SERINE PROTEASE INHIBITORS

INHIBITEURS DE LA SERINE PROTEASE

English Abstract

Bifunctional serine protease inhibitors and methods of preparing boron-
containing peptides are provided. The serine protease inhiitors comprise a
catalytic site-directed moiety, which binds to and inhibits the active site of
a serine protease, and an exosite associating moiety, which are joined by a
connector moiety. The catalytic site directed moiety and the exosite
associating moiety are capable of binding simultaneously to a molecule of the
serine protease.

French Abstract

L'invention concerne des inhibiteurs bifonctionnels de la sérine protéase et des procédés de préparation de peptides contenant du bore. Ces inhibiteurs comprennent une fraction catalytique dirigée qui se lie au site actif d'une sérine protéase et l'inhibe, et une fraction se liant à un exosite, ces deux fractions étant réunies par une fraction de couplage. La fraction dirigée catalytique et la fraction se liant à un exosite sont capables de se lier simultanément à une molécule de la sérine protéase.

Claims

41
CLAIMS
1. A method of making a peptide or a peptide-containing compound, comprising
performing the following steps to make a target amino acid sequence:
(i) 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;
(v) repeating step (iv) as often as necessary; and
(vi) cleaving a solid phase-linked compound prepared using steps (i)-(iv)
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 anester or other derivative thereof is incorporated in the solid phase-linked compound
prior to its cleavage from the solid phase.
2. A method of claim 1 in which step (ii) comprises reacting an amino group or
an optionally derivatised carboxy group of an amino acid with a functional group

42
coupled directly or indirectly to a solid phase as part of conventional solid phase
peptide synthesis, for example.
3. A method of claim 1 in which the compound comprising a boronic acid group
is an amino acid boronic acid, a peptide boronic ester 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. e g to form a solid phase having coupled thereto, suitably
via a linker. a moiety of the formula:
<IMG>
wherein R' a residue of a natural or unnatural amino acid or of an analogue thereof.
the analogue optionally having its amino group replaced by an alternative functional
group capable of forming a linkage other than a natural peptide linkage and/or its
.alpha.-hydrogen atom replaced.
4. A method for making a compound comprising a peptide boronic acid or
peptide boronate ester. the method comprising:
(i) 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 if the sequentially preceding amino acid coupled to the solid
phase;

43
(iv) repeating step (iii) as often as necessary;
(v) cleaving the resultant peptide boronate from the resin,
the method optionally comprising one or more further steps to make said compound.
5. A method of claim 4, wherein each amino acid coupled to the solid phase has aprotected amino group and step (iii) comprises deprotecting the amino group of the
sequentially preceding amino acid and/or the cleavage of step (v) is performed with
acid or by transesterification.
6. The use of amino acid or peptide boronic acids or boronate esters in the solid
phase synthesis of peptide-containing compounds.
7. The use in solid phase synthesis of a boronic acid residue attached to the solid
phase through by hydroxy residues.
8. A method for making a compound comprising a boron atom the method
comprising:
(i) 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.
9. A solid phase material having coupled thereto boronic acid residues through
hydroxy groups.

44
10. A solid phase material having coupled thereto a moiety of the formula:
<IMG>
wherein R is a residue bonded to the boron atom and the oxygen through which the
moiety is bonded to the solid phase is optionally bonded directly to our alkylene
residue, which may optionally be inertly substituted, or to a carbonyl group.
11. A bifunctional serine protease inhibitor comprising:
(a) a catalytic site-directed moitey (CSDM) which binds to and inhibits the
active site of a serine protease.
(b) an exosite associating moiety (EAM); and optionally.
(c) 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 provided that the inhibitor is not a thrombin inhibitor.
12. An inhibitor of claim 11, in which the CSDM has a P1 residue which is an
Arg, Lys or an analogue thereof or is hydrophobic and has a (P4)P3P2 residue
selected from
Residue Sequence
D-Phe-/substituted D-Phe-/D-Dpa-/Dba-/Pms-/.alpha.-Nal-/.beta.-Nal-
/TMSal-/Chg-/Phg-/D-Tiq-/para-ether of D-Tyr-/NaSO2-Pro
IleuGluGly, PyroGluGly, ArgGly, ChaGly, LeuArg
L-PhePhe, NalPhe, D-TiqPhe, NalThr, NalPhg

ValVal
.gamma.-BzlGluGly, Glu(OBzl)Ala, GlyArg, GlyLys
GlnGly
PhePro, GluGly
LeuSerThr
13. An inhibitor of claim 11 or claim 12. in which the EAM is an amino acid
sequence selected from:
YIDGR--IVEGSDAEIGMSPWQ
AIEGR--TATSEYQTFFNPRTFGS
SKPQGR--IVGGKVC
NLTRR--IVGGQECKDGEC
NLTRR--IVGGQECKDGEC
SKLTR--AEAVFPDVDYVN
FNDETR--VVGGEDAKPGQF
KIKPR--IVGGTASVRGE
KTSTR--IVGGTNSSWGE
ELR--MKNNEEAEDYDDDLTDSEMO
PKKCPGR--VVGGCVAHPHSWPWQVSLRT
14. An inhibitor of any of claims 1 to 3. wherein the connector moiety is of theformula:
-.lambda.-.sigma.- ,
wherein .lambda. is a residue of a non-peptide linker of the formula HO2C(CH2)h CO2H,
wherein h is from 2 to 6, and .sigma. is a peptide spacer comprising at least two adjacent
Gly residues.

46
15. A method of making an inhibitor of any of claims 11 to 14 comprising
performing the following steps to make a target amino acid sequence:
(i) 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, thecarboxyl group optionally being in the form of a reactive derivative thereof;
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.
16. A method of claim 15 which further includes a step (v) 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 acid and the other to the amino group of said following amino
acid.
17. A method of claim 14 or claim 15, wherein 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.
18. A method of any of claims 15 to 17, wherein the final amino acid of the
uninterrupted amino acid sequence coupled to the solid phase is reacted with a
compound having two carboxylate groups or reactive derivatives thereof, and the

47
unreacted carboxylate or carboxylate derivative is typically reacted with the amino
group of an amino acid which is the N-terminal amino acid of the CSDM.
19. A method of claim 18, wherein said N-terminal amino acid is already bonded to
the remainder of the CSDM and the CSDM has a heteroatom group in place of a
C-terminal carboxy group.
20. A pharmaceutical formulation comprising an inhibitor of any of claims 1 to 4formulated for use as a human or veterinary pharmaceutical, the formulation
optionally including a pharmaceutically acceptable diluent, excipient or carrier.
21 A method of treating by therapy or prophylaxis a bodily disease or disorder
capable of treatment by inhibition of a serine protease. comprising administering
orally or parenterally to a human or animal patient a therapeutically or
prophylactically effective amount of an inhibitor of any of claims 1 to 4.

Description

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I
SE~INE PROTI~ASE INHIBITORS
This invention relates to serine protease inhibitors and substrates. as well as to the
synthesis of such compounds and novel rnethods and materials for synthesis of boron-
cont~ining compounds.
Modulation or inhibition of serine protease inhibitors is useful intel alia to prevent
thrombosis.
The f'amil~- Ol serine protease enzymes clea~ es peptide bonds by a mechanism
10 involving the catalvtic triad of Asp-~is-Ser residues in the active site of the enzymes.
Serine protease inhibitors have been designed ~ hich use functional glroups to interact
with the triad and therebv blocl; activation of tlle enzyme s~lbstrates. It can be
desirable to mal;e inhibitors selective for one taroet protease. Discussion of the prior
art relating tO peptide inhibitors can be found in the specification of a UK patent
15 application entitled "Thrombin Inhibitors" filed on tlle 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 ~arious prior
20 documents referred to in that specification. The "Thrombin Inhibitors" specification
does not physicall~ forrn 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
25 ('ABE").
The arnino acid residue which provides the carbonyl group of the scissile bond of a
serine protease substrate is designated "Pl". Successive arnino acid residues on the N-
terrninal side of residue Pl are de.sign~ted P2, P3, P~ ... etc: arnino acid residues on the
30 C-terrninal side of residue Pl are designated Pl ' ~ P2 ' ~ P3 ' .... In fibrinogen, Pl ~ is
glycine and P2' is proline. The protease contains a "specificity pocket" which

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recognises the side chain of the Pl amino acid. Trypsin-like proteases normally
recognise P1 residues with arginine-like or serine-like side chains.
The present invention provides novel bifunctional serine protease inhibitors
5 comprising:
(a) a catalytic site-directed moiety (CSDM) which binds to and inhibits the
active site of a serine protease:
(b) an exosite associating moiety (EAM); and. optionally.
(c) a comlector moiety bonded between the EAM and the CSDM!
the CSDM and the EAM heing capable of binding simultaneousl~ to a molecule of the
serine protease.
15 In one class of inhibitors~ the serine protease is not thrombin. The serine protease is
preferably a trypsin-like protease. In any event, the inhibitor does not comprise a
thrombin inhibitor in which the connector moiety is bonded to the CSDM as a C-
terminal extension thereof, i.e. is not a compound as disclosed in US 5196404 and
corresponding International application No WO 91/02750.
In another aspect. the invention provides a novel method for preparing boron-
cont~ining peptides.
As used herein, ''natural" amino acid means an L-amino acid (or a residue thereof)
25 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 D-
30 isomers of natural L- amino acids and amino acids having side chain protecting
groups.

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The prefixes "D" and "L" are used as notmal to indicate amino acids of D- or L-
configuration respectively. A "D. L-" prefix indicates a racemic mixture whilst the
absence of a prefix indicates that the amino acid can be of either D- or 1,-
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.
Abbreviations and terms prefixed by boro indicate amino acids where in the
tenninal carboxyl ~roup -CO~I I has been replaced by a boron functionality.
Brief Description of the Drawings
Figure I is a Fourier l'ransform Infra Red (F.t.-I.R.) spectrum of a Merrifield
1 5 resin
Figure 2 is an F.t.-I.R. spectrum of the same resin after reaction with sodium
2.2-dimethyl- 1 ,3-dioxolane-4-methanolate.
Figure 3 is an F.t.-I.R. spectrum of the reacted resin after treatment with HCI
to deprotect the hydroxy groups of the dioxolane.
Figure 4 is an F.t.-I.R. spectrum of the deprotected resin after reaction with
phenylboronic acid.
Considering now the inventive compounds and processes in more details:
~5
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.
~t may comprise the amino acid sequence of any known inhibitor of a serine protease
catalytic site, for example.
. , , ~ ... . . .

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One class of CSDMs is included in the following Formula I:
X-(aa~)m-(aa3)n-(aa~)-(aal)-Z (I).
5 wherein aal~ aa2, and aa-' represent natural or unnatural acid residues and (aa4)m one or
more optional arnino acid residues linked to the arnino group of aa3. Alternatively any
one or more aa groups mav be analogues of amino acid residues in which the a-
hydrogen is replaced by a substituent. The sequence of amino acids andlor amino acid
analogues binds to the serine protease active site. Suitable sequences are described later
10 in this specification. ,~ represents H or a substituent on the N-terrninal amino group. Z
is -COOH or a C-terrninal extension group (carboxy replacement ~roup). for exarnple as
knov~n in the art. In preferred compounds Z is a heteroatom acid group. e.g. -B(OH),. -
P(O~I)2 or PO(OH),. or a derivative thereof. for exarnple a carboxylic acid ester, a
dioxo-boronate [-B(Osubstituent),] or a phosphate [-PO(Osubstituent),] or BF,.
15 Preferred heteroatom analogue groups are -B(OH)2 and -P(O)(OH),; a less preferred
heteroatom analogue group is S(O)2OH. Amongst other possible Z groups there may be
mentioned -CN, -COCH,CI and -COCH2F. In preferred embodiments m is from 0 to 7
and more usuallv 0 to 5, e.g. 0~ I or 2, especially 0. Normally. n=l .
20 In one class of compounds~ the (aa2)-(aal) 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
25 of serine protease inhibitors. i. e. inhibitors of the serine protease catalytic site, is to be
found in EP-B-145441, which patent 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 ~O 89/09612 (concerned particularly with inhibitors of Factor
30 VII/VIIa in the [TF:~'II/VIIa] complex).

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s
For inhibitors of trypsin-like enzymes~ preferred classes of Pl residues of CSDMs are
(i) Arg, Lys and their analogues, and (ii) hydrophobic residues; further description of
preferred Pl ~roups for thrombin which are also for other trypsin-like enzymes may
be found in the aforesaid specification entitled 'Thrombin Inhibitors" and in
PCT/GB96/00352. Chymotrypsin-like serine proteases bind preferentially to CSDMs
having phenylalanine-like and alanine-like side chains on the Pl residue. The
following table A indicates the most preferred (P4)P3P2 residues for eight particular
serine proteases:
Table A
Enzyme Residue Sequence
Thrombin D-Phe-/substituted D-Phe-/D-L)pa-/Dba-/Pms-ia-Nal-/~-Nal-
/TMSal-/Chg-/Phg-/D-Tiq-/para-ether of D-Tvr-/NaSO2-l'ro
Factor Xa lleuGluGly, PyroGluGly~ ArgGI~. ChaGI~. L,euArg
Factor VIla L-PhePhe, NalPhe, D-TiqPhe, NalThr, NalPhg
Factor IXa ValVal
Factor XIa ~-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~
15 The Exosite Associating Moiety (EAM)
The exosite associating moiety (EAM) is a moiety which binds to an exosite (ABE) of
a serine protease. Thrombin has a well defined exosite to which there binds, in
addition to a fibrinogen amino acid sequence C-terminal to the thrombin cleavage site,
20 non-substrate lie~ands of thrombin such as hirudin. Hirudin sequences such as Hir~3~64
- have 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
.. .... .. .

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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 (P~m~n~bhan K et al. "Structure
of ~Iuman Des (1-45) Factor Xa at 2.2A Resolution'' J.~l~fol Biol 1993, 232. 947-966)
are that the sequences 35-41 and 70-81. containing ~ acidic residues constitute a
cation binding exosite. The natural polypeptide inhibitors antistasin and ghilanten
contain the electropositive cation exosite associating sequences l08CRP~RKLIPRI17
and ~08CKPKRKI,VPR 117 respectivelv
It is clear that serine protease interactions in P' sites (C-terminal to the cleavage point)
can be as important to specificit~ as those at P sites (for example Ding. L.; Coombs
G.S.; Strandberg, L.; Navre, M.; Coreyu. D.R. and Madison. E.I.. Orioins of the
Specificitv of l'issue-tvpe Plasminogen Activator, I'roc.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 activitv where existin~ substrates were too insensitive.
Peptide libraries are showing great efficacv for screening for bioloL~ical activity in a
v ariety of applications (e.g Eichler, 1994) and are useful for the present invention.
The invention contemplates that sequences C-terminal to the cieavage point of other
serine proteinase substrates form an EAM. as follows
Table B
Enzyme Natural Substrate Cleavage Site (--)
Factor Xa Prothrombin YIDGR--IVEGSDAEIGMSPWQ
Factor Xa Prothrombin AIEGR--TATSEYQTFFNPRTFGS
Factor Xa Factor VII SKPQGR--IVGGKVC
Factor VIIa Factor X NLTRR--IVGGQECKDGEC

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Factor IXa Factor X NLTRR--IVGGQECKDGEC
Factor XIa Factor IX SKLTR--AEAVFPDVDYVN
FactorXIa Factor IX FNDFTR--VVGGEDAKPGQF
Factor XIIa Factor XI KIKPR--IVGGTASVRGE
Factor XIIa Plasma Kallikrein KTSTR--IVGGTNSSWGE
Protein Ca T~actor VIII ELR--MKNNEEAEDYDDDLTDSEMD
~ t-PA/UK Plasminogen PKKCPGR--VVGGCVAHPHSWPWQVSI,RT
The Connector Moiety
The compounds of the present invelltion may contain a connector moiety which
interconnects the CSDM and the EAM, the connector moiet! 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 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 extension or as or through a side chain. However, if
the compound is a thrombin inhibitor~ the connector moiety mav 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, e.g. at its N-terminal end. In one class
of compounds the connector preferably comprises a peptide "spacer" and a non-peptide
20 ' linker". A representative connector structure is:
- wherein ~ represents a non-peptide linker and ~ a spacer comprising a sequence of
25 arnino acids, ~ and ~ suitably being joined by a peptide bond. The spacer ~ is

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preferably linlced to the EAM and the linker ~ to the CSDM, although compounds in
which 6 is linked to the CSDM and ~ to the EAM form a less preferred embodiment
included in the invention.
5 The linker is typically a residue of a compound having functional groups to react with
the N-terminal arnino group of the spacer and a functional group of the CSDM. such as
the N-terminal group. for example. A preferred linker. therefore~ has t~o carbo~ylate
groups~ e.,~r. is a dicarboxylic acid which can form amide bonds with the N-te minal
amino groups of the CSDM and the spacer. Particularly preferred linkers are a residue
10 of glutaric acid (HO,C(CH2)3CO2H) and homologues thereof of the formula
(HO,C(CH,)~,CO,H) wherein h is an integer of 2 or from 4 to 6. The all;vlene residue [-
(CI-I,)2 6-] may be substituted by one or more substituents which do not stericall! hinder
the linker, ~vhereb~ the desirable flexibility of the linker is maintained.
15 Less preferably! the linker may comprise for example the 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 t~vo adjacent Gly residues. normally at its N-terminal end. The length
20 of the spacer is dependent upon inter alia the position on the CSDM to ~hich the linker
is attached.
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
25 the same day as the present application.
The connector moiety may have one or more natural amide bonds replaced by other
linkages.
SYNTHESIS

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The compounds of the invention can be prepared by using, for example, generally
known methods for peptide synthesis and for coupling peptides. Jn an exemplary
method, the novel compounds are made by a solid phase synthetic technique.
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 i The
Chemical Synthesis of Peptides '. John Jones Clarendon 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 a_ainst
10 reaction with itself and. after coupling with the solid phase-linked amino acid. is
deprotected for reaction with a furtl1er 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 =
15 fluorenylmethylcarbonyl). In Fmoc chemistry (also L~nown generallv 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 (PS), though other solids have been used that have suitable
swelling characteristics in solvent. since it is now known that the peptide chain grows in
~0 the pores on the inside of the bead. An example of arl alternative solid is the polvamide
called Kiesulguhr.
The linker can be many things, but we prefer to use PEG (i.e. a polyethylene glycol
linker), which has an alcohol 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 para-hydroxymethylbenzoic
acid linker. The HMBA is esterified onto the PEG, and then the peptide or amino acid
30 (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
typically removed by a secondary base (e.g. piperidine) and the resulting free amino

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group is reacted with a selected Fmoc-protected amino acid; the amino acid sequence is
extended by repetition of these steps.
Another solid phase ~ynthetic approach is the Boc technique (Boc =
5 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% divinylbenzel1e. 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
10 carefullv!) 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 arnino acid; as with Fmoc
chemistry. the amino acid sequence is extended by repetition of these steps.
15 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 theiractivated carboxy termini) to via the N-terrnini generated.
20 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. R.P.;
Jones, D.A.; Broadbridge, R.J.; Corina, D.L. and Akhtar, M. A Novel Method of
25 Solid Phase Synthesis Of Peptide Analogues, in Innovation and Perspectives in Solid
Phase Synthesis, ed., R.Epton. 1994, Mayflower Worldwide Limited, Birmingh~m,
page 353-356; Letsinger, R.L. and Kornet, M.J. J.Amer.Chem.Soc., 1963, 85, 3045.)
Such N-terminal coupling methods may be used in making the products of the
30 invention. In one embodiment the CSDM, including any directly attached amino
acid(s), is synthesised by N-terminal coupling. This technique is especially useiul if
the CSDM has C-terrninal heteroatom group; in this method the resin bound peptide
-

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Il
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 sequenceFinally the peptide boronate (comprising the CSDM) is clea~ed from the resin by
strong acid (e.g. HF or TFA) prior to being joined to the remainder of the final5 product.
When synthesising compounds whose CSDM contains a P I -P2 non-natural amide bond,
it is convenient to premake as interrnediates the bindin~ subsite affinitv moiety [X-
(aa4)m-(aa3)n-(aa~) of Forrnula I] and the specificit~ pocket affinity moiety with its
10 attached C-terrninal group L(aal)-Z of Forrnula I]. The tuo intermediates contain
suitable functional groups to react together to form the target non-natural arnide bond
[~l/] and are caused or allowed to react together to lorm the compound (or a precursor
thereof to undergo one or more further functional group transforrnations).
15 Suitable synthetic techniques for making peptides cont~ining a non-amide bond ~ are
described in PCT/GB96/00352.
We have unexpectedly successfully synthesised peptide boronate esters using solid
phase chemistry. For example~ in an exemplary process for synthesising a serine
20 proteinase inhibitor in which the cormector moietv and its attached EAM forrn an N-
terminal extension of the CSDM. the EAM is prepared by Fmoc solid phase peptide
chemistry, e.g using an Fmoc-polyarnide continuous flow method. A suitable solidphase for this purpose is the pre-derivatised solid support Fmoc-Leu-PEG-PS. Thepeptide-conjugated resin is subsequently treated with~ for example. glutaric anhydride,
25 one carboxyl group of which reacts with the N-terminal arnino 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 exarnple by
treatment with 100% TFA.
30 Another method of using peptide boronate esters in solid phase chemistry is acornpletely novel technique in which boronic acids [-B(OH)2] are directly esterified onto
diols coupled to a resin. Chain extension is continued from the arnino group of the
, . . , . . . . , .. _

CA 022~8634 1998-12-17
W 0 98/0044~ PCT/GB97/OlS74 12
amino acid by, for example, standard Fmoc chemistry. The boronic acid ester is cleaved
from the resin by acid (e.g. 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 e~ample.
The invention therefore includes a method of making a compound of the invention,comprising performing tl1e following steps to make a target amino acid sequence:
(i) providing a solid phase having coupled thereto functional _roups capable
of reacting with an amino group or, preferably~ with a carbo~ l group or a
reactive derivative thereof;
(ii) causing the amino or carbo~cyl group (which may be in the forrn of a
reactive derivative thereof~ of a terminal amino acid of an amino acid sequence
I S 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
~0
(iv) repeatin_ step (iii) as often as necessary.
In step (i), the functional groups coupled to a solid phase may be on a moiety ~vhich is
incorporated in the end product compound, e.g. may be an amino group (which may be
25 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 (v)
of coupling a said sequentially following amino acid of a step (iii) to said preceding
30 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

CA 022~8634 1998-12-17
W O 98/00442 PCT/GB97/01574 13
to the amino group of said preceding arnino acid and the other to the amino group of
said following arnino acid.
The sequentially following amino acid of a step (iii) may be part of a larger moiety. e.g.
5 of an amino acid sequence optionally containing a replacement for a natural peptide
bond.
In the method, any one or more carboxvlate groups reacted with an arnino group may be
in the forrn of a reactive carbonvl-containing derivative thereof~ such as an activated
10 carboxyl group for example an acid anhydride.
Before use. the final compound of the solid phase synthesis is cleaved from the solid
phase, for exarnple hl a manner l;nown per se. The cleaved compound may be subjected
to one or more further chemicai reactions before the end product compound is obtained.
1~
In a preferred embodiment, the terminal arnino acid reacted with the functional groups
attached to the solid phase is the C-terminal arnino acid of the EAM and step (iii) is
repeated to couple successive arnino acids of the EAM sequence and successive arnino
acids of any contiguous connector peptide. to forrn an uninterrupted amino acid
20 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 exarnple the anhydride of a dicarboxylic acid, to bond one of the
25 h~vo carboxylates to the arnino group of the final arnino acid. The unreacted carboxylate
or carboxylate derivative is typically reacted with the arnino group of an amino acid~
which is normally the N-terminal amino acid of the CSDM. In this latter case, the
amino acid mav already be bonded to the rem~in(ler of the CSDM, i.e. the CSDM may
be separately made in whole (or in part) for joining to the unreacted carboxylate
30 ~derivative). The compound having two carboxylate groups is preferably a linker as
described above.

CA 022~8634 1998-12-17
WO 98100442 PCT/GB97101574 14
In some prefèrred methods, there is used a preformed CSDM having a heteroatom group
in place of a C-terminal carboxy group. The heteroatom group is preferablv a boronate
or boronate derivative as described above.
5 An arnino 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
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
I O reaction.
A first preferred method. therefore. comprises:
~ (i) 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
~0 molecules to react with the funclional groups of the solid phase:
(iii~ deprotecting the amino groups of the reacted amino acid. whereby the
solid phase becomes provided with free amino groups;
(iv) repeating steps (ii) and (iii) with successive arnino acids of the EAi~I and
optionally of a contiguous spacer peptide to forrn on the solid phase an amino
acid sequence from the C-t~rrnin~l of the EAM to, at the free end of the
sequence~ the N-terminal of the spacer;
(v) contacting the solid phase with a linker compound having two carboxyl
groups or reactive residues thereof, and causing or allowing linker carboxy

CA 022~8634 1998-12-17
W O 98/00442 PCT/GB97/01~74
groups or reaetive carbox,v residues to react with the N-terminal amino groups of
the spacer sequence:
(vi) contaeting the solid phase havinP the linker compound coupled thereto
with the N-terminal arnino acid of a CSDM sequenee and causing or allowing
the amino groups of the amino aeid molecules to react with the carboxy groups
or reactive carboxy residues of the linker compound, the N-terminal amino acid
of the CSDM sequence optionally being part of a complete CSDM:
(vii) if necessar~. repeating steps (ii) and (iii~ with successive amino aeids 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:
(i) providing a solid phase having eoupled thereto functional groups capable
of reacting with a carboxyl group or a reactive derivative thereof;
(ii) contaeting the solid phase with the C-terminal amino aeid of an EAM.
the amino aeid having a protected amino group and optionally a derivatised
carboxy group~ and causing or allowing the carboxy groups of the amino acid
molecules to reaet with the functional groups of the solid phase:
(iii) deprotecting the amino group of the reacted amino acid, whereby the
solid phase becomes provided with free amino groups;
(iv) repeating steps (ii) and (iii) with successive amino acids of the EAM to
form on the solid phase an amino aeid sequence from the C-terminal ofthe EAM
to~ at the free end of the se~uence~ the N-terminal of the EAM;

CA 022~8634 1998-12-17
WO 98/00442 PCT/GB97/01574
16
(v) contacting the solid phase with a linker compound having two carboxyl
groups or reactive residues thereof, and 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 carbo~y 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 repcating 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.
In either of the preceding methods the synthesised compound is preferably cleaved from
the solid phase by acid.
The preceding methods preferably involve the use of a CSDM arnino acid or amino
acid sequence (e.g. a complete CSDM) having a C-terrninal 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
indirectlv to the solid phase.

CA 022~8634 1998-12-17
wO 98/00442 PCT/GBg7/01574
17
In one class of processes of the invention. the functional 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 of methods, an amino acid whose side chain has an amino or carboxyl
group is coupled to a soiid 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 svnthesis from the amino group. ~he other functional
l 0 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 boron-containin~ peptides is. however, of applicability to any such peptides~ and
not only to the serine protease inhibitors of the invention.
Solid Phase Synthesis of Boron-Containing Comr~ounds
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
20 which the C-terminal carbo~y group is replaced h~ a boronic acid group or a
derivative thereof. Representative compounds are of the Formula II:
(aa)lj-B(R2)(R3), (Il),
25 wherein:
(aa)~ represents a sequence of amino acids (e.g. as in Formula I); R2 and R3 are each
independently selected from halogen, -OH, -oR4 and -NR4Rs, where R4 and Rs are each
independently a group of the formula R6(CO)U-, wherein u is 0 or 1; R6 is H or an
30 optionally halo~enated alkyl, aryl or arylalkyl group cont~ining up to (10 - u) carbon
atoms and optionally substituted by one or more groups selected from -OH, R7(Co)Vo-

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W O 98/00442 PCT/GB97/01574 18
and R7(Co)V-, wherein v is 0 or 1; R7 is Cl-c6-v alkyl, or is an aryl. alkylary h arylalkyl
or alkylarylalkyl group cont~ining up to ( I 0-v) carbon atoms,
or R2 and R3 taken together represent a residue of a diol or a dithiol.
Such peptide boronates are described, for example, in WO 9~/07869 (equivalent toUSSN 08/317,387), EP 0471651 (which corresponds to US 5288707) and USSN
08/240,606~ the disclosures of which are incorporated herein b! ret'erence.
10 As already indicated. we have now surprisingly found that boron-containing peptides
may be synthesised bv solid phase chemistry without serious degradation. One aspect
of the invention. therefore~ is tlle use of a boron-containin(J amil1o acid analogue in
peptide s- nthesis using solid phase chemistrv~ especially Fmoc chemistry (also known
as the "Sheppard approach").
In another aspect. there is provided a method of making a peptide or a peptide-
containin~ compound~ comprising performing the followin steps to make a target
arnino acid sequence:
(i) providing a solid phase having coupled thereto funcrional groups;
(ii) causing a compound reactive with the functional groups selectively to
react therewith, the reacted compound having a functional group capable of
reacting v.~ith an amino group or with a carboxyl group or a reactive derivativethereof;
(iii) causing an arnino or carboxyl group (which may be in the form of a
reactive derivative thereof) of a terrninal amino acid of a target amino acid
sequence selectively to react with said functional groups of the reacted
compound;

CA 022~8634 1998-12-17
WO 98/00442 PCT/GB97/01574
19
(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;
(v) repeating step (i~) as often as necessary; and
(vi) cleaving a solid phase-linked compound prepared using steps (i)-(iv)
from the solid phase by the action of an acid or base~
I() characterised in that a compound comprising a boronic acid '~roup [-B(OH)2] or a
derivative thereof. especially an ester~ is incorporated in the solid phase-linked
compound prior to its clea~age from the solid phase.
The method of the invention for making a peptide or peptide-containing compound
15 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 ~i. e. 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-cont~ining compound
20 may comprise a step of couplillg to a solid phase-linked compound prepared byprevious 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
25 compound capable of reacting with an amino group or, as the case may be, a carboxyl
group could thus be linked to the solid phase-linked peptide. Diamines are useful for
interconnecting moieties having carboxyl groups (e.g. in the forrn of reactive
derivatives thereof). Extension of a solid phase-linked peptide by a dicarboxylic acid
especially glutaric acid, is useful in the preparation of bifunctional serine protease
30 inhibitors having a CSDM joined. typical through its N-terminal. to an EAM through
a connector moiety comprising a peptide spacer portion and dicarboxylic acid residue
linker portion.

CA 022~8634 1998-12-17
W 0 98/00442 20 PCT/GB97/OlS74
A solid phase-linked compound whose free end terminates with a dicarboxylic acidresidue 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
5 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-terrninal amino acid of a
CSDM.
Step (ii) of the method may comprise reacting an amino group or an optionally
10 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 carbo~cy 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 C-
15 terminal carboxy group of the amino acid attached to the solid phase through its sidechain may be replaced by the boronic acid residue [-B(O~)2] or an ester thereof.
The method may comprise N-terminal coupling in SPPS, in which the carbo~
terminus of a resin bound peptide is coupled to a free a-aminoboronate acid or ester.
20 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 (lIla)
\OH
or /O-EI
aak-B (IIIb)
\o-E2,
where El and E2 represent boronic ester-forming residues or may together
form a sin~le residue,

CA 022~8634 1998-12-17
W O 98/00442 21 PCT/GB97/01~74
with a diol coupled to the solid phase. The technique of linking a boron atom (e.g. as
part of an amino acid or peptide boronic acid or ester) to a solid phase throughhydroxy groups coupled (directly or indirectly) to the solid phase is novel and forms
an aspect of the invention.
s
If the compound comprising a boronic acid or ester is an amino acid boronate used in
step (ii) in either of the preceding embodiments~ the solid phase svnthetic 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 diol-containing resins, and then
chain extension continued from the N-terminal end by. for example~ standard Fmoc-
chemistry. Subsequently the boronic acid ester can be cleaved~ either by mineralacids, to give the free boronic acids [peptide-B(OH)2]. or by transesterification, e.g. by
15 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
20 acids/esters (e.g. Xu,Z.-H., 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.,
1 997, 42, 3203-3205).
~5
A general procedure is as follows:

CA 02258634 1998-12-17
WO 98/00442 22 PCTIGB97/01574
(~Linker + Diol (protected)
(e.g. 'Wang" Resin)
(~Linker--Diol (protected)
I ) Deprotect
Linker-- Diol
1) XN-CHR-B(OH)2
or XN-CHR-BO2-Ester
~Linker--Diol--B-CHR-NX
I ) remove X,
2) couple new amino acid
Linker--Diol--B-CHR-NHCO-(aa)Y
repeat steps of SPPs
B-CHR-NH-Peptide-Y
1 ) Base
2) Lewis acid (e.g. TFA)~ scavenger
HO\
B-CHR-NH-Peptide
HO/
The diol is a compounds having two or more alcoholic hydroxy groups.
X and Y are protecting groups.
R is the side chain of an amino acid boronatelboronic acid.
5 Typically, the resin is washed after each step. In suitable embodiments the diol is not
protected before it is reacted with the resin.
- A more specific procedure is set out below:

CA 02258634 1998-12-17
W O 98/00442 PCT/GB97/01574
23
-- C H2--C I Na 0~~~
~Resin) ~ X
\ O
(e.g. 'Wang' Resin) (Sodium salt of Solketal)
1 )24h, 2 fold excess sodium salt
2) wash
~Resin, Ox
\
1 )M ineral acid
2 )wash
' CH2--~~
Resin
''" --OH
1 )(TMS)2-N-CHR-3(0H)2
or (TMS)2-N-CHR-BO2-Ester
2) wash off excess
C H2 ~r ~B c HR-N(TMS )2
) Fmoc(aa)-OH, I-BuCOCI, Morpholine, Et 3N.
or Fmoc(aa)-OH, activating agent, (Bu)4NF.
2 )was h
~C H2 ~ u~
Resin I I B-CHR-NHCO-(aa)Fmoc
\ ~ ~O
repeat steps of SPPS.
C H2~ ~ B-C HR-NH-PeptideFmoc
1 ) Base
2) Lewis acid (e.g. TFA), scavenger.
HO
B-C HR-NH-Peptide
HO
TMS = Trimethylsilyl
SPPS = soiid phase peptide synthesis
SUBSTI~UTE SHEET (RULE 26)

CA 02258634 1998-12-17
W 0 98/00442 PCT/GB97/OlS74 24
The invention thereforc opens thc ~a!l to the preparation of peptide boronates by solid
phase chemistry, for e~;ample in preparing a library of peptide boronates by a
combinatorial method.
l he in~elltioll includes a nlethod tor mal~in~ ~ compound colllprisin~ ~ pcptide
boronic acid or peptide borollatc es~.r. the method comprisino:
pro~iciilll~ a solid nllase lla~in~ coupled tilereto alcollolic h~dro~-
~~roups:
I()
(ii) causill(~ an al~ lo ac~d borollic acid or peptidc borollic acid to react
\\ith the ll!dr()~ roups ~ilereb~ Tl~e borollic ~cid rcsi~uc becomcs esterified
to the solid pllasc:
I ~ (iii) causin~ lllc c~rbo~ roup oi the amino acid scquentiall~ follo~in~, in
the end producl r~eptide boronic acid or boronate ester selecti~el! to react with
the amillo ~roup of thc sequcntiall~ precedin~ amino acid coupled to the solid
phase:
'O (i~ ) lepCa~ill" StCr\ ( iii ) as oltcn as ncccssar!~;
(~) cle~ in~mhe resultant peptide boronate trom the resin
the metllod optionall! comprisin(~ one or more further steps to mal~e said compound
_. ~
The alcoholic hydro~ groups coupled to the solid phase are preferably arranged such
that pairs of the groups can be bonded to a boron atom. i.e. such that a boron atom can
be di-esterified by them:
-O\
~0 B
-0/
SUBSTmJTE SHEET (RULE 26)

CA 02258634 1998-12-17
W O 98/00442 PCT/GB97/01574
In some embodimen~s the hydro~;y groups are in a 1 2-arrangement (i c. on ad~acent
carbons); in other embodiments they are spaced apart on chains le. . a residue of'
NHfCl~,C~ OH)~)
I'referabl! eac}~ amillo acid coupled to thc solid pllase has ~ protected amino group
and step (iii) comprises deprolecting tl-e alllino ~roup of the sequelltiall~ preceding
amillo acid. I're~erablv the clea~age of step (~ ) is pelforllled ~ acid or h~
~ranses~erlllcanoll.
lore generall~. Ihere is pro~ided the use hl solid phase s!~ntllesis ~ horonic acid
resiciue a~tacile(~ to ~he so~id pllase ~hrou(11l hydro~;! gro~lr) residues
.~lso provici~d is ~l meth()d ~or mal;ima ;1 compound colllprisil~ oro~ a~om. ~he
nlethod COmprjsino:~
(i) providing a solid l hase havino coupled thereto alcoholic h!dro.
groups:
(ii ) causinn a boronic acid or boronate ester to react ~. ith the h~dro~
~() g1rouDs ~ hereb! the t oronic acid residue heconles esterified t~ thc solid phase:
and
(iii) performing one or more further stéps to ma~é said compound.
The aicoholic h! dro:~ groul-s are preferably arranged as deseribed above.
In other aspects. there are provided a solid phase material having coupled thereto
boronic acid residues through hydroxy groups~ as ~ell as a solid phase material
havino coupled thereto a moiety of the Formula IV:
SUBSTITUTE SHEET (FlULE 26
, , . . ~ . .. .~ .. . .. . . ~

CA 02258634 1998-12-17
W O 98t00442 PCT/GB97/01574
26
_oi \
B R (IV)
~0/
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 runctional groups reactive with alcoholic
hydroxv groups (but the materiai may conlaill such functional groups in prolected
forrn, e.g. prior to deprotection). In another class of material such functional groups
are unprotected. protecting groups ha~ lg previously heen removed. R is typicallv an
organic moiety ha~in~ one or more functional groups to enable R to under_o a
cl)clllic;ll rca~nion: ~n! prolccl~hle l'ul~clion;ll ~rour~ prolccled. In one ~:las.s
I (~ of malerials. the soli~ ph~e ~a~; cour)l~ Iherel~- a moic~! ot l:omlul;l V:
C--O
B 2
O (~')
One or both of the hydrogen aloms of the -CH,- group may be replaced by olher
groups compatible ~vith the use of the material, e.g. aikyl groups (for example methyl
20 or butyl).
In a yet further class of materials, the left hand o,Yygen of Formula IV is part of an
ester.
25 A first elass of solid phase synthetic methods comprises perforrning the following
steps to make a target amino aeid sequenee:
(i) providing a solid phase having coupled thereto functional groups
capable of reacting with a carboxyl group;
SUBSTITUTE SHEEr ~RULE 26

CA 02258634 1998-12-17
W O 98/00442 PCT/GB97/01574 27
(ii) causing a compound reactive with the functional groups and
comprising an amino group protected by a base-labile 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
pro~ecled by a base-labile protecting group to react with the deprotected amino
group resulting from step (iii);
(v) deprotecting the protected amino acid ~ a basc;
(vi) causing the amino acid sequentially t'ollo~in~l. in t~le tar~cl .~-~uellce.1() the sequentially preceding amino acid coupled to tile solid pl)~se to reacl ~illl
the deprotected amino group of the sequcnlially preccdin~ aminc) acid. Il~e
~;cquenliall! t'~ anlin(l aeid ~la~ing i~ a~ o ~rollr rr~ .t.~l h! ~ ha~.-
labilc pro~cc~ing ~roup~
(~ii) deproteclinP the prolected alnino a(:id ~rour~ ~ilh bas~:
I ~ (viii) repeating steps (vi) ~nd (viii ) as oJ'tcn as necess;lry; all(l
(i~;) clea~ ing thc acid labilc bond with acid or ! y tr~lls~sterilie~tlon.
characterised in thal ~ compound comprisin~ a horonic acid group [-B(OH)2] or a
derivative thereot~. especiallv an ester. is incorporalcd in the solid ~hasc-linlicd
~() compoun(l ~rior t(l clea~a c ot'thc acid lahilc bond.
As described above in relation lo the method of making a peplide or peptide-
containing compound. step (ii) of the method may comprise reacting an optionallyderivatised carbo:~y group of an amino acid with a functional group coupled directlv
25 or indirectly to a solid phase~ for example by a method known p~r- ~e 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. Alternalively, step (ii) may comprise
reacting the compound comprising a boronic acid or ester group in the forrn of an
amino acid or peptide boronic acid or ester with a diol coupled to the solid phase. In
30 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.
SUBStlTUTE SHEEr (RVLE 26~

CA 02258634 1998-12-17
W O 98/00442 PCT/GB97101S74
28
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 follo-ving
~ steps ~o make a targel amino acid sequence:
(i) providin~ a soiid phase having coupled thereto functional groups
capablc of reacting ~vith a carbox~l group;
(ii) causing 3 compound rcactiv~ ~ith ti~e lullctional groups and
conlprisillg an alllitlO group protectcd h~ arl acid-lahllc ~rotccting ~roup to
rcact with ~he functional groups. to form a base labile bond;
(iii) dcprotectillg the amino group ~vith an ~cid;
1~5 (iv) causing thc carbo~yl group of an amino acid ~vhose amino group is
pro~ectcd by an acid-labile protccting group to react ~ ith thc de~rotcctcd
amino group resulting from step (iii);
(v) dcprotcctillg ~hc protected amino ~cid ~-ith an acid:
~ i) causinsg the amino acid sc-lucllliall~ follo~vin in the target scquence~
'O thc scqucntiall~ l~rcccding anlino acid couplc(i to thc solid phase to react with
the deprotected amino group ol' the sequentially preceding arnino acid, the
sequentially follo~ving amino acid having its amino group protected by an
acid-labile protec~ing group;
(vii) deprotecting the protected amino acid oroup with acid;
(viii) repeating steps (vi) and (viii) as oRen 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~23 or a
derivative thereof, especially ester, is incorporated in the solid phase-linked
compound prior to cleavage of the acid labile bond.
SUBSTmlTE Sl IEET (RULE 26)

CA 02258634 1998-12-17
W O 98/00442 PCT/GB97/01574 29
The above-described varianls of the first class of soiid phase synthetic methods are
applicable also to the second class.
As described above in relation to the method of making a peptide or peptide-
containing compound, methods of the first and second classes of solid phase synthetic
methods mav comprise a step of coupling to a solid phase-linlied compound prepared
by previous s~eps of the process a compound other than an amillo acid or peptide
10 ~Svnthcsi.~ ncrall)
I ile e()mpouud~ of Ihe i~ enlloll do not have tc coluail~ horolu allhoul~h Ihe~ ma~ do
so Thc non-boron conlainino con-pounds may also be pr-:pared bv solid phase
svn~ sis. ~;ome conlpounds of the invenlioll have a nalural peptide bond replaced by
no~hcr lin~lo~ ~rour l:urthcr inronnalion o n mcttlc)~is suitahlc for the synthcsis of
hc con~r~-un~i~ o~ in~nlion mJy h~ found in t!~- ;Jlorcsai(~ patcnt applic;ltionlilcd today al~d entitle(! I hromhin Inhibi(ors''
-() USF,
The novel compounds accordhle to the present in~ention are useful as inhibitors or
substrales of ~ rine proteases, e,~ thrombin. an(l 111;1~' he used in vir~o or in vivo for
diagnostic and mechanistic studies of such enzymes. More generally, the novel peptides
2~ may be useful for research or synthetic purposes Furthemlore, because of their
inhibitory action, the inhibitors are useful in the prevention or treatment of diseases
caused by an excess of thrombin or another serine proteases in a regulatory system
particularly a mammalian system. e.g. the human or animal body, for example control of
the coagulation system. The pharmaceutically useful compounds have a
30 pharmaceutically acceptable group as any N-terrninal substituent (X).
SUBSTITUTE SHEET (RULE 26~
. .

CA 02258634 1998-12-17
W O 98100442 PCT/GB97101574
The anti-thrombotic compounds of tlte invention may be employed when an anti-
thrombogenic agent is needed. Generally these eompounds may be administered orally
or parenterally to a host in an effective amount to obtain an anti-thrombo~enic effect. In
the case of larger mammals such as humans. [he compounds mav be administered alone
or in combination ~vith one or more pharmaceutical carriers or diluents at a dose of from
0.02 to lOmg/K~ of bodv ~veislht and preferably l-lOOmg/Ko. to obtain the anti-
lhrombo~enic effecl~ and m;ly be given as a single dose or in divided doses or as a
sllstained releasc tonmll.ltion ~ en an e.~;tracorporeal blood loop is to be established
f~or a patienl 0.1-l()n~ ma~ be adminis~ered intravenousl~ l~or use ~ith whole
I () I-lood f'rom I -100 111~ pcr lilre ma~ be provided ~o pre\ ent coa~ulation.
Illlanllaeeutical dilllcl-ls or c;lrriers lor hun~;ln or ~el~nl~.lr~ use are ~ell ~;no~-n ~nd
mclude sllo~rs slar(:llcs a~ a~er. ~nd ma~ t-c used lo m~e acceptable fom~ulations of
-hannacculic31 comrosilioll~ (humaJI or ~elerinar!) colll;linill One or mort: of the
~uh!ect rertides in lhe required l-h;ln-n;~ceu~icall~ 3rprorn;lle or effeeti~e 3moun~ nr
.oncentrali(-n Th~ rh;~ml;lcelltie;~l fi-rnlul3tinni ~113~ tc in unil do~ll e f~-rm.
I omlulatiolls ot tllc con~ ullds includc tahicts. c;lrs~lles. injectable solutions and the
I i~e.
'O l~le anti-coa~ul~nt com~un~s ~ t thc ill~enlion ma~ o t)e added to bloo(i for the
purpose of preventillg coa~ulation of the blood in blood collecting or distribution
containers tubing or implantable apparatus ~hich comes in contact ~vith blood.
Advanta~es enabled by the compounds of the invention include oral activity rapid onset
~5 of activitv and low toxieitv. In addition. these compounds mav have special utility in
the treatment of individuals ~vho are hypersensitive to compoullds such as heparin or
other known inhibitors of thrombin or other serine proteases.
The methods of the invention are useful for the synthesis of serine protease inhibitors
~0 and other compounds. They are useful in combinatorial chemistry.
SUBSTITUTE S~IEET (12ULE 26)
.... ... .

CA 02258634 1998-12-17
W O 98/00442 PCT/GB97/01574 31
The invention will be fi~her described and illustrated by the E,Yamples whieh now
follow.
5 EXAMPLES
In Il1e e~;amples, amino acid residues are of L-conf Iguralion ~Inless other~vise stated.
1)-Phe-Pro-~orol3~g(~ CO(CI~ ('OC~ r~)Hir~ l '~
a. Gl~ G~yGln(Tyr6~)Hir'l~'J
Gl~Gl~Gll~ r'~')l3ir 1 \~hich h.~ lhe amino acid lormlll;l: H-Gl~-Glv-Gln-His-A~n-
Gly-~sp-l'he-C~lu-(ilu-lle-I'ro-~ilu-'l'~r-Leu-OI~ s r)re~ r~d h! ~olid phase r)e~ e
15 chemistr~ (-n ~ cll ()()~o ~ S~nlh~ cr ~l~in~ lloc-r~ mid~ conlin~
nO~ method ;~nd proprie~ 0~0 I'lus on column moni~on~ r~. I're-d~ d
solid support~ ~moc-Leu-~E(~-~S (1.6~, 0.~meq ~ u~ed t~lrou~ ou~ n1c)c-l.eu-
PEG-PS comprises polyeth~lene glyeol derivatised pol!st!rene ~ith 1~1BA lin~;er.Fmoe groups ~-~ere removed using 20% piperidine in 1)~ moc-amino acids (~
'-O equiv.) as their pen~atluorophel1vl esters ~ h side ch;lin rrotection ~here a~pro~n~te
(e~g. Fmoc-L-Tyr(tBu)OPfp~ Fmoc-L-Glu(tBu)OPfp, Fmoc-L-Asp(tBu)OPfp, Fmoc-L-
Asn(Trt)OPfp and Fmoc-His(boe)OPfp, were eoupled sequentiallv. Onee the requiredpeptide sequenee was eomplete the N-terminal Fmoe group was removed using 20%
piperidine in DMF. A positive ninhydrin test indieated that the Fmoc group had been
25 removed. The peptide-conjugated resin was subsequentlv decanted on a filter and
washed 'off line' with diehloromethane, methanol and diehloromethane before being
dried in-vacuo for a few hours.
b. HO,C(CH2)3COGlyGlyGln(Tyr63)Hirsl~64
The peptide obtained in Example la was suspended in DMF (5ml) and treated with
glutaric anhydride (300mg) and 4-methyl-morpholine (200mg) in a round bottomed
SUBSTITUTE S~IEET (RULE 26)

CA 02258634 1998-12-17
W O 98/00442 32 PCTtGB97/01574
flaslc (25ml). The reaction mixture was swirled overnight. The resin was washed with
DMF, DCM and MeOH, and then dried in-l~aCuo ovemiPht to obtain the target
compound.
5 c. H-D-Phe-ProBoroBpgOPin
il-D-Phe-~'roBoroBpgOPin was prepared by adding a 40% solution of HBr in acetic
acid (20ml) lo Cbz-D-Phe-r'ro-BoroBpgOPin (2~) in a round bottomcd fiask (lOOnll)
f~lle~l ~vitll a septum and fluslled wilh nilrogen. The flas~ was s~irled IO cffcct complcte
I () dissolu~iol) of the protected tripeptide. When tlle gas evolution ceased after
apr)roximatelv 30 minutes~ anhvdrous ether (200nll) was added and the reaction mixlurc
lorc~ efriger;llor for ~ ho-lr~ The re.lctioll ~oi.~;lurc \~a~ ere~ the rcsidlle
isSOI\~'d Ill l,tOI~ (llnl~ al~d dry eti~er \\as aJ ied 1~ rre~ ate Ihe inrodu~e(8()()111~,) a~ a ~ ile solid (~ 16~ Tlc (CIM/~. 9515/3)~ Rt-0.0~.
I ~
l)-I'hcl'roiUorol~ip~ inl~ O(C'l{.).lC()(;l!.(,ln(T! r ~)llir
'I-c .svnthesise [-D-Phci~rol3OroBpoOPin]CO(C'i ~)3COGly.Ciln(1'~r63)Hirs~ the dry
resil~ HOCC)(C~ CO~Ilv.Ciln(Tyr~'')i-~ir 1~ \~ai susr~7ndeti In 1)~ (10ml)~ before
3'1'1J (I''?nlg. ().~mn~ol) al~(l ll-i)-l'ile-l'roi3(!ro~3~0Pin ('~()n~ mmol) were
added to the reaction mi,~;lure. ~fter 5 nlinutes s(irring, ~nethylalnine (~(img. 0.04mmol)
was added alld tlle tlasl~ lel't stirring overni~hl.
The fully protecled peptide resin ~vas ~vashed ~ ith dichloromell~ane, methanol and
25 dichlorometllane and ~hell dried under vacuuln. CleavaPe from the resin with
simultaneous deprotection of side chain protecting groups was achieved by 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.
Purification of the crude peptide was carried out by reversed-phase HPLC using aVydac C-18 pl~)d~dli~te column (TP silica, 1011m, 25mm x 300mm). The column was
SUBSTITUTE SHEEr (RU~E 26)

CA 02258634 1998-12-17
WO 98/00442 PCT/GB97/01574
33
eluted with a 30-90% linear gradient of solvent A (0.1% TFA in water) and solvent B
(0.1 TFA in acetonitr'ile). The column eluants were monitored at 230nM, and fractions
were collected appropriately. The purity of the products were determined by analvtical
RP-HPLC and mass spectrometry
2 Cbz-D-Phe-Pro~ (CO,)-horoethvlglvcine pinanediol
~ . Cbz-D-Phc-~ro-~y(CO.)-Boro F,tg inin:lnc~iol
I-Chloroclhane-pinanediol boron~te estcr (0.321g,1.25xl0-3mol) added ~vith stimn~ to
Cb-~-D-l'l~c-Pro-OH (0.6~ 10 'n~ol). ~ cn l~e addi~ion l~ad bccn compleled~
DBU (0.23g. I.5~mmol) in CH~C'I~ ~as added to thc mi~;ture ~nd allo~ved to slir at room
lempcr~ture, before bcin lefi to slir for an c~;tcndcd ixriod ~ 4~C before ~vorkup. The
oi~aquc liquid ~ shcd ~ith ~iCI (o 1~ ;50 ml). I~'aHCO3 (1%. 50ml). The
or~nic la!er was dried i ! vi oro~ls stirring o~er anh~drou~ gSOJ, ~nd filtercd off. to
remove the desiccant. The filtrate ~vas concentrateci under reduced pressure on a rotary
cvaporator~ to afford a thici~, viscous rcsidue. i'reliminary c,Yamination by ~H N.M.R.
showed the required crude product Thc crude sampie ~vas dissolved in ;l small amount
~0 of MeO}i, applied to the sephadc~ L}i~0 column. ~n i then eluted ~ h a pump using
the sarne solvents. The elution profile ~as followcd ~vith the aid of a U.V. Iarnp
(226nM) and recorder. The void volumc, fraction 1-6~ and a further buli~ volume ~vere
collected. From the shape of the cluomatoeram it \~as dcellled that frzctions 1-6 ~-ould
be the most likely fractions in which the tripeptide m~y be found. The fractions ~ere
'5 concentrated individually to afford clear sliehtly coloured viscous residues. One
fraction containing the bulk of the material ~vhen placed under high vacuum was later
afforded as a slightly crystalline product (0~269 yield of 35%). N.M.R., FABMS (Fast
Atom Bombardment Mass Spectrometry) and C~ H, N were very strong (good)
indicators that the compound has been formed.
SUBSTITUTE SHEET (RULE 26

CA 02258634 1998-12-17
W O 98/00442 PCT/GB97101574
34
~ ~ <~ ~ CO~
~ CH,
1). ~/-Phc-l'ro-~lJ(~0.)-130roEtg l~in;lncdiol
Cb7-l)-Pl~e-PIo-l~J(CO.)-Bornl,tc pinanediol (~'rom E~;3mple ~a) ~-as dissolved in
0Inl ) .II)d Ir~ r~ 1 ()~~ô r(l/c . ~nd r)ur~r d \~ it~l ~r~on ~ h ~itimn~. lhc nas~
c~.lc~J;~ted an~ n.~.d ~"i. il. ~ l. stirrin~ l~or ~ inh~drin sl~ining indicated
dcpro~cted product ol~ TLC'. 'I'he sol-ltion ~v~ pur~ed ~ith ~r~on for 10 min. filtered
~n(J concc:ltr~ted und-r rcduc~d r)ressurc to af~ord a tl~icii blac~; oil. ~hich was dissolved
t~ in ('IICI;. I;ller~d all(i concen~raled. ~11 N.l~.R. of thc crude rroduct indicated no
protccled producl. 'rl~e residuc from above was chrom~tographed on a Sephade.~ LH20
chronlato~ra~ colullln 11~ (60 MHz) N.M.R. showed that the isolaled compound
dis~ e~l n);ln! ol' 11~ char~cterislics e.Ypected on the b~sis of ~I)e ~ 3tiv~ structure.
1~2nlE~ of t~lc ~'rce ~mino b,oron~ne ester ~as isol;~ted.
I ~
~. H-Phe-L-Glu-BoroBpgOPin
a. Fmoc-L-Glu(PEG-PS)OH
Tetrakistriphenylphosphine palladium(0) [PdP(Ph3)4] (Ig) was dissolved under Ar in a
solution of CH3CI containing 5% acetic acid and 2.5% N-methylmorpholine (30ml).
This mixture was transferred under Ar to a flask containing Fmoc-L-Glu(PEG-
PS)OAl ( I .6g). The resin was left to stand for 2 hours with occasional gentle
25 agitation. The resin was filtered on a sintered glass furmel and washed with 0.5%
SUBSTI~UTE SHEET (RULE 26~

CA 02258634 1998-12-17
WO 98/00442 PCT/GB97/0lS74
diisopropylethylarnine and sodium diethyldithiocarbamate (0.5%w/w)in DMF
(300ml) to remove the catalyst.
b. Fmoc-L-Glu(PEG-PS)NHBoroBpgOPin
S
The dry resin Fmoc-L-Glu(PEG-PS)OH (1.5g) was suspended in DMF(lOml) under
~r. TBT~ ( I 29mg, 0.4mmol ) alld ~I{.I30roBp~Ol'in ( I 65m~. O.5mmol)w were
;Idded to the reaction nli~tur~ 'ter 5 minutes ~irring. ~riethylamine (40mg,
().~mnlol) was addcd and Ihe t1as~ lcl-t stirring o~erni~ht. The resin was w~shed with
10 dichloronlethanc. melhanol and dichloronlethane and thcn dried under vacuum.
, - (; l u ( l~ r~ o ro ~ tl
ll-l'~c-l -(ilu(PEG-PS)NI1130roi~ 0Pin ~ S pre~;lred h! ~olid pi~ase chemislr~ on a
I 5 .~ l i lligcn 9n~0 r)eplide s~ nlhe~i~er
l:n~ Lr~)ur ~ r~mo~d ~rl~n~ e ~lid .~ur)por~ l'moc-L-Glu(PE(',-
30rOI~pL()I>ln U~ )~o ~lreri(iil~-' In ~ 1 . I nloc-l'~-Ol'fp ~-as coupled tO
~hc frce N-tenninu~.
~0 ~I~he l~rolcc~cd ~ep~ide r~;in ~ ashe~ dichloronl~h;lne. meth~nol and
dichloromelhane and then dricd under v~cuum.
(1. H-Phc-L-Glu-RoroBpgOPin
25 Cleavage of the peptide from the resin was achieved by treating the resin with 100%
TFA for two hours. TFA ~vas removed and the free peptide H-Phe-Glu-NH-
BoroBpgOPin was generated by precipitation with cold dry ether. The crude peptide
was collected by filtration and washed with further portions of ether.
30 Purification of the crude peptide was carried out by reversed-phase HP~C using a
Vydac C-18 preparative column (TP silica:particle size lOmm; 25mrnX300mm). The
column was eluted with a 30-90% linear gradient of solvent A(0.1%TFA in water)
SUBSTITUTE SHEEr (F~ULE 26~

CA 02258634 1998-12-17
W O 98/00442 PCT/GB97/01574
36
and solvent B(O. I %TFA in acetonitrile). The column eluants were monitored at
230nM, and fractions ~vere appropriately coliected. The purity of the products were
determined by analytical RP-HPLC and mass spectrometry. Product H-Phe-Glu N~
BoroBpgOPin, was obtained in 17% yield, 34mg, ES-MS: 626 [M+Na]; retention
S time analytical HPLC (4x250mm, Vydac, C-18 techsphere), eluled by 10-60% MeCNwith 0.1 % TFA in water with 0.1 % TFA over 25 minutes, gave Rt 23.1 minutes.
4 At~achmen~ of E~oroni~ Acid to l\lerrif~cld Resi
. I)criv:ltisation of Rcsin ~ith protcctc~3 Diol (2,~-dim~tl)~ io~;olanc-4-
mctll.lnol
~~\rC~-Cl ~;1 0 1 ~ /
0>~
11
(~CII.-O~
Na (solid, ~g? is added lO 2.2-dimetl1yl~ -dio~;olane-~-methanoi (~Omi ) under argon
gas, alld the mixture stirred until it gives a clear solution. Merrifield Resin (Sigma. I . I
MeQ. Cl per gram~ 20g) is added and thc mi~ture stirred overni~ht then hcated ~t 80~C
25 for 2~h.
Derivatised resin ~~as collected by filtration, washed by 1,4-dioxane (1 L), water
(3xSOOml), and I~ileOH:water (1: 1, 3~;500ml), MeOH (3xSOOml) and dry ether
(3xSOOml). An infra red spectrum was obtained by powdering of 1.5-2mg of resin with
~0 KBr (dry, 300mg) and compacting into a disc, then sc~nning on a Perkin~ 1600 Fourier
Transforrn I.R. rne derivatised resin (Fig. 2) compared to Merrifield resin ~Fig. I~
shows distinct stretching signals IOSO to I lSOcm '(s) for ether stretching frequencies
SUBST TUTE SHEET (RULE 26)

CA 02258634 1998-12-17
WO 98/00442 PCT/GB97/01574
characteristic of a five membered ring; and dialkyl ether stretching at 1060 to 1150cm~
I(s) for alkyl-alkyl stretching.
b. Deprotection
~ 1~--X (~ --~ OH
The derivatised r~sin ~vas mi:~ed with HCI (I SM, 250ml) and 1,4-dioxane (250ml) and
~he suspcn~io~ irrcd ~nd heated at 80~C. Atter 72~1 she resin ~v~s ~ashed by ~a~cr
(SOOml), ~c()~i (50()ml). DCM (-OOnll! and ~t.O (SOOml). thcn dri~d in lh~ air. ~
I.R. spec~rum o~ the resin shows distinc~ O-H stretching frequencies at 3400-3550cm '
I () (s), ~nd a main pca~; at 3413.6 (Fig. 3); this peak is substantially iargcr than the signal ~t
~917.6cnl l. In con1parison the ether (T~ig. ') and Mernftcld rcsin sho~ only a ~c~; _
3~00cn~ i onal for b~c~ground moistur~ .
c. Rcaction of thc Derivatisc~l Rcsin ~vith a boronic acid
~OH
~--CH~-O
Resin
/ --OH
~CH2-O~ \
~ B--R
0
The diol resin (Sg, 5.5mrnol of diol) was suspended in THF (dry, SOOml) and
phenylboronic acid (3.35g, 27.5mrnol, 5 equivalents), and 4A sieves (dried at 150~C).
SUBSTITV~E SHEET (RULE 26)

CA 02258634 1998-12-17
wo 98/00442 PCT/GB97/01574
38
After stirring under argon overnight, the resin was filtered under argon in a closed
system~ washed by THF (SOOml) and dried under vacuum. Ft-L.R. (Fig. 4) shows a
strong signal at 1026cm~l (aryl-alkyl stretching frequency) for the phenyl ring and a
weak signal at 341 7cm~l (compared to Fig 3, for the starting diol).
~ef. Leznot'f, C.C. and Wong, J.Y., The use of Polymer Supports in Organic Synthesis.
III. Selective Chcmical Reactions on One Aldehvde Group of Symmetrical
Dialdehvd~ n .I Chcm . 1973. 51. 3756-3764
I O
An,ll~ tic~ n(l ~cti~ t;l
~ ~ n.~ llo~ i"~ hl. I ~-)n~ain~ acti~ it\ d~l~ relalin~ to thc in~elltioIl In the Tablc, tl1e
n3~ r. ;~ nl~ hn~ lo~c;lrhon!i and ' ~ ir' refer~i lo normal hirudin.
'~iHir~ 6 l(d.~-.';) r.t~.r~i lo tilc amino a~id sequenc~ from amino acid 49 to amino acid
6~ of nonl~al hlnldin in ~hich Ih.~ nati~e l ! r(OSO3~1)63 is replaccd b~ T~r.
~() Tl~e COI~ In~ te(~ ~I'able I ~ rc r)r~ r~d b~ ~he s.~n~e or analol~ous mclhods to
~he compounds of thc prepar~tion E.~;~rnples I and ~ abo~e or, in the case of
interrnediates. ~ere obtained from sources
The following t~clmiqucs ~erc employed for activity measurement:
'5
Plasma thrombin time (1~
A volurne of 150~L1 of citrated norrnal human plasma and 20~LI of buffer or sarnple were
warrned at 37~C for 1 min. Coagulation was started by adding 1 501l1 of freshly prepared
3 0 bovine thrombin (5NI~u/ml saline) and the coagulation time was recorded on acoagulometer.
SUBSTITUTE SHEET ~ULE 26)

CA 02258634 1998-12-17
WO 98/00442 PCT/GB97/01574
39
A phosphate buffer, pH7.8, containing 0.1% bovine serum albumine and 0.02% sodiurn
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 concentratjon
as that used in the samples. The inhibitor concentrations were plotted against the
thrombin times in a semilogarilhmic graph from which the inhibitor concentration that
causcd a doublin(~ (40 sec) of the thrombin time was dclerrnined.
r)etcnnin~ltion of ~i
The inhibi~ion of human a-thrombin was detemlincd bv the inhibition of the en~yme
catalysed hydrol~sis ol~ thrcc dif~erent concentratiolls of thc chromo~enic substrate S-
~00~l1 of ~;anlpl.~ or bufl~r an~ 50~11 of ~S-~38 ~crc i~ ut~alcd at ~7~C for I min and
50~11 of human (~-thromhin (0 ~ ~'IH,~/ml) ~35 addcd. Tllc illiti;li ratc of inhibilcd and
uninhibit~ r~ ons ~r~ rc~or~cd a~ nn~ c ll-~r~ in ol.tical ~.nsit~ w~s
plotlc(l accon~illo to thc Illc~h-)J ol Llnc~ r aJl~ ur~c. Ihc ~;m alld ~r)parelll ~;n~
~ere determincd ~nd Ki ~vas calculated usin~ thc rclation!;hip.
~= Vma~;
I+Km . (l+~l)
[Sl Ki
The buffer used contained O.IM sodium phosphate. 0.2~1 NaCI, 0.5% PEG and 0.02%
sodium azidc. adJusted to pH 7.5 with orthophosphoric acid.
The sarnples consist of the compound disclosed in DMSO.
The reader is referred to Dixon, M and Webb, E. C., "Enzymes", third edition, 1979,
Academic Press, ~he disclosure of which is incorporated herein by reference, for a
further descnption of the measurement of Ki.
SUBSTITUTE SHEET (RULE 26

CA 02258634 1998-12-17
W O 98/00442 PCT/GB97/01574
T~blel
Ki~M TT
~M
98 GGGDFEPIPL n/e 100
99 [-BoroBrOPin]CO(CH2)3COGGGDFEPlPL n/e 58
105 GGGGDFEPIPL n/e 94.9
106 GGGGGDFEPIPL nle 15
107 [-BoroBrOPin]OC(CH2)3COGGGGDFEPlPL n/e 58.4
108 ~-BoroBrOPin]OC(CH2)3COGGGGGDFEPII I n/e 63.4
114 GGNSHNDGDFEEIPEEYL llir~ 0.613
1~1 HO.C(CH )lCOG( GGGDFI ~lrl 0.73~ .09
1 ~ L-L-l l cProl30roV~lOrin]OC'(CI1.)3COG D~ .I Ii 1.n i~ 11 7 ~7.1
129 [-D-PheProBoroV~lOPit1]OC(C'H~)3COG DI:I.I II I. 16.4 33.5
137 (-D-Phe-ProBoro~t~OPil-1OC(CI-3 )3C0G Dl:l.l ll II. 10.
166[-D-Phc-ProBoroBpgOPil1~C)C(CH.)iCOG~N~llr~ (~e-~in 0006140() 9
167 HiriY~(des-S) n c 0.1 ~ ~ T
175 [-D-Phe-ProBoroBpgOPinlC)C(CH2)3COG4Nliir~ ~((I:s-~;)0.00211 0.06~
176 Z-D-Phe-Pro-BoroBpgOPin + I iir~ 0.021 ~ 0.636
182 [-D-Phe-ProBoroCc .OI inlOC(Cli2)lCOGPGGNHir ~(des-S)0.00271 N.T.
IB4 HO C(CH2)3COGPGGNllir~ (des-S) 12.7 56.7
185 [-D-Phe-ProBoroCegOPin]OC(CH2)3COGPG3NHir ~ (des-S) nieO.75 7.36
186 [-D-Phe-Pro-BoroCegOPin~OC(CH2)3COGPG3NHir Y~(des-S) n/eO.9 4.61
267 [-PheProBoroCegPin]OC(CH )3COG2(EDFEPlPL) 0.762 4.9
268 [-PglP(OEt~2]0C(CH2)3COG2(EDFEPIPL) n/e88.8 66.5
[-D-PheProBorolrgOPinlOC(CH2)3COG2NHir~Y~b~(des-S)N.T. N.T.
n/e = no effect
n/e 11.7 = no effect up to a concentration of 11 .7~M
N.T. = not tested
SUBSTITUTE Sl IEEr (RULE 26)