WO2014020152A1

WO2014020152A1 - Ligands for apheresis and immunoabsorption

Info

Publication number: WO2014020152A1
Application number: PCT/EP2013/066280
Authority: WO
Inventors: Holger Bittermann; Marc Arnold; Thomas Neumann
Original assignee: Graffinity Pharmaceuticals Gmbh
Priority date: 2012-08-02
Filing date: 2013-08-02
Publication date: 2014-02-06

Abstract

The present invention relates to a method for separating one or more proteins from plasma by immunoadsorption IA, comprising • contacting the plasma with a ligand-substituted matrix; and • removing the plasma from the ligand-substituted matrix; • wherein the ligand-substituted matrix comprises a support material and at least one ligand covalently bonded to the support material.

Description

Ligands for Apheresis and Immunoabsorption

The present invention relates to the field of apheresis, preferably therapeutic apheresis, more preferably to plasmapheresis, and in particular to immunoadsorption of immunoglobulins, most preferably of the IgG type.

Apheresis is a process known to the person skilled in the art and comprises various subtypes. The term "apheresis" refers to a procedure in which blood of a patient or donor is passed through a medical device, which separates out one or more components of blood and returns remainder with or without extracorporeal treatment or replacement of the separated component

Therapeutic applications of apheresis (,. therapeutic apheresis") comprise plasmapheresis (removal or exchange of blood plasma or blood plasma components) and cytapheresis (a group of procedures for blood cell removal or exchange).

Examples of cytapheresis comprise the removal of excessive white blood cells (leukocytapheresis) or platelets (thrombocytapheresis), or the exchange of diseased red blood cells (erythrocytapheresis).

For the separation of the desired components of the blood in plasmapheresis, the method of choice is, in general, either centrifugation o membrane filtration, whereafter plasma is obtained. I n therapeutic plasmapheresis, the plasma is either treated to remove harmful constituents, or it is entirely discarded.

Therapeutic plasmapheresis (often referred to as therapeutic plasma exchange TPE) in the simplest form is a method, wherein repeated cycles of blood extraction and ex vivo centrifugation are carried out, thereafter discarding the plasma and returning the blood cells to the patient together with a suitable replacement solution (fresh frozen plasma, 5% albumin or similar colloidal solution). This method is also called„unselective plasmapheresis".

In a variant of TPE, the plasma that is removed can be replaced by the patient^'s own plasma after a secondary online purification procedure in order to remove antibodies or suspected antibodies implicated in the pathogenesis of autoimmune disease. Other important targets include circulating antigen-antibody complexes. This method is also called ...selective p lasmapheresis' ' .

The secondary plasma processing can be immunoadsorption (IA). This term, refers to a procedure in which plasma of the patient is passed through a medical device, which has a capacity to remove immunoglobulins Ig by specifically binding them, to a particular component present in the device. The method also separates circulating immune complexes (CIC). Immunoadsorption has been proven to be a useful treatment option in several autoimmune disorders or in renal transplant recipients. Among these, severe rheumatoid arthritis, systemic lupus erythematosus, acute renal transplant rejection, idiopathic dilated cardiomyopathy (DCM), and idiopathic thrombocytopenia are the best known diseases. The success of a IA therapy depends on the component employed in. the device.

Immunoglobulins are a class of soluble proteins found in body fluids of humans and other vertebrates. They are also termed "antibodies" and play a key role in the processes of recognition, binding and adhesion of cells. Antibodies are oligomeric glycoproteins which have a paramount role in the immune system by the recognition and elimination of antigens, in general bacteriae and viruses.

The polymeric chain of antibodies is constructed such that they comprise so-called heavy and light chains. The basic immunoglobulin unit consists of two identical heavy and two identical light chains connected by disulfide bridges. There are five types of heavy chains (α, γ, δ, ε, μ), which determine the immunoglobulin classes (IgA, IgG, IgD, IgE, IgM). The light chain group comprises two subtypes, λ and κ.

IgGs are soluble antibodies, that can be found in blood and other body fluids. They are built by B-cell derived plasma cells as response to and to neutralize bacterial or other pathogens. An IgG is an Y-shaped glycoprotein with an approximate molecular weight of 150 kDa, consisting of two heavy and two light chains. Each chain is distinguished in a constant and in a variable region. The two carboxy terminal domains of the heavy chains are forming the Fc fragment ("constant fragment"), the amino terminal domains of the heavy and light chains are recognizing the antigen and are named Fab fragment ("antigen-binding fragment").

In IA, the working principle of most commercially adsorber types for the treatment of antibody-mediated autoimmune diseases is the removal of Ig in a non-specific manner. Therapeutic immunoadsorption has most commonly been carried out using adsorbents on the basis of bacterial protein- A (Staphylococcal protein A, a major cell wall component of Staphylococcus aureus) or sheep antibodies directed to human immunoglobulin (Ig). Both ligands have to be purified from biological sources harboring potential infection risks due to cross-contamination of the purified ligand.

The Food and Drug Administration (FDA) approved IA for the treatment of rheumatoid arthritis, idiopathic thrombocytopenic purpura and hemophilia with inhibitors. Other off label indications include autoantibody mediated autoimmune disorders with a conceivable correlation between autoantibody titres and disease activity, such as systemic lupus erythematosus (SLE), myasthenia gravis (MG), Guillian-Barre-syndrome (GBS), idiopathic dilated cardiomyopathy (DCM), dermatomyositis and autoimmune bullous disorders. Moreover, in highly sensitized (HLA allo-antibodies) renal transplant recipients, IA has been successfully applied to prevent acute renal transplant rejection.

The molecular interaction between protein A and Ig is well characterized and the binding site on the Fc fragment involves residues in the CH2ZCH3 region of particularly IgGl, IgG2, IgG4 and to a lesser extent to IgG3. 1 A columns approved by the FDA include the protein- A silica column (Prosorba, Fresenius Medical Care, Redmont, CA, USA), for the treatment of refractory rheumatoid arthritis and for resistant idiopathic thrombocytopenic purpura. The protein-A sepharose column Immunosorba, Fresenius Medical Care, St Wendel, Saarland, Germany) received FDA-approval for patients with hemophelia developing inihibitors, i.e. allo-antibodies directed against factor concentrates.

However, the use of Protein A is limited by leaching from the column and poor stability under harsh conditions applied for regeneration and cleaning procedures. The chemical stability of Protein A can be improved by using genetically engineered Protein A. Yet, the high costs of Protein A resins have resulted in the search for suitable alternatives, in particular selected from small molecules

Other plasmaperfusion columns consist of crosslinked polyvinyl alcohol gel beads immobilizing specific hydrophobic amino acids such as tryptophan or phenylalanine as ligands. These adsorbers remove the target proteins by hydrophobic and electrostatic bonding between the adsorber and the target structures. Asahi Medical (Tokyo, Japan) offers a tryptophan column (Immusorba TR 350) and a phenylalanine column (Immusorba PH 350) which both have been applied in the autoimmune disorders, MG and GBS. The Therasorb adsorber system is based on coupling polyclonal sheep antibodies to a sepharose matrix which selectively bind to plasma components as human Ig (Ig-Therasorb) or low density lipoproteins (LDL-Therasorb). The Therasorb system is currently provided by Miltenyi Biotech, Bergisch Gladbach, Germany. Another plasmaperfusion column, Selesorb (Kaneka, Osaka, Japan) which is particularly used in SLE consists of dextran sulphate immobilized to cellulose. The dextran sulphate adsorber predominantly removes anti-DNA and anti-cardiolipin auto-Ab through electrostatic bonding between the negatively charged adsorbent and the Fab fragments of the autoantibodies, probably including positively charged aminoacids. More recently developed adsorber systems contain synthetic peptides covalently bound to a sepharose matrix as ligands for human Ig. Fresenius Medical Care provides two peptide-based adsorber systems, the Globaffin adsorber contains a peptide (PGAM146) which unspecifically binds to IgG, CIC and to a lesser extend to IgA and IgM. The working principle of most commercial adsorber types for the treatment of antibody-mediated autoimmune diseases is the removal of Ig in a non-specific manner.

From the foregoing, it is clear that the adsorbents currently used in IA bear potential drawbacks. As mentioned above, Protein A and anti-Ig sheep antibodies harbour potential infection risks due to cross-contamination of the purified ligand. Futhermore, raising antibodies in animals is expensive and time-consuming and variations in the quality between different antibody batches might be possible.

Synthetic small molecule affinity ligands are of particular interest for the purification of therapeutic proteins due to their generally higher chemical stability and their lower production costs. Synthetic affinity ligands that are more readily available, preferably cheaper than protein-based ligands, are more robust under stringent conditions and have a selectivity comparable to or even higher than Protein A would provide a suitable solution for selective antibody binding. Depending on the target protein, such affinity ligands should preferably offer the same broad applicability as Protein A, recognizing the constant Fc region of IgG type immunoglobulins.

The object underlying the present invention is the provision of methods for apheresis, in particular for immunoadsorption, not showing the above drawbacks. In particular, high infection risk due to cross contaminations, time-consuming production, high costs and possible batch related quality variations are to be avoided.

A synthetic adsorber small molecule ligands should combine safety advantages and an improved production technology.

The problem is solved by the embodiments of the present invention as laid out hereinafter. The present invention is directed to a method for separating one or more proteins from plasma by immunoadsorption (IA), comprising contacting the plasma with a ligand-substituted matrix; and

removing the plasma from the ligand-substituted matrix; with the ligand-substituted matrix comprising a support material and at least one ligand covalently bonded to the support material, the ligand being represented by formula (I)

L-(Sp)_v-Ar¹-Am-Ar² (I)

wherein

L is the linking point on the support material to which the ligand is attached;

Sp is a spacer group;

v is 0 or 1 ;

Am is an amide group -NR¹-C(0)-, and wherein either NR¹ is attached to Ar¹ and -C(O)- is attached to Ar², or -C(O)- is attached to Ar¹ and NR¹ is attached to Ar²; and

R¹ is hydrogen or Ci to C₄ alkyl, more preferably hydrogen or methyl; and most preferably hydrogen;

Ar¹ is a 5-, 6- or 7-membered mononuclear aromatic ring or partially saturated aromatic ring connected to Sp or L via a chemical bond and which is optionally furthermore

(a) attached to a further 5- or 6-membered mononuclear aromatic ring via a chemical bond; or

(b) fused to a mononuclear or binuclear aromatic ring as part of a multinuclear ring system wherein Ar¹ is directly connected to Am via a chemical bond present on the said 5-, 6- or 7- membered aromatic ring constituting Ar¹, or indirectly via a chemical bond which is either present at the further 5- or 6-membered aromatic ring attached to Ar¹, or on the further 5- or 6- membered aromatic ring fused to Ar¹;

and wherein Ar¹ is either not further substituted or attached to at least one substituent selected from Ci to C₄ alkyl; C₃ and C₄ cycloalkyl; C₂ to C₄ alkenyl; C₂ to C₄ alkynyl; a halogen; Ci to C₄ haloalkyl; hydroxyl-substituted Ci to C₄ alkyl; Ci to C₄ alkoxy; hydroxyl-substituted Ci to C₄ alkoxy; halogen- substituted Ci to C₄ alkoxy; Ci to C₄ alkylamino; Ci to C₄ alkylthio; - N0₂; =0; =S; =NH; -OH; and combinations thereof;

Ar² is a 5- or 6-membered mononuclear aromatic ring which is unsubstituted, or via a chemical bond attached to at least one substituent selected from Ci to C₆ alkyl; C3 to C₆ cycloalkyl; C₂ to C₆ alkenyl;Cs and C₆ cycloalkenyl; C₂ to C₆ alkynyl; a halogen; Ci to C₆ haloalkyl; hydroxyl-substituted Ci to C₆ alkyl; Ci to C₆ alkoxy; hydroxyl-substituted Ci to C₆ alkoxy; halogen-substituted Ci to C₆ alkoxy; Ci to C₆ alkylamino; Ci to C₆ alkylthio; carbamoyl; Ci to C₄ alkylenedioxy, preferably methylenedioxy and ethylenedioxy; -OH; -SH; a 5- or 6-membered mononuclear aromatic ring; and combinations thereof; and wherein Ar² optionally, further to the substituents to which it may be attached via a chemical bond as cited above, is fused to a 5- or 6-membered mononuclear aromatic ring as part of a multinuclear ring system.

The present invention is also directed to a method of therapeutic plasma exchange TPE in an individual, comprising separation of antibodies, preferably IgG type antibodies, from plasma by immunoadsorption (IA), comprising the following steps: a) providing a ligand-substituted matrix comprising a support material and at least one ligand covalently bonded to the support material, the ligand being represented by formula (I) as defined above; b) providing plasma from the individual; c) contacting the plasma with a ligand-substituted matrix; and d) removing the plasma from the ligand-substituted matrix.

In a preferred embodiment, the plasma from which the antibodies have been separated is returned to the individual, optionally together with other compounds present in the individual's blood from which the plasma was obtained.

The ligand-substituted matrix adsorbs antibodies of the IgG type present in the plasma of the respective indiviudual and, as the case may be, also circulating immune complexes (circulating antigen-antibody complexes). When employed in the methods of the present invention, these types of proteins can be removed from the patient's plasma. In an embodiment of the present invention, the plasma from, which the proteins have been separated is returned to the individual, without the need for providing a replacement solution for the plasma.

The plasma is contacted with the ligand-substituted matrix of the invention in a manner and for a time sufficient to remove the desired amount of protein. When the proteins have been separated to the desired degree, the plasma is removed from the ligand-substituted matrix. As the case may be, the plasma may then be further processed, see below.

The plasma which will be submitted to the separation of proteins, preferably antibodies, more preferably IgG type immunoglobulins, by IA, in particular within TPE, is in general obtained by methods known to the person skilled in the art, i.e. centrifugation of membrane filtration or even a mixture of both.

This plasma is subjected to a purification step, in order to separate IgG type immunoglobulins from the plasma before processing it, e.g. by returning it to the individual. This further processing is, in general, a purification step making use of the ligand-substituted matrix as defined in the present application, and wherein the ligand-substitueted matrix is contacted with the plasma in an appropriate way known to the person skilled in the art. To that end, the matrix is preferably arranged within a medical device capable of housing the matrix and having an inlet and outlet through which the plasma can enter into the device and get into contact with the matrix and thereafter leave the device. In general, the medical device will have a housing in which the matrix can be arranged in an appropriate manner by methods known to the person skilled in the art.

The way in which the plasma and the matrix are to be contacted to ensure the desired removal of the protein is known to the person skilled in the art. The person skilled in the art is also aware of the time necessary to ensure the desired removal of the protein. Reference is made e.g. to "Schmidt E, Klinker E, Opitz A, Herzog S, Sitaru C, Goebeler M, Mansouri Taleghoni B, Brocker EB, Zillikens D. Protein A immunoadsorption: a novel and effective adjuvant treatment of severe pemphigus. Br J Dermatol. 2003 Jun; 148(6): 1222-9."

"Shimanovich I, Herzog S, Schmidt E, Opitz A, Klinker E, Brocker EB, Goebeler M, Zillikens D. Improved protocol for treatment of pemphigus vulgaris with protein A immunoadsorption. Clin Exp Dermatol. 2006 Nov; 31(6):768-74." „Luftl M, Stauber A, Mainka A, Klingel R, Schuler G, Hertl M. Successful removal of pathogenic autoantibodies in pemphigus by immunoadsorption with a tryptophan-linked polyvinylalcohol adsorber. Br J Dermatol. 2003 Sep;149(3):598-605."

„Schmidt E, Zillikens D. Immunoadsorption in dermatology. Arch Dermatol Res. 2010 May; 302(4):241-53. "

The method of the present invention permits to treat a disease caused by pathological antibodies. This type of diseases is known to the person skilled in the art. The person skilled in the art is also aware which diseases are caused or can be caused by pathological antibodies. In one embodiment, these diseases are autoimmune disease. In another embodiment, these diseases are not caused by an autoimmune reaction and are, preferably, but not limited to, transplant rejections. Diseases which are preferably treated by the method of the present invention include rheumatoid arthritis, idiopathic thrombocytopenic purpura, hemophilia with inhibitors, systemic lupus erythematosus (SLE), myasthenia gravis (MG), Guillian-Barre- syndrome (GBS), idiopathic dilated cardiomyopathy (DCM), dermatomyositis, autoimmune bullous disorders, renal transplant rejection, in particular renal transplant rejection in highly sensitized (HLA allo-antibodies) renal transplant recipients, autoimmune hemolytic anemia, Sjogren's Syndrome, mixed connective tissue disease (MCTD), endocrine orbitopathy, pemphigus vulgaris.

The various terms used in the present application in connection with apheresis have the following meanings:

Apheresis: A procedure in which blood of the patient or donor is passed through a medical device, which separates out one or more components of blood and returns remainder with or without extracorporeal treatment or replacement of the separated component

Immunoadsorption (IA): A procedure in which plasma, after separation from the blood, is passed through a medical device, which has a capacity to remove immunoglobulins and, as the case may be, immune complexes (antibody-antrgene complexes) by specifically binding them to the active component (e.g., Staphylococcal, protein A. or the ligands of the inventio fixed to a matrix) of the device. In the present case, the antibodies and immune complexes are removed by adsorption at the ligand-substituted matrix, namely by interaction with the compounds/ligands disclosed in the present application fixed to a support material. The interaction preferably is molecular recognition. In general, the plasma is derived from the blood of an animal, preferably a mammal, in particular a human.

Therapeutic plasma exchange TPE (also referred to as therapeutical plasmapheresis): A procedure in which blood of the patient is passed through a medical, device, which separates out plasma from other components of blood. In one embodiment, the plasma may be removed and replaced with a replacement solution such as colloid solution (e.g., albumin and/or plasma) or combination of crystalloid/colloid solution (unselective TPE). In another embodiment (selective TPE) the plasma may be submitted to a further purification step, preferably immunoadsorption .

Plasmapheresis: A procedure in which blood of the patient or the donor is passed through a medical device, which separates out plasma from, other components of blood.

Therapeutic apheresis: A. therapeutic procedure in which a blood of the patient is passed through an. extracorporeal, medical device, which separates components of blood to treat a disease.

The ligand-substituted support material/matrix as used in the present invention, in accordance with the formula (I), has the structure according to formula (la), in one embodiment of the invention. In a further embodiment of the invention, the ligand-substituted matrix of the present invention, in accordance with the formula (I), has the structure according to formula (lb). In either formula (la) and (lb), L, Sp, N, R¹, Ar¹ and Ar² and the integer v have the meanings defined above in connection with formula (I)

L-(Sp)_v- Ar ¹ -N(Pvl )-C(0)- Ar² (la)

L- Sp Ar¹- C(O)- N(Pvl)-Ar² (lb)

Ligands according to the present invention bind to polyclonal and monoclonal IgG of human origin, in particular human IgGi, IgG₂, IgG₃ and IgG₄ and polyclonal and monoclonal IgGs from different animal species such as rabbit and mouse immunoglobulins. As L is an appropriate entity on the support material/matrix, the ligand-substitued matrix can also be depicted as in Fig (Ic), with M being the matrix/support material and the other variables having the meaning as laid out in connection with formula (I).

M-L-(Sp)_v-Ar¹-Am-Ar² (Ic)

The matrix comprises the support material, i.e. the matrix is entirely composed of the support material, or the support material forms a part of the matrix, which comprises components further to the support material.

The present invention thus makes use of a ligand or compound, wherein the compound binds to a protein, preferably an antibody or fragment of an antibody, the ligand or compound having the formula

(SpP)_v-Ar¹-Am-Ar² (II) wherein L, v, Ar¹, Am and Ar² have the meanings defined hereinafter in connection with formula (I), and wherein SpP is a spacer precursor, as specified below. The present invention is furthermore drawn to the use of the ligand for affinity purification of an antibody or an fragment of an antibody, preferably after attaching the ligand to an appropriate matrix as specified in the present application.

If v in formula (I) is 0, then the ligand is directly bonded to L. In other embodiments the ligand is bonded to the support material via a spacer group Sp. This applies when v in formula (I) is different from 0.

L in the above formula (I) is the linking point, also referred to as "point of attachment". The person skilled in the art is aware of appropriate linking points/points of attachment.

It is understood that the linking point L is either directly connected with the ligand/compound or via a spacer. L is an appropriate entity on the support material which lends itself for linking the ligands of the present invention to the support material. Appropriate entities are known to the person skilled in the art. Typically L is or may be part of an entity resulting from the reaction of an appropriate functional group on the support material with a corresponding functional group on the precursor compound of the ligand to form the matrix-bound ligand. The precursor compound of the ligand (which is reacted with the support matrix to form the ligand-substituted matrix) comprises, in one embodiment of the invention, a spacer precursor. "Spacer precursor" relates to the chemical entity which forms the part of the spacer remaining after formation of the ligand-substituted matrix and which contains an appropriate functional group (precursor group) for the formation of the linking point L by reaction with an appropriate group (precursor group) on the support material, see below. If the precursor compound of the ligand does not contain a spacer precursor (and will thus be directly connected to the support material via a bond), the appropriate functional group (precursor group) is attached to the ligand itself.

In one preferred embodiment, L is directly connected via a bond, preferably a single bond, to the ligand according to formula I. As L is an entity on the support material, the bound ligand is thus connected to the support material via L. In this context, the term "support material" refers to polymers known to the person skilled in the art and available on the market which lend themselves for the purposes of the present invention.

A "bond" in the context of the present invention is preferably a covalent chemical bond, for example a single, double or triple bond, preferably a single bond.

In a preferred embodiment, L is a functional group or a chemical entity which is either present on the support material as such and before attaching the ligands of the invention to the support material/matrix, or which is formed in the course of attaching the ligands of the invention to the support material/matrix in the course of a chemical reaction ("new functional group").

In general, the support material comprises functional groups for the attachment of molecules, preferably the ligands of the present invention. In the context of the present invention, the functional groups are regarded as a part of the support material; this also applies in cases where the ligands of the present invention are connected to the support material via a bond (i. e. L is connected to the ligand directly via a bond, without a spacer group Sp being present, see below), and wherein the bond is directly formed between the respective ligand according to the invention and either the said functional group for the attachment of molecules on the support material or with the support material itself under transformation of said functional group to a bond, preferably a single bond.

If the bond is directly formed between the respective ligand according to the invention and the functional group for the attachment of molecules on the support material, said functional group may be interconverted to a new functional group which is subsequently attached to the support material. In the context of the present invention, the new functional group is regarded as a part of the support material/matrix and forms L, linking the ligand with the matrix via a bond, preferably a single bond.

Appropriate functional groups on the support material (or "precursor groups" i.e. present before the ligands according to the invention are attached) and, independently from each other, on the ligands of the present invention which allow the direct connection of the ligand to the support material include, but are not restricted to, -OH, -SH, -NH₂, >NH, methanesulphonate, trifluoromethyl sulphonate, arylsulphonate, carboxylic acid, sulphonic acid, phosphoric acid, phosphoramidite, epoxide, N-hydroxysuccinimidyl carboxylate, 1- hydroxybenzotriazolyl carboxylate, l-hydroxy-7-azabenzotriazolyl carboxylate, fluoride, chloride, bromide, iodide, maleimide, acrylate, acrylamide, aldehyde, ketone, hydrazine, hydrazide, O-alkyl hydroxylamine, isocyanate, isothiocyanate, cyanate, thiocyanate, vinylsulphone.

Functional groups ("new functional group) to which the functional groups present on the support material may be interconverted upon linkage of the ligand include, but are not limited to, carbon-carbon single bond, carbon-nitrogen single bond, aryl alkyl ether, aryl alkyl thioether, diaryl ether, diaryl thioether, aryl alkyl amine, aryl dialkyl amine, amide, hydrazide, sulphonamide, sulphonic hydrazide, N-aryloxy amide, N-aryloxy sulphonamide, phosphate, phosphoric amide, phosphoric hydrazide, N-aryloxy phorphoric amide, hydrazone, oxime, urea, thiourea, isourea, imidocarbonate, isothiourea, imidothiocarbonate.

In a further embodiment of the invention, L is connected to a spacer group -Sp-. In this embodiment, L is a bond or a chemical unit resulting from the reaction of an appropriate functional group on the support material with an appropriate functional group (or a "precursor group") on the spacer group Sp. Accordingly, the chemical reaction between the functional group on the support material and the functional group (or "complementary group") on the spacer group Sp connects the ligand according to the invention with the support material via the linking point L.

The spacer group Sp preferably is a hydrocarbon group which may contain, in addition to C and H atoms, further atoms. Appropriate further atoms are known to the person skilled in the art. Preferred atoms include O, S, N, P, Si. The hydrocarbon group may be linear or branched or cyclic. A more detailed description of the spacer group Sp is provided below. Sp is linked to Ar¹ of the ligands according to the invention via a single, double or triple bond, preferably via a single bond. Furthermore, Sp is linked to a functional group (precursor group) by which the ligands of the invention can be covalently linked with functionals groups (precursor groups) present on the matrix in a chemical reaction under formation of a new functional group (also termed "final functional group", final chemical entity or connecting unit. Examples of such functional groups (precursor groups), which may be present, independently from each other, on the support material and on Sp include, but are not limited to, -OH, -SH, - NH₂, >NH, methanesulphonate, trif uoromethyl sulphonate, arylsulphonate, carboxylic acid, sulphonic acid, phosphoric acid, phosphoramidite, epoxide, N-hydroxysuccinimidyl carboxylate, 1-hydroxybenzotriazolyl carboxylate, l-hydroxy-7-azabenzotriazolyl carboxylate, fluoride, chloride, bromide, iodide, maleimide, acrylate, acrylamide, aldehyde, ketone, hydrazine, hydrazide, O-alkyl hydroxylamine, isocyanate, isothiocyanate, cyanate, thiocyanate, vinylsulphone. Examples of appropriate connecting units derived from the reaction of a functional group on the support material with a functional group present on the spacer Sp include, but are not restricted to, carbon-carbon single bond, ether, aryl alkyl ether, aryl alkyl thioether, diaryl ether, thioether, diaryl thioether, dialkylamine, trialkylamine, aryl alkyl amine, aryl dialkyl amine, amide, ester, hydrazide, sulphonamide, sulphonic hydrazide, N-aryloxy amide, N-aryloxy sulphonamide, phosphate, phosphoric amide, phosphoric hydrazide, N-aryloxy phorphoric amide, hydrazone, oxime, urea, thiourea, isourea, imidocarbonate, isothiourea, imidothiocarbonate.

The spacer group Sp, generally, connects Ar¹ of the ligand according to formula (I) and the functional group (precursor group) that can undergo a chemical reaction with a functional group (precursor group) present on the support material, forming a final functional group (connecting unit) as described before. Generally, Sp is a linear or branched hydrocarbon which may also contain cyclic subunits. The hydrocarbon may be saturated or unsaturated, i. e. it may contain double or triple bonds. If Sp is a linear hydrocarbon, it allows the connection of one ligand moiety to one precursor group present on the support material; in contrast, if Sp is a branched hydrocarbon, it may allow the connection of more than one ligand moieties to one precursor group present on the support material. Additionally to C and H, the hydrocarbon may contain other atoms such as N, O, P, and S, preferably N and O. The carbon chain may be interrupted by other atoms or atom groups. Sp may contain combinations of different atoms or atom groups interrupting the hydrocarbon chain. Examples of atoms or atom groups that may interrupt the carbon chain of the hydrocarbon are -0-, >N-, - C(=0)N(H)-, -C(=0)N<, -0-P(=0)(-0-)₂. It is understood that if the hydrocarbon is interrupted by a trivalent atom or atom group such as -N< (tertiary amino), -C(=0)-N< (tertiary amide) or -0-(P=0)(-0-)₂ (phosphoric ester), said atom group may serve as a branching point connecting more than one ligand-connected subunits of Sp with the subunit of Sp carrying the functional group which undergoes a chemical reaction with a functional group present on the support material, ultimately resulting in the formation of the connecting unit as described before. Examples of additional suitable atom groups that may allow the introduction of a branching point into Sp include natural or unnatural trifunctional amino acids such as glutamic acid, aspartic acid, aminomalonic acid, lysine, ornithine, and diaminopropionic acid. In these cases, one of the functional groups of said trivalent moieties may serve as the functional group intended to undergo a chemical reaction with the functional group present on the support material. Typically, the total length of Sp is below 100 atoms, preferably below 50 atoms and more preferably below 30 atoms. If Sp is branched, the length as defined by the distance from the atom connecting Sp with the functional group intended to undergo a chemical reaction with a functional group on the support material to the most distant atom directly connected to a Ar¹ moiety, is below 100 atoms, preferably below 50 atoms and more preferably below 40 atoms.

Examples of appropriate entities of linear Sp connected to a carbon-based functional group (FG) (like, for example, carboxylic acid, aldehyde, epoxide, carboxylic acid active esters) intended to undergo a chemical reaction with a chemical group present on the support material include, but are not restricted to: FG-(CH₂)_n- (alkylene) with 1 < n < 100, preferably 1 < n < 50, and more preferably 1 < n < 30;

FG-(CH₂)_n-0- (alkyleneoxy) with 1 < n < 99, preferably 1 < n < 49, and more preferably 1 < n < 29;

FG-(CH₂)_n-N(H)- (alkyleneamino) with 1 < n < 99, preferably 1 < n < 49, and more preferably 1 < n < 29;

FG-CH₂(-0-CH₂-CH₂)„- (oligoethyleneglycol) with 1 < n < 30, preferably 1 < n < 15, and more preferably 1 < n < 9;

FG-CH₂(-0-CH₂-CH₂)„-0- (oligoethyleneglycolyloxy) with 1 < n < 30, preferably 1 < n < 15, and more preferably 1 < n < 9;

FG-CH₂(-0-CH₂-CH₂)„-N(H)- (oligoethyleneglycolylamino) with 1 < n < 30, preferably 1 < n < 15, and more preferably 1 < n < 9;

FG-CH₂(-0-CH2-CH2)2-N(H)-C(=0)-CH2(-0-CH2-CH₂)2- (oligoethyleneglycol interrupted by amide);

FG-CH₂(-0-CH₂-CH₂)₂-N(H)-C(=0)-CH₂(-0-CH₂-CH₂)₂-0- (oligoethyleneglycolyloxy interrupted by amide);

FG-CH₂(-0-CH₂-CH₂)₂-N(H)-C(=0)-CH₂(-0-CH₂-CH₂)₂-N(H)- (oligoethyleneglycolylamino interrupted by amide).

Examples of appropriate entities of linear Sp attached to a non-carbon-based functional group (FG) (like, for example, NH₂, OH, SH, chloride, bromide, iodide, isocyanate, isothiocyanate) intended to undergo a chemical reaction with a chemical group present on the support material include, but are not restricted to:

FG-(CH₂)_n- (alkylene) with 1 < n < 100, preferably 1 < n < 50, and more preferably 1 < n < 30;

FG-CH₂CH₂(-0-CH₂-CH₂)„- (oligoethyleneglycol) with 1 < n < 30, preferably 1 < n < 15, and more preferably 1 < n < 9; FG-CH₂CH₂(-0-CH₂-CH₂)„-0- (oligoethyleneglycolyloxy) with 1 < n < 30, preferably 1 < n < 15, and more preferably 1 < n < 9;

FG-CH₂CH₂(-0-CH₂-CH₂)_n-N(H)- (oligoethyleneglycolylamino) with 1 < n < 30, preferably 1≤ n < 15, and more preferably 1 < n < 9;

FG-CH₂CH₂(-0-CH₂-CH₂)₂-N(H)-C(=0)-CH₂(-0-CH₂-CH₂)₂- (oligoethyleneglycol interrupted by amide);

FG-CH₂CH₂(-0-CH₂-CH₂)₂-N(H)-C(=0)-CH₂(-0-CH₂-CH₂)₂-0- (oligoethyleneglycolyloxy interrupted by amide);

FG-CH₂CH₂(-0-CH₂-CH₂)₂-N(H)-C(=0)-CH₂(-0-CH₂-CH₂)₂-N(H)- (oligoethyleneglycolylamino interrupted by amide).

Examples of appropriate entities of branched Sp including a trifunctional atom group of which one functional group serves as the functionality intended to react with the functional group present on the support material, include, but are not restricted to:

H₂N-C(H)(-C(=0)-N(H)-CH₂CH₂(-0-CH₂CH₂)₂-)(-CH₂-C(=0)-N(H)-CH₂CH₂(-0- CH₂CH₂)₂-) (glutamic acid bis(3,5-dioxa-l-octyl) amide; the NH₂ group is intended to react with the functional group present on the support material);

H₂N-C(H)(-C(=0)-N(H)-CH₂CH₂CH₂-)(-CH₂-C(=0)-N(H)-CH₂CH₂CH₂-) (glutamic acid bis(n-propyl) amide; the NH₂ group is intended to react with the functional group present on the support material);

HO-C(=0)-C(H)(-N(H)-C(=0)-CH₂(-0-CH₂CH₂)₂-)(-CH₂-N(H)-C(=0)-CH₂(-0-CH₂CH₂)₂-) (N,N'-bis(3,5-dioxaoctanoyl)diaminopropionic acid; the carboxylic acid group is intended to react with the functional group present on the support material);

In an embodiment of the present invention, R¹ is selected from: hydrogen; and Ci to C₄ alkyl, typically linear and branched Ci to C₄ alkyl which may comprise a cycloalkyl unit such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, cyclopropyl, methy Icy clopropy 1.

Preferably R¹ is selected from hydrogen, ethyl and methyl. More preferably, R¹ is hydrogen, or methyl. Most preferably, R¹ is hydrogen.

Ar¹ is an alicyclic or heterocyclic mononuclear aromatic ring or partially saturated aromatic ring which can have 5, 6 or 7 members. Ar¹ is connected to Sp or L via a chemical bond. The bond of Ar¹ to Sp or L can be a single bond, a double bond or a triple bond. Preferably, the bond is a single bond. Ar¹ can have bonds to substituents further to the chemical bond to Sp or L.

If Ar¹ is a heterocyclic aromatic ring or partially saturated heterocyclic aromatic ring, it contains at least one heteroatom from the group N, S and O, preferably from the group N and S, even more preferably one or more N-atoms. In all these embodiments, Ar¹ preferably is a 5- or 6-membered ring.

If Ar¹ is not attached to a further 5- or 6-membered mononuclear aromatic ring via a chemical bond or not fused to a further mononuclear or binuclear aromatic ring system, Ar¹ is preferably a 6-membered alicyclic aromatic ring (e.g. benzene) or a 6-membered heterocyclic ring having a N-atom (e.g. pyridine, pyrimidine, pyridazine). In this embodiment, Ar¹ is preferably benzene or pyridine, with pyridine being more preferred.

In one embodiment, Ar¹ is attached to a further 5- or 6-membered mononuclear aromatic ring via a chemical bond. The bond of Ar¹ to the further 5- or 6-membered aromatic ring can be a single bond or a double bond. Preferably, the bond is a single bond. The further 5- or 6- membered mononuclear aromatic ring can be alicylic or heterocyclic, i.e. it can contain one or more heteroatoms selected from N; O; and S.

If Ar¹ is attached to a 5- or 6-membered mononuclear aromatic ring via a chemical bond, Ar¹ is preferably thiazole, oxazole, isothiazole, isoxazole or triazole, with triazole and thiazole being more preferred. The 5- or 6-membered ring attached to Ar¹ is preferably benzene or pyridine. The pyridine or benzene ring may be unsubstituted or be substituted by at least one entity from the group -OH, methyl, ethyl, ethoxy, monofluoromethyl, difluoromethyl, trifluoromethyl, methoxy, monofluoromethoxy, difluoromethoxy, trifluoromethoxy. Preferred units for Ar¹ attached to a 5- or 6-membered mononuclear aromatic ring are 4- triazolylbenzene, 5-triazolylbenzene, 2-thiazolylbenzene, 4-thiazolylbenzene, 5- thiazolylbenzene, 2-oxazolylbenzene, 4-oxazolylbenzene, 5-oxazolylbenzene, 3- isothiazolylbenzene, 4-isothiazolylbenzene, 5-isothiazolylbenzene, 3-isoxazolylbenzene, 4- isoxazolylbenzene, 5-isoxazolylbenzene, 3-isothiazolylbenzene, 4-isothiazolylbenzene, 5- isothiazolylbenzene. Further preferred units for Ar¹ attached to a 5- or 6-membered mononuclear aromatic ring are triazolylpyridine, thiazolylpyridine, oxazolylpyridine, isoxazolylpyridine, isothiazolylpyridine, where the pyridine unit may be attached at different positions of Ar¹ and the pyridine nitrogen atom may be located at different positions. More preferred units for Ar¹ attached to a 5- or 6-membered mononuclear aromatic ring are 4- triazolylbenzene, 4-thiazolylbenzene, 2-(4-thiazolyl)pyridine and 3-(4-thiazolyl)pyridine, with 4-triazolylbenzene being most preferred.

In the present application, the term "partially saturated aromatic ring" designates a 5, 6- or 7- membered aromatic ring of which one or more double bonds are replaced by single bonds. It is understood that the ring is not entirely saturated, i. e. at least two sp² atoms remain. One or more atoms of the partially saturated ring system may be substituted by =0.

In another embodiment, Ar¹ is fused to a mononuclear or binuclear aromatic ring system as part of a multinuclear aromatic ring system ring system or a partially saturated multinuclear ring system. A multinuclear ring system is a binuclear or trinuclear ring system, preferably a binuclear ring system.

In the present application, the term "partially saturated multinuclear ring system" designates a 5, 6- or 7-membered aromatic ring being fused to another mono- or binuclear aromatic ring and of which one or more double bonds are replaced by single bonds. It is understood that the ring is not entirely saturated, i. e. at least two sp² atoms remain. As the aromatic ring system to which said ring is fused is unsaturated, at least those atoms of the partially saturated ring of Ar¹ that are also part of the additional ring system are sp² atoms. One or more atoms of the partially saturated ring system may be substituted by =0.

If Ar¹ is fused to a further mononuclear or binuclear aromatic ring system, Ar¹ is preferably imidazole, triazole, oxazole, thiazole, isoxazole, isothiazole, pyrazole, pyrrole, imidazoline, oxazoline, tetrahydropyrazine or tetrahydro[lH]-l,4-oxazepine, with imidazole, imidazoline and pyrazole being more preferred, and imidazole and imidazoline being most preferred. The further aromatic ring system is, preferably, mononuclear. The further aromatic ring system is preferably benzene, thiophene, pyridine, pyrimidine, pyridazine, furan, thiazole or oxazole, with benzene, thiophene and pyridine being more preferred, and benzene being most preferred. The preferred, more preferred and most preferred further aromatic ring system can be unsubstituted or be substituted by at least one entity from the group -OH, methyl, ethyl, ethoxy, monofluoromethyl, difluoromethyl, trifluoromethyl, methoxy, monofluoromethoxy, difluoromethoxy, trifluoromethoxy. Units formed from Ar¹ fused to a further aromatic ring system are binuclear or trinuclear ring systems, preferably binuclear ring systems.

Preferred units are benzimidazole, 2-methylbenzimidazole, 2-ethylbenzimidazole, 2- methoxybenzimidazole, benzotriazole, 2,3-dihydro[lH]benzimidazol-2-one, indazole, 1,2,3,4- tetrahydroquinoxalin-2-3-one, 2,3,4,5-tetrahydrobenzo[f][l,4]oxazepin-5-one, benzothiazole, and benzoxazole, with benzimidazole, 2-methylbenzimidazole, 2-ethylbenzimidazole, 2- methoxybenzimidazole, and 2,3-dihydro[lH]benzimidazol-2-one being more preferred and benzimidazole and 2-methylbenzimidazole being most preferred.

Ar¹ is directly connected to Am via a chemical bond present on the said 5-, 6- or 7-membered aromatic ring or the partially saturated aromatic ring constituting Ar¹, or indirectly via a chemical bond which is either present at the further 5- or 6-membered aromatic ring attached to Ar¹, or on the further aromatic ring fused to Ar¹. The direct connection of Ar¹ to Am can take place in all 3 alternatives described beforehand, i.e. when Ar¹ is attached to a further 5- or 6-membered mononuclear aromatic ring, or when Ar¹ is fused to a further 5- or 6- membered ring as part of a multinuclear ring system, or when none of these two alteratives applies.

In a further embodiment of the invention, Ar¹ is not further substituted. The term "not further substituted" denotes that besides the chemical bond to Sp or L and the one or more chemical bond(s) optionally present on Ar¹ (these are selected from the optional bond to Am, the bond to the further 5- or 6-membered aromatic ring and the bonds fusing Ar¹ to the aromatic ring), only hydrogen atoms are present on Ar¹ besides the above-specified bonded species.

In another embodiment, Ar¹ is attached to at least one substituent as specified below. The at least one substituent can be present in all alternatives described beforehand, i.e. when Ar¹ is directly connected to Am, or when Ar¹ is attached to a further 5- or 6-membered mononuclear aromatic ring, or when Ar¹ is fused to a further 5- or 6-membered ring as part of a multinuclear ring system, or when none of these alteratives applies (in which case Ar¹ must be directly connected to Am).

The at least one substituent is selected from Ci to C₄ alkyl; C₃ and C₄ cycloalkyl; C₂ to C₄ alkenyl; C₂ to C₄ alkynyl; a halogen; Ci to C₄ haloalkyl; hydroxyl-substituted Ci to C₄ alkyl; Ci to C₄ alkoxy; hydroxyl-substituted Ci to C₄ alkoxy; halogen-substituted Ci to C₄ alkoxy; Ci to C₄ alkylamino; Ci to C₄ alkylthio; -N0₂; =0; =S; =NH; -OH; and combinations thereof.

Ci to C₄ alkyl includes methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl. Ci to C₄ alkoxy includes methoxy, ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, sec-butyloxy, t-butyloxy. C₃ and C₄ cycloalkyl is cyclopropyl, cyclobutyl and methylcyclpropyl. Ci to C₄ haloalkyl includes: fluoro-, difluoro- and trifluoromethyl, and ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl substituted by one or more fluoro; chloro-, dichloro- and trichloromethyl; and ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec- butyl, t-butyl substituted by one or more chloro. Halogen- substituted Ci to C₄ alkoxy includes: fluoro-, difluoro- and trifluoromethoxy, and ethoxy, n-propyloxy, i-propyloxy, n- butyloxy, i-butyloxy, sec-butyloxy, t-butyloxy substituted by one or more fluoro; chloro-, dichloro- and trichloromethyl; and ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, sec-butyloxy, t-butyloxy substituted by one or more chloro.

Preferred substituents are methyl, ethyl, propyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy; cyclopropyl, hydroxymethyl, -N0₂, =0, with methyl, ethyl, methoxy and =0 being most preferred.

The bond of Ar¹ to the at least one substituent can be a single bond or a double bond.

Ar¹ or the unit resulting from attachment of a further 5- or 6-membered aromatic ring to Ar¹ or the unit resulting from fusing a further 5- or 6-membered aromatic ring to Ar¹ is attached to Sp or L, as well as to Am.

Depending on the orientation of Am, attachment to Ar¹ or the unit resulting from attachment or fusing a further 5- or 6-membered aromatic ring to Ar¹ may be accomplished via the C=0 group of Am or the NR¹ group of Am. Examples for appropriate entities for Ar¹ with no further 5- or 6-membered aromatic ring attached or fused to include but are not restricted to benzene, thiophene, pyridine, pyrimidine, pyrazine or pyridazine.

In a preferred embodiment, Ar¹ is benzene. In this embodiment, the NR¹ group or the C=0 group of Am and Sp or L may be oriented ortho, meta or para towards each other, with meta or para being preferred and para being more preferred. In another preferred embodiment, Ar¹ is pyridine. In this embodiment, the NR¹ group or the C=0 group of Am and Sp or L may be oriented ortho, meta or para towards each other, with meta or para being preferred and para being more preferred, while the pyridine N atom may be located in different positions.

Examples of appropriate entities resulting from Ar¹ attached to a further 5- or 6-membered aromatic ring include but are not restricted to 4-triazolylbenzene, 5-triazolylbenzene, 2- thiazolylbenzene, 4-thiazolylbenzene, 5-thiazolylbenzene, 2-oxazolylbenzene, 4- oxazolylbenzene, 5-oxazolylbenzene, 3-isothiazolylbenzene, 4-isothiazolylbenzene, 5- isothiazolylbenzene, 3-isoxazolylbenzene, 4-isoxazolylbenzene, 5-isoxazolylbenzene, 3- isothiazolylbenzene, 4-isothiazolylbenzene, 5-isothiazolylbenzene. In these cases the NR¹ group or the C=0 group of Am is typically attached to the benzene moiety and may be located ortho, meta or para, preferably meta or para, relative to the 5-membered aromatic ring of Ar¹, while Sp or L are typically attached to the 5-membered aromatic ring of Ar¹ via a ring atom of Ar¹ which is not adjacent to the ring atom to which the benzene moiety is attached.

Further examples of appropriate entities resulting from Ar¹ attached to a further 5- or 6- membered aromatic ring include but are not restricted to triazolylpyridine, thiazolylpyridine, oxazolylpyridine, isoxazolylpyridine, iso thiazolylpyridine, where the pyridine unit may be attached at different positions of Ar¹ and the pyridine nitrogen atom may be located at different positions. In these cases the NR¹ group or the C=0 group of Am is typically attached to the pyridine moiety and may be located ortho, meta or para, preferably meta or para, relative to the 5-membered aromatic ring of Ar¹, while Sp or L are typically attached to the 5- membered aromatic ring of Ar¹ via a ring atom of Ar¹ which is not adjacent to the ring atom to which the pyridine moiety is attached.

Examples of appropriate units resulting from Ar¹ fused to a further 5- or 6-membered aromatic ring include but are not restricted to benzimidazole, benzotriazole, 2,3- dihydro[lH]benzimidazole, indazole, 1,2,3,4-tetrahydroquinoxaline, 2,3,4,5- tetrahydrobenzo[f][l,4]oxazepine, benzothiazole, benzoxazole, with benzimidazole and 2,3- dihydro[lH]benzimidazole being more preferred and benzimidazole being most preferred.

If the unit resulting from fusing Ar¹ to a further 5- or 6-membered aromatic ring is benzimidazole, L or Sp are typically attached in the 1- or 2-position, while the NR¹ group or the C=0 group of Am is typically attached in the 5- or 6-position, preferably in the 5-position. If the unit resulting from fusing Ar¹ to a further 5- or 6-membered aromatic ring is benzotriazole, L or Sp are typically attached in the 1 -position, while the NR¹ group or the C=0 group of Am is typically attached in the 5-position. If the unit resulting from fusing Ar¹ to a further 5- or 6-membered aromatic ring is 2,3-dihydro[lH]benzimidazole, L or Sp are typically attached in the 1 -position, while the NR¹ group or the C=0 group of Am is typically attached in the 5-position. If the unit resulting from fusing Ar¹ to a further 5- or 6-membered aromatic ring is indazole, L or Sp are typically attached in the 1 -position, while the NR¹ group or the C=0 group of Am is typically attached in the 3- or in the 5-position. If the unit resulting from fusing Ar¹ to a further 5- or 6-membered aromatic ring is tetrahydroquinoxaline, L or Sp are typically attached in the 1 -position, while the NR¹ group or the C=0 group of Am is typically attached in the 6- or in the 7-position, preferably in the 6- position. If the unit resulting from fusing Ar¹ to a further 5- or 6-membered aromatic ring is 2,3,4,5-tetrahydrobenzo[fJ[l,4]oxazepine, L or Sp are typically attached in the 4-position, while the NR¹ group or the C=0 group of Am is typically attached in the 7-position. If the unit resulting from fusing Ar¹ to a further 5- or 6-membered aromatic ring is benzothiazole, L or Sp are typically attached in the 2-position, while the NR¹ group or the C=0 group of Am is typically attached in the 5- or 6-position. If the unit resulting from fusing Ar¹ to a further 5- or 6-membered aromatic ring is benzoxazole, L or Sp are typically attached in the 2-position, while the NR¹ group or the C=0 group of Am is typically attached in the 5- or 6-position.

Ar² is an alicyclic or heterocyclic mononuclear aromatic ring which can have 5 or 6 members. Ar² is connected to Am via a chemical bond. In one embodiment, Ar² is connected to the N atom of Am; in another embodiment, Ar² is connected to the C atom of Am. Preferably, Ar² is connected to the N atom of Am. Further to the chemical bond to Am, Ar² can be attached to at least one further chemical entity, namely to at least one substituent or fused to a 5- or 6- membered mononuclear aromatic ring as part of a multinuclear ring system. If Ar² is a heterocyclic aromatic ring, it contains at least one heteroatom from the group N, S and O, preferably from the group N and S, even more preferably at least one N-atom and at least one S-atom, most preferably two N-atoms and one S-atom. In all these embodiments, Ar² preferably is a 5- or 6- membered ring, even more preferably a 5-membered ring.

If Ar² is not fused to a further mononuclear or binuclear aromatic ring system, in one embodiment, Ar² is a 6-membered alicyclic aromatic ring (e.g. benzene) or a 6-membered heterocyclic ring having at least one N-atom (e.g. pyridine, pyrimidine, pyridazine, pyrazine or triazine). In this embodiment, Ar² is preferably benzene, pyridine, pyrazine, pyridazine or pyrimidine, with benzene and pyridazine being more preferred. In another embodiment, Ar² is a 5-membered heterocylic aromatic ring containing at least one heteroatom from the group N, S, and O, with N and S being preferred. Appropriate examples for Ar² include but are not restricted to oxazole, isoxazole, thiazole, isothiazole, 1,2,4-oxadiazole, 1,2,4-thiadiazole, 1,3,4-oxadiazole, 1,3,4-thiadiazole, pyrrole, pyrazole, imidazole, thiophene, furan. Preferred examples for Ar² are thiazole, 1,2,4-thiadiazole and 1,3,4-thiadiazole, with 1,2,4-thiadiazole and 1,3,4-thiadiazole being most preferred.

In another embodiment, Ar² is fused to a mononuclear or binuclear aromatic ring system as part of a multinuclear ring system. A multinuclear ring system is a binuclear or trinuclear ring system, preferably a binuclear ring system;

If Ar² is fused to a further mononuclear or binuclear aromatic ring system, Ar² is preferably lH-pyrazole, thiazole, oxazole, or imidazole. If Ar² is lH-pyrazole, the second aromatic ring system is fused to Ar² via the 4- and 5 -position, while Am is attached in the 3 -position. If Ar² is thiazole, the second aromatic ring is fused to Ar² via the 4- and 5 -position, while Am is attached in the 2-position. If Ar² is oxazole, the second aromatic ring system is fused to Ar² via the 4- and 5 -position, while Am is attached in the 2-position. If Ar² is imidazole, the second aromatic ring system is attached to Ar² via the 1- and 2-position, while Am is attached in the 4-position. Alternatively, the second aromatic ring system is attached to Ar² via the 4- and 5-position, while Am is attached in the 2-position. The further aromatic ring system is, preferably, mononuclear. The further aromatic ring system is preferably benzene, thiophene, pyridine, furan, thiazole, oxazole or imidazole. More preferably, the further aromatic ring system is benzene or thiazole. If the further aromatic ring system is thiazole, it is fused to Ar² via the 2- and 3 -position. The units formed from Ar² fused to a further aromatic ring system are preferably benzoxazole (Am attached to the 2-position), benzothiazole (Am attached to the 2-position), indazole (Am attached to the 3-position) or imidazo-[2,l-b]thiazole (Am attached to the 6-position).

Instead of being fused to a second aromatic ring system, if Ar² is oxazole or thiazole, it may be fused to cyclopentene via the 4- and 5-position. The resulting units, which are also preferred units, are 5,6-dihydro-4H-cyclopentaoxazole (Am attached to the 2-position) and 5,6-dihydro-4H-cyclopentathiazole (Am attached to the 2-position), with 5,6-dihydro-4H- cyclopentathiazole being more preferred..

In a further embodiment of the invention, Ar² is not further substituted. The term "not further substituted" denotes that besides the chemical bond to Am and optionally present bonds fusing Ar² to the aromatic ring, only hydrogen atoms are present on Ar² besides the above- specified bonded species.

In another embodiment, Ar² is attached to at least one substituent as specified below. The at least one substituent can be present in all 2 alternatives described beforehand, i.e. when Ar² is fused to a further 5- or 6-membered ring as part of a multinuclear ring system, or when Ar² is not fused to a further ring. The bond of Ar² to the at least one substituent can be a single bond or a double bond. Preferably, the bond is a single bond.

Ar², via a chemical bond, can be attached to at least one substituent selected from Ci to C₆ alkyl; C₃ to C₆ cycloalkyl; C₂ to C₆ alkenyl; C₅ and C₆ cycloalkenyl; C₂ to C₆ alkynyl; a halogen; Ci to C₆ haloalkyl; hydroxyl-substituted Ci to C₆ alkyl; Ci to C₆ alkoxy; hydroxyl- substituted Ci to C₆ alkoxy; halogen-substituted Ci to C₆ alkoxy; Ci to C₆ alkylamino; Ci to C₆ alkylthio; -OH; SH; carbamoyl; Ci to C₄ alkylenedioxy; a 5- or 6-membered mononuclear aromatic ring; and combinations thereof; and wherein Ar² optionally, further to the substituents to which it may be attached via a chemical bond as cited above, is fused to a 5- or 6-membered mononuclear aromatic ring as part of a multinuclear ring system.

Ci to C₆ alkyl includes methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, pentyl and hexyl. Ci to C₆ alkoxy includes methoxy, ethoxy, n-propyloxy, i-propyloxy, n- butyloxy, i-butyloxy, sec-butyloxy, t-butyloxy, pentyloxy and hexyloxy. C₃ to C₆ cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclpropyl, dimethylcyclopropyl, trimethylcyclopropyl, ethylcyclopropyl, propylcyclopropyl, methylethylcyclopropyl, methylcyclobutyl, dimethylcyclobutyl, ethylcyclobutyl, and methylcyclopentyl. Ci to C₆ haloalkyl includes: fluoro-, difluoro- and trifluoromethyl, and ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, propyl and hexyl substituted by one or more fluoro; chloro-, dichloro- and trichloromethyl; and ethyl, n-propyl, i-propyl, n- butyl, i-butyl, sec-butyl, t-butyl, propyl and hexyl substituted by one or more chloro. Halogen- substituted Ci to C₄ alkoxy includes: fluoro-, difluoro- and trifluoromethoxy, and ethoxy, n- propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, sec-butyloxy, t-butyloxy substituted by one or more fluoro; chloro-, dichloro- and trichloromethyl; and ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, sec-butyloxy, t-butyloxy substituted by one or more chloro; Ci to C₄ alkylenedioxy include methylenedioxy; ethylenedioxy, propylenedioxy, butylenedioxy, preferably methylenedioxy, ethylenedioxy.

Preferred substituents are methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy, cyclopropyl, hydroxymethyl, t-butyl, i-butyl, sec-butyl, fluro, chloro, carbamoyl, ethylthio, methylthio, with methyl, ethyl, carbamoyl, methoxy, ethylthio, and cyclopropyl being most preferred.

In one embodiment, the substituent to which Ar² is attached via a chemical bond is a 5- or 6- membered mononuclear aromatic ring. The bond of Ar² to the 5- or 6-membered aromatic ring can be a single bond or a double bond. Preferably, the bond is a single bond. The further 5- or 6-membered mononuclear aromatic ring can be alicylic or heterocyclic, i.e. it can contain one or more heteroatoms selected from N; O; and S.

If Ar² is attached to a 5- or 6-membered mononuclear aromatic ring via a chemical bond, Ar² is preferably oxazole, isoxazole, imidazole, pyrazole, pyrrole, thiazole, isothiazole, 1 ,2,4- thiadiazole, 1 ,3,4-thiadiazole, pyridine, pyridazine or pyrimidine. More preferably, Ar² is pyrazole, thiazole, isothiazole, 1 ,2,4-thiadiazole, 1 ,3,4-thiadiazole, or pyridazine. Most preferably, Ar² is pyrazole, thiazole, 1 ,2,4-thiadiazole or 1 ,3,4-thiadiazole. If Ar² is oxazole, Am is attached to the 2-position and the further mononuclear aromatic ring is attached to the 4- or 5 -position. Alternatively, Am is attached to the 4- or 5 -position, and the further mononuclear aromatic ring is attached to the 2-position. If Ar² is isoxazole, Am is attached to the 3-position, and the further mononuclear aromatic ring is attached to the 5-position. Alternatively, Am is attached to the 5-position and the further aromatic ring is attached to the 3-position. If Ar² is imidazole, Am is attached to the 2-position and the further mononuclear aromatic ring is attached to the 4-position. Alternatively, Am is attached to the 4-position and the further mononuclear aromatic ring is attached to the 2-position. If Ar² is pyrazole, Am is attached to the 3-position and the further mononuclear aromatic ring is attached to the 5- position. Alternatively, Am is attached to the 3- or 4-position and the further mononuclear aromatic ring is attached to the 1 -position. If Ar² is pyrrole, Am is attached to the 2-position and the further mononuclear aromatic ring is attached to the 4-position. Alternatively, Am is attached to the 4-position and the further mononuclear aromatic ring is attached to the 2- position. Still alternatively, Am is attached to the 3-position and the further mononuclear aromatic ring is attached to the 1 -position. If Ar² is thiazole, Am is attached to the 2-position and the further mononuclear aromatic ring is attached to the 4- or 5-position. Alternatively, Am is attached to the 4- or 5-position, and the further mononuclear aromatic ring is attached to the 2-position. If Ar² is isothiazole, Am is attached to the 3-position, and the further mononuclear aromatic ring is attached to the 5-position. Alernatively, Am is attached to the 5- position and the further aromatic ring is attached to the 3-position. If Ar² is 1,2,4-thiadiazole, Am is attached to the 3-position and the further mononuclear aromatic ring is attached to the 5-position. Alternatively, Am is attached to the 5-position and the further mononuclear aromatic ring is attached to the 3-position. If Ar² is 1,3,4-thiadiazole, Am is attached to the 2- position and the further mononuclear aromatic ring is attached to the 5-position. If Ar² is pyridine, Am and the further mononuclear aromatic ring are located meta or para, preferably para, relative to each other, while the nitrogen atom of the pyridine core may be located in different positions. If Ar² is pyridazine, Am is attached to the 3-position and the further mononuclear aromatic ring is attached to the 6-position. Alternatively, Am is attached to the 5-position and the futher mononuclear aromatic ring is attached to the 3-position. Still alternatively, Am is attached to the 3-position and the further mononuclear aromatic ring is attached to the 5-position. If Ar² is pyrimidine, Am and the further mononuclear aromatic ring are located meta or para, preferably para, relative to each other, while the nitrogen atoms of the pyrimidine core may have different positions.

The further mononuclear 5- or 6-membered aromatic ring attached to Ar² is preferably benzene, furan, thiophene, oxazole, isoxazole, thiazole, isothiazole, or pyridine. More preferably, the further mononuclear 5- or 6-membered aromatic ring attached to Ar² is furan, oxazole, isoxazole or pyridine, with furan being most preferred. If the further mononuclear aromatic ring is furan, it is attached to Ar² via the 2- or 3-position. If the further mononuclear aromatic ring is thiophene, it is attached to Ar² via the 2- or 3-position. If the further mononuclear aromatic ring is oxazole, it is attached to Ar² via the 2-, 4- or 5-position. If the further mononuclear aromatic ring is isoxazole, it is attached to Ar² via the 3-, 4- or 5- position. If the further mononuclear aromatic ring is thiazole, it is attached to Ar² via the 2-, 4- or 5-position. If the further mononuclear aromatic ring is isothiazole, it is attached to Ar² via the 3-, 4- or 5-position. If the further mononuclear aromatic ring is pyridine, it is attached to Ar² via the 2-, 3- or 4-position.

Preferred units for Ar² attached to a 5- or 6-membered mononuclear aromatic ring are 1- phenylpyrazole (attachment to Am via the 3-position), 2-(2-furyl)pyrazole (attachment to Am via the 5-position), 4-phenylthiazole (attachment to Am via the 2-position), 5-phenylthiazole (attachment to Am via the 2- position), 2-(2-furyl)-l,3,4-thiadiazole (attachment to Am via the 5-position), 2-(3-furyl)-l,3,4-thiadiazole (attachment to Am via the 5-position), 2-(2- pyridyl)-l,3,4-thiadiazole (attachment to Am via the 5-position), 2-(3-pyridyl)-l,3,4- thiadiazole (attachment to Am via the 5-position), 2-(4-pyridyl)-l,3,4-thiadiazole (attachment to Am via the 5-position), 3-(2-furyl)-l,2,4-thiadiazole (attachment to Am via the 5-position), 4-(2-furyl)thiazole (attachment to Am via the 2-position), 5-(2-furyl)thiazol (attachment to Am via the 2-position), 2-phenyl-l,3,4-thiadiazole (attachment to Am via the 5-position), 3- phenyl-l,2,4-thiadiazole (attachment to Am via the 5-position), 4-(2-pyridyl)thiazole (attachment to Am via the 2-position), 4-(3-pyridyl)thiazole (attachment to Am via the 2- position), 4-(4-pyridyl)thiazole (attachment to Am via the 2-position) 5-(2-pyridyl)thiazole (attachment to Am via the 2-position), 5-(3-pyridyl)thiazole (attachment to Am via the 2- position), 5-(4-pyridyl)thiazole (attachment to Am via the 2-position), 3-(2-furyl)pyridazine (attachment to Am via the 6-position), 2-(5-isoxazolyl)-l,3,4-thiadiazole (attachment to Am via the 5-position), 2-(5-oxazolyl)-l,3,4-thiadiazole (attachment to Am via the 5-position), 2- (2-oxazolyl)-l,3,4-thiadiazole (attachment to Am via the 5-position).

More preferred units for Ar² attached to a 5- or 6-membered mononuclear aromatic ring are 2- (2-furyl)-l,3,4-thiadiazole (attachment to Am via the 5-position), 2-(3-furyl)-l,3,4-thiadiazole (attachment to Am via the 5-position), 3-(2-furyl)-l,2,4-thiadiazole (attachment to Am via the 5-position), 4-(2-furyl)thiazole (attachment to Am via the 2-position), 5-(2-furyl)thiazole (attachment to Am via the 2-position), 4-(2-pyridyl)thiazole (attachment to Am via the 2- position), 4-(3-pyridyl)thiazole (attachment to Am via the 2-position), 4-(4-pyridyl)thiazole (attachment to Am via the 2-position), 3-(2-furyl)pyridazine (attachment to Am via the 6- position), 2-(5-isoxazolyl)-l,3,4-thiadiazole (attachment to Am via the 5-position), 2-(5- oxazolyl)-l,3,4-thiadiazole (attachment to Am via the 5-position), 2-(2 -oxazolyl)- 1,3,4- thiadiazole (attachment to Am via the 5-position). Most preferred units for Ar² attached to a 5- or 6-membered mononuclear aromatic ring are 2-(2-furyl)-l,3,4-thiadiazole (attachment to Am via the 5-position), 3-(2-furyl)-l,2,4-thiadiazole (attachment to Am via the 5-position), 4- (2-pyridyl)thiazole (attachment to Am via the 2-position), 4-(3-pyridyl)thiazole (attachment to Am via the 2-position), 4-(4-pyridyl)thiazole (attachment to Am via the 2-position).

The unit resulting from Ar² being attached to a further 5- or 6-membered mononuclear aromatic ring may be, in addition to the attachment to Am, be attached to one or more substituents selected from: methyl, ethyl, methoxy, methylenedioxy (in this case, the substituent is attached to two atoms of the unit resulting from Ar² being attached to a further 5- or 6-membered mononuclear aromatic ring), ethoxy, difluoromethyl, trifluoromethyl, difluoromethoxy, trifluoromethoxy, -F, -CI, -Br. Preferred substituents are methyl and methylenedio xy .

Ar² is either attached to a C=0 group (see formula la) or to the NR¹ group (see formula lb)

All the definitions cited beforehand in respect to various meanings of the variables L, Sp, Ar¹, Am, R¹ and Ar² depicted in formula (I) refer to embodiments of the present invention and include general embodiments, preferred embodiments, more preferred embodiments and even more preferred embodiments of each variable, independent of the fact if any one or all of the other variables besides the one under consideration refers to its general, preferred, more preferred or even more preferred embodiment. In consequence, a general embodiment of one variable can be combined with a preferred, more preferred, and even more preferred embodiment of any of the other variables.

There will now follow definitions of preferred definitions for the general formula (I). In all the embodiments as defined below (i.e. preferred, more preferred and even more preferred), R¹ is hydrogen or methyl; and most preferably hydrogen.

In a preferred embodiment of the present invention, the variables of formula (I) have the following meanings:

Ar¹ is a 5- or 6-membered mononuclear aromatic ring or partially saturated aromatic ring containing 1, 2 or 3 N atoms or benzene; wherein the said mononuclear ring or benzene is connected to Sp or L via a chemical bond, optionally furthermore

(a) attached to benzene or thiophene via a chemical bond; or

(b) fused to benzene or thiophene as part of a multinuclear ring system;

wherein Ar¹ is directly connected to Am via a chemical bond present on the said aromatic ring or benzene constituting Ar¹, or indirectly via a chemical bond which is either present at the benzene ring or thiophene ring attached to Ar¹, or on the benzene ring or thiophene ring fused to Ar¹;

and wherein Ar¹ is either not further substituted or attached to at least one substituent selected from: methyl, ethyl, propyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy; cyclopropyl, hydroxymethyl, -N0₂, =0, =S; and combinations thereof; and/or

Ar² is a 5-membered mononuclear aromatic ring which is unsubstituted, or via a chemical bond attached to at least one substituent selected from: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy, cyclopropyl, hydroxymethyl, t-butyl, i-butyl, sec-butyl, fluro, chloro, carbamoyl, ethylthio, methylthio, a 5- or 6-membered mononuclear aromatic ring; and combinations thereof; and wherein Ar² optionally, further to the substituents to which it may be attached via a chemical bond as cited above, is fused to benzene or thiazole as part of a multinuclear ring system.

In an even more preferred embodiment of the present invention, the variables of formula (I) have the following meanings:

Ar¹ is a 5- or 6-membered mononuclear aromatic ring or partially saturated aromatic ring selected from: imidazole, benzene, pyridine, 2,3-dihydro-lH-imidazole, and triazole, which is connected to Sp or L via a chemical bond and which is optionally furthermore

(a) attached to benzene via a chemical bond; or (b) fused to benzene as part of a multinuclear ring system

wherein Ar¹ is directly connected to Am via a chemical bond present on the 5- or 6-membered aromatic ring or partially saturated aromatic ring constituting Ar¹, or indirectly via a chemical bond which is either present at the benzene ring attached to Ar¹, or on the benzene ring fused to Ar¹;

Ar² is a 5-membered mononuclear aromatic ring containing at least one atom selected from N, S, O, preferably from N, S, which is unsubstituted, or via a chemical bond attached to at least one substituent selected from: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy, cyclopropyl, hydroxymethyl, t-butyl, i-butyl, sec-butyl, fluro, chloro, carbamoyl, ethylthio, methylthio, preferably methyl, ethyl, methoxy, ethylthio, and cyclopropyl; a 5- or 6-membered mononuclear aromatic ring; and combinations thereof; and wherein Ar² optionally, further to the substituents to which it may be attached via a chemical bond as cited above, is fused to benzene or thiazole as part of a multinuclear ring system.

In an even more preferred embodiment of the present invention, the variables of formula (I) have the following meanings: Ar¹ is selected from benzimidazole, 2,3-dihydrobenzimidazole, pyridine and 4-triazolylbenzene;

wherein Ar¹ is connected to Sp or L via a chemical bond attached to the 1- or 2-position of benzimidazole, the 1 -position of 2,3-dihydrobenzimidazole, the 2-position of pyridine or to the 1 -position of the triazole part of 4-triazolylbenzene;

wherein Ar¹ is connected to Am via a chemical bond attached to the 5- or 6-position of benzimidazole, the 5- or 6-position of 2,3-dihydrobenzimidazole, to the 5-position of pyridine or to the 4-position of the benzene part of 4-triazolylbenzene;

and wherein Ar¹ is either not further substituted or attached to at least one substituent selected from: methyl, ethyl, methoxy =S and =0; and combinations thereof; and/or

Ar² is a 5-membered mononuclear aromatic ring selected from 1,2,4-thiadiazole, 1,3,4- thiadiazole, and thiazole; which is unsubstituted, or via a chemical bond attached to at least one substituent selected from: methyl, ethyl, methoxy, ethylthio, and cyclopropyl; a 5- or 6- membered mononuclear aromatic ring selected from pyridine, furan, isoxazole, oxazole, isothiazole, thiazole; and combinations thereof.

The ligands used in the present invention (which, after being attached to the support material) form together with the support material the ligand-substituted matrix which is used in the method of the present invention. The ligands are in accordance with the following formulae wherein the symbols Sp, Ar¹, Ar² and Am have the meanings defined above, including the preferred meanings:

(SpP)_v-Ar¹-Am-Ar² (II)

(SpP)_v-Ar¹-N(R¹)-C(0)-Ar² (Ila)

(SpP)_v-Ar¹-C(0)-N(R¹)-Ar² (lib)

The ligands according to formulae (II), (Ila) and (lib) comprise the spacer precursor group SpP attached to the ligand.

The ligands according to formulae (II), (Ila) and (lib) can serve as precursors for the synthesis of further compounds not having the spacer group attached. The named compounds are generated after cleavage of the spacer group and, as the case may be, conversion of the functionality present in the ligands which is connected to SpP, to any other appropriate functionality known to the person skilled in the art. Appropriate reactions to that end are known to person skilled in the art. The resulting compounds are also included in the present invention.

Preferred ligands of the matrix according to the present invention are depicted below wherein in each formula the very left N atom is connected to the linking point L which is not shown. All formulae below show the spacer precursor group (SpP) attached to the ligands:

1 -(3 -Amino- 1 -propyl)-5 - [( 1 -methylindazol-3 -yl)carboxamido]benzimidazo le (LO 1 )

l-(3-Amino-l-propyl)benzimidazole-5-carboxylic acid N-(l-methyl-3-indazolyl) amide (L02)

4-(3-Amino-l-propyl)-7-[(l-methylindazol-3-yl)carboxamido]-2,3,4,5- tetrahydrobenzo[f][l,4]oxazepin-5-one (L03)

l-(3-Amino-l-propyl)indazole-3-carboxylic acid N-(3-carbamoyl-4-methoxyphenyl) amide (L04)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-[5-(2-furyl)- 1 ,3,4-thiadiazol-2-yl] amide (L05)

1 -(3-Amino- l-propyl)benzotriazole-5-carboxylic acid N-(l -methylindazo 1-3 -yl) amide (L06)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-(2-ethyl- 1 ,3,4-thiadiazol-5-yl) amide (L07)

1 -(3-Amino- l-propyl)benzimidazole-5-carboxylic acid N-(benzoxazol-2-yl) amide (L08)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-[2-(3,4-methylenedioxyphenyl)- l,3,4-thiadiazol-5-yl] amide (L09)

1 -(3-Amino- l-propyl)benzimidazole-5-carboxylic acid N-(benzothiazol-2-yl) amide (LIO)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-(5-methyl- 1 -phenylpyrazol-3-yl) amide (LI 1)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-(2-ethylthio- 1 ,3,4-thiadiazol-5-yl) amide (LI 2)

1 -(3-Amino- l-propyl)benzimidazole-5-carboxylic acid N-(l,3,4-thiadiazol-2-yl) amide (L13)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-(2-methyl- 1 ,3,4-thiadiazol-5-yl) amide (LI 4)

l-(3-Amino-l-propyl)benzimidazole-5-carboxylic acid N-(4-methylthiazol-2-yl) amide (L15)

l-(3-Amino-l-propyl)benzimidazole-5-carboxylic acid N-(thiazol-2-yl) amide (L16)

l-(3-Amino-l-propyl)benzimidazole-5-carboxylic acid N-(5-methylthiazol-2-yl) amide (L17)

4-[(3-Amino-l-propyl)amino]-3-nitrobenzoic acid N-[5-(2-furyl)-l,3,4-thiadiazol-2-yl]amide (L18)

6-[(3-Amino-l-propyl)amino]nicotinic acid N-[5-(2-furyl)-l,3,4-thiadiazol-2-yl]amide (L19)

4-[(l-Amino-3-propyl)oxy]benzoic acid N-[5-(2-furyl)-l,3,4-thiadiazol-2-yl] amide (L20)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-[5-(4-pyridyl)- 1 ,3,4-thiadiazol-2-yl] amide (L21)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-(3-phenyl- 1 ,2,4-thiadiazol-5-yl) amide (L22)

1 -(3-Amino- l-propyl)benzimidazole-5-carboxylic acid N-[4-(2-pyridyl)thiazol-2-yl] amide (L23)

1 -(3-Amino- 1 -propyl)-2-oxo-2,3-dihydrobenzimidazole-5-carboxylic acid N-[5-(2-fiiryl)- l,3,4-thiadiazol-2-yl] amide (L24)

1 -(8-Amino-3,5-dioxa- 1 -octyl)benzimidazole-5-carboxylic acid N-[5-(2-furyl)-l,3,4- thiadiazol-2-yl] amide (L25)

1- (8-Amino-3,5-dioxa-l-octyl)benzimidazole-5-carboxylic acid N-(5-methyl-l,3,4-thiadiazol-

2- yl) amide (L26)

l-(8-Amino-3,5-dioxa-l-octyl)benzimidazole-5-carboxylic acid N-(l,3,4-thiadiazol-2-yl) amide (L27)

1 -(3-Amino- 1 -propyl)-2,3-dioxo- 1 ,2,3,4-tetrahydroquinoxaline-6-carboxylic acid N-[5-(2- furyl)-l,3,4-thiadiazol-2-yl]amide (L28)

1 -(8-Amino-3,5-dioxa- 1 -octyl)benzimidazole-5-carboxylic acid N-(4-methylthiazol-2-yl) amide (L29)

1 -(3-Amino- l-propyl)-5-[(imidazo-[2,l-b]thiazol-6-yl)carboxamido]benzimidazole (L30)

2-(5-Amino- 1 -pentyl)- 1 -methylbenzimidazole-5-carboxyl

thiadiazol-2-yl]amide (L31)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-(5,6-dihydro cyclopentathiazol-2-yl) amide (L32)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-(2 -phenyl- 1 ,3,4-thiadiazol-5-yl) amide (L33)

1 -(3-Amino- l-propyl)benzimidazole-5-carboxylic acid N-(6-methylpyridazin-3-yl) amide

(L34)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-(2-cyclopropyl- 1 ,3,4-thiadiazol-5- yl) amide (L35)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-(3-methyl- 1 ,2,4-thiadiazol-5-yl) amide (L36)

4-(5-Amino-3-oxa-l-pentyloxy)benzoic acid N-[5-(2-furyl)-l,3,4-thiadiazol-2-yl] amide (L37) 4-[(l-Amino-3-propyl)oxy]benzoic acid N-[4-(2-pyridyl)thiazol-2-yl] amide (L38)

1 -(3-Amino- 1 -propyl)-2-methylbenzimidazole-5-carboxyl

thiadiazol-2-yl] amide (L39)

6-[(3-Amino-l-propyl)amino]nicotinic acid N-[4-(2-pyridyl)thiazol-2-yl]amide (L40)

l-(8-Amino-3,5-dioxa-l-octyl)benzimidazole-5-carboxylic acid N-[4-(2-pyridyl)thiazol-2-yl] amide (L41)

1 -(3-Amino- 1 -propyl)-2-methylbenzimidazole-5-carboxylic acid N-[4-(2-pyridyl)thiazol-2-yl] amide (L42)

1 -(3-Amino- 1 -propyl)-2-ethylbenzimidazole-5-carboxylic acid N-[4-(2-pyridyl)thiazol-2-yl] amide (L43)

1 -(3-Amino- 1 -propyl)-2-ethylbenzimidazole-5-carboxyl

thiadiazol-2-yl] amide (L44)

1 -(3-Amino- 1 -propyl)-2-methoxybenzimidazole-5-carboxylic acid N-[5-(2-fiiryl)- 1 ,3,4- thiadiazol-2-yl] amide (L45)

1 -(3-Amino- l-propyl)benzimidazole-5-carboxylic acid N-[4-(3-pyridyl)thiazol-2-yl] amide (L46)

1 -(3-Amino- l-propyl)benzimidazole-5-carboxylic acid N-[4-(4-pyridyl)thiazol-2-yl] amide (L47)

6-[ 1 -(3-Amino- 1 -propyl)triazol-4-yl]benzoic acid N-[5-(2-fiiryl)- 1 ,3,4-thiadiazol-2-yl]amide (L48)

1 -(8-Amino-3,5-dioxa- 1 -octyl)-2-methylbenzimidazole-5-carboxylic acid N-[5-(2-furyl)- l,3,4-thiadiazol-2-yl] amide (L49)

1 -(8-Amino-3,5-dioxa- 1 -octyl)-2-oxo-2,3-dihydrobenzimidazole-5-carboxylic acid N-[5-(2- furyl)-l,3,4-thiadiazol-2-yl] amide (L50)

1 -(3-Amino- 1 -propyl)-2-methylbenzimidazole-5-carboxylic acid N-[4-(4-pyridyl)thiazol-2-yl] amide (L51)

1 -(8-Amino-3,5-dioxa- 1 -octyl)-2-methylbenzimidazole-5-carboxyl

pyridyl)thiazol-2-yl] amide (L52)

(5)-Glutamic acid N,N'-bis {8- (5-[5-(2-furyl)- 1 ,3,4-thiadiazol-2-ylcarbamoyl]benzimidazol- 1 yl}-3,5-dioxa-l-octyl} amide (L53)

(S)-Glutamic acid N,N'-bis {8- {5-[5-(2-furyl)- 1 ,3,4-thiadiazol-2-ylcarb;

methylbenzimidazol-l-yl}-3,5-dioxa-l-octyl} amide (L54)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-[3-(2-furyl)- 1 ,2,4-thiadiazol-5-yl] amide (L55)

1 -(3-Amino- 1 -propyl)benzimidazole-5-carboxylic acid N-[5-(3-furyl)- 1 ,3,4-thiadiazol-2-yl] amide (L56)

1 -(3-Amino- l-propyl)benzimidazole-5-carboxylic acid N-[4-(2-furyl)thiazol-2-yl] amide (L57)

1 -(3-Amino- l-propyl)benzimidazole-5-carboxylic acid N-[5-(2-furyl)thiazol-2-yl] amide (L58)

1 -(3-Amino- l-propyl)benzimidazole-5-carboxylic acid N-[6-(2-furyl)pyridazin-3-yl] amide

(L59)

1 -(3-Amino- 1 -propyl)-2-methylbenzimidazole-5-carboxyl

thiadiazol-5-yl] amide (L60)

1 -(3-Amino- 1 -propyl)-2-methylbenzimidazole-5-carboxyl

thiadiazol-2-yl] amide (L61)

1 -(3-Amino- 1 -propyl)-2-methylbenzimidazole-5-carboxylic acid N-[4-(2-furyl)thiazol-2-yl] amide (L62)

1 -(3-Amino- 1 -propyl)-2-methylbenzimidazole-5-carboxylic acid N-[5-(2-furyl)thiazol-2-yl] amide (L63)

1 -(3-Amino- 1 -propyl)-2-methylbenzimidazole-5-carboxylic acid N-[6-(2-furyl)pyridazin-3- yl] amide (L64)

1 -(3-Amino- 1 -propyl)-2-ethylbenzimidazole-5-carboxyl

thiadiazol-5-yl] amide (L65)

1 -(3-Amino- 1 -propyl)-2-ethylbenzimidazole-5-carboxyl

thiadiazol-2-yl] amide (L66)

1 -(3-Amino- 1 -propyl)-2-ethylbenzimidazole-5-carboxylic acid N-[4-(2-furyl)thiazol-2-yl] amide (L67)

1 -(3-Amino- 1 -propyl)-2-ethylbenzimidazole-5-carboxylic acid N-[5-(2-furyl)thiazol-2-yl] amide (L68)

1 -(3-Amino- 1 -propyl)-2-ethylbenzimidazole-5 -carboxylic acid N-[6-(2-furyl)pyridazin-3-yl] amide (L69)

4-[2-(3-Amino-l-propylamino)thiazol-4-yl]benzoic acid N-[5-(2-furyl)-l,3,4-thiadiazol-2-yl] amide (L70)

1 -(3-Amino- 1 -propyl)-2-methylbenzimidazole-5 -carboxylic acid N-[5-(5-isoxazo thiadiazol-2-yl] amide (L71)

1 -(3-Amino- 1 -propyl)-2-methylbenzimidazole-5 -carboxylic acid N-[5-(5-oxazolyl)- 1 ,3,4- thiadiazol-2-yl] amide (L72)

1 -(3-Amino- 1 -propyl)-2-methylbenzimidazole-5 -carboxylic acid N-[5-(2-oxazolyl)- 1 ,3,4- thiadiazol-2-yl] amide (L73)

The synthesis of the ligands (II) can, for example, be carried out in a solution-phase synthesis by assembly of a carboxylic acid component and an amine component, ultimately forming the amide Am. The coupling can be accomplished by activation of the carboxylic acid component using an activating agent known to the person skilled in the art and by subsequent reaction of the activated carboxylic acid with the amine component, if necessary at elevated temperature which may be reached either by conventional heating of the reaction mixture or by microwave irradiation. In the course of this reaction, the amino group which, lateron, serves as the precursor group enabling the attachment of the ligands to the support material, needs to be protected by a suitable protecting group like tert-butoxycarbonyl. After successful coupling of the two components, the protecting group needs to be removed under suitable conditions. For the assembly of the individual components, additional solution phase synthesis steps may be applied which are known to the person skilled in the art. The deprotected ligands including the spacer group are purified by chromatographic methods also known to the person skilled in the art.

If a suitable solution-phase synthesis protocol should not be applicable to a particular ligand, the synthesis can alternatively be carried out on insoluble supports, also known as resins, e.g. polystyrene resins, preferably pre-loaded with a suitable spacer bearing a reactive group (functional group), e. g. a hydroxyl group, to which additional molecules may be attached by reactions known to the person skilled in the art. Finally, the ligands including the spacer group are released from the said insoluble support/resin by a suitable cleavage protocol known to the person skilled in the art and purified by chromatographic methods also known to the person skilled in the art.

The ligand-substituted support material/matrix of the present invention and the ligands attached thereto are the subject-matter of European Patent Application 12154471.2 as filed on February 8, 2012, of the applicant. The entire examples of this application are fully incorporated in the present application by reference.

The term "antibody" means an immunoglobulin. The term "immunoglobulin" as used in the context of the present invention refers to natural immunoglobulins and synthetic immunoglobulins, The term refers in particular to a natural immunoglobulin not being of synthetic origin and/or not containing any synthetic amino acid sequences. The antibody may be a member of any natural immunoglobulin class. These are known to the person skilled in the art and include IgA, IgE, IgG and IgM, The preferred immunoglobulin in the context of the present invention is IgG, in case of human antibodies more preferebly IgGi, IgG₂, IgG₃ and IgG₄. In a preferred embodiment of the present invention, the antibody is a human antibody. In an even more preferred embodiment of the present invention, the antibody is a member of an immunoglobulin class, preferably IgG, more preferably of human IgG in particular of human IgGi, IgG₂, IgG₃ and IgG₄.

The present invention is drawn to immunoadsorption, in particular within therapeutic plasma exchange, of antibodies, preferably of the IgG immunoglobulin class from complex mixtures, preferably plasma, more preferably human plasma, in particular human plasma from individuals suffering from a disease caused by pathological antibodies, making use of the affinity ligands of formula (I) and preferred embodiments thereof, as disclosed elsewhere in the specification. In one embodiment, these diseases are autoimmune diseases. In another embodiment, these diseases are not caused by an autoimmune reaction and are, preferably, but not limited to, transplant rejections

Accordingly, in a preferred embodiment, the present invention comprises a method for separating a protein, preferably an antibody, more preferably from the immunoglobulin classes IgA, IgE, IgG and IgM, even more preferably an IgG type immunoglobulin, in particular of human IgGi, IgG₂, IgG₃ and IgG₄, from plasma by immunoadsorption (IA). This method of the invention is used for purifying plasma, preferably plasma from a mammal, more preferably human plasma, in particular human plasma from individuals suffering from a disease caused by pathological antibodies, which may be an autoimmune disease or a disease not caused by an autoimmune reaction, which is preferably, but not limited to, transplant rejections.

The present invention also comprises the use of a ligand-substituted matrix according to formula (I) as defined hereinbefore, for separating proteins, preferably antibodies, from plasma, preferably the various plasma types cited beforehand. In this aspect, the separation of the protein occurs by immunoadsorption, preferably within therapeutic plasma exchange.

The present invention also comprises the use of a ligand-substituted matrix according to formula (I) as defined hereinbefore, for the manufacture of a medical device for separating proteins, preferably antibodies, from plasma. In a further aspect, the present invention also comprises a ligand-substituted matrix as defined hereinbefore for use in treating a disease caused by pathological antibodies. This type of diseases is known to the person skilled in the art. The person skilled in the art is also aware which diseases are caused or can be caused by pathological antibodies. In one embodiment, these diseases are autoimmune diseases. In another embodiment, these diseases are not caused by an autoimmune reaction and are, preferably, but not limited to, transplant rejections. Diseases which are preferably treated by the method of the present invention include rheumatoid arthritis, idiopathic thrombocytopenic purpura, hemophilia with inhibitors, systemic lupus erythematosus (SLE), myasthenia gravis (MG), Guillian-Barre-syndrome (GBS), idiopathic dilated cardiomyopathy (DCM), dermatomyositis, autoimmune bullous disorders, renal transplant rejection, in particular renal transplant rejection in highly sensitized (HLA allo-antibodies) renal transplant recipients, autoimmune hemolytic anemia, Sjogren's Syndrome, mixed connective tissue disease (MCTD), endocrine orbitopathy, pemphigus vulgaris.

Still, the present invention is directed to a medical device for separating proteins, preferably antibodies, from plasma, comprising a ligand-substituted matrix as defined hereinbefore.

When practicing the invention, the ligands according to the general formula (I) are attached to a support material of an appropriate support material, resulting in a ligand-substituted matrix, typically a matrix for plasmapheresis, preferably immunoadsorption (IA) (also referred to as affinity matrix in the context of the present invention) for protein separation. The ligands of the general formula are attached to the support material via L, optionally including a spacer -Sp-.

Accordingly, the present invention includes a ligand-substituted matrix (an affinity matrix) for protein separation, preferably antibody separation, more preferably IgG separation, in particular from the group IgGi, IgG₂, IgG₃ and IgG₄, from human plasma, comprising a support material and at least one ligand as specified in the specification beforehand, wherein the ligand is attached thereto via L.

The matrix - which may be denoted M - may comprise or consist of any appropriate support material which is known to the person skilled in the art. The material may be soluble or insoluble, particulate or non-particulate, or of a monolithic structure, including fibers and membranes, porous or non-porous. It provides a convenient means of separating ligands of the invention from solutes in a contacting solution. Examples of support materials include carbohydrate and crosslinked carbohydrate matrices such as agarose, Sepharose, Sephadex, cellulose, dextran, starch, alginate or carrageenan; synthetic polymer matrices such as polystyrene, styrene-divinylbenzene copolymers, polyacrylates, PEG polyacrylate copolymers, polymethacrylates, (e.g. poly(hydroxyethylmethacrylate), polyvinyl alcohol, polyamides or perfluorocarbons; inorganic matrices such as glass, silica or metal oxides; and composite materials.

The affinity matrix is prepared by providing a matrix of an appropriate support material and attaching a ligand of formula (I) thereto. Methods for attaching the ligand (I) to the support material are known to the person skilled in the art.

In the method according to the invention, a protein, preferably an antibody, more preferably an IgG type immunoglobilin, in particular from the group IgGi, IgG₂, IgG₃ and IgG₄, is contacted with the ligand-substituted matrix as described before.

The separation of the proteins, preferably the antibodies, even more preferably the antibodies cited beforehand, according to the invention involves molecular recognition of a protein by a ligand of formula (I). The ligand may be immobilized on a solid support material facilitating separation of the ligand-antibody complex later on. Separation techniques may include, but are not limited to, affinity chromatography on packed columns, monolithic structures or membranes. The term further includes adsorption in batch-mode or affinity precipitation.

Further embodiments of the methods an uses of the present invention include carrying them out in vitro, i.e. separating blood from the respective individual, optionally separating the plasma from the blood cells, and carrying out the methods and uses of the invention in a device.

Independently of the flavor, purification techniques are composed by an initial recognition phase where ligand is contacted with antibody in crude. In a second phase, either impurities are separated from the ligand-antibody complex (e.g. column chromatography) or ligand- antibody complex is separated from impurities (e.g. affinity precipitation). In a third step, the antibody may be released from the ligand-antibody complex by alteration of chemical and/or physical conditions like change in pH, ionic strength and/or addition of modifiers like organic solvents, detergents or chaotropes.

The invention will now be illustrated by the following examples, which shall not be construed to limit the invention.

Examples

Materials and Methods

If not otherwise stated, all chemicals and solvents were of analytical grade.

384-well filter plates having hydrophilic membrane filters were purchased from Pall GmbH (Dreieich/Germany). Top frits made from polyethylene with 10 μιη average pore size were provided by Porex (Bautzen/Germany). General purpose microtiterplates for collection of fractions were ordered from Greiner Bio One GmbH (Frickenhausen/Germany). Analytical assays were read out using a Fluostar Galaxy plate reader from BMG Labtech GmbH (Offenburg/ Germany) .

Analytical chromatography of ligands was conducted on a Shimadzu HPLC system (Shimadzu Deutschland GmbH, Duisburg/Germany) including a diode array detector and single-quad mass spectrometer. The monolithic CI 8 reversed phase column was purchased from Merck KGaA (Darmstadt/Germany). Solvents used in analyses were of mass spectrometry grade.

Antibodies used in the present invention were Palivizumab (IgGi) (Synagis, Abbott, USA), Cetuximab (IgGi) (Erbitux, Merck Serono GmbH, Germany), Denosumab (IgG₂) (Prolia, Amgen, USA), IgG3 from human plasma (Abeam, UK), IgG4 from human plasma (Abeam, UK) and poly-IgG from human serum (Sigma-Aldrich, USA). Furthermore, fatty acid free and globulin free serum albumin from human was used for binding studies (Sigma-Aldrich,

USA).

Coomassie brilliant blue dye reagent for Bradford assay was purchased from Thermo Scientific (Bonn/Germany).

Example 1: Alkaline Stability

A subset of the ligands of the invention 1 was tested for its alkaline stability. Ligands were treated with 0.5 M sodium hydroxide at 25 °C for 8 days. Hydrolysis was monitored by LC- MS analysis.

Ligand to.5

(h)

L01 840

L02 >999

L04 >999 L05 >999

L07 >999

L13 >999

L14 >999

L19 >999

L23 >999

L24 >999

L25 >999

L30 903

L36 >999

L39 >999

L40 782

L41 >999

L42 >999

L43 >999

L44 >999

L45 >999

L46 719

L47 >999

Most ligands showed half-lifes of more than 999 h in the presence of 0.5 M NaOH. Residual ligands showed half-lifes between 719 h and 903 h.

Example 2: Immobilization of Ligands

Ligands of the invention were immobilized on NHS-activated Sepharose 4 FF for subsequent binding experiments. Coupling was achieved by formation of an amide bond between the amino group of the spacer-precursor groups on the ligands and the NHS-activated carboxylic acid group of the pre-activated resin.

In coupling reactions, one volume of ligand dissolved at an approximate concentration of 20 to 30 mM in 90 % DMSO and 10 % N-Methyl-2-pyrrolidone containing 1 M N,N- Diisopropylethylamine was added to one volume of settled NHS-activated Sepharose 4 FF (GE Healthcare). Reactions were conducted for at least 3 h at 25 °C while shaking vigorously. The supernatant of reactions was withdrawn and resins were washed twice with appropriate solvent. Remaining active groups on resins were blocked with 1 M ethanolamine for 1 - 2 h at 25 °C. Final resins were washed and stored in 30 % ethanol at 4 °C until used in subsequent experiments. Example 3: Binding of antibody subclasses by immobilized ligands LI - L54

Experimental

Binding of different antibody subclasses by immobilized ligands was assessed in packed mode by microtiter plate chromatography. For each column, approximately 30 of resin were transferred to a 384-well filter plate and sealed with appropriate top frits. Columns with rProtein A Sepharose 4 FF was included as control.

Columns were equilibrated with 6.7 column volumes (cv) of phosphate buffered saline (0.15 M NaCl, 20 mM sodium phosphate, pH 7.3; PBS) prior to injection. 3.3 cv of whole IgGs dissolved at 0.5 mg ml^"1 in PBS were injected onto columns. Fatty acid free and globulin free albumin from human serum (HSA) served as control for specificity.

Unbound protein was washed from columns with 5 cv of PBS prior to elution with 5 cv of glycine buffer at pH 2.5. Transferred volumes were spun through columns at 50 g for 1 - 2 min. During injection of samples, speed was reduced to 10 g and centrifugation time increased to 5 - 10 min. Effluent fractions were collected in 384-well plates and concentrations of protein were analyzed by Bradford assay. Protein masses m, and m/t (see below) were calculated as the product of fraction volume and measured protein concentration.

Summary and Results

Binding and elution of several antibodies from different antibody subclasses was demonstrated. Among them were the two therapeutic antibodies Palivizumab and Cetuximab (both IgGi), the human antibody Denosumab (IgG₂), a human IgG₃ fraction, a human IgG₄ fraction and a human poly-IgG (h-poly-IgG) mixture isolated from human serum. Selectivity of ligands towards the antibodies was determined by investigation of the binding of fatty acid free and globulin free albumin from human serum (HSA). Results for commercial resin rProtein A Sepharose 4 FF (rProtein A) were included for comparison.

The fraction of bound protein was calculated as the difference between the total mass of protein injected ntj and the mass of protein detected in the flowthrough mfl, divided by the total mass of protein injected. m_i - m _ft

bound =

m_i Binding results for resins from Example 2 and reference resin are given in the table below.

Palivizumab Cetuximab Denosumab h-poly-lgG3 h-poly-lgG4 h-poly-lgG HSA

Ligand

(%) (%) (%) (%) (%) (%) (%)

L1 99 100 - - - 100 -

L2 91 100 - - - 100 -

L3 78 99 - - - - -

L4 85 100 - - - 100 -

L5 100 100 100 63 100 100 -

L6 74 - - - - - -

L7 90 100 - - - 94 -

L8 91 100 74 - - 95 -

L9 100 100 100 - - 100 -

L10 100 100 - - - 100 -

L1 1 61 98 - - - 88 -

L12 100 100 100 - - 100 -

L13 97 100 91 - - 97 -

L14 89 100 90 - - 92 -

L15 100 100 - - - 100 -

L16 87 100 71 - - 88 -

L17 88 100 82 - - 89 -

L18 100 100 100 - - 100 22

L19 99 100 - - - 98 10

L20 100 100 - - - 100 2

L21 85 100 72 - - 82 3

L22 100 100 100 - - 100 26

L23 100 100 100 18 - 100 10

L24 100 100 98 34 100 100 0

L25 100 99 100 - - 100 7

L26 63 94 - - - 63 4

L27 41 88 - - - 50 12

L28 58 89 - - - 63 9

L29 72 99 - - - 76 0

L30 93 100 - - - 94 0

L31 100 100 100 - - 100 10

L32 91 100 63 - - 91 32

L33 100 100 100 - - 100 14

L34 78 100 - - - 78 3

L35 100 100 99 - - 100 3

L36 93 99 - - - 81 0

L37 100 100 - - - 100 -

L38 95 100 - - - 99 7

L39 100 100 100 56 100 100 14

L40 89 100 - - - 98 -

L41 98 100 - - - 100 -

L42 97 100 - - - 100 -

L43 98 100 - - - 100 -

L44 100 100 - 55 - 100 -

L45 100 100 - - - 100 -

L46 98 100 - 32 - 100 - L47 98 100 - - - 100

L48 95 100 - - - 100

L49 100 100 - - - 100

L50 72 100 - - - 76

L51 94 100 - - 99 97

L52 80 99 - - - 83

L53 100 100 - - - 100

L54 100 100 - - - 100 rProtein A 100 100 100 6 99 95

Example 4: Binding of antibody subclasses by immobilized ligands L55 - L73

The binding behaviour of ligands L55-L73 is determined under the same conditions as in example 2. The following values are found:

L55 >80 100 >70 >30 >70 >80 <40

L56 >80 100 >70 >30 >70 >80 <40

L57 >80 100 >70 >30 >70 >80 <40

L58 >80 100 >70 >30 >70 >80 <40

L59 >60 >95 >70 >30 >70 >60 <30

L60 >80 100 >70 >30 >70 >80 <20

L61 >80 100 >70 >30 >70 >80 <20

L62 >80 100 >70 >30 >70 >80 <20

L63 >80 100 >70 >30 >70 >80 <20

L64 >60 >95 >50 >20 >50 >60 <20

L65 >80 100 >70 >30 >70 >80 <30

L66 >80 100 >70 >30 >70 >80 <30

L67 >80 100 >70 >30 >70 >80 <30

L68 >80 100 >70 >30 >70 >80 <30

L69 >60 >95 >50 >20 >50 >60 <30

L70 >50 >80 >50 >20 >50 >50 <40

L71 >60 >95 >50 >20 >50 >60 <20

L72 >60 >95 >50 >20 >50 >60 <20

L73 >60 >95 >50 >20 >50 >60 <20

Example 5: Binding of human immunoglobulins from human plasma

The IgG binding behavior of the resin- fixed ligands LI - L73 (example 2) for binding of human immunoglobulins from plasma upon chromatography on resins from example 2 is shown in the table below. IgG, |gG₂ lgG₃ lgG₄ Total IgG

Ligan Residu Residu Residu Residu Residu

Bound Bound d al Bound al Bound al Bound al al

(%) (%) (%) (%) (%) (%) (%) (%) (%) (%)

L1 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L2 >80 <20 >80 <20 >20 <80 >80 <20 >70 <30

L3 >70 <30 >70 <30 >10 <90 >70 <30 >60 <40

L4 >70 <30 >60 <40 >10 <90 >60 <40 >50 <50

L5 >90 <10 >90 <10 >50 <50 >90 <10 >80 <20

L6 >60 <40 >60 <40 >10 <90 >60 <40 >50 <50

L7 >80 <20 >80 <20 >20 <80 >80 <20 >70 <30

L8 >80 <20 >60 <40 >20 <80 >60 <40 >60 <40

L9 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L10 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L1 1 >50 <50 >50 <50 >10 <90 >50 <50 >40 <60

L12 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L13 >90 <10 >80 <20 >30 <70 >80 <20 >70 <30

L14 >80 <20 >80 <20 >20 <80 >80 <20 >70 <30

L15 >90 <10 >80 <20 >30 <70 >80 <20 >70 <30

L16 >80 <20 >60 <40 >20 <80 >60 <40 >60 <40

L17 >80 <20 >70 <30 >20 <80 >70 <30 >60 <40

L18 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L19 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L20 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L21 >70 <30 >60 <40 >10 <90 >60 <40 >50 <50

L22 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L23 >90 <10 >90 <10 >10 <90 >90 <10 >80 <20

L24 >90 <10 >90 <10 >20 <80 >90 <10 >80 <20

L25 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L26 >50 <50 >50 <50 >10 <90 >50 <50 >40 <60

L27 >30 <70 >30 <70 >10 <90 >30 <70 >20 <80

L28 >50 <50 >50 <50 >10 <90 >50 <50 >40 <60

L29 >60 <40 >60 <40 >10 <90 >60 <40 >50 <50

L30 >80 <20 >80 <20 >20 <80 >80 <20 >70 <70

L31 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L32 >80 <20 >50 <50 >20 <80 >50 <50 >60 <40

L33 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L34 >70 <30 >70 <30 >10 <90 >70 <30 >60 <40

L35 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L36 >80 <20 >80 <20 >20 <80 >80 <20 >70 <30

L37 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L38 >80 <20 >80 <20 >20 <80 >80 <20 >70 <30

L39 >90 <10 >90 <10 >50 <50 >90 <10 >80 <20

L40 >90 <10 >80 <20 >30 <70 >80 <20 >70 <20

L41 >90 <10 >90 <10 >30 <70 >90 <10 >80 <30

L42 >90 <10 >90 <10 >30 <70 >90 <10 >70 <30

L43 >90 <10 >80 <20 >30 <70 >80 <20 >70 <30

L44 >90 <10 >90 <10 >50 <50 >90 <10 >80 <20

L45 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L46 >90 <10 >90 <10 >20 <80 >90 <10 >80 <20

L47 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20 L48 >80 <20 >80 <20 >20 <80 >80 <20 >70 <30

L49 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L50 >60 <40 >60 <40 >10 <90 >60 <40 >50 <50

L51 >80 <20 >80 <20 >20 <80 >80 <20 >70 <30

L52 >70 <30 >70 <30 >10 <90 >70 <30 >60 <40

L53 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L54 >90 <10 >90 <10 >30 <70 >90 <10 >80 <20

L55 >70 <30 >60 <40 >30 <70 >60 <40 >60 <40

L56 >70 <30 >60 <40 >30 <70 >60 <40 >60 <40

L57 >70 <30 >60 <40 >30 <70 >60 <40 >60 <40

L58 >70 <30 >60 <40 >30 <70 >60 <40 >60 <40

L59 >50 <50 >60 <40 >30 <70 >60 <40 >40 <60

L60 >70 <30 >60 <40 >30 <70 >60 <40 >60 <40

L61 >70 <30 >60 <40 >30 <70 >60 <40 >60 <40

L62 >70 <30 >60 <40 >30 <70 >60 <40 >60 <40

L63 >70 <30 >60 <40 >30 <70 >60 <40 >60 <40

L64 >50 <50 >40 <60 >20 <80 >40 <60 >30 <70

L65 >70 <30 >60 <40 >30 <70 >60 <40 >60 <40

L66 >70 <30 >60 <40 >30 <70 >60 <40 >60 <40

L67 >70 <30 >60 <40 >30 <70 >60 <40 >60 <40

L68 >70 <30 >60 <40 >30 <70 >60 <40 >60 <40

L69 >50 <50 >40 <60 >20 <80 >40 <60 >30 <70

L70 >40 <60 >40 <60 >20 <80 >40 <60 >30 <70

L71 >50 <50 >40 <60 >20 <80 >40 <60 >30 <70

L72 >50 <50 >40 <60 >20 <80 >40 <60 >30 <70

L73 >50 <50 >40 <60 >20 <80 >40 <60 >30 <70 rProte

in A >90 <10 >90 <10 <10 >90 >90 <10 >80 <20

Claims

1. A method for separating one or more proteins from plasma by immunoadsorption IA, comprising contacting the plasma with a ligand-substituted matrix; and

removing the plasma from the ligand-substituted matrix; wherein the ligand-substituted matrix comprises a support material and at least one ligand covalently bonded to the support material, the ligand being represented by formula (I) formula (I)

L-(Sp)_v-Ar¹-Am-Ar²

(I) wherein

L is the linking point on the support material to which the ligand is attached;

Sp is a spacer group;

v is 0 or 1 ;

Am is an amide group -NR¹-C(0)-, and wherein either NR¹ is attached to Ar¹ and - C(O)- is attached to Ar², or -C(O)- is attached to Ar¹ and NR¹ is attached to Ar²; and R¹ is hydrogen or Ci to C₄ alkyl, more preferably hydrogen or methyl; and most preferably hydrogen;

(b) fused to a mononuclear or binuclear aromatic ring as part of a multinuclear ring system;

wherein Ar¹ is directly connected to Am via a chemical bond present on the said 5-, 6- or 7-membered aromatic ring or partially saturated aromatic ring constituting Ar¹, or indirectly via a chemical bond which is either present at the further 5- or 6-membered aromatic ring attached to Ar¹, or on the further 5- or 6- membered aromatic ring fused to Ar¹; and wherein Ar¹ is either not further substituted or attached to at least one substituent selected from: Ci to C₄ alkyl; C₃ and C₄ cycloalkyl; C₂ to C₄ alkenyl; C₂ to C₄ alkynyl; a halogen; Ci to C₄ haloalkyl; hydroxyl-substituted Ci to C₄ alkyl; Ci to C₄ alkoxy; hydroxyl-substituted Ci to C₄ alkoxy; halogen-substituted Ci to C₄ alkoxy; Ci to C₄ alkylamino; Ci to C₄ alkylthio; -N0₂; =0; =S; =NH; -OH; and combinations thereof; Ar² is a 5- or 6-membered mononuclear aromatic ring which is unsubstituted, or via a chemical bond attached to at least one substituent selected from: Ci to C₆ alkyl; C₃ to C₆ cycloalkyl; C₂ to C₆ alkenyl;C₅ and C₆ cycloalkenyl; C₂ to C₆ alkynyl; a halogen; Ci to C₆ haloalkyl; hydroxyl-substituted Ci to C₆ alkyl; Ci to C₆ alkoxy; hydroxyl-substituted Ci to C₆ alkoxy; halogen-substituted Ci to C₆ alkoxy; Ci to C₆ alkylamino; Ci to C₆ alkylthio; carbamoyl; Ci to C₄ alkylenedioxy, preferably methylenedioxy and ethylenedioxy; -OH; SH; a 5- or 6-membered mononuclear aromatic ring; and combinations thereof; and wherein Ar² optionally, further to the substituents to which it may be attached via a chemical bond as cited above, is fused to a 5- or 6-membered mononuclear aromatic ring as part of a multinuclear ring system;

2. The method according to claim 1, wherein

(a) attached to benzene or thiophene via a chemical bond; or

(b) fused to benzene or thiophene as part of a multinuclear ring system;

and wherein Ar¹ is either not further substituted or attached to at least one substituent selected from: methyl, ethyl, propyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy; cyclopropyl, hydroxymethyl, -N0₂, =0, =S; and combinations thereof;

3. The method according to claim 1 or 2, wherein

R¹ is hydrogen or methyl; and most preferably hydrogen

(a) attached to benzene via a chemical bond; or

(b) fused to benzene as part of a multinuclear ring system

wherein Ar¹ is directly connected to Am via a chemical bond present on the 5- or 6- membered aromatic ring or partially saturated aromatic ring constituting Ar¹, or indirectly via a chemical bond which is either present at the benzene ring attached to Ar¹, or on the benzene ring fused to Ar¹;

Ar² is a 5-membered mononuclear aromatic ring containing at least one atom selected from N, S, O, preferably from N, S, which is unsubstituted, or via a chemical bond attached to at least one substituent selected from: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy, cyclopropyl, hydroxymethyl, t-butyl, i-butyl, sec-butyl, fluoro, chloro, carbamoyl, ethylthio, methylthio, preferably methyl, ethyl, methoxy, ethylthio, and cyclopropyl; a 5- or 6-membered mononuclear aromatic ring; and combinations thereof; and wherein Ar² optionally, further to the substituents to which it may be attached via a chemical bond as cited above, is fused to benzene or thiazole as part of a multinuclear ring system.

4. The method according to any of claims 1 to 3, wherein

Ar¹ is selected from benzimidazole, 2,3-dihydrobenzimidazole, pyridine and 4- triazo lylbenzene;

wherein Ar¹ is connected to Sp or L via a chemical bond attached to the 1- or 2-position of benzimidazole, the 1 -position of 2,3-dihydrobenzimidazole, the 2-position of pyridine or to the 1 -position of the triazole part of 4-triazo lylbenzene;

wherein Ar¹ is connected to Am via a chemical bond attached to the 5- or 6-position of benzimidazole, the 5- or 6-position of 2,3-dihydrobenzimidazole, to the 5-position of pyridine or to the 4-position of the benzene part of 4-triazo lylbenzene;

and wherein Ar¹ is either not further substituted or attached to at least one substituent selected from: methyl, ethyl, methoxy =S and =0; and combinations thereof;

Ar² is a 5-membered mononuclear aromatic ring selected from 1,2,4-thiadiazole, 1,3,4- thiadiazole, and thiazole; which is unsubstituted, or via a chemical bond attached to at least one substituent selected from: methyl, ethyl, methoxy, ethylthio, and cyclopropyl; a 5- or 6-membered mononuclear aromatic ring selected from pyridine, furan, isoxazole, oxazole, isothiazole, thiazole; and combinations thereof.

5. The method according to any of claims 1 to 4 wherein the support material comprises a material selected from carbohydrates or crosslinked carbohydrates, preferably agarose, cellulose, dextran, starch, alginate and carrageenan, Sepharose, Sephadex; synthetic polymers, preferably polystyrene, styrene-divinylbenzene copolymers, polyacrylates, PEG-Polycacrylate copolymers polymethacrylates, polyvinyl alcohol, polyamides and perfluorocarbons; inorganic materials, preferably glass, silica and metal oxides; and composite materials.

6. The method according to any of claims 1 to 5 wherein the plasma is from a mammal, in particular from a human.

7. The method according to any of claims 1 to 6 wherein the protein is an antibody, preferably from the immunoglobulin class, even more preferably an IgG type immunoglobulin.

8. The method according to claim 7 wherein the IgG type protein is from the group human IgGi, IgG₂, IgG₃ and IgG₄

9. Use of a ligand-sustituted matrix according to formula (I) as defined in any of claims 1 to 5 for separating proteins, preferably antibodies, from plasma.

10. The use according to claim 9, where the separation of the protein occurs by immunoadsorption, preferably within therapeutic plasma exchange.

11. Use of a ligand-sustituted matrix according to formula (I) as defined in any of claims 1 to 5 for the manufacture of a medical device for separating proteins, preferably antibodies, from plasma.

12. A ligand- substituted matrix as defined in any of claims 1 to 5 for use in treating a disease caused by pathological antibodies, preferably an autoimmune disease or a transplant rejection, in particular rheumatoid arthritis, idiopathic thrombocytopenic purpura, hemophilia with inhibitors, systemic lupus erythematosus (SLE), myasthenia gravis (MG), Guillian-Barre-syndrome (GBS), idiopathic dilated cardiomyopathy (DCM), dermatomyositis, autoimmune bullous disorders, renal transplant rejection, in particular renal transplant rejection in highly sensitized (HLA allo-antibodies) renal transplant recipients, autoimmune hemolytic anemia, Sjogren's Syndrome, mixed connective tissue disease (MCTD), endocrine orbitopathy, pemphigus vulgaris.

13. A medical device for separating proteins, preferably antibodies, from plasma, comprising a ligand-substituted matrix as defined in any of claims 1 to 5

14. A ligand-substituted matrix a claimed in claim 9, wherein the matrix furthermore comprises at least one antibody bound to the ligand.