JP2000516202A - Serine protease inhibitor - Google Patents

Abstract

  (57) [Summary] Methods for making bifunctional serine proteases and boron-containing peptides are provided. Serine protease inhibitors include a catalytic site-directing moiety that binds to and inhibits the active site of serine protease and an external position-related moiety linked by a connector moiety. The catalytic site directing moiety and the exosite-related moiety can bind simultaneously to a serine protease molecule.

Description

DETAILED DESCRIPTION OF THE INVENTION                        Serine protease inhibitor   The present invention relates to serine protease inhibitors and substrates, The invention relates to methods and materials for the synthesis of compounds and for the synthesis of boron-containing compounds.   Modulation or inhibition of serine protease inhibitors may be particularly effective in preventing thrombosis Useful.   Serine protease family enzymes have three Asp-His-Ser residues in the active site of the enzyme. It cleaves peptide chemical bonds by a mechanism involving the ream. Serine Prote ASE inhibitors use functional groups that interact with the triplicate, thereby enzymatic substrate Designed to block activity. Selective for one target protease It is desirable to make inhibitors. Prior art papers on peptide inhibitors can be found in the book UK patent application filed on the same day as the application, entitled “Thrombin Inhibitor” and PCT / GB9 6/00352. Patent application for this title "thrombin inhibitor" A copy of the handbook is filed with this application. The content of this patent application is under the title Including the subject matter of the patent application entitled " It could be referenced along with the literature. The specification of this "thrombin inhibitor" It does not form a physical part of this patent specification.   Part 2 for binding to the anionic part of the substrate in serine proteases It is known that some have an order or "external position". This external position is Often referred to as the anion binding external position ("ABE").   Amino acid residues that provide the carbonyl group for serine / protease substrate cleavage bonds Expressed as “P1”. P2, P3, and P4 are consecutive amino acid residues located on the N-terminal side of the P1 residue. And the amino acid residues present on the C-terminal side of the P1 residue are represented by P1 ', P2', P3 ', etc. Expressed by In fibrinogen, P1 'is glycine and P2' is pro- This is Rin. Protease is a "specificity pocket" that recognizes the side chain of the P1 amino acid including. Trypsin-like proteases are usually arginine-like or serine-like Recognizes P1 residues with side chains.   The present invention provides novel bifunctional serine protease inhibitors. Its inhibition The agent is   (a) a catalytic site that binds to and inhibits the active site of serine protease Direction part (CSDM);   (b) an external position related part (EAM); and, optionally,   (c) Connector for connecting EAM and CSDM including. CSDM and EAM can bind simultaneously with serine protease molecules You.   In one class of inhibitors, the serine protease is not thrombin. Auction The protease is preferably a trypsin-like protease. Anyway, this obstacle The harmful agent is thrombin, where the connector part binds to the CSDM as its C-terminal extension No inhibitor, i.e. U.S. Patent No. 5,196,404 and corresponding international patent It is not a compound disclosed in Patent Application No. WO91 / 02750.   Another embodiment of the present invention is to provide a novel method for producing a boron-containing peptide.   As used herein, the “natural” amino acids are the 20 common amino acids found in proteins. That is, an L-amino acid (or the remainder thereof) selected from one of the "standard" α-amino acids. Group).   “Unnatural” amino acids are all α-amino acids except the 20 “standard” amino acids (Or its residue). Thus, an "unnatural" amino acid is a natural amino acid Includes D-isomers of acids and amino acids with side-chain protecting groups.   The prefixes “D” and “L” commonly refer to natural amino acids in the D or L configuration, respectively. Used to indicate. The prefix “D, L” indicates a racemic mixture and the prefix If the amino acid is D-type or Indicates that either of the L-shaped configurations is possible. Either D-type or L-type in the sentence In the case of non-specific groups that may also be present, the L-configuration is preferred.   Abbreviations and terms that begin with the prefix "boro" are terminal carboxy groups -CO_TwoH Represents an amino acid substituted with a boron function.BRIEF DESCRIPTION OF THE FIGURES   Figure 1 shows the Fourier transform infrared spectroscopy (Ft-IR) of Merrifield resin. It is a kutor.   FIG. 2 shows sodium 2,2-dimethyl-1,3-dioxolan-4-methanoate of the resin. F. after reaction with oleic acid. t.-I. R. It is a spectrum.   FIG. 3 shows the reaction resin after treatment of the hydroxy deprotection of dioxolane with HCl. F. t.-I. R. It is a spectrum.   FIG. 4 shows the F.V. of the deprotected resin after reaction with phenylboric acid. t.-I. R. In the spectrum is there.   The inventive compounds and methods will now be discussed in more detail.Catalytic site-directed part (CSDM)   The catalytic site directing moiety (CSDM) is located at the catalytic site of the serine protease enzyme. Binds and inactivates the site. The structure of CSDM is not important for the present invention. Absent. Is it a known inhibitor of the serine protease catalytic site? The amino acid sequence. For example, one class of CSDM is included in Formula I below. To get:         X- (aa^Four)_m− (Aa^Three)_n− (Aa^Two) − (Aa¹) -Z (I)   Where aa¹, Aa^Two, Aa^ThreeRepresents a natural or unnatural acid residue, (aa^Four)_mIs aa^Threeof Represents one or more optional amino acid residues linked to an amino group. Instead, one or more The aa group above is an analog of an amino acid residue in which the α-hydrogen is removed by a substituent. replace. The amino acid sequence and / or amino acid analog is a serine protease Binds to the active site. Suitable sequences are described later in this specification. X is H or N end And represents a substituent on the amino group. Z is, for example, -COOH or a C-terminal extension group (carbo Xy substituent). In preferred compounds, Z is a heteroatom acid group, For example, -B (OH)_Two, -P (OH)_Two, Or PO (OH)_TwoOr a derivative thereof, such as cal Boxylate, dioxo-boronate [-B (O substituent)_Two] Or phosphate Salt [-PO (O substituent)_Two] Or BF_TwoIt is. A preferred heteroatom analog group is -B (O H)_TwoAnd -P (O) (OH)_TwoAnd less preferred heteroatom analog groups are S (O)_TwoO H. Other possible Z groups include -CN, -COCH_TwoCl, -COCH_TwoF You. In a preferred embodiment, m is from 0 to 7 and more usual values are from 0 to 5. , For example, 1 or 2, especially 0. Usually, n = 1.   In one class of compounds, (aa^Two) − (Aa¹) The natural peptide bond is another bond (Ψ) Is replaced by Additionally or alternatively, other natural pepti The bond may also be replaced by another bond.   Catalytic site inhibitors of serine proteases are known in the art. Serine Pro A brief description of the protease inhibitors, ie, the catalytic site serine protease inhibitors A review is described in EP-B-145441, which is a patent which claims a cell having a C-terminal boron group. One class of proteases is disclosed. In addition, serine protease Patent specifications describing inhibitors include EP 293881, EP 471651 (equivalent to US 5288707), EP  235692, US 4963655, WO 89/09612 (especially VII / VII in [TF: VII / VIIa] complexes Factor VIIa inhibitors).   For inhibitors of trypsin-like enzymes, a preferred class of P1 residues in CSDM is (i) Arg, Lys and their analogs, (ii) hydrophobic residues, thrombin and Regarding the preferred P1 group for C and other trypsin-like enzymes, see the further title Thrombin inhibitors "and PCT / GB96 / 00352. Liver Trypsin-like serine proteases are phenylalanine analogs and alanine Preferentially binds to CSDM having the side chain of the analog on the P1 residue. Table A below The most preferred (P4) P3P2 residues for eight particular serine proteases are shown.   In each case, the preferred amino acids can be replaced with their analogs.External position related part (EAM)   External position-related part (EAM) binds to the external position of serine protease (ABE) Part. Thrombin has a clearly identifiable external position, whereas thrombin has In addition to the fibrinogen amino acid sequence c-terminal at the cleavage site, such as hirudin A non-thrombin substrate ligand binds. Hir^53-64A hirudin sequence such as Used in bifunctional peptides named "logs." 196404, and a review of thrombin EAM can be found in the aforementioned title “Tronbin EAM. Bin inhibitors "described in the UK patent application. The EAM for thrombin is Often referred to as "anion binding external position related moiety (ABEAM)".   FXa (Padmanabhan K et al, “Structure of Human Des (1-45) Factor Xa at 2.2 Å Resolution ”J Mol. Biol 1993, 232, 947-966) This means that sequences 35-41 and 70-81 containing eight acidic residues It is to configure. Natural polypeptide inhibitors antistasin and gira Is an electropositive cation external position-related sequence¹⁰⁸CRPKRKLIPR¹¹⁷and¹⁰⁸CKPKRKL VPR¹¹⁷Respectively.   The serine-protease interaction at the P 'site (C-terminal at the cleavage point) It is clear that the interaction is as important for specificity as ing, L .; Coombs, G .; S .; Strandberg, L .; Navre, M .; Coreyu, D.R. and Madi son, E.L. Origins of the Specificity of Tissue-type Plasminogen Activato r, Proc. Natl. Acad. Sci, 1995, 92, 7627-7631). Library Screenini Compare spans of both sites with larger ligands for faster use in Need to be Lawson recently published (1992) that P 'and P By screening for peptide sequences that contain sufficient Detection of Fvlla / TF activity that is too insensitive to the substrate. Peptide La Ibraries have a biological activity profile in a variety of applications (eg, Eichler, 1994). It shows great efficacy in cleaning and is useful for the present invention.   In the present invention, the C-terminal to the cleavage point of another serine proteinase substrate is Consider that end sequences form an EAM. Connector part   The compounds of the present invention include a connector portion that interconnects the CSDM and the EAM, The connector part is CSDM and EAM, respectively serine protease Simultaneously bind to the inhibitor molecule. For thrombin, the connector is CSD It binds to M as an N-terminal extension or as or through its side chain.   Connector part to CSDM, as C terminal extension or N terminal extension, or It may be attached as or through its side chain. However, if the compound is thrombin If it is an inhibitor, the connector portion cannot be the C-terminal extension of CSDM.   In particular, if the connector part is an N-terminal extension of CSDM or its side chain Preferably, the connector portion comprises at least two adjacent Gly residues, e.g., For example, it includes the amino acid sequence contained at its N-terminus.   In one class of compounds, preferably the connector is a peptide "spacer And non-peptide “linkers.” Typical connector structures are: And                       −λ−σ−   Wherein λ represents a non-peptide linker and σ is a spacer containing the sequence of amino acids. And λ and σ are connected by peptide bonds in the appropriate state. spacer σ is preferably linked to EAM, and linker λ is linked to CSDM. New However, if σ is linked to CSDM and linker λ is linked to CSDM in the compound, In this case, it is not preferable as an embodiment included in the present invention.   The linker typically comprises an N-terminal amino group of the spacer and a C-terminal such as an N-terminal group. It is a residue of a compound having a functional group that reacts with the functional group of SDM. Therefore, Preferred linkers have two carboxyl groups, eg, CSDM and a spacer. It has a dicarboxylic acid capable of forming an amide bond with an N-terminal amino group. Especially preferred A new linker is glutaric acid (HO_TwoC (CH_Two)_ThreeCO_TwoH) The formula of the residue and its homologues (HO_TwoC ( CH_Two)_hCO_TwoH), wherein h is an integer of 2 or 4, 5, or 6. Alkylene residue Group [(CH_Two)_2-6-] Is substituted with one or more substituents, which are sterically hindered by the linker. It does not harm and can maintain the desired flexibility of the linker. You.   Although less preferred, the linker may be, for example, a carboxyl group having 2 carbon atoms. Other compounds having two carboxyl groups separated by six atoms from one another Product residues.   The amino acid sequence of the spacer is not important in the present invention, but the sequence is small. It is preferred to include both two adjacent Gly residues, usually at the N-terminus of the sequence. S Pacer length depends on the position of the linker on the CSDM, especially I do.   Regarding the connector part suitable for peptide / thrombin inhibitor, It is further described in the UK application entitled "Thrombin Inhibitors" of the same day.   The connector portion may have one or more natural amide bonds replaced with other bonds .Synthesis   The compounds of the invention can be used, for example, for peptide synthesis and peptide coupling. It can be manufactured by a method known in the art. In an exemplary method, the novel compound is a solid phase Manufactured by synthetic techniques.   Because solid-phase synthesis is a well-known technique to peptide chemists, A fresh explanation is not needed here. An overview of this technique can be found in “The Chemical Syn thesis of Peptide ”, John Jones, Clarendon Press, Oxford, England, 1991 ". The general principle of solid phase synthesis is that the amino acid coupled to the solid phase An acid or peptide reacts with itself with a protected amino acid to form a solid phase. After a couple with a linked amino acid, the reaction with itself is counteracted with a protected amino acid. Is to be deprotected. Repeat these steps as many times as necessary You.   One solid phase synthesis technique is Fmoc technique (Fmoc = fluorenylmethylcarbonyl) . Fmoc chemical reaction (also commonly known as 'Sheppard approach') The carboxy terminus (or amino acid) of the peptide is terminated by a reactive function. It binds to the resin bead via the linker to be formed. The resin bead itself is typically Other solids that are polystyrene (PS) but have suitable swelling properties in solvents used. The reason is that peptide chains grow in pores inside the beads. Is currently known. Another example of a solid is a poly called Kiesulguhr. Amides.   There are a variety of linkers, but the preferred one is PEG (i.e. Cole Linker), which has an alcohol function.   The end of the linker is typically called a 'handle' and depends on the desired product. But the ends are ultimately cleaved by the acid for the Fmoc chemistry It is the part that can be done. The most common end (the one you use) is the HMBA It is a parahydroxyn methyl benzoic acid linker. HMBA esterified on PEG And then reacting with a peptide or amino acid (with Fmoc on the N-terminus) An ester linkage is generated on the final HMBA. This ester linkage is then cleaved with an acid . Fmoc protecting groups are base-labile and are typically removed with a secondary base (eg, piperidine) The resulting free amino group reacts with the selected Fmoc protected amino acid. These engineering Repeat the process to extend the amino acid sequence.   Another solid-phase synthesis method is the Boc technique (Boc = t-butyloxycarbonate). Le). Used for Boc chemistry (more commonly known as the Merrifield method) The resin is often a divinylbenzyl base, for example, 'Wang' resin has 2% It has chloromethylbenzene which copolymerizes with divinylbenzene. Chloromethylbe The benzene group reacts with an amino acid or peptide whose amino group is protected by Boc. To create a link on the resin. Connections on this resin are typically cleaved with dry HF liquid. (Very carefully!) This is called 'active' acid decomposition. Boc protecting group is acid labile , Typically prior to the reaction of the resulting free amino group with the selected Boc protected amino group Is cleaved by TFA. Repeat these steps as in the Fmoc chemistry To extend the amino acid sequence.   Two classic techniques of solid phase peptide synthesis (Sheppard and Merrifield) follow Amino acids to the solid phase resin particles via the carboxy terminus or its derivatives Doing, followed by new amino acids (via their active carboxy terminus) To the generated N-terminus.   Recent reports have shown that, for example, acid labile benzyloxy Release of the couple carboxy terminus to the resin via a bonyl bond, its activation, A couple through the N-terminus of an amino acid, a temporary Protection is disclosed (Sharma, R.P .; Jones, D.A .; Broadbridge, R.J .; C orina, D.L. and Akhtar, M.S. A Novel Method of Solid Phase Synhesis Of Pept ide Analogues, in Inovation and Perspectives in Solid Phase Synthesis, ed ., R. Epton. 1994, Mayflower World wide Limited, Birmingham, page 353-356 Letsinger, R .; L. and Komet, M .; J. Amer. Chem. Soc., 1963, 85, 3045).   The product of the present invention is produced using such an N-terminal coupling method. One implementation In an embodiment, the CSDM containing any directly connected amino acids is linked by an N-terminal couple. Combine. This technique is particularly useful when CSDM has a C-terminal heteroatom group It is. In this technique, the resin-bound peptide chain produced using the N-terminal couple is Derivatized to activate its carboxy terminus and then free α-aminoboro The ester or acid is coupled to the resin binding sequence. Finally, peptide boro Prior to coupling the catenate (including CSDM) to the end product residue, a strong acid (eg, For example, cleavage from the resin by HF or TFA).   When the CSDM of the synthetic compound contains a P1-P2 unnatural amide bond, X- (aa of formula I^Four)_m-(aa^Three)_n− (Aa^Two)] And the attached C-terminal group [(aa¹ ) -Z] is preliminarily prepared as an intermediate Flight It is profitable. These two intermediates contain the appropriate functional groups and can react together to target Form an amide bond [Ψ] and react together to form a compound (or one or more functional groups). (Precursors that cause deformation).   A suitable synthetic technique for producing peptides containing a non-amide bond は is PCT / G B96 / 00352.   We use solid-phase chemistry to create unexpected results for the synthesis of peptide boronate esters. It worked. For example, in the typical synthesis of a serine proteinase inhibitor The connector portion and its adhesive EAM form the N-terminal extension of the CSDM, EAM can be performed on Fmoc solid phase peptides using, for example, the Fmoc-polyamide continuous flow method. Manufactured by chemistry. Suitable solid phases for this purpose are pre-derivatized solid supported Fmoc-L eu-PEG-PS. The peptide conjugated resin is then treated, for example, with glutaric anhydride. You. One of the carboxyl groups reacts with the N-terminal amino group of EAM. Pre-synthetic peptide boronate CSDM is combined with resin / peptide / glutarate conjugate React to form the final compound. This compound is, for example, by 100% TFA Cleavage from the resin in the procedure.   Another method using peptide boronate esters in solid-phase chemistry Is a completely new technique in which boric acid [-B (OH)_Two] Coupled with resin Esterify directly with diol. Chain extension from amino groups of amino acids, for example Continue with standard Fmoc chemical coupling. Borate is a peptide boronate [pep Tide-B (OH)_Two] With an acid (eg, TFA) or transesterification By a concentrated solution of a tethered diol, such as pinanediol, Cleaved from   Accordingly, the present invention includes a method of producing a compound of the present invention to produce a target amino acid sequence. To perform the following steps.   (i) reaction with an amino group or preferably a carboxyl group or an active derivative thereof Providing a solid phase coupled to a functional group that can be   (ii) amino or carboxyl of the terminal amino acid of the amino acid sequence of the compound of the present invention Selectively reacting a group (which may be in the form of its active derivative) with said functional group;   (iii) the amino acid sequence sequence preceding the solid phase Continued Coupling the starting amino acid to the preceding amino acid;   (iv) Repeat step (iii) as many times as necessary.   In step (i), the functional group coupled to the solid phase is incorporated into the final compound Amino acids coupled directly or indirectly to the solid phase, for example Amino group (which can be derivatized).   Often, the process for obtaining the compounds of the present invention involves one or more additional steps. I will. The preferred method comprises, if desired, the sequence-wise following amino acid of step (iii). Acid to the preceding amino acid via a compound having a bifunctional group capable of reacting with an amino group. There is a step (v) of making a couple. Depending on the compound having two functional groups, One of the groups is attached to the amino group of the preceding amino acid, and one is Bond to the amino group.   The amino acid sequence following in step (iii) may be part of a larger portion, for example, It may be part of an amino acid sequence that optionally contains substitutions for peptide bonds.   In this method, any one or more carboxylate groups reacted with an amino group Also, their active carbonyl-containing derivatives, such as active carbonyl groups and acid anhydrides It can be in the form.   Before use, the final compound of the solid phase synthesis is cleaved from the solid phase, for example, in a known manner. Is done. The cleavage compound may be subjected to one or more additional chemical reactions before obtaining the final compound. You.   In a preferred embodiment, the terminal amino acids that react with the functional group attached to the solid phase are , The C-terminal amino acid of EAM, and step (iii) is repeated to obtain a contiguous sequence of EAM sequences. Non-disruptive by coupling a contiguous amino acid with a contiguous amino acid in the adjacent connector peptide Form an amino acid sequence.   The final amino acid of the non-interfering amino acid sequence coupled to the solid phase is two carboxy Compounds having a rate group or a reactive derivative thereof, such as dicarboxylic anhydride React and link one of the two carboxylate to the amino group of the final amino acid You. Unreacted carboxylate or its derivative is added to the amino group of one amino acid. Typically reacts, this amino acid is usually the N-terminal amino acid of CSDM. The latter In that case, the amino acid may already bind to the rest of the CSDM, ie (Or partially) to link to the corresponding carboxylate (derivative) Made into pieces. Compounds having two carboxylate groups are preferably as described above. Linker.   In some preferred methods, a heteroatom group is substituted for the C-terminal carboxy group. CSDM is used. The heteroatom group is preferably a boronate as defined above or Is a boronate derivative.   Amino acids or other moieties that have reacted with the solid phase material (solid phase and adhesion molecules) It preferably has other reactive functional groups protected from synthesis interferences besides the corresponding groups. The protected functional groups of all reactive amino acids or moieties may react by themselves. It is deprotected before it is subjected to the reaction.   Therefore, the first preferred method includes the following steps.   (i) coupling to a functional group capable of reacting with a carboxyl group or a reactive derivative thereof Preparing the solid phase;   (ii) The C-terminal amino acid of EAM is brought into contact with a solid phase, and the amino acid is protected amino group and And optionally has a derivatized carboxy group, and then the carboxy group of the amino acid molecule is Reacting or enabling the functional group of   (iii) deprotecting the amino group of the reacted amino acid, thereby releasing free Providing a mino group;   (iv) step (ii) with contiguous amino acids of the EAM and optionally adjacent spacer peptides And (iii) are repeated to form a stripe at the free end of the sequence from the C-terminus of EAM on the solid phase. Form an amino acid sequence up to the N-terminus of the pacer;   (v) Solid phase on a linker compound having two carboxy groups or its reactive residue To make a linker carboxy group or a reactive carboxy residue a spacer. React or enable the N-terminal amino group of the sequence;   (vi) The solid phase coupled with the linker compound is changed to the N-terminal amino acid of the CSDM sequence. The amino group of the amino acid molecule into the carboxy group of the linker compound or the reactive group. Reacts with or allows for the reaction of ruboxyl residues, Terminal amino acids are part of the complete CSDM;   (vii) If necessary, repeat steps (ii) and (iii) for the CSDM contiguous amino acids. Repetition To complete the CSDM sequence; and   (viii) Cleaving the compound obtained from the functional group on the solid phase.   A second preferred method includes the following steps.   (i) coupling to a functional group capable of reacting with a carboxyl group or a reactive derivative thereof Preparing the solid phase;   (ii) The C-terminal amino acid of EAM is brought into contact with a solid phase, and the amino acid is protected amino group and And optionally has a derivatized carboxy group, and then the carboxy group of the amino acid molecule is Reacting or enabling the functional group of   (iii) deprotecting the amino group of the reacted amino acid, thereby releasing free Providing a mino group;   (iv) repeating steps (ii) and (iii) for consecutive amino acids of EAM, Above is the amino acid sequence from the C-terminus of EAM to the N-terminus of EAM at the free end of the sequence Form;   (v) Solid phase on a linker compound having two carboxy groups or its reactive residue To form a linker carboxy group or a reactive carboxy residue on the N of the EAM. Reacting or allowing a reaction with an amino group;   (vi) The solid phase to which the linker compound is coupled is the N-terminal of the peptide spacer sequence. Selective contact with amino acids and the amino group of the amino acid molecule React or enable a xy group or a reactive carboxy residue;   (vii) Steps (ii) and (iii) are selectively repeated for consecutive amino acids of the spacer. And selectively repeat step (ii) for the N-terminal amino acid of the CSDM sequence Typically the N-terminal amino acid of the CSDM sequence is part of the complete CSDM;   (viii) If necessary, steps (ii) and (iii) for consecutive amino acids of CSDM To complete the CSDM sequence; and   (ix) cleaving the compound obtained from the functional group on the solid phase;   In any of the above methods, the synthetic compound is preferably cleaved from the solid phase by an acid. Is done.   The method is preferably a CSDM amino acid or an amino acid having a C-terminal boron group. (Eg, complete CSDM) sequences.   The functional groups coupled to the solid phase in the first and second preferred methods are Can be part of a moiety that is incorporated into the solid compound, for example, directly or indirectly on a solid phase. The amino group (which can be derived) of the pulled amino acid.   In one method of the present invention, the functional group attached to the solid phase is Part of the amino acid boronate that has been incorporated into the product. That is, the solid phase It has a diol bound to a noonic boronate.   In another class of this technique, the side chain has an amino or carboxyl group Amino acids bind to the solid phase through carboxyl or amino groups to the side chains You. Chain elongation is the reaction of amino acid functional groups, such as Fmoc synthesis from amino groups. Get up from one. Another functional group is then added to another component of the end product, e.g., amino Reacts with acid borates, which form the P1 residue of CSDM. However, Ho Solid-phase synthesis of elemental peptides is applicable to all such peptides, It is not only applicable to the serine protease inhibitors of the present invention.Solid-phase synthesis of boron-containing compounds   Peptide boronates are well defined in compounds made by solution chemistry In the category that did. Thus, peptide protease inhibitors are well known. And the C-terminal carboxy group is substituted by a boric acid group or a derivative thereof. Teens Representative compounds are represented by II:                 (aa)_k−B (R^Two) (R^Three), (II) Where (aa)_kRepresents a sequence of amino acids (eg, as in Formula I);^TwoAnd And R^ThreeAre each independently halogen, -OH, -OR^FourAnd -NR^FourR^FiveSelected from R^FourAnd R^FiveAre each independently of the formula R⁶(CO)_u-Is a group of which u is 0 or 1; R⁶Is H or optionally halogenated alkyl, aryl or Or arylalkyl groups, which contain up to (10-u) carbon atoms and In some cases, -OH, R⁷(CO)_vOR⁷(CO)_vSubstituted with one or more groups selected from Wherein v is 0 or 1;⁷Is C₁-C_6-vAlkyl or ally , Alkylaryl, arylalkyl or alkylarylalkyl groups And these groups contain up to (10-v) carbon atoms. Also R^TwoAnd R^ThreeIs together Represents a diol or dithiol residue.   Such peptide boric acids are described, for example, in WO 92/07869 (USSN0). 8 / 317,387), EP 0471651 (corresponding to US 5288707) and US SN 08 / 240,606 (herein incorporated by reference for reference) Incorporate).   As mentioned earlier, we have surprisingly identified boron-containing peptides as critical by solid-phase chemistry. It has been found that it can be synthesized without defects such as decomposition. Therefore, one aspect of the present invention The phase is a solid phase chemistry, especially the Fmoc chemistry (also known as the “Sheppard approach”). The use of boron-containing amino acid analogs in peptide synthesis according to   Another aspect of the invention involves performing the following steps to produce a target amino acid sequence. And providing a method for producing a peptide or a peptide-containing compound. And   (i) providing a solid phase coupled to a functional group;   (ii) selectively reacting it with a compound reactive with a functional group, The compound is a functional group that can react with amino group or carboxyl group or its reactive derivative. Having a group;   (iii) the amino or carboxyl group of the terminal amino acid of the target amino acid sequence (the opposite (Which may also be in the form of a derivative)). Shime;   (iv) sequence in the target sequence to the amino acid sequence-leading to the solid phase Coupling the following amino acid to the preceding amino acid;   (v) repeating step (iv) as many times as necessary;   (vi) The solid phase-linked compound produced by using steps (i) to (iv) is subjected to the action of an acid or a base. Cleavage from solid phase.   Characteristic point is the boric acid group [-B (OH)_Two] Or derivatives thereof, especially esters Compound into the solid phase-bound compound prior to its cleavage from the solid phase. It is.   The process of the present invention for producing a peptide or a peptide-containing compound In some cases, step (iv) [that is, in step (iv), The amino acid sequence following is part of a peptide] A step of binding the compound and the peptide.   The method of the invention for producing a peptide or peptide-containing compound comprises the steps of: Compounds other than amino acids or peptides may be added to the solid phase-linked compound produced in the previous step. A peptide, a peptide analog or an amino acid Having two carboxyl groups that are used to link the carboxylic acids in the liquid phase The step of coupling the compounds. Of course, amino groups or All other liquid phase compounds that can react with carboxyl groups It should be able to bind to the binding peptide. Diamine is a carboxyl group (for example, (In the form of their reactive derivatives). Zical Extension of the solid phase bound peptide by boxylic acid, especially glutaric acid, is typically Through the N-terminus of the peptide spacer moiety and dicarboxylic acid residue binding part Machine with CSDM connected to EAM via connector part containing It is useful for producing functional serine protease derivatives.   Solid-phase bound compounds whose free ends end with dicarboxylic acid residues are free carboxy The bond between a sil residue (in some cases in the form of a derivative) and the amino group of an amino acid Can be stretched further. For example, coupled with a solid phase bound EAM-Spencer moiety The dicarboxylate residue is combined with an amino acid of CSDM, for example, the N-terminal amino acid of CSDM. Can react.   Step (ii) of the method comprises the amino group of an amino acid or a selectively derived carboxy group. Directly or indirectly to, for example, a solid phase that is part of native solid phase peptide synthesis. Includes reaction with attached functional groups. Amino or carboxy groups attached to the solid phase It is often a terminal amino or carboxy group, but in some embodiments, for example, It is a side chain functional group such as a side chain carboxy group of rutaric acid. Solid phase via side chain The C-terminal carboxy group of the attached amino acid is a boric acid residue [-B (OH)_Two] Or its d Can be replaced by steles.   The method of the present invention involves N-terminal coupling in SPPS and The ruboxyl powder is free α-aminophosphoric acid prior to acid cleavage of the synthesized product from the resin. Couple with uric acid or ester.   In another embodiment, step (ii) is performed with amino acid or peptide boric acid or ester. The reaction of Or   Where E¹And E^TwoRepresents a boron ester forming residue, or together A single residue may be formed by the diol coupled to the solid phase. Boron atom (example (For example, as part of an amino acid or peptide boric acid or ester) and a solid phase Linkage is linked through hydroxyl groups (directly or indirectly) coupled to the solid phase. The tying technique is novel and forms a feature of the present invention.   If the compound containing boric acid or ester is a compound of any of the above embodiments, For the amino acid boronates used in step (ii), solid phase synthesis techniques can selectively Serine protease catalysis in the synthesis of highly functional serine protease inhibitors It can be used to produce site-specific peptide boronate inhibitors.   Thus, boric acid can be esterified directly on the diol-containing resin and then N 2 Subsequent chain extension from the ends can be performed, for example, by standard Fmoc chemistry. Then The borate ester is cleaved by a mineral acid to give free boric acid [peptide-B (OH)_TwoOr get Or by transesterification, such as diols and pinanediols It can be cleaved by a concentrated solution of diol.   Literature describes a method for producing a diol-containing solid phase resin, and the resin is Can be derivatized with aldehyde and derivatized with boric acid / ester (Eg, Xu, Z-H., McArthur, C.R and Leznoff, C.C. onoblocking of symmetrical Diketones on Insoluble Polymer Supports, Can. J. Chem., 1993, 61, 1405-1409. and Leznoff, C.C. and Sywanyk, W.S. ‘Use o f Polymer Supports in Organic Synthesis. 9． Synthesis of Unsymmetrical Car etenoids on Solid Phase ', J. et al. Org. Chem., 1997, 42, 3203-3205).   The outline of the manufacturing sequence is as follows:A diol is a compound having two or more alcoholic hydroxy groups. X and Y are protecting groups. R is the side chain of the amino acid boronate / boric acid.   Typically, the resin is washed after each step. In a suitable embodiment, the diol is Unprotected before reacting with the resin.   A more specific order is as follows:  Thus, the present invention provides, for example, the use of In the case of manufacturing a library, it is necessary to use solid-phase chemistry to produce peptide boronates. Open the way.   The present invention relates to a compound containing peptide boric acid or peptide boronate ester. Including the method of manufacturing the product, (i) providing a solid phase coupled to an alcoholic hydroxy group; (ii) reacting an amino acid boric acid or peptide boric acid with a hydroxy group, Thus, boric acid residues are esterified to the solid phase; (iii) Sequencely after peptide boric acid or boronate ester in the final product The carboxyl group of the following amino acid is added to the amino acid sequence Selectively reacting the amino group of the amino acid; (iv) repeating step (iii) as many times as necessary; (v) cleaving the resulting peptide boronate from the resin; Process.   In some cases, the method includes one or more additional steps in the preparation of the compound. No.   The alcoholic hydroxy group coupled to the solid phase is preferably a They are arranged so that they can bond with atoms. That is, the boron atom is Diesterified:   In some embodiments, the hydroxy group is in the 1,2-configuration (i.e., on adjacent carbons). ), And in another embodiment, are separated on a chain (e.g., NH (CH_TwoCH_TwoOH_Two) Remaining Base).   Preferably, each amino acid coupled to the solid phase has an amino protecting group, (iii) involves deprotecting the amino group that precedes the amino acid in sequence. Also, The cleavage in step (V) is preferably performed by an acid or transesterification reaction.   More generally, the present invention relates to the immobilization of boric acid residues attached to a solid phase with hydroxy residues. A use in a phase synthesis method is provided.   The present invention also provides a method for producing a compound containing a boron atom, the method comprising: (i) providing a solid phase coupled to an alcoholic hydroxy group; (ii) reacting boric acid or boronate ester with a hydroxy group, thereby To esterify the boron residue to the solid phase; (iii) performing one or more additional steps for the preparation of the compound; Including. The alcoholic hydroxy groups are preferably arranged as described above.   Another embodiment of the present invention provides a method for coupling a boric acid residue to a boric acid residue via a hydroxy group. Provide phase materials. It is also a solid phase material coupled to the portion of Formula IV below.   Wherein R is a residue attached to a boron atom, usually an organic moiety. Residue R Are a class of materials released from functional groups reactive to alcoholic hydroxy groups (However, the material may contain functional groups in a protected form, eg, before deprotection). Another category In the above material, the above-mentioned functional group is deprotected, and the protecting group is removed in advance. R is Typically, an organic moiety having one or more functional groups that enables a chemical reaction of R You. All functional groups that can be protected can be protected. In some classes of materials, the solid phase is Couple with the notation V part.   -CH_TwoOne or both of the hydrogen atoms of the group are other groups compatible with the use of the material, such as Alkyl (such as methyl or butyl).   In yet another class of materials, the oxygen on the left side of Formula IV is part of an ester.   The first class of solid-phase synthesis methods involves performing the following steps, which involves the targeted amino acid sequence An object of the present invention is to provide a method of manufacturing a row. (i) providing a solid phase coupled with a functional group capable of reacting with a carboxyl group; (ii) contains an amino group that is reactive to a functional group and protected by a base-labile protecting group. Reacting the compound with the functional group to form an acid labile bond; (iii) deprotecting the amino group with a base; (iv) Carboxy of amino acids wherein the amino group is protected by a base-labile protecting group To react with the deprotected amino group generated from step (iii); (v) deprotecting the protected amino acid with a base; (vi) Sequenceally preceding the amino acid sequence-coupled to the solid phase in the target sequence The subsequent amino acid is reacted with the deprotected amino group of the amino acid preceding the sequence. The amino acid following this sequence is protected by a base-labile protecting group. Having a amino group; (vii) deprotecting the protected amino acid group with a base; (viii) repeating steps (vi) and (vii) as many times as necessary; (ix) The acid labile bond is cleaved by an acid or by transesterification.   Characteristic point is the boric acid group [-B (OH)_Two] Or a derivative thereof, especially Incorporate the ester into the solid phase linking compound prior to cleavage from the acid labile bond It is to be.   As described above for the method for producing peptides or peptide-containing compounds, The step (ii) is performed by combining the amino group or the selectively derived carboxy group of the amino acid with, for example, The solid phase is directly or indirectly coupled to the solid phase by techniques known per se in solid phase chemistry. Includes reaction with pulled functional groups. This amino acid can be a boric acid or ester group, That is, it is a compound containing the amino acid boric acid. As an alternative, step (ii) comprises: Boric acid or ester group in the form of an amino acid or peptide boric acid or ester Reacting a compound containing a diol with a diol coupled to a solid phase. In both cases, an amino acid (or peptide) boric acid or ester is used, This method involves the production of a peptide boronate inhibitor at the serine protease catalytic site. Suitable for the synthesis of bifunctional serine protease inhibitors I have.   The methods for producing the various other peptides and peptide-containing compounds described above are described above. It is applicable to the first class of the solid phase synthesis method described above.   A second class of solid phase synthesis methods involves performing the following steps to produce the target amino acid sequence. No. (i) providing a solid phase coupled with a functional group capable of reacting with a carboxyl group; (ii) contains an amino acid group that is reactive to a functional group and protected by an acid labile protecting group. Reacting the compound with the functional group to form a base-labile bond; (iii) deprotecting the amino group with an acid; (iv) carboxyl of an amino acid whose amino group is protected by an acid labile protecting group Reacting the group with the deprotected amino group generated from step (iii); (v) deprotecting the protected amino acid with an acid; (vi) Sequenceally preceding the amino acid sequence-coupled to the solid phase in the target sequence The subsequent amino acid is reacted with the deprotected amino group of the amino acid preceding the sequence. The amino acid following this sequence is protected by a base-labile protecting group. Having a amino group; (vii) deprotecting the protected amino acid group with an acid; (viii) repeating steps (vi) and (viii) as many times as necessary; (ix) base-labile bonds are cleaved by bases or by transesterification .   Characteristic point is the boric acid group [-B (OH)_Two] Or a derivative thereof, especially The ester is assembled into a solid phase linking compound prior to cleavage from its acid labile bond. It is to put.   The various solid phase synthesis methods of the first class described above are also applicable to the second class.   Regarding the method for producing the peptide or the peptide-containing compound, as described above, And the second class of solid-phase synthesis methods are the solid-phase-linked compounds produced in the preceding steps. With a compound other than an amino acid or a peptide .Overview of synthesis   The compounds of the present invention should include boric acid, even though they may contain boron. Absent. Non-boron containing compounds can also be produced by solid phase synthesis. The present invention Some compounds have a natural peptide bond substituted with another linking group. Further information on suitable methods for the synthesis of all compounds of the present invention can be found in the foregoing. It is described in a patent application entitled "Thrombin Inhibitor" filed today.Use   The novel compounds of the present invention include inhibitors of serine proteases such as thrombin. Or as a substrate, and useful for diagnostic and metabolic studies of such enzymes. Used in vitro or in vivo. More generally, new peptides are studied or synthesized. Useful for purpose. Furthermore, due to its inhibitory effect, regulatory systems, especially in mammals Thrombin or other cells in a system, such as the human or animal body, such as the clotting system. It is useful for treating and preventing diseases caused by excess protease. Pharmaceutical Useful compounds have a pharmaceutically acceptable group such as an N-terminal substituent.   The antithrombotic compound of the present invention is used when an antithrombogenic agent is required . Generally, the compounds of the present invention can be administered orally or parenterally to a subject in an effective amount. It has antithrombogenic effect. In the case of large mammals such as humans, the compound 0.02-100 mg alone or in combination with one or more pharmaceutical carriers or diluents / Body weight, preferably 1-100 mg / body weight, to obtain antithrombogenic action . This administration can be made in a single dose or in divided doses or as a sustained release agent. Once an extracorporeal blood loop has been established for a patient, 0.1-10 mg / kg may be administered intravenously. For use in whole blood, 1-100 mg / l is provided to prevent clotting.   Pharmaceutical diluents and carriers for human and animal use are well known. , Sugar, starch and water and one or more target peptides Appropriate and effective amount or concentration of a pharmaceutical composition (human or veterinary) Formulation is made. The pharmaceutical preparation may be in unit dosage form. There are tablets , Capsules, injections and the like.   The anticoagulant compound of the present invention comprises a container for collecting or dispersing blood, Added to blood to prevent blood clotting in tubes or insertion devices Can be.   The advantages provided by the compounds of the present invention include oral potency, rapid onset of effect. Has current and low toxicity. The compounds of the present invention further include heparin and thrombin. Hypersensitive to other known inhibitors or other compounds such as serine proteases There are special advantages in the treatment about.   The method of the invention is useful for the synthesis of serine protease inhibitors and other compounds It is. It is also useful in combined chemistry.   The present invention is further described and illustrated by the following examples.Example   In the examples, the amino acid residues are in the L-configuration unless otherwise specified.   GlyGlyGln (Tyr⁶³) Hir^51-64Is an amino acid, the formula H-Gly-Gly-Gln-His-Asn-Gly-Asp- It has Phe-Glu-Glu-lle-Pro-Glu-Tyr-Leu-OH, and has the Fmoc-polyamide And Milligen ™ using 9050Plus on column monitor software Produced by solid phase peptide chemistry on a 9050 peptide synthesizer. Arakazi It was used through the induced solid support Fmoc-Leu-PEG-PS (1.6 g, 0.22 meq / g). Fmoc-Leu-PEG-PS is a polyethylene glycol derived police with HMBA linker Contains Tylene. The Fmoc group was removed using a 20% piperidine in DMF solution. Fmoc-amino acids (4 equivalents) are converted into their pentafluorophenyl esters as side chains. Protection (e.g., Fmoc-L-Tyr (tBu) 0Pfp, Fmoc-L-Glu (tBu) 0Pfp, Fmoc-L-Asp (tBU) 0Pfp , Fmoc-L-Asn (Trt) OPfp and Fmoc-His (boc) 0Pfp) Was. When the required peptide sequence is completed, the N-terminal Fmoc group is It was removed using a DMF solution. A positive ninhydrin test removes the Fmoc group. Indicates that The peptide conjugate resin is placed on the filter and Wash sequentially with chloromethane, methanol and dichloromethane, then several hours under reduced pressure While drying.   The peptide obtained in Example 1a was suspended in DMF (5 ml) and placed in a round bottom flask (25 ml). With glutaric anhydride (300 mg) and 4-methyl-morpholine (200 mg). I understood. The reaction mixture was stirred overnight. Wash the resin with DMF, DCM, Me0H and dry under reduced pressure overnight After drying, the target compound was obtained.   Cbz-D-Phe-Pro-BoroBp in a round-bottom flask (100 ml) with a septum and nitrogen flow A 40% HBr acetic acid solution (20 ml) was added to g0Pin (2 g), and HD-Phe-ProBoroBpgOPin was added. Manufactured. Rotate the flask and stir to completely dissolve the protected tripeptide. Was. When gas evolution ceased after about 30 minutes, anhydrous ether (200 ml) was added. The reaction mixture was stored in the refrigerator for 4 hours. The reaction mixture was filtered and the residue was washed with Et0H (1 The product precipitated upon addition of dry ether and a white solid (800 mg). ) (M-H) 516: Tlc (C / M / A 95/5/3), Rf = 0.05.   [-D-PheProBoroBpgOPin] CO (CH_Two)_ThreeCOGly_TwoGln (Tyr^51-64) Hir^51-64Compose First, dry resin HOCO (CH_Two)_ThreeCOGly_TwoGln (Tyr⁶³) Hir^51-64To the DMF solution (10 ml) After suspending, TBTU (129 mg, 0.4 mmol) and HD-Phe-ProBoroBpgOPin (2 30 mg, 0.4 mmol) was added to the reaction mixture. Stir for 5 minutes and add triethylamine (40 mg, 0.04 mmol) was added and the flask was left overnight with stirring.   Completely protected peptide resin is purified using dichloromethane, methanol, Washed with tan and dried under reduced pressure. Opening from resin by simultaneous deprotection of side chain protecting group Cracking was performed by treating the resin with 100% TFA for 2 hours. Remove TFA and remove C powder Free peptide with xylic acid was produced by precipitation with cold dry ether. Coarse pep The tide was collected by filtration and further washed with a portion of ether.   Purification of the crude peptide was performed using a Vydac ™ C-18 preparative column (TP silica, 10 μm, 25 mm X 300 mm) using reverse phase HPLC. Direct column gradient 30-90 Eluted with% A solvent (0.1% TFA in water) and B solvent (0.1% TFA in acetonitrile) Was. The column eluate was monitored at 230 nM and fractions were collected as appropriate. product Was measured by analytical RP-HPLC and mass spectrometer.   Chloroethane-pinanediol boronate ester (0.321 g, 1.25 × Cbz-D-Phe-Pro-OH (0.6 g, 1.52 × 10^-3mol) Was. When mixing is complete, the DBU CH_TwoCl_TwoSolution (0.23 g, 1.52 mmol) was added to the mixture, Stir at room temperature and then continue at 4 ° C. until testing. HCl ( 0.1M, 2 × 50ml), NaHCO_Three(1%, 50 ml). Organic phase MgSO_FourViolent in anhydrous It was dried with good stirring and filtered to remove the desiccant. Rotate evaporate the filtrate The mixture was concentrated under reduced pressure in a vessel to obtain a thick viscous residue.¹Required by preliminary inspection by H N.M.R The generation of a crude product was confirmed. Dissolve the crude sample in a small amount of Loaded on a Dex LH20 column, which was then pumped out using the same solvent. The content of the eluate was monitored with an ultraviolet lamp (226 nM) and a recorder. Void volume, Fractions 1-6, as well as bulk, were collected. Appearance in chromatogram Indicates that fractions 1 to 6 are the fractions most likely to have a tripeptide Was estimated. Concentrate the fractions to light and light colored sticky residue was gotten. Slightly crystalline from one fraction containing material bulk under strong vacuum (35% yield 0.269) was obtained. N.M.R., FABMS (Fast Atom Bombardm ent Mass Spectrometry), C.H.N is a very strong (good) indicator of compound formation. Was.  Cbz-D-Phe-Pro-Ψ (CO_Two) -BoroEtg pinanediol (from Example 2a) in MeOH solution (3 0 mg), treated with 10% Pd / C, distilled with argon while stirring, H while stirring for 5 hours_TwoWas pumped. Ninhydrin coloring on TLC The deprotected product was confirmed. The solution is flushed with argon for 10 minutes, filtered and Concentrated to give a dark black oil, which was extracted with CHCl_TwoDissolved in, filtered, Concentrated.¹H N.M.R. confirmed that the crude was not a protected product. From the above products Was separated on a Sephadex LH20 chromatography column.¹H (60MHz) N In the .M.R., The isolated compound exhibited features that could be considered from the proposed structure. 122mg The free aminoborate was isolated.   Tetrakistriphenylphosphine / palladium (O) [PdP (Ph_Three)_Four] (1g) Argo CH containing 5% acetic acid and 2.5% N-methylmorpholine_ThreeCl solution (30ml) And dissolved. This mixture was combined with Fmoc-L-Glu (PEG-PS) OA1 (1.6 g) under argon atmosphere. Transferred to flask containing. The resin was left undisturbed for 2 hours with occasional vibration . The resin was filtered through a sintered glass funnel with 0.5% diisopropylethylamine and Wash with a solution of sodium diethyldithiocarbamate (0.5% w / w) in DMF and touch The medium was removed. Dry resin Fmoc-L-Glu (PEG-PS) OH (1.5 g) was added to DMF solution (10 ml) under argon atmosphere. Suspended. TBTU (129 mg, 0.4 mmol) and NH_TwoBoroBpgPin (165mg, 0.5mmol) w Added to the reaction mixture. After stirring for 5 minutes, triethylamine (40 mg, 0.4 mmol) and the flask was left overnight with stirring. Resin Washed with chloromethane, methanol and dichloromethane and dried under reduced pressure.   H-Phe-L-Glu (PEG-PS) NHB by solid phase chemistry on a Milligen9050 peptide synthesizer oroBpgOPin was produced. Solid support Fmoc-L-Glu (PEG-PS) NHBoroBpgOPin from 20 The Fmoc group was removed using a solution of% piperidine in DMF. Releases Fmoc-Phe-OPfp Couple at the end of N. Dilute the protected peptide resin with dichloromethane, methanol, Washed with chloromethane and then dried under vacuum.   Peptide cleavage from the resin was achieved by treating the resin with TFA for 2 hours. T The FA was removed and the free peptide H-Phe-Glu-NH-BoroBpgOPin was precipitated with cold dry ether. Generated. The crude peptide was collected by filtration and washed with a portion of ether.   Purification of the crude peptide was performed using a VydacC-18 preparative column (TP silica: particle size 10 mm, (25 mm × 30 mm) using reverse phase HPLC. Straight column 30% to 90% A solvent (0.1% TFA aqueous solution) and B solvent (0.1% TFA acetonit Eluate). Monitor column eluate at 230 nM and filter fractions I collected it. The purity of the product was determined by analytical-HPLC and mass spectrometry. product H-Phe-Glu-NH-BoroBpgOPin was obtained in a yield of 17% and 34 mg. ES-MS: 626 [M + Na]; retention time analysis HPLC (4 × 250 mm, Vydac, C-18 techsphere), 0.1% TFA Eluted with water containing 10% to 60% MeCN containing 0.1% and 0.1% TFA for 25 minutes or more, and Rt 23. One minute was obtained. 4.Boric acid adhesion to Maryfield resin a. With a protected diol (2,2-dimethyl-1,3-dioxolan-4-methanol) Derivatization of resin  Under argon gas, Na (solid, 8 g, was converted to 2,2-dimethyl-1,3-dioxolane). -4- Add methanol (240 ml) and stir the mixture until a clear solution did. And Merrifield resin (Sigma, 1.1MeQ, Cl pergram, 20g) In addition, the mixture was stirred overnight and then heated at 80 ° C. for 24 hours.   The derivatized resin was collected by filtration, 1,4-dioxane (1 L), water (3 × 50). 0 ml), MeOH: water (1: 1, 3 x 500 ml), Me0H (3 x 500 ml), dry ether (3 x 500 ml). 1.5 to 2 mg of the resin is KBr (dry, 300 m g) powdering gives the infrared part of the spectrum, compresses it to disk and then n ™ 1600 Fourier Transform I.R. Induction resin (Fig. 2) is Melif Compared to the field resin (Fig. 1), the frequency of ether stretching characteristic of And clear stretch signal 1050-1150cm^-1(s) represents an alkyl-alkyl 1060-1150cm^-1(s) shows dialkyl ether stretching . b. Deprotection   HCl (1.5 M, 250 ml) and 1,4-dioxane (250 ml) were added to the derivation resin. Mix and the suspension was stirred and heated at 80 ° C. After 72 hours, the resin was washed with water (500 m l), Me0H (500 ml), DCM (500 ml), Et_TwoWash with O (500ml) and in air Dried. F. of resin t. -I. The R spectrum shows a clear O-H stretching frequency of 3400-3. 550cm^-1(s), the main peak is 3413.6 (FIG. 3), 917.6cm^-1Substantially larger than the signal at Ether in comparison 2) and Merrifield resin is 3400 cm weak against background moisture^-1Sig It just shows a null. c. Reaction of derivatized resin with boric acid   A diol resin (5 g, 5.5 mmol / diol) was added to a THF solution (dry 500 ml) and Phenylboric acid (3.35 g, 27.5 mmol, 5 eq) and 4 sieve (150 ° C) (Dried with). After stirring overnight under argon, the resin was stirred under argon. , Filtered in a closed system, washed with a THF solution (500 ml) and dried under reduced pressure. F. t . -L. R. (FIG. 4) is 1026 cm for the phenyl ring.^-1(Allyl-alkyl extension Strong signal and 3417 cm^-1Weak signal (for starting diol , FIG. 3).   Ref. Leznoff, C.C. and Wong, J.Y., The use of Polymer Supports in Orga nic Synthesis III. Selective Chemical Reactions on One Aldehyde Group of  Sylmmetrical Dialdeydes, Can. J. Chem., 193.51.5. 3756-3764.Analysis and activity data   Table 1 below shows activity data for the present invention. In the table, "Z" is benzoylo Xycarbonyl, "NHir" means normal pyrudine. “NHir49-64 (de s-S) "means the amino acid sequence from amino acid 49 to amino acid 64 of normal hirudin. , Where the wild Tyr (OSO_ThreeH)⁶³Is replaced by Try.   The compounds in Table 1 were prepared by a method for preparing the compound of Example 1 or 2 or a similar method. And the intermediates were obtained from appropriate sources.   The following is a method used for activity measurement.Plasma thrombin time (TT)   Add 150 μl of citrated normal human blood and 20 μl of buffer or sample to 37 Warm at 0 ° C. for 1 hour. 150 μl of newly prepared bovine thrombin (5 NIHu / ml salt Water) was added to start coagulation and the coagulation time was recorded.   Phosphate buffer containing 0.1% bovine serum albumin and 0.02% sodium azide The solution, pH 7.8, was used. Samples were dissolved in DMSO and diluted with buffer. Inhibition If no agent is used, add DMSO to the buffer to the same concentration as used in the sample. I got it. Inhibitor concentration plotted against thrombin time on a semi-log paper, The concentration of the inhibitor that doubled the time (40 seconds) was measured.Measurement of Ki   Inhibition of human α-thrombin was detected at three different concentrations of the chromogenic substrate S-2238. It was determined by elemental catalyzed hydrolysis.   200 μl of sample or buffer and 50 μl of S2238 at 37 ° C. for 1 minute Incubate and add 50 μl of human α-thrombin (0.25 NIH μ / ml) Was. The onset times of inhibition and non-inhibition were recorded at 4.5 nm. Lin optical density increase It plotted by the eweaver and Burke method. Km and apparent Km are measured Ki was calculated from the relationship.  0.1 sodium phosphate, 0.2 M NaCl, 0.5% PEG and 0.02% The pH was adjusted to 7.5 with orthophosphoric acid using a buffer containing sodium azide.   The specimen consists of a compound dissolved in DMSO.   For Ki measurement, see Dixon, M .; and Webb, E.C., "Enzymes", third edition 1979, Academic Press, which is hereby incorporated by reference. obtain.n / e = no effect n / e11.7 = No effect up to 11.7 μM concentration N.T. = not tested

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AL, AM, AT , AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, F I, GB, GE, GH, HU, IL, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, M X, NO, NZ, PL, PT, RO, RU, SD, SE , SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Green, Donovan             United Kingdom, e17.6Ei, Rondo             , Walthamstow, Gloucester Row             No. 50 (72) Inventor Skordalaquez, Emmanuel             United Kingdom, S14.5 Lyu, Ron             Don, Telegraph Hill, Waller Lo             No. 166 (72) Inventor Scully, Michael Fimber             United Kingdom, CM 11.2 N             Essex, Clay's Hill, Douglas             House (72) Goodwin, Christopher Andri             View             UK, B.A.2 SP, A             Bonn, Bass, Odd Down, Old             Fosse Road 67 (72) Inventor Kaka, Vijay Beer             United Kingdom, S.O.3.6 HN, Ha             Nmpshire, Warsash, Brooke             Avenue, Humbles Edge

Claims (1)

[Claims] 1. The following steps to create the target amino acid sequence: (i) providing a solid phase coupled to a functional group; (ii) selectively reacting it with a compound reactive with the functional group, the reacted compound being an amino group Or (iii) having a functional group capable of reacting with a carboxyl group or a reaction derivative thereof; (iii) the amino acid or carboxyl group of the terminal amino acid of the target amino acid sequence (which may also be in the form of a reaction derivative thereof), Reacting a functional group; (iv) coupling a sequencely preceding amino acid coupled to the solid phase with an amino acid sequencely succeeding in the target sequence to said preceding amino acid; (v) optionally performing step (iv). (Vi) cleaving the solid phase-linked compound produced using steps (i) to (iv) from the solid phase by the action of an acid or a base; of containing B (OH) _2] Objects or how the ester or derivative thereof, prior to cleavage from the solid phase, characterized in that incorporated into the solid-phase bound compound, to produce a peptide or peptide-containing compound. 2. In step (ii), reacting the amino group or selectively derived carboxy group of the amino acid with a functional group directly or indirectly coupled to a solid phase, for example as part of a conventional solid phase peptide synthesis. The method of claim 1, comprising: 3. The compound containing a boric acid group is an amino acid boric acid, a peptide boric acid or any boronate ester, and the step (ii) is performed by reacting the amino acid or the peptide boric acid or the ester with a diol coupled to a solid phase. Linker, part of the formula: (R ′ is a natural or unnatural amino acid, or a homolog thereof, wherein the homolog is an amino and / or substituted amino group optionally substituted with another functional group capable of forming a bond other than the natural peptide bond. 2. The method of claim 1, comprising forming a solid phase coupled thereto via the compound (having an alpha hydrogen atom). 4. The method comprises: (i) providing a solid phase coupled to an alcoholic hydroxy group; (ii) reacting the amino acid boric acid or peptide boric acid with the hydroxy group, thereby esterifying boric acid residues to the solid phase; iii) selectively reacting the carboxyl group of the amino acid sequence following the peptide boric acid or boronate ester in the final product with the amino group of the sequence preceding amino acid coupled to the solid phase; (iv) step (iv) (iii) repeating as many times as necessary; (v) cleaving the resulting peptide boronate from the resin; optionally, the method further comprises one or more steps of preparing the compound described above. A method for producing a compound containing a peptide boric acid or a peptide boronate ester, comprising the steps of: 5. Wherein each amino acid coupled to the solid phase has a protected amino group, step (iii) comprises deprotecting the amino group of the sequence preceding amino acid, and / or 5. The method according to claim 4, which is performed by a transesterification reaction. 6. Use of an amino acid or peptide boric acid or boronate ester in the solid phase synthesis of a peptide-containing compound. 7. 7. Use of boric acid residues attached to the solid phase via hydroxy residues in solid phase synthesis The method comprises: (i) providing a solid phase coupled to an alcoholic hydroxy group; (ii) reacting boric acid or a boronate ester with the hydroxy group, thereby esterifying boron residues to the solid phase; (iii) Carrying out one or more steps for the production of the above compound; and a method for producing a compound containing a boron atom. 9. Solid phase material coupled to boric acid residue via a hydroxy group. 10. The part of the following formula: Where R is a residue that binds to boron and the oxygen through which the moiety is attached to the solid phase binds directly to an optionally substituted alkylene residue or selectively to a carbonyl group. Solid phase material coupled to 11. (a) a catalytic site directing moiety (CSDM) that binds to and inhibits the active site of serine protease; (b) an external site-related moiety (EAM); and, optionally, (c) binds EAM to CSDM A dual-functional serine protease, which includes a connector moiety that binds to the CSDM and EAM molecules simultaneously with the serine protease molecule, except that the inhibitor is not thrombin. 12. CSDM is Arg, Lys or a homologue thereof, or a hydrophobic P1 residue and 12. The inhibitor of claim 11, which has a (P4) P3P2 residue selected from: 13. EAM is below 13. The inhibitor according to claim 11, which is an amino acid sequence selected from the group consisting of: 14． Wherein the connector moiety is -λ-σ-, where λ is the residue of a non-peptide linker of the formula HO ₂ (CH ₂ ) _h CO ₂ H, h is 2-6, and σ is at least two The peptide spacer containing adjacent Gly residues). 15. The following steps to create the target amino acid sequence: (i) providing a solid phase coupled to a functional group capable of reacting with an amino group or preferably a carboxyl group or an active derivative thereof; (ii) the terminal of the amino acid sequence of the compound of the invention Selectively reacting the amino or carboxyl group of the amino acid with said functional group, wherein the carboxyl group is selectively in the form of its active derivative; (iii) in the target sequence the amino acid preceding the sequence coupled to the solid phase 15. A method for producing the inhibitor of claims 11-14, comprising: coupling an amino acid that continues in sequence to the preceding amino acid; and (iv) repeating step (iii) as many times as necessary. 16. The method further comprises the step (v) of coupling the amino acid sequence following in step (iii) to the preceding amino acid in this step via a compound having two functional groups capable of reacting with an amino group. Whereby one of the functional groups binds to the amino group of the preceding amino acid and the other to the amino group of the subsequent amino acid). 17． 15. The terminal amino acid responsive to the functional group attached to the solid phase is the C-terminal amino acid of EAM, and step (iii) is repeated to couple the continuation amino acid of the EAM sequence with the continuation amino acid of the adjacent connector peptide. Or 15 methods. 18. The final amino acid of the non-interfering amino acid sequence coupled to the solid phase reacts with the compound having two carboxylate groups or a reactive derivative thereof, and the unreacted carboxylate or the carboxylate derivative is an N-terminal amino acid of CSDM. 18. The method of claims 15-17, which typically reacts with an amino group. 19. 19. The method of claim 18, wherein said N-terminal amino acid is already associated with a residue of CSDM, wherein CSDM has a heteroatom instead of a C-terminal carboxy group. 20. Pharmaceutical formulation comprising the inhibitor of claims 1-4 formulated for use as a human or veterinary medicament (the formulation optionally contains a pharmaceutically acceptable diluent, excipient or carrier) ). 21. Treating a disease or disorder treatable by inhibition of serine protease, comprising administering a therapeutically or prophylactically effective amount of the inhibitor of claims 1-4 to a patient or animal orally or parenterally. How to treat, either preventively or prophylactically.