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US20100317865A1 - Enzyme and receptor modulation - Google Patents

Enzyme and receptor modulation Download PDF

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Publication number
US20100317865A1
US20100317865A1 US12/867,455 US86745509A US2010317865A1 US 20100317865 A1 US20100317865 A1 US 20100317865A1 US 86745509 A US86745509 A US 86745509A US 2010317865 A1 US2010317865 A1 US 2010317865A1
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alkyl
modulator
ester
disubstituted glycine
glycine ester
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Inventor
Alan Hornsby Davidson
Alan Hastings Drummond
David Festus Charles Moffat
Alistair David Graham Donald
Stephen John Davies
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Chroma Therapeutics Ltd
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Chroma Therapeutics Ltd
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Assigned to CHROMA THERAPEUTICS LTD. reassignment CHROMA THERAPEUTICS LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DAVIDSON, ALAN HORNSBY, DAVIES, STEPHEN JOHN, DONALD, ALISTAIR DAVID GRAHAM, DRUMMOND, ALAN HASTINGS, MOFFAT, DAVID FESTUS CHARLES
Publication of US20100317865A1 publication Critical patent/US20100317865A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/73—Unsubstituted amino or imino radicals
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase

Definitions

  • This invention relates to a general method of increasing or prolonging the activity of a compound which modulates the activity of an intracellular enzyme or receptor by the covalent conjugation of an ⁇ , ⁇ -disubstituted glycine ester motif to the modulator.
  • the invention also relates to modulators to which an ⁇ , ⁇ -disubstituted glycine ester motif has been covalently conjugated, and to a method for the identification of such conjugates having superior properties relative to the parent non-conjugated modulator.
  • the invention further relates to the use of modulators containing ⁇ , ⁇ -disubstituted glycine ester motifs that allow the selective accumulation of amino acid conjugates inside cells of the monocyte-macrophage lineage.
  • Our International Patent Application WO 2006/117567 discloses a method for increasing the intracellular concentration of a given modulator of an intracellular enzyme or receptor by conjugating thereto an ⁇ -amino acid ester motif which is hydrolysed by one or more of the intracellular carboxylesterases hCE-1, hCE-2 and hCE-3. This results in increased potency by prolonging the residency of the modulator inside the cell, and leads to improved pharmacokinetic and pharmacodynamic properties. More consistent exposure and reduced dosing frequencies can be achieved. A further benefit is obtained when the ⁇ -amino acid ester motif is conjugated to the modulator such that the drug is targeted to the specific target cells responsible for its therapeutic action, reducing systemic exposure and hence side effects.
  • This invention is based on the novel finding that conjugates of ⁇ , ⁇ -disubstituted glycine esters can be hydrolysed by intracellular carboxyl esterases, and thus the methods and benefits described in WO 2006/117567 can also be obtained with such conjugates.
  • Such benefits include intracellular accumulation of the acid hydrolysis product, and in some cases selective accumulation in cells of a particular type.
  • the present invention takes advantage of the fact that, lipophilic (low polarity or charge neutral) molecules pass through the cell membrane and enter cells relatively easily, and hydrophilic (higher polarity, charged) molecules do not.
  • a lipophilic motif is attached to a given modulator, allowing the modulator to enter the cell, and if that motif is converted in the cell to one of higher polarity, it is to be expected that the modulator with the higher polarity motif attached would accumulate within the cell.
  • the accumulation of modulator with the higher polarity motif attached is therefore expected to result in prolonged and/or increased activity.
  • the present invention makes use of the fact that there are carboxylesterase enzymes within cells, which may be utilised to hydrolyse an ⁇ , ⁇ -disubstituted glycine ester motif attached to a given modulator to the parent acid. Therefore, a modulator may be administered as a covalent conjugate with an ⁇ , ⁇ -disubstituted glycine ester, in which form it readily enters the cell where it is hydrolysed efficiently by one or more intracellular carboxylesterases, and the resultant ⁇ , ⁇ -disubstituted glycine-modulator conjugate accumulates within the cell, increasing overall potency and/or active residence time.
  • modulators can be targeted to monocytes and macrophages.
  • monocyte or “monocytes”
  • macrophage or macrophages will be used to denote macrophages (including tumour associated macrophages) and/or monocytes.
  • the present invention provides a covalent conjugate of an ⁇ , ⁇ -disubstituted glycine ester and a modulator of the activity of a target intracellular enzyme or receptor, wherein: the ester group of the conjugate is hydrolysable by one or more intracellular carboxylesterase enzymes to the corresponding acid; and the ⁇ , ⁇ -disubstituted glycine ester is covalently attached to the modulator at a position remote from the binding interface between the modulator and the target enzyme or receptor, and/or is conjugated to the modulator such that the binding mode of the conjugated modulator and the said corresponding acid to the target enzyme or receptor is the same as that of the unconjugated modulator.
  • the invention provides a method of increasing or prolonging the intracellular potency and/or residence time of a modulator of the activity of a target intracellular enzyme or receptor comprising structural modification of the modulator by covalent attachment thereto of an ⁇ , ⁇ -disubstituted glycine ester at a position remote from the binding interface between the modulator and the target enzyme or receptor, and/or such that the binding mode of the conjugated modulator and the said corresponding acid to the target enzyme or receptor is the same as that of the unconjugated modulator, the ester group of the conjugate being hydrolysable by one or more intracellular carboxylesterase enzymes to the corresponding acid.
  • both the ester conjugate and the corresponding acidic ⁇ , ⁇ -disubstituted glycine conjugate retain the modulator activity of the parent unconjugated modulator.
  • the absolute enzyme inhibitory potencies or the receptor agonist or antagonist potencies of the ester and acid conjugates need not necessarily be as high as the corresponding potencies of the unconjugated modulator, since intracellular hydrolysis of the ester conjugate to the acid conjugate results in an accumulation of the latter within the cell, and the resultant concentration of the acid conjugate within the cell is higher than that achievable with the unconjugated modulator, thereby compensating for any intrinsically lower potency of the former relative to the latter. It will threfore be apparent that the conjugation strategy of the present invention differs from the traditional “pro-drug” approach.
  • the parent modulator would be modified by conversion to a derivative which is processed in vivo to release the original modulator, and it is the release of the original modulator which is responsible for the ultimate activity.
  • the original modulator is modified so that the eventual activity is the combined result of intracellular activity due to the ⁇ , ⁇ -disubstituted glycine conjugate of the modulator, and the accumulating ⁇ , ⁇ -disubstituted glycine conjugate of the modulator.
  • the active species is a conjugate, not the original unconjugated modulator.
  • the invention is concerned with modification of modulators of intracellular enzymes or receptors.
  • the principle of the invention is of general application, not restricted by the chemical identity of the modulator or the identity of the target enzyme or receptor, it is strongly preferred that the modulator be one that exerts its effect by reversible binding to the target enzyme or receptor, as opposed to those whose effect is due to covalent binding to the target enzyme or receptor.
  • the carboxylesterase-hydrolysed conjugate is required to retain the intracellular binding activity of the parent modulator with its target enzyme or receptor, attachment of the ester motif must take account of that requirement, which will be fulfilled if the carboxylesterase-hydrolysable ⁇ , ⁇ -disubstituted glycine ester motif is attached to the modulator such that the binding mode of the corresponding carboxylesterase hydrolysis product (ie the corresponding acid) to the target is essentially the same as the unconjugated modulator. In general this is achieved by covalent attachment of the carboxylesterase ester motif to the modulator at a position remote from the binding interface between the modulator and the target enzyme or receptor. In this way, the motif is arranged to extend into solvent, rather than potentially interfering with the binding mode,
  • the ⁇ , ⁇ -disubstituted glycine ester motif obviously must be a substrate for the carboxylesterase if the former is to be hydrolysed by the latter within the cell.
  • the ability of intracellular carboxylesterases appears not to depend on very strict structural requirements of the ⁇ , ⁇ -disubstituted glycine ester substrate.
  • most modes of covalent conjugation of the ⁇ , ⁇ -disubstituted glycine ester motif to a modulator will allow hydrolysis. Attachment by a flexible linker chain will usually be how this is achieved.
  • any chemical modification of a drug may subtly alter its binding geometry, and the chemistry strategy for linkage of the ⁇ , ⁇ -disubstituted glycine ester carboxylesterase motif may introduce additional binding interactions with the target, or may substitute for one or more such interactions.
  • the hydrolysed conjugate's binding mode to the target is the same as the unconjugated modulator is to be interpreted as requiring that there is no significant perturbation of the binding mode, in other words that the binding mode is essentially the same as that of the unconjugated modulator.
  • the main binding characteristics of the parent modulator are retained, and the modified and unmodified modulators have an overall common set of binding characteristics.
  • the esterase-hydrolysed carboxylic acid has a potency in an in vitro enzyme- or receptor-binding assay no less than one tenth of the potency of the parent modulator in that assay, and that the ester has a potency in a cellular activity assay at least as high as that of the parent modulator in the same assay.
  • Such information is in the vast majority of cases in the public domain, or can be modelled using computer based modelling methods, such as ligand docking and homology modelling, based on the known binding modes of structurally similar modulators, or the known structure of the active site of the target enzyme or receptor.
  • computer based modelling methods such as ligand docking and homology modelling, based on the known binding modes of structurally similar modulators, or the known structure of the active site of the target enzyme or receptor.
  • ligand docking and homology modelling based on the known binding modes of structurally similar modulators, or the known structure of the active site of the target enzyme or receptor.
  • Intracellular carboxylesterase enzymes capable of hydrolysing the ester group of the conjugated alpha amino acid to the corresponding acid include the three known human carboxylesterase (“hCE”) enzyme isotypes hCE-1 (also known as CES-1), hCE-2 (also known as CES-2) and hCE-3 (Drug Disc. Today 2005, 10, 313-325). Although these are considered to be the main enzymes other carboxylesterase enzymes such as biphenylhydrolase (BPH) may also have a role in hydrolysing the conjugates.
  • hCE human carboxylesterase
  • the broken cell assay described below is a simple method of confirming that a given conjugate of modulator and ⁇ , ⁇ -disubstituted glycine ester, or a given ⁇ , ⁇ -disubstituted glycine ester to be assessed as a possible carboxylesterase ester motif, is hydrolysed as required.
  • These enzymes can also be readily expressed using recombinant techniques, and the recombinant enzymes may be used to determine or confirm that hydrolysis occurs.
  • the desired conjugate retains the covalently linked ⁇ , ⁇ -disubstituted glycine acid motif when hydrolysed by the carboxylesterase(s) within the cell, since it is the polar carboxyl group of that motif which prevents or reduces clearance of the hydrolysed conjugate from the cell, and thereby contributes to its accumulation within the cell.
  • the cellular potency of the modified modulator is predominantly due to the accumulation of the acid and its modulation of the activity of the target (although the unhydrolysed ester also exerts its activity on the target for so long as it remains unhydrolysed).
  • the conjugate or more especially the hydrolysed conjugate (the corresponding acid), is not a substrate for such peptidases.
  • the ⁇ , ⁇ -disubstituted glycine ester group should not be the C-terminal element of a dipeptide motif in the conjugate.
  • the ⁇ , ⁇ -disubstituted glycine ester group may be covalently attached to the modulator via its amino group or via its one of the ⁇ -substituents. In some cases the modulator will have a convenient point of attachment for the carboxylesterase ester motif, and in other cases a synthetic strategy will have to be devised for its attachment.
  • hCE-2 and hCE-3 can be turned to advantage where it is required that the modulator should target enzymes or receptors in certain cell types only.
  • the relative amounts of these three carboxylesterase enzymes vary between cell types (see FIG. 1 of WO 2006/117567 and database at http:/symatlas.gnf.org/SymAtlas (note that in this database hCE3/CES3 is referred to by the symbol FLJ21736)).
  • the modulator is intended to act only in cell types where hCE-1 is found, attachment of the ⁇ , ⁇ -disubstituted glycine ester carboxylesterase ester motif in such a way that the amino group is not directly attached to a carbonyl, results in the hydrolysed modulator conjugate accumulating preferentially in cells with effective concentrations of hCE-1.
  • specific accumulation of the acid derived from the modulator conjugate in hCE-1 expressing cells can be achieved by linking the amino acid ester motif to the modulator in such a way that the nitrogen atom of the amino acid ester is not linked directly to a carbonyl, or is left unsubstituted.
  • Macrophages are known to play a key role in inflammatory disorders through the release of cytokines in particular TNF ⁇ and IL-1 (van Roon et al Arthritis and Rheumatism, 2003, 1229-1238). In rheumatoid arthritis they are major contributors to joint inflammation and joint destruction (Conell in NEng Mech 2004, 350, 2591-2602). Macrophages are also involved in tumour growth and development (Naldini and Carraro in Curr Drug Targets lnflamm Allergy,2005, 3-8). Hence agents that selectively target macrophage cells could be of value in the treatment of cancer, inflammation and autoimmune disease. Targeting specific cell types would be expected to lead to reduced side-effects.
  • the present invention enables a method of targeting modulators to macrophages, which is based on the above observation that the way in which the ⁇ , ⁇ -disubstituted glycine ester carboxylesterase ester motif is linked to the modulator determines whether it is hydrolysed by specific carboxylesterases, and hence whether or not the resultant acid accumulates in different cell types.
  • macrophages contain the human carboxylesterase hCE-1 whereas other cell types do not.
  • the ester motif when the nitrogen of the ester motif is substituted but not directly bonded to a carbonyl group moiety the ester will only be hydrolysed by hCE-1 and hence the esterase-hydrolysed modulator conjugates will only accumulate in macrophages.
  • the ⁇ , ⁇ -disubstituted glycine conjugates of the invention are in general more potent in cells containing the carboxylesterase (ie macrophages) than in cells which only contain the carboxylesterases HCE-2 and HCE-3.
  • conjugates of the invention in which the ⁇ , ⁇ -disubstituted glycine ester motif is linked to the modulator via one of the ⁇ substituents of the ⁇ , ⁇ -disubstituted glycine ester are in general more potent in cells containing the carboxylesterase HCE-1 (ie macrophages) than in cells which only contain the carboxylesterases HCE-2 and HCE-3.
  • the active hydrolysed acid conjugate therefore accumulates non-selectively in the case of such (generally) C-linked ester conjugates.
  • ⁇ , ⁇ -disubstituted glycine or “ ⁇ , ⁇ -disubstituted glycine acid” means a compound of formula H 2 N—C(R 2 R 3 )—COOH, wherein R 2 and R 3 represent the ⁇ -substituents (which of course are not hydrogen), and an “ ⁇ , ⁇ -disubstituted glycine ester is such a compound wherein the carboxylic acid group is esterified.
  • (C a -C b )alkyl wherein a and b are integers refers to a straight or branched chain alkyl radical having from a to b carbon atoms.
  • a is 1 and b is 6, for example, the term includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and n-hexyl.
  • divalent (C a -C b )alkylene radical wherein a and b are integers refers to a saturated hydrocarbon chain having from a to b carbon atoms and two unsatisfied valences.
  • (C a -C b )alkenyl wherein a and b are integers refers to a straight or branched chain alkenyl moiety having from a to b carbon atoms having at least one double bond of either E or Z stereochemistry where applicable.
  • the term includes, for example, vinyl, allyl, 1- and 2-butenyl and 2-methyl-2-propenyl.
  • divalent (C a -C b )alkenylene radical means a hydrocarbon chain having from a to b carbon atoms, at least one double bond, and two unsatisfied valences.
  • (C a -C b )alkynyl wherein a and b are integers refers to straight chain or branched chain hydrocarbon groups having from two to six carbon atoms and having in addition one triple bond. This term would include for example, ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl.
  • divalent (C a -C b )alkynylene radical wherein a and b are integers refers to a divalent hydrocarbon chain having from 2 to 6 carbon atoms, and at least one triple bond.
  • Carbocyclic refers to a mono-, bridged mono-, bi- or tricyclic radical having up to 16 ring atoms, all of which are carbon, and includes aryl and cycloalkyl.
  • cycloalkyl refers to a monocyclic saturated carbocyclic radical having from 3-8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and norbornyl.
  • aryl refers to a mono-, bridged mono-, bi- or tri-cyclic carbocyclic aromatic radical, and includes radicals having two monocyclic carbocyclic aromatic rings which are directly linked by a covalent bond.
  • Illustrative of such radicals are phenyl, biphenyl and napthyl.
  • heteroaryl refers to a mono-, bridged mono-, bi- or tri-cyclic aromatic radical containing one or more heteroatoms selected from S, N and O, and includes radicals having two such monocyclic rings, or one such monocyclic ring and one monocyclic aryl ring, which are directly linked by a covalent bond.
  • Illustrative of such radicals are thienyl, benzthienyl, furyl, benzfuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl, benztriazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl.
  • heterocyclyl or “heterocyclic” includes “heteroaryl” as defined above, and in its non-aromatic meaning relates to a mono-, bridged mono-, bi- or tri-cyclic non-aromatic radical containing one or more heteroatoms selected from S, N and O, and to groups consisting of a monocyclic non-aromatic radical containing one or more such heteroatoms which is covalently linked to another such radical or to a monocyclic carbocyclic radical.
  • radicals are pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido and succinimido groups.
  • any moiety herein means such moiety may be substituted with up to four compatible substituents, each of which independently may be, for example, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, hydroxy, hydroxy(C 1 -C 6 )alkyl, mercapto, mercapto(C 1 -C 6 )alkyl, (C 1 -C 6 )alkylthio, phenyl, halo (including fluoro, bromo and chloro), trifluoromethyl, trifluoromethoxy, nitro, nitrile (—CN), oxo, —COON, —COOR A , —COR A , —SO 2 R A , —CONH 2 , —SO 2 NH 2 , —CONHR A , —SO 2 NHR A , —CONR A R B ,
  • side chain of a natural or non-natural alpha-amino acid refers to the group R 1 in a natural or non-natural amino acid of formula NH 2 —CH(R 1 )—COOH, other than glycine, in which R 1 is hydrogen.
  • side chains of natural alpha amino acids include those of alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, histidine, 5-hydroxylysine, 4-hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, ⁇ -aminoadipic acid, ⁇ -amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine, ⁇ -methylserine, ornithine, pipecolic acid, and thyroxine.
  • Natural alpha-amino acids which contain functional substituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indolyl groups in their characteristic side chains include arginine, lysine, glutamic acid, aspartic acid, tryptophan, histidine, serine, threonine, tyrosine, and cysteine.
  • the ⁇ -substituents in the ⁇ , ⁇ -disubstituted glycine ester motif in the compounds of the invention is one of those side chains, the functional substituent may optionally be protected.
  • carboxyl groups may be esterified (for example as a C 1 -C 6 alkyl ester), amino groups may be converted to amides (for example as a NHCOC 1 -C 6 alkyl amide) or carbamates (for example as an NHC( ⁇ O)OC 1 -C 6 alkyl or NHC( ⁇ O)OCH 2 Ph carbamate), hydroxyl groups may be converted to ethers (for example an OC 1 -C 6 alkyl or a O(C 1 -C 6 alkyl)phenyl ether) or esters (for example a OC( ⁇ O)C 1 -C 6 alkyl ester) and thiol groups may be converted to thioethers (for example a tert-butyl or benzyl thio
  • ester groups which may in principle be present in the ⁇ , ⁇ -disubstituted glycine ester carboxylesterase ester motif for attachment to the modulator.
  • ⁇ , ⁇ -disubstituted glycines differing in the ⁇ -substituents R 2 and R 3 , which may be used as esters in the carboxylesterase ester motif.
  • Some ⁇ , ⁇ -disubstituted glycine esters are rapidly hydrolysed by one or more of the hCE-1, -2 and -3 isotypes or cells containing these enzymes, while others are more slowly hydrolysed, or hydrolysed only to a very small extent.
  • the carboxylesterase hydrolyses the free ⁇ , ⁇ -disubstituted glycine ester to the parent acid it will, subject to the N-carbonyl dependence of hCE-2 and hCE-3 discussed above, also hydrolyse the ester motif when covalently conjugated to the modulator.
  • the broken cell assay and/or the isolated carboxylesterase assay described herein provide a straightforward, quick and simple first screen for esters which have the required hydrolysis profile. Ester motifs selected in that way may then be re-assayed in the same carboxylesterase assay when conjugated to the modulator via the chosen conjugation chemistry, to confirm that it is still a carboxylesterase substrate in that background.
  • intracellular carboxylesterase enzymes capable of hydrolysing the ester group of a compound of the invention to the corresponding acid include the three known human enzyme isotypes hCE-1, hCE-2 and hCE-3. Although these are considered, to be the main enzymes, other enzymes such as biphenylhydrolase (BPH) may also have a role in, hydrolysing the ester.
  • BPH biphenylhydrolase
  • the carboxylesterase hydrolyses the free amino acid ester to the parent acid it will also hydrolyse the ester motif when covalently conjugated to the inhibitor.
  • the broken cell assay and/or the isolated carboxylesterase assay described herein provide a straightforward, quick, and simple first screen, for esters which have the required hydrolysis profile. Ester motifs selected in that way may then be re-assayed in the same carboxylesterase assay when conjugated to the inhibitor via the chosen conjugation chemistry, to confirm that it is still a carboxylesterase substrate in that background.
  • ester groups in the ⁇ , ⁇ -disubstituted glycine ester carboxylesterase ester motif include those of formula —(C ⁇ O)OR 14 wherein R 14 is R 8 R 9 R 10 C— wherein
  • alkyl includes fluoroalkyl
  • R 10 is often hydrogen.
  • R 14 include methyl, trifluoromethyl, ethyl, n- or iso-propyl, n-, sec- or tert-butyl, cyclohexyl, norbornyl, allyl, phenyl, benzyl, 2-, 3- or 4-pyridylmethyl, N-methylpiperidin-4-yl, tetrahydrofuran-3-yl or methoxyethyl.
  • R 14 is cyclopentyl.
  • Examples of ⁇ -substituents R 2 and R 3 of the ⁇ , ⁇ -disubstituted glycine ester conjugated to the modulator may be regarded as selected from the side chains of a natural or non-natural alpha-amino acid, and in such side chains any functional groups may be protected.
  • ⁇ -substituents R 2 and R 3 of the ⁇ , ⁇ -disubstituted glycine ester conjugated to the modulator include phenyl and groups of formula —CR a R b R c in which:
  • the ⁇ -substituents R 2 and R 3 of the ⁇ , ⁇ -disubstituted glycine ester conjugated to the modulator, taken together with the ⁇ -carbon itself, may form a 3-6 membered saturated cycloalkyl ring, such as a cyclopropyl, cyclopentyl or cyclohexyl ring or heterocyclyl ring such as a piperidin-4-yl ring.
  • At least one of the ⁇ -substitutents R 2 and R 3 of the ⁇ , ⁇ -disubstituted glycine ester conjugated to the modulator is a C 1 -C 6 alkyl substituent, for example methyl, ethyl, or n- or iso-propyl.
  • one of the ⁇ -substitutents R 2 and R 3 of the ⁇ , ⁇ -disubstituted glycine ester conjugated to the modulator is a C 1 -C 6 alkyl substituent, for example methyl, ethyl, or n- or iso-propyl, and the other is selected from the group consisting of methyl, ethyl, n- and iso-propyl, n-, sec- and tert-butyl, phenyl, benzyl, thienyl, cyclohexyl, and cyclohexylmethyl.
  • R 2 and R 3 are methyl. and the other is methyl, ethyl, or n- or iso-propyl; or where R 2 and R 3 taken together with the carbon to which they are attached form a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl rinng.
  • the ⁇ -substitutents R 2 and R 3 of the ⁇ , ⁇ -disubstituted glycine ester conjugated to the modulator are each methyl.
  • the ⁇ , ⁇ -disubstituted glycine ester may be conjugated to the modulator via its amino group, or via one of the ⁇ -substituents.
  • a linker radical may be present between the carboxylesterase ester motif and the modulator.
  • the structure of the radical linking the carboxylesterase ester motif to the rest of the modulator obviously depends on the particular chemistry strategy chosen for such linkage. Clearly the chemistry strategy for that coupling may vary widely, and thus many linkage structures are possible. The precise combination of variables making up the linking chemistry between the amino acid ester motif and the rest of the molecule will often be irrelevant to the primary binding mode of the compound as a whole. On the other hand, that linkage chemistry may in some cases pick up additional binding interactions with the enzyme, thereby enhancing binding.
  • the ⁇ , ⁇ -disubstituted glycine ester (formula H 2 N—C(R 2 R 3 )—COOH) may be conjugated to the modulator as
  • examples of Alk 1 and Alk 2 radicals when present, include —CH 2 —, —CH 2 CH 2 — —CH 2 CH 2 CH 2 —, —CH 2 CH 2 CH 2 CH 2 —, —CH ⁇ CH—, —CH ⁇ CHCH 2 —, —CH 2 CH ⁇ CH—, CH 2 CH ⁇ CHCH 2 —,—C ⁇ C—, —C ⁇ CCH 2 —, CH 2 C ⁇ C—, and CH 2 C ⁇ CCH 2 .
  • Alk 1 and Alk 2 include —CH 2 W—, —CH 2 CH 2 W— —CH 2 CH 2 WCH 2 —, —CH 2 CH 2 WCH(CH 3 )—, —CH 2 WCH 2 CH 2 —, —CH 2 WCH 2 CH 2 WCH 2 —, and —WCH 2 CH 2 — where W is —O—, —S—, —NH—, —N(CH 3 )—, or —CH 2 CH 2 N(CH 2 CH 2 OH)CH 2 —.
  • Further examples of Alk 1 and Alk 2 include divalent cyclopropyl, cyclopentyl and cyclohexyl radicals.
  • L 1 when n is 0, the radical is a hydrocarbon chain (optionally substituted and perhaps having an ether, thioether or amino linkage). Presently it is preferred that there be no optional substituents in L 1 .
  • L 1 is a divalent mono- or bicyclic carbocyclic or heterocyclic radical with 5-13 ring atoms (optionally substituted).
  • L 1 is a divalent radical including a hydrocarbon chain or chains and a mono- or bicyclic carbocyclic or heterocyclic radical with 5-13 ring, atoms (optionally substituted).
  • Q may be, for example, a divalent phenyl, naphthyl, cyclopropyl, cyclopentyl, or cyclohexyl radical, or a mono-, or bi-cyclic heterocyclic radical having 5 to 13 ring members, such as piperidinyl, piperazinyl, indolyl, pyridyl, thienyl, or pyrrolyl radical, but 1,4-phenylene is presently preferred.
  • a divalent phenyl, naphthyl, cyclopropyl, cyclopentyl, or cyclohexyl radical or a mono-, or bi-cyclic heterocyclic radical having 5 to 13 ring members, such as piperidinyl, piperazinyl, indolyl, pyridyl, thienyl, or pyrrolyl radical, but 1,4-phenylene is presently preferred.
  • m and p may be 0 with n being 1. In other embodiments, n and p may be 0 with m being 1. In further embodiments, m, n and p may be all 0. In still further embodiments m may be 0, n may be 1 with Q being a monocyclic heterocyclic radical, and p may be 0 or 1. Alk 1 and Alk 2 , when present, may be selected from —CH 2 —, —CH 2 CH 2 —, and —CH 2 CH 2 CH 2 — and Q may be 1,4-phenylene.
  • the principles of this invention can be applied to modulators of a wide range of intracellular targets which are implicated in a wide range of diseases.
  • the binding modes of known modulators to their targets are generally known soon after the modulators themselves become known.
  • modern techniques such as X-ray crystallography and NMR are capable of revealing such binding topologies and geometries, as are traditional medicinal chemistry methods of characterising structure-activity relationships.
  • it is straightforward to identify where in the structure of a given modulator an carboxylesterase ester motif could be attached without disrupting the binding of the modulator to the enzyme or receptor by use of structural data.
  • Table 1 lists some intracellular enzyme or receptor targets where there is published crystal structural data.
  • the method of the invention for increasing cellular potency and/or intracellular residence time of a modulator of the activity of a target intracellular enzyme or receptor, may involve several steps:
  • Step 1 Identify a position or positions on one or a plurality of modulator molecules sharing the same binding mode for the target enzyme or receptor, remote from the binding interface between the modulators and the target enzyme or receptor.
  • positions are identified from the X-ray co-crystal structure (or structure derived by nmr) of the target enzyme or receptor with a known modulator (or a close structural analogue thereof) bound to the enzyme or receptor, by inspection of the structure.
  • the X-ray crystal structure of the target enzyme or receptor with the modulator docked into the active site of the enzyme or receptor is modelled by computer graphics methods, and the model is inspected.
  • the presumption is that structural modification of the modulator at positions remote from the binding interface is unlikely to interfere significantly with the binding of the modulator to the active site of the enzyme or receptor. Suitable positions will normally appear from the co-crystal structure or docked model to be orientated towards solvent.
  • Step 2 Covalently modify the modulator(s) by attachment of an ⁇ , ⁇ -disubstituted glycine ester radical, or a range of different ⁇ , ⁇ -disubstituted glycine ester radicals at one or more of the positions identified in Step 1.
  • Attachment of ⁇ , ⁇ -disubstituted glycine ester radicals may be via an existing covalent coupling functionality on the modulator(s), or via a suitable functionality specifically introduced for that purpose.
  • the carboxylesterase motifs may be spaced from the main molecular bulk by a spacer or linker element, to position the motif deeper into solvent and thereby reduce still further any small effect of the motif on the binding mode of the modulator and/or to ensure that the motif is accessible to the carboxylesterase by reducing steric interference that may result from the main molecular bulk of the modulator.
  • Performance of Step 2 results in the preparation of one or, more usually, a small library of candidate modulators, each covalently modified relative to its parent inhibitor by the introduction of a variety of ⁇ , ⁇ -disubstituted glycine ester radicals, at one or more points of attachment identified in Step 1.
  • Step 3 Test the ⁇ , ⁇ -disubstituted glycine ester-conjugated modulator(s) prepared in step 2 to determine their activity against the target enzyme or receptor.
  • the carboxylesterase motif version(s) of the parent modulator(s), prepared as a result of performing Steps 1 and 2 are preferably tested in assays appropriate to determine whether the expected retention of modulator activity has in fact been retained, and to what degree and with what potency profile.
  • suitable assays will normally include assays in cell lines to assess degree of cellular activity, and potency profile, of the modified modulators.
  • Step 3 Other assays which may be employed in Step 3 include in vitro enzyme or receptor modulation assays to determine the intrinsic activity of the modified modulator and its putative carboxylesterase hydrolysis product; assays to determine the rate of conversion of the modified modulators to the corresponding carboxylic acid by carboxylesterases; and assays to determine the rate and or level of accumulation of the carboxylesterase hydrolysis product (the carboxylic acid) in cells.
  • both monocytic and non-monocytic cells, and/or a panel of isolated carboxylesterases can be used in order to identify compounds that show cell selectivity.
  • step 3 may be repeated with a different set of candidate alpha amino acid ester-conjugated versions of the parent modulator.
  • Step 4 From data acquired in Step 3, select one or more of the tested ⁇ , ⁇ -disubstituted glycine ester-conjugated versions of the parent modulator(s) which cause modulation of enzyme or receptor activity inside cells, are converted to and accumulate as the corresponding carboxylic acid inside cells, and which show increased or prolonged cellular potency.
  • Steps 1-4 represent a general algorithm for the implementation of the principles of the present invention.
  • the compounds of the invention may be prepared by a number of processes generally described below and more specifically in the Examples hereinafter.
  • reactive functional groups for example hydroxyl, amino and carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions [see for example Greene, T. W., “ Protecting Groups in Organic Synthesis ”, John Wiley and Sons, 1999].
  • Conventional protecting groups may be used in conjunction with standard practice.
  • deprotection may be the final step in the synthesis of a compound of general formula (I), and the processes according to the invention described herein after are understood to extend to such removal of protecting groups.
  • the compounds of the invention may be prepared according to the following Examples. All temperatures are in ° C. The following abbreviations are used
  • NBS N-bromosuccinimide
  • UV spectra were recorded at 220 and 254 nm using a G1315B DAD detector. Mass spectra were obtained over the range m/z 150 to 800 on a LC/MSD SL G1956B detector. Data were integrated and reported using ChemStation and ChemStation Data Browser softwares.
  • Examples 1, 3 and 5 below relate to the covalent conjugation of esterase motifs with di-substitution at the alpha carbon of the amino acid ester to these compounds, in a position remote from the binding interface between the inhibitor and the target enzyme (see the comments above concerning the binding mode of a model p38 MAP kinase inhibitor).
  • Examples 2, 4 and 6 below relate to the carboxylic acid esterase hydrolysis products of Examples 1, 3 and 5 respectively.
  • CDI 43.26 g, 267 mmol, 1.5 eq
  • anhydrous THF 1 I
  • Propiolic acid (16.43 mL, 267 mmol, 1.5 eq) was added dropwise and the mixture allowed to warm to room temperature and stirred for 1 hr.
  • a suspension of 2-(4- ⁇ [3-(2,4-difluorophenyl)-3-oxopropanimidoyl]-amino ⁇ phenyl)ethyl acetate (64.12 g, 178 mmol) in anhydrous THF (500 mL) was added and the mixture heated at 80° C. for 6 hours before being left to stir at room temperature overnight.
  • a dimethyl glycine cyclopentyl ester motif is covalently conjugated to the parent p38 MAP kinase inhibitor via the amino group of the dimethyl glycine cyclopentyl ester and through a —CH 2 CH 2 — linker radical.
  • This Example relates to the carboxylic acid hydrolysis product of the compound of Example 1.
  • Example 3 was synthesised using Intermediate 2 and Intermediate 5 in a similar manner to Example 1
  • Example 4 was synthesized using Intermediate 2 and Intermediate 6 in a similar manner to Example 2.
  • Example 5 was synthesised by the route shown in Scheme 7.
  • Step 5 Cyclopentyl N,N-bis(tert-butoxycarbonyl)-5- ⁇ [tert-butyl(dimethyl)silyl]oxy ⁇ -L-norvalinate
  • Step 6 Cyclopentyl N,N-bis(tert-butoxycarbonyl)-5- ⁇ [tert-butyl(dimethyl)silyl]oxy ⁇ -2-methylnorvalinate
  • Step 8 Cyclopentyl 5-bromo-N,N-bis(tert-butoxycarbonyl)-2-methylnorvalinate
  • NBS (3.95 g, 22.2 mmol) was suspended in DCM (30 mL) and triphenylphosphine added (5.44 g, 20.7 mmol). The reaction was stirred for 5 minutes before the addition of pyridine (0.94 mL, 8.9 mmol). Cyclopentyl N,N-bis(tert-butoxycarbonyl)-5-hydroxy-2-methylnorvalinate (3.08 g, 7.4 mmol) was then added as a solution in DCM (30 mL) and stirred overnight at room temperature.
  • Step 9 Cyclopentyl 5- ⁇ 4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenoxy ⁇ -N,N-bis(tert-butoxycarbonyl)-2-methylnorvalinate
  • Step 10 Cyclopentyl 5- ⁇ 4-[6-amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenoxy ⁇ -2-methylnorvalinate
  • Example 6 was synthesised by the route shown in Scheme 8.
  • Step 10 5- ⁇ -4-[6-Amino-5-(2,4-difluorobenzoyl)-2-oxopyridin-1(2H)-yl]-3,5-difluorophenoxy ⁇ -2-methylnorvaline
  • N-(1-Isobutoxyethoxy)-3-phenylpropionamide (58 mg) was dissolved in DCM (2 mL) and MeOH (0.5 mL) and stirred at RT under a nitrogen atmosphere. 4M HCl in dioxane (275 ⁇ L) was added to the solution and the reaction stirred for 2 hours. Solvent was then removed in vacuo and the residue purified by HPLC to give the product as a pink solid (9 mg).
  • the ability of compounds to inhibit histone deacetylase activities was measured using the commercially available HDAC fluorescent activity assay from Biomol.
  • the Fluor de LysTM substrate a lysine with an epsilon-amino acetylation
  • the source of histone deacetylase activity HeLa nuclear extract
  • Deacetylation of the substrate sensitises the substrate to Fluor de LysTM developer, which generates a fluorophore.
  • incubation of the substrate with a source of HDAC activity results in an increase in signal that is diminished in the presence of an HDAC inhibitor.
  • S i is the signal in the presence of substrate, enzyme and inhibitor
  • S o is the signal in the presence of substrate, enzyme and the vehicle in which the inhibitor is dissolved
  • B is the background signal measured in the absence of enzyme.
  • IC 50 values were determined by non-linear regression analysis, after fitting the results of eight data points to the equation for sigmoidal dose response with variable slope (% activity against log concentration of Compound), using Graphpad Prism software.
  • Histone deacetylase activity from crude nuclear extract derived from HeLa cells was used for screening.
  • the preparation purchased from 4C (Seneffe, Belgium), was prepared from HeLa cells harvested whilst in exponential growth phase.
  • the nuclear extract was prepared according to the methodology described by J. D. Dignam, Nucl. Acid. Res., 1983, 11, 1475-1489, snap frozen in liquid nitrogen and stored at ⁇ 80° C.
  • the final buffer composition was 20 mM Hepes, 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF and 20% (v/v) glycerol.
  • Cancer cell lines (U937 and HUT) growing in log phase were harvested and seeded at 1000-2000 cells/well (100 ⁇ l final volume) into 96-well tissue culture plates. Following 24 hours of growth cells were treated with compound. Plates were then re-incubated for a further 72-96 hours before a WST-1 cell viability assay was conducted according to the suppliers (Roche Applied Science) instructions.
  • S i is the signal in the presence of inhibitor and S o is the signal in the presence of DMSO.
  • Dose response curves were generated from 8 concentrations (top final concentration 10 ⁇ M, with 3-fold dilutions), using 6 replicates.
  • IC 50 values were determined by non-linear regression analysis, after fitting the results to the equation for sigmoidal dose response with variable slope (% activity against log concentration of compound), using Graphpad Prism software.
  • p38 MAP Kinase activity was measured in an assay performed by Upstate (Dundee UK).
  • p38 MAP Kinase ⁇ (5-10mU) is incubated with 25 mM Tris pH 7.5, 0.02 mM EGTA, 0.33 mg/mL myelin basic protein, 10 mM MgAcetate and [ ⁇ - 33 P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required).
  • the reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 ⁇ L of a 3% phosphoric acid solution. 10 ⁇ L of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
  • Duplicate data points are generated from a 1 ⁇ 3 log dilution series of a stock solution in DMSO. Nine dilutions steps are made from a top concentration of 10 ⁇ M, and a ‘no compound’ blank is included.
  • the standard radiometric filter-binding assay is performed at an ATP concentration at, or close to, the Km. Data from scintillation counts are collected and subjected to free-fit analysis by Prism software. From the curve generated, the concentration giving 50% inhibition is determined and reported.
  • U937 or HUT78 cells were plated in RPMI 1640, and were incubated at 37° C., 5% CO 2 for 18 hours. 10 mM stocks of compounds were diluted media/0.1% DMSO to give a log or semi-log dilution series. The wells for ‘no treatment’ and ‘anisomycin’ were treated with 0.1% DMSO only. The cells were incubated at 37° C., 5% CO 2 for a further 4 hours. Anisomycin was added to all wells except ‘no treatment’ at a final concentration of 10 ⁇ M. The cells were incubated at 37° C., 5% CO 2 for 30 minutes before harvest. Plates were stood on ice whilst harvesting, and all following steps were carried out at 4° C.
  • the cells were pelleted at 1000 rpm for 10 minutes at 4° C., the media aspirated, and the pellet washed with cold PBS.
  • the pellets were lysed in 120 ⁇ l of SDS lysis buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, with protease inhibitors and phosphatase inhibitors added according to the manufacturers' recommendations). After 30 minutes on ice, the samples were sonicated for 5 seconds before centrifugation at 13,000 rpm 4° C. for 10 minutes to remove cell debris. 10 ⁇ l of the resulting gel samples were loaded per lane on NOVEX 4-12% Bis-Tris MOPS gels.
  • Membranes from western transfer of gels were blotted with anti-phospho MAPKAP2, anti-phospho HSP27 and anti GAPDH according to the manufacturers' instructions. Signal was visualised using HRP-linked anti-rabbit or anti-mouse antibodies, ECL reagent and ECL film. IC50 values for the various compounds were visualised from the resulting photographic images, using both band-shift and signal intensity.
  • THP-1 cells were plated in 100 ⁇ l at a density of 4 ⁇ 10 4 cells/well in V-bottomed 96 well tissue culture treated plates and incubated at 37° C. in 5% CO 2 for 16 hours. 2 hours after the addition of the inhibitor in 100 ⁇ l of tissue culture media, the cells were stimulated with LPS( E coli strain 005:B5, Sigma) at a final concentration of 1 ⁇ g/mL and incubated at 37° C. in 5% CO 2 for 6 hours. TNF- ⁇ levels were measured from cell-free supernatants by sandwich ELISA (R&D Systems #QTA00B)
  • Hydrolysis of esters to the corresponding carboxylic acids by hCE-1 can be measured using the following procedure. At zero time, 100 ⁇ l of recombinant hCE-1 at a concentration of 6 ⁇ g/mL in phosphate assay buffer (K 2 PO 4 100 mM, KCl 40 mM, pH 7.4) was added to an equal volume of assay buffer containing 5 ⁇ M ester substrate. After thorough mixing, triplicate samples were incubated for 0, 20 or 80 minutes at 37° C. At the appropriate time, hydrolysis was stopped by the addition of 600 ⁇ l of acetonitrile. For zero time samples, the acetonitrile was added prior to the enzyme.
  • K 2 PO 4 100 mM, KCl 40 mM, pH 7.4 phosphate assay buffer
  • ester conjugate according to the invention may be tested to determine whether it meets the requirement that it be hydrolysed by intracellular esterases; by testing in the following assay.
  • the resulting supernatant was used as a source of esterase activity and was stored at ⁇ 80° C. until required.
  • Cells (8 ⁇ 10 4 /mL) were incubated at 37° C. in culture medium containing 6 ⁇ M compound in a 5% (v/v) CO 2 -humidified atmosphere. Incubations were terminated after 6h by centrifugation of 25 mL aliquots of the cell suspension at 300 g for 5 minutes at 4° C. 0.2 mL samples of the culture media supernatants were added to 4 volumes of acetonitrile (Sigma-Aldrich). After decanting the supernatant, the residual cell pellet (2 ⁇ 10 6 cells) was extracted into 1 mL of acetonitrile.
  • Example 1 shows that the acid of Example 1 has a similar IC50 in the enzyme assay to the parent compound (Compound I): (WO 03/076405) indicating that binding to the enzyme has not been disrupted by attachment of the esterase motif.
  • Di-substituted compounds, e.g. Example 1 are hydrolysed by hCE-1 and as a consequence the acid accumulates in cells. This accumulation of acid results in Example 1 being significantly more potent than the parent compound in the cellular assay.
  • Table 2 shows that the parent compound (Compound I) is equipotent in monocytic and non monocytic cell lines whereas Examples 1 and 3 are 100 times more potent in the monocytic cell line.
  • Example 1 only accumulates in the monocytic cell line showing that cell selectivity is achieved by the attachment of an esterase motif that is only cleaved in the monocytic cell line.
  • Table 3 indicates that a C-linked dialkyl compound (Example 5) is macrophage selective whereas the parent compound (Compound II; WO 03/076405) and a compound (Compound III; WO 2007/129036) lacking an alkyl group at the alpha carbon of the amino acid derivative are not. This illustrates that macrophage selectivity can be achieved by the introduction of a second substituent at the alpha carbon of the amino acid ester motif.
  • Table 4 shows that the acid of Examples 7 and 9 have similar IC50s in the enzyme assay to the parent compound (Compound IV): indicating that binding to the enzyme has not been disrupted by attachment of the esterase motif.
  • Di-substituted compounds, e.g. Example 7 are hydrolysed by hCE-1 and as a consequence the acid accumulates in monocytic cells. This accumulation of acid results in Examples 7 and 9 being significantly more potent than the parent compound in the cellular assay.
  • Table 5 shows that the parent compound (Compound IV) has similar potencies in monocytic (U937) and non monocytic (Hut78) cell lines whereas Examples 7 and 9 are 30 times more potent in the monocytic cell line than the non-monocytic cell line. This illustrates that a second substituent at the alpha position of the amino acid motif confers macrophage selectivity on the compounds.

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GB0903480D0 (en) * 2009-02-27 2009-04-08 Chroma Therapeutics Ltd Enzyme Inhibitors
GB201009853D0 (en) 2010-06-11 2010-07-21 Chroma Therapeutics Ltd HSP90 inhibitors
WO2012025701A1 (fr) * 2010-08-25 2012-03-01 Chroma Therapeutics Ltd. Dérivés d'ester de glycine alpha, alpha-disubstituée et leur utilisation comme inhibiteurs des hdac
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GB0803747D0 (en) 2008-04-09
ZA201006071B (en) 2011-05-25
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US20140088159A1 (en) 2014-03-27
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KR20160074017A (ko) 2016-06-27
US20160151509A1 (en) 2016-06-02
EA201001390A1 (ru) 2011-06-30
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IL207537A0 (en) 2010-12-30
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NZ587341A (en) 2012-07-27
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WO2009106848A3 (fr) 2010-05-14
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