LIGANDS FOR ANTIBODY AND Fc-FUSION PROTEIN PURIFICATION BY AFFINITY CHROMATOGRAPHY

Abstract

The present invention relates to the use for affinity purification of an antibody or a fragment of an antibody, of a ligand-substituted matrix comprising a support material and at least one ligand covalently bonded to the support material.

Claims

1-15. (canceled)

16. Use, for affinity purification of an antibody or a fragment of an antibody, of a ligand-substituted matrix comprising a support material and at least one ligand covalently bonded to the support material, the ligand being represented by formula (I) ##STR00161## wherein L is the linking point on the support material to which the ligand is attached; Sp is a spacer group; v is 0 or 1; Am is an amide group NR.sup.1C(O), and wherein either NR.sup.1 is attached to Ar.sup.1 and C(O) is attached to Ar.sup.2, or C(O) is attached to Ar.sup.1 and NR.sup.1 is attached to Ar.sup.2; and R.sup.1 is hydrogen or C.sub.1 to C.sub.4 alkyl, preferably hydrogen or methyl; and more preferably hydrogen; Ar.sup.1 is a divalent 5- or 6-membered substituted or unsubstituted aromatic ring, wherein the substituents are selected from C.sub.1 to C.sub.4 alkoxy, C.sub.1 to C.sub.4 alkyl, and combinations thereof; Ar.sup.2 is 5- or 6-membered unsubstituted or substituted heterocyclic aromatic ring which is (a) attached to a further 5- or 6-membered aromatic ring via a single bond; or (b) fused to a further 5- or 6-membered aromatic ring as part of a multicyclic ring system; or (c) attached to at least one substituent selected from C.sub.1 to C.sub.4 alkyl; C.sub.2 to C.sub.4 alkenyl; C.sub.2 to C.sub.4 alkynyl; a halogen; C.sub.1 to C.sub.4 haloalkyl; hydroxyl-substituted C.sub.1 to C.sub.4 alkyl; C.sub.1 to C.sub.4 alkoxy; hydroxyl-substituted C.sub.1 to C.sub.4 alkoxy; C.sub.1 to C.sub.4 alkylamino; C.sub.1 to C.sub.4 alkylthio; and combinations thereof; and wherein the first aromatic ring or the second aromatic ring or both, in alternative (a) and (b) may optionally carry one or more further substituents from those mentioned under (c).

17. The use of claim 16, wherein Ar.sup.1 is phenylene, preferably methoxy-substituted phenylene.

18. The use of claim 16, wherein the CO and the NH group are bonded to Ar.sup.1 in meta position to each other.

19. The use of claim 16, wherein the 5- or 6-membered heterocyclic aromatic ring of Ar.sup.2 is attached to the CO group via a carbon ring atom which is adjacent to a ring heteroatom, preferably a nitrogen or oxygen atom.

20. The use of claim 16, wherein the 5- or 6-membered heterocyclic aromatic ring of Ar.sup.2 contains two or more nitrogen atoms or one or more nitrogen atoms and an oxygen atom.

21. The use of claim 20, wherein the 5- or 6-membered heterocyclic aromatic ring of Ar.sup.2 is N-methyl-substituted pyrazole, pyridine, isoxazole or oxadiazole.

22. The use according to claim 16, wherein the support material comprises a material selected from carbohydrates or crosslinked carbohydrates, preferably agarose, cellulose, dextran, starch, alginate and carrageenan, Sepharose, Sephadex; synthetic polymers, preferably polystyrene, styrene-divinylbenzene copolymers, polyacrylates, PEG-Polyacrylate copolymers polymethacrylates, polyvinyl alcohol, polyamides and perfluorocarbons; inorganic materials, preferably glass, silica and metal oxides; and composite materials.

23. The use according to claim 16, wherein the protein is an antibody, preferably an IgG type antibody, or an Fc fusion protein.

24. The use of claim 23, wherein the purification is attained by binding of the ligand of the ligand-substituted matrix to an Fc fragment or domain of the antibody or the fusion protein.

25. The use according to claim 23, wherein the Fc fragment or domain or the antibody belongs to the IgG antibody class, more preferably to human, IgG or to polyclonal or monoclonal IgG of human origin, in particular to IgG.sub.1, IgG.sub.2 and IgG.sub.4.

26. A ligand-substituted matrix as defined in claim 16.

27. A method of synthesis of a ligand-substituted matrix according to claim 26, wherein a ligand according to formula (I) is attached to the support material.

28. A method for affinity purification, preferably affinity chromatography, of a protein, wherein a protein to be purified is contacted with a ligand-substituted matrix as defined in claim 16.

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

30. A ligand as shown in formula (III), wherein Sp, Ar.sup.1, Ar.sup.2 and Am have the meanings defined in claim 16. ##STR00162##

Description

EXAMPLES

[0204] Materials and Methods

[0205] If not otherwise stated, all chemicals and solvents were of analytical grade, with the exception of example 2. Reagents used in example 2 were of preparative to analytical grade depending on particulate requirements and availability.

[0206] 96 and 384-well filter plates having hydrophilic membrane filters with 0.45 m average pore size were purchased from Pall GmbH (Dreieich/Germany). Top frits made from polyethylene with 10 m average pore size were provided by Porex (Bautzen/Germany). General purpose microtiterplates for collection of fractions and analytical assays were ordered from Greiner Bio One GmbH (Frickenhausen/Germany). Analytical assays were read out using a Fluostar Galaxy plate reader from BMG Labtech GmbH (Offenburg/Germany).

[0207] Column chromatography with antibody and purification of antibody fragments was conducted on a Waters HPLC system (Waters GmbH, Eschborn/Germany). Omnifit column housings (Diba Industries Ltd, Cambridge/United Kingdom) were used for packing of columns. NHS-activated Sepharose 4 FF, rProtein A Sepharose FF and Superdex 70 chromatography media were bought from GE Healthcare (Uppsala/Sweden). Mabsorbent A2P HF was purchased from Prometic Life Sciences (Cambridge/United Kingdom) and MEP Hypercel from Pall Corporation (Port Washington N.Y., USA).

[0208] Analytical chromatography of ligands was conducted on a Shimadzu HPLC system (Shimadzu Deutschland GmbH, Duisburg/Germany) including a diode array detector and single-quad mass spectrometer. The monolithic C18 reversed phase column was purchased from Merck KGaA (Darmstadt/Germany). Solvents used in analyses were of mass spectrometry grade.

[0209] Antibodies used in the present invention were Bevacizumab (Avastin, F. Hoffmann-La Roche, Switzerland), Tocilizumab (RoActemra, F. Hoffmann-La Roche, 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 Thermo Scientific (Bonn/Germany).

[0210] Flowthrough from protein A chromatography (referred to as host cell proteins) was derived from the supernatant of antibody-producing CHO cell culture in serum-free medium.

[0211] Coomassie brilliant blue dye reagent for Bradford assay, was purchased from Thermo Scientific (Bonn/Germany).

Example 1

[0212] SPR Screening of Chemical Microarrays

[0213] Graffinity has developed high density chemical microarrays comprising thousands of small molecules immobilized onto gold chips. The arrays are constructed using maleimide-thiol coupling chemistry in combination with high density pintool spotting. For the construction, glass plates are microstructured by photolithographical methods in order to define up to 9,216 sensor fields per array. A subsequently applied gold coating provides the basis for the SPR effect and enables the formation of a binary, mixed self assembled monolayer (SAM) of two different thiols. One of the thiols exhibits a reactive maleimide moiety to which thiol-tagged array compounds can couple to during pintool spotting. The resulting microarrays comprise 9,216 sensor fields, each containing multiply copies of a defined and quality controlled array compound.

[0214] Surface plasmons are collective oscillations of electrons at a metal surface which can be resonantly excited by polarized light of appropriate wavelength and incidence angle. At surface plasmon resonance conditions, the light intensity reflected from the gold chip exhibits a sharp attenuation (SPR minimum). The angle and wavelength position of this reflectance minimum strongly depends on the refractive index of the medium adjacent to the metal surface. Processes which alter the local refractive index in close proximity to the metal surface (such as antibody binding to the immobilized ligands) can therefore be monitored sensitively. For SPR detection, the chemical microarray is incubated with the target protein under optimized screening conditions. If targets bind to the immobilized fragments a shift in the wavelength dependent SPR minimum (referred to as the SPR shift) occurs with respect to the buffer control and thus indicates protein-ligand interaction. The SPR imaging approach utilizes 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. While scanning over a range of wavelengths covering the SPR resonance conditions, reflection images are recorded stepwise. Automated spot finding routines and subsequent grey-scale analysis of the obtained pictures lead to SPR resonance curves of all 9,216 sensor fields per chip in parallel.

[0215] Screening of Antibodies and Fragments Thereof by SPR

[0216] The platform allowed screening of a proteins, antibodies and fragments thereof against Graffinity's immobilized compound library of 110,000 small molecules in a high-throughout fashion. For each of the targets, the SPR screening conditions were optimized individually before screening of the targets against the entire library. Among such optimized screening parameters were a) the composition of the screening buffer including appropriate detergents, b) the concentration of the antibody or protein target in solution and c) the surface density of immobilized ligands on the chip surface. Neumann et al. (1) summarizes the screened targets and the corresponding screening conditions applied. In total four different full-length antibodies were screened and compared. Additionally, the Fc and Fab fragments of two antibodies were obtained by proteolytic digestion and were subjected to array screening as well. In order to identify antibody specific ligands, the screening campaign included also antibody free host cell protein (HCP) of CHO cell lines as controls.

[0217] The experimental results of the microarray screening campaigns were analyzed by manual hit selection using in-house developed software routines as well as by an in-depth data analysis by commercially available data mining tools. The analysis resulted in a number of hit series which showed clear binding to antibodies and their Fc fragments but only weak to negligible binding to the HCP controls. Such primary hit series were used for hit confirmation in secondary assays and could be used as starting point for the synthesized of focused libraries around the primary hits. For example, the Fc specific ligand L1 showed distinct binding to the full length Bevacizumab and its Fc fragment but only minor binding to the Bevacizumab Fab fragment. Furthermore it clearly bound the other full length antibodies Anti RhD IgG, Tocilicumab and Palivizumab.

Example 2

[0218] Synthesis of Ligands

General Procedure A: Synthesis of 5-Arylcarboxamido-2-methoxybenzoic acid N-(3-aminopropyl)amides (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)

[0219] N-Fmoc-5-amino-2-methoxybenzoic acid (0.2-0.5 mmol) and HOAt (1 equivalent relative to the carboxylic acid) were dissolved in DMF, NMP, DMF/DMSO or NMP/DMSO mixtures (1-3 mL) and treated with DIC (1 equivalent relative to the carboxylic acid). After agitating for 2-5 min, the mixture was added to 0.1-0.15 mmol of 1,3-diaminopropane linked to either 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, each solvent several times) and treated with piperidine 25% in DMF (1-5 mL) for 15-30 min. Subsequently, the resin was washed thoroughly (DMF, dichloromethane or DMF, methanol, dichloromethane, each solvent several times), and dried at air, with a stream of nitrogen or in high vacuum.

[0220] The arylcarboxylic acid (0.2-0.5 mmol) and HOAt (1 equivalent relative to the carboxylic acid) were dissolved in DMF, NMP, DMF/DMSO or NMP/DMSO mixtures (1-3 mL) and treated with DIC (1 equivalent relative to the carboxylic acid). After agitating for 2-5 min, the mixture was added to the resin and shaken for several hours or overnight. After subsequent washing (DMF, dichloromethane or DMF, methanol, dichloromethane, each solvent several times), the target compound was cleaved from the support by treatment with a suitable cleavage mixture (dichloromethane, TFA, triethylsilane 85:10:5 for trityl resins, 45:45:10 for 2-chlorotrityl resins). Typically, the cleavage step was repeated once, followed by rinsing of the resin with dichloromethane. After evaporation of the solvents, the crude residue was purified by preparative reversed-phase HPLC.

[0221] With respect to the high robustness of amide coupling chemistry, other coupling protocols may be applied with comparable or identical results. Particularly, TBTU or HATU coupling (1 equivalent relative to the carboxylic acid; additionally 2 equivalents of DIPEA must be added) represents a reliable alternative to DIC/HOAt coupling which proceeds more rapidly (typical reaction times: 30 min to 3 h); however, for some carboxylic acids, particularly those with a free aryl NH like indoles or (benzo)pyrazoles, coupling under basic conditions might lead to unwanted side-products.

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

[0222] ##STR00083##

[0223] Prepared from 5-(2-furyl)-1-methylpyrazole-3-carboxylic acid following General Procedure A. ESI-MS: 398 (M+1).

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

[0224] ##STR00084##

[0225] Prepared from 5-(4-bromo-2-thienyl)-1-methylpyrazole-3-carboxylic acid following General Procedure A. ESI-MS: 493, 495 (M+1).

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

[0226] ##STR00085##

[0227] Prepared from 5-(2,5-dimethyl-3-thienyl)-1-methylpyrazole-3-carboxylic acid following General Procedure A. ESI-MS: 442 (M+1).

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

[0228] ##STR00086##

[0229] Prepared from 1-methyl-5-(2-thienyl)pyrazole-3-carboxylic acid following General Procedure A. ESI-MS: 414 (M+1).

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

[0230] ##STR00087##

[0231] Prepared from 1-methyl-5-(3-thienyl)pyrazole-3-carboxylic acid following General Procedure A. ESI-MS: 414 (M+1).

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

[0232] ##STR00088##

[0233] Prepared from 5-(3-chlorophenyl)-1-methylpyrazole-3-carboxylic acid following General Procedure A. ESI-MS: 442, 444 (M+1).

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

[0234] ##STR00089##

[0235] Prepared from 1-methyl-5-phenylpyrazole-3-carboxylic acid following General Procedure A. ESI-MS: 408 (M+1).

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

[0236] ##STR00090##

[0237] Prepared from 5-(1-methyl-2-pyrrolyl)-1-methylpyrazole-3-carboxylic acid following General Procedure A. ESI-MS: 411 (M+1).

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

[0238] ##STR00091##

[0239] Prepared from 3-(2-thienyl)pyridine-5-carboxylic acid following General Procedure A. ESI-MS: 411 (M+1).

2-Methoxy-5-(3-phenylpyridin-5-yl)carboxamidobenzoic acid N-(3-aminopropyl)amide (L11)

[0240] ##STR00092##

[0241] Prepared from 3-phenylpyridine-5-carboxylic acid following General Procedure A. ESI-MS: 405 (M+1).

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

[0242] ##STR00093##

[0243] Prepared from 5-(2-furyl)pyrazole-3-carboxylic acid following General Procedure A. ESI-MS: 384 (M+1).

2-Methoxy-5-(1-methylindazol-3-yl)carboxamidobenzoic acid N-(3-aminopropyl)amide (L27)

[0244] ##STR00094##

[0245] Prepared from 1-methylindazole-3-carboxylic acid following General Procedure A. ESI-MS: 382 (M+1).

2-Methoxy-5-(1-methylindol-3-yl)carboxamidobenzoic acid N-(3-aminopropyl)amide (L25)

[0246] ##STR00095##

[0247] Prepared from 1-methylindole-3-carboxylic acid following General Procedure A. ESI-MS: 381 (M+1).

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

[0248] ##STR00096##

[0249] Prepared from 5-chloro-1H-indazole-3-carboxylic acid following General Procedure A. ESI-MS: 402, 404 (M+1).

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

[0250] ##STR00097##

[0251] Prepared from indole-3-carboxylic acid following General Procedure A. Indole-3-carboxylic acid was coupled by activation with HOBt (1 equivalent) and DIC (1 equivalent, relative to the carboxylic acid); coupling time: 1 h. ESI-MS: 367 (M+1).

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

[0252] ##STR00098##

[0253] Prepared from benzo[c]isoxazole-3-carboxylic acid following General Procedure A. ESI-MS: 369 (M+1).

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

[0254] ##STR00099##

[0255] Prepared from benzo[c]furan-1-carboxylic acid following General Procedure A. ESI-MS: 368 (M+1).

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

[0256] ##STR00100##

[0257] Prepared from 1,5-dimethylpyrazole-3-carboxylic acid following General Procedure A. ESI-MS: 346 (M+1).

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

[0258] ##STR00101##

[0259] Prepared from 1-methyl-5-(trifluoromethyl)pyrazole-3-carboxylic acid following General Procedure A. ESI-MS: 400 (M+1).

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

[0260] ##STR00102##

[0261] Prepared from isoquinoline-1-carboxylic acid following General Procedure A. ESI-MS: 379 (M+1).

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

[0262] ##STR00103##

[0263] Prepared from indazole-3-carboxylic acid following General Procedure A. Indazole-3-carboxylic acid was coupled by activation with HOBt (1 equivalent) and DIC (1 equivalent, relative to the carboxylic acid); coupling time: 1 h. ESI-MS: 368 (M+1).

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

[0264] ##STR00104##

[0265] Prepared from 5-(2-methylthiazol-4-yl)isoxazole-3-carboxylic acid following General Procedure A. ESI-MS: 416 (M+1).

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

[0266] ##STR00105##

[0267] Prepared from 1-methylpyrazolo[3,4-b]pyridine-3-carboxylic acid following General Procedure A. ESI-MS: 383 (M+1).

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

[0268] ##STR00106##

[0269] Prepared from 5-(2-furyl)isoxazole-3-carboxylic acid following General Procedure A. ESI-MS: 385 (M+1).

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

[0270] ##STR00107##

[0271] Prepared from pyrazolo[1,5-a]pyridine-2-carboxylic acid following General Procedure A. ESI-MS: 368 (M+1).

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

[0272] ##STR00108##

[0273] Prepared from 1H-benzimidazole-2-carboxylic acid following General Procedure A. ESI-MS: 368 (M+1).

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

[0274] ##STR00109##

[0275] Prepared from imidazo[1,2-b]pyridazine-2-carboxylic acid following General Procedure A. ESI-MS: 369 (M+1).

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

[0276] ##STR00110##

[0277] Prepared from 3-phenylisoxazole-5-carboxylic acid following General Procedure A. ESI-MS: 395 (M+1).

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

[0278] ##STR00111##

[0279] Prepared from pyrazolo[1,5-a]pyrimidine-2-carboxylic acid following General Procedure A. ESI-MS: 369 (M+1).

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

[0280] ##STR00112##

[0281] Prepared from imidazo[1,2-a]pyridine-2-carboxylic acid hydrobromide following General Procedure A. One additional equivalent of DIPEA was added when the carboxylic acid hydrobromide was coupled using HATU/DIPEA. ESI-MS: 368 (M+1).

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

[0282] ##STR00113##

[0283] Prepared from imidazo[1,2-a]pyrimidine-2-carboxylic acid following General Procedure A. 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)amides by Suzuki coupling (L12, L13, L14, L22, L23)

[0284] To 0.1 mmol of 1,3-diaminopropane-trityl-polystyrene resin, N-Fmoc-5-amino-2-methoxybenzoic acid and 5-bromopyridine-3-carboxylic acid were coupled following General Procedure A. The resulting resin loaded with the bromopyridine carboxylic amide was dried thoroughly and transferred to a glass vial. After addition of caesium carbonate or potassium carbonate (0.3 mmol) and the respective boronic acid (1 mmol), absolute DMF (1 mL) was added and the mixture was extensively flushed with argon. Subsequently, tetrakis-triphenylphosphinepalladium(0) (10 mol) was added, the mixture was again flushed with argon and the vial was tightly closed. The mixture was heated to 100 C. and shaken overnight. Then the resin was washed thoroughly (DMF, dichloromethane, each solvent several times) and a small sample was cleaved (conditions, see below). When LCMS indicated incomplete conversion, the coupling step was repeated or conducted at higher temperatures. Otherwise, the resin was treated with dichloromethane-TFA-triethylsilane 85:10:5 (twofold cleavage), followed by rinsing of the resin with dichloromethane. After evaporation to dryness, the residue was purified by preparative reversed-phase HPLC.

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

[0285] ##STR00114##

[0286] Prepared from 4-(trifluoromethyl)phenylboronic acid following General Procedure B. ESI-MS: 473 (M+1).

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

[0287] ##STR00115##

[0288] Prepared from 2-furylboronic acid following General Procedure B. ESI-MS: 395 (M+1).

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

[0289] ##STR00116##

[0290] Prepared from 2-benzothienylboronic acid following General Procedure B. ESI-MS: 461 (M+1).

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

[0291] ##STR00117##

[0292] Prepared from 3-furylboronic acid following General Procedure B. ESI-MS: 395 (M+1).

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

[0293] ##STR00118##

[0294] Prepared from cyclopropylboronic acid following General Procedure B. ESI-MS: 369 (M+1).

5-(3-Ethynylpyridin-5-yl)carboxamido-2-methoxybenzoic acid N-(3-aminopropyl)amide (L24)

[0295] ##STR00119##

[0296] To 0.1 mmol of 1,3-diaminopropane-trityl-polystyrene resin, N-Fmoc-5-amino-2-methoxybenzoic acid and 5-bromopyridine-3-carboxylic acid were coupled following General Procedure A. The resulting resin loaded with the bromopyridine carboxylic amide was dried thoroughly and transferred to a glass vial. Copper(I) chloride (40 mol), triphenylphosphine (40 mol), bis(triphenylphosphine)palladium(II) dichloride (10 mol) and THF (1 mL) were added and the mixture was flushed with argon. Subsequently, trimethylsilylacetylene (1 mmol) and triethylamine (0.5 mL) were added, the mixture was flushed again with argon, then the vial was closed tightly and shaken overnight at 50 C. Then the resin was washed thoroughly with DMF and dichloromethane (each solvent several times). For TMS deprotection, THF (1 mL) and water (50 L) were added, followed by tetrabutylammonium fluoride (1 M solution in THF, 0.5 mL). The mixture was agitated for 2 h, then the resin was washed with DMF and dichloromethane (each solvent several times). Cleavage of the target compound from the resin was effected by treatment with dichloromethane-TFA-triethylsilane 85:10:5 (twofold cleavage), followed by rinsing with dichloromethane. After evaporation to dryness, the residue was purified by preparative reversed-phase HPLC. ESI-MS: 353 (M+1).

General Procedure C: Synthesis of 5-[5-(2-thienyl or 2-furyl)-1-methylpyrazol-3-yl]carboxamidoaryl carboxylic acid N-(3-aminopropyl)amides (L19, L9, L10, L17, L18)

[0297] The respective (N-Fmoc protected) aminoarylcarboxylic acid (0.2-0.5 mmol) and HOAt (1 equivalent relative to the carboxylic acid) were dissolved in DMF, NMP, DMF/DMSO or NMP/DMSO mixtures (1-3 mL) and treated with DIC (1 equivalent relative to the carboxylic acid). After agitating for 2-5 min, the mixture was added to 0.1-0.15 mmol of 1,3-diaminopropane linked to either 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, each solvent several times) and treated with piperidine 25% in DMF (1-5 mL) for 15-30 min. Subsequently, the resin was washed thoroughly (DMF, dichloromethane or DMF, methanol, dichloromethane, each solvent several times), and dried at air, with a stream of nitrogen or in high vacuum. 1-Methyl-5-(2-thienyl)pyrazole-3-carboxylic acid or 5-(2-furyl)-1-methylpyrazole-3-carboxylic acid (0.2-0.5 mmol) and HOAt (1 equivalent relative to the carboxylic acid) were dissolved in DMF, NMP, DMF/DMSO or NMP/DMSO mixtures (1-3 mL) and treated with DIC (1 equivalent relative to the carboxylic acid). After agitating for 2-5 min, the mixture was added to the resin and shaken for several hours or overnight. After subsequent washing (DMF, dichloromethane or DMF, methanol, dichloromethane, each solvent several times), the target compound was cleaved from the support by treatment with a suitable cleavage mixture (dichloromethane, TFA, triethylsilane 85:10:5 for trityl resins, 45:45:10 for 2-chlorotrityl resins). Typically, the cleavage step was repeated once, followed by rinsing of the resin with dichloromethane. After evaporation of the solvents, the crude residue was purified by preparative reversed-phase HPLC.

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

[0298] ##STR00120##

[0299] Prepared from N-Fmoc-2-aminothiazole-4-carboxylic acid and 1-methyl-5-(2-thienyl)pyrazole-3-carboxylic acid following General Procedure C. ESI-MS: 391 (M+1).

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

[0300] ##STR00121##

[0301] Prepared from N-Fmoc-3-amino-2-methylbenzoic acid and 1-methyl-5-(2-thienyl)pyrazole-3-carboxylic acid following General Procedure C. ESI-MS: 398 (M+1).

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

[0302] ##STR00122##

[0303] Prepared from N-Fmoc-3-amino-4-methylbenzoic acid and 1-methyl-5-(2-thienyl)pyrazole-3-carboxylic acid following General Procedure C. ESI-MS: 398 (M+1).

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

[0304] ##STR00123##

[0305] Prepared from N-Fmoc-3-aminobenzoic acid and 5-(2-furyl)-1-methylpyrazole-3-carboxylic acid following General Procedure C. ESI-MS: 368 (M+1).

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

[0306] ##STR00124##

[0307] Prepared from N-Fmoc-4-aminobenzoic acid and 5-(2-furyl)-1-methylpyrazole-3-carboxylic acid following General Procedure C. ESI-MS: 368 (M+1).

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

[0308] ##STR00125##

[0309] 1,3-Diaminopropane-trityl resin (0.1 mmol) was swollen in dichloromethane and treated with excess DIPEA and m-benzenedicarboxylic dichloride. After a few minutes, the resin was quickly washed with dichloromethane (3 times) and immediately treated with excess 3-amino-5-(2-furyl)pyrazole and DIPEA in NMP. When LCMS of a small resin sample which was cleaved (conditions, see below) showed complete conversion, the resin was washed (DMF, dichloromethane, each solvent several times) and treated with dichloromethane-TFA-triethylsilane (85:10:5; twofold cleavage, followed by rinsing of the resin with dichloromethane). The cleavage solution was evaporated to dryness and the residue was purified by preparative reversed-phase HPLC. ESI-MS: 354 (M+1).

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

[0310] ##STR00126##

[0311] 1,3-Diaminopropane-trityl-resin (0.15 mmol) was pre-swollen in NMP. N-Fmoc-5-amino-2-methoxybenzoic acid (0.2 mmol) and HATU (0.2 mmol) were dissolved in NMP (1.5 mL) and treated with DIPEA (0.4 mmol). After 2 min, the solution was added to the resin and the reaction mixture was shaken for 30 min. After washing the resin with DMF (3 times), 25% piperidine in DMF was added and shaking was continued for 30 min. Next, the resin was washed thoroughly with DMF, methanol and dichloromethane (each solvent 3 times) and dried at air.

[0312] Imidazo[2,1-b]thiazole-6-carboxylic acid hydrobromide (0.2 mmol) and HATU (0.2 mmol) were dissolved or suspended in NMP (1.5 mL) and treated with DIPEA (0.8 mmol). After 2 min, the solution was added to the resin and shaken overnight. After washing the resin with DMF, methanol and dichloromethane (each solvent 3 times), the target compound was cleaved from the resin by treatment with dichloromethane-TFA-triethylsilane 85:10:5 (twofold cleavage), followed by rinsing with dichloromethane. After evaporation to dryness, the residue was purified by preparative reversed-phase HPLC.

[0313] ESI-MS: 374 (M+1).

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

[0314] ##STR00127##

[0315] 2-Chlorotrityl chloride resin (150 mol) was pre-swollen in NMP and treated with NMP (1.5 mL), followed by 3-aminopropanol (1.5 mL). After shaking the resin for 2 h, it was washed with DMF, methanol and dichloromethane (each solvent several times) and dried at air. Then dichloromethane (2 mL) and DIPEA (2 mmol) were added, followed by dropwise addition of methanesulfonic chloride (1 mmol). The resin was agitated for 2 h, then it was washed quickly with dichloromethane (3 times) and swollen in NMP (1 mL). After addition of methylamine solution (8 M in ethanol, 1 mL), the resin was shaken overnight, then it was washed with DMF, methanol and dichloromethane (each solvent several times) and dried at air.

[0316] N-Fmoc-5-amino-2-methoxybenzoic acid (250 mol) and HOAt (250 mol) were dissolved in NMP (approx. 1.5 mL) and treated with DIC (250 mol). After 2 min, the solution was added to the resin and shaken for 5 h. After washing with DMF and dichloromethane (each solvent several times), the resin was treated with 25% piperidine in DMF (30 min, followed by washing as described before).

[0317] 5-(2-Furyl)-1-methylpyrazole-3-carboxylic acid (250 mol) and HOAt (250 mol) were dissolved in NMP (approx. 1.5 mL) and treated with DIC (250 mol). After 2 min, the solution was added to the resin and shaken overnight. Then the resin was washed as described before and the target compound was cleaved from the resin by treatment with dichloromethane-TFA-triethylsilane 45:45:10 (twofold cleavage), followed by rinsing with dichloromethane. After evaporation to dryness, the residue was purified by preparative reversed-phase HPLC. ESI-MS: 412 (M+1).

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

[0318] ##STR00128##

[0319] 5-Nitrosalicylic acid (0.3 mmol) and HOBt (0.3 mmol) were dissolved in DMSO (approx. 1.5 mL) and treated with DIC (0.3 mmol). After 2 min the solution was added to 1,3-diaminopropane-trityl resin (0.15 mmol) and shaken for 1 h, whereupon the resin was washed with DMF, water, diluted sodium carbonate solution, water, methanol and dichloromethane (each solvent several times). A small resin sample 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 min. In order to hydrolyze the amount of carbamoyl-nitrophenylester formed in the meantime, the resin was subsequently treated with THF (1 mL), methanol (0.5 mL) and aqueous NaOH (1 M, 0.5 mL) for 5 min, while the supernatant turning yellow indicated successful ester hydrolysis. After washing with DMF, methanol and dichloromethane (each solvent 3 times), DMSO (2 mL), caesium carbonate (1 mmol) and ethyl bromide (1 mmol) were added and the resin was shaken for approx. 2 h, then the reaction was repeated overnight (0.2 mL of water were added to dissolve the carbonate salt). As the conversion was still incomplete, the reaction was repeated using 0.5 mL of ethyl bromide and water (0.2 mL), leading to sufficient reaction progress after three days, then the resin was washed with DMF, water, methanol and dichloromethane (each solvent several times).

[0320] The nitro group was reduced by addition of NMP (1 mL), pyridine (0.5 mL) and a solution of tin(II) chloride (1 mmol) in NMP (1 mL). The mixture was shaken overnight, then the resin was washed with DMF, water and methanol, and insoluble side-products were carefully removed by flotation in methanol. After washing with dichloromethane, the resin was dried. 5-(2-Furyl)-1-methylpyrazole-3-carboxylic acid (0.25 mmol) and HOAt (0.25 mmol) were dissolved in NMP (approx. 1.5 mL) and treated with DIC (0.25 mmol). After 2 min, the solution was added to the resin and shaken overnight. Then the resin was washed with DMF and dichloromethane (each solvent several times) and the target compound was cleaved from the resin by treatment with dichloromethane-TFA-triethylsilane 85:10:5 (twofold cleavage), followed by rinsing with dichloromethane. After evaporation to dryness, the residue was purified by preparative reversed-phase HPLC. ESI-MS: 412 (M+1).

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

[0321] ##STR00129##

[0322] 5-Nitrosalicylic acid (0.3 mmol) and HOBt (0.3 mmol) were dissolved in DMSO (approx. 1.5 mL) and treated with DIC (0.3 mmol). After 2 min the solution was added to 1,3-diaminopropane-trityl resin (0.15 mmol) and shaken for 1 h, whereupon the resin was washed with DMF, water, diluted sodium carbonate solution, water, methanol and dichloromethane (each solvent several times). A small resin sample 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 min. In order to hydrolyze the amount of carbamoyl-nitrophenylester formed in the meantime, the resin was subsequently treated with THF (1 mL), methanol (0.5 mL) and aqueous NaOH (1 M, 0.5 mL) for 5 min, while the supernatant turning yellow indicated successful ester hydrolysis. After washing with DMF, methanol and dichloromethane (each solvent 3 times), DMSO (2 mL), caesium carbonate (1 mmol) and allyl bromide (1 mmol) were added and the resin was shaken for 1 h, then the reaction was repeated overnight (0.2 mL of water were added). As the conversion was still incomplete, the reaction was once again repeated for three days, then the resin was washed with DMF, water, methanol and dichloromethane (each solvent several times)

[0323] The nitro group was reduced by addition of NMP (1 mL), pyridine (0.5 mL) and a solution of tin(II) chloride (1 mmol) in NMP (1 mL). The mixture was shaken overnight, then the resin was washed with DMF, water and methanol, and insoluble side-products were carefully removed by flotation in methanol. After washing with dichloromethane, the resin was dried. 5-(2-Furyl)-1-methylpyrazole-3-carboxylic acid (0.25 mmol) and HOAt (0.25 mmol) were dissolved in NMP (approx. 1.5 mL) and treated with DIC (0.25 mmol). After 2 min, the solution was added to the resin and shaken overnight. Then the resin was washed with DMF and dichloromethane (each solvent several times).

[0324] The allyl ether was cleaved by treatment of the resin with dichloromethane (2 mL), piperidine (0.4 mL) and tetrakis-triphenylphosphinepalladium(0) (approx. 10 mg) for approx. 2 h. Then the resin was thoroughly washed with DMF, water, methanol and dichloromethane (each solvent several times) and the target compound was cleaved from the resin by treatment with dichloromethane-TFA-triethylsilane 85:10:5 (twofold cleavage), followed by rinsing with dichloromethane. After evaporation to dryness, the residue was purified by preparative reversed-phase HPLC. ESI-MS: 384 (M+1).

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

[0325] ##STR00130##

[0326] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was pre-swollen with NMP. Fmoc-5-amino-2-methoxybenzoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol). After two minutes, the solution was added to the resin and the mixture was shaken at room temperature for 3 h. After washing the resin with DMF, piperidine (25% in DMF, approx. 2 mL) was added and the resin was shaken for approx. 30 min, followed by thorough washing with DMF and dichloromethane. Indazole-3-carboxylic acid (0.5 mmol) and HOBt (0.5 mmol) were dissolved in NMP (1.5 mL). DIC (0.5 mmol) was added and the solution was added to the resin after 2 min. The mixture was shaken for 1 h, afterwards the resin was washed with DMF and dichloromethane. Caesium carbonate (1 mmol) and dry DMF (1.5 mL) were added to the resin, followed by ethyl bromide (1.5 mmol). The mixture was shaken over night, followed by washing with DMF, water, methanol and dichloromethane. The caesium carbonate/ethyl bromide step was repeated once over three days, then the resin was washed thoroughly. The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 396 (M+1).

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

[0327] ##STR00131##

[0328] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was pre-swollen in NMP. 5-Formylsalicylic acid (0.5 mmol) and HOAt (0.5 mmol) were dissolved in NMP (1.5 mL) and treated with DIC (0.5 mmol). After 2 min the solution was added to the resin and the mixture was agitated for 2 h, whereupon the resin was washed several times with DMF and DCM. Then the resin was treated with THF (1 mL), methanol (1 mL) and aqueous NaOH (2 M, 0.5 mL) for 1 h at room temperature, subsequently the resin was washed with methanol/water, DMF, methanol/water, DMF and DCM. After addition of acetonitrile (1 mL), tert-butanol (1 mL) and 2-methyl-2-butene (200 L), the resin was cooled to 0 C. and treated dropwise with a solution of sodium chlorite (0.25 mmol) and monosodium phosphate (0.2 mmol) in water (0.2 mL). After 30 min, acetonitrile (1 mL) and tert-butanol (1 mL) were added and sodium chlorite (1 mmol) and monosodium phosphate (0.8 mmol) in water (0.4 mL) were 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 (1 mmol), THF (2 mL) and methanol (0.2 mL) were added, followed by diethyl azodicarboxylate (1 mmol). The resin was agitated for 1 h at room temperature, then it was washed several times with DMF and dichloromethane. THF (1 mL), methanol (1 mL) and aqueous NaOH (2 M, 0.5 mL) were added and the mixture was shaken for four days at room temperature, subsequently the resin was washed with methanol/water, DMF, methanol/water, DMF and DCM. A solution of HATU (0.25 mmol) and DIPEA (0.5 mmol) in NMP (1 mL) was added and the mixture was shaken for 15 min at room temperature. Then 3-amino-1-methylindazole (0.25 mmol) in NMP (0.5 mL) was added and the reaction mixture was shaken over night, followed by washing with DMF and dichloromethane. The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 382 (M+1).

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

[0329] ##STR00132##

[0330] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was pre-swollen with NMP. Fmoc-5-amino-2-methoxybenzoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol). After two minutes, the solution was added to the resin and the mixture was shaken at room temperature for 3 h. After washing the resin with DMF, piperidine (25% in DMF, approx. 2 mL) was added and the resin was shaken for approx. 30 min, followed by thorough washing with DMF and dichloromethane. Indazole-3-carboxylic acid (0.5 mmol) and HOBt (0.5 mmol) were dissolved in NMP (1.5 mL). DIC (0.5 mmol) was added and the solution was added to the resin after 2 min. The mixture was shaken for 1 h, afterwards the resin was washed with DMF and dichloromethane. Caesium carbonate (1 mmol) and dry DMF (1.5 mL) were added to the resin, followed by allyl bromide (1 mmol). The mixture was shaken over night, followed by washing with DMF, water, methanol and dichloromethane. The caesium carbonate/allyl bromide step was repeated once over night, then the resin was washed thoroughly, dried and transferred to a glass vial. N-Methylmorpholine oxide (0.5 mmol) in acetone (2 mL) and water (0.5 mL) were added to the resin. Osmium-(VIII)-oxide (50 L of a 2.5% solution in tert-butanol) was added and the vial was closed firmly and shaken at room temperature over night. After addition of another portion of osmium-(VIII)-oxide (50 L of a 2.5% solution in tert-butanol) and further shaking for 2 h, the reaction was quenched with aqueous sodium dithionite (174 mg in 1 mL of water) and shaken for 30 min. Subsequently, the resin was washed several times with water, DMF, water, methanol, DMF and dichloromethane. The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 442 (M+1).

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

[0331] ##STR00133##

[0332] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was pre-swollen with NMP. Fmoc-5-amino-2-methoxybenzoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol). After two minutes, the solution was added to the resin and the mixture was shaken at room temperature for 2 h. After washing the resin with DMF and dichloromethane, piperidine (25% in DMF, approx. 2 mL) was added and the resin was shaken for approx. 30 min, followed by thorough washing with DMF and dichloromethane. The resin was swollen in dichloromethane (1 mL) and DIPEA (1 mmol) was added, followed by oxalic acid monoethyl ester monochloride (0.5 mmol) in dichloromethane (1 mL). After shaking for 5 min, the resin was washed with DMF, methanol and dichloromethane (three times each). 2,3-Diaminopyridine (0.5 mmol) in NMP (2 mL) was added and the mixture was shaken at approx. 100 C. for four days. After washing the resin (DMF, methanol, dichloromethane), the reaction was repeated at approx. 120 C. over night. After washing as described before, THF (1 mL), methanol (0.5 mL) and aqueous NaOH (2 M, 0.5 mL) were added to the resin and the mixture was agitated for 15 min, whereupon the resin was washed with methanol (five times) and dichloromethane (three times). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 369 (M+1).

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

[0333] ##STR00134##

[0334] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was pre-swollen in NMP. Fmoc-5-Amino-2-methoxybenzoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol) and after 2 min the mixture was added to the resin which was agitated for 3 h. After washing the resin with DMF, piperidine (25% in DMF, approx. 2 mL) was added and the resin was shaken for 30 min. Subsequently, the resin was washed thoroughly with DMF and dichloromethane and swollen in dichloromethane. Dichloromethane (1.5 mL), DIPEA (1 mmol) and oxalic acid monoethyl ester monochloride (0.5 mmol) were added and the mixture was agitated for 10 min, then the resin was washed several times with DMF and dichloromethane. Ethylene glycol (1 mL), 2-pyridylhydrazine (1 mmol) and DIPEA (1 mmol) were added and the mixture was heated to 100 C. for 4 h, then the resin was washed several times with DMF and dichloromethane. Triphenylphosphine (0.5 mmol), N-methylmorpholine (0.5 mmol) and dichloromethane (2 mL) were added to the resin, followed by drop-wise addition of trichloroacetonitrile (0.4 mmol). After shaking the mixture for 48 h, a solution of triphenylphosphine (0.5 mmol) and N-methylmorpholine (1 mmol) in dichloromethane (2 mL) were added along with tetrachlorocarbon (0.5 mL). The mixture was kept at 40 C. for 2 h, then THF (2 mL) and aqueous sodium carbonate (2 M, 0.5 mL) were added and the mixture was agitated over night. Finally, the resin was washed thoroughly with DMF, water, methanol and dichloromethane. The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 369 (M+1).

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

[0335] ##STR00135##

[0336] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was swollen in NMP. Then (S)-bis-Fmoc-lysine (200 mol) and HATU (200 mol) were dissolved in NMP (approx. 1.5 mL) and treated with DIPEA (400 mol) for 2 min, whereupon the solution was added to the resin and the mixture was agitated at room temperature. After 1 h the resin was washed with DMF and piperidine (25% in DMF, 2 mL) was added and the resin was agitated for 30 min. Subsequently, the resin was washed (DMF, methanol and dichloromethane). Fmoc-5-Amino-2-methoxybenzoic acid (400 mol) and HATU (400 mol) were dissolved in NMP (approx. 2 mL) and treated with DIPEA (800 mol) for 2 min, whereupon the solution was added to the resin and the mixture was shaken for 1 h. After washing the resin with DMF, deprotection was achieved by treatment with 25% piperidine in DMF, followed by washing as described before. Imidazo[2,1-b]thiazole-6-carboxylic acid (400 mol) and HATU (400 mol) were dissolved in NMP (approx. 2 mL) and treated with DIPEA (800 mol) for 2 min, whereupon the solution was added to the resin. After shaking the mixture for 1 h, the resin was washed with DMF, methanol and dichloromethane (three times each). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 801 (M+1), 401 ((M+2)/2).

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

[0337] ##STR00136##

[0338] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was swollen in NMP. Then (S)-bis-Fmoc-lysine (200 mol) and HATU (200 mol) were dissolved in NMP (approx. 1.5 mL) and treated with DIPEA (400 mol) for 2 min, whereupon the solution was added to the resin and the mixture was agitated at room temperature. After 1 h the resin was washed with DMF and piperidine (25% in DMF, 2 mL) was added and the resin was agitated for 30 min. Subsequently, the resin was washed (DMF, methanol and dichloromethane). Fmoc-8-Amino-3,6-dioxaoctanoic acid (400 mol) and HATU (400 mol) were dissolved in NMP (approx. 2 mL) and treated with DIPEA (800 mol) for 2 min, whereupon the solution was added to the resin and the mixture was shaken for 1 h. After washing the resin with DMF, deprotection was achieved by treatment with 25% piperidine in DMF, followed by washing as described before. Fmoc-5-Amino-2-methoxybenzoic acid (400 mol) and HATU (400 mol) were dissolved in NMP (approx. 2 mL) and treated with DIPEA (800 mol) for 2 min, whereupon the solution was added to the resin and the mixture was shaken for 1 h. After washing the resin with DMF, deprotection was achieved by treatment with 25% piperidine in DMF, followed by washing as described before. Imidazo[2,1-b]thiazole-6-carboxylic acid (400 mol) and HATU (400 mol) were dissolved in NMP (approx. 2 mL) and treated with DIPEA (800 mol) for 2 min, whereupon the solution was added to the resin. After shaking the mixture for 1 h, the resin was washed with DMF, methanol and dichloromethane (three times each). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 547 ((M+2)/2).

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

[0339] ##STR00137##

[0340] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was pre-swollen in NMP. Fmoc-5-Amino-2-methoxybenzoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol), and after 2 min the solution was added to the resin. After shaking for 105 min, the resin was washed with DMF and subsequently treated with piperidine (25% in DMF, 2 mL) for 1 h, followed by thorough washing with DMF, methanol and dichloromethane. 4-Bromopyridine-2-carboxylic acid (0.3 mmol) and HOAt (0.3 mmol) in NMP (3 mL) were treated with DIC (0.3 mmol). After 2 min the solution was added to the resin and the mixture was shaken for 1 h, whereupon the resin was washed as described before, dried thoroughly with a stream of dry nitrogen and transferred to a glass vial. Caesium carbonate (0.3 mmol) and 2-furylboronic acid (0.5 mmol) were added along with DMF (2 mL). The mixture was flushed thoroughly with argon. After addition of tetrakis-triphenylphosphinylpalladium-0 (10 mol), the mixture was once again flushed with argon, then the vial was closed tightly and the mixture was agitated at 100 C. over night. Subsequently, the resin was washed with DMF, water, methanol and dichloromethane (three times each). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 395 (M+1).

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

[0341] ##STR00138##

[0342] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was swollen in NMP. Then (S)-bis-Fmoc-lysine (200 mol) and HATU (200 mol) were dissolved in NMP (approx. 1.5 mL) and treated with DIPEA (400 mol) for 2 min, whereupon the solution was added to the resin and the mixture was agitated at room temperature. After 1 h the resin was washed with DMF and piperidine (25% in DMF, 2 mL) was added and the resin was agitated for 30 min. Subsequently, the resin was washed (DMF, methanol and dichloromethane). Fmoc-5-Amino-3-oxapentanoic acid (400 mol) and HATU (400 mol) were dissolved in NMP (approx. 2 mL) and treated with DIPEA (800 mol) for 2 min, whereupon the solution was added to the resin and the mixture was shaken for 1 h. After washing the resin with DMF, deprotection was achieved by treatment with 25% piperidine in DMF, followed by washing as described before. Fmoc-5-Amino-2-methoxybenzoic acid (400 mol) and HATU (400 mol) were dissolved in NMP (approx. 2 mL) and treated with DIPEA (800 mol) for 2 min, whereupon the solution was added to the resin and the mixture was shaken for 1 h. After washing the resin with DMF, deprotection was achieved by treatment with 25% piperidine in DMF, followed by washing as described before. Imidazo[2,1-b]thiazole-6-carboxylic acid (400 mol) and HATU (400 mol) were dissolved in NMP (approx. 2 mL) and treated with DIPEA (800 mol) for 2 min, whereupon the solution was added to the resin. After shaking the mixture for 1 h, the resin was washed with DMF, methanol and dichloromethane (three times each). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 503 ((M+2)/2).

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

[0343] ##STR00139##

[0344] Trityl polystyrene resin preloaded with 1,3-diaminopropane (450 mol, approx. 0.5 g) was pre-swollen in dichloromethane, followed by NMP. Fmoc-5-Amino-2-methoxybenzoic acid (0.6 mmol) and HATU (0.6 mmol) in NMP (4.5 mL) were treated with DIPEA (1.2 mmol) for 2 min. Subsequently the solution was added to the resin and the mixture was agitated for 40 min prior to threefold washing with DMF. After addition of piperidine (25% in DMF, approx. 6 mL), the resin was agitated for 30 min and subsequently washed with DMF, methanol and dichloromethane (three times each). After drying the resin in high vacuum, dichloromethane (3 mL) and DIPEA (3 mmol) were added and oxalic acid monoethyl ester monochloride (1.5 mmol) in dichloromethane was added cautiously. After shaking the mixture for 2 min, the resin was washed with dichloromethane, methanol and dichloromethane (three times each). After drying the resin, 150 mg were taken out. The remaining amount was treated with THF (4 mL), methanol (2 mL) and aqueous NaOH (2 M, 2 mL) for 2 h at room temperature. The resin was washed (DMF, acetic acid [2.5% in DMF], methanol and dichloromethane; three times each). Of this resin, 150 mg were reacted with HOAt (0.5 mmol) and DIC (0.5 mmol) in NMP (1 mL) for 2 min. Subsequently, 4-methoxy-N-hydroxybenzamidine (0.5 mmol) in NMP (1 mL) was added. After agitating the resin for 1 h, DIC (0.5 mmol) was added and the mixture was shaken over night. After washing the resin with DMF, methanol and dichloromethane (three times each), the coupling step was repeated. 3 h after the second addition of DIC, the resin was washed as described before, dried and transferred to a glass vial. NMP (2 mL) and DIC (0.5 mmol) were added and the mixture was heated to 120 C. for 30 min. Then the resin was washed as described before. The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 426 (M+1).

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

[0345] ##STR00140##

Synthesis of N-hydroxy-2-furylcarboxamidine

[0346] To a stirred solution of 2-cyanofuran (5 mmol) in ethanol (5 mL) was added aqueous hydroxylamine (50%, 5 mmol), whereupon slight heating of the reaction mixture was observed. After approx. 20 min, additional aqueous hydroxylamine (0.5 mmol) was added. After stirring for 1 h, the solvent was evaporated with a stream of nitrogen and the residue was purified by flash column chromatography (NH.sub.2-modified stationary phase, 0-5% methanol in dichloromethane, 1% triethylamine), furnishing a slightly turbid oil that solidified upon standing.

Synthesis of the Target Compound

[0347] Dry 2-chlorotrityl chloride resin (150 mol) was treated with 1,3-diaminopropane (2 mL), followed by dichloromethane (2 mL). After 5 min the resin was washed with DMF, methanol and dichloromethane (three times each) and swollen in NMP. Fmoc-5-Amino-2-methoxybenzoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol), and after 2 min the solution was added to the resin which was shaken at room temperature for 30 min. The resin was washed with DMF (three times) and treated with piperidine (25% in DMF, approx. 4 mL) for 30 min, whereupon the resin was washed with DMF, methanol and dichloromethane (three times each) and swollen in dichloromethane. After addition of dichloromethane (2 mL) and DIPEA (1 mmol), a solution of oxalic acid monoethyl ester monochloride (0.5 mmol) in dichloromethane (1 mL) was added cautiously and the mixture was agitated for 5 min, whereupon the resin was washed with DMF, methanol and dichloromethane (three times each), dried and transferred to a glass vial. Potassium carbonate (0.5 mmol), toluene (2 mL) and N-hydroxy-2-furylcarboxamidine (0.5 mmol; synthesis: see above) were added and the vial was firmly closed and agitated at 100 C. over night and at 110 C. for 2 h, whereupon the resin was washed with DMF, methanol and dichloromethane (three times each). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (45%) and triethylsilane (10%) in dichloromethane, followed by thorough rinsing of the resin with dichloromethane and removal of the solvents with a stream of nitrogen. The residue was purified by preparative reverse-phase HPLC. ESI-MS: 386 (M+1).

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

[0348] ##STR00141##

[0349] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was pre-swollen with NMP, followed by removal of excess solvent. Fmoc-5-amino-2-methoxybenzoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol). After two minutes, the solution was added to the swollen resin and the mixture was shaken at room temperature for 2 h. After washing the resin with DMF, piperidine (25% in DMF, approx. 2 mL) was added and the resin was shaken for approx. 30 min, followed by thorough washing with DMF and dichloromethane. Next, the resin was swollen in dichloromethane and treated with a solution of oxalic acid monoethyl ester monochloride (0.5 mmol) and DIPEA (1 mmol) in dichloromethane (1.5 mL) for 10 min, followed by repeated washing with DMF and dichloromethane. After swelling the resin in THF (1.5 mL), hydrazine hydrate (0.5 mL) was added and the resin was agitated for 3 h at room temperature, followed by washing with DMF and dichloromethane. Furan-2-carboxylic acid (0.5 mmol) and HOAt (0.5 mmol) were dissolved in NMP (1.5 mL) and treated with DIC (0.5 mmol). After 2 min the solution was added to the resin and the mixture was agitated at room temperature for 2 h, followed by washing with DMF and dichloromethane. Subsequently, a solution of triphenylphosphine (0.5 mmol) and N-methylmorpholine (1 mmol) in dichloromethane (2 mL) was added, followed by tetrachlorocarbon (0.5 mL). The mixture was heated to 40 C. for 2 h in a thoroughly closed glass vial. After addition of THF (2 mL) and aqueous Na.sub.2CO.sub.3 (2 M, 0.5 mL), the mixture was agitated over night at room temperature, followed by washing with DMF, water, methanol and dichloromethane. The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 386 (M+1).

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

[0350] ##STR00142##

[0351] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was pre-swollen with NMP. Fmoc-5-amino-2-methoxybenzoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol). After two minutes, the solution was added to the resin and the mixture was shaken at room temperature for 1 h. After washing the resin with DMF, piperidine (25% in DMF, approx. 2 mL) was added and the resin was shaken for 1 h, followed by thorough washing with DMF, methanol and dichloromethane. Indazole-3-carboxylic acid (0.5 mmol) and HOBt (0.5 mmol) were dissolved in NMP (1.5 mL). DIC (0.5 mmol) was added and after 2 min the solution was added to the resin. The mixture was shaken for 40 min, afterwards the resin was washed with DMF, methanol and dichloromethane (three times each). The resin was transferred to a glass vial and a solution of bromoacetic acid methyl ester (0.5 mmol) and DIPEA (1 mmol) in NMP (2 mL) was added. The vial was closed thoroughly and shaken at 80 C. for 75 min. Subsequently, the resin was washed (DMF, methanol, dichloromethane, three times each), swollen in THF (2 mL) and methanol (0.5 mL) and treated with sodium borohydride (large excess, added in portions). After the last addition, the mixture was shaken for 30 min at room temperature, then the resin was washed with methanol and dichloromethane (three times each). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 412 (M+1).

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

[0352] ##STR00143##

[0353] Trityl polystyrene preloaded with 1,3-diaminopropane (150 mol) was pre-swollen in NMP. Fmoc-8-Amino-3,6-dioxaoctanoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol) and after 2 min the solution was added to the resin which was shaken at room temperature for 1 h, whereupon it was washed with DMF (three times). Piperidine (25% in DMF, approx. 2 mL) was added and the mixture was shaken for approx. 30 min. The resin was washed with DMF, methanol and dichloromethane (three times each). Next, the coupling/deprotection protocol was applied to Fmoc-5-amino-2-methoxybenzoic acid (same amount as described before). Finally, 1-methylindazole-3-carboxylic acid was coupled using the HATU coupling protocol described above. The resin was washed with DMF, methanol and dichloromethane (three times each). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 527 (M+1).

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

[0354] ##STR00144##

[0355] Trityl polystyrene preloaded with 1,3-diaminopropane (150 mol) was pre-swollen in NMP. Fmoc-8-Amino-3,6-dioxaoctanoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol) and after 2 min the solution was added to the resin which was shaken at room temperature for 1 h, whereupon it was washed with DMF (three times). Piperidine (25% in DMF, approx. 2 mL) was added and the mixture was shaken for approx. 30 min. The resin was washed with DMF, methanol and dichloromethane (three times each). Next, the coupling/deprotection protocol was repeated with Fmoc-8-amino-3,6-dioxaoctanoic acid, afterwards it was applied to Fmoc-5-amino-2-methoxybenzoic acid (same amounts as described before). Finally, 1-methylindazole-3-carboxylic acid was coupled using the HATU coupling protocol described above. The resin was washed with DMF, methanol and dichloromethane (three times each). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 672 (M+1).

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

[0356] ##STR00145##

[0357] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was swollen in NMP. (S)-Bis-Fmoc-lysine (200 mol) and HATU (200 mol) were dissolved in NMP (approx. 1.5 mL) and treated with DIPEA (400 mol) for 2 min, whereupon the solution was added to the resin and the mixture was agitated at room temperature. After 1 h the resin was washed with DMF and piperidine (25% in DMF, 2 mL) was added and the resin was agitated for 30 min. Subsequently, the resin was washed (DMF, methanol and dichloromethane). Fmoc-8-Amino-3,6-dioxaoctanoic acid (400 mol) and HATU (400 mol) were dissolved in NMP (approx. 2 mL) and treated with DIPEA (800 mol) for 2 min, whereupon the solution was added to the resin and the mixture was shaken for 1 h. After washing the resin with DMF, deprotection was achieved by treatment with 25% piperidine in DMF, followed by washing as described before. Fmoc-5-Amino-2-methoxybenzoic acid (400 mol) and HATU (400 mol) were dissolved in NMP (approx. 2 mL) and treated with DIPEA (800 mol) for 2 min, whereupon the solution was added to the resin and the mixture was shaken for 1 h. After washing the resin with DMF, deprotection was achieved by treatment with 25% piperidine in DMF, followed by washing as described before. Imidazo[1,2-b]pyridazine-2-carboxylic acid (400 mol) and HATU (400 mol) were dissolved in NMP (approx. 2 mL) and treated with DIPEA (800 mol) for 2 min, whereupon the solution was added to the resin. After shaking the mixture for 1 h, the resin was washed with DMF, methanol and dichloromethane (three times each). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 542 ((M+2)/2).

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

[0358] ##STR00146##

Synthesis of 5-amino-2-methoxybenzoic acid methyl ester

[0359] 2-methoxy-5-nitrobenzoic acid methyl ester (1 mmol) and palladium(0) on charcoal (5%, 25 mg [containing approx. 50% w/w water]) in methanol (5 mL) were treated drop wise with triethylsilane (1 mL) over 10 min, followed by methanol (2 mL). After agitating for additional 5 min, the mixture was filtered through celite, followed by thorough rinsing of the celite layer. The filtrate was concentrated, dried in high vacuum and the residue was used without further purification.

Solution-Phase Synthesis of the Aminopropyl-Linked Ligand (Identical to L37)

[0360] imidazo[2,1-b]thiazole-6-carboxylic acid (1 mmol) and TBTU (1 mmol) in DMF (2 mL) were treated with DIPEA (2 mmol) for 5 min. Subsequently, the mixture was added to the complete amount of 5-amino-2-methoxybenzoic acid methyl ester obtained before, and the mixture was agitated for 30 min. After addition of saturated aqueous sodium carbonate (1 mL) and water (3 mL) 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 over night with a stream of dry nitrogen. 1,3-Diaminopropane (2 mL) was added to the residue and the mixture was heated to 80 C. for 4 h. Subsequently, the solution was concentrated to dryness, the residue was re-dissolved in methanol, concentrated and dried in high vacuum, furnishing an oil that solidified upon refrigerating. Yield: 453 mol (45%).

Synthesis of the Target Compound

[0361] N-Boc-glutamic acid (227 mol) and TBTU (453 mol) were dissolved in DMF (2 mL) and treated with DIPEA (906 mol) for 3 min, then the solution was added to the residue obtained before. After 90 min, another portion of TBTU-activated N-Boc-glutamic acid (0.2 mmol) in DMF (1 mL; activation for 2 min as described before) was added. After 45 min the solvent was evaporated over night with a stream of nitrogen. The residue was re-dissolved in methanol, evaporated to dryness and treated with trifluoroacetic acid (25% in dichloromethane), followed by evaporation to dryness and purification of the residue by preparative reverse-phase HPLC. ESI-MS: 858 (M+1), 430 ((M+2)/2).

General Procedure D: Synthesis of 5-Amino-2-Methoxybenzoic Acid Linked to Two Ado Units and One 1,3-Diaminopropane Unit (Base Resin)

[0362] Trityl polystyrene resin preloaded with 1,3-diaminopropane was pre-swollen in DCM and NMP. Fmoc-8-Amino-3,6-dioxaoctanoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol) for 2 min, then the solution was added to the resin and the mixture was agitated for 30 min to several hours. After washing the resin with DMF or DMF, methanol and dichloromethane, piperidine (25% in DMF, approx. 2 mL) was added and the resin was agitated for at least 20 min. Subsequently, the resin was washed thoroughly with DMF, methanol and dichloromethane (three times each) and the coupling/deprotection procedure was repeated once. Fmoc-5-Amino-2-methoxybenzoic acid (200 mol) was coupled to the resin employing the HATU-coupling protocol described before, followed by deprotection with piperidine as described before. The resin was dried at air prior to additional synthetic steps.

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

[0363] ##STR00147##

[0364] 5-(2-Furyl)-1-methylpyrazole-3-carboxylic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol) for 2 min. Subsequently, the solution was added to the base resin obtained by General Procedure D. After agitating the mixture for approx. 2 h, the resin was washed (DMF, methanol, dichloromethane, three times each). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 688 (M+1).

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

[0365] ##STR00148##

[0366] Imidazo[2,1-b]thiazole-6-carboxylic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol) for 2 min. Subsequently, the solution was added to the base resin obtained by General Procedure D. After agitating the mixture for approx. 2 h, the resin was washed (DMF, methanol, dichloromethane, three times each). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 664 (M+1).

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

[0367] ##STR00149##

[0368] The dry base resin obtained by General Procedure D was treated with dichloromethane (2 mL) and DIPEA (1 mmol), followed by drop-wise addition of oxalic acid monoethyl ester monochloride (0.5 mmol) in dichloromethane. The mixture was agitated for 10 min at room temperature, then the resin was washed with DMF, methanol and dichloromethane (three times each). Subsequently, the resin was swollen in NMP (2 mL) and treated with hydrazine hydrate (1 mmol) for 50 min at room temperature, followed by washing as described before. 2-Furoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol) for 2 min, then the solution was added to the resin. After approx. 1 h the resin was washed as described before. Next, 4-toluenesulfonyl chloride (0.5 mmol) and DIPEA (1 mmol) in dichloromethane (approx. 2 mL) was added and the mixture was agitated until cyclization was completed. The resin was washed as described before. The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 676 (M+1).

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

[0369] ##STR00150##

[0370] 5-(2-Methylthiazol-4-yl)isoxazole-3-carboxylic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol) for 2 min. Subsequently, the solution was added to the base resin obtained by General Procedure D. After agitating the mixture for approx. 2 h, the resin was washed (DMF, methanol, dichloromethane, three times each). The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 706 (M+1).

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

[0371] ##STR00151##

[0372] To 2-chlorotrityl chloride polystyrene resin (150 mol) was added 1,8-diamino-3,6-dioxaoctane (2 mL). After shaking the mixture for 5 min, dichloromethane (1 mL) was added to ensure sufficient swelling. After 25 min the resin was washed (DMF, methanol, dichloromethane, three times each). The resin was swollen in dichloromethane, then dichloromethane (2 mL) and DIPEA (1 mmol) were added, followed by chloroformic acid (4-nitrophenyl)ester (0.5 mmol) in dichloromethane (1 mL). The mixture was agitated for 5 min, then the resin was filtered off and immediately treated with 1,8-diamino-3,6-dioxaoctane (2 mL) for 20 min. Then the resin was washed as described before.

[0373] Fmoc-5-Amino-2-methoxybenzoic acid (200 mol) and HATU (200 mol) in NMP/DCM (1.5+1.5 mL) was treated with DIPEA (400 mol) for 2 min, subsequently the solution was added to the resin. After agitating the mixture for 5 h the resin was washed as described before and treated with piperidine (25% in DMF, approx. 2 mL) for 30 min, followed by washing as described before. Finally, imidazo[2,1-b]thiazole-6-carboxylic acid (200 mol) was coupled employing the HATU coupling protocol described before (neat NMP, approx. 1.5 mL; reaction time: over night), followed by washing of the resin as described before. The target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (45%) and triethylsilane (10%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, 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-aryl amides

[0374] Dry trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was treated with isophthaloyl chloride (1 mmol) and DIPEA (2 mmol) in dichloromethane (1.5 mL) for 3 min. The resin was washed quickly with dichloromethane or NMP (two or three times). Subsequently, the respective aromatic amine (0.3 mmol) and DIPEA (0.3 mmol) in NMP (1.5 mL) were added and the mixture was agitated for 1 h prior to washing with DMF, methanol and dichloromethane (three times each). The target compounds were cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. Due to the rather poor solubility of the compounds, methanol needed to be added to the cleavage or washing solutions in some cases. After evaporation of the solvents, the crude products were purified by reverse-phase HPLC.

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

[0375] ##STR00152##

[0376] Prepared from 2-amino-5-(2-furyl)-1,3,4-thiadiazole following General Procedure E. ESI-MS: 372 (M+1).

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

[0377] ##STR00153##

[0378] Prepared from 3-amino-1-methylindazole following General Procedure E. ESI-MS: 352 (M+1).

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

[0379] ##STR00154##

[0380] Prepared from 2-aminobenzothiazole following General Procedure E. ESI-MS: 355 (M+1).

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

[0381] ##STR00155##

[0382] Prepared from 2-amino-4-phenylthiazole following General Procedure E. ESI-MS: 381 (M+1).

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

[0383] ##STR00156##

[0384] Prepared from 2-amino-5-phenylthiazole following General Procedure E. ESI-MS: 381 (M+1).

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

[0385] ##STR00157##

[0386] 5-(Imidazo[2,1-b]thiazol-6-ylcarboxamido)-2-methoxybenzoic acid (3-amino-1-propyl)amide (L37) was prepared from imidazo[2,1-b]thiazole-6-carboxylic acid and 150 mol of 1,3-diaminopropane-preloaded trityl polystyrene resin following General Procedure A. The crude product that was obtained by acidic cleavage of the support bound ligand was dried with a stream of nitrogen. Saturated aqueous sodium carbonate (3 mL) was added and the mixture was extracted with ethyl acetate (three times) and dichloromethane (four times). The combined organic layers were evaporated to dryness. Boc-6-Aminohexanoic acid (100 mol) and HATU (100 mol) in DMF (1 mL) were treated with DIPEA (200 mol) and after 2 min the solution was added to the residue obtained before. After agitating the mixture for 20 min, water (2 mL) and saturated aqueous sodium carbonate (1 mL) 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 concentrated to dryness. The residue was treated with dichloromethane (1 mL) and trifluoroacetic acid (1 mL) for 40 min. After evaporation of the solvents the crude product was purified by reverse-phase preparative HPLC. ESI-MS: 487 (M+1).

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

[0387] ##STR00158##

[0388] 2-Chlorotrityl chloride polystyrene resin (150 mol) was treated with 1,8-diamino-3,6-dioxaoctane (500 L). After shaking the mixture for 3 min, dichloromethane (1 mL) was added and shaking was continued for 15 min prior to washing with DMF, methanol and dichloromethane (three times each). Subsequently the resin was swollen in NMP. Fmoc-6-Aminohexanoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol). After 2 min, the solution was added to the resin and the mixture was shaken for 2 h. Subsequently, the resin was washed as described before, followed by treatment with piperidine (25% in DMF, approx. 2 mL) for 1 h and washing as described before. Following this coupling/deprotection cycle, Fmoc-5-amino-2-methoxybenzoic acid was coupled to the resin (coupling time: over night; deprotection time: 30 min), followed by coupling of imidazo[2,1-b]thiazole-6-carboxylic acid (coupling time: 3 h). After final washing as described before, the target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (45%) and triethylsilane (10%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, 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)

[0389] ##STR00159##

[0390] Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 mol) was pre-swollen in NMP. Fmoc-8-Amino-3,6-dioxaoctanoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol). After 2 min, the solution was added to the resin and the mixture was shaken for 2 h. Subsequently, the resin was washed as described before, followed by treatment with piperidine (25% in DMF, approx. 2 mL) for 1 h and washing as described before. Following this coupling/deprotection cycle, Fmoc-5-amino-2-methoxybenzoic acid was coupled to the resin (coupling time: over night; deprotection time: 30 min), followed by coupling of imidazo[2,1-b]thiazole-6-carboxylic acid (coupling time: 3 h). After final washing as described before, the target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, 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)

[0391] ##STR00160##

[0392] 2-Chlorotrityl chloride polystyrene resin (150 mol) was treated with 1,6-diaminohexane (500 L). After shaking the mixture for 3 min, dichloromethane (1 mL) was added and shaking was continued for 15 min prior to washing with DMF, methanol and dichloromethane (three times each). Subsequently the resin was swollen in NMP. Fmoc-8-Amino-3,6-dioxaoctanoic acid (200 mol) and HATU (200 mol) in NMP (1.5 mL) were treated with DIPEA (400 mol). After 2 min, the solution was added to the resin and the mixture was shaken for 2 h. Subsequently, the resin was washed as described before, followed by treatment with piperidine (25% in DMF, approx. 2 mL) for 1 h and washing as described before. Following this coupling/deprotection cycle, Fmoc-5-amino-2-methoxybenzoic acid was coupled to the resin (coupling time: over night; deprotection time: 30 min), followed by coupling of imidazo[2,1-b]thiazole-6-carboxylic acid (coupling time: 3 h). After final washing as described before, the target compound was cleaved from the support by twofold treatment with trifluoroacetic acid (45%) and triethylsilane (10%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvents, the crude product was purified by reverse-phase HPLC. ESI-MS: 561 (M+1).

Example 3: Immobilization of Ligands

[0393] Experimental

[0394] Dried ligands were redissolved in DMSO at an approximate concentration of 100 mM based on gravimetric analyses. Precise concentrations of stock solutions were determined by the o-phthaldialdehyde assay (2). Dye-ligand molecules were quantitated by measuring absorbance at 340 nm. Measurements were calibrated with 1-(3-aminopropyl)imidazole.

[0395] In coupling reactions, one volume of ligand dissolved at an approximate concentration of 15 to 20 mM in 90% DMSO and 10% N-Methyl-2-pyrrolidone containing 1 M N,N-Diisopropylethylamine was added to one volume of settled NHS-activated Sepharose 4 FF (GE Healthcare) equilibrated with isopropanol. Reactions were conducted for 2 h at 25 C. while shaking vigorously. The supernatant of reactions was withdrawn and resins were washed twice with appropriate solvent. Remaining active groups on resins were blocked with 1 M Ethanolamine for 1 h at 25 C. Final resins were washed and stored in 20% ethanol at 4 C. until used in subsequent experiments. Reaction yields were determined based on mass balances after analysis of ligand concentrations in all fractions.

[0396] Summary and Results

[0397] Ligands from example 2 were immobilized on NHS-activated Sepharose 4 FF for subsequent chromatography and batch adsorption experiments. Coupling was achieved by formation of an amide bond between the amino group of the aminopropyl linker on ligands and the NHS-activated carboxylic group of the pre-activated resin.

[0398] The table below gives the results for coupling reactions with ligands from example 2. On average, an immobilized ligand density of 15.5 mol per ml of resin was obtained. Reaction yields averaged at 74.5%.

TABLE-US-00001 Initial Supernatant Immobilized Yield Ligand (mol per ml gel) (mol per ml gel) (mol per ml gel) (%) L1 18.8 7.8 11.0 58.5 L2 18.6 5.4 13.2 71.0 L3 18.0 6.1 11.9 66.1 L4 L5 20.6 6.8 13.8 67.1 L6 21.9 7.7 14.2 64.9 L7 22.1 8.4 13.7 62.2 L8 21.9 6.9 15.0 68.3 L9 20.7 7.9 12.8 62.0 L10 22.1 8.0 14.1 63.6 L11 19.0 4.4 14.6 76.8 L12 19.8 4.5 15.3 77.2 L13 20.1 4.7 15.4 76.8 L14 20.9 5.5 15.4 73.6 L15 19.5 4.5 15.0 77.0 L16 21.0 4.4 16.6 78.9 L17 20.8 4.4 16.4 78.8 L18 19.9 5.0 14.9 75.0 L19 19.6 6.1 13.5 68.8 L20 21.9 6.3 15.6 71.3 L21 11.8 5.1 6.7 56.7 L22 19.0 4.1 14.9 78.4 L23 19.5 3.8 15.7 80.6 L24 20.1 4.6 15.5 77.2 L25 19.3 4.3 15.0 77.7 L26 20.0 4.4 15.6 78.1 L27 20.5 5.2 15.3 74.7 L28 22.4 4.4 18.0 80.4 L29 22.6 2.5 20.1 88.9 L30 22.0 4.0 18.0 82.0 L31 24.7 2.7 22.0 89.0 L32 24.6 3.3 21.3 86.7 L33 22.7 5.7 17.0 74.9 L34 19.0 4.3 14.7 77.4 L35 23.1 2.7 20.4 88.2 L36 23.8 4.4 19.4 81.5 L37 23.8 6.8 17.0 71.3 Averages 15.5 74.5 L1 18.8 7.8 11.0 58.5 L2 18.6 5.4 13.2 71.0 L3 18.0 6.1 11.9 66.1 L4 L5 20.6 6.8 13.8 67.1 L6 21.9 7.7 14.2 64.9 L7 22.1 8.4 13.7 62.2 L8 21.9 6.9 15.0 68.3 L9 20.7 7.9 12.8 62.0 L10 22.1 8.0 14.1 63.6 L11 19.0 4.4 14.6 76.8 L12 19.8 4.5 15.3 77.2 L13 20.1 4.7 15.4 76.8 L14 20.9 5.5 15.4 73.6 L15 19.5 4.5 15.0 77.0 L16 21.0 4.4 16.6 78.9 L17 20.8 4.4 16.4 78.8 L18 19.9 5.0 14.9 75.0 L19 19.6 6.1 13.5 68.8 L20 21.9 6.3 15.6 71.3 L21 11.8 5.1 6.7 56.7 L22 19.0 4.1 14.9 78.4 L23 19.5 3.8 15.7 80.6 L24 20.1 4.6 15.5 77.2 L25 19.3 4.3 15.0 77.7 L26 20.0 4.4 15.6 78.1 L27 20.5 5.2 15.3 74.7 L28 22.4 4.4 18.0 80.4 L29 22.6 2.5 20.1 88.9 L30 22.0 4.0 18.0 82.0 L31 24.7 2.7 22.0 89.0 L32 24.6 3.3 21.3 86.7 L33 22.7 5.7 17.0 74.9 L34 19.0 4.3 14.7 77.4 L35 23.1 2.7 20.4 88.2 L36 23.8 4.4 19.4 81.5 L37 23.8 6.8 17.0 71.3 L38 24.9 5.0 19.9 79.7 L39 22.6 4.7 17.9 79.2 L40 26.2 5.9 20.3 77.7 L41 24.7 14.7 10.0 40.5 L42 27.5 8.1 19.4 70.4 L43 27.9 8.7 19.2 68.7 L44 22.8 4.1 18.7 82.0 L45 23.1 9.9 13.1 57.0 L46 26.0 10.2 15.8 60.9 L47 25.9 10.7 15.1 58.5 L48 23.5 12.1 11.4 48.5 L49 23.4 8.8 14.5 62.2 L50 25.9 9.0 16.9 65.1 L51 21.4 11.8 9.6 44.7 L52 23.6 5.3 18.3 77.6 L54 24.1 9.7 14.4 59.8 L55 26.0 6.6 19.4 74.6 L56 23.7 4.8 19.0 79.9 L57 23.8 7.2 16.6 69.7 L58 21.7 7.3 14.5 66.6 L59 23.8 5.6 18.2 76.4 L60 29.0 14.1 14.9 51.5 L61 31.9 15.7 16.2 50.9 L62 24.2 8.2 16.0 66.1 L63 23.7 10.6 13.0 55.1 L64 27.5 13.4 14.2 51.5 L65 30.1 13.5 16.6 55.0 L66 21.2 6.9 14.3 67.6 L67 23.1 8.8 14.3 61.8 L68 22.5 9.7 12.8 57.1 L69 24.0 11.9 12.1 50.3 L70 21.8 8.2 13.6 62.6 L71 29.5 12.6 16.9 57.2 L72 L73 L74 L75 23.5 8.6 14.9 63.5 L76 24.5 9.8 14.7 59.9 L77 24.3 8.4 15.9 65.4 L78 26.1 10.7 15.4 58.8 Averages 15.6 68.7

Example 4: Chromatographic Evaluation of Ligands (from Example 2)

[0399] Experimental

[0400] Resins were assessed in packed mode by microtiter plate chromatography. For each column, approximately 30 l of resin were transferred to a 384-well filter plate and sealed with appropriate top frits. Columns with rProtein A Sepharose FF and Mabsorbent A2P HF were included as controls.

[0401] Columns were equilibrated with 6.7 column volumes (cv) of phosphate buffered saline (0.15 M NaCl, 20 mM sodium phosphate, pH 7.3; PBS) prior to injection. Either 3.3 cv of whole IgGs dissolved at 0.75 mg ml.sup.1, Fab or Fc fragments dissolved at 0.25 mg ml.sup.1 (all three in PBS) or host cell proteins (HCP) were injected onto columns. Antibody fragments of Bevacizumab (Fab and Fc) were prepared by cleavage of whole IgG with papain protease (3) according to the protocol of the manufacturer. Unbound protein was washed from columns with 5 cv of PBS prior to elution with 5 cv of glycine buffer at pH 2.5. Volumes transferred were spun through columns at 50 g for 1-2 min. During injection of samples, acceleration was reduced to 10 g and centrifugation time increased to 5-10 min. Effluent fractions were collected in 384-well plates and concentrations of protein analyzed by Bradford assay. Protein masses m.sub.i, m.sub.ft and m.sub.e (see below) were calculated as the product of fraction volume and measured protein concentration.

[0402] Summary and Results

[0403] Binding and elution of several antibodies was demonstrated. Among them were the three humanized therapeutic antibodies Bevacizumab, Tocilizumab and Palivizumab and a human poly-IgG (h-poly-IgG) mixture isolated from human serum. In some cases, purified Bevacizumab Fab and Fc fragments or a mixture of rabbit IgGs (h-poly-IgG, isolated from serum) were used as additional feed. Selectivity of ligands toward antibodies was determined by investigation of binding behavior of host cell proteins. Results for commercial resins rProtein A Sepharose FF (rProtein A), Mabsorbent A2P (A2P) and MEP Hypercel (Hyper) were included for comparison.

[0404] The fraction of bound protein was calculated as the difference between the total mass of protein injected m, and the mass of protein detected in the flowthrough m.sub.ft, divided by the total mass of protein injected.

[00001]$\mathrm{bound}=\frac{m_i-m_{f.Math..Math.t}}{m_i}$

[0405] The yield of protein was calculated as the mass of protein eluted m.sub.e, divided by the mass of protein injected m.sub.i.

[00002]$\mathrm{yield}=\frac{m_e}{m_i}$

[0406] The selectivity of resins was calculated as the fraction of bound antibody B.sub.Ab, divided by the fraction of bound host cell proteins B.sub.HCP.

[00003]$\mathrm{selectivity}=\frac{B_{\mathrm{Ab}}}{B_{\mathrm{HCP}}}$

[0407] The fraction of bound protein and the yield of protein were calculated for each resin based on data for whole IgG-antibodies. In the case of Bevacizumab fragments and host cell proteins, only the fraction of bound protein is given. Selectivity of resins was calculated from data for Bevacizumab whole IgG and host cell proteins. Due to limited experimental precision, selectivity was truncated beyond values of 10.

[0408] Chromatography results for resins from example 3 and reference resins are given in the table below. Values for L53 are estimate values predicted on the basis of the close analogs L49 and L50.

TABLE-US-00002 Bevacizumab IgG Fab Fc Tocilizumab Palivizumab h-poly-IgG r-poly-IgG HCP Bound Yield Bound Bound Bound Yield Bound Yield Bound Yield Bound Yield Bound Ligand (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) Selectivity L1 100 91 18 100 100 97 100 92 100 90 100 100 30 3.3 L2 100 96 100 100 100 91 59 1.7 L3 100 89 97 95 100 93 102 72 62 1.4 L4 100 98 38 100 100 93 100 97 100 83 57 1.8 L5 100 96 77 84 100 80 71 72 97 69 48 2.0 L6 100 100 79 100 100 102 98 100 94 100 54 2.0 L7 100 110 92 109 100 99 100 91 70 1.4 L8 100 96 82 99 100 97 100 89 100 86 44 2.3 L9 100 91 71 64 41 33 65 53 58 1.7 L10 100 91 86 73 59 62 72 60 68 1.5 L11 95 94 100 89 78 75 89 79 56 1.7 L12 100 50 61 49 52 41 68 37 63 1.6 L13 100 94 9 64 91 79 65 58 76 75 38 2.7 L14 99 89 82 74 66 61 73 62 74 1.3 L15 99 94 100 98 100 90 98 83 64 1.6 L16 100 93 100 96 100 90 96 83 51 2.0 L17 100 94 97 89 81 70 91 87 36 2.8 L18 100 95 76 76 54 43 68 71 46 2.2 L19 100 99 47 48 18 16 49 48 56 1.8 L20 100 96 100 96 97 84 93 82 47 2.1 L21 99 95 52 53 26 26 54 61 50 2.0 L22 100 92 62 58 40 34 63 58 33 3.1 L23 100 98 12 67 86 85 61 57 75 74 32 3.1 L24 100 97 41 40 24 21 45 47 29 3.5 L25 100 95 100 93 82 78 93 81 46 2.2 L26 100 94 77 84 58 59 75 74 42 2.4 L27 100 94 6 96 100 104 100 94 98 89 35 2.9 L28 100 81 63 61 55 50 58 44 35 2.9 L29 100 84 100 96 100 88 95 70 43 2.4 L30 100 93 83 79 68 66 74 53 42 2.4 L31 100 96 100 98 99 94 95 74 23 4.4 L32 100 87 100 95 100 95 95 74 49 2.0 L33 99 83 48 51 33 31 51 39 17 5.8 L34 100 89 85 80 73 72 78 61 39 2.6 L35 100 96 100 92 100 95 95 76 51 2.0 L36 100 100 100 95 100 100 95 77 54 1.9 L37 100 92 99 95 92 87 94 70 29 3.4 rProtA 100 94 100 95 100 100 95 79 0 10.0 A2P 100 84 97 77 100 89 100 82 100 1.0 Hyper 100 83 100 71 100 59 95 57 77 1.3 L1 100 91 18 100 100 97 100 92 100 90 100 100 30 3.3 L2 100 96 100 100 100 91 59 1.7 L3 100 89 97 95 100 93 102 72 62 1.4 L4 100 98 38 100 100 93 100 97 100 83 57 1.8 L5 100 96 77 84 100 80 71 72 97 69 48 2.0 L6 100 100 79 100 100 102 98 100 94 100 54 2.0 L7 100 110 92 109 100 99 100 91 70 1.4 L8 100 96 82 99 100 97 100 89 100 86 44 2.3 L9 100 91 71 64 41 33 65 53 58 1.7 L10 100 91 86 73 59 62 72 60 68 1.5 L11 95 94 100 89 78 75 89 79 56 1.7 L12 100 50 61 49 52 41 68 37 63 1.6 L13 100 94 9 64 91 79 65 58 76 75 38 2.7 L14 99 89 82 74 66 61 73 62 74 1.3 L15 99 94 100 98 100 90 98 83 64 1.6 L16 100 93 100 96 100 90 96 83 51 2.0 L17 100 94 97 89 81 70 91 87 36 2.8 L18 100 95 76 76 54 43 68 71 46 2.2 L19 100 99 47 48 18 16 49 48 56 1.8 L20 100 96 100 96 97 84 93 82 47 2.1 L21 99 95 52 53 26 26 54 61 50 2.0 L22 100 92 62 58 40 34 63 58 33 3.1 L23 100 98 12 67 86 85 61 57 75 74 32 3.1 L24 100 97 41 40 24 21 45 47 29 3.5 L25 100 95 100 93 82 78 93 81 46 2.2 L26 100 94 77 84 58 59 75 74 42 2.4 L27 100 94 6 96 100 104 100 94 98 89 35 2.9 L28 100 81 63 61 55 50 58 44 35 2.9 L29 100 84 100 96 100 88 95 70 43 2.4 L30 100 93 83 79 68 66 74 53 42 2.4 L31 100 96 100 98 99 94 95 74 23 4.4 L32 100 87 100 95 100 95 95 74 49 2.0 L33 99 83 48 51 33 31 51 39 17 5.8 L34 100 89 85 80 73 72 78 61 39 2.6 L35 100 96 100 92 100 95 95 76 51 2.0 L36 100 100 100 95 100 100 95 77 54 1.9 L37 100 92 99 95 92 87 94 70 29 3.4 L38 100 100 100 99 91 79 100 99 45 2.2 L39 100 100 100 100 100 100 100 100 35 2.9 L40 100 100 90 90 62 62 98 97 22 4.5 L41 100 100 91 91 74 71 85 85 9 10.0 L42 100 92 100 99 99 99 100 100 32 3.1 L43 100 90 88 88 61 43 90 90 13 8.0 L44 100 100 78 78 56 56 73 73 4 10.0 L45 100 82 100 100 100 100 100 100 44 2.3 L46 100 82 100 97 85 85 93 93 20 5.0 L47 100 97 100 100 85 85 71 65 35 2.9 L48 100 97 87 87 58 54 84 84 8 10.0 L49 100 88 99 97 88 88 17 6.1 L50 100 92 100 100 91 91 15 6.9 L51 100 95 95 93 85 85 89 89 30 3.3 L52 100 99 100 100 100 83 100 100 41 2.4 L53 >95% >85% >90% >90% <20% >5 L54 100 100 100 100 100 84 100 98 36 2.8 L55 100 95 100 100 100 99 100 93 31 3.3 L56 100 95 95 95 81 79 83 83 21 4.9 L57 100 90 78 78 44 42 53 53 16 6.3 L58 100 90 100 100 97 83 98 93 23 4.3 L59 100 97 89 89 64 64 71 71 32 3.1 L60 100 82 93 93 95 94 30 3.3 L61 100 92 84 84 67 66 81 81 20 5.1 L62 100 100 95 91 84 77 89 89 3 10.0 L63 100 95 4 4 0 0 11 10 3 10.0 L64 100 93 99 99 100 97 36 2.8 L65 100 100 74 71 58 58 67 67 11 9.5 L66 100 100 54 53 41 27 46 46 0 10.0 L67 100 100 91 86 81 80 89 87 2 10.0 L68 100 100 71 71 65 65 65 65 4 10.0 L69 100 100 78 73 45 45 63 63 2 10.0 L70 100 83 100 100 55 55 64 64 19 5.4 L71 100 88 51 49 24 24 45 45 25 4.0 L72 100 83 93 93 91 91 90 90 40 2.5 L73 100 92 97 95 87 87 92 87 52 1.9 L74 100 96 97 91 88 86 91 90 53 1.9 L75 100 97 100 98 91 91 90 90 18 5.7 L76 100 98 93 89 59 59 73 73 9 10.0 L77 100 96 80 80 55 55 65 65 10 10.0 L78 100 94 94 94 61 61 76 76 14 7.4 rProtA 100 94 100 95 100 100 95 79 0 10.0 A2P 100 84 97 77 100 89 100 82 100 1.0 Hyper 100 83 100 71 100 59 95 57 77 1.3

[0409] Controls bound all antibodies to nearly 100%. Whereas Protein A bound no host cell proteins (HCP) at all, A2P and Hypercel showed high binding properties towards them. Resulting selectivity indices were 10.0 for Protein A, 1.0 for A2P and 1.3 for Hypercel.

[0410] Among the tested ligands from example 2, L1, L27, L31 and L37 showed best results in respect of antibody binding and selectivity. All of them bound all tested human whole IgG antibodies to more than 90% and thus have binding characteristics of generic Fc-binders. Consistent with this, L1 and L27 bound the Fc-fragment of Bevacizumab but not the Fab-fragment. Additionally, for L1 efficient binding of rabbit poly-IgGs was demonstrated. Yield of Bevacizumab, Tocilizumab and Palivizumab after chromatography varied between 87% and 100%. Yield of poly-IgG was slightly lower and ranged from 70% to 90%. Selectivity indices were 3.3 for L1, 2.9 for L27, 4.4 for L31 and 3.4 for L37.

[0411] Other ligands from example 2 showed either reduced binding of at least one of the tested antibodies or binding of HCP to more than 40% (resulting selectivity indices were 2.5 or lower).

Example 5: Isotherms and Time-Scales of Binding

[0412] Experimental

[0413] Experiments were performed with Bevacizumab as antibody. Parameters of Langmuir isotherms for purified antibody were determined by batch adsorption experiments in 96-well microtiter plates. In each well, 10 l of sorbent slurry (50% v/v) were mixed with 100 l of protein solution. Initial concentrations varied from 0.05-5 mg ml.sup.1 in phosphate-buffered saline (0.15 M NaCl, 20 mM phosphate buffer, pH 7.3; PBS). Reactions were agitated vigorously at 25 C. for at least 3 h. Afterwards, antibody concentration in the supernatents were measured by Bradford assay.

[0414] Uptake kinetics were investigated similarly by batch adsorption in 96-well filter plates. Again, 10 l of sorbent slurry (50% v/v) were mixed with 100 l of protein solution. However, a fixed initial concentration of 0.75 mg ml.sup.1 Bevacizumab in PBS was used. Reactions were agitated vigorously at 25 C. for up to 80 min. Supernatants were separated rapidly by spinning through filters after 2.5, 5, 10, 20, 40 and 80 minutes and sampled for analysis. Concentrations of the antibody were analyzed by Bradford assay.

[0415] Summary and Results

[0416] A subset of immobilized ligands from example 3 was characterized with respect to their affinity and maximum capacity towards Bevacizumab. Furthermore, for L1 the time-scale required for binding was determined. Commercial resins rProtein A Sepharose FF (rProtein A) and Mabsorbent A2P (A2P) were included for comparison.

[0417] Parameters of Langmuir isotherms, i.e. dissociation constants K.sub.d and maximum capacities q.sub.m, were determined from the measured concentrations in supernatants. Parameters were estimated by numerical fitting of the model equation derived by Chase (4).

[0418] Uptake kinetics were characterized by a time-scale of binding t.sub.0.8, which was defined as the time after which 80% of the equilibrium saturation of resin with antibody had occurred. For determination of t.sub.0.8, measured concentrations in the supernatant were interpolated by fitting of double-exponentials (5) and values of t.sub.0.8 were read out from graphs. Parameters of Langmuir isotherms and time-scales of binding for in-house resins as well as for reference resins are reported in the table below.

TABLE-US-00003 K.sub.d q.sub.m t.sub.0.8 Ligand (g ml.sup.1) (mg per ml resin) (min) L1 61 36 9.9 L13 162 37 L17 252 26 L22 147 29 L23 103 33 L24 210 33 L27 92 33 rProtein A 4.7 41 8.9 A2P 49 68 9.2

[0419] Highest affinity was observed for rProtein A, which had a K.sub.d of 4.7 g ml.sup.1. Dissociation constants of A2P (49 g ml.sup.1), resins L1 (61 g ml.sup.1) and L27 (92 g ml.sup.1) were lower by one order of magnitude. Dissociation constants of the residual resins were lower by two orders of magnitude and ranged from 103 g ml.sup.1 (L23) to 252 g ml.sup.1 (L17). Concerning maximum capacity, the highest capacity was measured for A2P (69 mg per ml of resin). rProtein A had a maximum capacity of 41 mg per ml of resin. Maximum capacities of in-house resins varied between 26 and 37 mg per ml of resin. Uptake of antibody was fastest with rProtein A (8.9 min), followed by A2P (9.3 min) and L1 (9.9 min).

Example 6: Dynamic Binding Capacity

[0420] Experimental

[0421] Dynamic binding capacities were determined by column chromatography with purified Bevacizumab. Resins were packed into analytical columns of 25 mm length and 3 mm inner diameter. Columns were fed with 1 mg ml.sup.1 antibody in phosphate-buffered saline (150 mM NaCl, 20 mM phosphate buffer, pH 7.3) at constant flow rate, while the concentration of antibody in effluents was monitored online via absorbance at 280 nm. Antibody was loaded until effluent concentration reached at least 10% of the feed concentration. Dynamic binding capacities were determined at a flow rate of 50 cm h.sup.1, corresponding to 3 min empty column residence time. Equilibrium capacities were also determined at a flow rate of 50 cm h.sup.1 by loading until complete breakthrough of antibody. Bound antibody was stripped from columns by washing with sodium citrate at pH 3.0 followed by glycine hydrochloride at pH 2.5.

[0422] Summary and Results

[0423] Dynamic (binding) capacity for Bevacizumab as a function of flow rate was determined for L1 (from example 3) and rProtein A Sepharose 4 FF (rProtein A).

[0424] Dynamic capacity was defined as the amount of antibody that can be injected onto a column at constant flow rate until the effluent concentration reaches 10% of the feed concentration. It was calculated as the time t.sub.0.1 after which 10% breakthrough occurred, multiplied by the flow rate F and feed concentration c.sub.f.

dynamic capacity=t.sub.0.1.Math.F.Math.c.sub.f

[0425] Equilibrium capacity is defined as the maximum amount of antibody that binds to a column for a given feed stream concentration. It was calculated by evaluation of the following integral

[00004]$\mathrm{equilibrium}.Math..Math.\mathrm{capacity}={}_0^{}.Math.\left[c_f-c\left(t\right)\right].Math.F.Math.t$

containing the feed concentration c.sub.f, effluent concentration over time c(t) and flow rate F.

[0426] The integral was evaluated numerically. Integration was limited to the time after which complete breakthrough had occurred. Calculations of dynamic capacity and equilibrium capacity were both corrected for the hold-up of columns and the chromatography system.

[0427] Capacities were Normalized by the Volume of Resin in Columns

[0428] Dynamic binding capacities for L1 and rProtein A Sepharose 4 FF as a function of flow rate and equilibrium binding capacities for a feed concentration of 1 mg ml.sup.1 Bevacizumab are given in the table below.

[0429] For rProtein A a dynamic binding capacity of 28.4 mg per ml or resin was determined. L1 had a dynamic capacity of 20.2 mg per ml of resin. The calculated equilibrium capacity was 40 mg per ml of resin for rProtein A and 25.7 mg per ml of resin for L1.

TABLE-US-00004 Dynamic Capacity Equilibrium Capacity Ligand (mg per ml of resin) (mg per ml of resin) L1 20.2 25.7 rProtein A 28.4 40.2

Example 7: Alkaline Stability

[0430] Ligand L1 from example 2 was tested for its alkaline stability over a period of 8 days. It was either treated with 0.1 M or 0.5 M sodium hydroxide at 25 C. Hydrolysis was monitored by LC-MS analysis.

[0431] At 0.5 M NaOH, half-life of L1 was 288 h. At 0.1 M NaOH, no degradation of the ligand was detected.

Example 8: Purification of Antibody from Cell Culture Supernatant

[0432] Experimental

[0433] Suitability of resins for the purification of antibody from cell culture supernatant was assessed either in packed mode by microtiter plate chromatography similar to example 3 or by regular column chromatography. In both cases, Bevacizumab spiked into host cell proteins at a concentration of 0.05 mg ml.sup.1 was used as feed. In addition, chromatography runs with host cell proteins (microtiter plate and column chromatography) and pure antibody alone (only microtiter plate chromatography) were conducted. For microtiter plate chromatography, 25 column volumes were injected per run. For column chromatography 100 column volumes were injected. Columns were equilibrated and washed with PBS before and after injection.

[0434] Bound protein was eluted with sodium citrate at pH 3.0. Protein concentrations in feeds and column effluents were analyzed either by Bradford assay or by UV-absorbance at 280 nm.

[0435] Summary and Results

[0436] Antibody was purified from cell culture supernatant by chromatography on resin L1 from example 3. Chromatographies on commercial resins rProtein A Sepharose FF (rProtein A) and Mabsorbent A2P (A2P) were included for comparison. For microtiter plate chromatography, three chromatography runs under identical conditions were conducted on each resin, either injecting the antibody, host cell proteins or a mixture of the latter. For column chromatography, only two runs were performed, either with antibody spiked into host cell proteins or host cell proteins alone.

[0437] Operation yield after chromatography of the mixture was calculated from the mass of total protein m.sub.mix,e recovered upon elution after injection of the mixture, the mass recovered after injection of host cell proteins alone m.sub.HCP,e and the mass of antibody injected m.sub.Ab,i.

[00005]$\mathrm{yield}=\frac{m_{\mathrm{mix},e}-m_{\mathrm{HCP},e}}{m_{\mathrm{Ab},i}}$

[0438] The purity of antibody after chromatography of the mixture was calculated from the mass of total protein recovered upon elution m.sub.mix,e after injection of the mixture and the mass m.sub.HCP,e recovered after injection of host cell proteins.

[00006]$\mathrm{purity}=\frac{m_{\mathrm{mix},e}-m_{\mathrm{HCP},e}}{m_{\mathrm{mix},e}}$

[0439] Purity and yield obtained after chromatography on resin L1 from example 3 and reference resins are given in the table below.

[0440] Within experimental precision, yields were 100% for rProtein A, 85% for L1 And 12% for A2P after microtiter plate chromatography. Recovery from A2P (12%) was low due to moderate acidic pH of 3.0 upon elution. Purity was highest after chromatography on rProtein A (100%), followed by chromatography on resin L1 (74%). Purity was lowest after chromatography on A2P with only 32%. Results after column chromatography were in similar range. Here, rProtein A showed a yield of 97% and a purity of 100%. L1 showed a yield of 90% and a purity of 83%.

TABLE-US-00005 Microtiter Plate Column Chromatography Chromatography Yield Purity Yield Purity Ligand (%) (%) (%) (%) L1 85 74 90 83 rProtein A 106 100 97 100 A2P 12 32

[0441] Documents cited in the examples: [0442] 1. T. Neumann, H D Junker, K. Schmidt, R. Sekul, SPR Based Fragment Screening: Advantages and Applications, Current Topics in Medicinal Chemistry 2007; 7: 1630-1642 [0443] 2. Roth M. Fluorescence reaction for amino acids. Anal Chem 197; 43(7):880-2. [0444] 3. Rousseaux J, Rousseaux-Prevost R, Bazin H. Optimal conditions for the preparation of Fab and F(ab).sub.2 fragments from monoclonal IgG of different rat IgG subclasses. J. Immunol. Methods 1983; 64(1-2):141-146. [0445] 4. Chase H A. Prediction of the performance of preparative affinity chromatography. J Chromatogr 1984; 297:179-202. [0446] 5. Coffman J L, Kramarczyk J F, Kelley B D. High-throughput screening of chromatographic separations: I. Method development and column modeling. Biotechnology and Bioengineering 2008; 100(4):605-618.