HK1185356B

HK1185356B - Ligands for antibody and fc-fusion protein purification by affinity chromatography

Info

Publication number: HK1185356B
Application number: HK13112735.0A
Authority: HK
Inventors: Holger Bittermann; Klaus Burkert; Marc Arnold; Oliver Keil; Thomas Neumann; Inge Ott; Kristina Schmidt; Daniel Schwizer; Renate Sekul
Original assignee: Graffinity Pharmaceuticals Gmbh
Priority date: 2010-08-03
Filing date: 2011-08-03
Publication date: 2017-07-14

Description

ligands for antibody and Fc-fusion protein purification by affinity chromatography

the present invention relates to the field of protein isolation, preferably to the purification of monoclonal and polyclonal antibodies and fusion proteins containing an immunoglobulin Fc fragment by affinity separation techniques, in particular chromatography using small molecule ligands.

Immunoglobulins are a class of soluble proteins found in the body fluids of humans and other vertebrates. They are also called "antibodies" and play a key role in the recognition, binding and adhesion processes of cells. Antibodies are oligomeric glycoproteins that have a critical role in the immune system by recognizing and clearing antigens, typically bacteria and viruses.

The polymeric chains of antibodies are constructed in such a way that they comprise so-called heavy and light chains. The basic immunoglobulin unit consists of two identical heavy chains and two identical light chains linked by disulfide bonds. There are 5 types of heavy chains (α, γ, μ) which define the immunoglobulin class (IgA, IgG, IgD, IgE, IgM). The light chain group includes two subtypes, λ and κ.

IgG is a soluble antibody, which can be found in blood and other body fluids. It is formed by B-cell derived plasma cells in response to and to neutralize bacteria or other pathogens. IgG is a Y-shaped glycoprotein with a molecular weight of about 150kDa, consisting of two heavy chains and two light chains. Each chain is distinguished into a constant region and a variable region. The two carboxy-terminal domains of the heavy chain form an Fc fragment ("constant fragment"), and the amino-terminal domains of the heavy and light chains recognize an antigen and are referred to as Fab fragments ("antigen-binding fragments").

Fc fusion proteins are formed by the binding of an antibody Fc fragment to a protein or protein domain that can provide specificity for a given drug target. Examples are domain antibody-Fc fusion proteins, in which the two heavy chains of the Fc segment are linked to the heavy chain variable domain (V) of a specific antibody_H) Or a light chain variable domain (V)_L) And (4) connecting. Other Fc fusion proteins are conjugates of an Fc fragment with any type of therapeutic protein or protein domain. The Fc portion is thought to increase the stability and deliverability (deliverability) of protein drugs.

Therapeutic antibodies and Fc fusion proteins are useful in the treatment of a variety of diseases, the primary examples include rheumatoid arthritis, psoriasis, multiple sclerosis, and many forms of cancer. The therapeutic antibody may be a monoclonal antibody or a polyclonal antibody. Monoclonal antibodies, derived from a cell line producing a single antibody, show the same specificity for a single antigen. Possible treatments for cancer involve antibodies that neutralize tumor cell specific antigens. Bevacizumab (Bevacizumab) (Avastin, Genentech) is a monoclonal antibody that neutralizes Vascular Endothelial Growth Factor (VEGF), thereby preventing the growth of new blood vessels into tumor tissue.

Therapeutic fusion proteins such as Etanercept (Enbrel, Amgen, TNF receptor domain linked to Fc segment) or Alefacept (Amevive, BiogenIdec, LFA-3 linked to the Fc portion of human IgG 1) are used or developed as drugs against autoimmune diseases.

Biological separation of proteins, meaning the recovery and purification of protein products from a variety of biological feed streams, is an important unit operation in the food, pharmaceutical and biotechnology industries. More and more therapeutic monoclonal antibodies (Mabs) and fusion proteins are entering the market or are currently in clinical development. Such proteins require a particularly high degree of purity, which is achieved by a well-designed multi-step purification scheme. Downstream processing and purification constitutes about 50-80% of the manufacturing cost, and therefore considerable efforts are underway to develop new or improved existing purification strategies (1).

Affinity chromatography is one of the most efficient chromatography methods for protein purification. It is based on highly specific protein-ligand interactions. The ligand is covalently immobilized on a stationary phase, which serves to capture the target protein from the feed solution. Affinity ligands can bind to their targets with high specificity and selectivity, enabling up to several thousand-fold high-yield enrichment even from complex mixtures.

In general, affinity chromatography on protein a is the first step in most Mab and Fc fusion protein purification protocols. Protein a is a cell wall-associated protein exposed to the surface of the bacterium staphylococcus aureus (staphylococcus aureus). It binds with nanomolar affinity to the constant part (Fc domain) of immunoglobulins of different species, in particular of the human subtypes IgG1, IgG2 and IgG4 (2). However, the use of protein a is limited by penetration into the product and poor stability under the harsh conditions of application in sanitary and cleaning in local regulations. The chemical stability of protein a can be improved by using genetically engineered protein a variants for Mab purification. However, the high cost of protein a resins has led to the search for suitable alternatives, particularly selected from small molecules.

MabsorbentA2P (prometics biosciences) is a small molecule ligand for purification of immunoglobulins. However, the ligand does not show adequate selectivity (3) and is not suitable for industrial chromatography.

Another approach is to use mixed mode chromatography as the primary capture step for antibody purification. The most common material is based on immobilized 2-mercaptoethylpyridine (mepyphercel, pall corporation), which can efficiently capture IgG from fermentation broth, but shows lower clearance of Host Cell Proteins (HCP) compared to protein a (4).

Synthetic affinity ligands that are stronger under stringent conditions and have comparable or even higher selectivity than protein a will provide suitable solutions for antibody and Fc fusion protein purification, which are more readily available (preferably less expensive than protein-based ligands).

Synthetic small molecule affinity ligands are of particular interest for purifying therapeutic proteins due to their generally higher chemical stability and their lower production cost. Synthetic affinity ligands that are more readily available (preferably less expensive than protein-based ligands), stronger under stringent conditions and have comparable or even higher selectivity than protein a would provide a suitable solution for antibody and Fc fusion protein purification. Depending on the target protein, such affinity ligands should preferably provide as broad a applicability as protein a, recognizing the constant Fc region of IgG-type immunoglobulins and Fc fusion proteins.

The problem underlying the present invention is to provide a matrix comprising small affinity ligands that bind to antibodies and the Fc region of Fc fusion proteins.

The problem is solved by the embodiments of the invention listed below.

The present invention relates to the use of a ligand-substituted matrix comprising a support material and at least one ligand covalently linked to said support material, said ligand being represented by formula (I)

Wherein

L is a point of attachment on the support material to the ligand;

sp is a spacer group;

v is 0 or 1;

am is an amide group-NR¹-C (O) -, and wherein NR¹And Ar¹Is linked and-C (O) -and Ar²Is linked, or-C (O) -is linked to Ar¹Linked and NR¹And Ar²Connecting; and is

R¹Is hydrogen or C₁-C₄Alkyl, preferably hydrogen or methyl; more preferably hydrogen;

Ar¹is a divalent 5 or 6 membered substituted or unsubstituted aromatic ring;

Ar²is a 5-or 6-membered heterocyclic aromatic ring, which

(a) To other 5-or 6-membered aromatic rings via single bonds; or

(b) Fused as part of a polycyclic ring system with other 5 or 6 membered aromatic rings; or

(c) With at least one substitution selected fromBase connection: c₁-C₄An alkyl group; c₂-C₄An alkenyl group; c₂-C₄An alkynyl group; halogen; c₁-C₄A haloalkyl group; hydroxy-substituted C₁-C₄An alkyl group; c₁-C₄An alkoxy group; hydroxy-substituted C₁-C₄An alkoxy group; c₁-C₄An alkylamino group; c₁-C₄An alkylthio group; and combinations thereof.

In one embodiment of the present invention, the ligand-substituted matrix according to formula (I) of the present invention has the structure of formula (Ia). In a further embodiment of the present invention, the ligand-substituted matrix according to formula (I) of the present invention has the structure of formula (Ib). In the formulae (Ia) and (Ib), L, Sp, N, R¹、Ar¹And Ar²And the integer v has the meaning as defined above for formula (I)

Ar¹May also be denoted as AR¹，Ar²May also be denoted as AR². Furthermore, S_pAnd may also be denoted as V. This is the designation used in patent application EP10008089.4, to which this application claims priority. Also, R defined in formula (I) and shown in formula (Ia) and (Ib)¹Corresponding to R4 defined and shown in the priority file.

The ligands of the invention can bind to polyclonal and monoclonal IgG of human origin, in particular human IgG₁、IgG₂And IgG₄And polyclonal and monoclonal iggs from different animal species, such as rabbit and mouse immunoglobulins.

The invention also relates to said ligand-substituted matrix and to a ligand (which is also referred to as "compound") attached to said matrix at a point of attachment L, optionally via a spacer group Sp.

In the above formula (I), L is a bonding point (linkingpoint), also referred to as a "bonding point". Suitable attachment/bond points are known to those skilled in the art.

It is to be understood that the attachment point L is attached to the support material either directly or via a spacer group. Generally, L is part of a group (moiety) generated by the reaction of an appropriate functional group on the support material with a corresponding functional group on a precursor compound of the ligand to form the ligand.

In one embodiment, L is a bond, preferably a single bond, which is directly attached to the support material, typically via an appropriate functional group on the material. In this context, the term "support material" means polymers known to the person skilled in the art and commercially available for the purposes of the present invention. The definition of "support material" is provided below. Typically, the support material comprises functional groups for linking molecules, typically those of the present invention. In the context of the present invention, a functional group on a support material is considered to be part of the support material; this also applies to the following cases: the ligands of the invention are linked to the support material via a bond (i.e. L is a bond, excluding the spacer group Sp, see below) and wherein the bond is formed directly between each ligand of the invention and a functional group on the support material.

In this context, "functional groups" refer on the one hand to suitable groups ("precursor groups") on the support material and on the other hand to suitable groups ("precursor groups") which are present in the ligands of the invention, for example on the C ═ O groups or on the spacer groups Sp. In this context "functional group" also means a suitable group ("precursor group") on the spacer group Sp. As used herein, a "precursor group" or "functional group" is a group that is capable of chemically reacting with a complementary "precursor group" or "functional group" under the formation of a chemical bond. Other reagents may be used to form the chemical bond, if necessary. As is clear from the foregoing, during the reaction, the functional group/precursor group is converted to a chemical bond under conversion of the initial functionality to a "final functionality" or "linking unit". As is clear from the foregoing, the functional group/precursor group is also or may be a "complementary group".

Examples of functional groups on the support material are known to those skilled in the art and include, but are not limited to, -OH, -SH, -NH₂、＞NH-COOH、-SO₃H、-CHO、-NHNH₂、-P(＝O)(OH)₂、-O-PH(＝O)(OH)、-P(OH)₂、-O-P(＝O)(OH)₂Esters, ethers, thioethers, epoxides, natural or unnatural amino acids, carboxylic acid amides, maleimides, ketones, sulfoxides, sulfones, ureas, isoureas, imidocarbonates, sulfonamides, sulfonyl hydrazides, sulfonates, phosphates, phosphonates, phosphoramides, phosphonamides, alkynes, oxime ethers, oximes, azides, alkoxyamines, semicarbazides, sulfonyl halides, phosphonyl halides, phosphoryl halides, carboxylic acid activated esters, sulfonic acid activated esters, phosphonic acid activated esters, phosphoric acid activated esters, phosphoramides, cyanates, thiocyanates, isocyanates, isothiocyanates, maleimides, vinylsulfonyls, Cl, Br, I, alkenes, alkylphosphonium.

Examples of functional groups attached to the-C ═ O group on the ligands of the present invention are known to those skilled in the art and include, but are not limited to, hydroxyl, mercapto, F, Cl, Br, ester, ether, thioether, carboxylic acid, epoxide, amine, amide, amino acid, carboxylic acid amide, maleimide, aldehyde, ketone, sulfonic acid, sulfoxide, sulfone, urea, isourea, imidocarbonate, sulfonamide, sulfonate, phosphate, phosphonate, phosphoramide, phosphonamide, hydrazine, oxime, azide, alkene, alkyne, hydroxylamine, thiourea, isocyanate, isothiocyanate, N-hydroxysuccinimide, 1-hydroxybenzotriazole, 7-aza-1-hydroxybenzotriazole, 6-chloro-1-hydroxybenzotriazole.

In this embodiment of the invention, the attachment point L is comprised of a bond or chemical unit resulting from the reaction of an appropriate functional group on the support material with an appropriate functional group (or "precursor group") on the-C ═ O group on the ligand of the invention, through which the ligand of the invention is attached to the support material/the matrix. Thus, a chemical reaction between a functional group on the support material and a functional group (or "complementary group") on the-C ═ O group on the ligand of the present invention, links the ligand of the present invention to the support material via the attachment point L.

In yet another embodiment of the invention, L is attached to a spacer group-Sp-. In this embodiment, L is a bond or chemical unit resulting from the reaction of an appropriate functional group on the support material with an appropriate functional group (or "precursor group") on the spacer group Sp. Thus, a chemical reaction between the functional group on the support material and the functional group (or "complementary group") on the spacer group Sp links the ligand of the invention to the support material via the attachment point L.

If v is 0 in formula (I), the ligand is directly bonded to L. In other embodiments, the ligand is bonded to the support material via a spacer group S. This applies when v is not 0 in formula (I).

The spacer group Sp is preferably a hydrocarbyl group which may contain other atoms in addition to the C and H atoms. Suitable other atoms are known to those skilled in the art. Preferred atoms include O, S, N, P, Si. The hydrocarbyl group may be linear, branched or cyclic. Sp is attached to the-C ═ O group of the ligands of the invention, and Sp is typically found alpha to the-C ═ O group. In addition, Sp contains functional groups (precursor groups) by which the ligands of the invention can be covalently linked to the matrix in a chemical reaction under the formation of the final functionality, which is also referred to as linking unit.

The spacer group Sp preferably contains a linking unit/final functionality that covalently links the ligand to the support material via a linking point L. Suitable linking units/final functionalities are known to those skilled in the art. The linking unit is preferably derived from a suitable precursor group, including-NH₂、＞NH、-SH、-COOH、-SO₃H、-CHO、-OH、-NHNH₂、-P(＝O)(OH)₂、-O-PH(＝O)(OH)、-P(OH)₂、-O-P(＝O)(OH)-NHNH₂Natural or unnatural amino acids, carboxylic acid amides, ethers, esters, thioethers, epoxides, maleimides, ketones, sulfoxides, sulfonesUrea, isourea, iminocarbonate, sulfonamide, sulfonylhydrazide, sulfonate ester, phosphate ester, phosphonate ester, phosphoramide, phosphonamide, hydrazine, oxime, azide, alkoxyamine, semicarbazide, sulfonyl halide, phosphonyl halide, phosphoryl halide, carboxylic acid activated ester, sulfonic acid activated ester, phosphonic acid activated ester, phosphoric acid activated ester, phosphoramidite, cyanate ester, thiocyanate ester, alkyl halide, alkyl sulfonate ester, isocyanate, isothiocyanate, vinyl sulfonyl, carboxylic acid halide, alkene and alkyne groups. The linking unit is typically attached to a carbon chain into which one or more heteroatoms, such as oxygen atoms or carboxamide groups, may be inserted. In one embodiment, the linking unit is attached to an alkene or alkene-polyoxyalkylene group-e.g. -CH₂-CH₂-(O-CH₂-CH₂) x-, x ranges from 1 to 10, preferably 1 to 6-linked.

The functional group on the spacer group is typically attached to a hydrocarbyl group, which may contain one or more heteroatoms, such as N, O, P, S, Si. Thus, the spacer group is a hydrocarbyl group containing one or more heteroatoms preferably selected from N, O, P, S, Si. Preferably, the atom attached to the C ═ O group of the ligands of the present invention is nitrogen, oxygen, carbon or sulfur. Suitable chemical entities/units comprised in Sp are known to the person skilled in the art. Sp typically contains at least one unit selected from the group consisting of alkene, alcohol, amine, amide, carbonyl, alkenyloxy, and phosphoric acid. The alkene units preferably have from 1 to 30, preferably from 1 to 12, in particular from 1 to 6, carbon atoms. The alcohol may be a primary, secondary or tertiary alcohol. The amine may be a non-organic or organic amine. Furthermore, the amine may be a diamine or a triamine, preferably a diamine. The amide may be a non-organic or organic amide and may be derived from a diamine or triamine, preferably a diamine. The alkenyloxy units may include linear or branched alkene units. The alkenyloxy units are preferably vinyloxy or propenyloxy units, preferably vinyloxy units. The units may also comprise two or more entities/units as exemplified above, e.g. amino acids. The amino acids may be natural or unnatural amino acids. The amino acids may be linear or branched. Branched chain amino acids may act as branch points, such that more than one ligand group is attached to one attachment point ("L"). Examples of preferred amino acids include 6-aminocaproic acid, 8-amino-3, 6-dioxaoctanoic acid, 5-amino-3-oxavaleric acid, glycine, β -alanine, lysine, ornithine, glutamic acid, 2, 3-diaminopropionic acid, 2, 4-diaminobutyric acid and serine.

The spacer group Sp may consist of one of the aforementioned units or of more than one unit. If the spacer group Sp consists of more than one of the aforementioned units, the units are connected to each other by a linking group. Such linking groups include, but are not limited to, amides, ureas, hydrazides, oxalic acid diamides, oximes, hydrazones, triazoles, thioureas, isoureas, ethers, preferably amides and ureas. Phosphate groups may also be used as linking groups. The phosphate group may be linked to one, two or three of the aforementioned units. If the phosphate group is attached to three of the aforementioned units, it may serve as a branch point, such that more than one ligand group is attached to one attachment point ("L").

Preferred units of the spacer group Sp include, but are not limited to, amines, preferably organic amines, diamines, such as ethylenediamine, piperazine, homopiperazine, - (CH)₂)_n-C(＝O)、-NH-((CH₂)₂O)_n-(CH₂)_n-NH-、-NH-(CH₂)_n-NH-、-NH-(CH₂)_n-、-NH-CH-(C(＝O)NH-、-NH-(O-C₂H₄)_n-CH₂-C (═ O) -, where n is an integer from 1 to 30, preferably from 1 to 12. In one embodiment, n is an integer from 1 to 6, i.e., 1, 2, 3, 4, 5, or 6. Other preferred units of the spacer group Sp include, but are not limited to, amino acids which may be natural or non-natural amino acids, in particular 6-aminocaproic acid, 8-amino-3, 6-dioxaoctanoic acid, 5-amino-3-oxapentanoic acid, lysine and glutamic acid. Thus, the named units and functional groups are part of the spacer group Sp and the attachment point L.

A particularly preferred example of a spacer group Sp is-NH- (CH)₂)_nNH-、-NH-((CH₂)₂O)_n-(CH₂)₂-NH-, wherein n is an integer from 1 to 30, preferably from 1 to 12. In one embodiment, n is an integer from 1 to 6, i.e., 1, 2, 3, 4, 5, or 6.

Particularly preferred examples of such spacer groups Sp are also:

-NH-CH₂-CH₂-CH₂-NH, wherein-NH-is attached to said point of attachment L;

-NH-(CH₂)₃-NH-, wherein N is linked to the linking point L and the other N is linked to C (═ O) of the ligand;

-N’H-CH(C(＝O)NH-(CH₂)₃-N”H-)((CH₂)₂C(＝O)NH-(CH₂)₃-N ' "H", wherein N ' is linked to said junction L, and N "and N '" are both linked to C (═ O) of two separate ligands;

-NH-(CH₂)₃-N’(CH₃) -, wherein N is attached to the attachment point L and N' is attached to C (═ O) of the ligand;

-N’H-(CH₂)₃-NH-C(＝O)-CH(-N”H-)(-(CH₂)₄-N ' "H", wherein N ' is linked to said junction L, and N "and N '" are both linked to C (═ O) of two separate ligands;

-N’H-(CH₂)₃-NH-C(＝O)-CH(-NH-C(＝O)-CH₂-(O-C₂H₄)₂-N”H-)(-(CH₂)₄-NH-C(＝O)-CH₂-(O-C₂H₄)₂-N ' "H", wherein N ' is linked to said junction L, and N "and N '" are both linked to C (═ O) of two separate ligands;

-N’H-(CH₂)₃-NH-C(＝O)-CH(-NH-C(＝O)-CH₂-O-C₂H₄-N”H-)(-(CH₂)₄-NH-C(＝O)-CH₂-O-C₂H₄-N ' "H-), wherein N ' is connected to the junction L, and N" and N ' "are each connected to two separate ligandsC (═ O) is attached;

-N’H-(CH₂)₃-NH-C(＝O)-CH₂-(O-C₂H₄)₂-N "H", wherein N' is linked to the point of attachment L and N "is linked to C (═ O) of the ligand;

-N’H-(CH₂)₃-NH-C(＝O)-CH₂-(O-C₂H₄)₂-NH-C(＝O)-CH₂-(O-C₂H₄)₂-N "H", wherein N' is linked to the point of attachment L and N "is linked to C (═ O) of the ligand;

-N’H-(C₂H₄-O)₂-C₂H₄-NH-C(＝O)-NH-C₂H₄-(O-C₂H₄)₂-N ' H-, wherein N ' is linked to the point of attachment L and the other N ' is linked to C (═ O) of the ligand;

-N’H-(CH₂)₅-C(＝O)-NH-(CH₂)₃-N "H", wherein N' is linked to the point of attachment L and N "is linked to C (═ O) of the ligand;

-N’H-C₂H₄-(O-C₂H₄)₂-NH-C(＝O)-(CH₂)₅-N "H", wherein N' is linked to the point of attachment L and N "is linked to C (═ O) of the ligand;

-N’H-(CH₂)₆-NH-C(＝O)-CH₂-(O-C₂H₄)₂-N "H", wherein N' is linked to the point of attachment L and N "is linked to C (═ O) of the ligand;

the total length of the spacer group Sp is generally less than 100 atoms, preferably less than 60 atoms, in particular less than 30 atoms.

In one embodiment of the invention, Sp may also be branched. The term "branched" means in this context that more than 1 ligand of formula (II) according to the invention is present on the spacer group Sp. This embodiment allows more than one ligand to be attached to one substrate attachment site (attachment point) L.

By "final functionality" or "linking unit" is meant herein a unit formed in the reaction between a precursor group (functional group) on the support material and a precursor group (functional group) on the ligand of the invention and/or on the spacer group Sp. The linking unit incorporates the bond between the support material and the ligand of the invention (which may include the spacer group Sp) to produce a ligand-substituted matrix.

The linking units incorporated in L and/or Sp are known to those skilled in the art and may include the following groups: ethers, thioethers, C-C single bonds, alkenyl groups, alkynyl groups, esters, amines, amides, carboxylic acid amides, sulfonic esters, sulfonamides, phosphonates, phosphonamides, phosphates, phosphoramides, thioesters phosphates, thiophosphates, secondary amines, secondary alcohols, tertiary alcohols, 2-hydroxyamines, ureas, isoureas, imidocarbonates, alkylthiosuccinimides, dialkyl succinimides, triazoles, ketones, sulfoxides, sulfones, oxime ethers, hydrazones, silyl ethers, diacylhydrazines, acyl sulfonylhydrazides, N-acyl sulfonamides, hydrazides, N-alkoxyamides, acyl ureas, acyloxy amides, sulfonyloxy amides, phosphoryloxy amides, phosphonooxy amides, acylhydrazones, N-carboxamidoisourea (N-carboxamidoisourea), N-carboxamidoiimidocarbonates, N-carboxamidoiisothioureas, N-carboxamidoimidothiocarbonates, phosphoniocarbonates, phosphonioamides, acylhydrazones, N-carboxamidoimides, N-carboxamidoimidothioureas, N-carboxamidoimidothiocarbonates, phosphon, Acylureas, acylthioureas, N-formylsemicarbazides, N-formylaminoisoureas, alkylthioethylsulfonyls, hydrazinylsuccinimides, acylhydrazinylsuccinimides, alkoxyaminosuccinimides, aminosuccinylsulphonyls, acylhydrazinethylsulphonyls, alkoxyaminoethylsulphonyl, aminoethylsulphonyl, tetrazoles, alkoxyamidines.

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

In one embodiment of the invention, R¹Selected from: hydrogen; and C₁To C₄Alkyl radicals, in generalC being straight-chain or branched₁To C₄Alkyl, which may comprise cycloalkyl units such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, methylcyclopropyl.

R¹Preferably selected from hydrogen and methyl. R¹More preferably hydrogen.

Ar¹An aromatic ring which may be alicyclic or heterocyclic; ar (Ar)¹Preferably an alicyclic aromatic ring, i.e. for example Ar¹Is phenylene. In an even more preferred embodiment, Ar¹Is phenylene. If Ar is present¹Is a heterocyclic aromatic ring containing one, two or more heteroatoms preferably selected from N, S, O and combinations thereof. Examples of suitable aromatic rings of the 5 or 6 membered heterocyclic group include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyrazole, 1, 2, 3-triazole, 1, 2, 4-triazole (4H-1, 2, 4-triazole and 1H-1, 2, 4-triazole), 1H-tetrazole, 2H-tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, 1, 2, 3-triazine, 1, 2, 4-triazine and 1, 3, 5-triazine. Preferred aromatic rings of the 5-or 6-membered heterocyclic group are imidazole, thiazole and pyridine.

In the formula (I), Ar¹Bonded to a C ═ O group and a NH group (see formula Ia) or bonded to 2C ═ O groups (see formula Ib).

In the cases depicted in formula Ia, in certain embodiments, the C ═ O and NH groups are meta to each other. If Ar is present¹Is phenylene, the C ═ O group and the NH group are then meta or para, preferably meta, to one another. If the aromatic ring is imidazole, the C ═ O group is generally bonded at the 2 position and the NH group is bonded at the 4 position. If the aromatic ring is a thiazole, the C ═ O group is generally bonded at the 4 position and the H group is bonded at the 2 position. If the aromatic ring is pyridine, the C ═ O group and the NH group are preferably meta to one another, and the nitrogen ring atoms can have different positions. In certain embodiments, the C ═ O group is typically bonded at the 3 position of the pyridine ring and the NH group is bonded at the 5 position. The divalent 5-or 6-membered aromatic ring comprising the preferred embodiments described above may be substituted orUnsubstituted. The 5-or 6-membered aromatic ring is generally substituted, preferably monosubstituted. An example of a suitable substituent is C₁To C₄Alkoxy, typically straight or branched C₁To C₄Alkoxy groups which may comprise cycloalkyl units such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, cyclopropoxy, methylcyclopropoxy and cyclobutoxy; c₁To C₄Alkyl, typically straight and branched C₁To C₄Alkyl groups which may contain cycloalkyl units such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, methylcyclopropyl and cyclobutyl; a hydroxyl group; and combinations thereof. Preferred substituents include methoxy, ethoxy and methyl, with methoxy being most preferred.

In the most preferred embodiment, Ar¹Is a methoxy-substituted phenylene radical in which the methoxy groups are located ortho to the C ═ O group and para to the NH group (the C ═ O group and the NH group are meta to one another). In other preferred embodiments, Ar¹Is a methoxy-substituted phenylene radical in which the methoxy group is located para to the C ═ O group and ortho to the NH group (the C ═ O group and the NH group are meta to each other). In yet other preferred embodiments, Ar¹Is an N-methyl substituted imidazole ring wherein the C ═ O group is bonded at the 2 position and the NH group is bonded at the 4 position. In still other preferred embodiments, Ar¹Is thiazole, wherein the C ═ O group is bonded at the 4 position and the NH group is bonded at the 2 position. In still other preferred embodiments, Ar¹Is a methyl-substituted phenylene radical in which the methyl radical is located in the ortho position to the C ═ O radical and in the ortho position to the NH radical (the C ═ O radical and the NH radical are in the meta position relative to one another). In still other preferred embodiments, Ar¹Is a methyl-substituted phenylene radical in which the methyl radical is located para to the C ═ O radical and ortho to the NH radical (the C ═ O radical and the NH radical are meta to one another). In still other preferred embodiments, Ar¹Is a pyridine ring, wherein the C ═ O group is bonded at the 3 position and the NH group is bonded at the 5 position. In still other preferred embodiments, Ar¹Is unsubstituted phenylene, in which the C ═ O group and the NH group are in the meta or para positions relative to one another.

In the cases depicted by formula Ib, in certain embodiments, the two C ═ O groups are meta to each other. If Ar is present¹Is phenylene, the two C ═ O groups are then meta or para to one another, preferably meta.

Ar²Is an aromatic ring of the 5-or 6-membered unsubstituted or substituted heterocyclic family, which

(a) To other 5-or 6-membered aromatic rings via single bonds; or

(b) As part of a polycyclic ring system fused to other 5-or 6-membered aromatic rings; or

(c) Is linked to at least one substituent selected from the group consisting of: c₁To C₄An alkyl group; c₂To C₄An alkenyl group; c₂To C₄An alkynyl group; halogen; c₁To C₄A haloalkyl group; hydroxy-substituted C₁To C₄An alkyl group; c₁To C₄An alkoxy group; hydroxy-substituted C₁To C₄An alkoxy group; c₁To C₄An alkylamino group; c₁To C₄An alkylthio group; and combinations thereof.

Ar²To a C ═ O group (cf. formula Ia) or to NR¹The groups are linked (see formula Ib).

Ar²The aromatic ring of the 5 or 6 membered heterocyclic group of (a) contains at least one heteroatom preferably selected from N, S, O and combinations thereof. More preferably, the aromatic ring of the 5 or 6 membered heterocyclic group contains two or more nitrogen atoms or a nitrogen atom and an oxygen atom. The C ═ O group can also be part of the first aromatic ring. In general, Ar²The aromatic ring of the 5 or 6 membered heterocyclic group of (a) is attached to the C ═ O group via a carbon ring atom which is adjacent to a ring heteroatom, preferably a nitrogen or oxygen atom. In certain embodiments, Ar²With an aromatic ring of the 5-or 6-membered heterocyclic group via a carbon ring atom and NR¹The carbon ring atoms being bound to a ring hetero atom, preferably a nitrogen, oxygen or sulfur atomSon, next to each other.

Ar²And with the C ═ O group of the formula (I) or with NR¹Examples of suitable aromatic rings of the 5 or 6 membered heterocyclic group to which the groups are directly bonded include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyrazole, 1, 2, 3-triazole, 1, 2, 4-triazole, (4H-1, 2, 4-triazole and 1H-1, 2, 4-triazole), 1H-tetrazole, 2H-tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, 1, 2, 3-triazine, 1, 2, 4-triazine, 1, 3, 5-triazine, 1, 2, 4-oxadiazole, 1, 3, 4-oxadiazole, 1, 2, 4-thiadiazole and 1, 3, 4-thiadiazole. Preferred aromatic rings of the 5 or 6 membered heterocyclic group are pyrazole, imidazole, isoxazole, pyrrole, furan, 1, 2, 4-triazole, pyridine, 1, 2, 4-triazine, pyridazine, pyrimidine, 1, 2, 4-oxadiazole, 1, 3, 4-oxadiazole, thiazole and 1, 3, 4-thiadiazole, most preferably pyrazole, imidazole, isoxazole, 1, 2, 4-oxadiazole, 1, 3, 4-oxadiazole, thiazole and 1, 3, 4-thiadiazole. In the case of the pyrazole ring, the C ═ O group is generally bonded to the 3, 4 or 5 position of the pyrazole ring, preferably to the 3 position. For the pyrazole ring, the NR¹The N atom of the radical is generally bonded to the 3-, 4-or 5-position of the pyrazole ring, preferably to the 3-position. For imidazole rings, the C ═ O group is typically bonded to the 4 or 2 position of the imidazole ring. For isoxazole rings, the C ═ O group is typically bonded to the 5 or 3 position of the isoxazole ring. For pyrrole rings, the C ═ O group is typically bonded to the 3 position of the pyrrole ring. For furan rings, the C ═ O group is typically bonded to the 2 position of the furan ring. For a 1, 2, 4-triazole ring, the C ═ O group is typically bonded to the 3 position of the 1, 2, 4-triazole ring. For pyridine rings, the C ═ O group is generally bonded to the 3 or 2 position of the pyridine ring, preferably to the 2 and 3 positions. For a 1, 2, 4-triazine ring, the C ═ O group is typically bonded to the 3 position of the 1, 2, 4-triazine ring. For pyridazine rings, the C ═ O group is typically bonded to the 4 position of the pyridazine ring. For a 1, 2, 4-oxadiazole ring, the C ═ O group is typically bonded to the 5 position of the 1, 2, 4-oxadiazole ring. For 1, 3, 4-oxadiazole rings, the C ═ O group is typically bonded to the 2 or 5 position of the 1, 3, 4-oxadiazole ringAnd (6) mixing. For the thiazole ring, the NR¹The N atom of the group is generally bonded to the 2-position of the thiazole ring. For 1, 3, 4-thiadiazole rings, the NR is¹The N atom of the group is typically bonded to the 2-position of the 1, 3, 4-thiadiazole ring.

Ar described hereinabove²In an alternative (a) to formula (I), the aromatic ring of the 5-or 6-membered heterocyclic group is bonded via a single bond to another 5-or 6-membered aromatic ring (hereinafter "Ar)²A second aromatic ring) to the aromatic ring. In these embodiments, with a C ═ O group (hereinafter "Ar ═ O ═ group)²The first aromatic ring of (a) is particularly preferably a pyrazole, pyridine, isoxazole, 1, 2, 4-oxadiazole or 1, 3, 4-oxadiazole ring. In the case of a pyrazole ring as the first aromatic ring, the C ═ O group is generally bonded to the 3 position of the pyrazole ring. The second aromatic ring is preferably bonded to the 5-position of the pyrazole ring. In other embodiments, the C ═ O group is bonded to the 5 position of the pyrazole ring, and the second aromatic ring is bonded to the 3 position of the pyrazole ring. If the first aromatic ring is pyridine, the C ═ O group and the second aromatic ring are preferably meta to each other, and the nitrogen ring atoms may have different positions. In certain embodiments, the C ═ O group is bonded to the 3 position of the pyridine ring, and the second aromatic ring is bonded to the 5 position of the pyridine ring. In other embodiments, the C ═ O group is bonded to the 2 position of the pyridine ring, and the second aromatic ring is bonded to the 4 position of the pyridine ring. In the case of an isoxazole ring as the first aromatic ring, the C ═ O group is generally bonded to the 3 position of the isoxazole ring. The second aromatic ring is preferably bonded to the 5-position of the isoxazole ring. In other embodiments, the C ═ O group is bonded to the 5 position of the isoxazole ring, and the second aromatic ring is bonded to the 3 position of the isoxazole ring. In the case of a 1, 2, 4-oxadiazole ring as the first aromatic ring, the C ═ O group is typically bonded to the 5 position of the 1, 2, 4-oxadiazole ring. The second aromatic ring is preferably bonded to the 3-position of the 1, 2, 4-oxadiazole ring. In the case of a 1, 3, 4-oxadiazole ring as the first aromatic ring, the C ═ O group is typically bonded to the 2 position of the 1, 3, 4-oxadiazole ring. The second aromatic ring is preferably bonded to the 5-position of the 1, 3, 4-oxadiazole ring. In certain embodiments, with NR¹Radical (hereinafter "Ar)²The first aromatic ring of (a) or (b) an aromatic ring of the 5-or 6-membered heterocyclic group of N atoms directly bonded theretoPyrazole, thiazole and 1, 3, 4-thiadiazole rings are particularly preferred. In the case of a pyrazole ring as the first aromatic ring, the NR is¹The N atom of the radical is generally bonded to the 5-position of the pyrazole ring. The second aromatic ring is preferably bonded to the 3-position of the pyrazole ring. If the first aromatic ring is a thiazole ring, the NR¹The N atom of the group is generally bonded to the 2-position of the thiazole ring. The second aromatic ring is preferably bonded to the 4-position of the thiazole ring. In other embodiments, the second aromatic ring is preferably bonded to the 5-position of the thiazole ring. In the case of a 1, 3, 4-thiadiazole ring as the first aromatic ring, the NR is¹The N atom of the group is typically bonded to the 2-position of the 1, 3, 4-thiadiazole ring. The second aromatic ring is preferably bonded to the 5-position of the 1, 3, 4-thiadiazole ring.

In an alternative (b), the first aromatic ring is fused with a second 5 or 6 membered aromatic ring as part of a polycyclic (preferably bicyclic) ring system. In the case of a pyrazole ring as the first aromatic ring and as part of a fused ring system, the C ═ O group is generally bonded to the 3 position of the pyrazole ring. The pyrazole ring is preferably fused to the second aromatic ring via positions 4 and 5. In other cases, the C ═ O group is bonded to the 4 position of the pyrazole ring, and the second aromatic ring is bonded to the pyrazole ring via the 1 and 5 positions. In other cases, the C ═ O group is bonded to the 3 position of the pyrazole ring, and the second aromatic ring is bonded to the pyrazole ring via the 1 and 5 positions. In the case of an isoxazole ring as the first aromatic ring and as part of a fused ring system, the C ═ O group is typically bonded to the 5 position of the isoxazole ring. The isoxazole ring is preferably fused to the second aromatic ring via positions 3 and 4. In the case of an imidazole ring as the first aromatic ring and as part of a fused ring system, the C ═ O group is typically bonded to the 4 position of the imidazole ring. The imidazole ring is preferably fused to the second aromatic ring via positions 1 and 2. In other cases, the C ═ O group is bonded to the 2 position of the imidazole ring, and the second aromatic ring is fused to the imidazole ring via the 4 and 5 positions. In the case where the 1, 2, 4-triazole ring is the first aromatic ring and is part of a fused ring system, the C ═ O group is typically bonded to the 3 position of the 1, 2, 4-triazole ring. The 1, 2, 4-triazole ring is preferably fused to the second aromatic ring via positions 4 and 5. In the case of pyridazines as the first aromatic ring and as part of a fused ring system, the C ═ CThe O group is typically bonded to the 4-position of the pyridazine ring. The pyridazine ring is preferably fused to the second aromatic ring via the 5-and 6-positions. In the case of a pyrrole ring as the first aromatic ring and as part of a fused ring system, the C ═ O group is typically bonded to the 3 position of the pyrrole ring. The pyrrole ring is preferably fused to the second aromatic ring via positions 4 and 5. In the case of a pyridine ring as the first aromatic ring and as part of a fused ring system, the C ═ O group is typically bonded to the 2 position of the pyridine ring. The pyridine ring is preferably fused to the second aromatic ring via the 3-and 4-positions. In the case where the 1, 2, 4-triazine ring is the first aromatic ring and is part of a fused ring system, the C ═ O group is typically bonded to the 6 position of the 1, 2, 4-triazine ring. The 1, 2, 4-triazine ring is preferably fused to the second aromatic ring via the 3-and 4-positions. In the case of a pyrazole ring as the first aromatic ring and being part of a fused ring system, the NR is¹The N atom of the radical is generally bonded to the 3-position of the pyrazole ring. The pyrazole ring is preferably fused to the second aromatic ring via positions 4 and 5. In the case of imidazole rings as the first aromatic ring and as part of a fused ring system, the NR is¹The N atom of the group is generally bonded to the 4-position of the imidazole ring. The imidazole ring is preferably fused to the second aromatic ring via positions 1 and 2. In the case of a thiazole ring as the first aromatic ring and being part of a fused ring system, the NR is¹The N atom of the group is generally bonded to the 2-position of the thiazole ring. The thiazole ring is preferably fused to the second aromatic ring via the 4-and 5-positions.

Ar in alternatives (a) and (b) and including the preferred embodiments described above²The second aromatic ring of (a) is typically a 5 or 6 membered alicyclic or heterocyclic ring. Examples of suitable 5 or 6 membered aromatic rings include, but are not limited to, benzene, pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyrazole, 1, 2, 3-triazole, 1, 2, 4-triazole (4H-1, 2, 4-triazole and 1H-1, 2, 4-triazole), 1H-tetrazole, 2H-tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, 1, 2, 3-triazine, 1, 2, 4-triazine and 1, 3, 5-triazine. Preferred second aromatic rings are benzene, furan, thiophene, pyrrole, pyridine, thiazole, pyrimidine and pyrazole, most preferred are benzene, thiazole, furan and thiophene.

In alternative (a), Ar²The second aromatic ring of (a) is typically furan, thiophene, pyrrole, benzene, pyridine or thiazole, preferably furan, thiophene, benzene and thiazole, most preferably furan and thiazole. In case of furan, thiophene, pyrrole, benzene or thiazole rings as the second aromatic ring in alternative (a), these rings are preferably bonded to the first aromatic ring via the 2-, 3-or 4-positions thereof, most preferably the 2-and 4-positions. In a representative embodiment of alternative (a), the first aromatic ring is a pyrazole ring and the second aromatic ring is a furan ring, i.e., Ar²Is a furanylpyrazolyl, for example 5- (2-furanyl) pyrazol-3-yl. In yet other representative embodiments, Ar²Is 5- (2-methylthiazol-4-yl) isoxazol-3-yl, 4- (2-furyl) pyridin-2-yl, 3- (4-methoxyphenyl) - [1, 2, 4]]Oxadiazol-5-yl, 3- (2-furyl) - [1, 2, 4]]Oxadiazol-5-yl or 5- (2-furyl) - [1, 3, 4]]Oxadiazol-2-yl.

In alternative (b), Ar²The second aromatic ring of (a) is preferably benzene, thiazole, pyrrole, pyrimidine or pyridine, most preferably benzene and thiazole. In a representative embodiment of alternative (b), Ar²Is benzo [ c]Isoxazol-3-yl, imidazo [2, 1-b]Thiazol-6-yl, cinnolin-4-yl, indol-3-yl, indazol-3-yl, benzo [ c ]]Furan-1-yl, 1-isoquinolinyl, 5-oxo-5H- [1, 3]Thiazole [3, 2-a ]]Pyrimidin-6-yl, pyrazolo [1, 5-a ]]Pyrimidin-3-yl, [1, 2, 4]]Triazolo [4, 3-a]Pyrimidin-3-yl and pyrazolo [5, 1-c)][1，2，4]Triazin-3-yl, preferably benzo [ c]Isoxazol-3-yl, indazol-3-yl and imidazo [2, 1-b ]]Thiazol-6-yl.

Ar described hereinabove for formula (I)²In alternative (c), the aromatic ring of the 5 or 6 membered heterocyclic group is linked to at least one (preferably one or two) substituents selected from: c₁To C₄Alkyl, typically straight or branched C₁To C₄Alkyl groups which may contain cycloalkyl units such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, methylcyclopropyl and cyclobutyl; c₂To C₄An alkenyl group; c₂To C₄Alkynyl groups such as ethynyl; halogen, e.g. fluorine, chlorineBromine and iodine; c₁To C₄Haloalkyl such as trifluoromethyl; hydroxy-substituted C₁To C₄An alkyl group; typically a mono-or dihydroxyalkyl group such as mono-or dihydroxyethyl or mono-or dihydroxypropyl; c₁To C₄Alkoxy, typically straight or branched C₁To C₄Alkoxy groups which may comprise cycloalkyl units such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, cyclopropoxy, methylcyclopropoxy and cyclobutoxy; hydroxy-substituted C₁To C₄Alkoxy, typically monohydroxy-substituted C₁To C₄An alkoxy group; c₁To C₄An alkylamino group; c₁To C₄Alkylthio groups, and combinations thereof. The substituents may be bonded to the ring via ring carbon atoms and/or ring heteroatoms, preferably via carbon and/or nitrogen ring atoms. Preferred substituents for the aromatic ring of the 5-or 6-membered heterocyclic group mentioned in alternative (c) are cyclopropyl, ethynyl, methyl and trifluoromethyl. The at least one substituent is preferably meta to the C ═ O group. In a representative embodiment of alternative (c), Ar²Is 3-cyclopropylpyridin-5-yl, 3-ethynylpyridin-5-yl, 5- (trifluoromethyl) pyrazol-3-yl or 1-methyl-5- (trifluoromethyl) pyrazol-3-yl.

It will be appreciated that Ar in alternatives (a) and (b)²The aromatic ring of (a), i.e. the first aromatic ring or the second aromatic ring or both, may optionally bear one or more further substituents, typically selected from the substituents mentioned above for alternative (c). The substituents may be bonded to the ring via a ring carbon atom or a ring heteroatom, preferably via a carbon or nitrogen atom. Ar (Ar)²The preferred substituent for the first and/or second aromatic ring of (a) is C₁To C₄Alkyl groups such as methyl; halogens such as bromine and chlorine; hydroxyalkyl such as methoxy, 2-hydroxyethyl or 2, 3-dihydroxypropyl, alkylamino such as aminopropyl and haloalkyl such as trifluoromethyl. If the first aromatic ring is a pyrazole ring, it is generally substituted by methyl, preferably by N-methyl, more preferably in position 1. In alternative (a)N-methyl substituents of pyrazoles are particularly preferred.

Ar substituted in alternative (a)²A representative example of (B) is 5- (2-furyl) -1-methylpyrazol-3-yl.

If the first aromatic ring is a pyridine ring, it typically bears no other substituents.

Ar substituted in alternative (b)²Representative examples of (a) are 5-chloro-1H-indazol-3-yl, 1-methylindol-3-yl, 1-methylindazol-3-yl, 5, 7-dimethyl- [1, 2, 4]Triazolo [4, 3-a]Pyrimidin-3-yl, 4, 7-dimethylpyrazolo [5, 1-c ]][1，2，4]Triazin-3-yl, 1-ethylindazol-3-yl, 1- (2-hydroxyethyl) indazol-3-yl, and 1- (2, 3-dihydroxypropyl) indazol-3-yl.

The invention also includes that the second aromatic ring in the alternatives (a) and (b) may optionally be fused with other aromatic rings such as benzene. A representative example of the second aromatic ring fused with other aromatic rings is benzothiophene.

In a preferred embodiment of the invention, the group at position α of the carbonyl function is an amino group NR²Wherein R is²May have a variety of meanings as defined below. In these cases, the ligand-substituted matrices of the invention can be depicted in the following figures (II), (IIa) and (IIb), which are encompassed by the above figures (I), (Ia) and (Ib).

In the above FIGS. (II), (IIa) and (IIb), for the sake of clarity, the NR is indicated²The groups are not shown as part of the attachment point L or the spacer group Sp, but are shown as part of such a ligand. But should be kept in mind, NR²Together with V, form a spacer group Sp as shown in figures (I), (Ia) and (Ib), or is part of the point of attachment L.

In one embodiment of the invention, R²Selected from: hydrogen; and C₁To C₄Alkyl radicalGenerally straight and branched C₁To C₄Alkyl, which may comprise cycloalkyl units such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, methylcyclopropyl.

R²Preferably selected from hydrogen and methyl. R¹More preferably hydrogen.

In the context of FIGS (I), (Ia) and (Ib), the other symbols Ar¹、Ar²And Am have the meanings and preferred meanings as defined above.

R defined in the context of formula (II) and shown in formulae (IIa) and (IIb)²R as defined and shown in patent application EP10008089.4 corresponding to the application for which priority is claimed³。

The invention also includes ligand-substituted matrices formed by the ligands of the invention (after attachment to the support material) together with the support material, which may be used in the methods of the invention. The ligand has the formula, wherein the symbols Sp and Ar¹、Ar²And Am have the meanings as defined above, including the preferred meanings:

the ligands of formulae (III), (IIIa) and (IIIb) comprise a spacer group Sp attached to the ligand.

The ligands of formulae (III), (IIIa) and (IIIb) can be used as precursors for the synthesis of other compounds to which no spacer group is attached. The compounds mentioned are generated after cleavage of the spacer group, which may be the case if the functionality comprising-C ═ O present in the part of the molecule to which the antibody is bound is converted to any other suitable functionality known to the person skilled in the art. Suitable reactions to produce such results are known to those skilled in the art. The resulting compounds are also included in the present invention.

Preferred ligands for the matrices of the invention are described below, wherein the leftmost N atom in each formula is attached to a point of attachment L (not shown). All formulae below show spacer groups attached to the ligands:

5- [5- (2-furyl) -1-methylpyrazol-3-yl ] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L1)

5- [5- (4-bromo-2-thienyl) -1-methylpyrazol-3-yl ] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L2)

5- [5- (2, 5-dimethyl-3-thienyl) -1-methylpyrazol-3-yl ] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L3)

5- (5-chloro-1H-indazol-3-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L4)

2-methoxy-5- [3- (2-thienyl) pyridin-5-yl ] carboxamidobenzoic acid N- (3-aminopropyl) amide (L5)

2-methoxy-5- [ 1-methyl-5- (2-thienyl) pyrazol-3-yl ] carboxamidobenzoic acid N- (3-aminopropyl) amide (L6)

5- [5- (3-chlorophenyl) -1-methylpyrazol-3-yl ] formylamino-2-methoxybenzoic acid N- (3-aminopropyl) amide (L7)

2-methoxy-5- [ 1-methyl-5- (3-thienyl) pyrazol-3-yl ] carboxamidobenzoic acid N- (3-aminopropyl) amide (L8)

2-methyl-3- [ 1-methyl-5- (2-thienyl) pyrazol-3-yl ] carboxamidobenzoic acid N- (3-aminopropyl) amide (L9)

4-methyl-3- [ 1-methyl-5- (2-thienyl) pyrazol-3-yl ] carboxamidobenzoic acid N- (3-aminopropyl) amide (L10)

2-methoxy-5- (3-phenylpyridin-5-yl) formylaminobenzoic acid N- (3-aminopropyl) amide (L11)

2-methoxy-5- {3- [4- (trifluoromethyl) phenyl ] pyridin-5-yl } carboxamidobenzoic acid N- (3-aminopropyl) amide (L12)

5- [3- (2-furyl) pyridin-5-yl ] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L13)

5- [3- (2-benzothienyl) pyridin-5-yl ] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L14)

2-methoxy-5- (1-methyl-5-phenylpyrazol-3-yl) carboxamidobenzoic acid N- (3-aminopropyl) amide (L15)

2-methoxy-5- [5- (1-methyl-2-pyrrolyl) -1-methylpyrazol-3-yl ] carboxamidobenzoic acid N- (3-aminopropyl) amide (L16)

3- [5- (2-furyl) -1-methylpyrazol-3-yl ] carboxamidobenzoic acid N- (3-aminopropyl) amide (L17)

4- [5- (2-furyl) -1-methylpyrazol-3-yl ] formylaminobenzoic acid N- (3-aminopropyl) amide (L18)

2- [ 1-methyl-5- (2-thienyl) pyrazol-3-yl ] carboxamidothiazole-4-carboxylic acid N- (3-aminopropyl) amide (L19)

5- [5- (2-furyl) pyrazol-3-yl ] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L20)

3- { N- [5- (2-furyl) pyrazol-3-yl ] carbamoyl } benzoic acid N' - (3-aminopropyl) amide (L21)

5- [3- (3-furyl) pyridin-5-yl ] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L22)

5- (3-Cyclopropylpyridin-5-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L23)

5- (3-ethynylpyridin-5-yl) formylamino-2-methoxybenzoic acid N- (3-aminopropyl) amide (L24)

2-methoxy-5- (1-methylindol-3-yl) formylaminobenzoic acid N- (3-aminopropyl) amide (L25)

5- (indol-3-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L26)

2-methoxy-5- (1-methylindazol-3-yl) formylaminobenzoic acid N- (3-aminopropyl) amide (L27)

5- [5- (2-furyl) -1-methylpyrazol-3-yl ] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) -N-methylamide (L28)

5- [5- (2-furyl) -1-methylpyrazol-3-yl ] carboxamido-2-ethoxybenzoic acid N- (3-aminopropyl) amide (L29)

5- [5- (2-furyl) -1-methylpyrazol-3-yl ] carboxamido-2-hydroxybenzoic acid N- (3-aminopropyl) amide (L30)

5- (benzo [ c ] isoxazol-3-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L31)

5- (benzo [ c ] furan-1-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L32)

5- (1, 5-dimethylpyrazol-3-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L33)

2-methoxy-5- [ 1-methyl-5- (trifluoromethyl) pyrazol-3-yl ] carboxamidobenzoic acid N- (3-aminopropyl) amide (L34)

5- (1-isoquinolinyl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L35)

5- (indazol-3-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L36)

5- (imidazo [2, 1-b ] thiazol-6-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L37)

5- (1-Ethylindazol-3-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L38)

5- [ N- (1-methylindazol-3-yl) carbamoyl ] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L39)

(RS) -5- [1- (2, 3-dihydroxy-1-propyl) indazol-3-ylcarboxamido ] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L40)

5- (3H-imidazo [4, 5-b ] pyridin-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L41)

2-methoxy-5- [5- (2-methylthiazol-4-yl) isoxazol-3-ylcarboxamido ] benzoic acid (3-amino-1-propyl) amide (L42)

2-methoxy-5- ([1, 2, 4] triazolo [4, 3-a ] pyridin-3-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L43)

2-methoxy-5- (1-methylpyrazolo [3, 4-b ] pyridin-3-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L44)

5- [5- (2-furyl) isoxazol-3-ylcarboxamido ] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L45)

2-methoxy-5- (pyrazolo [1, 5-a ] pyridin-2-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L46)

5- (1H-benzimidazol-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L47)

5- (imidazo [1, 2-b ] pyridazin-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L48)

(S) -2, 6-bis [5- (imidazo [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxyphenylcarboxamido ] hexanoic acid (3-amino-1-propyl) amide (L49)

(S) -2, 6-bis {8- [5- (imidazo [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxyphenylcarboxamido ] -3, 6-dioxaoctanoylamino } hexanoic acid (3-amino-1-propyl) amide (L50)

2-methoxy-5- (3-phenylisoxazol-5-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L51)

5- [4- (2-furyl) pyridin-2-ylcarboxamido ] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L52)

(S) -2, 6-bis {5- [5- (imidazo [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxyphenylcarboxamido ] -3-oxapentanoylamino } hexanoic acid (3-amino-1-propyl) amide (L53)

2-methoxy-5- [3- (4-methoxyphenyl) -1, 2, 4-oxadiazol-5-ylcarboxamido ] benzoic acid (3-amino-1-propyl) amide (L54)

5- [3- (2-furyl) -1, 2, 4-oxadiazol-5-ylcarboxamido ] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L55)

2-methoxy-5- (pyrazolo [1, 5-a ] pyrimidin-2-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L56)

5- (imidazo [1, 2-a ] pyridin-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L57)

5- [5- (2-furyl) -1, 3, 4-oxadiazol-2-yl ] carboxamido-2-methoxybenzoic acid (3-amino-1-propyl) amide (L58)

5- [1- (2-hydroxyethyl) indazol-3-ylcarboxamido ] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L59)

8- [ 2-methoxy-5- (1-methylindazol-3-ylcarboxamido) phenylcarboxamide ] -3, 6-dioxaoctanoic acid (3-amino-1-propyl) amide (L60)

8- {8- [ 2-methoxy-5- (1-methylindazol-3-ylcarboxamido) phenylcarboxamido ] -3, 6-dioxaoctanoylamino } -3, 6-dioxaoctanoic acid (3-amino-1-propyl) amide (L61)

(S) -2, 6-bis {8- [5- (imidazo [1, 2-b ] pyridazin-2-ylcarboxamido) -2-methoxyphenylcarboxamido ] -3, 6-dioxaoctanoylamino } hexanoic acid (3-amino-1-propyl) amide (L62)

5- (imidazo [1, 2-a ] pyrimidin-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L63)

(S) -1-aminopropane-1, 3-dicarboxylic acid bis {3- [5- (imidazo [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxyphenylcarboxamido ] propyl } amide (L64)

8- {8- {5- [5- (2-furyl) -1-methylpyrazol-3-ylcarboxamido ] -2-methoxyphenylcarboxamido } -3, 6-dioxaoctanoylamino } -3, 6-dioxaoctanoic acid (3-amino-1-propyl) amide (L65)

8- {8- [5- (imidazo [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxyphenylcarboxamido ] -3, 6-dioxaoctanoylamino } -3, 6-dioxaoctanoic acid (3-amino-1-propyl) amide (L66)

8- {8- {5- [5- (2-furyl) -1, 3, 4-oxadiazol-2-ylcarboxamido ] -2-methoxyphenylcarboxamido } -3, 6-dioxaoctanoylamino } -3, 6-dioxaoctanoic acid (3-amino-1-propyl) amide (L67)

8- {8- { 2-methoxy-5- [5- (2-methylthiazol-4-yl) isoxazol-3-ylcarboxamido ] phenylcarboxamido } -3, 6-dioxaoctanoylamino } -3, 6-dioxaoctanoic acid (3-amino-1-propyl) amide (L68)

N- (8-amino-3, 6-dioxaoctyl) -N' - {8- [5- (imidazo [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxyphenylcarboxamido ] -3, 6-dioxaoctyl } urea (L69)

Isophthalic acid N- (3-aminopropyl) -N' - [5- (2-furyl) -1, 3, 4-thiadiazol-2-yl ] amide (L70)

Isophthalic acid N- (3-aminopropyl) -N' - (1-methylindazol-3-yl) amide (L71)

Isophthalic acid N- (3-aminopropyl) -N' - (benzothiazol-2-yl) amide (L72)

Isophthalic acid N- (3-aminopropyl) -N' - (4-phenylthiazol-2-yl) amide (L73)

Isophthalic acid N- (3-aminopropyl) -N' - (5-phenylthiazol-2-yl) amide (L74)

5- (imidazo [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxybenzoic acid [3- (6-aminocaproylamino) -1-propyl ] amide (L75)

6- [5- (imidazo [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxyphenylcarboxamido ] hexanoic acid (8-amino-3, 6-dioxa-1-octyl) amide (L76)

8- [5- (imidazo [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxyphenylcarboxamido ] -3, 6-dioxaoctanoic acid (3-amino-1-propyl) amide (L77)

8- [5- (imidazo [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxyphenylcarboxamido ] -3, 6-dioxaoctanoic acid (6-amino-1-hexyl) amide (L78)

The synthesis of ligand (II) can be carried out, for example, on an insoluble support, also known as a resin, such as a polystyrene resin, preferably pre-filled with the desired linker bearing a reactive group such as an amino group to which a spacer group such as a 9-fluorenylmethyloxycarbonyl (Fmoc) -protected amino acid can be attached by amide formation. After deprotection of the spacer group, the remainder of the molecule is attached by any reaction that results in the formation of an appropriate bond, if desired, by an additional synthetic step, such as a nucleophilic substitution reaction. Finally, the ligand comprising the spacer group (linker moiety) is released from the insoluble support/resin by suitable cleavage protocols known to those skilled in the art and also purified by chromatographic methods known to those skilled in the art.

If a suitable solid phase synthesis scheme is not applicable to a particular ligand, the synthesis may alternatively be carried out in solution. Typically, such solution phase synthesis consists of the conjugation reaction between a ligand precursor and a suitable spacer group (e.g. to form an amide) and the synthetic steps necessary for the assembly of the ligand itself. In some cases, a protecting group is necessary to prevent side reactions.

The term "antibody" means an immunoglobulin, including natural or wholly or partially synthetically produced immunoglobulins, and also including all fragments and derivatives thereof which retain specific binding capacity. Representative fragments are Fc, Fab, heavy and light chains. The term also includes any polypeptide having a binding domain that is homologous or largely homologous, e.g., at least 95% identical when comparing amino acid sequences, to an immunoglobulin binding domain. These polypeptides may be derived from natural sources, or produced partially or wholly synthetically. The antibody may be monoclonal or polyclonal, and may be of human or non-human origin, or a chimeric protein in which the human Fc portion is fused to a murine Fab fragment (a therapeutic antibody ending in … … ximab, such as Rituximab (Rituximab)) or to a Fab fragment comprising human and murine sequences (a therapeutic antibody ending in … … zumab, such as Bevacizumab (Bevacizumab)) or linked to a different Fab fragment (a bispecific antibody). The antibody may be a member of any immunoglobulin class, preferably IgG, more preferably IgG for human antibodies₁、IgG₂And IgG₄。

In the most preferred embodiment of the invention, the antibody comprises an Fc fragment or domain, since it is assumed that the ligand of the invention is linked to the Fc portion of the antibody. Thus, the antibody of the invention is most preferably an antibody comprising an Fc fragment or domain of an immunoglobulin class, preferably an IgG, more preferably a human IgG or a polyclonal or monoclonal IgG of human origin, in particular an IgG₁、IgG₂And IgG₄。

The term "Fc fusion protein" refers to any combination of an Fc fragment and one or more proteins or protein domains. Examples of Fc fusion proteins includeBut are not limited to, human IgG₁Chimeras of Fc domains with soluble receptor domains (therapeutic proteins ending with … … cept), such as Etanercept (Etanercept, human IgG binding to two tumor necrosis factor receptor domains₁Fc) or rilocacept (Rilonacept, human IgG fused to the interleukin-1 receptor domain₁). The Fc fusion protein may be prepared by any route. For example, the Fc fusion protein may be prepared enzymatically or chemically by coupling the Fc fragment to an appropriate protein or protein domain, or it may be prepared recombinantly from genes encoding the Fc and protein/protein domain sequences. Alternatively, the Fc fusion protein may be prepared synthetically, in whole or in part. The Fc fusion protein can also optionally be a multimolecular complex. A functional Fc fusion protein will typically comprise at least about 50 amino acids, and more typically will comprise at least about 200 amino acids.

The present invention relates to affinity purification of antibodies or Fc-fusion proteins against plasma from complex mixtures such as fermentation broths or human or animal sources, preferably by affinity chromatography, using affinity ligands of formula (I) and preferred embodiments thereof as disclosed elsewhere in the specification.

Thus, in a preferred embodiment, the invention comprises a method for purifying a protein, preferably an immunoglobulin or Fc fusion protein, by affinity purification, preferably affinity chromatography. The affinity ligands of the invention bind to the Fc region of an antibody.

Bevacizumab (Genentech/Roche) is a humanized monoclonal antibody. It arrests the formation of new blood vessels by targeting and inhibiting the function of the vascular endothelial growth factor-a (VEGF-a) protein that stimulates neovascularization (angiogenesis) to stop tumor growth.

Toslizumab (Tocilizumab,Genentech/Roche) is a humanized monoclonal antibody. It targets the interleukin-6 receptor and blocks the action of the proinflammatory cytokine interleukin-6. It is approved for the treatment of rheumatoid arthritis.

Palivizumab (Palivizumab,abbott) is a humanized monoclonal antibody directed against the a epitope of the Respiratory Syncytial Virus (RSV) F protein. It is used for preventing RSV infection.

Polyclonal human and rabbit IgG were studied to demonstrate the general applicability of the ligands for affinity purification of immunoglobulins via the Fc portion.

In the practice of the present invention, the ligands of formula (I) are attached to a support matrix of a suitable support material to produce a ligand-substituted matrix for protein separation, typically a matrix for affinity purification, preferably affinity chromatography (in the context of the present invention also referred to as affinity matrix). The ligand of the formula is attached to a supporting matrix via L (optionally including a spacer-Sp-).

Thus, the present invention comprises a ligand-substituted matrix (affinity matrix) for protein separation comprising a support material and at least one ligand as detailed earlier in the specification, wherein the ligand is linked to the support material via L.

The support matrix, which may be denoted as M, may comprise any suitable support material known to those skilled in the art. The materials may be soluble or insoluble, particulate or non-particulate, or monolithic structures, including porous or non-porous fibers and membranes. Which provides a convenient way of isolating the ligands of the invention from the solute in the contact solution. Examples of support matrices include carbohydrates and cross-linked carbohydrate matrices such as agarose, Sepharose (Sepharose), Sephadex (Sephadex), cellulose, dextran, starch, alginate or carrageenan; synthetic polymer matrices such as polystyrene, styrene-divinylbenzene copolymers, polyacrylates, PEG-polyacrylate copolymers, polymethacrylates (e.g. poly (hydroxyethyl methacrylate)), polyvinyl alcohols, polyamides or perfluorocarbons; inorganic substrates such as glass, silica or metal oxides; and a composite material.

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

The invention also relates to a method for affinity purification, preferably affinity chromatography, of a protein, wherein the protein to be purified is contacted with the aforementioned ligand-substituted matrix.

The term "affinity purification" (which may be used interchangeably with the term "affinity separation") refers to any separation technique involving molecular recognition of a protein by a ligand of formula (I). The ligand may be immobilized on a solid support that facilitates later separation of the ligand-antibody complex. Separation techniques may include, but are not limited to, affinity chromatography on packed columns, monolithic structures, or membranes. The term also includes adsorption or affinity precipitation in batch mode.

Regardless of preference, the purification technique includes an initial recognition stage, in which the ligand is contacted with the unprocessed antibody. In the second stage, the impurities are separated from the ligand-antibody complex (e.g., column chromatography) or the ligand-antibody complex is separated from the impurities (e.g., affinity precipitation). In a third step, the antibody is released from the ligand-antibody complex by changing chemical and/or physical conditions, such as pH change, ionic strength change and/or addition of modifiers such as organic solvents, detergents or chaotropes (chaotropes).

The invention will be illustrated by the following examples, which should not be construed as limiting the invention.

Documents cited in this specification:

1.A.Cecilia，A.Roque，C.R.Lowc，M.A.Taipa：AntibodiesandGeneticallyEngineeredRelatedMolecules：ProductionandPurification，Biotechnol.Prog.2004，20，639-654

2.K.L.Carson：Flexibility-theguidingprincipleforantibodymanufacturing，NatureBiotechnology，2005，23，1054-1058；S.Hober，K.Nord，M.Linhult：ProteinAchromatographyforantibodypurification，J.Chromatogr.B.2007，848，40-47

3.T.Arakawa，Y.Kita，H.Sato，D.Ejima，ProteinExpressionandPurification.2009，63，158-163

4.S.Ghose，B.Hubbard，S.M.Cramer，JournalofChromatographyA，2006，1122，144-152

examples

Materials and methods

Unless otherwise stated, all chemicals and solvents were of analytical grade except example 2. The reagents used in example 2 range from preparative to analytical grade depending on the specific requirements and availability.

96 and 384 well filter plates with hydrophilic membrane filters with an average pore size of 0.45 μm were purchased from pall GmbH (Dreieich/Germany). A top sieve plate (Topfrit) made of polyethylene and having an average pore size of 10 μm was supplied by Porex (Bautzen/Germany). Universal microtiter plates for fraction collection and analytical assays were purchased from Greiner BioOneeGmbH (Frickenhausen/Germany). The assay was read using a FluostarGalaxyplaterdelder microplate reader from BMGLAbtech GmbH (Offenburg/Germany).

Column chromatography of the antibodies and purification of the antibody fragments were carried out on a Waters HPLCSystem (Waters GmbH, Eschborn/Germany). Omnifit column housing (Diba Industies Ltd, Cambridge/United kingdom) was used to house the column. NHS-activated Sepharose 4FF, rProteinA (recombinant protein A) Sepharose FF and Superdex70 chromatography media were purchased from GEHealthcare (Uppsala/Sweden). MabsorbentA2PHF is available from PrometicicLifeSciences (Cambridge/United kingdom), and MEPHypercel is available from pall corporation (PortWashingtonNY, USA).

Analytical chromatography of the ligand was performed on shimadzu hplcsystem (shimadzu deutschlandgmbh, Duisburg/Germany) including a diode array detector and a single quadrupole mass spectrometer. Monolithic C18 reverse phase columns were purchased from MerckkKGaA (Darmstadt/Germany). The solvents used for the analysis were of mass spectral order.

The antibodies used in the present invention are bevacizumab (Avastin, f. hoffmann-LaRoche, Switzerland), tositumumab (RoActemra, f. hoffmann-LaRoche, Switzerland), palivizumab (Synagis, Abbott, USA), poly IgG from human serum (Sigma-Aldrich, USA) and poly IgG from rabbit serum (Sigma-Aldrich, USA). Immobilized papain for preparation of antibody fragments was purchased from ThermoScientific (Bonn/Germany).

The flow-through of the protein a chromatography (fiowthrough), referred to as host cell protein, was derived from the supernatant of antibody-producing CHO cell cultures in serum-free medium.

Coomassie Brilliant blue stain for Bradford assay was purchased from Thermoscientific (Bonn/Germany).

Example 1

SPR screening of chemical microarrays

Graffity developed high density chemical microarrays that contained thousands of small molecules immobilized on gold chips. The array was constructed using maleimide-thiol coupled chemistry in conjunction with high density pin spotting (pintolspoting). For the construction, the glass plate was microstructured by means of photolithography (photolithographical) to define up to 9216 sensor areas per array. The subsequently applied gold coating may provide the basis for the SPR effect and enable the formation of a binary, mixed self-assembled monolayer (SAM) of two different thiol groups. One of the thiol groups presents a reactive maleimide moiety to which thiol-labeled array compounds can be coupled during stitch spotting. The resulting microarray includes 9216 sensor areas each containing multiple copies of defined and controlled quality array compounds.

Surface plasmons are collective oscillations of electrons at a metal surface that can be resonantly excited by polarized light of appropriate wavelength and angle of incidence. Under the condition of surface plasmon resonance, the intensity of light reflected from the gold chip exhibits a sharp attenuation (SPR minimum). The angle and wavelength position of this reflection minimum strongly depend on the refractive index of the medium adjacent to the metal surface. Thus, processes that change the local refractive index in close proximity to the metal surface (e.g., binding of an antibody to an immobilized ligand) can be sensitively monitored. For SPR detection, the chemical microarray is incubated with the target protein under optimized screening conditions. If the target binds to the immobilized fragment, a shift in the wavelength-dependent SPR minimum relative to the buffer control (referred to as SPR shift) occurs and thus indicates protein-ligand interaction. SPR imaging methods utilize an expanded beam of parallel light to illuminate the entire microarray sensor area at a fixed angle. The reflected light is then captured by means of a CCD camera. The reflectance images are recorded in steps as they are scanned over a range of wavelengths encompassing the SPR resonance condition. The automated spot finding procedure and subsequent grey scale analysis of the acquired images produced SPR resonance curves for all 9216 sensor areas per chip in parallel.

Screening of antibodies and fragments thereof by SPR

This platform allows screening proteins, antibodies and fragments thereof in a high throughput manner against a library of graffity immobilized compounds of 110,000 small molecules. For each target, SPR screening conditions were optimized individually before target screening was performed against the entire library. Wherein the optimized screening parameters are a) the composition of the screening buffer comprising a suitable detergent, b) the concentration of the antibody or protein target in solution, and c) the surface density of the immobilized ligand on the chip surface. Neumannetal (1) summarizes the targets screened and the corresponding screening conditions applied. A total of 4 different full length antibodies were screened and compared. In addition, Fc and Fab fragments of 2 antibodies were obtained by proteolytic digestion and then also subjected to array screening. For the recognition of antibody-specific ligands, the screening procedure also included antibody-free CHO cell line Host Cell Protein (HCP) as a control.

The experimental results of the microarray screening activities were analyzed by manual hit selection and in-depth data analysis by commercially available data mining tools using an in-house developed software flow. This analysis yielded a series of hits (hitgerie) that showed significant binding to the antibody and its Fc fragment, while binding to the HCP control was only weak to negligible. These first hit series are used for hit confirmation for the second measurement, and can be used as a starting point for synthesizing a focus library (focusedlibrary) in the vicinity of the first hit series. For example, Fc-specific ligand L1 showed clear binding to full-length bevacizumab and its Fc fragment, while only minor binding to bevacizumab Fab fragment. In addition, it clearly binds to the other full length antibodies anti hdigg, toslizumab and palivizumab.

Example 2

Synthesis of ligands

General procedure a: synthesis of 5-Arylcarboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L1, L2, L3, L6, L8, L7, L15, L16, L5, L11, L20, L27, L25, L4, L26, L31, L32, L33, L34, L35, L36, L42, L44, L45, L46, L47, L48, L51, L56, L57, L63)

N-Fmoc-5-amino-2-methoxybenzoic acid (0.2-0.5mmol) and HOAt (1 equivalent relative to the carboxylic acid) were dissolved in DMF, NMP, a DMF/DMSO mixture or a NMP/DMSO mixture (1-3mL) and treated with DIC (1 equivalent relative to the carboxylic acid). After stirring for 2-5 minutes, the mixture was added to 0.1-0.15mmol of 1, 3-diaminopropane with trityl-polystyrene resin or 2-chlorotrityl-polystyrene resin. The mixture was shaken for several hours or overnight. Next, the resin was washed (DMF or DMF, dichloromethane or DMF, methanol, dichloromethane, several times with each solvent) and then treated with 25% piperidine in DMF (1-5mL) for 15-30 minutes. Subsequently, the resin was washed thoroughly (DMF, dichloromethane or DMF, methanol, dichloromethane, several times with each solvent) and then air-dried with a stream of nitrogen or dried under high vacuum.

Aryl carboxylic acid (0.2-0.5mmol) and HOAt (1 equivalent relative to the carboxylic acid) were dissolved in DMF, NMP, a DMF/DMSO mixture, or a NMP/DMSO mixture (1-3mL) and treated with DIC (1 equivalent relative to the carboxylic acid). After stirring for 2-5 minutes, the mixture was added to the resin and shaken for several hours or overnight. After subsequent washing (DMF, dichloromethane or DMF, methanol, dichloromethane, several times in each solvent), the target compound is cleaved from the support by treatment with a suitable cleavage mixture (85: 10: 5 dichloromethane, TFA, triethylsilane for trityl resins, 45: 10 for 2-chlorotrityl resins). Typically, the cleavage step is repeated once and the resin is then rinsed with dichloromethane. After evaporation of the solvent, the crude residue was purified by preparative reverse phase HPLC.

Due to the high stability of the amide coupling chemistry, other coupling methods can be applied to obtain similar or identical results. In particular, the DIC/HOAt coupling can be reliably replaced by TBTU or HATU couplings (1 equivalent relative to the carboxylic acid; 2 equivalents of DIPEA must additionally be added), which proceed more rapidly (typical reaction times: 30 minutes to 3 hours); however, for certain carboxylic acids, especially those having a free aryl NH, such as indole or (benzo) pyrazole, coupling under basic conditions may lead to unwanted side products.

Prepared according to general procedure a from 5- (2-furyl) -1-methylpyrazole-3-carboxylic acid. ESI-MS: 398(M + 1).

Prepared according to general procedure a from 5- (4-bromo-2-thienyl) -1-methylpyrazole-3-carboxylic acid. ESI-MS: 493, 495(M + 1).

Prepared according to general procedure a from 5- (2, 5-dimethyl-3-thienyl) -1-methylpyrazole-3-carboxylic acid. ESI-MS: 442(M + 1).

Prepared according to general procedure a from 1-methyl-5- (2-thienyl) pyrazole-3-carboxylic acid. ESI-MS: 414(M + 1).

Prepared according to general procedure a from 1-methyl-5- (3-thienyl) pyrazole-3-carboxylic acid. ESI-MS: 414(M + 1).

Prepared according to general procedure a from 5- (3-chlorophenyl) -1-methylpyrazole-3-carboxylic acid. ESI-MS: 442, 444(M + 1).

2-methoxy-5- (1-methyl-5-phenylpyrazol-3-yl) formyloxybenzoic acid N- (3-aminopropyl) amide (L15)

Prepared according to general procedure a from 1-methyl-5-phenylpyrazole-3-carboxylic acid. ESI-MS: 408(M + 1).

Prepared according to general procedure a from 5- (1-methyl-2-pyrrolyl) -1-methylpyrazole-3-carboxylic acid. ESI-MS: 411(M + 1).

Prepared according to general procedure a from 3- (2-thienyl) pyridine-5-carboxylic acid. ESI-MS: 411(M + 1).

Prepared according to general procedure a from 3-phenylpyridine-5-carboxylic acid. ESI-MS: 405(M + 1).

Prepared from 5- (2-furyl) pyrazole-3-carboxylic acid according to general procedure a. ESI-MS: 384(M + 1).

Prepared from 1-methylindazole-3-carboxylic acid according to general procedure a. ESI-MS: 382(M + 1).

Prepared from 1-methylindole-3-carboxylic acid according to general procedure a. ESI-MS: 381(M + 1).

Prepared from 5-chloro-1H-indazole-3-carboxylic acid according to general procedure a. ESI-MS: 402, 404(M + 1).

Prepared from indole-3-carboxylic acid according to general procedure a. Indole-3-carboxylic acid was coupled by activation with HOBt (1 equivalent) and DIC (1 equivalent relative to carboxylic acid); coupling time: for 1 hour. ESI-MS: 367(M + 1).

Prepared according to general procedure a from benzo [ c ] isoxazole-3-carboxylic acid. ESI-MS: 369(M + 1).

Prepared according to general procedure A from benzo [ c ] furan-1-carboxylic acid. ESI-MS: 368(M + 1).

Prepared according to general procedure a from 1, 5-dimethylpyrazole-3-carboxylic acid. ESI-MS: 346(M + 1).

Prepared according to general procedure a from 1-methyl-5- (trifluoromethyl) pyrazole-3-carboxylic acid. ESI-MS: 400(M + 1).

Prepared from isoquinoline-1-carboxylic acid according to general procedure a. ESI-MS: 379(M + 1).

Prepared from indazole-3-carboxylic acid according to general procedure a. Indazole-3-carboxylic acid was coupled by activation with HOBt (1 equivalent) and DIC (1 equivalent relative to carboxylic acid); coupling time: for 1 hour. ESI-MS: 368(M + 1).

Prepared according to general procedure a from 5- (2-methylthiazol-4-yl) isoxazole-3-carboxylic acid. ESI-MS: 416(M + 1).

Prepared according to general procedure a from 1-methylpyrazolo [3, 4-b ] pyridine-3-carboxylic acid. ESI-MS: 383(M + 1).

Prepared from 5- (2-furyl) isoxazole-3-carboxylic acid according to general procedure a. ESI-MS: 385(M + 1).

Prepared according to general procedure a from pyrazolo [1, 5-a ] pyridine-2-carboxylic acid. ESI-MS: 368(M + 1).

Prepared from 1H-benzimidazole-2-carboxylic acid according to general procedure a. ESI-MS: 368(M + 1).

Prepared from imidazo [1, 2-b ] pyridazine-2-carboxylic acid according to general procedure a. ESI-MS: 369(M + 1).

Prepared according to general procedure a from 3-phenylisoxazole-5-carboxylic acid. ESI-MS: 395(M + 1).

Prepared according to general procedure a from pyrazolo [1, 5-a ] pyrimidine-2-carboxylic acid. ESI-MS: 369(M + 1).

Prepared from imidazo [1, 2-a ] pyridine-2-carboxylic acid hydrobromide according to general procedure a. Additional 1 equivalent of DIPEA was added when HATU/DIPEA was used to couple the formic acid hydrobromide. ESI-MS: 368(M + 1).

Prepared according to general procedure a from imidazo [1, 2-a ] pyrimidine-2-carboxylic acid. ESI-MS: 369(M + 1).

General procedure B: synthesis of 5- [3- (aryl or cyclopropyl) pyridin-5-yl ] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L12, L13, L14, L22, L23) by Suzuki coupling

N-Fmoc-5-amino-2-methoxybenzoic acid and 5-bromopyridine-3-carboxylic acid were coupled with 0.1mmol of 1, 3-diaminopropane-trityl-polystyrene resin according to general procedure A. The resulting resin loaded with bromopicolinic acid amide was thoroughly dried and then transferred to a glass vial. After addition of cesium carbonate or potassium carbonate (0.3mmol), boric acid (1mmol) and anhydrous DMF (1mL) were added, respectively, and the mixture was purged thoroughly with argon. Subsequently, tetrakis-triphenylphosphine palladium (0) (10. mu. mol) was added, the mixture was again flushed with argon, and the vial was sealed. The mixture was heated to 100 ℃ and shaken overnight. The resin was then washed thoroughly (DMF, dichloromethane, several times with each solvent) and a small sample was cut (conditions see below). When LCMS indicates incomplete conversion, the coupling step is repeated or performed at a higher temperature. In addition, the resin was treated with dichloromethane-TFA-triethylsilane 85: 10: 5 (double cleavage) and then the resin was washed with dichloromethane. After evaporation to dryness, the residue was purified by preparative reverse phase HPLC.

Prepared from 4- (trifluoromethyl) phenylboronic acid according to general procedure B. ESI-MS: 473(M + 1).

Prepared from 2-furylboronic acid according to general procedure B. ESI-MS: 395(M + 1).

Prepared from 2-benzothienylboronic acid according to general procedure B. ESI-MS: 461(M + 1).

Prepared from 3-furylboronic acid according to general procedure B. ESI-MS: 395(M + 1).

Prepared from cyclopropylboronic acid according to general procedure B. ESI-MS: 369(M + 1).

N-Fmoc-5-amino-2-methoxybenzoic acid and 5-bromopyridine-3-carboxylic acid were coupled with 0.1mmol of 1, 3-diaminopropane-trityl-polystyrene resin according to general procedure A. The resulting resin loaded with bromopicolinic acid amide was thoroughly dried and then transferred to a glass vial. Copper (I) chloride (40. mu. mol), triphenylphosphine (40. mu. mol), bis (triphenylphosphine) palladium (II) dichloride (10. mu. mol) and THF (1mL) were added, and the mixture was flushed with argon. Subsequently, trimethylsilylacetylene (1mmol) and triethylamine (0.5mL) were added, the mixture was again flushed with argon, then the vial was sealed and shaken overnight at 50 ℃. The resin was then washed thoroughly with DMF and dichloromethane (several times with each solvent). For TMS deprotection, THF (1mL) and water (50 μ L) were added followed by tetrabutylammonium fluoride (1M solution in THF, 0.5 mL). The mixture was stirred for 2 hours, then the resin was washed with DMF and dichloromethane (several times with each solvent). Cleavage of the target compound from the resin was performed by treatment with dichloromethane-TFA-triethylsilane 85: 10: 5 (double cleavage) followed by washing with dichloromethane. After evaporation to dryness, the residue was purified by preparative reverse phase HPLC. ESI-MS: 353(M + 1).

General procedure C: synthesis of 5- [5- (2-thienyl or 2-furyl) -1-methylpyrazol-3-yl ] formylcarbamic acid N- (3-aminopropyl) amide (L19, L9, L10, L17, L18)

Each (N-Fmoc-protected) aminoarylcarboxylic acid (0.2-0.5mmol) and HOAt (1 equivalent relative to carboxylic acid) was dissolved in DMF, NMP, a DMF/DMSO mixture, or a NMP/DMSO mixture (1-3mL) and treated with DIC (1 equivalent relative to carboxylic acid). After stirring for 2-5 minutes, the mixture was added to 0.1-0.15mmol of 1, 3-diaminopropane attached to trityl-polystyrene resin or 2-chlorotrityl-polystyrene resin. The mixture was shaken for several hours or overnight. The resin was then washed (several times with DMF or DMF, dichloromethane or DMF, methanol, dichloromethane, each solvent) and treated with 25% piperidine in DMF (1-5mL) for 15-30 min. Subsequently, the resin was washed thoroughly (DMF, dichloromethane or DMF, methanol, dichloromethane, several times with each solvent) and air dried with a stream of nitrogen or dried under high vacuum.

1-methyl-5- (2-thienyl) pyrazole-3-carboxylic acid or 5- (2-furyl) -1-methylpyrazole-3-carboxylic acid (0.2-0.5mmol) and HOAt (1 equivalent relative to carboxylic acid) were dissolved in DMF, NMP, a DMF/DMSO mixture or a NMP/DMSO mixture (1-3mL) and treated with DIC (1 equivalent relative to carboxylic acid). After stirring for 2-5 minutes, the mixture was added to the resin and shaken for several hours or overnight. After subsequent washing (DMF, dichloromethane or DMF, methanol, dichloromethane, several times in each solvent), the target compound is cleaved from the support by treatment with a suitable cleavage mixture (85: 10: 5 dichloromethane, TFA, triethylsilane for trityl resins and 45: 10 dichloromethane, TFA, triethylsilane for 2-chlorotrityl resins). Typically, the cleavage step is repeated once and the resin is then rinsed with dichloromethane. After evaporation of the solvent, the crude residue was purified by preparative reverse phase HPLC.

Prepared according to general procedure C from N-Fmoc-2-aminothiazole-4-carboxylic acid and 1-methyl-5- (2-thienyl) pyrazole-3-carboxylic acid. ESI-MS: 391(M + 1).

Prepared according to general procedure C from N-Fmoc-3-amino-2-methylbenzoic acid and 1-methyl-5- (2-thienyl) pyrazole-3-carboxylic acid. ESI-MS: 398(M + 1).

Prepared according to general procedure C from N-Fmoc-3-amino-4-methylbenzoic acid and 1-methyl-5- (2-thienyl) pyrazole-3-carboxylic acid. ESI-MS: 398(M + 1).

Prepared according to general procedure C from N-Fmoc-3-aminobenzoic acid and 5- (2-furyl) -1-methylpyrazole-3-carboxylic acid. ESI-MS: 368(M + 1).

Prepared according to general procedure C from N-Fmoc-4-aminobenzoic acid and 5- (2-furyl) -1-methylpyrazole-3-carboxylic acid. ESI-MS: 368(M + 1).

1, 3-diaminopropane-trityl resin (0.1mmol) was swollen in dichloromethane and then treated with excess DIPEA and isophthaloyl chloride. After a few minutes, the resin was washed rapidly with dichloromethane (three times) and immediately treated with excess 3-amino-5- (2-furyl) pyrazole in NMP and DIPEA. When LCMS of a small sample of the resin cleaved (conditions see below) showed complete conversion, the resin was washed (DMF, dichloromethane, several times each solvent) and treated with dichloromethane-TFA-triethylsilane (85: 10: 5; double cleavage followed by washing of the resin with dichloromethane). The cleavage solution was evaporated to dryness and the residue was purified by preparative reverse phase HPLC. ESI-MS: 354(M + 1).

The 1, 3-diaminopropane-trityl-resin (0.15mmol) was pre-swollen in NMP. N-Fmoc-5-amino-2-methoxybenzoic acid (0.2mmol) and HATU (0.2mmol) were dissolved in NMP (1.5mL) and treated with DIPEA (0.4 mmol). After 2 minutes, the solution was added to the resin and the reaction mixture was shaken for 30 minutes. After washing the resin with DMF (three times), DMF containing 25% piperidine was added and shaking was continued for 30 minutes. The resin was then washed thoroughly with DMF, methanol and dichloromethane (three times for each solvent) and then dried in air.

Imidazo [2, 1-b ] thiazole-6-carboxylic acid hydrobromide (0.2mmol) and HATU (0.2mmol) were dissolved or resuspended in NMP (1.5mL) and treated with DIPEA (0.8 mmol). After 2 minutes, the solution was added to the resin and shaken overnight. After washing the resin with DMF, methanol and dichloromethane (three times for each solvent), the target compound was cleaved from the resin by treatment with dichloromethane-TFA-triethylsilane 85: 10: 5 (double cleavage) followed by washing with dichloromethane. After evaporation to dryness, the residue was purified by preparative reverse phase HPLC.

ESI-MS：374(M+1)。

The 2-chlorotrityl chloride resin (150. mu. mol) was pre-swollen in NMP and treated with NMP (1.5mL) followed by 3-aminopropanol (1.5 mL). After shaking the resin for 2 hours, it was washed with DMF, methanol and dichloromethane (several times with each solvent) and dried in air. Then, dichloromethane (2mL) and DIPEA (2mmol) were added, followed by dropwise addition of methanesulfonyl chloride (1 mmol). The resin was stirred for 2 hours, then washed rapidly with dichloromethane (three times) and swollen in NMP (1 mL). After addition of methylamine solution (8M in ethanol, 1mL), the resin was shaken overnight, then washed with DMF, methanol and dichloromethane (several times with each solvent) and dried in air.

N-Fmoc-5-amino-2-methoxybenzoic acid (250. mu. mol) and HOAt (250. mu. mol) were dissolved in NMP (ca. 1.5mL) and treated with DIC (250. mu. mol). After 2 minutes, the solution was added to the resin and shaken for 5 hours. After washing with DMF and dichloromethane (several times for each solvent), the resin was treated with DMF containing 25% piperidine (30 min, then washed as before).

5- (2-furyl) -1-methylpyrazole-3-carboxylic acid (250. mu. mol) and HOAt (250. mu. mol) were dissolved in NMP (about 1.5mL) and treated with DIC (250. mu. mol). After 2 minutes, the solution was added to the resin and shaken overnight. The resin was then washed as before and the target compound cleaved from the resin by treatment with dichloromethane-TFA-triethylsilane 45: 10 (double cleavage) followed by washing with dichloromethane. After evaporation to dryness, the residue was purified by preparative reverse phase HPLC. ESI-MS: 412(M + 1).

5-Nitrosalicylic acid (0.3mmol) and HOBt (0.3mmol) were dissolved in DMSO (about 1.5mL) and treated with DIC (0.3 mmol). After 2 min, the solution was added to 1, 3-diaminopropane-trityl resin (0.15mmol) and shaken for 1h, then the resin was washed with DMF, water, dilute sodium carbonate solution, water, methanol and dichloromethane (several times for each solvent). A small sample of the resin was treated with benzoyl chloride and DIPEA, washed and cleaved (conditions see below), and the resulting product was analyzed by HPLC-MS, indicating incomplete loading. Thus, the coupling step was repeated for 30 minutes. To hydrolyze the amount of carbamoyl-nitrophenyl ester formed concurrently, the resin was then treated with THF (1mL), methanol (0.5mL), and aqueous NaOH (1M, 0.5mL) for 5 minutes, indicating successful ester hydrolysis when the supernatant turned yellow. After washing with DMF, methanol and dichloromethane (three times for each solvent), DMSO (2mL), cesium carbonate (1mmol) and bromoethane (1mmol) were added and the resin was shaken for about 2 hours, then the reaction was repeated overnight (0.2mL of water was added to dissolve the carbonate). Because the conversion was still incomplete, the reaction was repeated using 0.5mL of bromoethane and water (0.2mL) so that the reaction proceeded well after three days, and then the resin was washed with DMF, water, methanol and dichloromethane (several times with each solvent).

The nitro group was reduced by the addition of NMP (1mL), pyridine (0.5mL) and a solution of tin (II) chloride (1mmol) in NMP (1 mL). The mixture was shaken overnight, then the resin was washed with DMF, water and methanol, and insoluble byproducts were carefully removed by flotation in methanol. After washing with dichloromethane, the resin was dried.

5- (2-furyl) -1-methylpyrazole-3-carboxylic acid (0.25mmol) and HOAt (0.25mmol) were dissolved in NMP (about 1.5mL) and treated with DIC (0.25 mmol). After 2 minutes, the solution was added to the resin and shaken overnight. The resin was then washed with DMF and dichloromethane (several times with each solvent) and the target compound was cleaved from the resin by treatment with dichloromethane-TFA-triethylsilane 85: 10: 5 (double cleavage) followed by washing with dichloromethane. After evaporation to dryness, the residue was purified by preparative reverse phase HPLC. ESI-MS: 412(M + 1).

5-Nitrosalicylic acid (0.3mmol) and HOBt (0.3mmol) were dissolved in DMSO (about 1.5mL) and treated with DIC (0.3 mmol). After 2 min, the solution was added to 1, 3-diaminopropane-trityl resin (0.15mmol) and shaken for 1h, then the resin was washed with DMF, water, dilute sodium carbonate solution, water, methanol and dichloromethane (several times for each solvent). A small sample of the resin was treated with benzoyl chloride and DIPEA, washed and cleaved (conditions see below), and the resulting product was analyzed by HPLC-MS, indicating incomplete loading. Thus, the coupling step was repeated for 30 minutes. To hydrolyze the amount of carbamoyl-nitrophenyl ester formed concurrently, the resin was then treated with THF (1mL), methanol (0.5mL), and aqueous NaOH (1M, 0.5mL) for 5 minutes, indicating successful ester hydrolysis when the supernatant turned yellow. After washing with DMF, methanol and dichloromethane (three times for each solvent), DMSO (2mL), cesium carbonate (1mmol) and allyl bromide (1mmol) were added and the resin shaken for 1 hour, then the reaction was repeated overnight (0.2mL water was added). Since the conversion was still incomplete, the reaction was repeated again for three days, and then the resin was washed with DMF, water, methanol and dichloromethane (several times with each solvent).

5- (2-furyl) -1-methylpyrazole-3-carboxylic acid (0.25mmol) and HOAt (0.25mmol) were dissolved in NMP (about 1.5mL) and treated with DIC (0.25 mmol). After 2 minutes, the solution was added to the resin and shaken overnight. The resin was then washed with DMF and dichloromethane (several times with each solvent).

The allyl ether was cleaved by treating the resin with dichloromethane (2mL), piperidine (0.4mL), and tetrakis-triphenylphosphine palladium (0) (about 10mg) for about 2 hours. The resin was then washed thoroughly with DMF, water, methanol and dichloromethane (several times with each solvent), and the target compound was cleaved from the resin by treatment with dichloromethane-TFA-triethylsilane 85: 10: 5 (double cleavage) followed by washing with dichloromethane. After evaporation to dryness, the residue was purified by preparative reverse phase HPLC. ESI-MS: 384(M + 1).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen with NMP. A solution of Fmoc-5-amino-2-methoxybenzoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol). After 2 minutes, the solution was added to the resin and the mixture was shaken at room temperature for 3 hours. After washing the resin with DMF, piperidine (25% in DMF, about 2mL) was added, the resin was shaken for about 30 minutes, and then washed thoroughly with DMF and dichloromethane. Indazole-3-carboxylic acid (0.5mmol) and HOBt (0.5mmol) were solubilized in NMP (1.5 mL). DIC (0.5mmol) was added and after 2 min the solution was added to the resin. The mixture was shaken for 1 hour, and then the resin was washed with DMF and dichloromethane. Cesium carbonate (1mmol) and dry DMF (1.5mL) were added to the resin followed by ethyl bromide (1.5 mmol). The mixture was shaken overnight and then washed with DMF, water, methanol and dichloromethane. After three days the cesium carbonate/ethyl bromide procedure was repeated once and the resin was then washed thoroughly. The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 396(M + 1).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen in NMP. 5-formyl salicylic acid (0.5mmol) and HOAt (0.5mmol) were dissolved in NMP (1.5mL) and treated with DIC (0.5 mmol). After 2 min, the solution was added to the resin and the mixture was stirred for 2h, then the resin was washed several times with DMF and DCM. The resin was then treated with THF (1mL), methanol (1mL) and aqueous NaOH (2M, 0.5mL) at room temperature for 1 hour, followed by washing the resin with methanol/water, DMF, methanol/water, DMF and DCM. After addition of acetonitrile (1mL), t-butanol (1mL) and 2-methyl-2-butene (200. mu.L), the resin was cooled to 0 ℃ and treated dropwise with an aqueous solution (0.2mL) of sodium chlorite (0.25mmol) and sodium monohydrogen phosphate (0.2 mmol). After 30 minutes, acetonitrile (1mL) and tert-butanol (1mL) were added, an aqueous solution (0.4mL) of sodium chlorite (1mmol) and sodium monohydrogen phosphate (0.8mmol) was added dropwise, and the mixture was allowed to warm to room temperature. The resin was washed with methanol/water, DMF, methanol/water, DMF and DCM. After thorough drying, triphenylphosphine (1mmol), THF (2mL) and methanol (0.2mL) were added followed by diethyl azodicarboxylate (1 mmol). The resin was shaken at room temperature for 1 hour and then washed several times with DMF and dichloromethane. THF (1mL), methanol (1mL) and aqueous NaOH (2M, 0.5mL) were added, the mixture was shaken at room temperature for 4 days, and the resin was then washed with methanol/water, DMF, methanol/water, DMF and DCM. A solution of HATU (0.25mmol) and DIPEA (0.5mmol) in NMP (1mL) was added and the mixture was shaken at room temperature for 15 min. Then, a solution of 3-amino-1-methylindazole (0.25mmol) in NMP (0.5mL) was added and the reaction mixture was shaken overnight and then washed with DMF and dichloromethane. The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 382(M + 1).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen with NMP. A solution of Fmoc-5-amino-2-methoxybenzoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol). After 2 minutes, the solution was added to the resin and the mixture was shaken at room temperature for 3 hours. After washing the resin with DMF, piperidine (25% in DMF, about 2mL) was added, the resin was shaken for about 30 minutes, and then washed thoroughly with DMF and dichloromethane. Indazole-3-carboxylic acid (0.5mmol) and HOBt (0.5mmol) were dissolved in NMP (1.5 mL). DIC (0.5mmol) was added and after 2 min the solution was added to the resin. The mixture was shaken for 1 hour, and then the resin was washed with DMF and dichloromethane. Cesium carbonate (1mmol) and dry DMF (1.5mL) were added to the resin followed by allyl bromide (1 mmol). The mixture was shaken overnight and then washed with DMF, water, methanol and dichloromethane. The cesium carbonate/allyl bromide procedure was repeated once overnight, and then the resin was washed thoroughly, dried and transferred to a glass vial. A solution of methylmorpholine N-oxide (0.5mmol) in acetone (2mL) and water (0.5mL) were added to the resin. Osmium (VIII) oxide (50 μ L of a 2.5% solution in tert-butanol) was added, the vial was sealed and shaken overnight at room temperature. After adding another portion of osmium (VIII) oxide (50. mu.L of a 2.5% solution in tert-butanol) and shaking for a further 2 hours, the reaction was quenched with an aqueous solution of sodium dithionite (174 mg in 1mL of water) and shaken for 30 minutes. Subsequently, the resin was washed several times with water, DMF, water, methanol, DMF and dichloromethane. The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 442(M + 1).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen with NMP. A solution of Fmoc-5-amino-2-methoxybenzoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol). After 2 minutes, the solution was added to the resin and the mixture was shaken at room temperature for 2 hours. After washing the resin with DMF and dichloromethane, piperidine (25% in DMF, about 2mL) was added, the resin was shaken for about 30 minutes, and then thoroughly washed with DMF and dichloromethane. The resin was swollen in dichloromethane (1mL), DIPEA (1mmol) was added, followed by a solution of oxalic acid monoethyl ester chloride (0.5mmol) in dichloromethane (1 mL). After shaking for 5 minutes, the resin was washed with DMF, methanol and dichloromethane (three times for each solvent). A solution of 2, 3-diaminopyridine (0.5mmol) in NMP (2mL) was added and the mixture was shaken at about 100 ℃ for 4 days. After washing the resin (DMF, methanol, dichloromethane), the reaction was repeated overnight at about 120 ℃. After washing as before, THF (1mL), methanol (0.5mL) and aqueous NaOH (2M, 0.5mL) were added to the resin and the mixture was stirred for 15 minutes, then the resin was washed with methanol (5 times) and dichloromethane (three times). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 369(M + 1).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen in NMP. A solution of Fmoc-5-amino-2-methoxybenzoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol), and after 2 minutes the mixture was added to the resin and stirred for 3 hours. After washing the resin with DMF, piperidine (25% in DMF, ca. 2mL) was added and the resin was shaken for 30 minutes. Subsequently, the resin was washed thoroughly with DMF and dichloromethane and swelled in dichloromethane. Dichloromethane (1.5mL), DIPEA (1mmol) and oxalic acid monoethyl chloride (0.5mmol) were added, the mixture was stirred for 10 min, and then the resin was washed several times with DMF and dichloromethane. Ethylene glycol (1mL), 2-pyridylhydrazine (1mmol) and DIPEA (1mmol) were added, the mixture was heated to 100 ℃ for 4 hours, and then the resin was washed several times with DMF and dichloromethane. Triphenylphosphine (0.5mmol), N-methylmorpholine (0.5mmol) and dichloromethane (2mL) were added to the resin, followed by dropwise addition of trichloroacetonitrile (0.4 mmol). After the mixture was shaken for 48 hours, triphenylphosphine (0.5mmol) and a solution of N-methylmorpholine (1mmol) in dichloromethane (2mL) were added along with carbon tetrachloride (0.5 mL). The mixture was held at 40 ℃ for 2 hours, then THF (2mL) and an aqueous solution of sodium carbonate (2M, 0.5mL) were added and the mixture was stirred overnight. Finally, the resin was washed thoroughly with DMF, water, methanol and dichloromethane. The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 369(M + 1).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen in NMP. Then (S) -bis-Fmoc-lysine (200 μmol) and HATU (200 μmol) were dissolved in NMP (about 1.5mL) and treated with DIPEA (400 μmol) for 2 minutes, then the solution was added to the resin and the mixture was stirred at room temperature. After 1 hour the resin was washed with DMF, piperidine (25% in DMF, ca. 2mL) was added and the resin was stirred for 30 minutes. The resin was then washed (DMF, methanol and dichloromethane). Fmoc-5-amino-2-methoxybenzoic acid (400. mu. mol) and HATU (400. mu. mol) were dissolved in NMP (ca. 2mL) and treated with DIPEA (800. mu. mol) for 2 min, then the solution was added to the resin and the mixture was shaken for 1 h. After washing the resin with DMF, deprotection was accomplished by treatment with 25% piperidine in DMF followed by washing as described previously. Imidazo [2, 1-b ] thiazole-6-carboxylic acid (400. mu. mol) and HATU (400. mu. mol) were dissolved in NMP (ca. 2mL) and treated with DIPEA (800. mu. mol) for 2 minutes, and then the solution was added to the resin. After shaking the mixture for 1 hour, the resin was washed with DMF, methanol and dichloromethane (three times for each solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 801(M +1), 401((M + 2)/2).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen in NMP. Then, (S) -bis-Fmoc-lysine (200. mu. mol) and HATU (200. mu. mol) were dissolved in NMP (about 1.5mL) and treated with DIPEA (400. mu. mol) for 2 minutes, then the solution was added to the resin, and the mixture was stirred at room temperature. After 1 hour the resin was washed with DMF, piperidine (25% in DMF, ca. 2mL) was added and the resin was stirred for 30 minutes. The resin was then washed (DMF, methanol and dichloromethane). Fmoc-8-amino-3, 6-dioxaoctanoic acid (400. mu. mol) and HATU (400. mu. mol) were dissolved in NMP (ca. 2mL) and treated with DIPEA (800. mu. mol) for 2 minutes, then the solution was added to the resin and the mixture was shaken for 1 hour. After washing the resin with DMF, deprotection was accomplished by treatment with 25% piperidine in DMF followed by washing as described previously. Fmoc-5-amino-2-methoxybenzoic acid (400. mu. mol) and HATU (400. mu. mol) were dissolved in NMP (ca. 2mL) and treated with DIPEA (800. mu. mol) for 2 min, then the solution was added to the resin and the mixture was shaken for 1 h. After washing the resin with DMF, deprotection was accomplished by treatment with 25% piperidine in DMF followed by washing as described previously. Imidazo [2, 1-b ] thiazole-6-carboxylic acid (400. mu. mol) and HATU (400. mu. mol) were dissolved in NMP (ca. 2mL) and treated with DIPEA (800. mu. mol) for 2 minutes, and then the solution was added to the resin. After shaking the mixture for 1 hour, the resin was washed with DMF, methanol and dichloromethane (three times for each solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 547((M + 2)/2).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen in NMP. A solution of Fmoc-5-amino-2-methoxybenzoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol) and after 2 min the solution was added to the resin. After shaking for 105 minutes, the resin was washed with DMF, followed by treatment with piperidine (25% in DMF, 2mL) for 1 hour, followed by thorough washing with DMF, methanol and dichloromethane. A solution (3mL) of 4-bromopyridine-2-carboxylic acid (0.3mmol) and HOAt (0.3mmol) in NMP was treated with DIC (0.3 mmol). After 2 minutes, the solution was added to the resin and the mixture was shaken for 1 hour, then the resin was washed as before, thoroughly dried with a stream of dry nitrogen and transferred to a glass vial. Cesium carbonate (0.3mmol) and 2-furylboronic acid (0.5mmol) were added along with DMF (2 mL). The mixture was flushed thoroughly with argon. After addition of tetrakis-triphenylphosphine palladium-0 (10. mu. mol), the mixture was again flushed with argon, then the vial was sealed, and the mixture was stirred at 100 ℃ overnight. The resin was then washed with DMF, water, methanol and dichloromethane (three times for each solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 395(M + 1).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen in NMP. Then, (S) -bis-Fmoc-lysine (200. mu. mol) and HATU (200. mu. mol) were dissolved in NMP (about 1.5mL) and treated with DIPEA (400. mu. mol) for 2 minutes, then the solution was added to the resin, and the mixture was stirred at room temperature. After 1 hour the resin was washed with DMF, piperidine (25% in DMF, ca. 2mL) was added and the resin was stirred for 30 minutes. The resin was then washed (DMF, methanol and dichloromethane). Fmoc-5-amino-3-oxapentanoic acid (400. mu. mol) and HATU (400. mu. mol) were dissolved in NMP (ca. 2mL) and treated with DIPEA (800. mu. mol) for 2 min, then the solution was added to the resin and the mixture was shaken for 1 h. After washing the resin with DMF, deprotection was accomplished by treatment with 25% piperidine in DMF followed by washing as described previously. Fmoc-5-amino-2-methoxybenzoic acid (400. mu. mol) and HATU (400. mu. mol) were dissolved in NMP (ca. 2mL) and treated with DIPEA (800. mu. mol) for 2 min, then the solution was added to the resin and the mixture was shaken for 1 h. After washing the resin with DMF, deprotection was accomplished by treatment with 25% piperidine in DMF followed by washing as described previously. Imidazo [2, 1-b ] thiazole-6-carboxylic acid (400. mu. mol) and HATU (400. mu. mol) were dissolved in NMP (ca. 2mL) and treated with DIPEA (800. mu. mol) for 2 minutes, and then the solution was added to the resin. After shaking the mixture for 1 hour, the resin was washed with DMF, methanol and dichloromethane (three times for each solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 503((M + 2)/2).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (450. mu. mol, ca. 0.5g) was pre-swollen in dichloromethane and then pre-swollen with NMP. A solution of Fmoc-5-amino-2-methoxybenzoic acid (0.6mmol) and HATU (0.6mmol) in NMP (4.5mL) was treated with DIPEA (1.2mmol) for 2 min. The solution was then added to the resin and the mixture was stirred for 40 minutes and then washed three times with DMF. After addition of piperidine (25% in DMF, about 6mL), the resin was stirred for 30 min, then washed with DMF, methanol and dichloromethane (three times for each solvent). After the resin was dried under high vacuum, dichloromethane (3mL) and DIPEA (3mmol) were added, and oxalic acid monoethyl chloride in dichloromethane (1.5mmol) was added carefully. After shaking the mixture for 2 minutes, the resin was washed with dichloromethane, methanol and dichloromethane (three times for each solvent). After the resin was dried, 150mg was taken out. The remaining amount was treated with THF (4mL), methanol (2mL) and aqueous NaOH (2M, 2mL) for 2 hours at room temperature. The resin was washed (DMF, acetic acid [ 2.5% in DMF ], methanol and dichloromethane; three times for each solvent). 150mg of the resin was reacted with a solution of HOAt (0.5mmol) and DIC (0.5mmol) in NMP (1mL) for 2 min. A solution of 4-methoxy-N-hydroxybenzamidine (0.5mmol) in NMP (1mL) was then added. After stirring the resin for 1 hour, DIC (0.5mmol) was added and the mixture was shaken overnight. After washing the resin with DMF, methanol and dichloromethane (three times for each solvent), the coupling step was repeated. 3 hours after the second addition of DIC, the resin was washed, dried and transferred to a glass vial as described previously. NMP (2mL) and DIC (0.5mmol) were added and the mixture was heated to 120 ℃ for 30 min. The resin was then washed as described previously. The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 426(M + 1).

Synthesis of N-hydroxy-2-furylformamidine: aqueous hydroxylamine (50%, 5mmol) was added to a stirred solution of 2-cyanofuran (5mmol) in ethanol (5mL) at which time a slight warming of the reaction mixture was observed. After about 20 minutes, additional aqueous hydroxylamine (0.5mmol) was added. After stirring for 1 hour, the solvent was evaporated with a stream of nitrogen and the residue was purified by flash column chromatography (NH)₂Modified stationary phase, dichloromethane with 0-5% methanol, 1% triethylamine) yielding a slightly cloudy oil which solidifies upon standing.

Synthesis of target compound: the dried 2-chlorotrityl chloride resin (150. mu. mol) was treated with 1, 3-diaminopropane (2mL) and then dichloromethane (2 mL). After 5 minutes the resin was washed with DMF, methanol and dichloromethane (three times for each solvent) and swollen in NMP. A solution of Fmoc-5-amino-2-methoxybenzoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol), and after 2 minutes the solution was added to the resin and shaken at room temperature for 30 minutes. The resin was washed with DMF (three times) and treated with piperidine (25% in DMF, about 4mL) for 30 minutes, then the resin was washed with DMF, methanol and dichloromethane (three times for each solvent) and swollen in dichloromethane. After addition of dichloromethane (2mL) and DIPEA (1mmol), a solution of oxalic acid monoethyl ester acid chloride (0.5mmol) in dichloromethane (1mL) was added carefully, the mixture was stirred for 5 minutes, and then the resin was washed with DMF, methanol and dichloromethane (three times with each solvent), dried and transferred to a glass vial. Potassium carbonate (0.5mmol), toluene (2mL) and N-hydroxy-2-furanylformamidine (0.5 mmol; synthesis: see above) were added, the vial was sealed and stirred overnight at 100 ℃ then at 110 ℃ for 2 hours, then the resin was washed with DMF, methanol and dichloromethane (three times for each solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (45%) and triethylsilane (10%) in dichloromethane, followed by thorough washing of the resin with dichloromethane and removal of the solvent with a stream of nitrogen. The residue was purified by preparative reverse phase HPLC. ESI-MS: 386(M + 1).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen with NMP and then excess solvent was removed. A solution of Fmoc-5-amino-2-methoxybenzoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol). After 2 minutes, the solution was added to the swollen resin and the mixture was shaken at room temperature for 2 hours. After washing the resin with DMF, piperidine (25% in DMF, about 2mL) was added and the resin was shaken for about 30 minutes and then washed thoroughly with DMF and dichloromethane. The resin was then swollen in dichloromethane and treated with oxalic acid monoethyl chloride (0.5mmol) and a solution of DIPEA (1mmol) in dichloromethane (1.5mL) for 10 min, then washed repeatedly with DMF and dichloromethane. After swelling the resin in THF (1.5mL), hydrazine hydrate (0.5mL) was added and the resin was stirred at room temperature for 3 hours and then washed with DMF and dichloromethane. Furan-2-carboxylic acid (0.5mmol) and HOAt (0.5mmol) were dissolved in NMP (1.5mL) and treated with DIC (0.5 mmol). After 2 minutes, the solution was added to the resin and the mixture was stirred at room temperature for 2 hours, then washed with DMF and dichloromethane. Subsequently, triphenylphosphine (0.5mmol) and a solution of N-methylmorpholine (1mmol) in dichloromethane (2mL) were added followed by carbon tetrachloride (0.5 mL). The mixture was heated to 40 ℃ for 2 hours in a fully closed glass vial. After addition of THF (2mL) and Na₂CO₃After an aqueous solution (2M, 0.5mL), the mixture was stirred at room temperature overnight and then washed with DMF, water, methanol and dichloromethane. By using in dichloro chlorideThe target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in methane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 386(M + 1).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen with NMP. A solution of Fmoc-5-amino-2-methoxybenzoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol). After 2 minutes, the solution was added to the resin and the mixture was shaken at room temperature for 1 hour. After washing the resin with DMF, piperidine (25% in DMF, about 2mL) was added and the resin was shaken for about 1 hour and then washed thoroughly with DMF, methanol and dichloromethane. Indazole-3-carboxylic acid (0.5mmol) and HOBt (0.5mmol) were solubilized in NMP (1.5 mL). DIC (0.5mmol) was added and after 2 min the solution was added to the resin. The mixture was shaken for 40 minutes, then the resin was washed with DMF, methanol and dichloromethane (three times for each solvent). The resin was transferred to a glass vial and a solution of methyl bromoacetate (0.5mmol) and DIPEA (1mmol) in NMP (2mL) was added. The vial was completely closed and shaken at 80 ℃ for 75 minutes. Subsequently, the resin was washed (DMF, methanol, dichloromethane, three times each solvent), swollen in THF (2mL) and methanol (0.5mL) and treated with sodium borohydride (large excess, split addition). After the last addition, the mixture was shaken at room temperature for 30 minutes, and then the resin was washed with methanol and dichloromethane (three times for each solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 412(M + 1).

Trityl polystyrene pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen in NMP. A solution of Fmoc-8-amino-3, 6-dioxaoctanoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol), and after 2 minutes the solution was added to the resin and shaken at room temperature for 1 hour, then washed with DMF (three times). Piperidine (25% in DMF, about 2mL) was added and the mixture was shaken for about 30 minutes. The resin was washed with DMF, methanol and dichloromethane (three times for each solvent). The coupling/deprotection method was then applied to Fmoc-5-amino-2-methoxybenzoic acid (same amount as before). Finally, the scheme for HATU coupling described above was used to couple 1-methylindazole-3-carboxylic acid. The resin was washed with DMF, methanol and dichloromethane (three times for each solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 527(M + 1).

Trityl polystyrene pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen in NMP. A solution of Fmoc-8-amino-3, 6-dioxaoctanoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol), and after 2 minutes the solution was added to the resin and shaken at room temperature for 1 hour, then washed with DMF (three times). Piperidine (25% in DMF, about 2mL) was added and the mixture was shaken for about 30 minutes. The resin was washed with DMF, methanol and dichloromethane (three times for each solvent). The coupling/deprotection protocol was then repeated with Fmoc-8-amino-3, 6-dioxaoctanoic acid, which was then applied to Fmoc-5-amino-2-methoxybenzoic acid (same amount as before). Finally, the scheme for HATU coupling described above was used to couple 1-methylindazole-3-carboxylic acid. The resin was washed with DMF, methanol and dichloromethane (three times for each solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 672(M + 1).

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen in NMP. Then (S) -bis-Fmoc-lysine (200 μmol) and HATU (200 μmol) were dissolved in NMP (about 1.5mL) and treated with DIPEA (400 μmol) for 2 minutes, then the solution was added to the resin and the mixture was stirred at room temperature. After 1 hour the resin was washed with DMF, piperidine (25% in DMF, ca. 2mL) was added and the resin was stirred for 30 minutes. Subsequently, the resin was washed (DMF, methanol and dichloromethane). Fmoc-8-amino-3, 6-dioxaoctanoic acid (400. mu. mol) and HATU (400. mu. mol) were dissolved in NMP (ca. 2mL) and treated with DIPEA (800. mu. mol) for 2 minutes, then the solution was added to the resin and the mixture was shaken for 1 hour. After washing the resin with DMF, deprotection was accomplished by treatment with 25% piperidine in DMF followed by washing as previously described. Fmoc-5-amino-2-methoxybenzoic acid (400. mu. mol) and HATU (400. mu. mol) were dissolved in NMP (ca. 2mL) and treated with DIPEA (800. mu. mol) for 2 min, then the solution was added to the resin and the mixture was shaken for 1 h. After washing the resin with DMF, deprotection was accomplished by treatment with 25% piperidine in DMF followed by washing as described previously. Imidazo [1, 2-b ] pyridazine-2-carboxylic acid (400. mu. mol) and HATU (400. mu. mol) were dissolved in NMP (ca. 2mL) and treated with DIPEA (800. mu. mol) for 2 min, then the solution was added to the resin. After shaking the mixture for 1 hour, the resin was washed with DMF, methanol and dichloromethane (three times for each solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 542((M + 2)/2).

Synthesis of methyl 5-amino-2-methoxybenzoate: methyl 2-methoxy-5-nitrobenzoate (1mmol) and palladium on activated carbon (0) (5%, 25mg [ containing about 50% w/w water ]) were treated dropwise with triethylsilane (1mL) in methanol (5mL) over 10 minutes, followed by methanol (2 mL). After stirring for a further 5 minutes, the mixture was filtered through celite and the celite layer was rinsed thoroughly. The filtrate was concentrated, dried under high vacuum and the residue was used without further purification.

Liquid phase synthesis of aminopropyl-linked ligands (same as L37): a solution of imidazo [2, 1-b ] thiazole-6-carboxylic acid (1mmol) and TBTU (1mmol) in NMP (2mL) was treated with DIPEA (2mmol) for 5 min. Subsequently, the mixture was added to the whole amount of methyl 5-amino-2-methoxybenzoate previously obtained, and the mixture was stirred for 30 minutes. After addition of saturated aqueous sodium carbonate (1mL) and water (3mL), the mixture was extracted four times with ethyl acetate. The combined organic layers were washed twice with aqueous citric acid (5%), once with saturated aqueous sodium carbonate, and twice with water. The remaining organic layer was concentrated and dried overnight with a stream of dry nitrogen. 1, 3-diaminopropane (2mL) was added to the residue and the mixture was heated to 80 ℃ for 4 hours. Subsequently, the solution was concentrated to dryness, the residue was redissolved in methanol, concentrated and dried under high vacuum to produce a cold solidified oil. Yield: 453 μmol (45%).

Synthesis of target compound: N-Boc-glutamic acid (227. mu. mol) and TBTU (453. mu. mol) were dissolved in DMF (2mL) and treated with DIPEA (906. mu. mol) for 3 minutes, and then the solution was added to the residue obtained previously. After 90 min, another portion of TBTU activated N-Boc-glutamic acid (0.2mmol) in DMF (1 mL; activated for 2 min as before) was added. After 45 minutes the solvent was evaporated with a stream of nitrogen overnight. The residue was redissolved in methanol, evaporated to dryness and treated with trifluoroacetic acid (25% in dichloromethane) then evaporated to dryness before the residue was purified by preparative reverse phase HPLC. ESI-MS: 858(M +1), 430((M + 2)/2).

General procedure D: synthesis of 5-amino-2-methoxybenzoic acid (base resin) linked to two adenosine units and one 1, 3-diaminopropane unit

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane was pre-swollen in DCM and NMP. A solution of Fmoc-8-amino-3, 6-dioxaoctanoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol) for 2 minutes, then the solution was added to the resin and the mixture was stirred for 30 minutes to several hours. After washing with DMF or DMF, methanol and dichloromethane, piperidine (25% in DMF, ca. 2mL) was added and the resin was stirred for at least 20 minutes. Subsequently, the resin was washed thoroughly with DMF, methanol and dichloromethane (three times for each solvent) and the coupling/deprotection steps were repeated once. Fmoc-5-amino-2-methoxybenzoic acid (200. mu. mol) was coupled to the resin using the protocol for HATU coupling described previously, followed by deprotection with piperidine as described previously. The resin was dried in air and then subjected to additional synthesis steps.

A solution of 5- (2-furyl) -1-methylpyrazole-3-carboxylic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol) for 2 min. Subsequently, the solution was added to the base resin obtained by general procedure D. After stirring the mixture for about 2 hours, the resin was washed (DMF, methanol, dichloromethane, three times for each solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 688(M + 1).

A solution of imidazo [2, 1-b ] thiazole-6-carboxylic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol) for 2 min. Subsequently, the solution was added to the base resin obtained by general procedure D. After stirring the mixture for about 2 hours, the resin was washed (DMF, methanol, dichloromethane, three times for each solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 664(M + 1).

The dried base resin obtained by general procedure D was treated with dichloromethane (2mL) and DIPEA (1mmol) and then oxalic acid monoethyl ester acid chloride (0.5mmol) in dichloromethane was added dropwise. The mixture was stirred at room temperature for 10 minutes, then the resin was washed with DMF, methanol and dichloromethane (three times for each solvent). Subsequently, the resin was swollen in NMP (2mL) and treated with hydrazine hydrate (1mmol) at room temperature for 50 minutes, followed by washing as before. A solution of 2-furoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol) for 2 minutes and then the solution was added to the resin. After about 1 hour the resin was washed as described previously. Then 4-tosylchloride (0.5mmol) and a solution of DIPEA (1mmol) in dichloromethane (ca. 2mL) were added and the mixture was stirred until the cyclization was complete. The resin was washed as described above. The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 676(M + 1).

A solution of 5- (2-methylthiazol-4-yl) isoxazole-3-carboxylic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol) for 2 min. Subsequently, the solution was added to the base resin obtained by general procedure D. After stirring the mixture for about 2 hours, the resin was washed (DMF, methanol, dichloromethane, three times for each solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 706(M + 1).

1, 8-diamino-3, 6-dioxaoctane (2mL) was added to 2-chlorotrityl chloride polystyrene resin (150. mu. mol). After shaking the mixture for 5 minutes, dichloromethane (1mL) was added to ensure adequate swelling. After 25 min, the resin was washed (DMF, methanol, dichloromethane, three times each solvent). The resin was swollen in dichloromethane, then dichloromethane (2mL) and DIPEA (1mmol) were added, followed by a solution of chloroformate (4-nitrophenyl) ester (0.5mmol) in dichloromethane (1 mL). The mixture was stirred for 5 minutes, then the resin was filtered off and immediately treated with 1, 8-diamino-3, 6-dioxaoctane (2mL) for 20 minutes. The resin was then washed as before.

A solution of Fmoc-5-amino-2-methoxybenzoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP/DCM (1.5+1.5mL) was treated with DIPEA (400. mu. mol) for 2 min before the solution was added to the resin. After stirring the mixture for 5 hours, the resin was washed as before and treated with piperidine (25% in DMF, about 2mL) for 30 minutes, then washed as before. Finally, imidazo [2, 1-b ] thiazole-6-carboxylic acid (200. mu. mol) (anhydrous NMP, about 1.5 mL; reaction time: overnight) was coupled using the HATU coupling protocol described previously, and the resin was washed as described previously. The target compound was cleaved from the support by double treatment with trifluoroacetic acid (45%) and triethylsilane (10%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 622(M +1), 312((M + 2)/2).

General procedure E: synthesis of isophthalic acid N- (3-amino-1-propyl) -N' -arylamide

A dry trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was treated with a solution of isophthaloyl chloride (1mmol) and DIPEA (2mmol) in dichloromethane (1.5mL) for 3 min. The resin was washed rapidly (two or three times) with either dichloromethane or NMP. Subsequently, a solution of aromatic amine (0.3mmol) and DIPEA (0.3mmol) in NMP (1.5mL) were added, respectively, and the mixture was stirred for 1 hour, then washed with DMF, methanol and dichloromethane (three times per solvent). The target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. Since the solubility of the resulting compound is rather poor, in some cases it is necessary to add methanol to the cleavage solution or to the washing solution. After evaporation of the solvent, the crude product was purified by reverse phase HPLC.

Prepared from 2-amino-5- (2-furyl) -1, 3, 4-thiadiazole according to general procedure E. ESI-MS: 372(M + 1).

Isophthalic acid N- (3-aminopropyl) -N' - (1-methylindazol-3-yl) amide (L71)

Prepared from 3-amino-1-methylindazole according to general procedure E. ESI-MS: 352(M + 1).

Isophthalic acid N- (3-aminopropyl) -N' - (benzothiazol-2-yl) amide (L72)

Prepared from 2-aminobenzothiazole according to general procedure E. ESI-MS: 355(M + 1).

Isophthalic acid N- (3-aminopropyl) -N' - (4-phenylthiazol-2-yl) amide (L73)

Prepared from 2-amino-4-phenylthiazole according to general procedure E. ESI-MS: 381(M + 1).

Isophthalic acid N- (3-aminopropyl) -N' - (5-phenylthiazol-2-yl) amide (L74)

Prepared from 2-amino-5-phenylthiazole according to general procedure E. ESI-MS: 381(M + 1).

5- (imidazo [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L37) was prepared according to general procedure A from imidazo [2, 1-b ] thiazol-6-carboxylic acid and 150. mu. mol of 1, 3-diaminopropane preloaded trityl polystyrene resin. The crude product obtained by acid cleavage of the support-bound ligand was dried with a stream of nitrogen. Saturated aqueous sodium carbonate (3mL) was added and the mixture was extracted with ethyl acetate (three times) and dichloromethane (four times). The combined organic layers were evaporated to dryness. A solution of Boc-6-aminocaproic acid (100. mu. mol) and HATU (100. mu. mol) in DMF (1mL) was treated with DIPEA (200. mu. mol) and after 2 min the solution was added to the resin obtained previously. After the mixture was stirred for 20 minutes, water (2mL) and saturated aqueous sodium carbonate solution (1mL) were added, and the mixture was extracted twice with ethyl acetate. The combined organic layers were washed with aqueous citric acid (5%, twice), saturated sodium carbonate and water, and then concentrated to dryness. The residue was treated with dichloromethane (1mL) and trifluoroacetic acid (1mL) for 40 min. After evaporation of the solvent, the crude product was purified by reverse phase preparative HPLC. ESI-MS: 487(M + 1).

2-Chlorotriphenylmethylchloride polystyrene resin (150. mu. mol) was treated with 1, 8-diamino-3, 6-dioxaoctane (500. mu.L). After shaking the mixture for 3 minutes, dichloromethane (1mL) was added and shaking was continued for 15 minutes, then washed with DMF, methanol and dichloromethane (three times for each solvent). Subsequently, the resin was swollen in NMP. A solution of Fmoc-6-aminocaproic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol). After 2 minutes, the solution was added to the resin and the mixture was shaken for 2 hours. Subsequently, the resin was washed as before, then treated with piperidine (25% in DMF, about 2mL) for 1 hour and washed as before. Fmoc-5-amino-2-methoxybenzoic acid was coupled to the resin (coupling time: overnight; deprotection time: 30 min) followed by the coupling of imidazo [2, 1-b ] thiazole-6-carboxylic acid (coupling time: 3 h) according to the coupling/deprotection cycle. After the final wash as described above, the target compound was cleaved from the support by double treatment with trifluoroacetic acid (45%) and triethylsilane (10%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 561(M + 1).

8- [5- (imidazo- [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxyphenylcarboxamido ] -3, 6-dioxaoctanoic acid (3-amino-1-propyl) amide (L77)

Trityl polystyrene resin pre-loaded with 1, 3-diaminopropane (150. mu. mol) was pre-swollen in NMP. A solution of Fmoc-8-amino-3, 6-dioxaoctanoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol). After 2 minutes, the solution was added to the resin and the mixture was shaken for 2 hours. Subsequently, the resin was washed as before, then treated with piperidine (25% in DMF, ca 2mL) for 1 hour and washed as before. Fmoc-5-amino-2-methoxybenzoic acid was coupled to the resin (coupling time: overnight; deprotection time: 30 min) followed by the coupling of imidazo [2, 1-b ] thiazole-6-carboxylic acid (coupling time: 3 h) according to the coupling/deprotection cycle. After the final wash as described above, the target compound was cleaved from the support by double treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 519(M + 1).

8- [5- (imidazo- [2, 1-b ] thiazol-6-ylcarboxamido) -2-methoxyphenylcarboxamido ] -3, 6-dioxaoctanoic acid (6-amino-1-hexyl) amide (L78)

2-Chlorotriphenylmethylchloride polystyrene resin (150. mu. mol) was treated with 1, 6-diaminohexane (500. mu.L). After shaking the mixture for 3 minutes, dichloromethane (1mL) was added and shaking was continued for 15 minutes, then washed with DMF, methanol and dichloromethane (three times for each solvent). Subsequently, the resin was swollen in NMP. A solution of Fmoc-8-amino-3, 6-dioxaoctanoic acid (200. mu. mol) and HATU (200. mu. mol) in NMP (1.5mL) was treated with DIPEA (400. mu. mol). After 2 minutes, the solution was added to the resin and the mixture was shaken for 2 hours. Subsequently, the resin was washed as before, then treated with piperidine (25% in DMF, about 2mL) for 1 hour and washed as before. Fmoc-5-amino-2-methoxybenzoic acid was coupled to the resin (coupling time: overnight; deprotection time: 30 min) followed by the coupling of imidazo [2, 1-b ] thiazole-6-carboxylic acid (coupling time: 3 h) according to the coupling/deprotection cycle. After the final wash as described above, the target compound was cleaved from the support by double treatment with trifluoroacetic acid (45%) and triethylsilane (10%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 561(M + 1).

Example 3: immobilization of ligands

Experiment of

The dried ligand was redissolved in DMSO at a concentration of approximately 100mM based on gravimetric method. The exact concentration of the stock solution was determined by o-phthaldialdehyde assay (2). The dye-ligand molecules were quantified by measuring absorbance at 340 nm. The measurement values were corrected with 1- (3-aminopropyl) imidazole.

In the coupling reaction, 1 volume of ligand was dissolved in 90% DMSO at a concentration of approximately 15-20mM, and then 10% N-methyl-2-pyrrolidone containing 1MN, N-diisopropylethylamine was added to 1 volume of a fixed NHS-activated Sepharose 4FF (GEHealthcare) equilibrated with isopropanol. The reaction was carried out at 25 ℃ for 2 hours while shaking vigorously. The reaction supernatant was removed and the resin was washed twice with the appropriate solvent. The remaining reactive groups on the resin were blocked with 1M ethanolamine at 25 ℃ for 1 hour. The resin was finally washed and stored in 20% ethanol at 4 ℃ until used in subsequent experiments. After analysis of the ligand concentration in all fractions, the yield of the reaction was determined based on mass balance.

Summary and results

The ligand from example 2 was immobilized on NHS activated sepharose 4FF for subsequent chromatography and batch adsorption experiments. Coupling is achieved by formation of an amide bond between the amino group of the aminopropyl linker on the ligand and the NHS activated carboxyl group of the preactivated resin.

The table below gives the results of the coupling reaction with the ligand from example 2. On average, an immobilized ligand density of 15.5. mu. mol per ml of resin was obtained. The reaction yield averaged 74.5%.

Example 4: chromatographic evaluation of the ligand (from example 2)

Experiment of

The resin was assessed by microtiter plate chromatography in packed mode. For each column, approximately 30 μ Ι of resin was transferred to a 384 well filter plate and sealed with an appropriate top sieve plate. Columns with rpoteina sepharose FF and MabsorbentA a2PHF were included as controls.

Prior to injection, the column was equilibrated with 6.7 column volumes (cv) of phosphate buffered saline (0.15M NaCl, 20mM sodium phosphate, pH 7.3; PBS). Will be added in an amount of 0.75mgml^-1Dissolved 3.3cv of intact IgG in 0.25mgml^-1The dissolved Fab or Fc fragment (all three in PBS) or the Host Cell Protein (HCP) was injected into the column. Antibody fragments of bevacizumab (Fab and Fc) were prepared according to the manufacturer's protocol by cleaving intact IgG with papain (3). Unbound protein was washed off the column with 5cv of PBS and then eluted with 5cv of glycine buffer pH 2.5. The transferred volume was spun through the column at 50g for 1-2 minutes. During the injection of the sample, the acceleration is reduced to 10g and the centrifugation time is increased to 5-10 minutes. The eluted fractions were collected in 384-well plates and analyzed for protein concentration by Bradford assay. Calculation of protein Mass m_i、m_ftAnd m_e(see below) as fraction volume product and protein concentration was measured.

Summary and results

Binding and elution of several antibodies was demonstrated. Among them are three humanized therapeutic antibodies bevacizumab, toslizumab and palivizumab and a human poly-IgG (h-poly-IgG) mixture isolated from human serum. In some cases, a mixture of purified bevacizumab Fab and Fc fragments or rabbit IgG (h-poly-IgG, isolated from serum) was used as additional feed. The selectivity of the ligand for the antibody is determined by studying the binding behavior of host cell proteins. Results for commercially available resins rproteinA Sepharose FF (rproteinA), MabsorbentA2P (A2P) and MEPHypercel (hyper) were included for comparison.

The "bound" protein fraction was calculated as follows: total mass m of injected protein_iWith the mass m of protein detected in the flow-through_ftDivided by the total mass of protein injected.

The "yield" of protein was calculated as follows: mass m of eluted protein_eDivided by the mass m of the protein injected_i.。

The "selectivity" of the resin is calculated as follows: bound antibody fraction B_AbDivided by the bound host cell protein fraction B_HCP。

For each resin, the "bound" protein fraction and protein "yield" were calculated based on data for intact IgG antibodies. For the bevacizumab fragments and host cell proteins, only the "bound" protein fractions are given. The selectivity of the resin was calculated from the data for bevacizumab whole IgG and host cell proteins. Selectivity takes values over 10 due to limited experimental accuracy.

The chromatographic results for the resin from example 3 and the reference resin are given in the table below. The value of L53 is an estimate predicted based on the close analogs L49 and L50.

The control group bound almost 100% of all antibodies. However, protein a did not bind at all to Host Cell Protein (HCP), and A2P and Hypercel showed superior binding properties to HCP. The resulting selectivity index was 10.0 for protein a, 1.0 for A2P, and 1.3 for Hypercel.

Of the ligands from example 2 tested, L1, L27, L31 and L37 showed the best results with respect to antibody binding and selectivity. They all bound more than 90% of all human intact IgG antibodies tested and therefore had the binding characteristics of a generic Fc binder. Consistent with this, L1 and L27 bound the Fc fragment of bevacizumab and not the Fab fragment. In addition, efficient binding of L1 to rabbit poly IgG was demonstrated. Yields of bevacizumab, toslizumab and palivizumab after chromatographic separation varied between 87% and 100%. The yield of poly-IgG was slightly lower, ranging from 70% to 90%. Selectivity index: l1 was 3.3, L27 was 2.9, L31 was 4.4, and L37 was 3.4.

Other ligands from example 2 showed reduced binding to at least one of the test antibodies, or more than 40% binding to HCP (resulting in a selectivity index of 2.5 or less).

Example 5: combined Isotherms (Isotherms) and time-scales (time-scale)

Experiment of

The experiments were performed with bevacizumab as antibody. Langmuir isotherm (Langmuiristherms) parameters for purification of antibodies were determined by batch adsorption experiments in 96-well microtiter plates. In each well, 10. mu.l of the adsorbent slurry (50% v/v) was mixed with 100. mu.l of the protein solution. Initial concentration in phosphate buffered saline (0.15M NaCl, 20mM phosphate buffer, pH 7.3; PBS) was 0.05-5mgml^-1To change between. The reaction was stirred vigorously at 25 ℃ for at least 3 hours. The antibody concentration in the supernatant was then measured by Bradford assay.

The kinetics of absorption were similarly studied by batch adsorption in 96-well titer plates. Mu.l of the adsorbent slurry (50% v/v) was again mixed with 100. mu.l of the protein solution. However, a PBS containing 0.75mgml was used^-1A fixed initial concentration of bevacizumab. The reaction was stirred vigorously at 25 ℃ for up to 80 minutes. Supernatants were quickly separated by spin through filters at 2.5, 5, 10, 20, 40 and 80 minutes before injection for analysis. Antibody concentrations were analyzed by Bradford assay.

Summary and results

The subset of immobilized ligands from example 3 was characterized for its affinity and maximum capacity for bevacizumab. In addition, the time course required for binding was determined for L1. Commercially available resins rproteinA Sepharose FF (rproteinA) and MabsorbentA2P (A2P) were included for comparison.

Determination of the parameters of the Langmuir isotherm, i.e.the dissociation constant K, from the measured concentrations in the supernatant_dAnd a maximum capacity q_m. Estimating parameters by numerical fitting of model equations derived by Chase (4)And (4) counting.

By combined time intervals t_0.8The absorption kinetics are characterized, said t_0.8The time is defined as: after which there was 80% equilibrium saturation of the resin with the antibody. For t_0.8The measured concentration in the supernatant is interpolated by fitting a double exponential (5) and then reading t from the graph_0.8The value of (c). Parameters for langmuir isotherms and time courses for the bound internal resin and the reference resin are reported in the table below.

The highest affinity was observed for rProteinA, whose K_dIs 4.7. mu.g ml^-1。A2P(49μgml^-1) Resin L1 (61. mu.g ml)^-1) And L27 (92. mu.g ml)^-1) Is an order of magnitude lower. The dissociation constant of the residual resin was two orders of magnitude lower, ranging from 103. mu.g ml^-1(L23) to 252. mu. gml^-1(L17). With respect to the maximum capacity, the highest capacity (69 mg per ml resin) was measured for A2P. The maximum capacity of rProteinA was 41mg per ml resin. The maximum capacity of the internal resin varies between 26mg and 37mg per ml of resin. Antibody uptake was fastest with rproteinA (8.9min), followed by A2P (9.3min) and L1(9.9 min).

Example 6: dynamic binding capacity

Experiment of

Dynamic binding capacity was determined by column chromatography with purified bevacizumab. The resin was packed into an analytical column having a length of 25mm and an internal diameter of 3 mm. Will contain 1mgml at a constant flow rate^-1Antibody phosphate buffered saline (150mM NaCl, 20mM phosphate buffer, pH7.3) was fed to the column while the antibody concentration in the effluent was monitored on-line by absorbance at 280 nm. The antibody is loaded until the effluent concentration reaches at least 10% of the feed concentration. To be provided with50cmh^-1The flow rate of (d) corresponds to a 3 minute empty column residence time to determine the dynamic binding capacity. Also by loading until the antibody completely passed through at 50cmh^-1To determine the equilibrium capacity. Bound antibody was eluted from the column by washing with sodium citrate, ph3.0, followed by glycine hydrochloride, ph 2.5.

Summary and results

The dynamic (binding) capacity of L1 (from example 3) and rProteinA sepharose 4ff (rProteinA) for bevacizumab was determined as a function of flow rate.

"dynamic capacity" is defined as the amount of antibody as follows: the column can be fed with a constant flow rate until the effluent concentration reaches the feed concentration of 10%. It is calculated as follows: time t_0.1(10% breakthrough occurred after this time), multiplied by the flow rate F and feed concentration c_f。

Dynamic capacity of t_0.1·F·c_f

The equilibrium capacity is defined as: the maximum amount of antibody bound to the column for a given concentration of feed stream. Which is obtained by a process comprising the feed concentration c_fThe integral of the outflow concentration c (t) and the flow rate F over time is evaluated to calculate

The integral is evaluated numerically. Integration is limited by the time that full penetration occurs. The calculated values for dynamic and equilibrium capacities were corrected for retention of the column and the chromatography system. The capacity was normalized by the volume of resin in the column.

Dynamic binding Capacity of L1 and rProteinA Sepharose 4FF as a function of flow Rate and for 1mgml^-1The equilibrium binding capacities for the feed concentrations of bevacizumab are given in the table below.

For rpoteina, the dynamic binding capacity per ml resin was determined to be 28.4 mg. The dynamic capacity of L1 was 20.2mg per ml of resin. For the calculated equilibrium capacities, rProteinA was 40mg per ml resin and L1 was 25.7mg per ml resin.

Example 7: stability to alkali

The base stability of ligand L1 from example 2 was tested over a period of 8 days. It is treated with 0.1M or 0.5M sodium hydroxide at 25 ℃. Hydrolysis was monitored by LC-MS analysis.

The half-life of L1 was 288 hours at 0.5M Na 0H. No degradation of the ligand was detected at 0.1M NaOH.

Example 8: purification of antibodies from cell culture supernatants

Experiment of

The suitability of the resin for purification of antibodies from cell culture supernatants was assessed by microtiter plate chromatography in packed mode similar to example 3 or by conventional column chromatography. In both cases, this will be at 0.05mgml^-1Bevacizumab incorporated into the host cell protein at a concentration was used as feed. Furthermore, chromatography runs with host cell proteins (microtiter plates and column chromatography) and with pure antibodies only (microtiter plate chromatography only) were performed. For microtiter plate chromatography, 25 column volumes were injected per run. 100 column volumes were injected for column chromatography. The column was equilibrated and washed with PBS before and after injection. Bound proteins were eluted with sodium citrate ph 3.0. The protein concentration in the feed and in the column effluent was analyzed by Bradford assay or by UV-absorbance at 280 nm.

Summary and results

The antibody was purified from the cell culture supernatant by chromatography on resin L1 from example 3. Chromatograms on commercially available resins rProteinA sepharose ff (rProteinA) and MabsorbentA2P (A2P) were included for comparison. For microtiter plate chromatography, three chromatographs run under identical conditions were performed on each resin, with injection of antibody, host cell protein or host cell protein mixture. For column chromatography, two runs were performed with only the antibody incorporated into the host cell protein or with only the host cell protein.

Total mass m of protein recovered from elution after injection of the mixture_mix，eMass m recovered after injection of host cell protein alone_HCP，eAnd the mass m of the injected antibody_Ab，iTo calculate the "yield" of the operation of the mixture after chromatographic separation.

Total mass m of protein recovered by elution after injection of the mixture_mix，eAnd mass m recovered after injection of host cell protein_HCP，eThe "purity" of the antibody after chromatographic separation of the mixture was calculated.

The following table gives the purity and yield obtained after chromatographic separation on resin L1 from example 3 and the reference resin.

Within the accuracy of the experiment, the yield of rProteinA after microtiter plate chromatography was 100%, L1 was 85%, A2P was 12%. Recovery from A2P was low (12%), due to the moderate acidity of ph3.0 after elution. The purity was highest after chromatography on rpoteina (100%) followed by the purity after chromatography on resin L1 (74%). The purity after chromatography on A2P was the lowest, only 32%. The results after performing column chromatography were in a similar range. In this regard, rpoteina showed a yield of 97% and a purity of 100%. L1 showed a yield of 90% and a purity of 83%.

Documents cited in the examples:

1.T.Neumann，HDJunker，K.Schmidt，R.Sekul，SPRBasedFragmentScreening：

AdvantagesandApplications，CurrentTopicsinMedicinalChemistry2007；7：1630-1642

2.RothM.Fluorescencereactionforaminoacids.AnalChem197；43(7)：880-2.

3.RousseauxJ，Rousseaux-PrévostR，BazinH.OptimalconditionsforthepreparationofFabandF(ab′)2fragmentsfrommonoclonalIgGofdifferentratIgGsubclasses.J.Immunol.Methods1983；64(1-2)：141-146.

4.ChaseHA.Predictionoftheperformanceofpreparativeaffinitychromatography.JChromatogr1984；297：179-202.

5.CoffmanJL，KramarczykJF，KelleyBD.High-throughputscreeningofchromatographicseparations：I.Methoddevelopmentandcolumnmodeling.BiotechnologyandBioengineering2008；100(4)：605-618.

Claims

1. Use of a ligand-substituted matrix comprising a support material and at least one ligand covalently bound to the support material, said ligand being represented by formula (I) for affinity purification of an antibody or an Fc fusion protein

Wherein

L is a point of attachment of a ligand to the support material;

sp is a spacer group;

v is 0 or 1;

R¹Is hydrogen or C₁To C₄An alkyl group;

Ar¹is a divalent 5-or 6-membered substituted or unsubstituted aromatic ring, wherein NR is¹And Ar¹Is linked and-C (O) -and Ar²When attached, the substituents are selected from C₁To C₄Alkoxy radical, C₁To C₄Alkyl, hydroxy, and combinations thereof;

(a) To other 5-or 6-membered aromatic rings via single bonds; or

(c) Is linked to at least one substituent selected from the group consisting of: c₁To C₄An alkyl group; c₂To C₄An alkenyl group; c₂To C₄An alkynyl group; halogen; c₁To C₄A haloalkyl group; hydroxy-substituted C₁To C₄An alkyl group; c₁To C₄An alkoxy group; hydroxy-substituted C₁To C₄An alkoxy group; c₁To C₄An alkylamino group; c₁To C₄An alkylthio group; and combinations thereof;

and wherein the first aromatic ring or the second aromatic ring or both in alternatives (a) and (b) may optionally bear one or more further substituents from those mentioned in (c).

2. The use of claim 1, wherein R¹Is hydrogen or methyl.

3. The use of claim 1, wherein R¹Is hydrogen.

4. The method of claim 1, wherein Ar¹Is phenylene.

5. The method of claim 1, wherein Ar¹Is a methoxy substituted phenylene group.

6. Use according to any one of claims 1 to 5, wherein the C ═ O and NH groups are meta to each other to Ar¹And (4) bonding.

7. Use according to any one of claims 1 to 5, wherein Ar²The aromatic ring of the 5 or 6 membered heterocyclic group of (a) is attached to the C ═ O group via a carbon ring atom which is adjacent to the ring heteroatom.

8. Use according to claim 7, wherein the ring heteroatom is a nitrogen or oxygen atom.

9. Use according to any one of claims 1 to 5, wherein Ar²The aromatic ring of the 5 or 6 membered heterocyclic group of (a) contains two or more nitrogen atoms, or a nitrogen atom and an oxygen atom.

10. Use according to any one of claims 1 to 5, wherein Ar²The aromatic ring of the 5 or 6 membered heterocyclic group of (a) is an N-methyl-substituted pyrazole, pyridine, isoxazole or oxadiazole.

11. The use of claim 1, wherein the support material comprises a material selected from the group consisting of carbohydrates, synthetic polymers, inorganic materials, and composite materials.

12. The use of claim 11, wherein the carbohydrate is a cross-linked carbohydrate.

13. The use of claim 11, wherein the carbohydrate is agarose, dextran, alginate and carrageenan.

14. The use of claim 13, wherein the agarose is agarose gel.

15. The use of claim 13, wherein the glucan is starch, cellulose or sephadex.

16. The use of claim 11, wherein the synthetic polymer is polystyrene, styrene-divinylbenzene copolymer, polyacrylate, PEG-polyacrylate copolymer, polymethacrylate, polyvinyl alcohol, polyamide, or perfluorocarbon.

17. The use of claim 11, wherein the inorganic material is glass, silica or a metal oxide.

18. The use of claim 1, wherein the antibody is an IgG-type antibody.

19. The use of claim 18, wherein said purification is achieved by binding of the ligand of said ligand-substituted matrix to the Fc-fragment of an antibody or Fc-fusion protein.

20. The use of claim 18 or 19, wherein the antibody is an antibody comprising an Fc fragment of an immunoglobulin class.

21. The use of claim 20, wherein the immunoglobulin class is IgG.

22. The use of claim 20, wherein the immunoglobulin class is human IgG.

23. The use of claim 20, wherein the immunoglobulin class is polyclonal IgG or monoclonal IgG of human origin.

24. The use of claim 21, wherein the immunoglobulin class is IgG₁、IgG₂Or IgG₄。

25. A ligand-substituted matrix as defined in claim 1.

26. A method of synthesizing a ligand-substituted matrix according to claim 25, wherein the ligand of formula (I) is attached to the support material as described in the use of claim 1.

27. A method for affinity purification of an antibody or Fc-fusion protein, wherein the antibody or Fc-fusion protein to be purified is contacted with a ligand-substituted matrix as defined in the use of claim 1.

28. The method of claim 27, wherein the ligand binds to the Fc region of an antibody or Fc fusion protein.

29. A ligand shown as a formula (III), wherein Sp and Ar¹、Ar²And Am has the meaning defined in the use of claim 1: