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WO1998000443A1 - Inhibiteurs de thrombine - Google Patents

Inhibiteurs de thrombine Download PDF

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Publication number
WO1998000443A1
WO1998000443A1 PCT/GB1997/001575 GB9701575W WO9800443A1 WO 1998000443 A1 WO1998000443 A1 WO 1998000443A1 GB 9701575 W GB9701575 W GB 9701575W WO 9800443 A1 WO9800443 A1 WO 9800443A1
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WO
WIPO (PCT)
Prior art keywords
group
amino acid
inhibitor
groups
amino
Prior art date
Application number
PCT/GB1997/001575
Other languages
English (en)
Inventor
John Joseph Deadman
Said Elgendy
Donovan Green
Emmanuel Skordalakes
Michael Finbarr Scully
Christopher Andrew Goodwin
Vijay Vir Kakkar
Original Assignee
Trigen Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Trigen Limited filed Critical Trigen Limited
Priority to AU30426/97A priority Critical patent/AU3042697A/en
Priority to JP10503898A priority patent/JP2000516203A/ja
Priority to EP97925198A priority patent/EP0910583A1/fr
Publication of WO1998000443A1 publication Critical patent/WO1998000443A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to thrombin inhibitors and thrombin substrates, and to their use for example in the treatment or prevention of thrombosis.
  • Cardiovascular disease is a major cause of mortality, with incidence across the world higher than that of cancer.
  • Acute events in the disease state such as myocardial infarction, stroke, peripheral arterial occlusion and venous thromboembolic disease have recently been understood to be precipitated by formation of thromboembolic clots.
  • This clot formation, as well as the aetiology of the disease state, e.g. formation of atheromatous plaque has been shown to be mediated by the coagulation serine protease enzymes which control also the normal haemostatic balance of the blood. Modulation of any one coagulation protease, especially thrombin, has been shown to control thrombogenesis. This has led to the development of inhibitors of thrombin to prevent thrombotic events in the clinic.
  • Thrombin cleaves peptide bonds by a mechanism involving the catalytic triad of Asp- His-Ser residues in the active site of the enzyme.
  • Thrombin inhibitors have been designed which use functional groups, e.g. CO-H, B(OH) 2 . P(0)(OR) 2 , beta lactam, hloromethylketone. to interact with the triad and thereby block activation of the substrates.
  • Inhibitors selective for thrombin have been prepared by making peptide inhibitors comprising peptide sequences that bind preferentially to subsites unique in the thrombin. Typically these sequences mimic the structure around the scissile bond of the natural substrate of thrombin, which is fibrinogen.
  • selective peptide inhibitors of thrombin typically incorporate a sequence based on Phe-Pro, or more generally (aa)-Pro, where (aa) is some hydrophobic amino acid or analogue thereof.
  • the amino acid residue which provides the carbonyl group of the scissile bond of a peptide sequence is designated "PI ".
  • PI 1 is glycinc and P2 ' is proline.
  • the protease contains a "specificity pocket" which recognises the side chain of the PI amino acid. Thrombin normally recognises PI residues with arginine-like or serine-like side chains.
  • Peptide inhibitors of thrombin have been made in which the P 1 terminal carboxy group is replaced by another acid group, e.g. a boronic acid group or a phosphorus oxyacid function.
  • the PI terminal carboxy or heteroatom analogue group may be derivatised. lor example to form an ester, an alcohol, a thiol or an amine or to replace the OH groups of boronic acid with fluorine.
  • the identity of the derivative moiety is not critical and may be selected according to the desired use of the target compound.
  • Peptide inhibitors having a boron or phosphorus heteroatom analogue group at the PI residue are described in, for example.
  • WO 92/07869 (equivalent to USSN 08/317.837), EP 0471651 (which corresponds to US 5288707) and USSN 08/240,606, the disclosures of which are incorporated herein by reference.
  • Inhibitors having a PI sulphonic acid group and derivatives thereof are described in Wong, S.C., Green. G.D.J.. and Shaw, E.. J.Med.Chem., 1978, 21, 456-459, ' Inactivation of trypsin-Iike serine proteases by sulfonylation. Variation of the positively charged group and inhibitor length'.
  • ⁇ -amino boronic acid peptides have been prepared because of the favourable binding energy of the interaction of boron with a nucleophile. such as the lone pair of the Ser hydroxyl or His imidazole group, to give a tetrahcdral boronate intermediate which mimics the shape of the "transition state" formed during substrate cleavage and so is tightly bound to the enzyme.
  • the ⁇ -amino group of such ⁇ -amino boronic acid compounds forms the P1-P2 amide link of the peptide.
  • thrombin is strongly inhibited by ⁇ -aminoboronic acid- containing peptides: Tapparelli, C; Metternich, R.; Erhardt, C; Zurini, M.; Claeson, G. Scully; M.F.; Stone, S.R. "In Vitro and In Vivo Characterisation of a Neutral Boron- containing Thrombin Inhibitor” J.Biol.Chem. 1993, 268, 4734-4741; ' Boroarginine Thrombin Inhibitors' Kettner, C. Mersinger, L.. & Knabb, R. (1990) J .Biol.Chem. 265, 18289-18297; Taparelli.
  • EP 01 18280 and equivalent US patents US 4638047 and 4772686 describe peptide thrombin inhibitors comprising amino acid residues on the C-terminal side of the scissile bond in which the Pl-Pl ' scissile peptide bond is replaced b ⁇ a non-hydrolysable isosteric linkage, namely -COCH , -CHOHCH r or CH 2 NH-.
  • peptide thrombin inhibitors include peptide analogues.
  • Such inhibitors are known in which there is at the carboxy position of the PI residue an optionally derivatised carboxy group or an optionally derivatised heteroatom analogue of a carboxy group, for example.
  • aa , aa . and aa represent natural or unnatural acid residues and (aa ) m one or more optional amino acid residues linked to the amino group of aa'.
  • any one or more aa groups may be analogues of amino acid residues in which the ⁇ - hydrogen is replaced by a substituent.
  • the sequence of amino acids and/or amino acid analogues binds to the thrombin active site. Suitable sequences are described later in this specification.
  • X represents H or a substituent on the N-terminal amino group.
  • Z is - COOH or a C-terminal extension group (carboxy replacement group), for example as known in the art. In preferred compounds Z is a heteroatom acid group, e.g.
  • -B(OH) 2 - P(OH) 2 or PO(OH) 2 , or a derivative thereof, for example a carboxylic acid ester, a dioxo-boronate [-B(Osubstituent) 2 ] or a phosphate f-PO(Osubstituent) 2 ] or BF 2 .
  • Preferred heteroatom analogue groups are -B(OH) 2 and -P(0)(OH) 2 , a less preferred heteroatom analogue group is S(0) 2 OH
  • -CN -COCH 2 Cl and -COCH 2 F
  • Derivatives of the acid groups include those in which inert organic groups, typically containing no more than 20 carbon and hetero-atoms, replace the hydrogen of any acid -OH group, the inert organic groups may be joined to the acid group through the intermediary of a functional group, such as carbonyl or amino.
  • a functional group such as carbonyl or amino.
  • an -OH group is replaced by a substituent which may, for example, be an inert organic group or halogen, notably fluorine
  • substituents aie hydrocarbyl and hydrocarbyl substituted by halogen or -OH.
  • the hydrocarbyl moietv may contain an ether or ester linkage, for example
  • A is a group selected to have affinit) for the specificity pocket of thrombin.
  • W is a moiety selected to have affinity for the thrombin binding subsite and Z is -COOH or a replacement group therefor.
  • Deadman et al provide in particular compounds of the formula III-
  • A is a group selected to have affinity for the specificity pocket of thrombin.
  • W is a moiety selected to have affinity for the thrombin binding subsite, ⁇ is a linker between W and A other than a natural peptide group and Z is -COOH or a replacement group therefor.
  • hirudin a family of isoproteins isolated from the glandular secretion of the leech Hirudo medicinalis.
  • This single chain protein containing 65 residues has a high affinity for ⁇ -thrombin (K D 10 ⁇ " to 10 " ? M) and does not inhibit other haemocoagulant enzymes. It consists of an active site blocking moiety, (Hirudin ), a fibrinogen-recognition exo-site binding moiety, (often regarded as Hirudin ' ). and a linker (often regarded as Hirudin "" ), connecting these inhibitor moieties.
  • hirudin binds to a low affinity site on thrombin which is separate from the catalytic site. This binding involves interaction of a domain from the C-terminus of hirudin with an "anion-binding exosite" (ABE) in thrombin. Following the low affinity binding, the hirudin-thrombin complex undergoes a conformational change and the hirudin then binds to a high affinity site on the thrombin. This latter site corresponds to the active site of thrombin.
  • ABE anion-binding exosite
  • hirulogs bifunctional peptide thrombin inhibitors based upon the structure of hirudin. These bifunctional peptides are named "hirulogs".
  • the hirulogs are described as mimicking the action of hirudin by binding to both the low affinity ABE and the catalytic site of ⁇ -thrombin. They contain a catalytic site directed moiety ("CSDM") joined by a linker region to an anion exosite associating moiety (“ABEAM”).
  • CSDM catalytic site directed moiety
  • ABEAM anion exosite associating moiety
  • the CSDM which forms the N-terminus of a hirulog molecule, is a low molecular weight peptide which most typically is of the structure H-D-Phe-Pro-Arg-Pro. Peptides containing the P3-P2-P1 sequence D-Phe-Pro-Arg were already known in the art to be thrombin-specific.
  • the CSDM of a hirulog is joined at its C-terminus to a linker of suitable length (18A- 42A) to enable the CSDM to bind to the catalytic site and the ABEAM to the anion- binding exosite.
  • the linker is a peptide having the amino acid sequence (Gly) and is linked to an ABEAM comprising amino acid residues 53-64 of hirudin.
  • Hirulog 8 therefore has the sequence:
  • the ABEAM may comprise the ABE binding domain of any molecule known to bind the anion binding site of thrombin.
  • the present invention provides novel bifunctional peptide thrombin inhibitors, in which the ABEAM does not form a C-terminal extension of the CSDM but preferably is joined to the CSDM at its N-terminal amino acid.
  • the ABEAM is linked to the CSDM through a spacer peptide and a non-peptide linker moiety.
  • the CSDM functionally comprises a moiety of any of formula I, II, III and IV but is preferably such a moiety in which Z is a heteroatom acid group or a derivative thereof.
  • the ABEAM may be the ABE binding domain of any molecule known to bind to the anion binding site of thrombin and. for example, may comprise any ABEAM mentioned in US 5196404 or WO 91/02750. or an exosite-binding analogue thereof.
  • the two binding domains are connected by a connector moiety which enables both groups to bind simultaneously to a thrombin molecule.
  • the invention provides a method for preparing compounds of the invention by solid phase synthesis
  • natural amino acid means an L-amino acid (or a residue thereof) selected from the group consisting of
  • Trp tryptophan
  • unnatural ammo acid any ⁇ -amino acid (or residue thereof) other than the natural amino acids listed above Unnatural amino acids therefore include the D- isomers of the natural L-amino acids.
  • unnatural amino acids include for instance: D-Phe, norleucine, hydroxyproline, ⁇ -carboxyglutamic acid, pyroglutamic acid, and other amino acids having side chain protecting groups and which are capable of incorporation into the peptides of the invention.
  • analogue when used in reference to amino acid residues or other moieties denotes an alternative to another group without implying that analogous groups impart the same properties to a compound. To the contrary, biological properties of compounds can be significantly changed by replacing a moiety with an analogue thereof.
  • aryl as used herein includes aryl groups containing heteroato s. i.e. heteroaryl groups.
  • alkyl includes cycloalkyl and alkyl containing cycloalkyl. where cycloalkyl is in particular cyclohexyl or cyclopentyl.
  • amino protecting group means any amino protecting group employable in peptide synthesis. Examples include: alkyl (especially methyl or other C,-C 6 alkyl), acetyl. benzoyl, BPoc, formyl. morpholinocarbonyl, trifluoroacetyl, methoxysuccinyl. aromatic urethane protecting groups such as benzyloxycarbonyl, aliphatic urethane protecting groups such as tert-butyloxycarbonyl or adamantyloxycarbonyl. Amino protecting groups are described in Gross and Meinhoffer, eds.. The Peptides. Vol. 3, 3-88, and exemplified in D. W. Greene, "Protecting Groups in Organic Synthesis".
  • Preferred amino protecting groups include: R (CH 2 ) e OCO- or R 1 (CH 2 ) c S0 2 -, where R is a C 5 -C 12 , preferably C 6 -C !0 , aryl, arylalkyl or alkylaryl group optionally substituted by halogen or -OH, especially phenyl, naphthyl or C r C 4 alkylphenyl, and e is 0 to 3.
  • additional N-protecting groups are desirable in the compound structure during synthesis.
  • protecting groups arc optionally removed or exchanged in the final structure.
  • Mtr (4-methoxy-2,3,6-trimethyl- benzenesulphonyl) or Pmc (2,2,5,7.8-pentamethyl-chroman-6-sulphate) may be used to protect Arg and Dtt (dithiothreitol) to protect Lys.
  • amino acid residues having acidic or hydroxy side chains may be suitably protected in the form of t-butyl, benzyl or other suitable esters or ethers, as is known in the art (e.g. Sheppard - "Solid Phase Peptide Synthesis. E. Atherton. R.C. Sheppard. IRL Press. Oxford. 1989).
  • salts include acid addition salts, e.g. salts of benzene sulphonic acid (BSA), hydrochloric acid (HCI), hydrobromic acid (HBr), acetic acid, trifluoroacetic acid (TFA). succinic acid, citric acid and other addition salt-forming acids known in the art.
  • BSA benzene sulphonic acid
  • HCI hydrochloric acid
  • HBr hydrobromic acid
  • acetic acid acetic acid
  • TFA trifluoroacetic acid
  • succinic acid citric acid and other addition salt-forming acids known in the art.
  • inhibitors modified by, in particular. isosteric replacement of one or more peptide bonds by -CO-CH 2 -. -CH(OH)-CH 2 or - CH 2 -NH- linkages, or by N 4 .
  • the peptides may be in the free form or in a form protected at one or more remaining functional groups, e.g. amino, imino or amide (including peptide), nitro. carboxyl, hydroxyl. guanidino or nitrile. Examples of. and synthetic routes to.
  • the present invention provides thrombin inhibitors comprising: a) a catalytic site-directed moiety (CSDM) that binds to and inhibits the active site of thrombin: b) an anion binding exosite associating moiety (ABEAM); and c) a connector moiety bonded to the CSDM as an N-terminal extension or as or through a side chain thereof and to the ABEAM, the connector moiety being capable of permitting the CSDM and the ABEAM to bind simultaneously to a molecule of thrombin.
  • CSDM catalytic site-directed moiety
  • ABEAM anion binding exosite associating moiety
  • the invention also provides a preferred class of compounds of the formula:
  • the moiety ⁇ , .... ⁇ represents an ABEAM and is preferably as defined herein under the heading "The Anion Binding Exosite Associating Moiety (ABEAM)"; ⁇ is a peptide spacer and ⁇ is a non-peptide linker, each preferably being as defined herein under the heading "The Connector Moiety”; and (aa ) m ... Z represents the CSDM and is preferably as defined herein under the heading "The Catalytic Site-Directed Moiety (CSDM) '* .
  • the Figure is a schematic representation of the binding of an inhibitor of the invention to thrombin.
  • the catalytic site directed moiety binds to and inactivates the catalytic site of thrombin.
  • the structure of the CSDM is not critical to the invention. It may comprise the amino acid sequence of any known inhibitor of the thrombin catalytic site, for example.
  • Suitable CSDMs may comprise a peptide inhibitor of formulae I or II. in which one or more natural amide linkages may be replaced by an alternative linkage, for e.xample the P1 -P2 link as described in International patent application No PCT/GB96/00352 and shown in Formulae III and IV.
  • Preferred CSDMs are peptides in which the C-terminus carboxy group is replaced by a heteroatom acid group or a derivative thereof, especially a boron-containing group.
  • Suitable CSDMs of this class are described in WO 92/07869 (and corresponding USSN 08/317,837), US 5288707, US 5196404 and PCT/GB96/00352 and corresponding cases.
  • the most preferred CSDMs contain the amino acid sequence Phe-Pro-Arg or sequences in which one or more of Phe. Pro and Arg is replaced by. for example, a known functional alternative or analogue.
  • Phe may be replaced by another hydrophobic amino acid, such as Dpa, Dba or Nal.
  • Arg may be replaced by a residue with an alkoxvalkvl side chain or a side chain having a terminal amino. amidino. imidazole. guanidino. N or isothioureido group. Mpg is a preferred Arg replacement.
  • CSDMs comprise, amongst other structures, peptides falling within formulae I. II, III and IV:
  • peptides are in the compounds of the invention bonded to a connector moiety other than through Z. which is C-terminal carboxy group (residue) or a replacement therefor.
  • m is normally from 0 to 7 and n is 0 or. most usually. 1.
  • the moiety on the C-terminal side of the PI residue (groups Z of formulae I and II) is not critical to the invention and may be any such moiety known in the art. for example. It is a moiety which interacts with the active site triad residues (Asp-His-Ser) of thrombin.
  • the PI residue is linked on its C-terminal side to a functional group which may be a carboxyl group (-COOH) or a derivative thereof, such as an ester, an amide or a ketone. for example, or even a nitrile group.
  • the natural carboxy group is replaced by a heteroatom acid group, of which the preferred examples are boron or phosphorus acid groups, notably boronic acid residues [-B(OH) 2 J, phosphonic acid residues [-P(0)(OH) 2 ], phosphorous acid residues [-P(OH 2 )] or phosphinic acid residues [-P(0)(OH)(H)].
  • boron groups are most preferred.
  • a less preferred heteroatom acid group is sulphonyl [-S(0) 2 OH].
  • heteroatom acid group there may be used a derivative thereof.
  • the invention is not primarily concerned with selection of derivatives of the carboxy or heteroatom acid groups: in principle, any derivative group may be used which does not prevent the inhibiting function of the compound.
  • Substituent groups include inert organic groups, generally containing a total number of carbon atoms and heteroatoms not exceeding 20. Representative inert groups are hydrocarbyl. optionally containing an ether or ester linkage and or substituted by halogen or -OH.
  • the acid derivatives have the hydrogen of an -OH group replaced by a substituent group, which may be linked to the oxygen by a functional group, for example a carbonyl or amino group.
  • substituents are diol residues, as further described below.
  • an -OH group is replaced by an amino group, which may be mono- or di- substituted.
  • An alternative replacement functional group is thiol. especially substituted thiol.
  • an -OH group is replaced by an inert organic group (e.g. a hydrocarbyl group as described above) or by a halogen atom, especially fluorine.
  • an inert organic group e.g. a hydrocarbyl group as described above
  • a halogen atom especially fluorine.
  • One class of CSDMs has a C-terminal group (Z of formulae I - IV) of the formula V:
  • Het is a heteroatom; s is 0, 1 or 2; t is the valency of Het, t-2s being an integer of at least 1 ; and each Y is independently hydrogen, halogen, hydroxy, substituted hydroxy, substituted thiol, amino or substituted amino, wherein two hydroxy groups, two thiol groups or an amino group are/is optionally substituted by a single divalent substituent.
  • Het is preferably boron or phosphorus, and is most preferably boron.
  • each Y is independently F or other halogen, O ⁇ 1 or N ⁇ ' ⁇ , wherein ⁇ and ⁇ 2 are independently selected from H, hydrocarbyl and hydrocarbylcarbonyl, the hydrocarbyl groups optionally being substituted by one or more moieties selected from halogen, -OH or alkoxy and or containing an ether or ester linkage (-0- or -COO-), which groups contain up to 20 carbon atoms, or wherein two Y groups taken together form the residue of a diol or a dithiol.
  • Particularly preferred C-terminal groups are of the formula
  • R and R are each independently selected from halogen, -OH, -OR and -NR R ⁇ where R 4 and R ""1 are each independently a group of the formula R (CO) u -, wherein u is 0 or 1.
  • R 6 is H or an optionally halogenated alkyl. aryl or arylalkyl group containing up to (10 - u) carbon atoms and optionally substituted by one or more groups selected from -OH, R 7 (CO) v O- and R 7 (CO) v -. wherein v is 0 or 1.
  • R 7 is C,-C 6 . v alkyl, or is an aryl. alkylaryl, arylalkyl or alkylarylalkyl group containing up to (10-v) carbon atoms.
  • R and R taken together represent a residue of a diol or a dithiol
  • R and R are each independently selected from the group consisting of R , R 3 , R 4 and R " ;
  • R is a group selected from the following: -H, -OR , -OR " .
  • R 2 and R 3 are preferably -OR 4 in which R is preferably a said optionally halogenated alkyl, aryl or arylalkyl group optionally substituted as aforesaid.
  • R is preferably a said optionally halogenated alkyl, aryl or arylalkyl group optionally substituted as aforesaid.
  • the diol or dithiol preferably comprises two or more -OH or, as the case may be, -SH groups connected by at least two connecting atoms.
  • the connecting atoms arc preferably in an organic moiety containing up to 20 and. more preferably, up to 10 carbon atoms.
  • the organic moiety may be a hydrocarbyl group optionally containing between the members of one or two pairs of adjacent carbon atoms an N, S or O atom.
  • the organic moiety may be inertly substituted. Normally the substituted compounds are mono- or di- substituted, exemplar.' substituents being halogen especially -F. and -OH.
  • Preferred diol residues are of pinanediol, pinacol, perfluoropinacol, ethylene glycol, diethylene glycol. catechol. 1.2-cyclohexanediol, 1,2-cyclohexaneethanediol, 1 ,3- propanediol. 2.3-butanediol. 1 ,2-butanediol, 1,4-butanediol, 2,3-dimethylbutane-2-3- diol, glycerol, or diethanolamine or another amino dihydroxy alcohol.
  • pinanediol and especially pinacol are most preferred.
  • the most preferred compounds comprise a boronic acid residue substituted with a diol residue.
  • the compounds of the invention optionally may contain a CSDM in which a natural peptide linkage (-NHCO-) is replaced by an alternative linker group.
  • the replaced peptide link may be the P1-P2 link or another link and is represented by ⁇ in formulae III and IV.
  • ⁇ will hereafter be used.
  • ⁇ is a group which may be included in a compound of the invention without the inhibiting activity of the compound being lost .
  • Preferred ⁇ groups enhance the inhibitory activity of the compound. If ⁇ is long, there is a tendency for binding of the peptide inhibitor to the target enzyme to be weakened. Typically, therefore, ⁇ has a chain length of no more than 5 atoms, i.e. no more than 5 atoms separate the carbon atoms of the residues linked by ⁇ . More preferred ⁇ groups have a chain length of 2 or 3 atoms, a chain length of two atoms being most preferred.
  • ⁇ is preferably not isoelectronic with -NHCO- of it forms the P1-P2 linkage.
  • One less preferred class of embodiments does not have ⁇ groups of the so-called isosteric (to - CONH-) type, such as -COCH 2 -, -CH(OH)-CH 2 -. -CH 2 -NX- or -NHCO-. for example.
  • -COCH 2 - and -CH(OH)-CH - are very acceptable in some compounds.
  • Representative ⁇ groups include -C0 2 -, -CH 2 0-, -NHCO-, -CHYCH 2 -.
  • -CH CH- CO(CH 2 ) p CO- where p is 1. 2 or 3.
  • -COCHY- -C0 2 -CH 2 NH-, -CHY-NX-, -N(X)CH 2 - N(X)CO-.
  • -CH C(CN)CO-.
  • X is H. an amino protecting group (e.g. CH 3 ) and Y is H or halogen (especially F).
  • exemplary' Y-containing groups are -CH 2 CH 2 -, -COCHF- and -CH 2 NX-.
  • the most preferred ⁇ groups are -C0 2 - and -CH 2 0-.
  • the invention encompasses peptides in which one or more amide linkages other than the P1-P2 linkage are also replaced by an unnatural linker ⁇ , e.g. a preferred ⁇ group of this invention or, more preferably, a so-called isosteric group, e.g. -COCH 2 -. -CH(OH)-CH 2 - or -CI -NH-.
  • an unnatural linker ⁇ e.g. a preferred ⁇ group of this invention or, more preferably, a so-called isosteric group, e.g. -COCH 2 -. -CH(OH)-CH 2 - or -CI -NH-.
  • Such replacement of peptide bonds other than the P1 -P2 bond is described, for example, in EP 01 18280.
  • the N-terminal group (X of the Formulae) is in many compounds of the invention replaced by the connector moiety and its attached ABEAM. Where present, X may be hydrogen (to form an -NH 2 group) or an amino protecting group.
  • the amino protecting group of the pharmaceutical compounds may be any pharmaceutically acceptable group, for example as described hereinbefore.
  • Alkyl groups, e.g. C,-C ft alkyl such as methyl, for example, are suitable.
  • a preferred class of protecting groups are those of the formula R l ⁇ (CH 2 ) e OCO- and R 10 (CH 2 ) e SO 2 -. wherein e is 0, 1. 2 or 3 and R 10 is a C 5 - C
  • R ° groups when m and n are 0. or when m is 0 and n is i are phenyl. naphthyl, C,-C 4 alkylphenyl or phenyl C,-C alkyl. In preferred embodiments, e is O.
  • N-terminal groups can enhance bioavailability of active compounds, although not necessarily effecting potency against the isolated target enzyme.
  • Typical groups of the active compounds include morpholin-N-alkyl or N-carbonyl derivatives, succinimidyl, alkyl or aryl-alkyl-sulphonyl.
  • N-methylpiperazine or groups as known in the art, such as Rosenberg, et al. J.Med.Chem., 1993, 36, 449-459 or Hashimoto. N. et al. Pharm.Res,. 1994, 1 1, 1443-1451. or Bernstein. P.R., et al. J.Med.Chem., 1994, 37. 3313-3326.
  • These groups can be introduced to the peptides by hydrogenation to remove urethane protecting groups used for synthesis to give the free amino terminus (see Example 2) and reacylation or acetylation with a derivative of the appropriate X group.
  • Peptide inhibitors of the thrombin catalytic site comprise a sequence of amino acid residues and are commonly tripeptides.
  • the specific sequence is not critical to the invention.
  • the amino acids may be natural or unnatural, e.g. the D-isomer or racemate of a natural amino acid; they may be modified amino acids in which the ⁇ -H is replaced by a substituent, for example hydrophobic or hydrophilic groups containing up to about 20 or even more, e.g. 22. carbon atoms. More preferred substituents contain up to 15, or preferably up to 10, carbon atoms.
  • Preferred classes of replacements for the ⁇ -hydrogen of the amino acid residues are:
  • Q amino. amidino. imidazole, guanidino, N or isothioureido. and q is an integer of from 1 to 5;
  • D is O or S
  • E is H, C r C 6 alkyl, or a saturated or unsaturated cyclic group which normally contains up to 14 members and preferably is a 5-6 membered ring or an 8-14 membered fused ring system, which alkyl or cyclic group is optionally substituted by up to 3 groups (e.g. 1 group) independently selected from -R 13 ,
  • R 13 is -(CH 2 ) 2 H or a moiety which has a total number of carbon and heteroatoms from 5 to 10 and which contains a ring system (e.g. an aryl group) and optionally an alkyl and/or alkylene group, wherein f and g are each independently from 0 to 10. g preferably being at least 1 except that -OH is a preferred substituent, provided that (f+g) does not exceed 10. preferably does not exceed 6 and more preferably is 1 , 2.
  • a is 0. If a is 1. c is preferably 0. Preferably, (a+b+c+d) and (a+b+c+e) are no more than 4 and are more preferably 1. 2 or 3. (a+b+c+d) may be 0.
  • Exemplary groups for E, E and E include aromatic rings such as phenyl, naphthyl. pyridyl, quinolinyl and furanyl, for example; non-aromatic unsaturated rings, for example cyclohexenyl; saturated rings such as cyclohexyl, for example: and fused ring systems containing both aromatic and non-aromatic rings, for example fluorenyl.
  • a preferred class of E. Ei and E ⁇ groups are aromatic rings, especially 6- membered aromatic rings.
  • E and E are preferably phenyl.
  • the phenyl or other aryl groups may be substituted by nitro or cyano. preferably at the 4-position.
  • E contains a substituent which is C C b alkyl, (C C 5 alkyl)carbonyl, carboxy C,-C 5 alkyl, aryl, especially 5-membered or preferably 6- membered aryl (e.g. phenyl or pyridyl), or arylalkyl (e.g. arylmethyl or arylethyl where aryl is preferably 6-membered).
  • E contains a substituent which is OR , wherein R preferably is a 6-membered ring, which may be aromatic (e.g. phenyl) or non-aromatic (e.g. morpholine or piperazine) or is alkyl (e.g. methyl or ethyl) substituted by such a 6- membered ring.
  • R preferably is a 6-membered ring, which may be aromatic (e.g. phenyl) or non-aromatic (e.g. morpholine or piperazine) or is alkyl (e.g. methyl or ethyl) substituted by such a 6- membered ring.
  • a particularly preferred class of moieties of formula V or VI are those in which E is a 6- membered aromatic ring substituted, preferably at the 2-position or 4-position, by -R 13 or -OR 13
  • a further preferred class of substituents of formula V or VI are of the formula C q H 2q T or
  • q is as defined above and T is hydrogen, halogen (e.g. F).
  • Said moiety is preferably 5-membered or more preferably 6-membered aryl (e.g. phenyl or pyridyl) or arylalkyl (e.g. arylmethyl or arylethyl) where aryl has 5 or preferably 6 members.
  • T is at the 2-position of the phenyl group and is -R 1 . -COR , -C0 R ' or -0 2 CR . and R 13 is C r C
  • a class of residues which includes certain natural amino acid residues as well as many unnatural amino acid residues is of the formula
  • W and W may be the same or different and are selected from hydrogen and hydrogen replacement groups (i), (ii) and (iii) described above in relation to amino acid residues in which the ⁇ -hydrogen is replaced by a substituent; preferably, one of W 1 and
  • W is hydrogen.
  • W and W together with the carbon atom to which they are bonded form a ring system, especially a hydrophobic ring system
  • -HNC(W')(W 2 )CO- is the residue of an amino acid in which W is H and W is a group which together with the ⁇ -amino group forms a cyclic group, i.e. the amino acid is of the formula IX:
  • U is a moiety forming a cyclic structure, which may be substituted or unsubstituted.
  • the cyclic structure is preferably a 4-6 membered ring or an 8-10 membered fused ring system optionally substituted by up to 3 groups independently selected from -R 13 , -R'OR 13 . -R'COR 13 , -R l C0 2 R 13 and -RO 2 CR 13 .
  • R 1 and R 13 are as hereinbefore defined.
  • Exemplary substituents are C,-C 3 alkyl. Any one or more hydrogen atoms bonded to a carbon atom may optionally be replaced by halogen, especially F.
  • the cyclic structure may contain additional heteroatoms, for example sulphur, such as in a 5- or 6- member ring, for example.
  • a ring carbon atom may be a member of a carbonyl group, for example as part of an amide linkage in the cyclic structure, as in pyroglutamic acid, for example.
  • the cyclic structure preferably contains no heteroatom in addition to the ⁇ -amino nitrogen.
  • the ring fused to that containing the ⁇ -amino nitrogen is preferably aromatic and most preferably phenyl, as in D-Tiq.
  • PI Residue Residues analogous to Lys or Arg and amongst the residues favoured by thrombin at PI are those with group (i) side chains and an ⁇ -hydrogen. that is, residues of the formula
  • Q includes amino. amidino, imidazole, guanidino. N 3 or isothioureido.
  • Specific analogy residues to Lys and Arg include Gpa. amidinoPgl or amidinopiperidylglycine. Also very acceptable PI residues are those with hydrophobic side chains, including Phe and its analogues.
  • Suitable hydrophobic side chains for the PI residue include group (iii) side chains of Formula V, especially those in which a is 0, D is O or is absent and/or E is H, C r C 6 alkyl, C,-C 6 trialkylsilyl or C 6 -C, 0 aryl optionally substituted by up to three groups selected from C,-C 4 alkyl. halogen and C,-C 4 alkoxy, of which H is less preferred.
  • the Formula V groups contain a total number of carbon atoms and heteroatoms not exceeding 14. more preferably not exceeding 10 and most preferably not exceeding 8.
  • E is H, C r C 6 alkyl.
  • C,-C 6 haloalkyl is a preferred E group.
  • Particularly preferred hydrophobic PI side chains are C r C 8 , preferably C,-C 6 , alkyl (e.g. ethyl, isopropyl, pentyl), alkoxvalkvl containing from 2 to 6 carbon atoms (e.g. methoxypropyl) and moieties containing a 5-10 membered aryl or heteroaryl group and optionally a total number of alkyl and/or alkylene carbon atoms not exceeding 4. especially phenyl C r C alkyl (e.g. phenylmethyl). Any of the aforesaid alkyl or alkylene groups may be substituted by one. or more than one, halo atom. e.g.
  • bromopropyl especially 3-bromopropyl, or other bromoalkyl (usually substituted by Br at the terminal carbon) is a preferred PI side chain.
  • Methoxyalkyl is a particularly preferred side chain.
  • the PI side chain is C r C 6 hydroxyalkyl. 3 -methoxypropyl. 3-halopropyl and -hydroxy propyl and alkyl homologues thereof are particularly preferred.
  • W of formula II normally comprises a sequence of up to 9 amino acids, and more usually of up to 7 amino acids, wherein at least one amino acid has a hydrophobic side chain, e.g. Phe or a Phe analogue.
  • the P3 residue (aa ) is desirably hydrophobic; the P2 residue (aa ) is also preferably hydrophobic and more preferably is Pro or a ring homologue thereof.
  • Any P4 residue of a thrombin inhibitor is preferably also hydrophobic.
  • Residues analogous to Phe include those with group (iii) side chains, those of formula IX and those in W 1 and W 2 from a hydrophobic ring system or an alkenyl or aralkenyl group.
  • Ar and Ar " are each independently selected from the group consisting of H; phenyl; phenyl substituted by halogen (e.g. p-halophenyl, especially p-iodophenyl), a C r C 6 group which is alkyl or alkyl substituted or interrupted by a carbonyl or carbonyloxy group (e.g.
  • alkylcarbonyl or alkoxy carbonyl or substituted by -R or -OR wherein R is a 5- or 6-membered aromatic or non-aromatic ring or is C C alkyl substituted by such a 6-membered ring; bipyridyk furanyl; chromanyl; quinolinyl; thienyi; pyridyl; ⁇ - or ⁇ -naphthyl; thionaphthyl; indolyl; p-iodophenylalanyl; diphenyl-methyl; or fluorenyl; or are wholly or partially saturated groups corresponding to any of these (e.g. cyclohexyl.
  • L, and L 2 are each independently selected from the group consisting of CH 2 , CH 2 -CH 2 , 0-CH 2 , S-CH 2 , and a bond.
  • V is H, or -NHV and one of Ar'-L 1 and Ar 2 -L 2 together form a group of the formula
  • L 1 or L 2 is a single bond, its attached Ar group be diphenylmethyl, fluorenyl or cyclohexyl.
  • Ar -L is H.
  • the P2 (aa " ) residue is normally proline or an analogous residue.
  • Residues analogous to proline are preferably those ring homologues included in the formula VIII
  • R -CH 2 -, -CH 2 -CH 2 -, -S-CH 2 -, -S-C(CH 3 ) 2 - or -CH 2 -CH 2 -CH 2 -.
  • R -CH 2 -, -CH 2 -CH 2 -, -S-CH 2 -, -S-C(CH 3 ) 2 - or -CH 2 -CH 2 -CH 2 -.
  • proline analogues are 2- and 3-thioproline and pipecolic acid.
  • Particularly preferred Phe analogues for the P3 residues are D-Phe substituted at the phenyl 2-position (i) by a C r C 6 group which is alkyl or alkyl substituted or interrupted by a carbonyl or carbonyloxy group (e.g. is alkylcarbonyl or alkyloxycarbonyl) or (ii) by a 5 or 6 membered aryl group; D-Dpa; Dba; Pms; ⁇ - or ⁇ -Nal; TMSal; Chg; Phg; D- Tiq or a para ether of D-Tyr.
  • An exemplary substituted phenylalanine residue is D- phenylalanine-2-carboxylic acid methyl ester.
  • Exemplary tyrosine-para-ethcrs are D- tyrosine-O-phenyl. D-tyrosine-0-ethyl-2-(N-morpholine) and D-tyrosine-O-ethyl-2- N(piperazine).
  • the most preferred Phe analogues are Dpa. Nal and Dba. Other preferred Phe analogues for. in particular, the P3 residue have side chain c), d), e) f), g). or h) of US 5288707 and EP 0471651.
  • Particularly preferred R groups are C 9 -C, fused ring systems containing a phenyl ring, especially naphthyl.
  • R l is a fused ring system
  • e is preferably 0; if R is a single ring, e may suitably be 1.
  • the residue of the acid function -COO- or -S0 2 - is preferably -S0 2 -.
  • Particularly preferred amino protecting group analogues for Phe are benzyloxycarbonyl (Cbz) and naphthylsulfonyl.
  • Arg or Lys preferably have an ⁇ - hydrogen, but the hydrogen may be replaced by another group, e.g. a W moiety.
  • preferred classes of PI (aa ) residues of the inventive compounds are (i) Arg, Lys and their analogues as described above, and (ii) hydrophobic residues.
  • Preferred P2 (aa " ) residues are Pro and its analogues as described above.
  • Preferred P3 (aa ) residues are Phe and its analogues as described above.
  • Tripeptide inhibitors are preferred, especially tripeptide boronates. and a particularly preferred sequence is PheProBoroMpg, in which one or more natural peptide bonds may be replaced by another link.
  • the PI Mpg residue may be replaced by Pgl or Bpg.
  • Residues may be in either D- or L-configuration. D-configuration is preferred for the P3 residue of thrombin inhibitors.
  • ABSEAM Anion Binding Exosite Associating Moiety
  • the anion binding exosite associating moiety is a moiety which binds to the anion binding exosite (ABE) of thrombin. It preferably comprises an amino acid sequence of the ABE binding domain of hirudin or an ABE binding modification thereof.
  • the ABEAM comprises a sequence of up to 15 amino acids of which at least four are anionic amino acids and more normally at least five or six are anionic.
  • the ABEAM of this class comprises a sequence of up to 13 amino acids, e.g. of 10, 1 1, 12 or 13 amino acids.
  • the ABEAM comprises a sequence of up to 10 amino acids e.g. of 8, 9 or 10 amino acids of which at least 3 amino acids are anionic and preferably 4 or 5 are anionic.
  • An exemplary sequence is EDFEPIPL.
  • the anionic amino acids preferably include residues 3 and 4 from the C-terminus. or residues 4 and 5 from the C-terminus.
  • the invention includes compounds in which all of residues 3. 4 and 5 from the C-terminus are anionic.
  • ABEAMs comprise a moiety of the following formula:
  • to ⁇ 1 is an amino acid residue
  • wl , ⁇ v2 and w3 are each independently 1 or 0 (but w3 is preferably 0) and ⁇ is a carboxy terminal residue (attached to the terminal carbonyl group) preferably selected from hydroxy, C,-C 6 alkoxy, amino, mono- or di (C r C 4 ) alkyl substituted amino or benzylamino, in which moiety ⁇ 4 , ⁇ 8 and ⁇ ( , and two or three other amino acid residues are anionic.
  • said other anionic amino acid residues include ⁇ ;, or ⁇ 2 .
  • said moiety includes at least two pairs of adjacent anionic amino acid residues.
  • exemplary ABEAM moieties have anionic amino acids as ⁇ , ⁇ 5 , ⁇ g , ⁇ 9 and preferably, ⁇ 2 .
  • Other exemplary ABEAM moieties have anionic amino acids as ⁇ 8 - ⁇ , ,, ⁇ , and ⁇ .
  • the anionic amino acids are desirably Gly or Asp. ⁇ is preferably hydroxy.
  • the invention includes compounds containing an ABEAM comprising the ⁇ 4 - ⁇ M sequence in which ⁇ 4 is an anionic amino acid; ⁇ 5 is any amino acid; ⁇ 6 is lie, Val, Leu, Nle or Phe; ⁇ 7 is Pro, Hyp, 3.4-dehydroPro, thiazolidine-4-carboxylate.
  • Sar any N- methyl amino acid or D-Ala; ⁇ 8 is an anionic amino acid; ⁇ 9 is an anionic amino acid; ⁇ i0 is a lipophilic amino acid selected from the group consisting of Tyr, T ⁇ , Phe, Leu, Nle, He, Val, Cha, Pro: and ⁇ , , is any amino acid.
  • ABEAM preferably comprises a sequence homologous to amino acids 57-64 of hirudin, i.e. ⁇ 4 is Glu; ⁇ is Glu; ⁇ 6 is He; ⁇ 7 is Pro; ⁇ 8 is Glu; ⁇ 9 is Glu; ⁇ i0 is Tyr, Tyr(S0 3 H), Tyr(OS0 3 H) or (3-,5-diiodoTyr), and ⁇ ⁇ is Leu and ⁇ is preferably OH.
  • native hirudin contains Try(OS0 3 H) at position 63.
  • carboxy terminal hirudin peptides which contain Tyr(S0 3 H) are reported to have identical anticoagulant activity as those which contain the native Tyr(OS0 3 H).
  • ABEAM components within the scope of this invention may comprise those portions of any molecule known to bind to the anion binding site of thrombin. These include amino acids 1675-1686 of Factor V, amino acids 272-285 of platelet glycoprotein lb, amino acids 415-428 of thrombomodulin, amino acids 245-259 of prothrombin Fragment 2 and amino acids 30 to 44 of fibrinogen A ⁇ chain.
  • the ABEAM component may be selected from any of the hirudin peptide analogues described by J.L.Krstenasky et al., "Development of MDL-28,050, A Small Stable Antithrombin Agent Based On A Functional Domain of the Leech Protein, Hirudin", Thromb. Haemostas.. 63. pp. 208-14 (1990).
  • the ABEAM may comprise hirudin variants synthesised by chemical and/or biological means, such as, for example, chemically modified, amino acid substituted analogues as described in WO 92/01712.
  • Residue ⁇ ! 2 is absent in preferred embodiments.
  • the ⁇ residue and optionally the ⁇ ., residue may be absent if the ABEAM comprises a Hirudin sequence.
  • the ⁇ and preceding residues may be absent if the ABEAM comprises a thrombin platelet receptor sequence.
  • ABEAM sequences include DFEPIPL.
  • the hirulogs of Maraganore et al contain a linker between the C-terminal carboxy residue of the CSDM and the N-terminal amino residue of the ABEAM.
  • the CSDM and the ABEAM are interconnected by a connector bonded to the CSDM at its N-terminal amino residue or as a side chain of a CSDM residue. It has unexpectedly been found that this invention enables the provision of highly active thrombin inhibitors.
  • the connector is coupled to the N-terminal amino group of the CSDM.
  • the amino group typically forms an amide bond with a carbonyl group of the ABEAM.
  • the connector is coupled to the CSDM as (or through) a side chain thereof.
  • the connector is bonded to a side chain group of a CSDM amino acid residue: this arrangement is convenient when the side chain has a functional group, for example amino or carboxyl, which can react with a complementary functionality on the connector, e.g. to form an amide or ester linkage.
  • the connector is bonded to the ⁇ -carbon of a CSDM amino acid residue directly or through a functional group.
  • the connector, or a side chain linked thereto replaces the ⁇ -hydrogen of a CSDM amino acid residue.
  • the connector when arranged as a side chain or, one may say, a side chain extension of the CSDM does not have to be coupled to an ⁇ -carbon of an amino acid residue but may, for example, be coupled to a constituent of a non-peptide bond between two amino acid residues.
  • compounds of the invention in which the connector does not form a C-terminal extension of the CSDM. are capable of adopting a configuration in which the ABEAM and CSDM can simultaneously bind to a thrombin molecule. Even more smprisingly, the invention enables provision of thrombin inhibitors having an improved Ki value over those of the exemplified hirulogs of WO 91/02750.
  • the connector is selected to be capable of permitting simultaneous binding of the CSDM and the ABEAM to thrombin.
  • the connector preferably comprises a peptide "spacer” and a non-peptide "linker".
  • a representative connector structure is:
  • ⁇ represents a non-peptide linker and ⁇ a spacer comprising a sequence of amino acids, ⁇ and ⁇ suitably being joined by a peptide bond.
  • the spacer ⁇ is preferably linked to the ABEAM and the linker ⁇ to the CSDM, although compounds in which ⁇ is linked to the CSDM and ⁇ to the ABEAM 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, is a dicarboxylic acid which can form amide bonds with the N-terminal amino groups of the CSDM and the spacer.
  • Particular preferred linkers are a residue of glutaric acid (H0 2 C(CH 2 ) C0 2 H and homologues thereof of the formula (H0 2 C(CH 2 ) h C0 2 H wherein h is an integer of 2 or from 4 to 6.
  • the alkylene residue [-(CH 2 ) 2 . 6 -] may be substituted by one or more substituents which do not sterically hinder the linker, whereby the desirable flexibility of the linker is maintained..
  • 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 two adjacent Gly residues, normally at its N-terminal end.
  • the length of the spacer is dependent upon inter alia the position on the CSDM to which the linker is attached. A particular problem which arises in specifying the length of the spacer is that of defining the junction between the ABEAM and the spacer.
  • a glutaric acid linker is bonded to the P3 N-terminal amino group of a tripeptide (P3P2P1) CSDM
  • one class of compounds is of the formula:
  • ⁇ is a sequence of from 5 to 12 amino acids, preferably a sequence of from 5 to 10 amino acids and most preferably a sequence of from 5 to 7 amino acids, ⁇ preferably includes at least 2 adjacent Gly residues an may include one or more consecutive Hir residues on the C-terminal side of Hir 54 , e.g. Hir " or Hir ' ; h is from 2 to 6 and is preferably 3; and the remaining symbols are as defined previously.
  • Alternative connector moieties having such lengths may be used.
  • the spacer ( ⁇ ) may have one or more natural amide bonds replaced by other linkages, as described above in relation to the CSDM.
  • the essential feature of the inventive inhibitors is their possession of interlinked CSDMs and ABEAMs as discussed above. Other features of the compounds are not of the essence.
  • the compounds of the invention may therefore be in the form of a pharmaceutically acceptable salt thereof and/or comprise one or more protectable functional groups (e.g. -OH or -NH 2 ) protected by a pharmaceutically acceptable protecting group.
  • Suitable salts include acid addition salts, as described above, and those of acid groups with Group I or Group II metal cations (e.g. Na + , K + , Mg + , Ca ).
  • protecting groups of protectable functional groups there may be mentioned t-butyl and benzyl as protecting groups for -OH and -COOH functions.
  • the inhibitory compounds of the invention have affinity for thrombin.
  • Compounds which have affinity for an enzyme significantly inhibit or retard the enzyme's activity. It is desirable for the compounds to have an inhibition constant (Ki) for thrombin of 0.5 ⁇ M or less, preferably of 0.3 ⁇ M or less and most preferably of 0.1 ⁇ M or less.
  • Particularly preferred compounds have a Ki of 0.05 ⁇ M or less is obtained, e.g. of InM or less, especially of less than 0.75nM.
  • Ki values herein refer to values determined at 37°C.
  • the inhibiting compounds it is often preferred for the inhibiting compounds to be selective towards thrombin.
  • the ratio of Ki for non selected enzymes: Ki for thrombin is preferably at least 2 and more preferably at least 3.
  • the Ki ratio may be at least 5.
  • the compounds of the invention can be prepared by using, for example, generally known methods for peptide synthesis and for coupling peptides.
  • the novel compounds are made by a solid phase synthetic technique.
  • the invention therefore includes a method of making a compound of the invention, comprising the following steps: (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;
  • step (iv) repeating step (iii) as often as necessary.
  • the functional groups coupled to a solid phase may be on a moiety which is inco ⁇ orated 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 normally are. included in the method in order to obtain the compound of the invention.
  • preferred methods include, when desired, 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.
  • any sequentially following amino acid of step (iii) may be part of a larger moiety, e.g. of an amino acid sequence optionally containing a replacement for a natural peptide bond.
  • 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 and acid anhydride.
  • 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.
  • the terminal amino acid reacted with the functional groups attached to the solid phase is the C-terminai amino acid of the ABEAM and step (iii) is repeated to couple successive amino acids of the ABEAM sequence and successive amino acids of any contiguous connector peptide.
  • the final amino acid of the uninterrupted amino acid sequence thereby coupled to the solid phase is normally reacted with a compound having two carboxylate groups or reactive derivatives thereof, for example the anhydride of a dicarboxy ⁇ c 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.
  • 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.
  • 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 desirably has all its reactive functional groups protected other 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:
  • a second preferred method comprises:
  • the synthesised compound is preferably cleaved from the solid phase by acid.
  • the preceding methods preferably involve the use of a CSDM amino acid or amino acid sequence (e.g. a complete CSDM) having a C-terminal boron group.
  • a CSDM amino acid or amino acid sequence e.g. a complete CSDM having a C-terminal boron group.
  • the functional groups coupled to a solid phase may be part of a moiety which is inco ⁇ orated 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.
  • Fmoc chemistry also known generally as the 'Sheppard approach'
  • the carboxy terminus of a peptide 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 the pores on the inside of the bead.
  • PS polystyrene
  • An example of an alternative solid is the polyamide called Kiesulguhr.
  • the linker can be many things, but we prefer to use PEG (i.e. a polyethylene glycol linker), which terminates in an alcohol function.
  • PEG i.e. a polyethylene glycol linker
  • the terminus of the linker typically called a 'handle', depending 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 used) is HMBA or para-hydroxymethylbenzoic acid linker.
  • the HMBA is esterified onto the PEG, and then the 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 resin used in Boc chemistry is often divinylbenzyl based, for instance a 'Wang " resin has chloromethyl benzene co- polymerised to 2% divinylbenzene.
  • the link to the resin is typically cleaved (very carefully!) by dry, liquid HF. This is described as 'vigorous ' acidolysis.
  • N-terminal coupling methods may be used in making the products of the invention.
  • 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 ⁇ -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 (e.g. HF or TFA) prior to being joined to the remainder of the final product.
  • strong acid e.g. HF or TFA
  • the two intermediates contain suitable functional groups to react together to form the target non-natural amide bond [ ⁇ of Formula I] 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).
  • Suitable synthetic techniques for making peptides containing non-amide bond ⁇ are described in PCT/GB96/00352.
  • Suitable intermediates for such peptides are of the formula X-(aa 4 ) m -(aa 3 ) n -(aa 2 )-G' or W-G 1 and G 2 -(aa')-Z or G 2 -A-Z.
  • G 1 and G 2 are groups which may be reacted together to form a linking group other than a natural amide bond, optionally after "working up” (e.g. hydrogenation) of the direct product.
  • ⁇ (CH,O) Species (aa 2 ) which contain a free hydroxyl group (OH), and G is a leaving group, especially halogen, e.g. Cl, Br and the base DBU or an organolithium (e.g. Butyl lithium) are used.
  • Ketomethylene bonds can be prepared by reaction of a unit X-(aa ) n ⁇ -(aa ', ) n - (aa " )- carbonyldiimidazole and the lithium salt of tert-butyl acetate to give a beta- diketone X-(aa ) m (aa ) n (aa 2 )-COCH 2 COOtBU. and alkylation with NaH and a halomethylketone (Hoffman, R.V. and Kim, H.O.. Tet.Lett., 1992, 33. 3597-3582) or ⁇ - haloboronate (e.g. Hal-CHRB0 2 Pin) or ⁇ -halophosphonate and subsequent hydrolysis.
  • ⁇ - haloboronate e.g. Hal-CHRB0 2 Pin
  • ⁇ (CH-CH) could be prepared by the methods described by Ibuka, T., Yoshizawa, H., Habashita, H.. Fuji, N.,Chounan, Y., Tanaka, M., and Yamamoto, Y., Tet.Lett., 1992, 33, 3783-3786 or Ibuka, T., Habashita, H., Otaka, A., Fuji, N., Oguchi, Y., Uyehara. T. and Yamamoto, Y..
  • the reaction is preferably carried out in a dry. aprotic. polar solvent for example tetrahydrofuran. at a temperature between about -79°C and room temperature (typically, 20°C).
  • the intermediates may be obtained by the methods disclosed herein or alternatively by general methods as described in Matteson et al, Organometallics, 2, 1284-8 (1984), or as in Elgendy et al, Tet.Lett. 1992, 23, 4209-4212 or Tetrahedron 1994, 5_ ⁇ , 3803-3812 or Rangaishenvi et al, J.Org.Chem 1991, 5 ⁇ , 3286-3294, or in EP-A-0599633.
  • Suitable replaceable protecting groups may be used, for example as outlined for instance in Greene, T.W. and Wuts, P.G.M.. "Protective Groups in Organic Chemistry". Wiley- Interscience. 1991 .
  • the starting amino acid(s) for the preparation of the protected peptide of intermediate may be prepared by standard, well-known methods such as those described for example in Angew. Chem. 21, 793 (1981), J.Am Chem. Soc, 102, 6881 (1987) and J Jones, "The Chemical Synthesis of Peptides", Oxford Science Publications. No. 23. Clarendon Press, Oxford 1992, or may be obtained from a variety of well known commercial sources.
  • a non-peptide bond may be inco ⁇ orated during solid phase synthesis, for example using a method described in EP 257742 and equivalent US 4803261 (which is included herein by reference).
  • novel compounds according to the present invention are useful as inhibitors or substrates of thrombin, and may be used in vitro or in vivo for diagnostic and mechanistic studies of thrombin. More generally, the novel peptides may be useful for research or synthetic pu ⁇ oses. Furthermore, because of their inhibitory action, the inhibitors are useful in the prevention or treatment of diseases caused by an excess of thrombin 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 pharmaceutically acceptable group as any N-terminal substituent (X).
  • the compounds of the invention may be employed when an anti-thrombogenic agent is needed. Generally, these compounds may be administered orally or parenterally to a host in an 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 lOmg/Kg of body weight and preferably l -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 extraco ⁇ oreal 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 compounds of the invention may also be added to blood for the pu ⁇ ose of preventing coagulation of the blood in blood collecting or distribution containers, tubing or implantable apparatus which comes in contact with blood.
  • Advantages enabled by the invention include oral activity, rapid onset of activity and low toxicity.
  • these compounds may have special utility in the treatment of individuals who are hypersensitive to compounds such as heparin or other known inhibitors of thrombin or other serine proteases.
  • amino acid residues are of L-configuration unless otherwise stated.
  • GlyGlyGlnfTyr 6 Hir " * which has the amino acid formula: H-Gly-Gly-Gln-His-Asn- Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu-OH. was prepared by solid phase peptide chemistry on a Milligen 9050 PepSynthesizer using an Fmoc-polyamide continuous 5 flow method and proprietary 9050 Plus on column monitoring software.
  • 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 20% piperidine in DMF. Fmoc-amino acids (4 equiv.) as their pentafluorophenyl esters with side chain protection where appropriate 0 (e.g.
  • 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 i -vacuo for a few hours.
  • Example la The peptide obtained in Example la was suspended in DMF (5ml) and treated with glutaric anhydride (300mg) and 4-methyl-mo ⁇ holine (200mg) in a round bottomed flask (25ml). 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.
  • 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 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 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.
  • 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 J LH20 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. From the shape of the chromatogram. it was deemed that 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.
  • Table 1 contains activity data relating to the invention.
  • the 5 designation “Z” denotes benzoyloxy carbonyl 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(OS0 3 H) is replaced by Tyr.
  • 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.
  • a volume of 150 ⁇ l of citrated normal human plasma and 20 ⁇ l of buffer or sample were warmed at 37°C for 1 min. Coagulation was started by adding 150 ⁇ l of freshly prepared bovine thrombin (5NIIIu/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.
  • the inhibition of human ⁇ -thrombin was determined by the inhibition of the enzyme catalysed hydrolysis of three different concentrations of the chromogenic substrate S-
  • the buffer used contained 0.1M sodium phosphate. 0.2M NaCl. 0.5% PEG and 0.02% sodium azide. adjusted to pH 7.5 with orthophosphoric acid.
  • the samples consist of the compound disclosed in DMSO.

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Abstract

L'invention concerne des inhibiteurs peptidiques bifonctionnels de thrombine et leurs procédés de fabrication. Ces inhibiteurs possèdent une fraction d'association à l'exosite de fixation d'anions reliée à une fraction dirigée catalytique. Un peptide espaceur et une fraction de séquence de liaison non peptidique permettent à la fois à la fraction d'association à l'exosite de fixation d'anions et à la fraction dirigée catalytique de se lier simultanément à une molécule de thrombine, facilitant ainsi le traitement de la thrombose.
PCT/GB1997/001575 1996-06-29 1997-06-11 Inhibiteurs de thrombine WO1998000443A1 (fr)

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AU30426/97A AU3042697A (en) 1996-06-29 1997-06-11 Thrombin inhibitors
JP10503898A JP2000516203A (ja) 1996-06-29 1997-06-11 トロンビン阻害剤
EP97925198A EP0910583A1 (fr) 1996-06-29 1997-06-11 Inhibiteurs de thrombine

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GB9613718.7 1996-06-29

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1466917A1 (fr) 2002-09-09 2004-10-13 Trigen Limited Procédé der préparation des acides boroniques peptidiques et acides obtenus
US7112572B2 (en) 2002-09-09 2006-09-26 Trigen Limited Multivalent metal salts of boronic acids
GB2442187A (en) * 2005-07-28 2008-03-26 Tenneco Automotive Operating Wind deflector with symmetrical geoometry
US9217027B2 (en) 2007-06-18 2015-12-22 Isis Innovation Limited Thrombin inhibitor
US9518129B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9518128B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules
WO2019030706A1 (fr) 2017-08-10 2019-02-14 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et méthodes d'utilisation en chirurgie orthopédique
WO2019035055A1 (fr) 2017-08-16 2019-02-21 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et procédés d'utilisation avec des agents antiagrégants plaquettaires

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2353619C2 (ru) * 2007-06-28 2009-04-27 Общество С Ограниченной Ответственностью "Бионика" Новые соединения, обладающие функцией антикоагулянтов, фармацевтические композиции на их основе для лечения тромботических состояний и плазмозамещающий раствор для коррекции гиперкоагуляционных нарушений при гемодилюции

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WO1991002750A1 (fr) * 1989-08-18 1991-03-07 Biogen, Inc. Nouveaux inhibiteurs de thrombine
WO1992013952A1 (fr) * 1991-02-08 1992-08-20 Biogen, Inc. Inhibiteurs ameliores de thrombine
WO1996029347A1 (fr) * 1995-03-20 1996-09-26 National Research Council Of Canada Inhibiteurs de thrombine fondes sur la sequence de l'acide amine de l'hirudine
WO1996030407A1 (fr) * 1995-03-27 1996-10-03 National Research Council Of Canada Inhibiteurs de thrombine bifonctionnels a composant de liaison d'exosite de reconnaissance du fibrinogene fortement tronque

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WO1991002750A1 (fr) * 1989-08-18 1991-03-07 Biogen, Inc. Nouveaux inhibiteurs de thrombine
WO1992013952A1 (fr) * 1991-02-08 1992-08-20 Biogen, Inc. Inhibiteurs ameliores de thrombine
WO1996029347A1 (fr) * 1995-03-20 1996-09-26 National Research Council Of Canada Inhibiteurs de thrombine fondes sur la sequence de l'acide amine de l'hirudine
WO1996030407A1 (fr) * 1995-03-27 1996-10-03 National Research Council Of Canada Inhibiteurs de thrombine bifonctionnels a composant de liaison d'exosite de reconnaissance du fibrinogene fortement tronque

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DIMAIO J ET AL: "A NEW CLASS OF POTENT THROMBIN INHIBITORS THAT INCORPORATES A SCISSILE PSEUDOPEPTIDE BOND", FEBS LETTERS, vol. 282, no. 1, 22 April 1991 (1991-04-22), pages 47 - 52, XP000147169 *
J. DIMAIO ET AL.: "Synthesis of a Homologous Series of Ketomethylene Arginyl Pseudopeptides and Application to Low Molecular Weight Hirudin-like Thrombin Inhibitors", JOURNAL OF MEDICINAL CHEMISTRY, vol. 35, no. 18, 4 September 1992 (1992-09-04), WASHINGTON US, pages 3331 - 3341, XP002043788 *
S. ELGENDY ET AL.: "Design of a novel class of bifunctional thrombin inhibitors, synthesised by the first application of peptide boronates in solid phase chemistry", TETRAHEDRON LETTERS, vol. 38, no. 18, 5 May 1997 (1997-05-05), OXFORD GB, pages 3305 - 3308, XP002043733 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1466917A1 (fr) 2002-09-09 2004-10-13 Trigen Limited Procédé der préparation des acides boroniques peptidiques et acides obtenus
EP1466916A1 (fr) * 2002-09-09 2004-10-13 Trigen Limited Acides boroniques peptidiques utilises pour préparer leurs sels
US7112572B2 (en) 2002-09-09 2006-09-26 Trigen Limited Multivalent metal salts of boronic acids
US7371729B2 (en) 2002-09-09 2008-05-13 Trigen Limited Boronic acid salts useful in parenteral formulations
GB2442187A (en) * 2005-07-28 2008-03-26 Tenneco Automotive Operating Wind deflector with symmetrical geoometry
US9217027B2 (en) 2007-06-18 2015-12-22 Isis Innovation Limited Thrombin inhibitor
US9605082B2 (en) 2011-12-14 2017-03-28 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9518128B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules
US9518129B2 (en) 2011-12-14 2016-12-13 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9988461B2 (en) 2011-12-14 2018-06-05 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US9988463B2 (en) 2011-12-14 2018-06-05 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US10287363B2 (en) 2011-12-14 2019-05-14 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US10370454B2 (en) 2011-12-14 2019-08-06 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
US11155637B2 (en) 2011-12-14 2021-10-26 Janssen Pharmaceuticals, Inc. Thrombin-binding antibody molecules and uses thereof
WO2019030706A1 (fr) 2017-08-10 2019-02-14 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et méthodes d'utilisation en chirurgie orthopédique
WO2019035055A1 (fr) 2017-08-16 2019-02-21 Janssen Pharmaceutica Nv Molécules d'anticorps anti-thrombine et procédés d'utilisation avec des agents antiagrégants plaquettaires

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