AU729393B2 - Serine protease inhibitors - Google Patents

Description

WO 98/00442 PCT/GB97/01574 1 SERINE PROTEASE INHIBITORS This invention relates to serine protease inhibitors and substrates, as well as to the synthesis of such compounds and novel methods and materials for synthesis of boroncontaining compounds.

Modulation or inhibition of serine protease inhibitors is useful inter alia to prevent thrombosis.

The family of serine protease enzymes cleaves peptide bonds by a mechanism involving the catalytic triad of Asp-His-Ser residues in the active site of the enzymes.

Serine protease inhibitors have been designed which use functional groups to interact with the triad and thereby block activation of the enzyme substrates. It can be desirable to make inhibitors selective for one target protease. Discussion of the prior art relating to peptide inhibitors can be found in the specification of a UK patent application entitled "Thrombin Inhibitors" filed on the same date as this application and in PCT/GB96/00352. A copy of the specification of the application entitled "Thrombin Inhibitors" is filed herewith. The content of this application does include the subject matter of the application entitled "Thrombin Inhibitors", whose specification the skilled reader may wish to consult, along with various prior documents referred to in that specification. The "Thrombin Inhibitors" specification does not physically form part of the published specification of this application.

Certain serine proteases are known to have a second site or "exosite" for binding to an anionic portion of the substrate. This exosite is often called the anion binding exosite The amino acid residue which provides the carbonyl group of the scissile bond of a serine protease substrate is designated Successive amino acid residues on the Nterminal side of residue PI are designated P2, P3, P4 etc; amino acid residues on the C-terminal side of residue PI are designated PI P2', P3' In fibrinogen, PI is glycine and P2' is proline. The protease contains a "specificity pocket" which I f, 2 to the synthesis of bifunctional serine protease inhibitors comprising: a catalytic site-directed moiety (CSDM) which binds to and inhibits the active site of a serine protease; an exosite associating moiety (EAM); and, optionally, a connector moiety bonded between the EAM and the

CSDM,

the CSDM and the EAM being capable of binding simultaneously to a molecule of the serine protease.

Peptide boronates are a well established class of compounds which have hitherto been made by solution chemistry. Thus, peptide serine protease inhibitors are known in which the C-terminal carboxy group is replaced by a boronic acid group or a derivative thereof. Representative compounds are of the Formula II: (aa)k-B(R 2

(II),

wherein: 25 (aa)k represents a sequence of amino acids (eg as in Formula R 2 and R 3 are each independently selected from halogen, -OH, OR 4 and -NR 4

R

5 where R 4 and R 5 are each independently a group of the formula R wherein us is 0 or 1; R 6 is H or an optionally halogenated alkyl, aryl 30 or arylalkyl group containing up to (10 u) carbon atoms and optionally substituted by one or more groups selected 7 7 7 from -OH, R (CO)vO- and wherein v is 0 or 1; R 7 is Ci-C 6 -v alkyl, or is an aryl, alkylaryl, arylalkyl or alkylarylalkyl group containing up to (10-v) carbon atoms, or R 2 and R 3 taken together represent a residue of a diol Sor a dithiol.

Hs\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 3 Such peptide boronates are described, for example, in WO 92/07869 (equivalent to USSN 08/317,387), EP 0471651 (which corresponds to US 5288707) and USSN 08/240,606, the disclosures of which are incorporated herein by reference.

We have now surprisingly found that boron-containing peptides may be synthesised by solid phase chemistry without serious degradation. One aspect of the invention, therefore, is the use of a boron-containing amino acid analogue in peptide synthesis using solid phase chemistry, especially Fmoc chemistry (also known as the "Sheppard approach").

In another aspect, there is provided a method of making a peptide or a peptide-containing compound, comprising performing the following steps to make a target amino acid sequence: providing a solid phase having coupled thereto functional groups; (ii) causing a compound reactive with the .2 functional groups selectively to react therewith, the 25 reacted compound having a functional group capable of reacting with an amino group or with a carboxyl group or a reactive derivative thereof; (iii)causing an amino or carboxyl group (which 0g"" 30 may be in the form of a reactive derivative thereof) of a terminal amino acid of a target amino acid sequence selectively to react with said functional groups of the reacted compound; 35 (iv) coupling the amino acid sequentially following, in the target sequence, the sequentially preceding amino acid coupled to the solid phase to said H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 4 preceding amino acid; repeating step (iv) as often as necessary; and (vi) cleaving a solid phase-lined compound prepared using steps from the solid phase by the action of an acid or base, characterised in that a compound comprising a boronic acid group [-B(OH) 2 or a derivative thereof, especially an ester, is incorporated in the solid phase-linked compound prior to its cleavage from the solid phase.

A particular method of using peptide boronate esters in solid phase chemistry is a completely novel technique in which boronic acids [-B(OH) 2 are directly esterified onto diols coupled to a resin. Chain extension is continued from the amino group of the amino acid by, for example, standard Fmoc chemistry. The boronic acid ester is cleaved from the resin by acid TFA) to give the peptide boronate [peptide-B(OH) 2 or by transesterification, for example, by concentrated solution of a hindered diol, such as pinanediol, for example.

*e As used herein, "natural" amino acid means an L-amino acid (or a residue thereof) selected from one of the twenty common or "standard" a-amino acids found in proteins.

By "unnatural" amino acid is meant any a-amino acid (or residue thereof) other than the twenty "standard" amino acids. Unnatural amino acids therefore include the Disomers of natural L- amino acids and amino acids having side chain protecting groups.

The prefixes and are used as normal to indicate amino acids of D- or L- configuration respectively. A "D, H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 5 prefix indicates a racemic mixture whilst the absence of a prefix indicates that the amino acid can be of either D- or L- configuration, except in the examples where residues are of L-configuration unless otherwise states.

For those groups of unspecified configuration in the text which can be of D- or L- configuration, L- configuration is preferred.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", means "including but not limited to", and is not intended to exclude other additives, components, integers or steps.

Abbreviations and terms prefixed by "boro" indicate amino acids where in the terminal carboxyl group -CO 2 H has been replaced by a boron functionality.

Brief Description of the Drawings Figure 1 is a Fourier Transform Infra Red spectrum of a Merrifield resin Figure 2 is an spectrum of the same resin after reaction with sodium 2,2-dimethyl-1,3- 25 dioxolane-4-methanolate.

Figure 3 is an spectrum of the reacted resin after treatment with HC1 to deprotect the hydroxy 1* groups of the dioxolane.

Figure 4 is an spectrum of the 30 deprotected resin after reaction with phenylboronic acid.

o Considering now the inventive processes in more detail: Solid phase synthesis is a technique familiar to peptide chemists and detailed elucidation is therefore not required here. An introduction to the technique may be found in "The Chemical Synthesis of Peptides", John Jones, Clarendon H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 6 Press, Oxford, England, 1991. The principle of conventional solid phase synthesis is that an amino acid or peptide coupled to a solid phase is reacted with an amino acid which is protected against reaction with itself and, after coupling with the solid phase-linked amino acid, is deprotected for reaction with a further amino acid protected against reaction with itself. These steps are repeated as often as necessary.

One solid phase synthesis technique is the Fmoc technique (Fmoc fluorenylmethylcarbonyl). In Fmoc chemistry (also known generally as the 'Sheppard approach'), the carboxy terminus of a peptide (or an amino acid) is coupled to a resin bead via a linker which is terminated by a reactive function. The resin bead itself is typically polystyrene though other solids have been used that have suitable swelling characteristics in solvent, since it is now known that the peptide chain grows in the pores on the inside of the bead. An example of an alternative solid is the polyamide called Kiesulguhr.

The linker can be many things, but we prefer to use PEG a polyethylene glycol linker), which has an alcohol 25 function.

The terminus of the linker, typically called a 'handle', depends on the desired product, but for Fmoc chemistry will be a moiety such that it can finally be cleaved by acid.

The most common terminus (which we have used) is HMBA or 30 para-hydroxymethylbenzoic acid linker. The HMBA is esterified onto the PEG, and then the peptide or amino acid (with Fmoc on its N-terminus) is reacted to give also an ester link to the HMBA. The ester links are then cleavable by acid. The Fmoc protecting group is base labile and 35 typically removed by a secondary base piperidine) and the resulting free amino group is reacted with a selected RAJ- Fmoc-protected amino acid; the amino acid sequence is H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 7 extended by repetition of these steps.

Another solid phase synthetic approach is the Boc technique (Boc tertiarybutyloxycarbonyl). The resin used in Boc chemistry (also known more generally as the Merrifield method) is often divinylbenzyl based, for instance a 'Wang' resin has chloromethyl benzene co-polymerised to 2% divinylbenzene. The chloromethyl benzene group is reacted with an amino acid or peptide whose amino group is protected by Boc, to give a link to the resin. The link to the resin is typically cleaved (very carefully!) by dry, liquid HF.

This is described as 'vigorous' acidolysis. The Boc protecting group is acid labile and typically cleaved by TFA, prior to reaction of the resultant free amino group with a selected Boc-protected amino acid; as with Fmoc chemistry, the amino acid sequence is extended by repetition of these steps.

The two classical methods of solid phase peptide synthesis (Sheppard and Merrifield), therefore, involve coupling amino acids via their carboxy-termini or their derivatives to a solid resin particle, then sequentially coupling new amino acids (via their activated carboxy termini) to via the N-termini generated.

Alternatively recent reports have shown coupling to the resin via the N-termini, for example via an acid labile benzyloxycarbonyl linkage, subsequent liberating of the 'carboxy termini, activating of these and coupling of amino acids via their N-termini, the carboxy termini of the amino acids temporarily being protected. (Sharma, Jones, Broadbridge, Corina, D.L. and Akhtar, M. A Novel Method of Solid Phase Synthesis Of Peptide. Analogues, in Innovation and Perspectives in Solid Phase Synthesis, ed., R.Epton, 1994, Mayflower Worldwide Limited, Birmingham, page 353-356; Letsinger, R.L. and Kornet, M.J.

1 RA4- J.Amer.Chem.Soc., 1963, 85, 3045.) H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 8 The invention provides a method of making a peptide or a peptide-containing compound, comprising performing the following steps to make a target amino acid sequence: providing a solid phase having coupled thereto functional groups; (ii) causing a compound reactive with the functional groups selectively to react therewith, the reacted compound having a functional group capable of reacting with an amino group or with a carboxyl group or a reactive derivative thereof; (iii) causing an amino or carboxyl group (which may be in the form of a reactive derivative thereof) of a terminal amino acid of a target amino acid sequence selectively to react with said functional groups of the reacted compound; (iv) coupling the amino acid sequentially following, in the target sequence, the sequentially preceding amino acid coupled to the solid phase to said preceding amino acid; repeating step (iv) as often as necessary; and (vi) cleaving a solid phase-linked compound prepared using steps from the solid phase by the action of an acid or base, characterised in that a compound comprising a boronic acid group [-B(OH) 2 1 or a derivative thereof, especially an ester, is incorporated in the solid phase-linked compound 35 prior to its cleavage from the solid phase.

The method of the invention for making a peptide or H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 9 peptide-containing compound may comprise a step of coupling a peptide to a solid phase-linked compound prepared by previous steps of the process, optionally as a step (iv) of the process a step (iv) of the process in which the sequentially following amino acid is part of a peptide].

The method of the invention for making a peptide or peptide-containing compound may comprise a step of coupling to a solid phase-linked compound prepared by previous steps of the process a compound other than an amino acid or peptide, such as, for example, a compound having two carboxyl groups for use in linking an amino group of a solid phase-linked peptide with an amino group of a peptide, peptide analogue or amino acid in the liquid phase. Of course, any other liquid phase compound capable of reacting with an amino group or, as the case may be, a carboxyl group could thus be linked to the solid phaselinked peptide. Diamines are useful for interconnecting moieties having carboxyl groups in the form of reactive derivatives thereof). Extension of a solid phaselinked peptide by a dicarboxylic acid, especially glutaric acid, is useful in the preparation of bifunctional serine protease inhibitors having a CSDM joined, typical through its N-terminal, to an EAM through a connector moiety 25 comprising a peptide spacer portion and dicarboxylic acid residue linker portion.

A solid phase-linked compound whose free end terminates with a dicarboxylic acid residue may be further extended by reaction of the free carboxyl residue (optionally in the form of a derivative thereof) with the amino group of an amino acid, e.g. a dicarboxylic acid residue coupled to a solid phase-linked EAM-spacer moiety may be reacted with an amino acid of a CSDM, for example the N-terminal amino acid 35 of a CSDM.

Step (ii) of the method may comprise reacting an amino A\ Step (ii) of the method may comprise reacting an amino H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 10 group or an optionally derivatised carboxy group of an amino acid with a functional group coupled directly or indirectly to a solid phase, as part of conventional solid phase peptide synthesis, for example. The amino or carboxy group coupled to the solid phase is often the terminal amino or carboxy group but in some embodiments is a functional group of a side chain, such as the side chain carboxyl group of glutaric acid, for example; the Cterminal carboxy group of the amino acid attached to the solid phase through its side chain may be replaced by the boronic acid residue [-B(OH) 2 or an ester thereof.

The method may comprise N-terminal coupling in SPPS, in which the carboxy terminus of a resin bound peptide is coupled to a free a-aminoboronate acid or ester, prior to acid cleavage of the resultant product from the resin.

In-another embodiment, step (ii) comprises reacting an amino acid or peptide boronic acid or ester

OH

aak-B (Ilia)

OH

or O-E' aak-B (IIIb) 2 where E and E 2 represent boronic ester-forming residues or may together form a single residue, with a diol coupled to the solid phase. The technique of linking a boron atom as part of an amino acid or peptide boronic acid or ester) to a solid phase through hydroxy groups coupled (directly or indirectly) to the solid phase is novel and forms an aspect of the invention.

If the compound comprising a boronic acid or ester is an CO 447 amino acid boronate used in step (ii) in either of the H:\Bkrot\Keepxspeci\30425-97-2.doc 14/04/00 11 preceding embodiments, the solid phase synthetic method may be used to make a peptide boronate inhibitor of a serine protease catalytic site, optionally.in the synthesis of a bifunctional serine protease inhibitor.

Thus, boronic acids can be directly esterified onto diolcontaining resins, and then chain extension continued from the N-terminal end by, for example, standard Fmocchemistry. Subsequently the boronic acid ester can be cleaved, either by mineral acids, to give the free boronic acids [peptide-B(OH) 2 or by transesterification, e.g. by a concentrated solution of a diol, especially a hindered diol such as pinanediol, for example.

The literature describes ways of preparing diol-containing solid phase resins, which can be derivatised by aldehydes and are suitable also to be derivatised by boronic acids/esters McArthur,C.R and Leznoff,C.C.

'The monoblocking of symmetrical Diketones on Insoluble Polymer Supports', Can.J.Chem., 1993, 61,1405-1409. and Leznoff,C.C. and Sywanyk,W. 'Use of Polymer Supports in Organic Synthesis.9. Synthesis of Unsymmetrical Caretenoids on Solid Phase', J.Org.Chem., 1997, 42, 3203- 3205).

A general procedure is as follows: eoee* o°° o**oo *~o *oo H:\Bkro\Keep\speci\3425-97-2.doc 14/04/00 12 SLinker Diol (protected) Wang" Resin) Linker Diol(protected) Resin 1) Deprotect N Linker Diol Resin 1) XN-CHR-B(OH) 2 orXN-CHR-BO 2 -Ester SLinker Diol B-CHR-NX 1) remove 2) couple new amino acid Linker Diol B-CHR-NHCO-(aa)Y 3 0 repeat steps of SPPs B-CHR-NH-Peptide-Y 1) Base 2) Lewis acid TFA), scavenger 3

HO

SB-CHR-NH-Peptide

HO

.H•

The diol is a compound having two or more alcoholic hydroxy 40 groups.

X and Y are protecting groups.

R is the side chain of an amino acid boronate/boronic acid.

Typically, the resin is washed after each step. In suitable embodiments the diol is not protected before it is reacted with the resin.

H:\Bkrot\Keep\speci\3042597-2.doc 14/04/00 13 A more specific procedure is set out below: Rsn

CH

2 -C1 Na+O 0> 0 Wang" Resin) (Sodium salt of Solketal) 1) 24h, 2 fold excess sodium salt 0V 2) wash Resin 20 Oi) mineral acid 2) wash

OH

H

2 Resin

OH

I1) (TM S) 2

-N-CHR-B(OH)

2 o(TMS) 2 -N-CHR-B0 2 -Ester 2) wash off excess

H

2

OH\

Resin H20

OH

I1) Fmoc(aa)-OH, I-BuCOCL, Morpholine, Et 3

N

or Fmoc(aa)-OH, activating agent, (Bu) 4

NF

2 -OH\1 2) wash Resin

OH

reetsteps of SPPS Resin _H 2 OHB-CHR-NH -PeptideFmoc 1) Base HO~ 2) Lewis acid TFA), scavenger B-CHR-NH-Peptide %see:~HO110 H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 14 TMS Trimethylsilyl SPPS solid phase peptide synthesis The invention therefore opens the way to the preparation of peptide boronates by solid phase chemistry, for example in preparing a library of peptide boronates by a combinatorial method.

The invention includes a method for making a compound comprising a peptide boronic acid or peptide boronate ester, the method comprising: providing a solid phase having coupled thereto alcoholic hydroxy groups; (ii) causing an amino acid boronic acid or peptide boronic acid to react with the hydroxy groups whereby the boronic acid residue becomes esterified to the solid phase; (iii) causing the carboxyl group of the amino acid sequentially following, in the end product peptide boronic acid or boronate ester, selectively to react with the amino group of the sequentially preceding amino acid coupled to 25 the solid phase; (iv) repeating step (iii) as often as necessary; cleaving the resultant peptide boronate from the 30 resin, the method optionally comprising one or more further steps to make said compound.

35 The alcoholic hydroxy groups coupled to the solid phase are preferably arranged such that pairs of the groups can be r/ "bonded to a boron atom, i.e. such that a boron atom can be HA\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 15 di-esterified by them:

B-

-0o In some embodiments the hydroxy groups are in a 1,2arrangement on adjacent carbons); in other embodiments they are spaced apart on chains a residue of NH(CH 2

CH

2 0H) 2 Preferably each amino acid coupled to the solid phase has a protected amino group and step (iii) comprises deprotecting the amino group of the sequentially preceding amino acid.

Preferably the cleavage of step is performed with acid or by transesterification.

More generally, there is provided the use in solid phase synthesis of a boronic acid residue attached to the solid phase through hydroxy group residues.

Also provided is a method for making a compound comprising a boron atom, the method comprising: providing a solid phase having coupled thereto alcoholic hydroxy groups; (ii) causing a boronic acid or boronate ester to react with the hydroxy groups whereby the boronic acid residue "becomes esterified to the solid phase; and (iii) performing one or more further steps to make said compound.

The alcoholic hydroxy groups are preferably arranged as described above.

In other aspects, there are provided a solid phase material .c having coupled thereto boronic acid residues through SH:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 16 hydroxy groups, as well as a solid phase material having coupled thereto a moiety of the Formula IV: 0

B-R

0

(IV)

wherein R is a residue bonded to the boron atom, and is usually an organic moiety. Residue R is in one class of material free of functional groups reactive with alcoholic hydroxy groups (but the material may contain such functional groups in protected form, e.g. prior to deprotection). In another class of material, such functional groups are unprotected, protecting groups having previously been removed. R is typically an organic moiety having one or more functional groups to enable R to undergo a chemical reaction; any protectable functional groups may be protected. In one class of materials, the solid phase has coupled thereto a moiety of Formula V: :-C-0 *e H2

B-R

0

(V)

0*oo0 One or both of the hydrogen atoms of the -CH2- group may be 30 replaced by other groups compatible with the use of the material, e.g. alkyl groups (for example methyl or butyl).

In a yet further class of materials, the left hand oxygen of Formula IV is part of an ester.

H:\Bkrot\Keep\speci\3O425-97-2.doc 14/04/00 17 A first class of solid phase synthetic methods comprises performing the following steps to make a target amino acid sequence: providing a solid phase having coupled thereto functional groups capable of reacting with a carboxyl group; (ii) causing a compound reactive with the functional groups and comprising an amino group protected by a baselabile protecting group to react with the functional groups to form an acid labile bond; (iii) deprotecting the amino group with a base; (iv) causing the carboxyl group of an amino acid whose amino group is protected by a base-labile protecting group to react with the deprotected amino group resulting from step (iii); deprotecting the protected amino acid with a base; (vi) causing the amino acid sequentially following, in the target sequence, the sequentially preceding amino acid coupled to the solid phase to react with the deprotected amino group of the sequentially preceding amino acid, the sequentially following amino acid having its amino group 25 protected by a base-labile protecting group; (vii) deprotecting the protected amino acid group with o: base; (viii) repeating steps (vi) and (viii) as often as necessary; and (ix) cleaving the acid labile bond with acid or by transesterification,

S

characterised in that a compound comprising a boronic acid group [-B(OH) 2 or a derivative thereof, especially an 35 ester, is incorporated in the solid phase-linked compound a prior to cleavage of the acid labile bond.

H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 18 As described above in relation to the method of making a peptide or peptide-containing compound, step (ii) of the method may comprise reacting an optionally derivatised carboxy group of an amino acid with a functional group coupled directly or indirectly to a solid phase, for example by a method known per se in solid phase chemistry.

The amino acid may be the compound comprising a boronic acid or ester group, i.e. an amino acid boronic acid or ester. Alternatively, step (ii) may comprise reacting the compound comprising a boronic acid or ester group in the form of an amino acid or peptide boronic acid or ester with a diol coupled to the solid phase. In either case that an amino acid (or peptide) boronic acid or ester is used, the process is suitable for making a peptide boronate inhibitor of a serine protease catalytic site, optionally in the synthesis of a bifunctional serine protease inhibitor.

The other variants described above of the method of making a peptide or peptide-containing compound are applicable to said first class of solid phase synthetic methods.

A second class of solid phase synthetic methods comprises performing the following steps to make a target amino acid sequence: providing a solid phase having coupled thereto functional groups capable of reacting with a carboxyl group; (ii) causing a compound reactive with the functional groups and comprising an amino group protected by an acido0o0 labile protecting group to react with the functional f groups, to form a base labile bond; (iii) deprotecting the amino group with an acid; (iv) causing the carboxyl group of an amino acid whose 35 amino group is protected by an acid-labile protecting group to react with the deprotected amino group resulting from Sstep (iii); 1:\Bkrot\Keep\speci\30425-97-2 .doc 14/04/00 19 deprotecting the protected amino acid with an acid; (vi) causing the amino acid sequentially following, in the target sequence, the sequentially preceding amino acid coupled to the solid phase to react with the deprotected amino group of the sequentially preceding amino acid, the sequentially following amino acid having its amino group protected by an acid-labile protecting group; (vii) deprotecting the protected amino acid group with acid; (viii) repeating steps (vi) and (viii) as often as necessary; and (ix) cleaving the base labile bond with base or by transesterification, characterised in that a compound comprising a boronic acid group [-B(OH) 2 or a derivative thereof, especially ester, is-incorporated in the solid phase-linked compound prior to cleavage of the acid labile bond.

The above-described variants of the first class of solid phase synthetic methods are applicable also to the second class.

S

o* o 25 As described above in relation to the method of making a peptide or peptide-containing compound, methods of the *000 first and second classes of solid phase synthetic methods may comprise a step of coupling to a solid phase-linked compound prepared by previous steps of the process a 30 compound other than an amino acid or peptide.

The invention may be applied to the synthesis of bifunctional serine protease inhibitors, as described below.

S*o** S A BI-FUNCTIONAL SERINE PROTEASE INHIBITORS HA\Bkrot\Keep>\speci\30425-97-2.doc 14/04/00 20 The bifunctional serine protease inhibitors comprise: a catalytic site-directed moiety (CSDM) which binds to and inhibits the active site of a serine protease; an exosite associating moiety (EAM); and, optionally, a.connector moiety bonded between the EAM and the CSDM, the CSDM and the EAM being capable of binding simultaneously to a molecule of the serine protease.

The Catalytic Site-Directed Moiety (CSDM) The catalytic site-directed moiety (CSDM) binds to and inactivates the catalytic site of a serine protease enzyme.

The structure of the CSDM is not critical to the invention.

It -may comprise the amino acid sequence of any known inhibitor of a serine protease catalytic site, for example.

One class of CSDMs is included in the following Formula I: 2. 3 25 wherein aa, aa 2 and aa 3 represent natural or unnatural 'acid residues and (aa 4 )m one or more optional amino acid *3 residues linked to the amino group of aa 3 Alternatively wheresidues in which the a-hydrogen is replaced by a substituent. The sequence of amino acids and/or amino acid analogues binds to the serine protease active site.

Suitable sequences are described later in this specification. X represents H or a substituent on the N- 0 "terminal amino group. Z is -COOH or a C-terminal extension group (carboxy replacement group), for example as known in 1the art. In preferred compounds Z is a heteroatom acid AV group, e.g. -B(OH) 2

-P(OH)

2 or PO(OH) 2 or a derivative H:\Bkrot\Keep\spci\30425-97-2.doc 14/04/00 21 thereof, for example a carboxylic acid ester, a dioxoboronate [-B(Osubstituent) 2 or a phosphate PO(Osubstituent) 2 or BF 2 Preferred heteroatom analogue groups are -B(OH) 2 and (OH) 2 a less preferred heteroatom analogue group is S(O) 2 0H. Amongst other possible Z groups there may be mentioned -CN, -COCH 2 C1 and

COCH

2 F. In preferred embodiments m is from 0 to 7 and more usually 0 to 5, e.g. 0, 1 or 2, especially 0. Normally, n=l.

In one class of compounds, the (aa2)-(aa l natural peptide linkage is replaced by another linkage Additionally or alternatively other natural peptide linkages may be replaced by another linkage.

Catalytic site inhibitors of serine proteases are well known in the art. A short review of serine protease inhibitors, i.e. inhibitors of the serine protease catalytic site, is to be found in EP-B-145441, which patent 20 discloses a class of serine proteases having a C-terminal boron group. Other patent specifications describing serine protease inhibitors include EP 293881, EP 471651 (equivalent to US 5288707), EP 235692, US 4963655 and WO 89/09612 (concerned particularly with inhibitors of Factor VII/VIIa in the [TF:VII/VIIa] complex).

For inhibitors of trypsin-like enzymes, preferred classes of P1 residues of CSDMs are Arg, Lys and their analogues, and (ii) hydrophobic residues; further description of preferred P1 groups for thrombin which are also for other trypsin-like enzymes may be found in the aforesaid specification entitled "Thrombin Inhibitors" and in Australian patent 707059 (derived from PCT/GB96/00352).

Chymotrypsin-like serine proteases bind preferentially to CSDMs having phenylalanine-like and alanine-like side chains on the P1 residue. The following table A indicates the most preferred (P4)P3P2 residues for eight particular C serine proteases: \\melb£iles\homeS\LJColes\Keep\Speci\ 3 0 4 2 5 9 7 2 .doc 23/11/00 22 Table A Enzyme Residue Sequence Thrombin D-Phe-/substituted D-Phe-/D-Dpa-/Dba-/Pms-/ a-Nal-/P-Nal-/TMSal-/Chg-/Phg-/D-Tiq-/paraether of D-Tyr-/NaSO 2 -Pro Factor Xa IleuGluGly, PyroGluGly, ArgGly, ChaGly, LeuArg Factor VIIa L-PhePhe, NalPhe, D-TiqPhe, NalThr, NalPhg Factor IXa ValVal Factor XIa K-BzlGluGly, Glu(OBzl)Ala, GlyArg, GlyLys Factor XIIa GlnGly Urokinase PhePro, GluGly Protein Ca LeuSerThr In each case, a preferred amino acid may be replaced by an analogue thereof.

The Exosite Associating Moiety (EAM) The exosite associating moiety (EAM) is a moiety which 10 binds to an exosite (ABE) of a serine protease. Thrombin has a well defined exosite to which this binds, in addition to a fibrinogen amino acid sequence C-terminal to the thrombin cleavage site, non-substrate ligands of thrombin such as hirudin. Hirudin sequences such as Hir 53 64 have 15 been used in bifunctional peptides named "hirulogs". The hirulogs are described in US 5196404; a further description of thrombin EAMs may be found in the aforesaid UK patent application entitled "Thrombin Inhibitors". EAMs for thrombin are often termed "anion binding exosite associating moieties" (ABEAMs).

The implications from the crystal structure of FXa (Padmanabhan K et al, "Structure of Human Des (1-45) Factor k Xa at 2.2A Resolution" J Mol.Biol 1993, 232, 947-966) are .o .o l o oa* o* H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 23 that the sequences 35-41 and 70-81, containing 8 acidic residues constitute a cation binding exosite. The natural polypeptide inhibitors antistasin and ghilanten contain the electropositive cation exosite associating sequences 'CRPKRKLIPR117 and 8 CKPKRKLVPR117 respectively.

It is clear that serine protease interactions in P' sites (C-terminal to the cleavage point) can be as important to specificity as those at P sites (for example Ding, L.; Coombs, Strandberg, Navre, Coreyu, D.R. and Madison, E.L. Origins of the Specificity of Tissue-type Plasminogen Activator, Proc.Natl.Acad.Sci. 1995, 92, 7627- 7631), and larger ligands need to be compared spanning both sites, as are available rapidly from library screening.

Recently (Lawson 1992) has shown that screening of peptide sequences, containing significantly P' as well as P binding units, allows detection of FVIIa/TF activity where existing substrates were too insensitive. Peptide libraries are showing great efficacy for screening for biological activity in a variety of applications (e.g Eichler, 1994) and are useful for the present invention.

The invention contemplates that sequences C-terminal to the .cleavage point of other serine proteinase substrates form 25 an EAM, as follows: *SSo

*S

*ooe H: \Bkrot\Keep\speci\30425-97-2 .doc 14/04/00 24 Table B Enzyme Natural Cleavage Site Substrate Factor Xa Prothrombin YIDGR--IVEGSDAEIGMSPWQ Factor Xa Prothrombin AIEGR--TATSEYQTFFNPRTFGS Factor Xa Factor VII SKPQGR--IVGGKVC Factor VIIa Factor X NLTRR--IVGGQECKDGEC Factor IXa Factor X NLTRR--IVGGQECKDGEC Factor XIa Factor IX SKLTR--AEAVFPDVDYVN Factor XIa Factor IX FNDFTR--VVGGEDAKPGQF Factor XIIa Factor XI KIKPR--IVGGTASVRGE Factor XIIa Plasma KTSTR--IVGGTNSSWGE Kallikrein Protein Ca Factor VIII ELR--MKNNEEAEDYDDDLTDSEMD t-PA/UK Plasminogen PKKCPGR--VVGGCVAHPHSWPWQVSLRT The Connector Moiety

S.

S

S p.

S

S

**Se

S

S

S

S

S

The compounds of the present invention may contain a connector moiety which interconnects the CSDM and the EAM, the connector moiety being capable of permitting the CSDM and the EAM to bind simultaneously to a molecule of the respective serine proteinase inhibitor. In the case of 10 thrombin, the connector moiety is bonded to the CSDM as an N-terminal extension or as or through a side chain thereof.

The connector moiety may be bonded to the CSDM either as a C-terminal extension or, alternatively, as an N-terminal 15 extension or as or through a side chain. However, if the compound is a thrombin inhibitor, the connector moiety may not be a C-terminal extension of the CSDM.

Especially if the connector moiety is an N-terminal extension of the CSDM, or if it is comprised in a side chain thereof, it desirably comprises an amino acid sequence containing at least two adjacent Gly residues, H,\Bkrot\JKeep\speci\30425-97-2.doc 14/04/00 25 e.g. at its N-terminal end. In one class of compounds the connector preferably comprises a peptide "spacer" and a nonpeptide "linker". A representative connector structure is: wherein represents a non-peptide linker and a a spacer comprising a sequence of amino acids, X and suitably being joined by a peptide bond. The spacer a is preferably linked to the EAM and the linker X to the CSDM, although compounds in which a is linked to the CSDM and X to the EAM form a less preferred embodiment included in the invention.

The linker is typically a residue of a compound having functional groups to react with the N-terminal amino group of the spacer and a functional group of the CSDM, such as the N-terminal group, for example. A preferred linker, therefore, has two carboxylate groups, e.g. is a dicarboxylic acid which can form amide bonds with the Nterminal amino groups of the CSDM and the spacer.

Particularly preferred linkers are a residue of glutaric acid (H0 2

C(CH

2 3

CO

2 H) and homologues thereof of the formula (HO2C(CH 2 )hCO 2 H) wherein h is an integer of 2 or from 4 to 6.

The alkylene residue [-(CH 2 2 6 -1 may be substituted by one 25 or more substituents which do not sterically hinder the linker, whereby the desirable flexibility of the linker is maintained.

Less preferably, the linker may comprise for example the 30 residue of another compound having two carboxyl groups whose carbon atoms are separated by from 2 to 6 atoms.

The amino acid sequence of the spacer is not critical to the invention but it preferably comprises at least two adjacent Gly residues, normally at its N-terminal end. The length of the spacer is dependent upon inter alia the position on the As-/ CSDM to which the linker is attached.

H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 26 A further description of connector moieties suitable for peptide thrombin inhibitors can be found in the aforesaid UK patent application entitled "Thrombin Inhibitors" filed on the same day as the present application.

The connector moiety may have one or more natural amide bonds replaced by other linkages.

SYNTHESIS

N-terminal coupling methods may be used in making the bifunctional inhibitors described above. In one embodiment the CSDM, including any directly attached amino acid(s), is synthesised by N-terminal coupling. This technique is especially useful if the CSDM has C-terminal heteroatom group; in this method the resin bound peptide chain made using N-terminal coupling is derivatized to activate its carboxy termini, then a free a-aminoboronate ester or acid is coupled to the resin bound sequence. Finally the peptide boronate (comprising the CSDM) is cleaved from the resin by strong acid HF or TFA) prior to being joined to the remainder of the final product.

25 When synthesising compounds whose CSDM contains a Pl-P2 nonnatural amide bond, it is convenient to premake as intermediates the binding subsite affinity moiety [X-(aa 4 )m- (aa 3 )n-(aa 2 of Formula I] and the specificity pocket affinity moiety with its attached C-terminal group [(aa 1

)-Z

30 of Formula The two intermediates contain suitable functional groups to react together to form the target nonnatural amide bond [Iy] and are caused or allowed to react together to form the compound (or a precursor thereof to undergo one or more further functional group transformations).

R0 Suitable synthetic techniques for making peptides containing H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 1 27 a non-amide bond W are described in PCT/GB96/00352.

In an exemplary process for synthesising a serine proteinase inhibitor in which the connector moiety and its attached EAM form an N-terminal extension of the CSDM, the EAM is prepared by Fmoc solid phase peptide chemistry, e.g. using an Fmoc-polyamide continuous flow method. A suitable solid phase for this purpose is the pre-derivatised solid support Fmoc-Leu-PEG-PS. The peptide-conjugated resin is subsequently treated with, for example, glutaric anhydride, one carboxyl group of which reacts with the N-terminal amino group of the EAM. A pre-synthesised peptide boronate CSDM is reacted with the resin/peptide/glutaric acid conjugate to form the final compound, which is cleaved from the resin, for example by treatment with 100% TFA.

Another method of using peptide boronate esters in making the inhibitors is a completely novel technique in which boronic acids [-B(OH) 2 are directly esterified onto diols coupled to a resin. Chain extension is continued from the amino group of the amino acid by, for example, standard Fmoc chemistry. The boronic acid ester is cleaved from the resin :by acid TFA) to give the peptide boronate [peptide-

B(OH)

2 or by transesterification, for example, by 25 concentrated solution of a hindered diol, such as pinanediol, for example.

S: The invention therefore includes a method of making a said bifunctional inhibitor comprising performing the following 30 steps to make a target amino acid sequence: providing a solid phase having coupled thereto functional groups capable of reacting with an amino group or, preferably, with a carboxyl group or a reactive derivative thereof; S (ii) causing the amino or carboxyl group (which may be H:\Bkrot\Keep\speci\3'0425-97-2.doc 14/04/00 28 in the form of a reactive derivative thereof) of a terminal amino acid of an amino acid sequence of a compound of the invention selectively to react with said functional groups; (iii) coupling the amino acid sequentially following, in the target sequence, the sequentially preceding amino acid coupled to the solid phase to said preceding amino acid; and (iv) repeating step (iii) as often as necessary.

In step the functional groups coupled to a solid phase may be on a moiety which is incorporated in the end product compound, e.g. may be an amino group (which may be derivatised) of an amino acid coupled directly or indirectly to the solid phase.

One or more additional steps may be, and often are, included in-the method to obtain the compound of the invention.

Thus, preferred methods include, when desired, a step of coupling a said sequentially following amino acid of a step (iii) to said preceding amino acid of the step through a compound having two functional groups capable of reacting with an amino group, whereby one of said functional groups becomes bonded to the amino group of said preceding amino 25 acid and the other to the amino group of said following S* amino acid.

0000 eeo The sequentially following amino acid of a step (iii) may be 00o0 0 part of a larger moiety, e.g. of an amino acid sequence 30 optionally containing a replacement for a natural peptide bond.

In the method, any one or more carboxylate groups reacted with an amino group may be in the form of a reactive carbonyl-containing derivative thereof, such as an activated carboxyl group, for example an acid anhydride.

H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 29 Before use, the final compound of the solid phase synthesis is cleaved from the solid phase, for example in a manner known per se. The cleaved compound may be subjected to one or more further chemical reactions before the end product compound is obtained.

In a preferred embodiment, the terminal amino acid reacted with the functional groups attached to the solid phase is the C-terminal amino acid of the EAM and step (iii) is repeated to couple successive amino acids of the EAM sequence and successive amino acids of any contiguous connector peptide, to form an uninterrupted amino acid sequence.

The final amino acid of the uninterrupted amino acid sequence coupled to the solid phase may be reacted with a compound having two carboxylate groups or reactive derivatives thereof, for example the anhydride of a dicarboxylic acid, to bond one of the two carboxylates to the amino group of the final amino acid. The unreacted carboxylate or carboxylate derivative is typically reacted with the amino group of an amino acid, which is normally the N-terminal amino acid of the CSDM. In this latter case, the amino acid may already be bonded to the remainder of the CSDM, i.e. the CSDM may be separately made in whole (or in part) for joining to the unreacted carboxylate (derivative).

The compound having two carboxylate groups is preferably a linker as described above.

30 In some preferred methods, there is used a preformed CSDM having a heteroatom group in place of a C-terminal carboxy group. The heteroatom group is preferably a boronate or boronate derivative as described above.

An amino acid or other moiety reacted with the solid phase material (the solid phase and any attached molecules) desirably has all its reactive functional groups which could H:\Bkrot\Keep\speci\30425-91-2.doc 14/04/00 30 interfere with the synthesis protected, other of course than the group to be reacted with the solid phase material. Any protected functional group of the reacted amino acid or moiety which is subsequently itself to be reacted is deprotected before it is subjected to reaction.

A first preferred method, therefore, comprises: providing a solid phase having coupled thereto functional groups capable of reacting with a carboxyl group or a reactive derivative thereof; (ii) contacting the solid phase with the C-terminal amino acid of an EAM, the amino acid having a protected amino group and optionally a derivatised carboxy group, and causing or allowing the carboxy groups of the amino acid molecules to react with the functional groups of the solid phase; (iii) deprotecting the amino groups of the reacted amino acid, whereby the solid phase becomes provided with free o amino groups;

S

(iv) repeating steps (ii) and (iii) with successive 25 amino acids of the EAM and optionally of a contiguous spacer peptide to form on the solid phase an amino acid sequence from the C-terminal of the EAM to, at the free end of the sequence, the N-terminal of the spacer; 30 contacting the solid phase with a linker compound having two carboxyl groups or reactive residues thereof, and causing or allowing linker carboxy groups or reactive carboxy residues to react with the N-terminal amino groups of the spacer sequence; (vi) contacting the solid phase having the linker compound coupled thereto with the N-terminal amino acid of a H:ABkrot\Keep\speci\30425-97-2.doc 14/04/00 1 I 31 CSDM sequence and causing or allowing the amino groups of the amino acid molecules to react with the carboxy groups or reactive carboxy residues of the linker compound, the Nterminal amino acid of the CSDM sequence optionally being part of a complete CSDM; (vii) if necessary, repeating steps (ii) and (iii) with successive amino acids of the CSDM to complete the CSDM sequence; and (viii) cleaving the resultant compound from the functional groups of the solid phase.

A second preferred method comprises: providing a solid phase having coupled thereto functional groups capable of reacting with a carboxyl group ora reactive derivative thereof; (ii) .contacting the solid phase with the C-terminal amino acid of an EAM, the amino acid having a protected amino group and optionally a derivatised carboxy group, and causing or allowing the carboxy groups of the amino acid .molecules to react with the functional groups of the solid 25 phase; (iii) deprotecting the amino group of the reacted amino acid, whereby the solid phase becomes provided with free oo: amino groups; (iv) repeating steps (ii) and (iii) with successive amino acids of the EAM to form on the solid phase an amino acid sequence from the C-terminal of the EAM to, at the free end of the sequence, the N-terminal of the EAM; contacting the solid phase with a linker compound having two carboxyl groups or reactive residues thereof, and H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 32 causing or allowing linker carboxy residues to react with the N-terminal amino groups of the EAM; (vi) optionally contacting the solid phase having the linker compound coupled thereto with the N-terminal amino acid of a peptide spacer sequence and causing or allowing the amino groups of the amino acid molecules to react with the carboxy groups or reactive carboxy residues of the linker compound; (vii) optionally repeating steps (ii) and (iii) with successive amino acids of the spacer and then repeating step (ii) with the N-terminal amino acid of a CSDM sequence, the N-terminal amino acid of the CSDM sequence optionally being part of a complete CSDM; (viii) if necessary, repeating steps (ii) and (iii) with successive amino acids of the CSDM to complete the CSDM sequence; and (ix) cleaving the resultant compound from the functional groups of the solid phase.

*e In either of the preceding methods the synthesised compound 25 is preferably cleaved from the solid phase by acid.

The preceding methods preferably involve the use of a CSDM oo amino acid or amino acid sequence a complete CSDM) having a C-terminal boron group.

In the first and second preferred methods the functional groups coupled to a solid phase may be part of a moiety which is incorporated in the end product compound, e.g. may be an amino group (which may be derivatised) of an amino acid coupled directly. or indirectly to the solid phase.

In one class of processes of the invention, the functional H: \Bkrot\Keep\speci\3O425-97-2.doC 14/04/00 33 groups coupled to the solid phase are part of an amino acid boronate which is incorporated in the end product compound, i.e. the solid phase has coupled thereto a diol to which is bound an amino acid boronate.

In another class o.f methods, an amino acid whose side chain has an amino or carboxyl group is coupled to a solid phase through the carboxyl group or amino group to the side chain. Chain extension is carried out from one of the functional groups of the amino acid, for example Fmoc synthesis from the amino group. The other functional group is then reacted with some other constituent part of the end product, for example an amino acid boronate (to form the Pl residue of the CSDM). Solid phase synthesis of boroncontaining peptides is, however, of applicability to any such peptides, and not only to the serine protease inhibitors of the invention.

Use The bifunctional inhibitors made according to the present invention are useful as inhibitors or substrates of serine proteases, e.g. thrombin, and may be used in vitro or in vivo for diagnostic and mechanistic studies of such enzymes.

25 More generally, the novel peptides may be useful for research or synthetic purposes. Furthermore, because of their inhibitory action, the inhibitors are useful in the *o o prevention or treatment of diseases caused by an excess of thrombin or another serine proteases in a regulatory system 30 particularly a mammalian system, e.g. the human or animal body, for example control of the coagulation system. The pharmaceutically useful compoinds have a pharmaceutically acceptable group as any N-terminal substituent The anti-thrombotic compounds may be employed when an antithrombogenic agent is needed. Generally, these compounds may be administered orally or parenterally to a host in an HAkBkrot\Keepkspeci\30425-97-2.doc 14/04/00 34 effective amount to obtain an anti-thrombogenic effect. In the case of larger mammals such as humans, the compounds may be administered alone or in combination with one or more pharmaceutical carriers or diluents at a dose of from 0.02 to 10mg/Kg of body weight and preferably 1-100mg/Kg, to obtain the anti-thrombogenic effect, and may be given as a single dose or in divided doses or as a sustained release formulation. When an extracorporeal blood loop is to be established for a patient, 0.1-10mg/Kg may be administered intravenously. For use with whole blood, from 1-100 mg per litre may be provided to prevent coagulation.

Pharmaceutical diluents or carriers for human or veterinary use are well known.and include sugars, starches and water, and may be used to make acceptable formulations of pharmaceutical compositions (human or veterinary) containing one or more of the subject peptides in the required pharmaceutically appropriate or effective amount or concentration. The pharmaceutical formulations may be in unit dosage form. Formulations of the compounds include tablets, capsules, injectable solutions and the like.

The anti-coagulant compounds may also be added to blood for the purpose of preventing coagulation of the blood in blood o* 25 collecting or distribution containers, tubing or implantable apparatus which comes in contact with blood.

The methods of the invention are useful for the synthesis of serine protease inhibitors and other compounds. They are 30 useful in combinatorial chemistry.

The invention will be further described and illustrated by gas:. the Examples which now follow.

EXAMPLES

i In the examples, amino acid residues are of L-configuration H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 35 unless otherwise stated.

1. [-D-Phe-Pro-BoroBpgOPin] CO(CH 2 3 COGly 2 -Gln (Tyr 63 Hir51-64 a. GlyGlyGln (Tyr 3 Hir 51 64 GlyGlyGln(Tyr 63 )Hir 5 1 64 which has the amino acid formula: H- Gly-Gly-Gln-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu- Tyr-Leu-OH, was prepared by solid phase peptide chemistry on a Milligen® 9050 PepSynthesizer using an Fmoc-polyamide continuous flow method and proprietary 9050 Plus on column monitoring software. Pre-derivatised solid support, Fmoc- Leu-PEG-PS (1.6g, 0.22meq/g) was used throughout; Fmoc-Leu- PEG-PS comprises polyethylene glycol derivatised polystyrene with HMBA linker. Fmoc groups were removed using piperidine in DMF. Fmoc-amino acids (4 equiv.) as their ~pentafluorophenyl esters with side chain protection where appropriate Fmoc-L-Tyr(tBu)OPfp, Fmoc-L-Glu(tBu)OPfp, Fmoc-L-Asp(tBu)OPfp, Fmoc-L-Asn(Trt)OPfp and Fmoc- 20 His(boc)OPfp, were coupled sequentially. Once the required S* peptide sequence was complete the N-terminal Fmoc group was removed using 20% piperidine in DMF. A positive ninhydrin test indicated that the Fmoc group had been removed. The peptide-conjugated resin was subsequently decanted on a filter and washed 'off line' with dichloromethane, methanol and dichloromethane before being dried in-vacuo for a few hours.

b. HO 2 C (CH 2 3 COGlyGlyGln (Tyr 6 Hir 5 1 64 The peptide obtained in Example la was suspended in DMF and treated with glutaric anhydride (300mg) and 4methyl-morpholine (200mg) in a round bottomed flask The reaction mixture was swirled overnight. The resin was washed with DMF, DCM and MeOH, and then dried in-vacuo overnight to obtain the target compound.

\melb-files \home$\IaColes\Keep\ Speci \3 042 5- 97-2. doc 23/11/00 36 H-D-Phe-ProBoroBpgOPin H-D-Phe-ProBoroBpgOPin was prepared by adding a 40% solution of HBr in acetic acid (20ml) to Cbz-D-Phe-Pro-BoroBpgOPin (2g) in a round bottomed flask (100ml) fitted with a septum and flushed with nitrogen. The flask was swirled to effect complete dissolution of the protected tripeptide. When the gas evolution ceased after approximately 30 minutes, anhydrous ether (200ml) was added and the reaction mixture was stored in a refrigerator for 4 hours. The reaction mixture was filtered, the residue was dissolved in EtOH (1ml) and dry ether was added to precipitate the produce (800mg) as a white solid 516; Tlc 95/5/3), Rf=0.05.

d. PheProBoroBpgOPin] CO (CH 2 3 COGly 2 G1n (Tyr 63 Hir 51 64 To -synthesise -D-PheProBoroBpgOPin] CO (CH2) 3 COGly 2 G1n (Tyr 6 3 Hir 51 64, the dry resin HOCO(CH 2 3 COGly 2 Gln(Tyr 63 )Hir 51 64 was suspended:in DMF (10ml), before TBTU (129mg, 0.4mmol) and H- D-Phe-ProBoroBpgOPin (230mg, 0.4mmol) were added to the reaction mixture. After 5 minutes stirring, triethylamine (40mg, 0.04mmol) was added and the flask left stirring overnight.

The fully protected peptide resin was washed with dichloromethane, methanol and dichloromethane and then dried under vacuum. Cleavage from the resin with simultaneous deprotection of side chain protecting groups was achieved by 30 treating the resin with 100% TFA for two hours. TFA was removed and the free peptide with a C-terminal carboxylic acid was generated by precipitation with cold dry ether.

The crude peptide was collected by filtration and washed with further portions of ether.

c.

o* a a *o oooo a a* a ooo.

a a a

RA*

*z.

I

Purification of the crude peptide was carried out by reversed-phase HPLC using a Vydac" C-18 preparative column H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 37 (TP silica, 10pm, 25mm x 300mm) The column was eluted with a 30-90% linear gradient of solvent A TFA in water) and solvent B (0.1 TFA in acetonitrile). The column eluants were monitored at 230nM, and fractions were collected appropriately. The purity of the products were determined by analytical RP-HPLC and mass spectrometry.

2. Cbz-D-Phe-Pro-W (C0 2 -boroethylglycine pinanediol This example, although performed in the liquid phase, serves to illustrate formation of a peptide in which the natural peptide linkage between the C-terminal amino acid residue (BoroEtg) and the adjacent residue (Pro) is replaced by a C0 2 -linkage.

a. Cbz-D-Phe-Pro-xg(CO 2 )-BoroEtg pinanediol l-Chloroethane-pinanediol boronate ester (0.321g,1.25x10o 20 3mol) added with stirring to Cbz-D-Phe-Pro-OH (0.6g, 1.52x10-mol) When the addition had been completed, DBU (0.23g, 1.52mmol) in CH 2 C12 was added to the mixture and allowed to stir at room temperature, before being left to stir for an extended period at 40C before workup. The 25 opaque liquid was washed with HC1 (0.1M, 2x50 ml), NaHCO 3 50ml). The organic layer was dried by vigorous stirring over anhydrous MgS0 4 and filtered off, to remove the desiccant. The filtrate was concentrated under reduced pressure on a rotary evaporator, to afford a thick, viscous residue. Preliminary examination by 'H N.M.R. showed the required crude product. The crude sample was dissolved in a small amount of MeOH, applied to the sephadex® column, and then eluted with a pump using the same solvents. The elution profile was followed with the aid of a U.V. lamp (226nM) and recorder. The void volume, fraction 1-6, and a further bulk volume were collected.

SFrom the shape of the chromatogram, it was deemed that \\melb_files\home$\LJColes\Keep\Speci\30425-97-2.doc 23/11/00 37a fractions 1-6 would be the most likely fractions in which the tripeptide may be found. The fractions were concentrated individually to afford clear slightly coloured viscous residues. One fraction containing the bulk of the material when placed under high vacuum was later afforded as a slightly crystalline product (0.269 yield of *o* \\melbfiles\home$\taColes\Keep\Speci\30425-97-2.doc 23/11/00 I 38 FABMS (Fast Atom Bombardment Mass Spectrometry) and C, H, N were very strong (good) indicators that the compound has been formed.

C2 B O D N NNO CH2OCO-NH-CH-CO

CH

2 b. H-Phe-Pro-y (C0 2 -BoroEtg pinanediol Cbz-D-Phe-Pro-y(C0 2 )-BoroEtg pinanediol (from Example 2a) was dissolved in MeOH (30ml) and treated with 10% Pd/C, and purged with argon with stirring, the flask evacuated and pumped with H 2 with stirring for 5H. Ninhydrin staining indicated deprotected product on TLC. The solution was purged with argon for 10 min, filtered and concentrated under reduced pressure to afford a thick black.oil, which was dissolved in CHC1 3 filtered and concentrated. H N.M.R. of the crude product indicated no protected product.

The residue from above was chromatographed on a Sephadex LH20 chromatography column. 1H (60 MHz) N.M.R. showed that 20 the isolated compound displayed many of the characteristics expected on the basis of the putative structure. 122mg of the free amino boronate ester was isolated.

3. H-Phe-L-Glu-BoroBpgOPin a. Fmoc-L-Glu(PEG-PS)OH Tetrakistriphenylphosphine palladium(0) [PdP(Ph 3 (ig) was dissolved under Ar in a solution of CH 3 C1 containing acetic acid and 2.5% N-methylmorpholine (30ml). This S mixture was transferred under Ar to a flask containing IH:\Bkrot\Keep\spci\30425-97-2.doc 14/04/00 39 Fmoc-L-Glu(PEG-PS)OAl The resin was left to stand for 2 hours with occasional gentle agitation. The resin was filtered on a sintered glass funnel-and washed with diisopropylethylamine and sodium diethyldithiocarbamate (0.5%w/w)in DMF (300ml) to remove the catalyst.

b. Fmoc-L-Glu(PEG-PS)NHBoroBpgOPin The dry resin Fmoc-L-Glu(PEG-PS)OH (1.5g) was suspended in DMF(10ml) under Ar. TBTU (129mg, 0.4mmol) and

NH

2 BoroBpgOPin (165mg, 0.5mmol)w were added to the reaction mixture. After 5 minutes stirring, triethylamine 0.4mmol) was added and the flask left stirring overnight.

The resin was washed with dichloromethane, methanol and dichloromethane and then dried under vacuum.

c. H-Phe-L-Glu(PEG-PS)NHBoroBpgOPin H-Phe-L-Glu(PEG-PS)NHBoroBpgOPin was prepared by solid phase chemistry on a Milligen 9050 peptide synthesiser.

Fmoc group was removed from the solid support Fmoc-L- Glu(PEG-PS)NHBoroBpgOPin using 20% piperidine in DMF. Fmoc- Phe-OPfp was coupled to the free N-terminus.

25 The protected peptide resin was washed with dichloromethane, methanol and dichloromethane and then dried under vacuum.

d. H-Phe-L-Glu-BoroBpgOPin 0 Cleavage of the peptide from the resin was achieved by treating the resin with 100% TFA for two hours. TFA was removed and the free peptide H-Phe-Glu-NH-BoroBpgOPin was generated by precipitation with cold dry ether. The crude 35 peptide was collected by filtration and washed with further portions of ether portions of ether.

HA\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 40 Purification of the crude peptide was carried out by reversed-phase HPLC using a Vydac C-18 preparative column (TP silica:particle size 10mm; 25mmX300mm). The column was eluted with a 30-90% linear gradient of solvent A(0.1%TFA in water) and solvent B(0.1%TFA in acetonitrile). The column eluants were monitored at 230nM, and fractions were appropriately collected. The purity of the products were determined by analytical RP-HPLC and mass spectrometry.

Product H-Phe-Glu-NH-BoroBpgOPin, was obtained in 17% yield, 34mg, ES-MS: 626 retention time analytical HPLC (4x250mm, Vydac, C-18 techsphere), eluted by 10-60% MeCN with 0.1% TFA in water with 0.1% TFA over 25 minutes, gave Rt 23.1 minutes.

4. Attachment of Boronic Acid to Merrifield Resin a. Derivatisation of Resin with protected Diol (2,2dimethyl-1,3-dioxolane-4-methanol a a.

a a a Resin CH 2

-O

0 o.oo Na (solid, 8g) is added to 2,2-dimethyl-1,3-dioxolane-4- '1 methanol (240ml), under argon gas, and the mixture stirred until it gives a clear solution. Merrifield Resin (Sigma, 1.1 MeQ. Cl per gram, 20g) is added and the mixture stirred overnight, then heated at 80 0 C for 24h.

S Derivatised resin was collected by filtration, washed by 441)I H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 V" o 41 1,4-dioxane water (3x500ml), and MeOH:water (1:1, 3x500ml), MeOH (3x500ml) and dry ether (3x500ml). An infra red spectrum was obtained by powdering of 1.5-2mg of resin with KBr (dry, 300mg) and compacting into a disc, then scanning on a Perkin® 1600 Fourier Transform I.R. The derivatised resin (Fig. 2) compared to Merrifield resin (Fig. 1) shows distinct stretching signals 1050 to 1150cm- 1(s) for ether stretching frequencies characteristic of a five membered ring; and dialkyl ether stretching at 1060 to 1150cm- 1 for alkyl-alkyl stretching.

b. Deprotection

OH

Resin

CH

2 -O Re sin

CH

2 OH

TOH

The derivatised resin was mixed with HC1 (1.5M, 250ml) and 1,4-dioxane (250ml) and the suspension stirred and heated at 0 C. After 72h the resin was washed by water (500ml), MeOH 20 (500ml), DCM (500ml) and Et 2 0 (500ml), then dried in the air. spectrum of the resin shows distinct O-H stretching frequencies at 3400-3550cm 1 and a main peak at 3413.6 (Fig. this peak is substantially larger than the signal at 2917.6cm 1 In comparison the ether (Fig. 2) and Merrifield resin show only a weak 3400cm 1 signal for background moisture.

c. Reaction of the Derivatised Resin with a boronic acid HA\BkroC\Keep\speci\30425-97-2.doc 14/04/00 42

OH

CH

2 -OC O H Resin

SOOH

100

R

0 The diol resin (5g, 5.5mmol of diol) was suspended in THF (dry, 500ml) and phenylboronic acid (3.35g, 27.5mmol, equivalents), and 4A sieves (dried at 150 0 After stirring under argon overnight, the resin was filtered under argon in a closed system, washed by THF (500ml) and dried under vacuum. Ft-L.R. (Fig. 4) shows a strong signal at 1026cm 1 (aryl-alkyl stretching frequency) for the phenyl ring and a weak signal at 3417cm 1 (compared to Fig 3, for the starting diol).

Ref. Leznoff, C.C. and Wong, The use of Polymer Supports in Organic Synthesis. III. Selective Chemical Reactions on One Aldehyde Group of Symmetrical Dialdehydes., Can.J.Chem., 1973, 51, 3756-3764.

Analytical and Activity Data o The following Table 1 contains activity data relating to the invention. In the Table, the designation denotes benzoyloxycarbonyl and "NHir" refers to normal hirudin.

"NHir49-64(des-S) refers to the amino acid sequence from amino acid 49 to amino acid 64 of normal hirudin in which the native Tyr(OS 3

H)

63 is replaced by Tyr.

H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 43 The compounds listed in Table 1 were prepared by the same or analogous methods to the compounds of the preparation Examples 1 and 2 above or, in the case of intermediates, were obtained from sources.

The following techniques were employed for activity measurement: Plasma thrombin time (TT) A volume of 150pl of citrated normal human plasma and of buffer or sample were warmed at 37 0 C for 1 min.

Coagulation was started by adding 150pl of freshly prepared bovine thrombin (5NIHu/ml saline) and the coagulation time was recorded on a coagulometer.

A phosphate buffer, pH7.8, containing 0.1% bovine serum albumine and 0.02% sodium azide was used. The samples were dissolved in DMSO and diluted with the buffer. When no inhibitor was used DMSO was added to the buffer to the same concentration as that used in the samples. The inhibitor concentrations were plotted against the thrombin times in a semilogarithmic graph from which the inhibitor concentration that caused a doubling (40 sec) of the thrombin time was determined.

Determination of Ki o.o The inhibition of human a-thrombin was determined by the inhibition of the enzyme catalysed hydrolysis of three different concentrations of the chromogenic substrate S- 30 2238.

20011 of sample or buffer and 50l1 of S-2238 were incubated at 37 0 °C for 1 min and 50pl of human a-thrombin (0.25 NIHg /ml) was added. The initial rate of inhibited and 35 uninhibited reactions were recorded at 4.5nm. The increase in optical density was plotted according to the method of A Lineweaver and Burke. The Km and apparent Km were HA\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 44 determined and Ki was calculated using the relationship.

V= Vmax 1 Km (1 Ki The buffer used contained 0.1M sodium phosphate, 0.2M NaCl, PEG and 0.02% sodium azide, adjusted to pH 7.5 with orthophosphoric acid.

The samples consist of the compound disclosed in DMSO.

The reader is referred to Dixon, M and Webb, E. C., "Enzymes", third edition, 1979, Academic Press, the disclosure of which is incorporated herein by reference, for a further description of the measurement of Ki.

0 0 0°00 0000 0* -o H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 45 Table 1 Ki PM

TTI

98 GGGDFEPIPL n/e 1001 99 [-BoroBrOPin]CO(CH 2 3 COGGGDFEPIPL n/e 58 105 GGGGDFEPIPL n/e 94.9 106 GGGGGDFEPIPL n/e 107 [E-BoroBrOPin ]OC (CH2) 3 COGGGGDFEPIPL n/e 58.4 108 -BoroBrOPin OC (CH 2 3 COGGGGGDFEPI PL rile 63.41 114 GGNSHNDGDFEEIPEEYL Hir 4 9 "1 0.613 2 121 H0 2 C (CH 2 3 COGGGGGDFEPIPL 0.738 30.9 128 -L-PheProBoroValOPin] OC (CH 2 3

COG

5 DFEPIPL n/e 11.7 27.1 129 -D-PheProBoroValOPin] OC (dH 2 3

COG

5 DFEPIPL 16.4 33.5 137 (-D-Phe-ProBoroEtgOPin] OC (CH 2 3

COG

5 DFEPIPL 1.23 10.2 166 -D-Phe-ProBoroBpgOPin OC (CH 2 3

COG

2 N~ir 9 0.000649 0.02 14 (des-S) 167 Hir 49 64 (des-S) n/e 0.155 N.T.1 175 [-D-Phe-ProBoroBpgOPin]OC (CH 2 3 COGNHir 4 9 0.00211 00 64 (des-S) 176 Z-D-Phe-Pro-BoroBpgOPin Hir 49 -6 0.0218 0.63 61 182 [-D-Phe-ProBoroCegOPin]OC (CH 2 3 COGPGGNHir"- 0.00271 N.T.1 64 184 HO 2

C(CH

2 3 COGPGGNHir 4 1 64 (des-S) 12.7 185 [-D-Phe-ProBoroCegOPinj OC (CH 2 3

COGPG

3 Hir~ 9- le 0.75 73 64 (des-S) 186 Phe- Pro -BoroCegOPin]OC (CH 2 3

COGPG

3 Nir 49 nle 0.9 4.61 64 (des-S) 267 f-PheProBoroCegPin]OC(CH 2 3 C0G 2 (EDFEPIPL) 0.762 4.9 268 (-Pgl 9 (OEt) 2 ]0C(CH 2 3 C0G 2 (EDFEPIPL) n/e 88.8 66.5 -D-PheProBorolrgOPin] OC (CH 2 3

COG

2 N~ir 49 6 (des N.T. NT rile no effect n/e 11.7 =no effect up to a concentration of 11.7p.M N.T. not tested

S

S

S. S S S @0

S

55 S S

S

S

*S*S

5.5.

S

S

SOSS

H:\Bkrot\Keep\speci\30425-97-2 .doc 14/04/00

Claims (18)

1. A method of making a peptide or a peptide- containing compound, comprising performing the following steps to make a target amino acid sequence: providing a solid phase having coupled thereto functional groups; (ii) causing a compound reactive with the functional groups selectively to react therewith, the reacted compound having a functional group capable of reacting with an amino group or with a carboxyl group or a reactive derivative thereof: (iii) causing the amino or carboxyl group (which may be in the form of a reactive derivative thereof) of a terminal amino acid of a target amino acid sequence selectively to react with said functional groups of the reacted compound; (iv) coupling the amino acid sequentially following, in the target sequence, the sequentially preceding amino acid coupled to the solid phase to said preceding amino acid; *o repeating step (iv) as often as necessary; and (vi) cleaving a solid phase-linked compound prepared using steps from the solid phase by the action of 30 an acid or base, *D wherein a compound comprising a boronic acid group [-B(OH 2 or an ester or other derivative thereof is incorporated in the solid phase-linked compound prior to its cleavage from 35 the solid phase.

2. A method according to claim 1, in which step (ii) H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 47 comprises reacting an amino group or an optionally derivatised carboxy group of an amino acid with a functional group coupled directly or indirectly to a solid phase, for example as part of a conventional solid phase peptide synthesis.

3. A method according to claim 1, in which the compound comprising a boronic acid group is an amino acid boronic acid, a peptide boronic acid or a boronate ester of either and step (ii) comprises reacting the amino acid or peptide boronic acid or ester with a diol coupled to the solid phase.

4. A method according to claim 3, in which the diol comprises a residue of the formula 0 OH OH and the step results in formation of a solid phase having coupled thereto, directly or via a linker, a moiety of the formula: B-R' O O g wherein R' is selected from the group consisting of residues of natural and unnatural amino acid and analogues thereof. A method according to claim 4, in which the analogues are amino acids having their amino groups replaced by an alternative functional group capable of forming a linkage other than a natural peptide linkage. H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 48

6. A method according to claim 1, in which step (iii) comprises causing the carboxyl group of said terminal amino acid to react with said functional groups, or causing a reactive derivative of the carboxyl group to do so.

7. A method according to claim 6, wherein said compound comprising a boronic acid group or an ester or other derivative thereof comprises an amino acid sequence having a C-terminal boronic acid group or ester or other derivative thereof, and the method further comprises the following additional steps between steps and (vi): (va) contacting the solid-phase linked compound prepared using steps with a linker compound having two carboxyl groups or reactive residues thereof, and causing or allowing the linker carboxy groups or reactive carboxy residues to react with the N-terminal amino group of-said compound; and (vb) contacting the solid phase having the linker compound coupled thereto with the N-terminal amino acid of said amino acid sequence having a C-terminal boronic acid group or ester or other derivative thereof, and causing or allowing the amino group of the N-terminal amino acid to *e 25 react with the carboxy groups or reactive carboxy residues .e of the linker compound.

8. A method according to claim 1, in which step (iii) comprises causing the amino group of said terminal 30 amino acid to react with said functional groups.

9. A method according to claim 8 in which, between steps and the carboxy terminus of a resin bound peptide is coupled to a free c-aminoboronate acid or ester 35 and step (vi) comprises cleavage by the action of an acid. A method for making a compound comprising a H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 49 peptide boronic acid or peptide boronate ester, the method comprising: providing a solid phase having coupled thereto alcoholic hydroxy groups; (ii) causing an amino acid boronic acid or peptide boronic acid to react with the hydroxy groups whereby the boronic acid residue becomes esterified to the solid phase; (iii) causing the carboxyl group of the amino acid sequentially following, in the end product peptide boronic acid or boronate ester, selectively to react with the amino group of the sequentially preceding amino acid coupled to the solid phase; (iv) repeating step (iii) as often as necessary; cleaving the resultant peptide boronate from the resin; the method optionally comprising one or more further steps to make said compound. S 25 11. A method according to claim 10, wherein each amino acid coupled to the solid phase has a protected amino *group and step (iii) comprises deprotecting the amino group of the sequentially preceding amino acid and/or the cleavage of step is performed with acid or by 30 transesterification. o

12. The use of amino acid or peptide boronic acids or boronate esters in the solid phase synthesis of peptide- containing compounds.

13. The use in solid phase synthesis of a boronic (acid residue attached to the solid phase through hydroxy H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 50 residues.

14. A method for making a compound comprising a boron atom, the method comprising: providing a solid phase having coupled thereto alcoholic hydroxy groups; (ii) causing a boronic acid or boronate ester to react with the hydroxy groups whereby the boronic acid residue becomes esterified to the solid phase; and (iii) performing one or more further steps to make said compound. A solid phase material having coupled thereto boronic acid residues through hydroxy group residues.

16. A solid phase material having coupled thereto a moiety of the formula: -0 .B-R may optionally be inertly substituted, or to a carbonyl group wherein R is a residue bonded to the boron atom, and the *25 oxygen through which the moiety is bonded to the solid eh. according to claim 12.

18. A method of making a bifunctional serine protease w 1 35 inhibitor comprising: 1 0 inhibitor comprising: 42 5-97-2.doc 14/04/00 4 51 a catalytic site-directed moiety (CSDM) which binds to and inhibits the active site of a serine protease; an exosite associating moiety (EAM); and, optionally, a connector moiety bonded between the EAM and CSDM, the CSDM and the EAM being capable of binding simultaneously to a molecule of the serine protease, the method comprising performing the following steps to make a target amino acid sequence: providing a solid phase having coupled thereto functional groups capable of reacting with an amino group or, preferably, with a carboxyl group or a reactive derivative thereof; (ii) causing the amino or carboxyl group of a terminal amino acid of the target amino acid sequence selectively to react with said functional groups, the carboxyl group optionally being in the form of a reactive derivative thereof; 0 25 (iii) coupling the amino acid sequentially following, in the target sequence, the sequentially preceding amino acid coupled to the solid phase to said preceding amino 0 acid; 30 (iv) repeating step (iii) as often as necessary to form an uninterrupted amino acid sequence coupled to the solid phase; reacting the final amino acid of the uninterrupted 35 amino acid sequence coupled to the solid phase with a compound having two carboxylate groups or reactive zi derivatives thereof; and H:\Bkrot\Keep\speci\30425-97-2.doc 14/04/00 52 (vi) reacting the unreacted carboxylate or carboxylate derivative with the amino group of an amino acid which is the N-terminal amino acid of the CSDM and is already bonded to the remainder of the CSDM and the CSDM has a boronic acid group or an ester or other derivative thereof in place of a C-terminal carboxy group.

19. A method according to claim 18, in which the terminal amino acid reacted with the functional groups attached to the solid phase is the C-terminal amino acid of the EAM and step (iii) is repeated to couple successive amino acids of the EAM sequence and successive amino acids of any contiguous connector peptide. A bifunctional serine protease inhibitor whenever made using a method according to claim 18 or claim 19.

21. A pharmaceutical formulation comprising two or 20 more inhibitors according to claim 20, formulated for use as a human or veterinary pharmaceutical.

22. A formulation according to claim 21, further comprising a pharmaceutically acceptable diluent, excipient 25 or carrier.

23. A method of identifying a serine protease inhibitor comprising the step of screening for the ability of said inhibitor to inhibit serine protease activity by a peptide boronate prepared by a method according to any one of claims 1 to 11 or the use according to claim 12. \\melb_files\home$\LJColes\Keep\Speci\30425-97-2.doc 23/11/00 53

24. A method according to claim 1 substantially as hereinbefore described with reference to any one of the examples. Dated this 22nd day of November 2000 TRIGEN LIMITED By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia 0*0 \\melb-files\home$\LTColes\Keep\Speci\30425-97-2.doc 23/11/00