JPH09508355A - How to make a combinatorial compound library - Google Patents

Abstract

  (57) [Summary] The present invention relates to the preparation of a plurality of different units consisting of solid or semi-solid supports (beads), synthetic oligomers (ligands) and identification structures (tags) by which the monomers of the ligands are encoded, and a new class of compounds and It relates to the use of that library for the search of individual compounds. The invention further relates to compounds discovered using the novel process and their use as thrombin inhibitors.

Description

Detailed Description of the Invention How to make a combinatorial compound library   The present invention relates to solid or semi-solid carriers (beads), synthetic oligomers (ligands) and A number of different, composed of identifying structures (tags) encoding the monomers of these ligands. For manufacturing units, and for searching new classes of compounds and individual compounds. About the use of the library. Furthermore, the present invention provides detection by the above novel process. Compound and its use as a thrombin inhibitor.   In recent years, there has been a continuous increase in the number of chemical substances that selectively bind to specific receptors. There is a great demand. These compounds are then, inter alia, inhibitors, Can be used as a gonist, antagonist, or for marking Wear. In this regard, the possibility is to duplicate a large number (different libraries) of different compounds. Sometimes they are synthesized, and they are linked to their ability to bind to specific receptors. It has been developed by testing. These libraries (combination compound live Rally (combinatorial compound libraries = CCL) is usually a natural amino acid or From nucleotides, and even more often, modified amino acids, modified nucleotidies And other chemicals used as monomer building blocks. You.   The individual different ligands of these libraries are usually run in parallel, in mixtures or Are synthesized as physically separate individuals. These parallel composites are In the meantime, it is possible to produce a library containing a large number of different ligands. Tolerate.   The literature consists of two documents for chemical synthesis of libraries of multiple test candidates. It describes the different causal strategies. 1. The ligands are obtained together in solution after their synthesis (Houghten et al. Nature ( 1991) 354, 84-86). 2. The ligand is synthesized on a solid support and the synthesis is performed by only one species of ligand. To be bound to the carrier (1 bead, 1 sequence; Lam et al. Nature (1991) , 354, 82-84; Furka et al., 14^thIntern. Congr. Biochem. FR 013, 1988).   In the first strategy, the problem is that all the ligands are in solution simultaneously In that case, it consists in unambiguously identifying the desired ligand. With many variable positions Each of them is occupied by 1 in 20 amino acids to identify one ligand For ligands with 4 variable positions capable of 4 × 20 = 80 pools were synthesized , And each possible monomer per variable position, as it must be calibrated. It is necessary to synthesize 1 pool for each.   The second strategy creates all the desired possibilities in one pool and Simple separation of the solid support containing the desired ligand and hence identification of that ligand Tolerate. However, their composition must then be identified As such, there are limitations on the type of ligand used. Therefore, Clearly determined by sequencing (peptides from natural amino acids, DNA and RNA) Only those ligands that can be identified as are suitable. A further drawback is the solid Attachment of the ligand to the body can lead to false positive results, ie many assays Can not be done with a ligand attached to a solid support It is.   Contrary to these known possibilities, the present invention provides only one of each solid support. Identification of a type of ligand (1 bead, 1 sequence) and 1 encoding this ligand Provides a library with attached structures (tags). 1 to 1 tag The clear designation of Gand is In the binding assay, in the amount they are present on the carrier, known methods and / or Cannot be unequivocally identified by an automated procedure It makes it possible to identify such ligands. The more surprising advantage is that The results should not be affected by the solid support in any way. The ligand is the carrier without loss of identification ability in whole or in part. It can be separated from the. Therefore, the same With a defined ligand, not with a carrier-bound ligand Performing an assay, eg, further enzymatic or purity assay by IR, NMR or MS Can be.   Accordingly, the present invention is directed to solid or semi-solid carriers (beads), synthetic oligomers, respectively. (Ligand) and thereby the monomer of that ligand can be identified A library comprising a number of different units of an identified structure:   a) each carrier carries only one type of ligand,   b) the ligand and the tag are attached to the same carrier at different positions,   c) The ligand, regardless of the tag and without changing its information content, Can be separated from the carrier,   d) the tag is a sequenceable polypeptide, and   e) to be removed under mild acid conditions to synthesize the above tag or ligand. Such a library is used in which a protecting group capable of protecting is used.   Therefore, the inventive process described above allows two different tests to be performed. You can:   a) a first for preselecting a ligand that binds to its acceptor 1 assay, the ligand of which is the solid or semi-solid described above Bound to a carrier,   b) its carrier and its information structure for further characterization of its ligand Second assay, after separation of its ligand from. This second test is also The attachment of its ligand to the sceptor can be investigated or the attached ligase It can be a completely different assay that cannot be performed with a handset.   Protecting groups that can be removed under mildly acidic conditions are, for example, 10% of acetic acid. Can be completely removed at concentrations of up to, preferably up to 5%. This These preferred protecting groups are dimethyl-dimethoxybenzyloxycarbonyl protected It will be at least 10 times more unstable than the group (Ddz). A particularly preferred group is triti Of the red type, for example, unsubstituted or alkoxy-substituted trityl.   The term ligand specifically includes all compounds that comprise a large number of monomers. You. Typically these monomers contain at least two reactive groups. this Illustrative examples of such reactive groups are amino, azide, isocyanide, isocyanato. , Hydrazino, carbonyl, carboxyl, acylhalogeno, hydroxyl, su Luffhydryl, sulfonyl chloride, phosphate groups, and halogeno groups. this The reactivity of these groups is modified by protecting or activating groups to facilitate synthesis. Can be done. Typical examples of monomers containing more than one reactive group are natural and natural. And unnatural amino acids, ω-aminocarboxylic acids, sugars, nucleotides and nucleotidies De analog. When using a monomer containing two or more reactive groups, the Optional Gandh branching and cyclization can also be inserted. Only one reactive group The containing monomers can be used as end groups.   The inventive process, which is characterized in detail below, is as follows: Not only consecutively, but in certain combinations with each other Library of ligands that bind to a basic compound It is also possible to create. This is a basic building block that contains a large number of reactive groups. Attached to the carrier directly or via a linker, and then this basic structure is attached. Individual monomers in building blocks, this time containing one or more reactive groups Can be carried out by attaching. Within the context of the present invention, the basic building block Locks (Basic building blocks) are typically steroid nuclei, penicillin Or penem nucleus, soraphens, benzodiazipine (benzodiazip) ines), sugars or desferoxiamine. Then the different Reactive groups can be attached to these nuclei by standard chemical methods.   Preferred ligands are, for example, known automation without further experimental effort. The sequence can be unambiguously identified by sequencing. Contains no monomer.   Monomers that cannot be unequivocally identified by sequencing are the natural 20 amino acids. Acids and all chemicals other than naturally occurring nucleotides in DNA and RNA You. Typical examples are modified amino acids or nucleotides, ω-aminocarboxylic Acids, D-amino acids, sugars, amino acids with sugar side chains and only one reactive group, eg For example, an end group carrying acetyl or benzyl.   The ligand is usually attached to the solid or semi-solid support through a linker. The linker for this ligand typically contains at least two reactive groups. It is a chemical substance. Preferred linkers are those in which the linker and tag are synthesized on the carrier. Mildly basic and mildly acidic conditions so that not only can it be assayed Endures. Therefore, the preferred linker is cleaved by a particular reaction. Can be cleaved by, for example, cyanogen bromide Methionine, or only under strong base or strongly acidic conditions, by photolysis, or reductively or Is a linker that can be cleaved under oxidative conditions. Particularly preferred linker The basic conditions are such that the ligand can be selectively removed from the carrier. It is what creates a cleavable bond underneath and a bond which is stable under acidic conditions. Preferred phosphorus An exemplary example of Kerr is p-hydroxymethyl-benzoic acid, 4-hydroxymethyl. Phenylacetic acid, benzhydrylamino, alkyl, hydroxycrotonylamino Methyl, 3-nitro-4-hydroxymethylbenzoic acid, p-nitrobenzhydrido Luamine, 4- [4,4'-bis (methylsulfinyl) -2-oxybenzhi Drill amino] butyric acid and a disulfide linker, which is then used for solid phase synthesis. It can also be linked through a group called a "handle" Of. These linkers suitable for use in the inventive process include: In addition to the cleavable building blocks described above, they may or may not affect the cleavage reaction. A building block spatially from the cleavable group of the variable part of the ligand Suitable for segregating minutes can be included.   Proteases such as trypsin, yscα, yscF or V8-protease Short peptides with specific sites can also be used as linkers.   Particularly preferred linkers are such that under suitable conditions only certain parts of the linker are cleavable. Are cleaved, so that in some parts, for example, for further assay The cleavage allows for the possibility of separating the ligand from the solid support. It can also be trolled. These linkers are either photoluminescent or A spout-like agent, for example a linker that can be cleaved by ammonia Very particularly preferred. Because these linkers are The residue can be removed completely or with little residue prior to calibration. Because it can.   They are precisely defined and, despite the heterogeneity of their ligands, are in equal parts. And the ability to cleave different ligands The linker between is the same for the cleavable moiety as described above, and for all ligands Can consist of parts. Suitable building blocks for the constant part are generally In addition to the cleavable portion of the linker, attachment to other chemical building blocks or ligands It can be a chemical agent that allows for wear. An illustrative example is a bifunctional chemical , For example, the constant part of an amino acid, a nucleic acid or its ligand.   An information structure (tag) is a natural amino acid (Ala, A rg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser , Thr, Trp, Tyr, and Val). Reactivity Amino acids that do not carry any side groups of and / or are easily identifiable during sequencing , Typically using Ala, Asn, Asp, Gly, Ile, Leu, Phe, Pro, Trp and Val Is preferred. Particularly preferred amino acids are Ala, Asn, Asp, Gly, Leu and Phe. And Pro. This tag, in turn, is a linker that is cleavable under certain conditions. It can be bound to the carrier through one of the Important about this And the separation of the linker and tag can be independent of the other, and from the carrier That is, the separation of the ligands does not change the information of the tag.   When all 20 natural amino acids are used, 20 different monomers per variable site are coded. Can be loaded. Encode more than 20 different monomers per variable site , Is used, one or more amino acids are used for each coding unit. Therefore, using dipeptides By using 20 different amino acids²Up to different monomers You can   To avoid translation errors when sequencing tags, the preferences listed above are preferred. Only amino acids can be used. These amino acids are the coding monomers. Can be used as a di- or tripeptide to increase the number of . That is, each monomer of the ligand is encoded by one or more amino acids in the tag. Is done.   The tag is as small as possible and affects the test result, and its ligand Liquid on a solid or semi-solid support to ensure that the amount of The ratio of code to tag is desirably greater than 1, preferably 2-100, and better. It is preferably 4 to 10.   To be considered for implementation, the tag will be labeled with its ligand when sequencing the tag. So that we can accurately identify the beginning and end of the Start and / or end sequences that do not encode Gand's monomer can be provided. Wear. Illustrative examples of such starting and ending sequences include the monomer of the ligand Unencoded amino acids as well as di- or tripeptides of the same amino acids, eg Val -Val or Leu-Leu-Leu.   The term "solid or semi-solid support" means that it is insoluble in the reaction medium and A macroscopic particle that allows both the gand and the tag to be bound in sufficient quantity. It will be understood to mean. The amount of tag that can be bound to the carrier is Signals that can be unequivocally identified when sequencing tags in (eg> 1 pm ol) must be large enough to produce The tag is radioactive Less than 1 pmol of tag per carrier will also be released if it contains monomer marked by the substance. It can be identified because of the lower detection limit for radiopharmaceuticals.   The binding of the ligand to the tag is linked to the reactive group on the surface of the carrier, such as an amino group. , Carboxyl, hydroxyl or halogen groups. These reactivity The groups are usually already a constituent of the carrier, but they are applied subsequently or It can also be modified. The resin commonly used in solid phase synthesis is used , For example, they are used in Merrifield peptide synthesis. That Have a polystyrene content that is mostly crosslinked by copolymerization with divinylbenzene. It consists of children. These molecules were attached to their reactants in solid phase synthesis. For further derivatization.   The present invention allows the synthesis of ligands and tags by orthogoral and other syntheses. It also relates to a method of producing the above library, which comprises.   The synthesis of the library of the present invention comprises the following steps:   a) the first unit of the tag (preferably less than equal, ie reactive on the carrier) Less than 50 mol% based on the group) and the first monomer of the ligand or its building block or Attaches a linker for each to a solid or semi-solid support;   b) optionally further non-variable monomers to the ligand or further A non-variable coding unit attached to the tag;   c) a portion of the solid or semi-solid support for the variable monomer of the ligand. Split into;   d) In each part separately, within the coding sequence, further in the ligand Modification, or other possible variable models at this site of its ligand. Attaching a unit of a nomer and a tag encoding this step;   e) mixing these parts;   f) Step b until the variable part of the ligand is completely synthesized ) -E) are repeated; and   g) optionally one or more further constant monomers to the ligand, Or a further non-coding unit is attached to the tag (reactive ski (See pages 1 and 2); A tag or ligand containing a protecting group that can be removed under mildly acidic conditions. It is used for the synthesis of the strand.   The first unit of the tag and the first monomer of the ligand and the first monomer The type of conjugation with the linker depends on the type of reactive group chosen, and Then by standard methods for such groups.   Further syntheses are standard known methods for solid phase synthesis (Fields et al., Int. ern. J. Pept. Prot. Res. (1990), 35, 161-214). Ligand and And transient protecting groups used for tag synthesis (removed before each synthetic step) The protective end groups which can be) are orthogonal protecting groups. That is, the ligand and the tag The syntheses can be performed independently of each other. For example, two groups of protecting groups Can be used. The first group is removed under mildly acidic conditions, and The other group is removed by the action of light under basic conditions under oxidizing or reducing conditions. You. Protective groups that can be removed under mildly acidic conditions and removed under basic conditions. It is particularly preferred to use a combination with protecting groups which can be   Ensures compatibility with a large number of protecting groups that will remain intact during ligand synthesis In order to do so, the acid-removing transient protecting groups are those with particularly pronounced acid lability. Suitable acid labile groups are dimethyldimethoxybenzyloxycarbonyl protecting groups (D It is at least 10 times more unstable than dz).   In order to achieve sufficient orthogonality, base-removability Fluorenylmethoxycarbonyl (Fmoc) is preferably a compound of the structure of Acid-scavenging trityl used for the synthesis of other structures and for the synthesis of other structures. (Trt) or a substituted trityl protecting group such as alkoxy-substituted trityl is used. You.   Illustrative examples of protecting group type combinations are:   The β-elimination type group is typically a fluorenylmethyl type protecting group. Yes; the above NPS represents a nitrophenylsulfenyl type group. Eligible group For a general description of Fields et al. (Intern. J. Pept. Prot. Res. (1 990), 35, 161-214).   Also, protecting groups that can be removed by photolysis, such as nitrobenzyl. Or a protecting group for a ligand or tag with a nitroveratryloxycarbonyl group A combination of one of the above groups of is also suitable.   For example, the reaction steps required for the synthesis of amides are widely used in the art. Types of activation of carboxylic acid groups that are known and usually participate in the reaction Depends on. This reaction is usually carried out in the presence of a condensing agent or in the form of an anhydride. When activating carboxylic acid And in the presence of an agent that binds to the carboxylic acid formed. In some cases, A chaotropic reagent such as LiF in NB-methylpyrrolidone It can also be added. These reactions are carried out at -30 ° C to + 150 ° C, preferably + 10 ° C. Within a temperature range of ℃ to +70 ℃, and most preferably +20 ℃ to +50 ℃, and It is also carried out in an inert gas atmosphere as appropriate.   Illustrative examples of useful condensing agents are carbodiimides such as N, N'-diethyl. -, N, N'-diisopropyl-, N, N'-dicyclohexyl- or N-ethyl Ru-N '-(3-dimethylaminopropyl) -carbodiimide; carbonyl compound Such as carbonyl diimidazole; 1,2-oxazolium compounds, eg For example, 2-ethyl-5-phenyl-1,2-oxazolium-3'-sulfone And 2-tert-butyl-5-methylisoxazolium perchlorate; reed Ruamino compounds, such as 2-ethoxy-1-ethoxycarbonyl-1,2-di -Hydroquinoline; and uronium compounds, such as 2- (1H-benzotria Zol-1-yl) -1,1,3,3-tetra-methyluronium tetraflu Oroborate (TBTU); or phosphonium compounds, eg benzotriazole -1-yloxy-tris (dimethylamino) phosphonium hexafluorophoh Sulfate (BOP) or benzotriazol-1-yl-oxy-pyrrolidinophos Fonium hexafluorophosphate (PyBOP).   Useful acid acceptors are typically alkali metals, carbonates or bicarbs. Carbonates, such as sodium or potassium carbonate or bicarbone (Usually with sulphate) or organic bases, eg sterically hindered Tri-lower alkylamines such as N-diisopropyl-N-ethylamine It is.   The reactive side chains of the ligand and tag monomers that do not participate in the reaction are It can be protected by a protecting group. Helpful protection for their introduction and removal The groups and processes are, among others, “Protective Groups in Organic Chemistry”, Pl enum Press, London, New York 1973; “Methoden der organischen Chemie”, H ouben-Weyl, 4th edition, Vol.15 / 1, Georg-Thieme-Verlag, Stuttgart 1974;  Th. W. Greene, “Protective Groups in Organic Synthesis”, JohnWiley & Sons, New York 1981; Atherton et al., “Solid phase peptide synthesis-A practical approach ”IRL Press Oxford University, 1984; Jones,“ The chem ical synthesis of peptides ”, Oxford Science Publications, Clavendon Pre ss Oxford, 1991; and Bodanszky, "Peptide Chemistry", Springer Verlag B. erlin, 1988.   Typical examples of hydroxy protecting groups are acyl groups such as tert-butoxycarbo. Nyl groups, etherified groups such as tert-butyl groups, and silyl or tin groups, eg For example, it is a tri-n-butyltin group or tert-butyldimethylsilyl.   Carboxyl groups are the above-mentioned tert-butyl type, benzyl, trimethylsilyl Can be protected by ester formation with ethyl or 2-triphenylsilyl groups it can.   The amino group is conveniently a removable acylamino, arylmethylamino, ester Mercaptoamino, 2-acylalk-1-enylamino, silylamino, Tin-amino or azido group, tert-butoxycarbonyl, allyloxy Carbonyl, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl , Diphenylmethoxycarbonyl, nitrophenylsulfenyl, 2,2,2- Trichloroethoxycarbonyl, pentamethylchromanosulfonyl (PMC) or those containing a methoxytrimethylbenzylsulfonyl (Mtr) protecting group Can be protected.   The thiol can be protected by the acetamidomethyl group.   These protecting groups are usually conveniently found in 95% triflour by conventional methods of peptide chemistry. Oloacetic acid removes the ligand and tag together after complete synthesis. If there is In addition, strong nucleophilic groups, such as 1,2-ethanedithiol, are also generated Can be added to capture the protecting groups.   Further methods of removal of these protecting groups are known and, inter alia, β- Desorption, solvolysis, hydrolysis, alcoholysis, acidolysis, treatment with base Or comprising a reduction.   The number of moieties for the reaction described in d) is preferably in each case a It corresponds to the number of possible attachments of the next monomer in Gandh. As mentioned above, tags One unit of can consist of one or more amino acids. 1 unit If they consist of several amino acids, each of these is, in turn, attached to its growth tag, Or synthesized separately as a unit and then attached to the tag in one reaction Can be done. Tag sequencing carries that information at its start and end One or more amino acids that indicate the start or end (start sequence / termination sequence) of the It can be more easily carried out by attaching it to.   Prepared as described above to identify ligands that bind to the desired acceptor Processed library with an acceptor to be investigated, and acceptor The carrier with the adherents is washed and isolated.   Possible acceptors within the scope of the present invention are one or more It is a macromolecular unit that has the affinity to bind to a ligand. Illustrative example Is a serotonin receptor, GABA receptor and benzodiazepine receptor -Containing receptors, transport proteins such as Na and K channels, antibodies and Enzymes such as protease, thrombin, renin, ACE, aromatase And reverse transcriptase; or fragments thereof having identical binding properties for the ligand. You. Ligands that bind to these acceptors are antagonists, inhibitors -, As an agonist or as a marker for an acceptor.   When not already present in nature, these acceptors are identifiable groups, such as For example, fluorescent, chemiluminescent or radioactive groups, avidin, biotin, reporter enzymes, Immunologically detectable groups (ELISA) etc. are provided. Mark with the above marker In general, the method of baking and the method of affinity chromatography are Known in the art.   Isolation of the carrier to which the acceptor binds can be accomplished by, for example, UV or fluorescent acceptor Automatic, typically used under blue light or for sorting cells This is done manually by sorting the carrier with the help of a solubilizer.   The tag of each carrier isolated in this manner can be labeled using conventional peptide analysis methods such as Edma. A ligand that can be sequenced by n-degradation and is attached to its carrier Is clearly identified. Sequencing is performed on the carrier before or after separation of the ligand. On the attached tag, it can be done, for example, by an automated sequencing device. Wear. Sequencing of the ligands after separation is preferred. In this case, open The ligand monomer does not interfere with its sequencing. The other possibility is that they A good practice so that it is not broken down by tag analysis (Eg, acetylation) to derivatize the ligand at the end.   A further possibility is to separate the tag from the carrier and then sequence the tag. It is to be.   To rule out false-positive results from the above binding tests, these ligands were It can be wholly or partly separated from the carrier, and in a homogeneous liquid solution, To assay, for example, a second loudness assay for their attachment to the acceptor. Can be served. Separation of ligands can be achieved, for example, by Optionally by cleavage at the linker between the carrier and the ligand. Re If the linker is p-hydroxybenzoic acid, this cleavage will result in gaseous ammonia, Ammonia / THF saturated solution or liquid ammonia (eg microtitre Can be performed by treating the individual carriers (in the solution plate) . The separated ligand is then rinsed from the carrier and once again its acceptor. Can be tested for its binding 1 to That cannot be done by the ligand bound to the carrier, typically Are subjected to binding tests, tests in which light absorption is measured, or MS, IR or NMR studies. You.   A suitable choice of linker is for the first assay by treatment with gaseous ammonia. First part of the ligand from the isolated carrier, and ammonia / THF or liquid ammonia To separate the second portion of the ligand for further assay by treatment with To be able to. Suitable linkers in this regard are typically 4-hydroxymeth Chill benzoic acid.   A typical example is a potential thrombi containing unnatural amino acids and other building blocks. Synthesis of a library of protein inhibitors Therefore, it is more protease-resistant.   The library has, for example, 2 constant and 3 variable sites and has the following set: Success:   X-Y-Z-Pro-GABA X = 2-cyanobenzenesulfonyl, D-Phe, N-benzylglycine, β-Ala or Is acetyl Y = L-Pro, D-Pro, β-Ala or L-Asp Z = L-Arg, D-Arg, β-Ala or Sarcosyl It consists of a pentamer with.   This composition gives a possibility of 5 × 4 × 5 = 100 for the composition of the ligand.   Each individual monomer is encoded by a dipeptide in the identified structure.   The individual monomers are encoded by dipeptides within the above identified structure.   After screening this library, the pentamer D-Phe-D-Pro-Arg-Pro- GABA and D-Phe-Pro-D-Arg-Pro-GABA are all effective thrombin inhibitors Is invented.   Therefore, a further object of the present invention is to inhibit the thrombin of the formula D-Phe-D- Pro-Arg-Pro-GABA, D-Phe-Pro-D-Arg-Pro-GABA compound, or pharmaceutically acceptable And their salts; alone or in combination with further optional excipients A pharmaceutical composition comprising a thrombin inhibitor.   The present invention further provides a novel method for the therapeutic and prophylactic treatment of the human or animal body. It relates to each compound detected by the method of use. The preferred field of use is thrombin Is in the field of therapeutic and prophylactic treatment of, for example, embolism and thrombosis.   The pharmaceutical composition of the present invention may be applied to warm-blooded animals enterally, such as orally, and also rectally. And parenteral administration, such as subcutaneous, intravenous, or intraperitoneal administration, and They are pharmacologically active compounds alone or with pharmaceutically acceptable carriers. Including together. The daily dose will depend on the age and individual condition of the patient and the mode of administration. Will depend.   The new pharmaceutical composition comprises about 10-80%, preferably about 20-60%, of the active compound. Pharmaceutical compositions for enteral or parenteral administration are typically in unit dosage form, eg For example, in sugars, tablets, capsules or suppositories, and also in ampoules. These dosage forms are in a manner known per se, typically by conventional mixing, granulation. It can be prepared by the method of derivatization, confectioning, dissolution or freeze-drying. Wear.   Pharmaceutical compositions for oral administration are preferred. Suitable carriers are especially bulking agents, for example , Sugar, conveniently lactose, saccharose, mannitol or sorbitol, Lulose preparation and / or calcium phosphate, typically tricalcium Phosphates or calcium hydrogen phosphate, and also binders, such as den Pun paste conveniently for maize, corn, rice or potato starch , Gelatin, tragacanth, methyl cellulose and / or polyvinyl pyrrolide And / or, if desired, a disintegrant, such as the starches mentioned above, Also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, algin An acid or salt thereof, for example sodium alginate. Excipients are especially lubricants , Flow regulators and lubricants, conveniently silica, talc, stearic acid or their A salt of, typically magnesium stearate or calcium stearate, and And / or polyethylene glycol. Sugar coating Is typically arabic with a suitable parenteral or enteric coating. Rum, talc, polyvinylpyrrolidone, polyethylene glycol and / or diacid A concentrated sugar solution, which may include titanium oxide, in a suitable organic solvent or solvent mixture. Suitable cellulose preparations for the preparation of shellac solutions or enteral coatings Products such as acetyl cellulose phthalate or hydroxypropylmethyl -Can be provided using a solution of cellulose phthalate. Dye Is a pigment or a tablet to conveniently identify or indicate different doses of the active compound. It can be added to dragee coatings.   Additional pharmaceutical compositions for oral administration include dry-filled caps made from gelatin. Cells and also gelatine and plasticizers such as glycerol or sorbitol Is a soft-seal capsule. This dry-filled capsule is In the form, conveniently a bulking agent, eg lactose, a binder, eg Starch, and / or glidants such as talc or stearic acid The active ingredient is admixed with gnesium and with or without stabilizers. Can be included. In soft capsules, the active ingredients preferably are In liquids, typically fatty oils, paraffin oils or liquid polyethylene glycols Can also be dissolved or suspended in and the stabilizer added.   Pharmaceutical compositions suitable for rectal administration typically include suppository bases and the above-described activation compounds. It is a suppository consisting of a combination of products. Examples of suitable suppository bases are natural or synthetic triglycerides. Cerides, paraffin hydrocarbons, polyethylene glycols and higher alkanols It is. Gelatin caps for rectal administration containing a combination of base material and active compound Cells can also be used. Suitable substrate materials are typically liquid triglycerides. Lido, polyethylene glycol or paraffin hydrocarbon.   Pharmaceutical compositions for parenteral administration include aqueous solutions or suspensions of the active compounds, conveniently , A suitable lipophilic solvent or medium, such as fatty oils, typically sesame oil, or synthetic fats Oil injection using acid ester, eg ethyl oleate or triglyceride Spray suspension or viscosity-increasing substance, conveniently sodium carboxymethyl cellulose Aqueous injection suspension which may contain glucose, sorbitol and / or dextran And also with or without stabilizers.   The present invention provides a thrombin inhibitor as described above, preferably in the form of a pharmaceutical composition. Also related to use. The dose of the active compound depends on the species of warm-blooded animal, the age of the patient and the individual. It will depend on the particular symptoms and also on the mode of administration. For patients weighing approximately 75 kg A contemplated daily dose for oral administration is about 0.1 mg to 500 mg, preferably about 1 mg. Will be ~ 50 mg.   The invention is explained in more detail by the examples below.   The following abbreviations are used.   DCCI = dicyclohexylcarbodiimide   DCE = dichloroethane   DICD = diisopropylcarbodiimide   DIPEA = diisopropylethylamine   DMA = dimethylacetamide   DMF = dimethylformamide   DMAP = dimethylaminopyridine   DMSO = dimethylsulfoquinimide   Fmoc = fluorenylmethyloxycarbonyl   GABA = γ-aminobutyric acid   HOBT = hydroxybenzotriazole   NEt_Three  = Triethylamine   OSu = O-succinimide   Pip = piperidine   PMC = pentamethylchromansulfonyl   Trt = Trityl   Iterative processing is performed according to the common-phase technology procedures commonly used in peptide chemistry. J. Process (Fields et al., Intern. J. Pept. Prot Res. (1990), 35, 161. -214), for example, adding the reagent solution to the resin and subsequent filtration (weak Isolation of the filtrate under vacuum).   Is it a pentamer with two constants and three variable positions and with the following composition: The preparation of a library consisting of (see Reaction Scheme 1) is described:   X-Y-Z-Pro-GABA X = 2-cyanobenzenesulfonyl, D-Phe, N-benzylglycine, β-Ala, Acetyl Y = L-Pro, D-Pro, β-Ala, L-Asp Z = L-Arg, D-Arg, β-Ala, L-Asp, sarcosyl.   The individual monomers listed above are encoded by the following dipeptides within the identified structure :       Building block dipeptide       2-Dianobenzenesulfonyl chloride DG       D-phe GD       N-benzylglycine GF       β-Ala AA       Acetyl AG       L-Pro DA       D-Pro GA       L- Arg GG       D-Arg NG       L-Asp AN       Sarkosyl NAExample 1: Preparation of trityl protected amino acids and dipeptides   10 mmol of each dipeptide or amino acid was added to 36 ml of chloroform / acetonitrile. Suspend in (4: 1). Next, slowly add 10 mmol of trimethylsilyl chloride dropwise. And the reaction mixture is refluxed at about 65 ° C. for 2 hours. Then 20 mmol Liethylamine was added dropwise, and finally 10 mmol of chloride chloride in 10 ml of chloroform. Add a solution of lysyl. After 60 minutes, excess methanol was added and an additional 5 After minutes, the reaction mixture is concentrated by evaporation and the residue is dried. this The crude material was dissolved in a 2M solution of ice cold potassium hydrogen sulfate (or citric acid) and then The solution is extracted with ethyl acetate. With aqueous alkali (at about 5 ° C) After reverse extraction, the aqueous phase is adjusted to pH 6-7 with potassium hydrogen sulfate solution.  Precipitate Trt-dipeptide or Trt-amino acid and extract with ethyl acetate I do. 10 mmol triethylamine are added to the organic phase and the batch is steamed. Concentrates by release (Trt-dipeptide or Trt-amino Acid triethylammonium).    Trt-dipeptides DG, GD, GF, AA, AG, DA, GA, GG, NG, AN and NA and A, D, F, G and N Trt-amino acid triethylammonium salts in this manner Prepare.Example 2: Partial derivatization of a carrier resin with less than an equivalent amount of constant units of the tag. (Novabiochem) 180μmol of reactive alkylamino group, 20% Pip / D Wash 5 times for 40 seconds with MA, then 10 times 60 seconds with DMA at room temperature .   Loading: about 480 μmol / g is equivalent to about 2000 pmol / resin particles (beads) It is.   For pre-activation, 60 μmol of Trt-Gly-OH NEt of Example 1_Three66 μmol (HO (Based on BT) dissolved in a 0.5 M solution of HOBT / DMA, and 66 μmol in this solution Add a 2M solution of DICD / DMA (based on DICD). After 40 minutes at room temperature, The mixture was diluted with 2 ml DMA and the solution pipetted onto the resin. Add Immediately afterwards, add 66 μmol DIPEA. 60 minutes for this coupling reaction to continue. The resin is rinsed with DMA for 10 x 45 seconds.Example 2.1: Measurement of tag loading by measurement of free amino groups   The reporter group Fmoc is bound to the resin, washed off with excess and then cleaved again. Let me measure. procedure;    275 μmol Fmoc-OSu is dissolved in 2.1 ml DMA and added to the resin. Immediately afterwards, 413 μmol (based on DIPEA) of a 1.5 M solution of DIPEA / DMA was pipetted onto the resin. Add by pet. After 80 minutes at room temperature, wash the resin with DMA for 10 x 18 seconds I do. The resin was treated with 20% Pip / DMA at room temperature to remove the Fmoc group. 15 × 40 seconds To process. The collected filtrates are diluted in a defined manner and their absorbance (extinc measurement) at 299.8 nm. The total number of free amino groups is about 154 μmol, That is 85% of the total loading of the technique (percentage of ligand). Therefore, the tag loading of the resin is about 15%.Example 3: Coupling of a linker for a ligand to a resin   For pre-activation, 769 μmol 4-hydroxymethylbenzoic acid 845 μmol Dissolved in a 0.5M solution of HOBT / DMA (based on HOBT) and added to this solution 84 Add 5 μmol (based on DICD) of a 2M solution of DICD / DMA. 40 at room temperature After minutes, the reaction mixture was diluted with 0.3 ml DMA and pipetted onto the resin. Added. Immediately after, 845 μmol DIPEA was added and the coupling reaction For 60 minutes at room temperature. Then rinsing is performed as follows:    10 × 45 seconds by DMA   5 x 60 seconds with isopropanol    6 × 45 seconds by DMA.Example 4: Linkage of the first constant C-end component of the ligand by a linker Batch: 154 μmol   The modification technique of Example 3 was as follows:    615 μmol Fmoc-GABA-OH    600 μl DMA and   3 ml of cycloloethane Pipette to the solution of.   In this suspension,    646 μmol DCCI and    307 μl of dichloroethane Is added in 2 portions at room temperature over 5 minutes, and Finally the following:   30.7 μmol DMAP and   65 μl of dichloroethane Add the solution. After a total of 20 minutes, add 154 μmol N-methylmorphine I do. The reaction is continued for 4 hours and then rinsed as follows:    4 × 45 seconds by DMA   3 × 45 seconds with dichloroethane    4 × 45 seconds by DMA.Example 5: Chain extension of the constant part of the ligand   The Fmoc group is removed as described in Example 2. 122 μmol by measuring absorbance Was obtained. For pre-activation, 367 μmol Fmoc-Pro-OH (based on HOBT Dissolve in 404 μmol HOBT / DMA 0.5M solution, and add to this solution (DICD (Based on) 404 μmol of a 2M solution of DICD / DMA is added. After 40 minutes at room temperature, Was diluted with 1.3 ml of DMA and 122 μmol of the resin of Example 4 was added. To be added. Immediately after, 404 μmol DIPEA was added and the coupling reaction was The reaction is continued for 45 minutes at room temperature. Rear-cup with the same amount of Fmoc-Pro (as above) Post-coupling to increase its yield (additional 35 minutes). This Of the resin and rinsed with acetic anhydride / pyridine / DMA 1: 1: 8 (v: v: v). Treatment for 1 x 4 minutes to block unreacted amino groups, and then: rinse:    5 × 45 seconds by DMA   5 x 45 seconds with isopropanol.   The resin is dried and divided into pools suitable for variable position introduction. The introduction of the variable Z in this example is based on five different constructions. Separate reactions by building blocks (“split synthe sis) ”) requires 5 parts.Example 6: Synthesis of variable part of ligand and its corresponding tag   5 pools are divided into 19 μmol each (1 pool for each is about this It can be changed in the position. ).Example 6.1: Synthesis of the first variable position of the ligand   First, the Fmoc group is removed in the ligand as described in Example 2. next , Perform individual coupling reactions for the first variable position (Z position):   Each of the Fmoc building blocks listed above was treated with 84 μmol HOBT for 40 minutes at room temperature. Pre-activated with solution (0.5M in DMA) and 84 μmol DICD solution (2M in DMA), Then, after diluting with 230 μl of DMA, add to 1 resin part. Immediately after, 84 μmol  Add DIPEA. This coupling is carried out for 45 minutes at room temperature. Rear-Cupuri For another 35 minutes with the same mixture. Acetylation and rinsing Example 5 Do as described in.Example 6.2: Tag chain extension via dipeptides   According to the code table for each pool of Example 6.1, according to the attached monomers , The tag portion having the Trt peptide of Example 1 encoding the monomer, Further synthesis (see reaction scheme 2).   First, the trityl group of the tag irreversibly linked to the resin was added to 5% of dichloroethane. Cleavage with formic acid (twice for 1 minute) and rinse:    5 × 45 seconds by DMA    3% for 45 seconds with 3% triethylamine in DMA    3 x 45 seconds by DMA.   The dipeptide is then linked to the corresponding tag in each reaction:   1 Trt-GG 129 μmol   2 Trt-NG 129 μmol   3 Trt-AA 129 μmol   4 Trt-AN 129 μmol   5 Trt-NA 129μmol   The above-mentioned Trt-dipeptide was added to 142 μmol HOBT and 142 μmol DICD in DMA. Preactivate in solution for about 40 minutes. Then add this solution to its corresponding resin part , And immediately treat with 142 μmol DIPEA. This coupling reaction Continue for 60 minutes at room temperature and then rinse as follows:    2 × 45 seconds by DMA   Acetic anhydride: pyridine: DMA = 1: 1: 8 (v: v: v)                                                 1 x 5 minutes    5 × 45 seconds by DMA.Example 6.3: Chain extension of the tag via individual amino acids (denaturation of Example 6.2)   First, the trityl group of the tag that is irreversibly linked to the resin is described in Example 6.2. Remove as by cleavage with 5% formic acid in dichloromethane.    The five parts of each tag component of 4.3 μmol are as follows:   1: Trt-G (I) 129 μmol          Trt-G (II) 129 μmol   2: Trt-G (I) 129 μmol          Trt-N (II) 129 μmol   3: Trt-A (I) 129 μmol          Trt-A (II) 129 μmol   4: Trt-N (I) 129 μmol          Trt-A (II) 129 μmol   5: Trt-A (I) 129 μmol          Trt-N (II) 129 μmol   The Trt-amino acids listed in (I) were added to 142 μmol of HOBT and 142 in DMA. Preactivate for about 40 minutes in μmol DCID solution. Next, add this solution to the corresponding resin part. Add in minutes and immediately treat with 142 μmol DIPEA. This coupling The reaction is continued for 60 minutes at room temperature and then rinsed as follows:    2 × 45 seconds by DMA   Acetic anhydride: pyridine: DMA = 1: 1: 8 (v: v: v)                                                 1 x 5 minutes    5 × 45 seconds by DMA.   Thereafter, the Trt group was removed as indicated above and listed under (II).  Each of the Trt amino acids was coupled as described above.Example 6.4: Synthesis of the second variable position of the ligand   Position Y is synthesized generally according to the introduction of Z (for different coding). (See reaction scheme 2). This was done for all samples of Example 6.2 or 6.3. By mixing and dividing them into 4 parts of equal size (4Y parts) To do.   In these four individual parts, possible monomers at this position Each of these was attached as described in Example 6.1:   As described in Example 6.2 or 6.3, the tag unit belonging to this monomer Then attach the tray.Example 6.5: Synthesis of the third variable position of the ligand   Position X is generally according to the introduction of Y or Z (for different coding) Synthesize (see Reaction Scheme 2). This was mixed with all the samples of Example 6.4. And then divided into 5 parts (5X parts) of equal size.   Possible monomers at this position in these five individual moieties Of each was attached as described in Example 6.1:   Coupling with 2-cyanobenzenesulfonyl chloride at room temperature And through its reaction with a solution of its sulfonyl chloride in pyridine / DMA; The coupling of acetic anhydride is carried out as described in Example 5.   A unit of tags belonging to this monomer, as described in Example 6.2 or 6.3. Are then attached.Example 7: Deprotection of ligand   A suitable number of resin particles (provided that The number of components) was subjected to deprotection reactions commonly used in peptide chemistry to obtain Fmoc And the tert-butyl protecting group is removed. To remove the Fmoc protecting group, Example 6 . Resin 5 was again treated repeatedly with 20% Pip / DMA (see Example 2), And then 40 minutes at room temperature to remove the t-butyl and Pmc protecting groups. , 95% trifluoroacetic acid (5% water + 2% ethanedithiol). Tree If the linkage of the ligand to the lipid is intact, all the functional groups in the ligand and in the tag are resolved. Let go. Then a rinse is performed as follows: 4x45 seconds by DCE 4 x 45 seconds with isopropanol Isopropanol: H₂O = 1: 1 (v: v) for 4 x 45 seconds H₂5 x 45 seconds depending on O.Example 8: Marking of thrombin with fluorescein   Isothiocyanate was added to a solution of human thrombin (100 ml borate buffer). -0.53 mg in pH 8) (10 μl of a 2.9 mg solution in 290 μmol DMSO). this The reaction is continued for 60 minutes at room temperature and the reaction mixture is subjected to gel chromatography. Ruffy Column (Sephadex   Dilute 20 μl of fluorescein-thrombin fraction (approximately 3% fraction volume) to 500 μl. And measured photometrically at 495 nm. This is about 2 μm Gives the concentration of rescein. Therefore, the fluorescein / thrombin ratio is about 1: 1. You.Example 9: Identification of thrombin binding ligand in heterogeneous phase   Approximately 500 beads of the resin of Example 7 were treated with fluorescein of Example 8 for 10 minutes at room temperature. Incubate in a 2.2 μm solution of in-labeled thrombin. below:   68 mg imidazole   876 mg NaCl   147 mg CaCl₂× 2H₂O   3.35g PEG (3350)   H up to 100 ml₂O After rinsing with the solution for 3 x 1 min, the beads were exposed to longwave light (~ 470 (blue in nm). Sort the particles, which are obviously fluorescent And   5 × 3 minutes with formamide, and   H₂10 × by O Rinse and then dry.Example 10: Assay for thrombin inhibition of individual bead ligands in the wave phase   The beads sorted in Example 9 were treated with saturated anion in THF for 24 hours at room temperature. Keep in monia solution. Then fill them Numbered microtitration plates using size 0.65 μm) It was divided into individual cavities. Tetrahydrofuran from these ligands Second numbered microtitration play with a water / water mixture Can be rinsed in place, where they can be directly used in the thrombin inhibition assay. Certify. 2.3 × 10 per cavity^-2 NIH unit of thrombin and 9.3 μg Chromogenic substrate S-2302 (D-Pro-Phe-Arg-p-nitroanilide 2HCl; ex Chromogenix ) Is added in a final volume of 150 μl. About 75 minutes Color development over a period of time at the wavelength of 405 nm with the help of the Multiwell-Platereader And the corresponding absorbance value is plotted as a function of time. Different inhibition A cross-comparison of the drug diagram gradients shows that the ligands with inhibitor activity are the same. Serve as a basis for setting.   The composition of the two thus identified thrombin inhibitors is identified from the tags. This in an automated sequencer (ABI477 available from Applied Biosystems) Belongs to each ligand separated by solid-phase protein sequencing (Edman degradation) Screen each of the bead tags, and the following:   D-Phe-D-Pro-Arg-Pro-GABA   as well as   D-Phe-Pro-D-Arg-Pro-GABA (Molecular weight in each case (MALDI-MS M + H⁺) Is 600.7. ) As above This is done by identifying the composition of the two ligands from the code table. Reaction scheme 1: Reaction scheme 2: The unit encoding the first variable monomer into the ligand and into the tag. Introduction of Introducing a second variable monomer and a second unit encoding it: Introducing a third variable monomer, and a third unit encoding it:

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI G01N 33/566 A61K 37/64 AED // C07K 105: 00 (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, MW, SD, SZ), AM, AU, BB, BG, BR, BY, CA, CN, CZ, EE, FI, GE, HU, JP, KG , KP, KR, KZ, LK, LR, LT, LV, MD, MG, MN, NO, NZ, PL, RO, RU, SI, SK, TJ, TT, UA, US, UZ VN (72) inventor Matthew, Ian, Timothy, Wiria-time UK, West Sussex Senior Rueichi 12 4 Ie, Horsham, c Inburu Hurst Road 7

Claims (1)

[Claims]   1. Group or semi-group carriers (beads), synthetic oligomers (ligands) and their Each from the identified structure (tag) by which the Gand's monomer can be identified. A library comprising a plurality of different units, each of which is:   a) each carrier unit carries only one type of ligand,   b) the ligand and tag are attached to the same carrier at different positions,   c) The ligand is irrespective of the tag and does not change its information content, Can be separated from the carrier,   d) the tag is a polypeptide that can be sequenced, and   e) removed under mild acidic conditions to synthesize the above tag or ligand Such a library using protecting groups that are capable.   2. A protecting group that can be removed under mildly acidic conditions is dimethyldimethoxide. At least 10 times more labile than the cibenzyloxycarbonyl protecting group, The library according to claim 1.   3. Protecting groups that can be removed under mildly acidic conditions are trityl type The library according to claim 1, which is a group of   4. Known and / or automated in the amount they are obtained on the carrier unit Monomers that cannot be clearly identified by the procedure are labeled with their ligands. The library of claim 1, which comprises:   5. The ligand according to claim 1, wherein the ligand is attached to the carrier through a cleavable linker. Library.   6. The ligand should be cleaved under basic, acidic, photolytic, oxidative or reducing conditions. The library according to claim 1, wherein the library is attached to a carrier through a linker that can be attached. .   7. The ligand is carried through a linker that can be cleaved under basic conditions. The library according to claim 1, which is attached to the body.   8. The ligand can be removed from the carrier in several parts. The library according to Item 1.   9. The linker is p-hydroxymethylbenzoic acid, 4-hydroxymethylphene. Nylacetic acid, benzhydrylamino, allyl, hydroxycrotonylaminomethyl , 3-nitro-4-hydroxymethylbenzoic acid, p-nitrobenzhydrylami 4- [4,4'-bis (methylsulfinyl) -2-oxybenzhydryl 6. Amino] butyric acid and a disulfide linker selected from the group consisting of: The listed library.   Ten. The lysine according to claim 5, wherein the linker is 4-hydroxymethylbenzoic acid. Ibrary.   11. The each monomer of the ligand is encoded by one or more amino acids. The library according to 1.   12. The library of claim 1, wherein the tag is provided with a constant starting sequence.   13. The library of claim 1, wherein the tag is provided with a constant termination sequence.   14. The library of claim 1, which provides a constant starting sequence for the ligand.   15． The library according to claim 1, wherein the group or semi-group carrier is a polymer resin. Lee.   16. The polymeric resin of claim 1, wherein the polymeric resin comprises free amino groups. Library.   17． The live according to claim 1, wherein the ratio of ligand to tag on the carrier is greater than 1. Rally.   18． The lyase of claim 1, wherein the ratio of ligand to tag on the carrier is 2-100. Braly.   19. Live according to claim 1, wherein the ratio of ligand to tag on the carrier is 4-10. Rally.   20. Ligand by orthogonal and alternating synthesis The method for producing a library according to claim 1, which comprises synthesizing a tag and a tag.   twenty one. The following stages:   a) the first unit of the tag and the first monomer of the ligand or its building block or Attaches a linker for each to the group or semi-group carrier;   b) optionally further invariant monomers to the ligand or further non- Attaching a variable coding unit to the tag;   c) The above-mentioned group or semi-group carrier is attached to the portion of the ligand for the variable monomer. Split;   d) In each part separately, within the coding sequence, the Modification or other variable monomer possible at this site of its ligand, And attaching a unit of tag encoding this step;   e) mixing the above parts;   f) repeating steps b) to e) until the variable part of the ligand is completely synthesized And;   g) optionally one or more additional non-variable monomers to the ligand Adds additional non-coding units to the above tag Attach, A protecting group that comprises a tag or a protecting group that can be removed under mildly acidic conditions. A method of making a library according to claim 20, as used for the synthesis of a ligand. .   twenty two. The orthogonal protecting group is used for the synthesis of a ligand and a tag. Recipe.   23.2 protecting groups were used for the synthesis of ligands and tags, the first group being mild Can be removed under various acidic conditions, and other groups can The function of, or can be removed under oxidizing or reducing conditions. The manufacturing method described in 20.   24.2 transient protecting groups were used for the synthesis of the ligand and tag, and the first group was , Under mild acidic conditions and other groups may be removed under basic conditions. The production method according to claim 20, which is capable.   twenty five. 22. The method according to claim 21, wherein one of the protecting groups belongs to the trityl type (Trt). Manufacturing method.   26. Fluorenylmethoxy-carbonyl type (Fmoc) and trityl -The process according to claim 21, wherein a protecting group of type (Trt) is used.   27. 21. The method according to claim 20, wherein the synthesis of the ligand is performed using an Fmoc protecting group.   28. The method according to claim 20, wherein the tag is synthesized using a trityl protecting group.   29. A third reactive side chain of the ligand or tag monomer that should not participate in the reaction 21. The method according to claim 20, wherein the group is protected by a protecting group.   30. Reactive side chains of the ligand or monomer of the tag that should not participate in the reaction are N-methylmethyl chromansulfonyl (PMC), Mtr or tert-butyl type protecting groups The method according to claim 20, which is more protected. .   31. Processing the library of claim 1 with an acceptor to be investigated And isolating the components of the library that bind to its acceptor. A structure detection method consisting of:   32. Acceptor is a receptor, transport protein, antibody, enzyme or fragment thereof 32. The method of claim 31, wherein   33. 32. The method of claim 31, wherein the acceptor carries an identifiable group.   34. Identifiable groups are fluorescent, chemiluminescent or radioactive groups, avidin, biotin 34. The method according to claim 33, which is a protein, a reporter enzyme or an immunologically detectable group. .   35. 34. The method of claim 33, wherein the identifiable group is a fluorescent group.   36. The library according to claim 1 comprises an acceptor carrying a fluorescent group. The components of the library that are mixed, washed, and exhibit fluorescence are isolated. The method according to paragraph 31.   37. The ligand is wholly or partially separated from the isolated carrier. The method according to paragraph 31.   38. Further assays are performed with the ligand bound to a group or semi-group carrier. 38. The method of claim 37, which cannot be performed.   39. 38. The method of claim 37, wherein the further assay is a binding test, NMR or MS. .   40. The ligand was readily isolated by treatment with volatile or gaseous agents. 38. The method of claim 37, which is separated from the carrier.   41. Ligand is separated from the isolated carrier by treatment with acid or alkali 38. The method of claim 37, wherein the method is performed.   42. Is the ligand a carrier isolated by treatment with alkali? 38. The method of claim 37, which is separated from   43. Ligand is dissolved in gaseous ammonia, liquid ammonia or ammonia / THF 38. The method of claim 37, wherein the treatment with the liquid separates from the isolated carrier.   44. The first part of the ligand is treated with gaseous ammonia and the second part Minutes were isolated by treatment with ammonia / THF solution or liquid ammonia. 38. The method of claim 37, wherein the method is separated from the carrier.   45. The formula D-Phe-D-Pro-Arg-Pro-GABA or D-Phe-Pr for limiting thrombin a compound represented by o-D-Arg-Pro-GABA, or a pharmaceutically acceptable compound thereof salt.   46. Represented by the formula D-Phe-D-Pro-Arg-Pro-GABA or D-Phe-Pro-D-Arg-Pro-GABA Or a pharmaceutically acceptable salt thereof, and further optional excipients A pharmaceutical composition comprising an agent.