WO2007129020A1 - Dérivés de la pyrimidine utiles en tant qu'inhibiteurs de la dhfr - Google Patents

Dérivés de la pyrimidine utiles en tant qu'inhibiteurs de la dhfr Download PDF

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Publication number
WO2007129020A1
WO2007129020A1 PCT/GB2007/001475 GB2007001475W WO2007129020A1 WO 2007129020 A1 WO2007129020 A1 WO 2007129020A1 GB 2007001475 W GB2007001475 W GB 2007001475W WO 2007129020 A1 WO2007129020 A1 WO 2007129020A1
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compound
alkyl
hydrogen
radical
optionally substituted
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PCT/GB2007/001475
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English (en)
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Alan Hornsby Davidson
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Chroma Therapeutics Ltd.
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Publication of WO2007129020A1 publication Critical patent/WO2007129020A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/95—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in positions 2 and 4
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • This invention relates to compounds which inhibit the dihydrofolate reductase family of enzymes and to their use in the treatment of cell proliferative diseases, including cancer and inflammation, and to their use as anti-infective agents for bacterial, fungal or parasitic infection.
  • Folate is a vitamin which is a key component in the biosynthesis of purine and pyrimidine nucleotides. Following absorption, dietary folate is reduced to dihydrofolate and then further reduced to tetrahydrofolate by the enzyme dihydrofolate reductase (DHFR). Inhibition of DHFR leads to a reduction in nucleotide biosynthesis resulting in inhibition of DNA biosynthesis and reduced cell division. DHFR inhibitors are widely used in the treatment of cancer (Bertino J, J.Clin. Oncol. 1993, 11 , 5-14), cell proliferative diseases such as rheumatoid arthritis (Cronstein N., Pharmacol. Rev.
  • DHFR inhibitors have also found use as anti-infective (Salter A., Rev. Infect. Dis. 1982, 4,196-236,) and anti-parasitic agents (Plowe C. 2004,BMJ 328, 545-548). Many types of DHFR inhibitor compounds have been suggested, and several such compounds are used as anti-cancer, anti-inflammatory, anti-infective and anti-parasitic agents. The two general templates for DHFR inhibitors are shown below:
  • Methotrexate is the most widely used DHFR inhibitor and contains a glutamate functionality which enables it to be actively transported into, and retained inside, cells.
  • cancer cells can become resistant to methotrexate by modifying this active transport mechanism.
  • non-mammalian cells lack the active transport system and methotrexate has limited utility as an anti-infective agent.
  • Lipophilic DHFR inhibitors which can be taken up by passive diffusion have therefore been developed both to circumvent cancer cell resistance and for use as anti- infective agents. The following publications disclose examples of such compounds:
  • DHFR inhibitors that accumulate in cells in a way that does not depend on the active transport mechanism of methotrexate would be of value.
  • methotrexate based and lipophilic inhibitors give rise to side effects hence agents that target specific cell types would also be of value.
  • This invention is based on the finding that the introduction of an alpha amino acid ester grouping into the DHFR inhibitor molecular templates (A) and (B) facilitates penetration of the agent through the cell membrane, and thereby allows intracellular esterase activity to hydrolyse the ester to release the parent acid. Being charged, the acid is not readily transported out of the cell, wherein it therefore accumulates to increase the intracellular concentration of the active DHFR inhibitor. This leads to increases in potency and duration of action.
  • the invention therefore makes available a class of compounds whose structures are characterised by having an alpha amino acid ester moiety which is a substrate for intracellular carboxylesterase (also referred to herein as an "esterase motif) covalently linked to a DHFR inhibitor molecular template, and to the corresponding de-esterified parent acids, such compounds having pharmaceutical utility in the treatment of diseases such as cancers and other cell proliferative diseases which benefit from intracellular inhibition of DHFR.
  • carboxylesterase also referred to herein as an "esterase motif
  • a further aspect of the invention is that attachment of particular alpha amino acids and their mode of attachment results in the selective accumulation of DHFR inhibitors in specific cell types.
  • a and D are independently -CHR 6 - or -NR 6 -;
  • Each R 2 independently represents hydrogen or -OR 6 ;
  • Re is hydrogen or CrC 6 alkyl
  • L 1 is a divalent radical of formula -(Alk 1 ) m (Q) n (Alk 2 ) p - wherein m, n and p are independently 0 or 1
  • Q is (i) an optionally substituted divalent mono- or bicyclic carbocyclic or heterocyclic radical having 5 - 13 ring members, or (ii), in the case where p is 0, a divalent radical of formula -Q 1 -X 2 - wherein X 2 is -O-, -S- or NR A - wherein R A is hydrogen or optionally substituted Ci-C 3 alkyl
  • Q 1 is an optionally substituted divalent mono- or bicyclic carbocyclic or heterocyclic radical having 5 - 13 ring members
  • AIk 1 and AIk 2 independently represent optionally substituted divalent C 3 -C 7 cycloalkyl radicals, or optionally substituted straight or branched, C 1 -C 6 alkylene, C 2 -C 6 alkenylene, or C 2 -C 6 alkynylene radicals which may optionally contain or terminate in an ether (-O-), thioether (-S-) or amino (-NR A -) link wherein R A is hydrogen or optionally substituted C 1 -C 3 alkyl;
  • z is 0 or 1 ;
  • R is a radical of formula (X) or (Y)
  • R 1 is a carboxylic acid group (-COOH), or an ester group which is hydrolysable by one or more intracellular carboxylesterase enzymes to a carboxylic acid group;
  • Ring B is a monocyclic heterocyclic ring of 5 or 6 ring atoms wherein R 1 is linked to a ring carbon adjacent the ring nitrogen shown, and ring B is optionally fused to a second carbocyclic or heterocyclic ring of 5 or 6 ring atoms in which case the bond shown intersected by a wavy line may be from a ring atom in said second ring.
  • Compounds of formula (I) and (II) above may be prepared in the form of salts, especially pharmaceutically acceptable salts, N-oxides, hydrates, and solvates thereof.
  • the invention provides the use of a compound of the invention in the preparation of a composition for inhibiting the activity of the DHFR enzyme.
  • the compounds with which the invention is concerned may be used for the inhibition of DHFR activity, in vitro or in vivo.
  • the compounds of the invention may be used in the preparation of a composition for the treatment of cell-proliferation disease, for example cancer cell proliferation; of autoimmune diseases; and of bacterial, fungal or parasitic infection.
  • cell-proliferation disease for example cancer cell proliferation; of autoimmune diseases; and of bacterial, fungal or parasitic infection.
  • the invention provides a method for the treatment of the foregoing disease types, which comprises administering to a subject suffering such disease an effective amount of a compound of the invention.
  • R x is the group characterising the ester, notionally derived from the alcohol R X OH.
  • (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 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 3 -C b alkynyl wherein a and b are integers refers to straight chain or branched chain hydrocarbon groups having from a to b 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 a to b carbon atoms, and at least one triple bond.
  • Carbocyclic refers to a 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 and cyclooctyl.
  • aryl refers to a 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-, 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-, 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, benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido and succinimido groups.
  • substituent is phenyl, phenoxy or monocyclic heteroaryl or heteroaryloxy with 5 or 6 ring atoms
  • the phenyl or heteroaryl ring thereof may itself be substituted by any of the above substituents except phenyl phenoxy, heteroaryl or heteroaryloxy.
  • An "optional substituent” or “substituent” may be one of the foregoing specified groups.
  • salt includes base addition, acid addition and quaternary salts.
  • Compounds of the invention which are acidic can form salts, including pharmaceutically acceptable salts, with bases such as alkali metal hydroxides, e.g. sodium and potassium hydroxides; alkaline earth metal hydroxides e.g. calcium, barium and magnesium hydroxides; with organic bases e.g. N-methyl-D-glucamine, choline tris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethyl piperidine, dibenzylamine and the like.
  • bases such as alkali metal hydroxides, e.g. sodium and potassium hydroxides; alkaline earth metal hydroxides e.g. calcium, barium and magnesium hydroxides; with organic bases e.g. N-methyl-D-glucamine, choline tris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethyl pipe
  • salts including pharmaceutically acceptable salts with inorganic acids, e.g. with hydrohalic acids such as hydrochloric or hydrobromic acids, sulphuric acid, nitric acid or phosphoric acid and the like, and with organic acids e.g. with acetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic, p- toluenesulphonic, benzoic, benzenesunfonic, glutamic, lactic, and mandelic acids and the like.
  • hydrohalic acids such as hydrochloric or hydrobromic acids, sulphuric acid, nitric acid or phosphoric acid and the like
  • organic acids e.g. with acetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic, p- toluenesulphonic, benzoic, benzenesunfonic, glut
  • 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol.
  • solvent molecules for example, ethanol.
  • 'hydrate' is employed when said solvent is water.
  • esters of the invention are converted by intracellular esterases to the carboxylic acid. Both the esters and carboxylic acids may have DHFR inhibitory activity in their own right.
  • the compounds of the invention include not only the ester, but also the corresponding carboxylic acid hydrolysis products.
  • the ester group Ri must be one which in the compound of the invention is hydrolysable by one or more intracellular carboxylesterase enzymes to a carboxylic acid group.
  • 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 conjugates.
  • BPH biphenylhydrolase
  • the carboxylesterase hydrolyses the free amino acid ester to the parent acid it will, also hydrolyse the ester motif when covalently linked to the rest of the molecule.
  • the broken cell assay described herein provides 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 incorporated in the DHFR inhibitor of the invention via the chosen conjugation chemistry, to confirm that it is still a carboxylesterase substrate in that background.
  • R 8 is hydrogen or optionally substituted (C 1 -C 3 )alkyl-(Z 1 ) a -[(C 1 - C 3 )alkyl] b - or (C 2 -C 3 )alkenyl-(Z 1 ) a -[(Ci-C 3 )alkyl] b - wherein a and b are independently 0 or 1 and Z 1 is -O-, -S-, or -NRn- wherein Ri 1 is hydrogen or (C r C 3 )alkyl; and R 9 and Ri 0 are independently hydrogen or (C r C 3 )alkyl-;
  • R 8 is hydrogen or optionally substituted Ri 2 Ri 3 N-(Ci-C 3 )alkyl- wherein Ri 2 is hydrogen or (C r C 3 )alkyl and Ri 3 is hydrogen or (C r C 3 )alkyl; or Ri 2 and Ri 3 together with the nitrogen to which they are attached form an optionally substituted monocyclic heterocyclic ring of 5- or 6- ring atoms or bicyclic heterocyclic ring system of 8 to 10 ring atoms, and R 9 and R 10 are independently hydrogen or (C r C 3 )alkyl-;or
  • R 8 and R 9 taken together with the carbon to which they are attached form an optionally substituted monocyclic carbocyclic ring of from 3 to 7 ring atoms or bicyclic carbocyclic ring system of 8 to 10 ring atoms, and Ri 0 is hydrogen. Within these classes, R 10 is often hydrogen. Specific examples of R 7 include methyl, ethyl, n- or iso-propyl, n-, sec- or tert-butyl, cyclohexyl, allyl, phenyl, benzyl, 2-, 3- or 4-pyridylmethyl, N-methylpiperidin-4-yl, tetrahydrofuran-3-yl or methoxyethyl. Currently preferred is where R 7 is cyclopentyl.
  • 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 the maintenance of joint inflammation and joint destruction. Macrophages are also involved in tumour growth and development (Naldini and Carraro, Curr Drug Targets lnflamm Allergy ,2005, 3-8 ). Hence agents that selectively target macrophage cell proliferation could be of value in the treatment of cancer and autoimmune disease. Targeting specific cell types would be expected to lead to reduced side-effects.
  • the inventors have discovered a method of targeting DHFR inhibitors to macrophages which is based on the observation that the way in which the esterase motif is linked to the DHFR inhibitor determines whether it is hydrolysed, and hence whether or not it accumulates in different cell types. Specifically it has been found that macrophages contain the human carboxylesterase hCE-1 whereas other cell types do not. In the general formula (I) or (II) when the nitrogen of the esterase motif is substituted but not directly bonded to a carbonyl group, the ester will only be hydrolysed by hCE-1 and hence the DHFR inhibitors will only accumulate in macrophages.
  • macrophage or macrophages will be used to denote macrophages (including tumour associated macrophages) and/or monocytes.
  • R 4 is present in the compounds of the invention when R in formula (I) is a radical of formula (X)
  • R 4 may be optionally substituted C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl, aryl or heteroaryl such monocyclic heteroaryl having 5 or 6 ring atoms, for example methyl, ethyl, n-or isopropyl, cyclopropyl, cyclopentyl, cyclohexyl, phenyl, or pyridyl.
  • R 3 is optionally substituted CrC 6 alkyl such as methyl, ethyl, n-or isopropyl, or n-, iso- or sec-but
  • esters with a slow rate of esterase cleavage are preferred, since they are less susceptible to pre-systemic metabolism. Their ability to reach their target tissue intact is therefore increased, and the ester can be converted inside the cells of the target tissue into the acid product.
  • ester is either directly applied to the target tissue or directed there by, for example, inhalation, it will often be desirable that the ester has a rapid rate of esterase cleavage, to minimise systemic exposure and consequent unwanted side effects.
  • esters tend to be cleaved more rapidly than if that carbon is substituted, or is part of a ring system such as a phenyl or cyclohexyl ring.
  • This radical arises from the particular chemistry strategy chosen to link the amino acid ester motif R to the aromatic ring system.
  • the chemistry strategy for that coupling may vary widely, and thus many combinations of the variables Y 1 , L 1 , and z are possible.
  • the precise combination of variable making up the linking chemistry between the amino acid ester motif and the aromatic ring system will often be irrelevant to the primary binding mode of the compound as a whole.
  • z may be O or 1 , so that a methylene radical linked to the phenyl ring is optional;
  • AIk 1 and AIk 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 AIk 1 and AIk 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 heterocyclicl radical having 5 to13 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 heterocyclicl radical having 5 to13 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.
  • AIk 1 and AIk 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.
  • R 2 is hydrogen; halogen, for example fluoro or chloro; or C 1 -C 4 alkoxy for example methoxy, ethoxy or n- or iso-propoxy. Presently it is preferred that it be hydrogen or methoxy.
  • the group R 6 in variables A, D, E and G.
  • R 6 may be hydrogen or C r C 6 alkyl, for example methyl, ethyl or n- or iso-propyl. Specifically, R 6 may be hydrogen or methyl.
  • R is a radical of formula (Y), it may be selected from, for example, the following:
  • the compounds of the invention may be synthesised as described below and in the Examples herein.
  • the compounds with which the invention is concerned are inhibitors of DHFR and are therefore of use in the treatment of cell proliferative disease, such as cancer, in treatment of inflammation, and in the treatment of bacterial, fungal or parasitic infection in humans and other mammals.
  • the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing treatment. Optimum dose levels and frequency of dosing will be determined by clinical trial.
  • the compounds with which the invention is concerned may be prepared for administration by any route consistent with their pharmacokinetic properties.
  • the orally administrable compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions.
  • Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate.
  • the tablets may be coated according to methods well known in normal pharmaceutical practice.
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; nonaqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.
  • the drug may be made up into a cream, lotion or ointment. Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.
  • the drug may be formulated for aerosol delivery for example, by pressure-driven jet atomizers or ultrasonic atomizers, or preferably by propellant-driven metered aerosols or propellant-free administration of micronized powders, for example, inhalation capsules or other "dry powder" delivery systems.
  • Excipients such as, for example, propellants (e.g. Frigen in the case of metered aerosols), surface-active substances, emulsifiers, stabilizers, preservatives, flavorings, and fillers (e.g. lactose in the case of powder inhalers) may be present in such inhaled formulations.
  • the drug may be made up into a solution or suspension in a suitable sterile aqueous or non aqueous vehicle.
  • Additives for instance buffers such as sodium metabisulphite or disodium edeate; preservatives including bactericidal and fungicidal agents such as phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents such as hypromellose may also be included.
  • the active ingredient may also be administered parenterally in a sterile medium.
  • the drug can either be suspended or dissolved in the vehicle.
  • adjuvants such as a local anaesthetic, . preservative and buffering agents can be dissolved in the vehicle.
  • one advantage lies in their property of accumulating in lung tissue, resulting in reduced systemic exposure relative to the analogous DHFR inhibitor not conjugated to the amino acid ester motif.
  • agents can be given directly to the lung using inhalation methodologies, such agents still enter the systemic circulation. This can result in undesirable side effects, and can limit the dose and range of agents that can be used to treat lung disorders.
  • the neutral ester species is taken up by lung tissue where, depending on the nature of the esterase motif, it is rapidly cleaved to the acid which, as a consequence of it being a charged species, is retained in the lung tissue for a longer period of time than the neutral ester.
  • the agent is concentrated in the lung tissue and systemic exposure is reduced.
  • Microwave irradiation was carried out using a CEM Discover focused microwave reactor.
  • UV spectra were recorded at 215 nm using a Gilson G1315A Diode Array Detector, G1214A single wavelength UV detector, Waters 2487 dual wavelength UV detector, Waters 2488 dual wavelength UV detector, or Waters 2996 diode array UV detector.
  • Mass spectra were obtained over the range m/z 150 to 850 at a sampling rate of 2 scans per second or 1 scan per 1.2 seconds using Micromass LCT with Z-spray interface or Micromass LCT with Z-spray or MUX interface. Data were integrated and reported using OpenLynx and OpenLynx Browser software
  • Boc fert-butoxycarbonyl
  • DIPEA diisopropylethylamine
  • NaHCO 3 sodium hydrogen carbonate
  • the title compound can be obtained by the methods described in J.Med.Chem., 1986, 29, 1080-1087.
  • Stage 3 product (1.57g, 3.9mmol) was dissolved in acetic acid:THF:water (3:1 :1 , 100ml). The reaction mixture was stirred at 3O 0 C for 16 h for complete reaction. EtOAc (200ml) was added and washed with 1 M Na 2 CO 3 , 1 M HCI and brine. The EtOAc extracts were dried over MgSO 4 and evaporated under reduced pressure to give the product as a clear oil which crystallised on standing (1.Og, 95%).
  • stage 1 product (5g, 14.82mmol) in MeOH (25ml) was treated with 1 M NaOH (22.23ml, 22.23mmol) and allowed to stir at RT for 2h. After this time, the reaction mixture was concentrated to ⁇ 1ml and partitioned between water and Et 2 O. The layers were separated and the aqueous extracted with a further portion of Et 2 O. The aqueous layer was then layered with further Et 2 O and then acidified to pH 2 with cone. HCI. The layers separated, the aqueous extracted with Et 2 O and the combined extracts washed with brine, then dried over MgSO 4 and filtered and evaporated to give the product as a colourless oil (4.1g, 86%). m/z 324 [M+H] + .
  • Stage 2 product (3.5g, 10.82mmol) was dissolved in anhydrous THF (30ml), cooled to -10 0 C and N-methyl morpholine (1.428ml, 12.99mmol) added. After stirring for a few moments, isopropyl chloroformate (12.99ml, 12.99mmol) was added dropwise. The solution was stirred at -10 0 C for 10 min and then allowed to warm to RT. In a separate flask, sodium borohydride (1.024g, 27.1mmol) was dissolved in water (20ml) and MeOH (5ml), cooled to -5 0 C and stirred for 30 min.
  • the mixed anhydride solution was added via cannula to the borohydride solution, and once the addition was complete, the reaction was allowed to warm to RT. After 3 h at RT, AcOH (6.22ml, 108mmol) was added and the solution allowed to stir at RT for 30 min. Et 2 O was added, the layers separated and the aqueous extracted with Et 2 O. The combined organic layers were washed with sat. NaHCO 3 (x 2), brine and then dried over MgSO 4 , filtered and evaporated to give the crude product (2.5g, 75%). This was used without further purification or characterization.
  • stage 3 product (2g, 8.08mmol) in a mixture of Et 2 O (50ml) and acetonitrile (50ml), at -10 0 C, were added successively imidazole (0.99Og, 14.55mmol) and triphenyl phosphine (3.6Og, 13.74mmol). Iodine (3.49g, 13.74mmol) was then added in small portions over 20 min. Once the addition was complete the reaction was maintained at -10 0 C and stirred for a further 1.5 h. The reaction was then diluted with Et 2 O and washed successively with sat. Na 2 CO 3 , sat. CuSO 4 and water, and then dried over MgSO 4 , filtered and evaporated. The residue was purified by column chromatography eluting with 5 to 10% EtOAc in hexanes to afford the title compound (1.8g, 53%). m/z 420 [M+H] + . ⁇ Synthesis of example compounds
  • Triethylamine (0.77ml, 5.2 mmol) and formic acid (0.19ml, 5.2 mmol) were dissolved in EtOH (4ml) and added to a solution of stage 1 product (0.7g, 1.7mmol) in EtOH (4ml).
  • 10% Pd/C (approximately 10 mol%) was added and the mixture was heated to reflux. After 2 h the hot reaction mixture was filtered through celite and the residue was washed with MeOH. The filtrate and washings were combined and evaporated and the residue was partitioned between DCM and sat. aq. NaHCO 3 . The organic layer was washed with brine, dried over MgSO 4 and concentrated under reduced pressure.
  • Examples (3) and (4) were prepared by the same method described for examples (1) and (2) in scheme 5, using 2,5-dimethoxy-3-hydroxynitrobenzene and 2,4-diamino-5- methylquinazoline-6-carbonitrile as the starting materials.
  • Example (5) was prepared by the same method described for examples (1) and (2) in scheme 5, using 2,5-dimethoxy-3-hydroxynitrobenzene, tert-butyl (2S)-2- ⁇ [(benzyloxy)carbonyl]amino ⁇ -4-iodobutanoate and 2, 4-diamino-5-methylquinazoline- 6-carbonitrile as the starting materials.
  • Stage 1 is the same as described for example (1).
  • Stage 2 Synthesis of (S)-cyclopentyl 2-amino-4-(4-nitrophenoxy)butanoate
  • Stage 1 product (4.Og, 9.8mmol) in DCM (12ml) was added trifluoroacetic acid (12ml). After stirring at RT for 1 h the reaction was diluted with DCM, cooled in ice and neutralised by the addition of aq. ammonia. The organic layer was collected and washed with water, aq. sodium hydrogen carbonate and brine, then dried over MgSO 4 and concentrated under reduced pressure to afford the title compound as a yellow oil (3.Og, 100%).
  • Triethylamine (1.29ml, 9.3mmol) and formic acid (348 ⁇ l, 9.3mmol) were dissolved in EtOH (10ml) and added to a solution of Stage 3 product (1.2g, 3.1mmol) in EtOH (10ml).
  • 10% Pd/C approximately 10 mol% was added and the mixture was heated to reflux. After 30 min the hot reaction mixture was filtered through celite and the residue was washed with MeOH. The filtrate and washings were combined and evaporated and the residue was partitioned between DCM and sat. aq. NaHCO 3 . The organic layer was washed with brine, dried over MgSO 4 and concentrated under reduced pressure to afford the title compound (1.01g, 92%).
  • Stage 4 product (21 mg, 38 ⁇ M) was suspended in ethanol (1.0 ml). A solution of lithium hydroxide 1M (380 ⁇ l) was added to the above and the suspension allowed to stir over the weekend. The ethanol was subsequently removed under reduced pressure, the residual diluted with water and taken down to pH 4 with dilute acetic acid. The resulting precipitate was collected by filtration and washed with water, ethanol and diethyl ether to furnish the title compound (7 mg, 38%).
  • Examples (8) and (9) were prepared by the same method described for examples (6) and (7) in scheme 6, using 2,5-dimethoxy-3-hydroxynitrobenzene and 2,4-diamino-5- methylquinazoline-6-carbonitrile as the starting materials.
  • Example (10) was prepared by the same method described for examples (6) and (7) in scheme 6, using 2,5-dimethoxy-3-hydroxynitrobenzene, tert-butyl (2S)-2- ⁇ [(benzyloxy)carbonyl]amino ⁇ -4-iodobutanoate and 2, 4-diamino-5-methylquinazoline- 6-carbonitrile as the starting materials.
  • the ability of compounds to inhibit DHFR activity was measured in an assay based on the ability of DHFR to catalyse the reversible NADPH-dependent reduction of dihydrofolic acid to tetrahydrofolic acid using a Sigma kit (Catalogue number CS0340). This uses proprietary assay buffer and recombinant human DHFR at 7.5 x 10 "4 Unit per reaction, NADPH at 60 ⁇ M and dihydrofolic acid at 50 ⁇ M. The reaction was followed by monitoring the decrease in absorbance at 340 nm, for a 2 minute period, at room temperature, and the enzyme activity was calculated as the rate of decrease in absorbance.
  • Cancer cell lines (U937, HCT 116, THP 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 h of growth cells were treated with compound. Plates were then re-incubated for a further 72 - 96 h before a WST-1 cell viability assay was conducted according to the suppliers (Roche Applied Science) instructions.
  • S 1 is the signal in the presence of inhibitor and S 0 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.
  • IC50 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.
  • IC50 values were allocated to one of the three ranges as follows:
  • Range B 100 nM ⁇ IC50 ⁇ 1000 nM
  • Range C IC50 > 1000 nM
  • Rates of hydrolysis are expressed in pg/ml/min.
  • Table 2 presents data showing that several amino acid ester motifs, conjugated to various intracellular enzyme inhibitors by several different linker chemistries are all hydrolysed by intracellular carboxyesterases to the corresponding acid.

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Abstract

Composés de formule (I) ou (II) qui sont des inhibiteurs de la dihydrofolate réductase et qui sont utiles pour le traitement, par exemple, de maladies prolifératives des cellules : A et D étant chacun indépendamment -CHR6- ou -NR6 ; E et G étant chacun indépendamment =CR6- ou =N- ; chaque R2 représente indépendamment un hydrogène ou -OR6, où R6 est un hydrogène ou un alkyle en C1-C6 ; Y1 est une liaison, -(C=O)-, -S(O2)-(C=O)NR3-, -NR3(C=O)-, -S(O2)NR3-, -NR3S(O2)- ou -NR3(C=O)NR5-, où R3 et R5 sont chacun indépendamment un hydrogène ou un alkyle en C1-C6 éventuellement substitué ; L1 est un radical divalent de formule -(Alk1)m(Q)n(Alk2)p- dans lequel m, n, p, Q, Alk1 et Alk2 sont tels que définis dans les revendications ; z est 0 ou 1 ; et R est un radical de formule (X) ou (Y) dans lequel R1 est un groupe acide carboxylique (-COOH) ou un groupe ester qui est hydrolysable par une ou plusieurs enzymes de type carboxylestérases intracellulaires en un groupe acide carboxylique ; et R4 et le cycle B sont tels que définis dans les revendications.
PCT/GB2007/001475 2006-05-04 2007-04-23 Dérivés de la pyrimidine utiles en tant qu'inhibiteurs de la dhfr WO2007129020A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010097586A1 (fr) 2009-02-27 2010-09-02 Chroma Therapeutics Ltd. Inhibiteurs d'enzymes

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002068397A1 (fr) * 2001-02-28 2002-09-06 Melacure Therapeutics Ab Esters de diaminoquinazoline esters destines a etre utilises avec des inhibiteurs de la dihydrofolate reductase
WO2003002064A2 (fr) * 2001-06-28 2003-01-09 Essential Therapeutics, Inc. Composes heterocycliques tenant lieu d'inhibiteurs selectifs de dhfr bacterienne, et utilisations
WO2006117567A2 (fr) * 2005-05-05 2006-11-09 Chroma Therapeutics Ltd Modulation d'enzyme et de recepteur

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002068397A1 (fr) * 2001-02-28 2002-09-06 Melacure Therapeutics Ab Esters de diaminoquinazoline esters destines a etre utilises avec des inhibiteurs de la dihydrofolate reductase
WO2003002064A2 (fr) * 2001-06-28 2003-01-09 Essential Therapeutics, Inc. Composes heterocycliques tenant lieu d'inhibiteurs selectifs de dhfr bacterienne, et utilisations
WO2006117567A2 (fr) * 2005-05-05 2006-11-09 Chroma Therapeutics Ltd Modulation d'enzyme et de recepteur

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LAU, HOLLIS ET AL: "Efficacies of lipophilic inhibitors of dihydrofolate reductase against parasitic protozoa", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY , 45(1), 187-195 CODEN: AMACCQ; ISSN: 0066-4804, 2001, XP002442717 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010097586A1 (fr) 2009-02-27 2010-09-02 Chroma Therapeutics Ltd. Inhibiteurs d'enzymes

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