic acid tert-butyl ester (12)

To compound (11) (215 mg, 0.58 mmol) and triethylamine (1.27 mmol) in 4 ml of THF was added dropwise (BOC) 2O (252 mg, 1.15 mmol) at 0-4 ° C. The mixture was then stirred overnight at room temperature. The solvent was evaporated under reduced pressure to give a yellow oil to which was added a saturated NaCl solution (2 ml) and extraction was performed with ethyl acetate. The extract was dried over MgSO4 and evaporated on a vacuum rotary evaporator. Obtained (12) as a yellow oil (139 mg).

¹ H NMR (CDCl3) δ 1.4 and 1.5 (m, 9H), 1.6-1.8 (m, 2H), 2.3 (m, 2H), 3.2-3.7 ( m, 14H), 3.6 (s, 2H), 7.4 and 7.7 (s, 5H);

ESI-TOF MS: calcd for C23H35F3N4O3 473.56 (M + H) +, is 473.243.

3.

4- (3-Amino-propyl) -7-benzyl- [1,4,7] triazonane-1-carboxylic acid tert-butyl ester (13)

1M NaOH was added to a solution of (12) (139 mg, 0.32 mmol) in 5 mL of methanol and stirred for hours at room temperature. The reaction mixture was then washed with methanol and filtered. The solvent was evaporated under reduced pressure on a rotary evaporator. Yield 15 mg (13) as a pale yellow oil.

GB ¹ 228 280 B1 H NMR (CDCl3) δ 1.43 and 1.46 (m, 9H), 1.6-2.0 (m, 2H), 2.3 (m, 2H), 2,8- 4.0 (m, 18H), 7.4 and 7.7 (s, 5H);

ESI-TOF MS: calcd for C21H36N4O2 377.55 (M + H) +, is 377.313.

P r x l a d IV.

This example illustrates the preparation of 2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -N- (3- [1,4,7] triazonan-1-yl -propyl) -acetamide (17) according to the procedure in Scheme IV.

1.

{2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy] -ethoxy} -anoic acid (14)

The synthesis was carried out according to: J. Phys. Chem. B (2004) 108, 7665-73 (Lee J.K. et al.) To give 318 mg (14) as a colorless oil.

¹ H NMR (CDCl3) δ 1,24-1,4 (m, 12H), 1.54-1.62 (m, 2H), 2,0-2,06 (dd, 2H), 3.46 ( t, 2H), 3.56-3.76 (m, 12H), 4.14 (s, 2H), 4.9-5.0 (m, 2H), 5.74-5.84 (m, 1H);

ESI-TOF MS: calcd for C19H36O6 361.5 (M + H) +, is 361.233; calcd for C19H35O6 359 (MH) ^- , is 359.314.

2.

N- [3- (4,7-diformyl- [1,4,7] triazonan-1-yl) propyl] -2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy ] -ethoxy} -acetamide (15)

Compound (14) (56 mg, 0.15 mmol) and N-hydroxysuccinimide (17 mg, 0.15 mmol) were dissolved in 1 mL of CH2Cl2 and cooled to 0 ° C. Then dicyclohexylcarbodiimide (32 mg, 0.15 mmol) was added. After 12 hours After stirring at 0 ° C, the formed white precipitate was filtered off. The filtrate was rotary evaporated under reduced pressure to yield a yellow oil. This oil was added directly to (9) (45 mg, 0.19 mmol) with triethylamine (0.19 mmol) in 1 mL of DMF. The mixture was stirred for 5 hours and then the solvent was removed by rotary evaporation under reduced pressure. An orange oil was obtained which was separated by column chromatography (silica gel, hexane-ethyl acetate 2: 1) to give 26 mg (15) as a white solid.

¹ H NMR (CDCl3) δ 1.2-1.4 (m, 12H), 1.54-1.62 (m, 4H), 1,9-2,06 (m, 2H), 2,24- 2.5 (m, 6H), 3.3-3.7 (m, 26H), 3.97-4.0 (s, 2H), 4.9-5.0 (m, 2H), 5. 34 (br, 1H), 5.77-5.84 (m, 1H), 8.1 and 8.2 (s, 2H);

gHSQC {1H / 13C} NMR (CDCl3) δ (rotamers) 1.295 / 26.59, 1.297 / 23.34, 1.56 / 30.04, 1.62 / 25.36, 1.91 / 28.05, 2.01 / 27.83, 2.04 / 34.23, 2.31 / 34.50, 2.50 / 31.07, 3.39 / 73.34, 3.441 / 49.23, 3.422 / 36, 64, 3.44 / 71.87, 3.52 / 73.36, 3.56 / 75.66, 3.58 / 70.31, 3.67 / 51.62, 3.67 / 70.76, 3.73 / 47.62, 3.73 / 62.18, 3.97 / 70.81, 4.01 / 70.83, 4.96 / na, 5.80 / na, 8.1 / na, 8.2 / na;

ESI-TOF MS: calcd for C30H56N4O7, 585.80 (M + H) +, is 585.325.

3.

S- (11- {2- [2- (2 - {[3- (4,7-diformyl- [1,4,7] triazonan-1-yl) propylcarbamyl] methoxy} ethoxy) ethoxy ester ] -ethoxy) -undecyl) thioacetic acid (16)

The synthesis was carried out according to J. Am. Chem. Soc. (1991) 113, 12-20 (Pale-Grosdemange C.E.S. et al.). From 20 mg (0.034 mmol) (15), there was obtained 4 mg of the product (16) as a white solid after separation by column chromatography (silica gel, hexane-ethyl acetate 1: 1).

¹ H NMR (CDCl3) δ 1.2-1.4 (m, 14H), 1.6 (m, 4H), 1.9-2.1 (m, 4H), 2.2-2.4 ( m, 5H), 3.2-4.0 (m, 32H), 8.1 and 8.2 (s, 2H);

ESI-TOF MS: calcd for C32H60N4O8S 661.92 (M + H) +, is 661.408.

4.

2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -N- (3-triazonan-1-yl-propyl) -acetamide (17)

To 0.1 mL of 0.1M HCl in methanol was added (16) (1 mg, 0.002 mmol) and stirred at room temperature for 12 hours. It was then evaporated under reduced pressure on a rotary evaporator to yield 0.96 mg (17) as a pale yellow oil.

ESI-TOF MS: calcd for C28H58N4O5S 563.86 (M + H) +, is 563.391.

P r z k ł a d V.

This example illustrates a general method of forming mixed SAM on gold. The components forming the monolayer are thiols of general formula IIa together with 2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] ethoxy} -ethanol.

The gold coated substrate was washed with ethanol and dried under nitrogen. The gold plated plates were immersed in an ethanol solution containing a mixture of thiols HS- (CH2) 11-triethylene glycol-OH and HS (CH2) 11-triethylene glycol-azakorona (total thiols concentration: 2 mM) for 8 hours at room temperature. The SAM coated plates were then washed with ethanol and dried.

Claims (9)

1. New, asymmetrically substituted 1,4,7-triazacyclononane derivatives having the ability to complex metal ions, especially Ni and Cu, of the general formula I, possibly in the form of salts, hydrates or complexes with metals, in which R2 and R3 are the same or different and represent, independently of each other, a tert-butyloxycarbonylmethyl, formyl or benzyl group. Z is a group -CH2p is an integer of 3, with R2 or R3 being the same only when they represent a formyl group. 2. Derivatives according to claim A method as claimed in claim 1, selected from the group consisting of: 4- (3-Amino-propyl) -7-tert-butoxycarbonylmethyl- [1,4,7] triazonane-1-carboxylic acid tert-butyl ester, (3-Amino-propyl) -7-benzyl- [1,4,7] triazonane-1-carboxylic acid tert-butyl ester, {4- (3-Amino-propyl) -7-formyl- acid tert-butyl ester [1,4,7] triazonan-1-yl} -acetic, 4- (3-Amino-propyl) -7-benzyl- [1,4,7] triazonan-1-carbaldehyde, [4- (3-Amino-propyl) -7-benzyl- [1,4,7] triazonan-1-yl] -acetic acid; 3. New, asymmetrically substituted 1,4,7-triazacyclononan derivatives with oligo (oxyethylene) fragment of general formula 2: AND R3 formula 2 optionally in the form of salts, hydrates and metal complexes in which: R2 and R3 is hydrogen, Z is an integer -CH2-, p is an integer 3, n is an integer 11, m is an integer 3, k is an integer 1, A is -O-, -C (O) -NH- or -NH-C (O) -, -C (O) O-, -OC (O) -, B is -OC (O) NH-, -C (O) NH-, -CH2NH-, -NH-, -N (CH ₃ ) -, -N = N-, Y is CH2 = CH-, -SH, CH3C (O) S- or vial protecting groups. 4. Derivatives according to p. 3. The process of claim 3, which are selected from the group consisting of: N- [3- (4-benzyl-7-formyl- [1,4,7] triazonan-1-yl) -propyl] -2- {2- [2 - (2-undec-10-enyloxy-ethoxy) -ethoxy-ethoxy-acetamide, 4-Benzyl-7- [3- (2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy] -ethoxy} -acetylamino) -propyl] - [1, 4,7] triazonane-1-carboxylic acid, S- (11- {2- [2- (2 - {[3- (4-benzyl-7-formyl- [1,4,7] triazonan-1-yl) propylcarbamoyl] methoxy} ethoxy) ester -ethoxy] -ethoxy} -undecyl) thioacetic acid, 4- {3- [2- (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy] -ethoxy} ethoxy) -acetylamino] -propyl} -7-benzyl-triazonan- acid tert-butyl ester 1-carboxylic acid, PL 228 280 Β1 N- [3- (4-benzyl- [1,4,7] triazonan-1-yl) propyl] -2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] ethoxy} -ethoxy) -acetamide, {4-Formyl-7- [3- (2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy] -ethoxy} -acetylamino) -propyl] - [1 , 4,7] triazonan-1-yl} -acetic, 4-tert-Butoxycarbonylmethyl-7- [3- (2- {2- [2- (2-undec-10-enyloxyethoxy) -ethoxy] -ethoxy} -acetylamino) -propyl] -triazonan-1 acid tert-butyl ester -carboxylic acid, (4- {3- [2- (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy] -ethoxy} ethoxy) -acetylamino] -propyl} -7-formyl-triazonane -1-yl) -acetic, 4- {3- [2- (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy] -ethoxy} ethoxy) -acetylamino] -propyl} -7-tert-butoxycarbonylmethyl- acid tert-butyl ester [1,4,7] triazonane-1-carboxylic acid, (4- {3- [2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -acetylamino ] -propyl} - [1,4,7] triazonan-1-yl) -acetic, N- [3- (4,7-diformyl- [1,4,7] triazonan-1-yl) propyl] -2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy ] -ethoxy-acetamide, S- (11 - {2- [2- (2 - {[3- (4,7-diformyl- [1,4,7] triazonan-1-yl) propylcarbamoyl] methoxy} -ethoxy) ethoxy ester ] -ethoxy} -undecyl) thioacetic acid, 2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -N- (3-triazonan-1-yl-propyl) -acetamide, N- (3- [1,4,7] triazonan-1-yl-propyl) -2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy] -ethoxy} -acetamide, N- [3- (4-benzyl- [1,4,7] triazonan-1-yl) propyl] -2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy] - ethoxy} -acetamide, {4- [3- (2- {2- [2- (2-undec-10-enyloxy-ethoxy) -ethoxy] -ethoxy} -acetylamino) -propyl] - [1,4, 7] triazonan-1-yl} -acetic. 5. Derivatives according to claim 1 3. The compounds according to claim 3, characterized in that they are amide-bond derivatives of general formula 2a: R4 ° tHk NN 1 ^R2 \ R3 formula 2a being a special case of derivatives of formula 2 and their possible salts, hydrates and metal complexes where R2 and R3 are as defined in the claim 3, p is an integer 3, n is an integer 11, m is an integer 3, k is an integer 1, R4 is hydrogen. 6. Derivatives according to claim 1 The compounds of claim 3, characterized in that they are compounds of general formula 2a, selected from the group consisting of: S- (11- {2- [2- (2 - {[3- (4-benzyl-7-formyl- [1,4,7] triazonan-1-yl) propylcarbamoyl] methoxy} ethoxy) ester -ethoxy] -ethoxy} -undecyl) thioacetic acid, 4- {3- [2- (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy] -ethoxy} ethoxy) -acetylamino] -propyl} -7-benzyl-triazonan- acid tert-butyl ester 1-carboxylic acid, N- [3- (4-benzyl- [1,4,7] triazonan-1-yl) propyl] -2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxyyethoxy} -ethoxy ) -acetamide, (4- {3- [2- (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy] -ethoxy} ethoxy) -acetylamino] -propyl} -7-formyl-triazonane -1-yl) -acetic, PL 228 280 Β1 4- {3- [2- (2- {2- [2- (11-acetylsulfanyl-undecyloxy) -ethoxy] -ethoxy} ethoxy) -acetylamino] -propyl} -7-tert-butoxycarbonylmethyl- acid tert-butyl ester [1,4,7] triazonane-1-carboxylic acid, (4- {3- [2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -acetylamino ] -propyl °} - [1,4,7] triazonan-1-yl) -acetic, S- (11 - {2- [2- (2 - {[3- (4,7-diformyl- [1,4,7] triazonan-1-yl) propylcarbamoyl] methoxy) ethoxy) ethoxy ester ] -ethoxy} -undecyl) thioacetic acid, 2- (2- {2- [2- (11-mercapto-undecyloxy) -ethoxy] -ethoxy} -ethoxy) -N- (3-triazonan-1-yl-propyl) -acetamide. 7. A method for the preparation of new 1,4,7-triazacyclononan (tacn) derivatives of the general formula 1, characterized in that to the 1-formyl-1,4,7-triazacyclononane derivative with a protected linker of formula 1 and formula 1 and which is a special case of the derivatives of formula I, or their optional salts, hydrates and metal complexes, in which R1 is a tert-butyloxycarbonylmethyl, formyl or benzyl group, Z is a -CH2- group, and p is an integer 3, selectively introduced by a substitution reaction R2, selected from benzyl, tert-butyl or formyl, to give the tacn derivative of general formula I ii R1 R2 and ^{J of} formula 1 and in which R1, Z and p are as defined above, from which the compound of formula I is released by selective deprotection. 8. A method for the preparation of new 1,4,7-triazacyclononan (tacn) derivatives of the general formula 1 or 2, characterized in that the derivative of the tacn compound having an amine terminated linker and the R2 substituent introduced on the azacorona secondary amine group, represented by the formula liii, '\, R2 h ₂ n4 and _p nn / \ \ —- NV H formula liii PL 228 280 Β1 where the Z, p substituents are as defined above, selectively introducing the trifluoroacetyl group on the primary amine group of the linker, at reduced temperature, from -5 ° C to 5 ° C, with ethyl or methyl trifluoroacetate, formula 1 iv to give the tacn derivative of formula 1 iv in which the substituents Z, p are as defined above, and then a substituent R3 is introduced in a substitution reaction, a tert-butyloxymethyl group, in a basic environment in which the trifluoroacetyl group is withdrawn from the amino group to give the tacn derivative of formula 1v in which R2, R3, Z, p are as defined above, and then ABOUT Ν N G-N-y i R3 -R2 formula 1v from the derivative of formula 1v above releases the compound of formula 1 by selective deprotection. 9. Use of a compound according to claim 1 1 or 3 for the production of self-assembled monolayers (SAMs).