WO1994012507A1 - Organophosphorus compounds - Google Patents

Organophosphorus compounds Download PDF

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WO1994012507A1
WO1994012507A1 PCT/GB1993/002466 GB9302466W WO9412507A1 WO 1994012507 A1 WO1994012507 A1 WO 1994012507A1 GB 9302466 W GB9302466 W GB 9302466W WO 9412507 A1 WO9412507 A1 WO 9412507A1
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group
compound
reaction
alkyl
aryl
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PCT/GB1993/002466
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French (fr)
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Martin Keith Johnson
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Medical Research Council
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Priority claimed from GB929225003A external-priority patent/GB9225003D0/en
Priority claimed from GB929225000A external-priority patent/GB9225000D0/en
Application filed by Medical Research Council filed Critical Medical Research Council
Priority to AU55714/94A priority Critical patent/AU5571494A/en
Publication of WO1994012507A1 publication Critical patent/WO1994012507A1/en

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/657109—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms esters of oxyacids of phosphorus in which one or more exocyclic oxygen atoms have been replaced by (a) sulfur atom(s)
    • C07F9/657127—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms esters of oxyacids of phosphorus in which one or more exocyclic oxygen atoms have been replaced by (a) sulfur atom(s) condensed with carbocyclic or heterocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/657154—Cyclic esteramides of oxyacids of phosphorus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/6574—Esters of oxyacids of phosphorus
    • C07F9/65744—Esters of oxyacids of phosphorus condensed with carbocyclic or heterocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase

Definitions

  • the present invention relates to an intermediate in the preparation of a class of compounds which may be employed as inhibitors of serine esterase enzymes and which may be employed in the identification and purification of serine esterase enzymes.
  • organophosphorus (OP) esters are known to inhibit the catalytic activity of a class of enzymes known as "serine esterases" such as acetylcholinesterase (AChE), chymotrypsin, various chemotactic factors and blood-clotting enzymes and neuropathy target esterase (NTE).
  • serine esterase as used herein covers tissue proteases and esterases.
  • the enzymes of interest can be inhibited by progressive covalent reaction with a variety of organophosphorus (OP) esters.
  • the enzymes of interest typically comprise the serine esterase consensus sequence
  • OP inhibitors those which inhibit AChE within the neurotransmitter systems of insects may be effective insecticides and more than 100 OP insecticides are marketed worldwide (Organophosphorus Insecticides: A General Introduction IPCS Environmental Health Criteria Series No.63 (1986) WHO, Geneva).
  • OP inhibitors of serine esterases are normally esters, thiol esters or acid anhydride derivatives of phosphorus- containing acids.
  • the general structural formula is:
  • R' and R'' are usually simple alkyl or aryl groups both of which may be bonded directly to the phosphorus atom (in phosphinates), or linked via -O- or -S- (in phosphates), or R' may be bonded directly and R'' via one of the above groups (phosphonates).
  • phosphoroamidates carbon is linked to phosphorus through an amino-group.
  • the group L is referred to as the leaving group and can be any one of a wide variety of substituted and/or branched aliphatic, aromatic, or heterocyclic groups, linked to phosphorus via a bond of some lability (usually -O- or -S-).
  • the double- bonded atom Y may be oxygen or sulphur, and related compounds would be called phosphates or phosphorothioates, respectively.
  • the P O analogue of a thioate ester is referred to as the oxon.
  • reaction 1 a specific serine in the protein is phosphorylated with loss of the leaving group L (reaction 2).
  • reaction 2 This is a progressive reaction leading to formation of a reasonably stable covalent bond between OP compound and enzyme with consequent inhibition of catalytic activity: the binding of OP is not reversed by removal of excess inhibitor as would be the case for many reversible pharmacologic agents acting on receptors or for reversible inhibitors of enzymes.
  • the rate of reaction (2) is a specific property of both the chemical and of the enzyme. The overall "reactivity" can be measured in the laboratory as the second order rate constant of the progressive reaction. After inhibition, two further reactions are then possible.
  • Reaction 3 (reactivation) may occur spontaneously but rather slowly at a rate that can be influenced by added nucleophilic reagents, such as oximes, which may catalyse the reaction and thereby act as an antidote.
  • Reaction 4 (aging) involves cleavage of one or other bond in the R-O-P chain with the loss of R and the formation of a charged monosubstituted phosphoric acid residue still attached to protein. This reaction is called aging because it is a slow progressive process and the product is no longer responsive to nucleophilic reactivating agents.
  • One particular class of organophosphorus compound associated with serine esterase inhibitory activity possesses a six- membered phosphorus-containing ring structure (4-H-1,3,2- benzo-dioxophosphorane) (II).
  • R''' simple alkyl or aryl
  • OPIDP organophosphate induced delayed polyneuropathy
  • esters may be synthesised by a condensation reaction of the appropriate phosphorodichloridate (III) with 2-hydroxy-benzyl alcohol (saligenin, IV):
  • inhibited esterases VI proceeds as follows:
  • Eto and coworkers have synthesised compounds related to compound (II) as putative inhibitors by modifying the precursors (III) and (IV) (Eto M. as above and Sasaki et al.
  • esters of structure (V) A further disadvantage of the conventional synthetic route to esters of structure (V) is that the preparation of compounds with different R''' group requires the individual preparation of suitable precursors (III) containing the desired R''' group.
  • Hal may be the same or different and are selected from F, Cl and Br
  • R 1 hydrogen or an alkyl, aryl, alkoxy, acyl, halogen or nitro group or a fused aromatic ring
  • R 2 hydrogen or an alkyl, aryl or cyano group.
  • A an alkyl, aryl, peptide, heterocyclic or reporter group.
  • B-R 2 is CHR 2 .
  • R 1 may be any substituent selected from the group comprising hydrogen, and alkyl, aryl, alkoxy, acyl, halogen or nitro groups or may be a fused aromatic ring such that the compound VIII is an inhibitor of a serine esterase enzyme.
  • R 1 may comprise a fused aromatic ring such as phenyl (such that structure VII comprises a naphthalene ring system), pyridine (quinoline, isoquinoline), pyrrole (indole), furan or thiophene.
  • phenyl such that structure VII comprises a naphthalene ring system
  • pyridine such that structure VII comprises a naphthalene ring system
  • pyridine quinoline, isoquinoline
  • pyrrole indole
  • furan or thiophene such as a fused aromatic ring such as phenyl (such that structure VII comprises a naphthalene ring system), pyridine (quinoline, isoquinoline), pyrrole (indole), furan or thiophene.
  • R 1 is selected from the group comprising hydrogen and C 1-5 alkyl, C 6-10 aryl, C 2-5 acyl, C 1-5 alkoxy, chlorine and nitro groups.
  • R 1 is hydrogen.
  • R 1 may comprise one or more, preferably one, substituents appearing at any of the positions C-5, C-6, C-7 or C-8 of the aromatic ring of structure VII. Preferably the R 1 substituent appears at the C-6 or C-8 position.
  • R 2 may be any substituent selected from the group comprising hydrogen and alkyl, aryl and cyano groups such that compound VII is an inhibitor of a serine esterase enzyme.
  • R 2 is selected from the group comprising hydrogen, C 1-5 alkyl, C 6-10 aryl or cyano.
  • R 2 is hydrogen.
  • both R 1 and R 2 are hydrogen.
  • reference to an alkyl group means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e.g. alkenyl or alkynyl) hydrocarbyl radical.
  • the alkyl group is preferably C 3 to C 12 , more preferably C 5 to C 10 , more preferably C 5 to C 7 .
  • the alkyl group is preferably C 1 to C 10 , more preferably C 1 to C 5 , more preferably methyl.
  • aryl group means an aromatic group, such as phenyl or naphthyl, or a heteroaromatic group containing one or more, preferably one, heteratom, such as pyridyl, pyrrolyl, furanyl and thiophenyl.
  • the aryl group comprises phenyl.
  • the alkyl and aryl groups may be substituted or unsubstituted, preferably unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent.
  • Substituents may include halogen atoms; oxygen containing groups such as oxo, hydroxy, carboxy, carboxyalkyl, alkoxy, alkoyl, alkoyloxy; nitrogen containing groups such as amino, alkylamino, dialkylamino, cyano, azide and nitro; sulphur containing groups such as thiol, alkylthiol, sulphonyl and sulphoxide; heterocyclic groups containing one or more, preferably one, heteroatom, such as thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, pyrrolidinyl, pyrrolinyl
  • Alkyl includes substituted and unsubstituted benzyl.
  • Hal may be the same or different and are selected from fluorine, chlorine or bromine.
  • Hal are the same.
  • Hal are chlorine.
  • R is a substituted or unsubstituted alkyl or aryl group.
  • the reaction is conducted by stepwise addition of compound (IX) to a suspension of PHal 5 in a dry organic solvent, preferably toluene.
  • a dry organic solvent preferably toluene.
  • the temperature of the reaction is maintained in the range -10°C to 10°C, preferably 0°C to 5°C.
  • the product (VII) may be isolated and purified by distillation.
  • a compound of formula (VII) in the preparation of a serine esterase inhibitor or thionate analogue thereof.
  • the serine esterase inhibitor or thionate analogue thereof is of the formula (VIII) as hereinbefore defined.
  • the use and applications of compounds of formula (VIII) are further described in United Kingdom Patent Application No. 9225000.0 and in an International patent application having the same filing date as the present application.
  • the present invention further provides a process for the preparation of a compound of formula (VIII) comprising the steps of reaction of compound (VII) with a compound A-X-H followed by reaction with compound H 2 Y.
  • a feature of the present invention is that the trihalo compound (VII) gives substantially only a mono-reaction product with a stoichiometric amount of A-X-H. Di- or tri- substitution of the halogen atoms by the group A-X- is not observed.
  • Y may be oxygen or sulphur such that the compound H 2 Y comprises water or hydrogen sulphide, respectively.
  • compounds of formula (VIII) may possess potent serine esterase inhibitory activity.
  • compounds of formula VIII are generally not significant inhibitors of serine esterase activity, but may be metabolically activated in vivo to the corresponding oxons
  • X may be O, NH, NR or S such that the compound A-X-H comprises an alcohol, amine, secondary amine or thiol respectively.
  • X may comprise groups which include NH and NR, such as substituted or unsubstituted hydrazine and hydrazone derivatives.
  • X is O or NH.
  • X is NH.
  • the reaction between compound (VII) and A-X-H may be conducted in a dry organic solvent such as acetonitrile, dimethyl formamide, dimethyl sulphoxide, trichloroethylene or diethyl ether in the presence of a base.
  • a dry organic solvent such as acetonitrile, dimethyl formamide, dimethyl sulphoxide, trichloroethylene or diethyl ether
  • any weak base is suitable, but a sterically hindered base, preferably 2,6-Lutidine, is preferred.
  • the reaction is conducted in the presence of a reagent to trap the hydrogen halide produced in the reaction.
  • an inorganic base such as K 2 CO 3 , MgO or PbO may be used.
  • the group A may be selected from a wide variety of alkyl, aryl, peptide, heterocyclic or reporter groups.
  • the level and selectivity of the serine esterase inhibitory activity of compound (VIII) is dependent in part upon the structure of the group A. It will be appreciated that the level and selectivity of the serine esterase inhibitory activity of
  • the group A may comprise a C 1-20 alkyl or C 6-20 aryl group.
  • the alkyl group may be straight chain or branched.
  • the alkyl and aryl groups may be substituted or unsubstituted.
  • the alkyl and aryl groups may contain one or more hetero atoms, for example O, N or S.
  • the group A may comprise a heterocyclic group or a polypeptide of up to 10 amino acid residues, preferably 3 to 5 residues. Selection of a polypeptide may be advantageous in improving the selectivity of the inhibitor for a particular serine esterase.
  • the group A comprises a C 1-12 straight chain alkyl group, a phenyl group or a benzyl group.
  • the group A is substituted with an amino group, preferably a ⁇ - amino group.
  • the group A may comprise a reporter group.
  • reporter group is defined as a labelling or affinity group facilitating identification and/or purification of any compound to which it is attached.
  • the group A may comprise, for example, a fluorescent or radio-label.
  • An isotopic radio-label may, for example, comprise tritium, [ 14 C], [ 32 P] or [ 125 I].
  • the group A may be derived from the well known Bolton Hunter reagent (N-succinimidyl 3-(4-hydroxy-5-[ 125 I]iodophenyl) propionate) (Amersham Life Sciences). Such labelling groups may be identified and assayed according to conventional techniques.
  • A is an affinity group.
  • an "affinity group” is a molecule or functional group suitable for affinity labelling or chromatography.
  • the affinity group comprises one member of a binding pair.
  • Binding pairs are pairs of molecules capable of a non- covalent binding interaction and examples are well known to persons skilled in the art. They may comprise, for example, an antigen-antibody binding pair such as digoxigenin- (digoxigenin antibody). Alternatively the binding pair may comprise a non-antibody binding pair such as the biotin- avidin binding pair.
  • A is biotin.
  • the reporter group may be attached directly to X.
  • the reporter group is attached to X via a linker.
  • the linker may comprise any suitable spacer group such that the reporter group and the organophosphorus serine esterase inhibitor part of the molecule (VIII) are sufficiently separated so as not to adversely affect each other's function.
  • the preferred linker comprises a C 1-20 alkyl chain, preferably a C 7-12 alkyl chain.
  • the alkyl chain is unbranched and unsubstituted.
  • the linker may comprise a peptide of up to 10 amino acid residues, preferably 3 to 5 residues.
  • the linker may be chosen to optimise the selectivity of the organophosphorus inhibitor for a particular serine esterase.
  • a compound according to the present invention as an inhibitor of a serine esterase enzyme.
  • Mass-spectral analysis using a probe-applied sample in a VG tandem mass spectrometer operating in the electron-impact mode showed a spectrum of primary ion and fragmentation products indicative of a trichlorocompound of the predicted MW.
  • 4-H-1,3,2-Benzodioxaphosphorin-3,3,3-tribromide or trifluoride may be prepared by analogous reaction using PBr 5 or PBr 5 , respectively.
  • Mixed trihalides may be prepared using mixed-halide PHal 5 (PHal x Hal' 5-x ) reagents.
  • inorganic bases K 2 CO 3 , MgO, PbO
  • inorganic bases K 2 CO 3 , MgO, PbO
  • SCPCl 3 solution [10 -1 -10 -3 M]
  • second reactant a range of base each dissolved in equivalent volumes of dry solvent.
  • Traces of fumes of HCl were observed to be formed when the more concentrated solutions were used. After 10-30 min a crystalline precipitate was observed in some cases but this was not prognostic of a successful reaction: this dissolved (or decomposed) instantly on addition of water.
  • the product solution was vacuum-dried, dissolved in a suitable solvent (such as acetonitrile or DMF) and subjected to HPLC separation, generally using acetonitrile/ triethylammonium acetate buffer gradients with monitoring of u/v absorbtion at 210 nm and bioassay of interesting fractions. Fractions shown to contain inhibitors were vacuum-dried, stored at -20° and analysed at intervals by standard methods. Specific examples
  • 1-N-biotinyl, 1,9-diaminononane was synthesised from N- hydroxylsuccinimidylbiotin (Pierce Chem. Co.) and 1,9- diaminononane by standard reaction in dimethylformamide and purified by HPLC (see general procedure). It was dissolved in dry dimethylformamide (0.23 ⁇ mole in 29 ⁇ l) and lutidene and SCPCl 3 (each 47 ⁇ mole in 23 ⁇ l dry acetonitrile) were added. After 15 min reaction at 37°C reaction was completed by addition of pH 5 buffer (25 ⁇ l). Aliquots of the solution were vacuum dried and stored at 5° for varying times prior to purification by HPLC and storage.
  • NTE neuropathy target esterase
  • the membranes were dissolved by boiling in sodium dodecyl sulphate (SDS) solution (0.15%) in neutral buffer containing EDTA (1 mM) and dithiothreitol (10 mM).
  • SDS sodium dodecyl sulphate
  • the affinity-labelled NTE was selectively adsorbed onto avidin bound covalently to a matrix such as Agarose or Polyacrylic beads. Adsorbtion was monitored analytically by assay of biotin-carrying protein remaining in the liquid medium at different times. No significant adsorbtion of non-biotinyl proteins was confirmed by assay of total protein. After the beads had been washed twice with dilute SDS the adsorbed S9B-NTE was released by boiling the beads in 5% SDS in buffer for 5 min.
  • affinity-labelled NTE was achieved by preparative polyacrylamide gel electrophoresis to separate NTE from traces of other labelled esterases and from two endogenous biotin-containing proteins of much lower molecular weight than NTE. Quantitative analysis (assay) of affinity purified NTE was carried out using enzyme enhanced chemiluminescence assay for biotin. The pure protein was then available for determination of amino-acid sequence, raising of antibodies etc. using conventional techniques. The serine esterase may be released from the inhibitor by treatment with alkali at pH 11 at ambient temperature.
  • the S9B reagent and technique applied above to label NTE in broken membranes can equally be applied to selectively label NTE in fresh sections of intact animal tissue. Standard techniques can then be applied using enzyme-linked antibodies to visualise NTE at the light microscopic or EM level. Alternatively a digoxigenin-linked reagent derived from SCPCl 3 can be used. Table I: Inhibitors of hen brain NTE produced from reaction of SCPCI 3 with amines
  • Bolton-Hunter Reagent (BHR; N-succinimidyl 3-(4- hydroxyphenyl) propionate, Sigma Chem. Co.) (2 ⁇ moles) was iodinated by the well-known standard procedure (Bolton, A.E. & Hunter, W.M. (1973), Biochem, J. 133, 529-539), using NaI (10 ⁇ mole) plus a trace of [ 125 I] -Nal (Amersham International). The product was extracted from quenched aqueous reaction mixture (135 ⁇ l) into benzene (2 x 400 ⁇ l).
  • Iodo-BHR 1.0 ⁇ mole in MeCN/DMSO (220 ⁇ l) was mixed with 1,9-diaminononane (5.0 ⁇ mole in MeCN/DMSO (100 ⁇ L)) at room temperature. An immediate reaction was observed and, after vortexing and centrifugation, a yellow-green liquor was obtained above a white sediment. TLC analysis was performed on samples of liquor (silica strips : Isopropanol/ water/0.880 ammonia (8:1:1)). The developed strips were sprayed with fluorescamine reagent, (Sigma Chem. Co.) and viewed under long-wave U/V illumination. Strips were then cut into slices for ⁇ -counting.
  • the pooled fractions 4-6 were fluorescamine-positive and considered likely to contain pure product ⁇ -(w-amino-nonyl) 3-(4-hydroxy, 3,5-di-iodophenyl) proprionamide.
  • the pool was concentrated and used for coupling to the OP reagent.
  • the specific radioactivity of this preparation was adequate for tracing progress of the steps of the synthesis.
  • Increases of specific radioactivity of the sodium iodide used in initial labelling are possible up to at least 4 orders of magnitude so that detectable labelling of even minute amounts of enzyme are feasible in gels or histochemical localisation in tissue sections.
  • intermediate levels of specific radioactivity would be quite adequate for tracing an enzyme purification on the scale at which such procedures are normally performed.

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Abstract

Compounds of formulas (a) or (b) wherein Hal may be the same or different and are selected from fluorine, chlorine and bromine; R1 is hydrogen or an alkyl, aryl, alkoxy, acyl, halogen or nitro group or a fused aromatic ring; R2 is hydrogen or an alkyl, aryl or cyano group. The compounds are useful in the preparation of inhibitors of serine esterase enzymes.

Description

ORGANOPHOSPHORUS COMPOUNDS
INTRODUCTION
The present invention relates to an intermediate in the preparation of a class of compounds which may be employed as inhibitors of serine esterase enzymes and which may be employed in the identification and purification of serine esterase enzymes.
BACKGROUND OF THE INVENTION
Some organophosphorus (OP) esters are known to inhibit the catalytic activity of a class of enzymes known as "serine esterases" such as acetylcholinesterase (AChE), chymotrypsin, various chemotactic factors and blood-clotting enzymes and neuropathy target esterase (NTE). The term "serine esterase" as used herein covers tissue proteases and esterases. The enzymes of interest can be inhibited by progressive covalent reaction with a variety of organophosphorus (OP) esters. The enzymes of interest typically comprise the serine esterase consensus sequence
(-glycine-glutamic acid-serine-X-glycine-, wherein X is an amino acid and is variable) at their catalytic centre. It should be noted that chymotrypsin, trypsin and various other typical proteases, which are sensitive to inhibition by organophosphorus esters, are included within the definition of "serine esterase". Among the OP inhibitors, those which inhibit AChE within the neurotransmitter systems of insects may be effective insecticides and more than 100 OP insecticides are marketed worldwide (Organophosphorus Insecticides: A General Introduction IPCS Environmental Health Criteria Series No.63 (1986) WHO, Geneva). OP inhibitors of serine esterases are normally esters, thiol esters or acid anhydride derivatives of phosphorus- containing acids. The general structural formula is:
Figure imgf000004_0001
R' and R'' are usually simple alkyl or aryl groups both of which may be bonded directly to the phosphorus atom (in phosphinates), or linked via -O- or -S- (in phosphates), or R' may be bonded directly and R'' via one of the above groups (phosphonates). In phosphoroamidates, carbon is linked to phosphorus through an amino-group. The group L is referred to as the leaving group and can be any one of a wide variety of substituted and/or branched aliphatic, aromatic, or heterocyclic groups, linked to phosphorus via a bond of some lability (usually -O- or -S-). The double- bonded atom Y may be oxygen or sulphur, and related compounds would be called phosphates or phosphorothioates, respectively. The P = O analogue of a thioate ester is referred to as the oxon.
The interactions of OP inhibitors with serine esterases generally all proceed in analogous fashion. However, the rates of each step may differ according to the structure of the OP compound and may also differ from enzyme to enzyme. In each case, the initial step of inhibition requires that the OP compound is in the oxon (P = O) form. Pure thionate (P = S) compounds are generally not significant inhibitors in their original form but are metabolically activated in vivo to oxons.
The four stages of interaction of OP oxons with an esterase are illustrated in Scheme 1.
Figure imgf000005_0001
Scheme 1
Following the formation of a Michaelis complex (reaction 1), a specific serine in the protein is phosphorylated with loss of the leaving group L (reaction 2). This is a progressive reaction leading to formation of a reasonably stable covalent bond between OP compound and enzyme with consequent inhibition of catalytic activity: the binding of OP is not reversed by removal of excess inhibitor as would be the case for many reversible pharmacologic agents acting on receptors or for reversible inhibitors of enzymes. The rate of reaction (2) is a specific property of both the chemical and of the enzyme. The overall "reactivity" can be measured in the laboratory as the second order rate constant of the progressive reaction. After inhibition, two further reactions are then possible. Reaction 3 (reactivation) may occur spontaneously but rather slowly at a rate that can be influenced by added nucleophilic reagents, such as oximes, which may catalyse the reaction and thereby act as an antidote. Reaction 4 (aging) involves cleavage of one or other bond in the R-O-P chain with the loss of R and the formation of a charged monosubstituted phosphoric acid residue still attached to protein. This reaction is called aging because it is a slow progressive process and the product is no longer responsive to nucleophilic reactivating agents. One particular class of organophosphorus compound associated with serine esterase inhibitory activity possesses a six- membered phosphorus-containing ring structure (4-H-1,3,2- benzo-dioxophosphorane) (II).
Figure imgf000006_0001
wherein Y = O or S
X = O, S or NH
R''' = simple alkyl or aryl
Both the 2-methyl ester and 2-methylamide (R''' -X=CH3O- and CH3NH-, respectively) of the 2-sulphide (Y = S) are known pesticides and are converted to the anti-esterase 2-oxides in vivo. (Eto M. (1969) Specificity and Mechanism in the Action of Saligenin Cyclic Phosphorus Esters, Residue Reviews, 25,187-200; Eto M. (1974) Organophosphorus Pesticides: Organic and Biological Chemistry, CRC Press, Cleveland, Ohio). The phenyl and 2-tolyl (R''' X = PhO- and CH3PhO-, respectively) of the 2-oxide are known to cause a toxic syndrome known as organophosphate induced delayed polyneuropathy (OPIDP) and the phenyl ester is known to be a potent progressive inhibitor of NTE, the target for initiation of OPIDP (Johnson et al . , Archives of Toxicology (1975) 34, 259-288).
Conventionally, these esters may be synthesised by a condensation reaction of the appropriate phosphorodichloridate (III) with 2-hydroxy-benzyl alcohol (saligenin, IV):
Figure imgf000007_0001
The formation of inhibited esterases VI proceeds as follows:
Figure imgf000007_0002
Eto and coworkers have synthesised compounds related to compound (II) as putative inhibitors by modifying the precursors (III) and (IV) (Eto M. as above and Sasaki et al.
(1988) Synthesis of 2-methoxy-4-H-1,3,2-benzodioxaphosphorin-2-sulphide and Related Compounds
Utilising Intra-molecular Cyclisation Reactions, Agric. Biol. Chem., 52, 159-168). Thus products substituted in positions 6 or 8 are mentioned as well as several simple alkyl alternatives to the R''' group.
However, the conventional synthetic route to esters of structure (V) suffers from a number of disadvantages. In the first instance the phosphorodichloridate compounds (III) are highly reactive such that preparation, purification and use of these compounds requires special precautions. These compounds are particularly prone to polymerisation. Additionally, the high reactivity of these compounds severely limits the range of R''' substituents that may be employed. Typically R''' is limited to the simplest alkyl
(e.g. methyl) or aryl (e.g. phenyl) groups. A further disadvantage of the conventional synthetic route to esters of structure (V) is that the preparation of compounds with different R''' group requires the individual preparation of suitable precursors (III) containing the desired R''' group.
There is therefore a need for an improved synthetic route to compounds of structure (II), which avoids the need to prepare highly reactive phosphorochloridate precursors (III) and which facilitates preparation of compounds of structure (II) with a variety of substituents XR'''. SUMMARY OF THE INVENTION
According to the present invention there is provided a compound of the formula (VII):
Figure imgf000008_0001
wherein Hal may be the same or different and are selected from F, Cl and Br
R1 = hydrogen or an alkyl, aryl, alkoxy, acyl, halogen or nitro group or a fused aromatic ring
B-R2 is C=O or CHR2
R2 = hydrogen or an alkyl, aryl or cyano group.
It has been found that the compound of the present invention provides a convenient and widely applicable intermediate in the preparation of compounds of structure (VIII):-
Figure imgf000008_0002
wherein B, R1 and R2 are as hereinbefore defined,
Y = O or S X = O, S, NH or NR
A = an alkyl, aryl, peptide, heterocyclic or reporter group. Preferably B-R2 is CHR2.
R1 may be any substituent selected from the group comprising hydrogen, and alkyl, aryl, alkoxy, acyl, halogen or nitro groups or may be a fused aromatic ring such that the compound VIII is an inhibitor of a serine esterase enzyme.
R1 may comprise a fused aromatic ring such as phenyl (such that structure VII comprises a naphthalene ring system), pyridine (quinoline, isoquinoline), pyrrole (indole), furan or thiophene.
Preferably, R1 is selected from the group comprising hydrogen and C1-5 alkyl, C6-10 aryl, C2-5 acyl, C1-5 alkoxy, chlorine and nitro groups. Preferably, R1 is hydrogen.
R1 may comprise one or more, preferably one, substituents appearing at any of the positions C-5, C-6, C-7 or C-8 of the aromatic ring of structure VII. Preferably the R1 substituent appears at the C-6 or C-8 position.
R2 may be any substituent selected from the group comprising hydrogen and alkyl, aryl and cyano groups such that compound VII is an inhibitor of a serine esterase enzyme. Preferably, R2 is selected from the group comprising hydrogen, C1-5 alkyl, C6-10 aryl or cyano. Preferably, R2 is hydrogen.
Preferably, both R1 and R2 are hydrogen. In general, as used herein, reference to an alkyl group means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e.g. alkenyl or alkynyl) hydrocarbyl radical. Where cyclic, the alkyl group is preferably C3 to C12, more preferably C5 to C10, more preferably C5 to C7. Where acyclic, the alkyl group is preferably C1 to C10, more preferably C1 to C5, more preferably methyl.
Reference to an aryl group means an aromatic group, such as phenyl or naphthyl, or a heteroaromatic group containing one or more, preferably one, heteratom, such as pyridyl, pyrrolyl, furanyl and thiophenyl. Preferably, the aryl group comprises phenyl.
The alkyl and aryl groups may be substituted or unsubstituted, preferably unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent. Substituents may include halogen atoms; oxygen containing groups such as oxo, hydroxy, carboxy, carboxyalkyl, alkoxy, alkoyl, alkoyloxy; nitrogen containing groups such as amino, alkylamino, dialkylamino, cyano, azide and nitro; sulphur containing groups such as thiol, alkylthiol, sulphonyl and sulphoxide; heterocyclic groups containing one or more, preferably one, heteroatom, such as thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, piperidyl, piperazinyl, morpholinyl, thionaphthyl, benzofuranyl, isobenzofuryl, indolyl, oxyindoyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, isoindazolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolyl, isoquinolyl, naphthridinyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxadinyl, chromenyl, chromanyl, isochromanyl and carbolinyl; and aryl groups such as phenyl and substituted phenyl. Alkyl includes substituted and unsubstituted benzyl. Hal may be the same or different and are selected from fluorine, chlorine or bromine. Preferably, Hal are the same. Preferably, Hal are chlorine. R is a substituted or unsubstituted alkyl or aryl group.
According to a further aspect of the present invention there is provided a process for the preparation of compound (VII) comprising reaction of a compound of formula (IX):-
Figure imgf000011_0001
with PHal5.
Preferably, the reaction is conducted by stepwise addition of compound (IX) to a suspension of PHal5 in a dry organic solvent, preferably toluene. Preferably, the temperature of the reaction is maintained in the range -10°C to 10°C, preferably 0°C to 5°C. The product (VII) may be isolated and purified by distillation.
According to a further aspect of the present invention there is provided use of a compound of formula (VII) in the preparation of a serine esterase inhibitor or thionate analogue thereof. Preferably, the serine esterase inhibitor or thionate analogue thereof is of the formula (VIII) as hereinbefore defined. The use and applications of compounds of formula (VIII) are further described in United Kingdom Patent Application No. 9225000.0 and in an International patent application having the same filing date as the present application. Thus, the present invention further provides a process for the preparation of a compound of formula (VIII) comprising the steps of reaction of compound (VII) with a compound A-X-H followed by reaction with compound H2Y. A feature of the present invention is that the trihalo compound (VII) gives substantially only a mono-reaction product with a stoichiometric amount of A-X-H. Di- or tri- substitution of the halogen atoms by the group A-X- is not observed.
Y may be oxygen or sulphur such that the compound H2Y comprises water or hydrogen sulphide, respectively. When Y is oxygen, compounds of formula (VIII) may possess potent serine esterase inhibitory activity. When Y is sulphur, compounds of formula VIII are generally not significant inhibitors of serine esterase activity, but may be metabolically activated in vivo to the corresponding oxons
(Y = O), which are inhibitors of serine esterase enzymes.
Thus selection of Y = sulphur may be particularly advantageous in the preparation of precursor serine esterase inhibitors possessing good stability and capable of in vivo activation to a serine esterase inhibitor. Selection of
Y = O may be particularly advantagous if a compound with direct serine esterase inhibitory activity (i.e. not requiring in vivo activation) is required.
X may be O, NH, NR or S such that the compound A-X-H comprises an alcohol, amine, secondary amine or thiol respectively. X may comprise groups which include NH and NR, such as substituted or unsubstituted hydrazine and hydrazone derivatives. Preferably X is O or NH. Preferably X is NH.
The reaction between compound (VII) and A-X-H may be conducted in a dry organic solvent such as acetonitrile, dimethyl formamide, dimethyl sulphoxide, trichloroethylene or diethyl ether in the presence of a base. In principle any weak base is suitable, but a sterically hindered base, preferably 2,6-Lutidine, is preferred. Preferably the reaction is conducted in the presence of a reagent to trap the hydrogen halide produced in the reaction. Suitably an inorganic base such as K2CO3, MgO or PbO may be used. Alternatively if the reagent A-X-H comprises X = NH or NR, an excess of this reagent may be used.
If A-X-H is an alcohol, phenol or thiol (i.e. X = O or S), the alkali metal derivatives of these compounds may be employed.
The group A may be selected from a wide variety of alkyl, aryl, peptide, heterocyclic or reporter groups. The level and selectivity of the serine esterase inhibitory activity of compound (VIII) is dependent in part upon the structure of the group A. It will be appreciated that the level and selectivity of the serine esterase inhibitory activity of
(VIII) required for any particular application may be optimised without undue experimentation by a person skilled in the art by modifying group A according to the teachings of the present specification and according to common general knowledge.
The group A may comprise a C1-20 alkyl or C6-20 aryl group. The alkyl group may be straight chain or branched. The alkyl and aryl groups may be substituted or unsubstituted. The alkyl and aryl groups may contain one or more hetero atoms, for example O, N or S. The group A may comprise a heterocyclic group or a polypeptide of up to 10 amino acid residues, preferably 3 to 5 residues. Selection of a polypeptide may be advantageous in improving the selectivity of the inhibitor for a particular serine esterase.
Preferably the group A comprises a C1-12 straight chain alkyl group, a phenyl group or a benzyl group. Preferably, the group A is substituted with an amino group, preferably a ω- amino group.
According to a preferred embodiment of the present invention the group A may comprise a reporter group. As used herein a "reporter group" is defined as a labelling or affinity group facilitating identification and/or purification of any compound to which it is attached.
Of particular advantage is the use of a compound of formula (VIII) (where A comprises a reporter group) to react with a serine esterase enzyme according to the inhibitory pathway outlined above to yield the serine esterase enzyme to which is covalently attached the reporter group. The reporter group facilitates identification and/or purification of the serine esterase enzyme.
Thus the group A may comprise, for example, a fluorescent or radio-label. An isotopic radio-label may, for example, comprise tritium, [14C], [32P] or [125I]. For example the group A may be derived from the well known Bolton Hunter reagent (N-succinimidyl 3-(4-hydroxy-5-[125I]iodophenyl) propionate) (Amersham Life Sciences). Such labelling groups may be identified and assayed according to conventional techniques.
Preferably A is an affinity group. As used herein an "affinity group" is a molecule or functional group suitable for affinity labelling or chromatography. Preferably, the affinity group comprises one member of a binding pair. Binding pairs are pairs of molecules capable of a non- covalent binding interaction and examples are well known to persons skilled in the art. They may comprise, for example, an antigen-antibody binding pair such as digoxigenin- (digoxigenin antibody). Alternatively the binding pair may comprise a non-antibody binding pair such as the biotin- avidin binding pair.
Preferably A is biotin. The reporter group may be attached directly to X. Preferably, the reporter group is attached to X via a linker. The linker may comprise any suitable spacer group such that the reporter group and the organophosphorus serine esterase inhibitor part of the molecule (VIII) are sufficiently separated so as not to adversely affect each other's function. For example, in the purification of NTE the preferred linker comprises a C1-20 alkyl chain, preferably a C7-12 alkyl chain. Preferably the alkyl chain is unbranched and unsubstituted.
Alternatively, the linker may comprise a peptide of up to 10 amino acid residues, preferably 3 to 5 residues. The linker may be chosen to optimise the selectivity of the organophosphorus inhibitor for a particular serine esterase.
According to a further aspect of the present invention there is provided use of a compound according to the present invention as an inhibitor of a serine esterase enzyme.
The invention will now be described by way of example. It will be appreciated that the following description is by way of example only and that modification of detail may be made within the scope of the invention.
EXPERIMENTAL
Synthesis of 4-H-1,3,2-Benzodioxaphosphorin-3,3,3-trichloride (Saligenin cyclic phosphoro trichloride SCPCl3)
In a well-ventilated fume cupboard sodium-dried toluene (400 ml) was cooled to 0° under an atmosphere of argon in a 2L flask placed in an external ice/water bath. Powdered PCl5 (BDH, 84 g:0.4 mole) was sprinkled into the stirred toluene to obtain a suspension. While vigorous stirring was continued saligenin (2-hydroxybenzyl alcohol, Aldrich Chem. Co.; 42 g:0.35 mole) was added slowly over 2 hours by intermittent additions of aliquots (about 1 g) while maintaining the suspension temperature below 5°C.
At each addition fumes of HCl (and possibly some PCl5 entrained) were produced and were allowed to disperse before the next addition. After a further 30 min no powdered material remained and the solution was decanted away from a small amount of sticky orange solid and warmed under a flow of nitrogen to 27° during 2 hours. Solvent was stripped using a rotary evaporator at water-pump vacuum and water bath at 40-55°: a small amount of white solid was deposited in the cool condenser. The colourless residual oil with a few pieces of suspended white solid was left under argon at ambient temperature overnight. Dry toluene (150 ml) was added and the stripping procedure was repeated with the bath temperature raised eventually to 60° in order to remove any residual PCl5. The flask contents ( ca . 55 ml, straw- coloured) were allowed to cool under a stream of argon and transferred to a round-bottom flask fitted for vacuum distillation using a silicone-oil bath as heat source. Reduction of pressure (rotary evaporation pump) was carried out slowly to assist in avoiding vigorous degassing and the bath temperature was raised to 140° during 30 min. An initial distillate fraction (1 g; 108-112°; 0.15 mM Hg) was contaminated with charred material which had settled on the walls of the air-condenser. Thereafter steady distillation was maintained and three fractions distilling in the range 108-110°; 0.12-0.15 mm were collected while the bath temperature was raised through the range 147-164°. At this point distillation was slowing and it appeared that about 30 ml of darkening liquid remained undistilled. For the sake of obtaining quality product distillation was halted at this point and the residue was cooled under vacuum: it solidified to a reddish intractable mass.
Yield: Fn 1 (green/black: 1 g), Fn 2 (very pale yellow: 9.3 g), Fn 3 (colourless: 21.4 g), Fn 4 (colourless : 3.8 g): total = 35.5 g:0.13 mole=40% based on saligenin. The distillates were stored in the distillation receivers with carefully greased (silicone) stoppers kept in a desiccator at ca . 15°. The products appeared unchanged over many months provided that moist air was carefully excluded by a gentle flow of nitrogen or argon whenever a sample was removed for further study. The compound reacted with unprotected ground glass and stoppers were carefully regreased after every sampling.
Identification of product
Mass-spectral analysis using a probe-applied sample in a VG tandem mass spectrometer operating in the electron-impact mode showed a spectrum of primary ion and fragmentation products indicative of a trichlorocompound of the predicted MW.
4-H-1,3,2-Benzodioxaphosphorin-3,3,3-tribromide or trifluoride may be prepared by analogous reaction using PBr5 or PBr5, respectively. Mixed trihalides may be prepared using mixed-halide PHal5 (PHalxHal'5-x) reagents.
Synthesis of inhibitors of esterase activity: general procedure
The desired reaction was:-
SCPCl3 + R[NH2, NHR,SH or OH]
base
SCPCl2. [NH,NR,S or O] R
Figure imgf000017_0001
H2O
Figure imgf000017_0002
→ SCP.O. [NH,NR,S or O] R where SCP represents the saligenin cyclic phosphoryl moiety.
Solutions of SCPCl3 were made fresh in dry solvents (usually acetonitrile, dimethyl formamide, dimethyl sulphoxide, trichloroethylene or diethyl ether) on the day of use. Solutions of the other reactant and of base were usually made fresh but appeared to be satisfactory for longer periods provided atmospheric moisture and CO2 were excluded. Preferred base was 2,6-lutidene which, because of steric hindrance on nitrogen, is believed to be less inclined than, say, pyridine or triethylamine to attack the phosphorus atom, but in principle any weak base is acceptable. Also when the second reactant was an amine, an excess of this could also be used to trap HCl. Alternatively inorganic bases (K2CO3, MgO, PbO) could be used to trap HCl. For phenols, alcohols, and thiols, alkali metal derivatives could be used in suspension as reactants. Reaction was initiated at room temperature by addition of the SCPCl3 solution [10-1-10-3M] to 0.1 to 3 molar equivalents of second reactant plus a range of base each dissolved in equivalent volumes of dry solvent. Traces of fumes of HCl were observed to be formed when the more concentrated solutions were used. After 10-30 min a crystalline precipitate was observed in some cases but this was not prognostic of a successful reaction: this dissolved (or decomposed) instantly on addition of water. Reaction was completed by addition of an excess of aqueous buffer (usually citric acid/sodium citrate in the pH range 3-7) with or without a suitable water-miscible solvent (acetonitrile or others) and the clear homogeneous solution was processed according to requirements. For bioassay the solution was simply diluted in assay buffer and tested for inhibitory power by standard procedures (Johnson 1977, Arch. Toxicol, 37, 113-115). Inhibitory power is recorded as I50, being that concentration of compound required to halve the catalytic activity of an enzyme as a result of incubation of enzyme and compound together under defined conditions of time and temperature. For purification and identification the product solution was vacuum-dried, dissolved in a suitable solvent (such as acetonitrile or DMF) and subjected to HPLC separation, generally using acetonitrile/ triethylammonium acetate buffer gradients with monitoring of u/v absorbtion at 210 nm and bioassay of interesting fractions. Fractions shown to contain inhibitors were vacuum-dried, stored at -20° and analysed at intervals by standard methods. Specific examples
The following examples describe preparation of organophosphorus serine esterase inhibitors with an amino linkage (X = NH). However, it will be appreciated that inhibitors with secondary amino, oxygen or sulphur linkages (X = NR, O or S) may be prepared by analogous reactions with secondary amines, alcohols, phenols or thiols in the presence of base or as their alkali metal derivatives. Furthermore, the corresponding 2-sulphide inhibitors (Y = S) may be prepared by analogous reaction using H2S rather than aqueous work up. Synthesis of Esterase Inhibitors from SCPCl3
(1) From aniline
Dry potassium carbonate (2-5 mg) was suspended in dry acetonitrile (0.1 ml) and aniline (10 μmole) in dry acetonitrile (0.1 ml) was added followed by SCPCl3 (5 μmole) freshly dissolved in dry acetonitrile (0.1 ml) and the mixture was shaken gently at ambient temperature in a closed container for 15 min. Nine ml of citrate buffer pH 5.5
(mixture of sodium citrate/citric acid, 0.2 M) containing EDTA (1 mM) was added with shaking to complete the reaction. At intervals thereafter aliquots of the solution were tested for presence of a progressive inhibitor of phenyl valerate esterase by the standard assay procedure (Johnson (1977) Archives of Toxicology, vol. 37, 113-115): an inhibitor was detected. Assuming a 100% conversion of aniline the I50 (15 min/37°) for the product was approximately 22 x 10-6M .
(2) From n-pentylamine To a mixture of n-pentylamine (1.0 μmole) and 2,6-lutidene
(1.2 μmole) in dry acetonitrile (0.2 ml) was added SCPCl3
(0.5 μmole in 0.1 ml dry acetonitrile). Reaction was allowed to proceed for 15 min at ambient temperature and completed by addition of citrate buffer (9 ml of pH 4.0) followed by 3 ml of acetonitrile to redissolve a trace of precipitated product. An inhibitor with apparent I50 approximately 7 x 10-6 M was detected. When the reaction was repeated with ten-fold higher concentrations of reagents initially, conversion was more efficient and the calculated I50 from several experiments ranged from 0.5 to 3.5 x 10-6 M. Optimisation of temperature and time of reaction was not attempted. Solutions of the inhibitor left in pH 4 buffer for 4 weeks had 15-20% original activity on re-assay.
(3) From 1,12-diamino-dodecane To a solution of 1,12-diamino-dodecane (10μmoles) in dry dimethyl sulphoxide/acetonitrile (0.2 ml of 50/50 mixture) was added 2,6-lutidiene (15 μmoles in 0.1 ml dry acetonitrile) followed by SCPCl3 (15 μmole in 0.1 ml dry acetonitrile). After 12 min reaction at ambient temperature reaction was completed as before. The inhibitory product had I50 < < 10-6M assuming complete conversion of starting material.
(4) From peptides SCPCl3 (20 μl, 215 mM in dry acetonitrile; 2.08 equivalents) was added to a mixture of 2,6-Lutidene (20 μl, 430 mM in dry acetonitrile; 4.15 equivalents) and the peptide or alternative of choice (80 μl, 50 mM in dry dimethylformamide/acetonitrile 1:1 v/v; 1.0 equivs.) at 15°C. After 20 min. reaction was completed by addition of a mixture of Na-citrate buffer (25 mM, pH 5.0) and acetonitrile (4:1 v/v). In some cases the final mixture was opalescent indicating formation of an insoluble product. In all cases a further 0.8 ml of acetonitrile was added to give a clear solution of crude product at a nominal concentration of 809 μm based on assumption of 100% conversion of peptide.
Seventeen compounds listed in Table II below were reacted with SCPCl3 and a representative 5 products were selected for screening as inhibitors of trypsin (a typical protease and model for other proteases such as plasminogen activator, elastase and dipeptidyl peptidase IV known to be relevant to various disease states. Inhibitors were preincubated with enzyme at a concentration of 16 μm for up to 60 min at 18°C prior to addition of assay substrate.
Preparation of affinity-agent-labelled organophosphorus inhibitors - From 1-N-biotinyl, 1,9-diaminononane
1-N-biotinyl, 1,9-diaminononane was synthesised from N- hydroxylsuccinimidylbiotin (Pierce Chem. Co.) and 1,9- diaminononane by standard reaction in dimethylformamide and purified by HPLC (see general procedure). It was dissolved in dry dimethylformamide (0.23 μmole in 29 μl) and lutidene and SCPCl3 (each 47 μmole in 23 μl dry acetonitrile) were added. After 15 min reaction at 37°C reaction was completed by addition of pH 5 buffer (25 μl). Aliquots of the solution were vacuum dried and stored at 5° for varying times prior to purification by HPLC and storage. By specific analysis for biotin content the yield of purified product was 17% and the I50 was approximately 10-9M. The structure of this compound (S9B) was confirmed by mass- spectrometry using a tandem MS in the FAB mode. S9B was used for affinity labelling and purification of neuropathy target esterase. It can also be used for affinity labelling histochemistry to determine the location of NTE in animal tissue sections, cultured cells etc. An alternative affinity histochemical reagent can be made by substituting digoxigenin for biotin in the synthesis.
Affinity purification of neuropathy target esterase (NTE) Hen brain microsomal membranes were preincubated with paraoxon (diethyl 4-nitrophenyl phosphate) to block some irrelevant sites and then incubated, usually at 37° but at any convenient temperature, for a period of time (10-60 min) with a concentration of S9B sufficient to achieve progressive covalent organophosphorylation of the enzyme with concomitant inhibition of its catalytic activity. Typically the reaction was performed at 37°C for 20 minutes with a S9B concentration of 160 nM. Excess inhibitor was removed by washing the membranes with buffer. The membranes were dissolved by boiling in sodium dodecyl sulphate (SDS) solution (0.15%) in neutral buffer containing EDTA (1 mM) and dithiothreitol (10 mM). The affinity-labelled NTE was selectively adsorbed onto avidin bound covalently to a matrix such as Agarose or Polyacrylic beads. Adsorbtion was monitored analytically by assay of biotin-carrying protein remaining in the liquid medium at different times. No significant adsorbtion of non-biotinyl proteins was confirmed by assay of total protein. After the beads had been washed twice with dilute SDS the adsorbed S9B-NTE was released by boiling the beads in 5% SDS in buffer for 5 min. Final purification of the affinity-labelled NTE was achieved by preparative polyacrylamide gel electrophoresis to separate NTE from traces of other labelled esterases and from two endogenous biotin-containing proteins of much lower molecular weight than NTE. Quantitative analysis (assay) of affinity purified NTE was carried out using enzyme enhanced chemiluminescence assay for biotin. The pure protein was then available for determination of amino-acid sequence, raising of antibodies etc. using conventional techniques. The serine esterase may be released from the inhibitor by treatment with alkali at pH 11 at ambient temperature. Use of affinity-labelled orgeuiophosphorus inhibitors for histochemistry
The S9B reagent and technique applied above to label NTE in broken membranes can equally be applied to selectively label NTE in fresh sections of intact animal tissue. Standard techniques can then be applied using enzyme-linked antibodies to visualise NTE at the light microscopic or EM level. Alternatively a digoxigenin-linked reagent derived from SCPCl3 can be used. Table I: Inhibitors of hen brain NTE produced from reaction of SCPCI3 with amines
Amine Estimated I50( M)a Monoamines
Aniline 2.2 x 10-5
Benzylamine ~1 x 10-6
n-pentylamine 0.5 - 3.5 x 10-6
n-octylamine 1.6 x 10-6
n-dodecylamine 4 x 10-6
Diamines
1,7-diaminopentane 10-6
1,9-diaminononane < 10-6
1,12-diaminododecane < < 10-6
1-N-biotinyl-ω-amino- alkanes (n-carbon atoms) n = 9 (Reagent S9B) 10-6 (PURE product ) b
Activity of other 1-N-biotinyl diamines were ranked by comparison of unpurified products as follows :
C-9≃ C-10 > C-12 > C- 8 > C-7 > > C-5
Notes a. Syntheses were NOT optimised for yield but, unless shown otherwise, I50 was measured on an unpurified product and is calculated assuming 100% yield based on amine. b. This pure compound has been used for affinity purification of NTE. Table II: Peptides reacted with SCPCl3
NAME RESULT OF TEST FOR
INHIBITORY POWER
L-Alanine methyl ester. HCl.
D-Alanine benzyl ester. Tosylate. (-)
L-Arginine methyl ester. Di HCl.
L-Glycine ethyl ester. HCl.
L-Phenylalanine methyl ester. HCl.
L-Tryptophanamide. HCl.
(H)-L-Tyrosine (Z)-benzyl ester. HCl.
L-Valine methyl ester. HCl. (+)
Gly-Ala-benzyl ester. HCl. (-)
L-Phenylalanine benzyl ester. HCl.
L-Proline benzyl ester. HCl. (+)
Z-Arginine-OH.
N-E-Z Lysine benzyl ester. HCl. (+)
L-Prolyl-L-Proline benzyl ester. HCl.
L-Proline methyl ester. HCl.
L-Glutamyl dibenzyl ester. Tosylate.
S-benzyl L-cysteinyl-7-
(amido-4-trifluoromethyl coumarin). Preparation of radiolabelled organophosphorus inhibitors
Bolton-Hunter Reagent (BHR; N-succinimidyl 3-(4- hydroxyphenyl) propionate, Sigma Chem. Co.) (2 μmoles) was iodinated by the well-known standard procedure (Bolton, A.E. & Hunter, W.M. (1973), Biochem, J. 133, 529-539), using NaI (10 μmole) plus a trace of [125I] -Nal (Amersham International). The product was extracted from quenched aqueous reaction mixture (135 μl) into benzene (2 x 400 μl). TLC analysis (silica strips with ethyl acetate/toluene (1:1) as solvent) showed that approximately 80% of extracted material was di-iodo BHR and 20% was mono-iodo BHR. The extracts were concentrated under nitrogen and redissolved in acetonitrile/dimethyl sulphoxide (3:1). After processing and sampling losses, about 1.1 μmoles of iodo-BHR (mixed mono and di) was obtained with average specific radio activity of 2 x 107 cpm/μmole (about 9μCi/μmole). Iodo-BHR (1.0 μmole in MeCN/DMSO (220 μl) was mixed with 1,9-diaminononane (5.0 μmole in MeCN/DMSO (100 μL)) at room temperature. An immediate reaction was observed and, after vortexing and centrifugation, a yellow-green liquor was obtained above a white sediment. TLC analysis was performed on samples of liquor (silica strips : Isopropanol/ water/0.880 ammonia (8:1:1)). The developed strips were sprayed with fluorescamine reagent, (Sigma Chem. Co.) and viewed under long-wave U/V illumination. Strips were then cut into slices for γ-counting. From the reaction mixture a spot with RF about 0.4 was found to fluoresce and to be associated with radiolabel. A sample containing diamine but no iodo-BHR gave a spot with RF about 0.5 and unchanged iodo-BHR migrated with RF about 0.6. The bulk of product was then purified by HPLC on a C-18 column with stepwise increases in concentration of methanol/water (60% to 90% plus 0.1% trifluoracetic acid). The 45 fractions were screened by TLC analysis (as above). About 36% of total radioactivity eluted in fractions 4-6 with a further 21% in fractions 7-12. The pooled fractions 4-6 were fluorescamine-positive and considered likely to contain pure product Ν-(w-amino-nonyl) 3-(4-hydroxy, 3,5-di-iodophenyl) proprionamide. The pool was concentrated and used for coupling to the OP reagent. Purified (see above) [125I] -BHR-diaminononane conjugate [162 nmoles (3.2 x 106 cpm) in 225 μl MeCΝ/DMSO (3:1)] was mixed with high-purity 1,6-Lutidene (Aldrich Chem. Co.) (1 μmole in 100 μl dry MeCΝ), and SCPCl3 (1 μmole in dry MeCΝ (100 μl)) was added. The mixture was left overnight at room temperature. Citrate buffer (25mM;pH5;200 μl) was added to hydrolyse excess SCPCl3 and to convert the expected dichlorophosphamidate to the phosphoramidate [as in previous example (s)]. Biochemical assay of the ability of the unpurified product solution to inhibit hen brain microsomal esterase activity using phenyl valerate as substrate [as in previous example (s)] indicated that a covalently reacting inhibitor had been synthesised with I50 approximately 2.7 μM (20 min. preincubation at ambient temperature).
The specific radioactivity of this preparation (presumed same as the iodo BHR, i.e. 9 μCi/μmole) was adequate for tracing progress of the steps of the synthesis. Increases of specific radioactivity of the sodium iodide used in initial labelling are possible up to at least 4 orders of magnitude so that detectable labelling of even minute amounts of enzyme are feasible in gels or histochemical localisation in tissue sections. Also, intermediate levels of specific radioactivity would be quite adequate for tracing an enzyme purification on the scale at which such procedures are normally performed.
It has been found that in the reaction coupling SCPCl3 to a labelled compound a much shorter reaction time (5-20 min. rather than overnight) and approximately 1:1 molar concentration of reactants (rather than 6:1) leads to much higher yield of labelled inhibitory product.
Figure imgf000027_0001

Claims

CLAIMS : 1. A compound of the formula:
Figure imgf000028_0001
wherein Hal may be the same or different and are selected from fluorine, chlorine and bromine
R1 = hydrogen or an alkyl, aryl, alkoxy, acyl, halogen or nitro group or a fused aromatic ring
B-R2 is C=O or CHR2
R2 = hydrogen or an alkyl, aryl or cyano group.
2. A compound according to claim 1 wherein Hal are the same and are chlorine.
3. A compound according to claim 1 or 2 wherein R1 and R2 are hydrogen.
4. A process for the preparation of a compound according to any one of claims 1 to 3, comprising reaction of a compound of the formula
Figure imgf000028_0002
with PHal5.
5. Use of a compound according to any one of claims 1 to 3 in the preparation of a serine esterase inhibitor.
6. Use of a compound according to any one of claims 1 to 3 in the preparation of a compound of the formula:
Figure imgf000029_0001
wherein R1 and R2 are as hereinbefore defined,
Y = O or S
X = O, S or NH
A = an alkyl, aryl, peptide, heterocyclic or reporter group.
7. A process for the preparation of a compound of the formula:
Figure imgf000029_0002
wherein R1 and R2 are as hereinbefore defined,
Y = O or S
X = O, S or NH
A = an alkyl, aryl, peptide, heterocyclic or reporter group the process comprising the steps of
1) reaction of a compound according to any one of claims 1 to 3 with a compound A-X-H, 2) followed by treatment with H2Y.
8. A process according to claim 7 wherein A comprises an affinity group.
9. A process according to claim 8 wherein A comprises biotin.
10. A process according to claim 8 wherein A comprises digoxigenin.
PCT/GB1993/002466 1992-11-30 1993-11-30 Organophosphorus compounds WO1994012507A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1360130A (en) * 1962-06-12 1964-04-30 Sumitomo Chemical Co Cyclic phosphate esters having insecticidal and nematicidal effects and their production process
FR1502154A (en) * 1965-10-29 1967-11-18 Sumitomo Chemical Co Manufacturing process of cyclic phosphate esters
FR1527848A (en) * 1966-03-04 1968-06-07 Battelle Development Corp Process for the manufacture of substituted pyrocatechin esters, with fungicidal action, of phosphoric or thiophosphoric acids, as well as products conforming to those obtained by the present process or similar process
US4087490A (en) * 1975-07-31 1978-05-02 Makhteshim Chemical Works, Ltd. Cyclic phosphoryl and thiophosphoryl halides

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Publication number Priority date Publication date Assignee Title
US5021346A (en) * 1987-06-15 1991-06-04 Biopool International, Inc. Solid phase reactant for inhibition and removal of serine proteases and method of use
DE3813278A1 (en) * 1988-01-12 1989-07-20 Boehringer Mannheim Gmbh METHOD FOR DETECTING NUCLEIC ACIDS

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
FR1360130A (en) * 1962-06-12 1964-04-30 Sumitomo Chemical Co Cyclic phosphate esters having insecticidal and nematicidal effects and their production process
FR1502154A (en) * 1965-10-29 1967-11-18 Sumitomo Chemical Co Manufacturing process of cyclic phosphate esters
FR1527848A (en) * 1966-03-04 1968-06-07 Battelle Development Corp Process for the manufacture of substituted pyrocatechin esters, with fungicidal action, of phosphoric or thiophosphoric acids, as well as products conforming to those obtained by the present process or similar process
US4087490A (en) * 1975-07-31 1978-05-02 Makhteshim Chemical Works, Ltd. Cyclic phosphoryl and thiophosphoryl halides

Non-Patent Citations (2)

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Title
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JOURNAL FÜR PRAKTISCHE CHEMIE vol. 318, no. 4, 1976, LEIPZIG DD pages 607-612 J. GLOEDE ' Reaktion von Orthosäurechloriden mit *

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