WO2017168137A1 - Pyrimidobenzimidazoles pour utilisation dans le traitement et la prévention de troubles neurodégénératifs - Google Patents

Pyrimidobenzimidazoles pour utilisation dans le traitement et la prévention de troubles neurodégénératifs Download PDF

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WO2017168137A1
WO2017168137A1 PCT/GB2017/050866 GB2017050866W WO2017168137A1 WO 2017168137 A1 WO2017168137 A1 WO 2017168137A1 GB 2017050866 W GB2017050866 W GB 2017050866W WO 2017168137 A1 WO2017168137 A1 WO 2017168137A1
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optionally substituted
compound
alkyl
halo
ring
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PCT/GB2017/050866
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Pavel Pavlov
Bengt Winblad
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Great Matter Pharma Ab
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    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to the treatment and prevention of neurodegenerative disorders.
  • it relates to the use of pyrimido[1 ,2-a]benzimidazoles, and analogues thereof, in the treatment of disorders such as Alzheimer's disease and other neurodegenerative disorders.
  • Neurodegeneration is a general term referring to conditions characterised by the progressive loss of structure or function of neurons, which may also be characterised by neuron death.
  • Many diseases are characterised by neurodegeneration, including well- known conditions such as Alzheimer's disease and Parkinson's disease. At present, such diseases are thought to be incurable, with the progressive worsening of neuron damage eventually leading to death of the patient.
  • NDDs Neurodegenerative diseases
  • CNS central nervous system
  • NDDs share many common characteristics. Targeting these similarities provides the potential for the development of treatments that could ameliorate many diseases simultaneously. For example, there are many parallels between different neurodegenerative disorders including atypical protein assemblies as well as induced cell death. Nonetheless, due to the complexity of the pathogenesis of NDDs, the identification of suitable therapeutic targets has provided troublesome and several aspects of the disease pathology have been investigated, such as the following. Mitochondrial dysfunction in NDD
  • the neuronal function is critically dependent on the energy supply provided by mitochondrial oxidative phosphorylation.
  • the mitochondrial dysfunction and oxidative stress are one of the earliest events that occur in brain as well as in peripheral tissues of NDD patients. Proteins implicated in various NDDs as causative factors were found to be physically associated with mitochondria resulting in impairment of mitochondrial function (see, for example, Anandatheerthavarada, H. K, et. a/., J. Cell Biol., 161 , 41-54 (2003), Pavlov, P.F. et. ai , FASEB J., 25, 78-88 (201 1), Devi, L. et al. , J Biol Chem., 283, 9089- 1000 (2008), Yano, H. et. ai , Nat Neurosci., 17, 822-831 (2014) and Liu, J. et al., Neuron, 43, 5-17 (2004)).
  • TOM outer membrane
  • Molecular chaperones Hsp70 and Hsp90, as well as specific factors in cytosol participate in targeting of newly synthesised proteins to mitochondrial TOM complex.
  • the specificity of Hsp70 and Hsp90 action is mediated by their co-chaperones and adaptor proteins.
  • TPR tetratricopeptide
  • NDD neurodegeneration
  • Molecular chaperones are primarily responsible for the maintenance of correct conformation of other protein molecules.
  • molecular chaperones are responsible for the intracellular protein transport, degradation of damaged or misfolded proteins.
  • An increasing body of evidence suggests an important role for Hsp70/Hsp90 chaperones in various neurodegenerative disorders, particularly in buffering the transition state between soluble and aggregated state of disease related proteins. This may explain the neuroprotective effect of molecular chaperone inhibitors in different models of neurodegeneration.
  • PP5 protein phosphatase 5
  • AD patients Liu F, et al., J Biol Chem., 280, 1790-1796 (2005).
  • PP5 resides in cytoplasm in an inactive form and is activated upon binding to Hsp90 (Yang J, et al., EMBO J., 12, 1-10 (2005)).
  • SGTA Small glutamine-rich TPR protein
  • Altered metal homeostasis contributes to the loss of neurons from a complex interplay of factors including oxidative injury, excitotoxic stimulation, dysfunction of critical proteins and mitochondrial failure. Attempts to modulate metal homeostasis in NDDs are an emerging therapeutic strategy. Series of 8-hydroxyquinoline analogues have shown the greatest potential for the treatment of several NDDs, including Alzheimer's disease and Parkinson's disease. Two of these, clioquinol and PBT2, have shown promising results in several phase 2 clinical trials for NDDs (see Adlard, P. A. et ai, Neuron., 59, 43-55 (2008)).
  • 8-hydroxyquinoline analogues are able to bind virtually all transition metals with high potency, providing a mechanism for potential undesirable side effects associated with depletion of essential trace metals.
  • the mechanism of toxicity of clioquinol was attributed to the depletion of vitamin B12 (cobalamin) (see Yassin, M.S. et al., J Neurol Sci., 173, 40-44 (2000)). Therefore chronic, potentially life-long treatment of NDDs would require more specific modulation of metal levels in the brain.
  • NDDs neurodegenerative diseases
  • these targets should allow for treatments that target a wide range of NDDs, safely and with low levels of side effects.
  • X represents NR 3 and the dashed bond by which it is bound represents a single bond
  • Y represents N and the dashed bond by which it is bound represents a double bond
  • X represents N and the dashed bond by which it is bound represents a double bond
  • Y represents NR 4 and the dashed bond by which it is bound represents a single bond
  • R 1 represents H, halo, -CN, -N0 2 , -OR a1 , -S(0) P R a2 , -N(R a3 )R a4 , -S(0) q N(R a5 )R a6 , -C(0)N(R a7 )R a8 , -OC(0)N(R a9 )R a1 °, -C(0)OR a11 , Ci-e alkyl optional
  • R 3 represents H, halo, -CN, -N0 2 , -OR b1 , -S(0) P R b2 , -N(R b3 )R b4 , -S(0) q N(R b5 )R b6 , -C(0)N(R b7 )R b8 , -OC(0)N(R b9 )R b1 °, -C(0)OR b11 , Ci-e alkyl optionally substituted with one or more R b12 , aryl optionally substituted with one or more G 5 or heteroaryl optionally substituted with one or more G 6 ; where present, R 4 represents H, halo, -CN, -N0 2 , -OR c1 , -S(0) P R c2 , -NCR ⁇ R 04 , -S(0) q N(R c5 )R c6 , -C(0)N(
  • R d3 and R d4 , R d5 and R d6 , R d7 and R d8 , and R d9 and R d1 ° may be linked together to form, together with the nitrogen atom to which they are attached, a 3- to 6-membered alkyl ring, which ring optionally contains one further heteroatom and which ring is optionally substituted by one or more E 3 ; each R a12 , R b12 , R c12 and R d12 independently represents oxy, halo, -CN, -N0 2 , -OR e1 , -SR e2 , -N(R e2 )R e3 , -C(0)OR e5 , aryl optionally substituted with one or more G 13 or heteroaryl optionally substituted with one or more G 14 ; each R e1 to R e5 independently represent Ci-e alkyl optionally substituted with one or more E 4 , aryl optionally substitute
  • a method of treating or preventing a neurodegenerative disorder comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula I (as described herein), or a pharmaceutically acceptable salt thereof.
  • references herein to compounds of particular aspects of the invention will include references to all embodiments and particular features thereof, which embodiments and particular features may be taken in combination to form further embodiments.
  • salts include acid addition salts and base addition salts.
  • Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of the invention with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of the invention in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
  • carboxylate salts e.g. formate, acetate, trifluoroacetate, propionate, isobutyrate, heptanoate, decanoate, caprate, caprylate, stearate, acrylate, caproate, propiolate, ascorbate, citrate, glucuronate, glutamate, glycolate, a-hydroxybutyrate, lactate, tartrate, phenylacetate, mandelate, phenylpropionate, phenylbutyrate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, dinitrobenzoate, o-acetoxybenzoate, salicylate, nicotinate, isonicotinate, cinnamate, oxalate, malonate, succinate, suberate, sebacate, fumarate, malate, maleate, hydroxymaleate, hippurate, phthalate or ter
  • sulphonate salts e.g. benzenesulphonate, methyl-, bromo- or chloro-benzenesulphonate, xylenesulphonate, methanesulphonate, ethanesulphonate, propanesulphonate, hydroxyethanesulphonate, 1- or 2- naphthalene-sulphonate or 1 ,5-naphthalenedisulphonate salts
  • base addition salts include salts formed with alkali metals (such as Na and K salts), alkaline earth metals (such as Mg and Ca salts), organic bases (such as ethanolamine, diethanolamine, triethanolamine, tromethamine and lysine) and inorganic bases (such as ammonia and aluminium hydroxide). More particularly, base addition salts that may be mentioned include Mg, Ca and, most particularly, K and Na salts.
  • compounds of the first aspect of the invention may exist as solids, and thus the scope of the invention includes all amorphous, crystalline and part crystalline forms thereof, and may also exist as oils. Where compounds of the first aspect of the invention exist in crystalline and part crystalline forms, such forms may include solvates, which are included in the scope of the invention. Compounds of the first aspect of the invention may also exist in solution.
  • Compounds of the first aspect of the invention may contain double bonds and may thus exist as E (entadel) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.
  • Compounds of the first aspect of the invention may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism.
  • Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation.
  • the various stereoisomers i.e. enantiomers
  • the desired optical isomers may be obtained from appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e.
  • a 'chiral pool' method by reaction of the appropriate starting material with a 'chiral auxiliary' which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution); for example, with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.
  • references to halo and/or halogen groups will each independently refer to fluoro, chloro, bromo and iodo (for example, fluoro (F) and chloro (CI)), such as flouro.
  • oxy groups may be present as substituents on any suitable carbon atom (i.e. a carbon atom able to form a double bond with the required oxygen atom).
  • Ci -Z alkyl groups (where z is the upper limit of the range) defined herein may be straight-chain or, when there is a sufficient number (i.e. a minimum of three) of carbon atoms, be branched-chain, and/or cyclic (so forming a C3- z -cycloalkyl group).
  • a sufficient number i.e. a minimum of four
  • such groups may also be part cyclic.
  • part cyclic alkyl groups may include cyclopropylmethyl and the like.
  • such groups may also be multicyclic (e.g.
  • alkyl groups may also be saturated or, when there is a sufficient number (i.e. a minimum of two) of carbon atoms, be unsaturated (forming, for example, a C2 alkenyl or a C2 alkynyl group).
  • references to polycyclic (e.g. bicyclic or tricyclic) groups e.g. when employed in the context of cycloalkyi groups
  • references to polycyclic (e.g. bicyclic or tricyclic) groups will refer to ring systems wherein at least two scissions would be required to convert such rings into a straight chain, with the minimum number of such scissions corresponding to the number of rings defined (e.g. the term bicyclic may indicate that a minimum of two scissions would be required to convert the rings into a straight chain).
  • bicyclic e.g.
  • aryl when employed in the context of alkyl groups may refer to groups in which the second ring of a two-ring system is formed between two adjacent atoms of the first ring, and may also refer to groups in which two non-adjacent atoms are linked by an alkylene group, which later groups may be referred to as bridged.
  • aryl includes references to Ce-14 (e.g. Ce-io) aromatic groups. Such groups may be monocyclic or bicyclic and, when bicyclic, be either wholly or partly aromatic.
  • Ce-io aryl groups that may be mentioned include phenyl, naphthyl, 1 ,2,3,4- tetrahydronaphthyl, indanyl, and the like (e.g.
  • aryl phenyl, naphthyl and the like, such as phenyl).
  • particular aryl groups that may be mentioned include phenyl.
  • the point of attachment of substituents on aryl groups may be via any carbon atom of the ring system.
  • heteroaryl includes references to 5- to 14- (e.g. 5- to 10-) membered heteroaromatic groups containing one or more heteroatoms selected from oxygen, nitrogen and/or sulphur.
  • Such heteroaryl groups may comprise one, two, or three rings, of which at least one is aromatic.
  • Substituents on heteroaryl/heteroaromatic groups may, where appropriate, be located on any atom in the ring system including a heteroatom.
  • the point of attachment of heteroaryl/heteroaromatic groups may be via any atom in the ring system including (where appropriate) a heteroatom.
  • Bicyclic heteroaryl/heteroaromatic groups may comprise a benzene ring fused to one or more further aromatic or non-aromatic heterocyclic rings, in which instances, the point of attachment of the polycyclic heteroaryl/heteroaromatic group may be via any ring including the benzene ring or the heteroaryl/heteroaromatic or heterocycloalkyl ring.
  • heteroaryl/heteroaromatic groups examples include pyridinyl, pyrrolyl, furanyl, thiophenyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, imidazolyl, imidazopyrimidinyl, imidazothiazolyl, thienothiophenyl, pyrimidinyl, furopyridinyl, indolyl, azaindolyl, pyrazinyl, pyrazolopyrimidinyl, indazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, quinazolinyl, benzofuranyl, benzothiophenyl, benzoimidazolyl, benzoxazolyl, benzothiazolyl, benzoxazoly
  • heteroaryl includes polycyclic (e.g. bicyclic) groups in which one ring is aromatic (and the other may or may not be aromatic).
  • heteroaryl groups that may be mentioned include e.g.
  • heteroaryl groups that may be mentioned include pyridinyl (e.g. pyridine-2-yl), thiadiazolyl (e.g. 1 ,3,4-thiadiazolyl, such as 1 ,3,4-thiadiazol-2-yl) and benzothiazolyl (e.g. 1 ,3-benzothiazolyl, such as 1 ,3-benzothiazol-2-yl).
  • pyridinyl e.g. pyridine-2-yl
  • thiadiazolyl e.g. 1 ,3,4-thiadiazolyl, such as 1 ,3,4-thiadiazol-2-yl
  • benzothiazolyl e.g. 1 ,3-benzothiazolyl, such as 1 ,3-benzothiazol-2-yl.
  • certain substituents may be joined, together with the atoms to which they are attached, to form rings alkyl rings having a particular number of ring members, which rings may necessary or
  • references to such rings as alkyl rings will indicate the components of the ring other than the relevant heteroatom(s) are alkyl.
  • all atoms linked so as to form the ring may be counted as a ring member.
  • heteroatoms will take their normal meaning as understood by one skilled in the art.
  • Particular heteroatoms that may be mentioned include phosphorus, selenium, tellurium, silicon, boron, oxygen, nitrogen and sulphur (e.g. oxygen, nitrogen and sulphur, such as oxygen and nitrogen).
  • the present invention also embraces isotopically-labelled compounds of the present invention which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (or the most abundant one found in nature). All isotopes of any particular atom or element as specified herein are contemplated within the scope of the compounds of the invention.
  • the compounds of the invention also include deuterated compounds, i.e. in which one or more hydrogen atoms are replaced by the hydrogen isotope deuterium.
  • compounds of the invention that are the subject of this invention include those that are stable. That is, compounds of the invention include those that are sufficiently robust to survive isolation, e.g. from a reaction mixture, to a useful degree of purity.
  • R 1 and R 2 each independently represent H, halo, -CN, -N0 2 , -OR a1 , -S(0) P R a2 , -N(R a3 )R a4 , -S(0) q N(R a5 )R a6 , -C(0)N(R a7 )R a8 , -OC(0)N(R a9 )R a1 °, -C(0)OR a11 , Ci-e alkyl optionally substituted with one or more R a12 , aryl optionally substituted with one or more G 1 or heteroaryl optionally substituted with one or more G 2 , or alternatively R 1 and R 2 may be linked together to form, together with the carbon atoms to which they are attached, aryl optionally substituted by one or more G 3 , heteroaryl substituted by one or more G 4 , or a 5- to 6-membered alkyl ring, which ring optionally contains one
  • R 1 and R 2 each independently represent H, halo, -CN, -N0 2 , -OR a1 , -S(0) P R a2 , -N(R a3 )R a4 , -S(0) q N(R a5 )R a6 , -C(0)N(R a7 )R a8 , -OC(0)N(R a9 )R a1 °, -C(0)OR a11 , Ci-e alkyl optionally substituted with one or more R a12 or aryl optionally substituted with one or more G 1 , or alternatively R 1 and R 2 may be linked together to form, together with the carbon atoms to which they are attached, aryl optionally substituted by one or more G 3 , heteroaryl substituted by one or more G 4 , or a 5- to 6-membered alkyl ring, which ring optionally contains one or two heteroatom
  • R 1 and R 2 each independently represent H, halo (e.g. fluoro), -OR a1 , -C(0)OR a1 ⁇ Ci -6 alkyl optionally substituted with one or more R a12 or aryl optionally substituted with one or more G ⁇ or alternatively R 1 and R 2 may be linked together to form, together with the carbon atoms to which they are attached, a 6-membered aryl (i.e. a phenyl ring) optionally substituted by one or more G 3 .
  • halo e.g. fluoro
  • R 1 and R 2 each independently represent H, halo (e.g. fluoro), -OR a1 , -C(0)OR a11 , Ci-e alkyl (e.g. Ci-5 alkyl) optionally substituted with one or more R a12 or aryl optionally substituted with one or more G 1 , or alternatively R 1 and R 2 may be linked together to form, together with the carbon atoms to which they are attached, a 6-membered aryl (i.e. a phenyl ring).
  • halo e.g. fluoro
  • -OR a1 e.g. Ci-5 alkyl
  • R a12 or aryl optionally substituted with one or more G 1
  • R 1 and R 2 may be linked together to form, together with the carbon atoms to which they are attached, a 6-membered aryl (i.e. a phenyl ring).
  • R a12 represents oxy, halo (e.g. fluoro), -CN, -OR e1 , -SR e2 , -C(0)OR e5 or aryl optionally substituted with one or more G 13 .
  • R 1 and R 2 each independently represent H, -OH, -C(0)OCi- 3 alkyl (e.g. -C(0)OCi- 2 alkyl), C1-6 alkyl (e.g.
  • R 1 and R 2 may be linked together to form, together with the carbon atoms to which they are attached, a 6-membered aryl (i.e. a phenyl ring);
  • R a12 represents oxy, halo (e.g. fluoro), -CN, -OR 91 , -SR e2 , -C(0)OR e5 or phenyl optionally substituted with one or more G 13 ;
  • R e1 represents C1-3 alkyl optionally substituted with one or more fluoro (e.g. methyl);
  • R e2 represents phenyl optionally substituted with one or more halo (e.g. chloro) or C1-3 alkyl (e.g. methyl) group, or heteroaryl optionally substituted with one or more halo (e.g. chloro) or C1-3 alkyl (e.g. methyl) group;
  • R e5 represents H or C1-3 alkyl (e.g. C1-2 alkyl, such as ethyl); and/or (e.g. and)
  • G 13 represents a halo (e.g. fluoro or chloro) or -OC1-3 alkyl (e.g. -OCH3) group.
  • R 1 and R 2 each independently represent H or C1-6 alkyl (e.g. C1-3 alkyl, such as Ci alkyl) optionally substituted with one or more R a12 ;
  • R a12 represents halo (e.g. fluoro) or -OR e1 ;
  • R e1 represents C1-3 alkyl optionally substituted with one or more fluoro (e.g. methyl). Particular R 1 groups that may be mentioned include H.
  • R 2 groups that may be mentioned include H, methyl and -CH2OCH3. More particular R 2 groups that may be mentioned include -CH2OCH3.
  • Particular compounds of formula I that may be mentioned include those in which X represents NR 3 and the dashed bond by which it is bound represents a single bond, and Y represents N and the dashed bond by which it is bound represents a double bond.
  • the compound of formula I is represented by a compound of formula la
  • R 1 to R 3 , Z and n are as defined herein.
  • R 3 represents H or Ci-e alkyl optionally substituted with one or more R b12 .
  • R 3 represents H or Ci-e alkyl optionally substituted with one or more R b12 .
  • R 3 represents H or Ci-e alkyl (e.g. C1-3 alkyl) optionally substituted with one or more R b12 ; and R b12 represents oxy, halo (e.g. fluoro), -CN or phenyl optionally substituted by one or more halo (e.g fluoro).
  • R b12 represents oxy, halo (e.g. fluoro), -CN or phenyl optionally substituted by one or more halo (e.g fluoro).
  • R 3 represents H or C1-3 alkyl optionally substituted with one or more R b12 ;
  • R b12 represents oxy, -CN or phenyl.
  • R 3 groups that may be mentioned include H.
  • Particular compounds of formula I include those in which X represents N and the dashed bond by which it is bound represents a double bond, and Y represents NR 4 and the dashed bond by which it is bound represents a single bond.
  • the compound of formula I is represented by a compound of formula lb
  • R 1 , R 2 , R 4 , Z and n are as defined herein.
  • R 4 represents Ci-e alkyi (e.g. C1-5 alkyi) optionally substituted with one or more R c12 .
  • R 4 represents C1-6 alkyi (e.g. C1-5 alkyi) optionally substituted with one or more R c12 ; and R c12 represents phenyl optionally substituted with one or more halo (e.g. fluoro) or C1-3 alkyi (Ci alkyi) optionally substituted with one or more halo (e.g. fluoro).
  • R c12 represents phenyl optionally substituted with one or more halo (e.g. fluoro) or C1-3 alkyi (Ci alkyi) optionally substituted with one or more halo (e.g. fluoro).
  • Z represents halo (e.g. fluoro) or C1-3 alkyi (e.g. methyl) optionally substituted with one or more halo (e.g. fluoro); and/or n represents 0 or 1.
  • halo e.g. fluoro
  • C1-3 alkyi e.g. methyl
  • n 0 or 1.
  • Z represents fluoro; and/or n represents 0 or 1.
  • n represents 0.
  • p represents 0.
  • all embodiments of the invention and particular features mentioned herein may be taken in isolation or in combination with any other embodiments and/or particular features mentioned herein (hence describing more particular embodiments and particular features as disclosed herein) without departing from the disclosure of the invention.
  • R 1 and R 2 each independently represent H, -OH, -C(0)OCi- 3 alkyl (e.g. -C(0)OCi- 2 alkyl), Ci-6 alkyl (e.g. C1-5 alkyl) optionally substituted with one or more R a12 or aryl optionally substituted with one or more G ⁇ or alternatively R 1 and R 2 may be linked together to form, together with the carbon atoms to which they are attached, a 6-membered aryl (i.e. a phenyl ring);
  • R a12 represents oxy, halo (e.g. fluoro), -CN, -OR 91 , -SR e2 , -C(0)OR e5 or phenyl optionally substituted with one or more G 13 ;
  • R e1 represents C1-3 alkyl optionally substituted with one or more fluoro (e.g. methyl);
  • R e2 represents phenyl optionally substituted with one or more halo (e.g. chloro) or C1-3 alkyl (e.g. methyl) group, or heteroaryl optionally substituted with one or more halo (e.g. chloro) or C1-3 alkyl (e.g. methyl) group;
  • R e5 represents H or C1-3 alkyl (e.g. C1-2 alkyl, such as ethyl);
  • G 13 represents a halo (e.g. fluoro or chloro) or -OC1-3 alkyl (e.g. -OCH3) group;
  • R 3 represents H or C1-6 alkyl (e.g. C1-3 alkyl) optionally substituted with one or more R b12 ;
  • R b12 represents oxy, halo (e.g. fluoro), -CN or phenyl optionally substituted by one or more halo (e.g fluoro);
  • R 4 represents C1-6 alkyl (e.g. C1-5 alkyl) optionally substituted with one or more R c12 ;
  • R c12 represents phenyl optionally substituted with one or more halo (e.g. fluoro) or C1-3 alkyl (Ci alkyl) optionally substituted with one or more halo (e.g. fluoro); Z represents halo (e.g. fluoro); and/or (e.g. and) n represents 0 or 1.
  • R 1 and R 2 each independently represent H or Ci-e alkyi (e.g. C1-3 alkyi, such as Ci alkyi) optionally substituted with one or more R a12 ;
  • R a12 represents halo (e.g. fluoro) or -OR e1 ;
  • R e1 represents C1-3 alkyi optionally substituted with one or more fluoro (e.g. methyl);
  • R 3 represents H or C1-3 alkyi optionally substituted with one or more R b6 ;
  • R b12 represents oxy, -CN or phenyl;
  • R 4 represents C1-6 alkyi (e.g. C1-5 alkyi) optionally substituted with one or more R c12 ;
  • R c12 represents phenyl optionally substituted with one or more halo (e.g. fluoro) or C1-3 alkyi (Ci alkyi) optionally substituted with one or more halo (e.g. fluoro); and/or (e.g. and) n represents 0.
  • halo e.g. fluoro
  • C1-3 alkyi Ci alkyi
  • n e.g. fluoro
  • X represents NR 3 and the dashed bond by which it is bound represents a single bond, and Y represents N and the dashed bond by which it is bound represents a double bond (i.e. the compound of formula I is a compound formula la);
  • R 1 and R 2 each independently represent H or C1-6 alkyi (e.g. C1-3 alkyi, such as Ci alkyi) optionally substituted with one or more R a12 ;
  • R a12 represents halo (e.g. fluoro) or -OR e1 ;
  • R e1 represents C1-3 alkyi optionally substituted with one or more fluoro (e.g. methyl); R 3 represents H; and n represents 0.
  • references to the treatment of a particular medical condition take their normal meanings in the field of medicine.
  • the term may refer to achieving a reduction in the severity of one or more clinical symptom associated with the relevant condition.
  • references to treating the condition may refer to achieving a reduction in cognitive decline, which may be measured using cognitive tests as known to those skilled in the art.
  • such treatment may also be measured through analysis of relevant biomarkers as known to those skilled in the art, such as through analysis of brain oxygen consumption using routine techniques (e.g. PET scanning).
  • relevant biomarkers as known to those skilled in the art, such as through analysis of brain oxygen consumption using routine techniques (e.g. PET scanning).
  • PET scanning routine techniques
  • references to the prevention of a particular condition take their normal meanings in the field of medicine.
  • the term may refer to achieving a reduction in the likelihood of a subject developing the condition (which may be observed as the development of one or more clinical symptom associated with the relevant condition), such as a reduction of at least 10% (e.g. at least 30%, such as at least 50%).
  • references to prevention may also be referred to as prophylaxis.
  • references to the treatment or prevention of a particular condition will refer in particular to the treatment of the condition.
  • references to treating or preventing a particular condition will refer in particular to treating the condition.
  • references to patients will refer to a living subject in which the relevant treatment or prevention (e.g. treatment) occurs, including mammalian (e.g. human) patients.
  • the treatment or prevention e.g. the treatment
  • the term therapeutically effective amount will refer to an amount of a compound that confers a therapeutic effect on the treated patient.
  • the effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of and/or feels an effect).
  • compounds of the invention may possess pharmacological activity as such, certain pharmaceutically-acceptable (e.g. "protected") derivatives of compounds of the invention may exist or be prepared which may not possess such activity, but may be administered (e.g. parenterally or orally) and thereafter be metabolised in the body to form compounds of the invention.
  • Such compounds (which may possess some pharmacological activity, provided that such activity is appreciably lower than that of the active compounds to which they are metabolised) may therefore be described as "prodrugs" of compounds of the invention.
  • references to prodrugs will include compounds that form a compound of the invention, in an experimentally-detectable amount, within a predetermined time, following enteral or parenteral administration (e.g. oral or parenteral administration). All prodrugs of the compounds of the first aspect of the invention are included within the scope of the invention.
  • compounds of the invention are useful because they possess pharmacological activity, and/or are metabolised in the body following oral or parenteral administration to form compounds that possess pharmacological activity.
  • compounds of the first aspect of the invention are useful in the treatment or prevention of neurodegenerative disorders, which term will be readily understood by one of skill in the art.
  • neurodegenertive disorder may refer to disorders (which may also be referred to as diseases, medical conditions, conditions, or the like) characterised by the progressive loss of structure or function of neurons, which may also be characterised by neuron death.
  • Neurodegenerative disorders that may be mentioned include Alzheimer's Disease and other dementias (such as fronto-temporal dementia and dementia with Lewy bodies), vascular dementia, traumatic brain injury, brain cancers, degenerative nerve diseases, encephalitis, epilepsy, genetic brain disorders, head and brain malformations, hydrocephalus, stroke, Parkinson's Disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS or Lou Gehrig's Disease), Huntington's Disease and prion diseases (such as Creutzfeld-Jacob disease (CJD)).
  • dementias such as fronto-temporal dementia and dementia with Lewy bodies
  • vascular dementia traumatic brain injury
  • brain cancers degenerative nerve diseases
  • encephalitis epilepsy
  • genetic brain disorders head and brain malformations
  • hydrocephalus stroke
  • Parkinson's Disease multiple sclerosis
  • MS amyotrophic lateral sclerosis
  • Huntington's Disease and prion diseases such as Creutzfeld-Jacob disease (CJD)
  • Particular neurodegenerative disorders include Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.
  • the neurodegenerative disorder is Alzheimer's disease.
  • Certain compounds of the invention as disclosed herein may be novel and/or not previously disclosed for use in medicine.
  • a compound as described in the first aspect of the invention (such as in any embodiment or combination of embodiments thereof) for use in medicine (or as a pharmaceutical).
  • a pharmaceutical composition comprising a compound as defined in first aspect of the invention, and optionally one or more pharmaceutically acceptable adjuvant, diluent and/or carrier, for use in the treatment or prevention of a neurodegenerative disorder (as described herein).
  • a method of treating or preventing a neurodegenerative disorder comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound as defined in first aspect of the invention, and optionally one or more pharmaceutically acceptable adjuvant, diluent and/or carrier.
  • compounds of the invention may act systemically and/or locally (i.e. at a particular site). In particular, compounds of the invention may act systemically.
  • compositions as described herein will normally be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, sublingually, intranasally, topically, by any other parenteral route or via inhalation, in a pharmaceutically acceptable dosage form.
  • Pharmaceutical compositions as described herein will include compositions in the form of tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for parenteral or intramuscular administration, and the like.
  • the pharmaceutical formulation is provided in a pharmaceutically acceptable dosage form, including tablets or capsules, liquid forms to be taken orally or by injection, suppositories, creams, gels, foams, inhalants (e.g. to be applied intranasally), or forms suitable for topical administration.
  • a pharmaceutically acceptable dosage form including tablets or capsules, liquid forms to be taken orally or by injection, suppositories, creams, gels, foams, inhalants (e.g. to be applied intranasally), or forms suitable for topical administration.
  • compounds of the invention may be present as a solid (e.g. a solid dispersion), liquid (e.g. in solution) or in other forms, such as in the form of micelles.
  • the compound in the preparation of pharmaceutical formulations for oral administration, may be mixed with solid, powdered ingredients such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives, gelatin, or another suitable ingredient, as well as with disintegrating agents and lubricating agents such as magnesium stearate, calcium stearate, sodium stearyl fumarate and polyethylene glycol waxes.
  • the mixture may then be processed into granules or compressed into tablets.
  • Soft gelatin capsules may be prepared with capsules containing one or more active compounds (e.g. compounds of the first and, therefore, second and third aspects of the invention, and optionally additional therapeutic agents), together with, for example, vegetable oil, fat, or other suitable vehicle for soft gelatin capsules.
  • active compounds e.g. compounds of the first and, therefore, second and third aspects of the invention, and optionally additional therapeutic agents
  • hard gelatine capsules may contain such compound(s) in combination with solid powdered ingredients such as lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives or gelatin.
  • Dosage units for rectal administration may be prepared (i) in the form of suppositories which contain the compound(s) mixed with a neutral fat base; (ii) in the form of a gelatin rectal capsule which contains the active substance in a mixture with a vegetable oil, paraffin oil, or other suitable vehicle for gelatin rectal capsules; (iii) in the form of a ready- made micro enema; or (iv) in the form of a dry micro enema formulation to be reconstituted in a suitable solvent just prior to administration.
  • Liquid preparations for oral administration may be prepared in the form of syrups or suspensions, e.g. solutions or suspensions, containing the compound(s) and the remainder of the formulation consisting of sugar or sugar alcohols, and a mixture of ethanol, water, glycerol, propylene glycol and polyethylene glycol. If desired, such liquid preparations may contain colouring agents, flavouring agents, saccharine and carboxymethyl cellulose or other thickening agent.
  • Liquid preparations for oral administration may also be prepared in the form of a dry powder to be reconstituted with a suitable solvent prior to use. Solutions for parenteral administration may be prepared as a solution of the compound(s) in a pharmaceutically acceptable solvent.
  • solutions may also contain stabilizing ingredients and/or buffering ingredients and are dispensed into unit doses in the form of ampoules or vials.
  • Solutions for parenteral administration may also be prepared as a dry preparation to be reconstituted with a suitable solvent extemporaneously before use.
  • compositions as described in the fourth aspect of the invention may be administered at varying doses, with suitable doses being readily determined by one of skill in the art.
  • Oral, pulmonary and topical dosages may range from between about 0.01 mg/kg of body weight per day (mg/kg/day) to about 200 mg/kg/day, preferably about 0.01 to about 10 mg/kg/day, and more preferably about 0.1 to about 5.0 mg/kg/day.
  • treatment with such compounds and compositions may comprise administration of a compositions typically containing between about 0.01 mg to about 2000 mg, for example between about 0.1 mg to about 500 mg, or between 1 mg to about 100 mg, of the active ingredient.
  • a compositions typically containing between about 0.01 mg to about 2000 mg, for example between about 0.1 mg to about 500 mg, or between 1 mg to about 100 mg, of the active ingredient.
  • the most preferred doses will range from about 0.001 to about 10 mg/kg/hour during constant rate infusion.
  • treatment may comprise administration of such compounds and compositions in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily (with reference to the doses described herein).
  • the skilled person e.g.
  • the physician will be able to determine the actual dosage which will be most suitable for an individual patient, which is likely to vary with the route of administration, the type and severity of the condition that is to be treated, as well as the species, age, weight, sex, renal function, hepatic function and response of the particular patient to be treated.
  • the above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • treatment with compounds of the invention may further comprise (i.e. be combined with) further (i.e. additional/other) treatment(s) for the same condition.
  • treatment with compounds of the invention may be combined with other means for the treatment or prevention of neurodegenerative disorders (as defined herein).
  • the treatment may also comprise treatment with one or more additional therapeutic agent suitable for the treatment or prevention of a neurodegenerative disorder (as defined herein).
  • the pharmaceutical composition may further comprise one or more additional therapeutic agent suitable for the treatment or prevention of a neurodegenerative disorder (as defined herein).
  • pharmaceutical compositions comprising compounds of the invention and one or more additional therapeutic agent suitable for the treatment or prevention of a neurodegenerative disorder may be novel.
  • a pharmaceutical composition comprising a compound as defined in first aspect of the invention, and optionally one or more pharmaceutically acceptable adjuvant, diluent and/or carrier, and an additional therapeutic agent suitable for the treatment or prevention of a neurodegenerative disorder (as defined herein).
  • an additional therapeutic agent suitable for the treatment or prevention of a neurodegenerative disorder (as defined herein).
  • combinations of therapeutic agents may also described as a combination product and/or provided as a kit-of-parts.
  • a combination product comprising: (A) a compound as defined in the first aspect of the invention.
  • kits-of-parts comprising:
  • one or more other therapeutic agent suitable for the treatment or prevention of a neurodegenerative disorder (as defined herein), optionally in admixture with one or more pharmaceutically-acceptable adjuvant, diluent or carrier,
  • components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other.
  • references to an additional therapeutic agent suitable for the treatment or prevention of a neurodegenerative disorder will refer to a therapeutic agent that is suitable for the treatment or prevention of the neurodegenerative disorder for which the compound or composition is for use in treating or preventing (or the neurodegenerative disorder for which the compound or formulation is to be used in a method of treating or preventing).
  • the additional therapeutic agent may be a therapeutic agent that is suitable for treating or preventing (e.g. treating) Alzheimer's disease, as known to those skilled in the art, such as acetylcholinesterase inhibitors (e.g. tacrine, rivastigmine, galantamine and donepezil) and NMDA receptor antagonists (e.g. memantine).
  • acetylcholinesterase inhibitors e.g. tacrine, rivastigmine, galantamine and donepezil
  • NMDA receptor antagonists e.g. memantine
  • compositions/formulations, combination products and kits as described herein may be prepared in accordance with standard and/or accepted pharmaceutical practice.
  • a process for the preparation of a pharmaceutical composition/formulation as hereinbefore defined, which process comprises bringing into association a compound of the invention, as hereinbefore defined, with one or more pharmaceutically-acceptable adjuvant, diluent or carrier.
  • a process for the preparation of a combination product or kit-of-parts as hereinbefore defined comprises bringing into association a compound of the invention, as hereinbefore defined, or a pharmaceutically acceptable salt thereof with the other therapeutic agent that is useful in the treatment or prevention of a neurodegenerative disorder (as defined herein), and at least one pharmaceutically-acceptable adjuvant, diluent or carrier.
  • references to bringing into association will mean that the two components are rendered suitable for administration in conjunction with each other.
  • the two components of the kit of parts may be:
  • compounds of the invention may be prepared in accordance with, or by analogy to, the techniques described in US 4,072,679 (the contents of which are incorporated herein by reference) and/or by conventional synthetic procedures, in accordance with standard techniques, from available starting materials using appropriate reagents and reaction conditions.
  • the skilled person may refer to inter alia the general synthetic techniques described in: "Comprehensive Organic Synthesis” by B. M. Trost and I. Fleming, Pergamon Press, 1991 ; “Comprehensive Organic Functional Group Transformations” by A. R. Katritzky, O. Meth-Cohn and C. W. Rees, Pergamon Press, 1995; and/or “Comprehensive Organic Transformations” by R. C. Larock, Wiley- VCH, 1999.
  • compounds of the invention are thought to be particularly useful in the treatment or prevention of neurodegenerative disorders, and so may be useful in the preparation of pharmaceutical formulations for treating or preventing such disorders. In doing so, compounds of the invention may be more effective and/or deliver low levels of side effects than compounds of the prior art known for the same use.
  • compounds of the invention as described herein are thought to be useful in the treatment or prevention of neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (AMS), as they may inhibit interactions between recombinant human Tom34 and the C- terminal amino acid residues of human Hsp90 protein, which may in turn act to reduce accumulation of amyloid precursor protein (APP) inside mitochondria, or other proteins shown to be mis-targeted to mitochondria (for example, alpha-synuclein or superoxide dismutase (SOD1)).
  • compounds of the invention may act to bind transition metals, particularly Cu 2+ and Zn 2+ , which in turn may restore Cu 2+ inhibited ⁇ 4 2 fibrillization.
  • Figure 1A represents a dot-blot assay as described in Example 2.
  • Figure 1 B represents a concentration-dependent inhibition of Tom34-C90-HRP interactions as described Example 3.
  • Figure 2A represents results of co-immunoprecipitation assay of SH-SY5Y cell lysate as described in Example 4.
  • Figure 2B represents immunostaining results of fractionated SH-SY5Y cells with 22c1 1 antibodies as described in Example 5.
  • FIG. 3A schematically presents copper/zinc binding site on pyrimido[1 ,2-a]benzimidazol- 4(1 H)-one (Compound 4, also referred to herein as GMP-1), as described below, as described in Example 6
  • Figure 3B represents absorbance scan of Compound 4, also referred to herein as GMP- 1 , as described in Example 6.
  • Figure 3C represents pH-dependent binding of Compound 4, also referred to herein as GMP-1 , as described in Example 6.
  • Figure 4A represents SDS-PAGE results of ⁇ -casein cleavage as described in Example 7.
  • Figure 4B presents cytochrome oxidase activity measurement in isolated mouse brain mitochondria as described in Example 8.
  • Figure 4C represents time course of thioflavin T fluorescence measurement of ⁇ 4 2 as described in Example 9.
  • Figure 5 provides the results of MTT viability assay of SH-SY5Y cells as described in Example 10.
  • Figure 6A represents results of mobility assay of transgenic drosophila flies as described in Example 11.
  • Figure 6B represents results of a viability assay using flies expressing in the neurons dimer ⁇ 4 2 peptide connected via linker of 12 amino acids, as described in Example 12.
  • Figure 7 A represents results of open field test with transgenic AD model (5xFAD) mice as described in Example 13.
  • Figure 7B represents results of a contextual fear conditioning test as described in Example 14.
  • Figure 8A represents immunohistochemical results of brain cortex and hippocampus sections stained with antibodies directed to ⁇ 4 2 (6E10), as described in Example 15.
  • Figure 8B represents results of western blot analysis of total brain extract or purified mitochondria with 6E10 antibody, as described in Example 16.
  • Figure 9A represents immunohistochemical results of brain cortex and hippocampus sections stained with antibodies directed GFAP protein, as described in Example 17.
  • Figure 9B represents immunohistochemical results of brain cortex and hippocampus sections stained with antibodies directed CD1 1 b protein, as described in Example 18.
  • Figure 10 represents analysis of cytochrome oxidase activity in purified mouse brain mitochondria, as described in Example 19.
  • Figure 1 1 represents survival analysis of transgenic drosophila flies expressing the mutant form of human FUS protein, as described in Example 20.
  • TBS-T Tris-buffered saline with 0.1 % Tween-20
  • ⁇ 4 2 peptide was purchased from Bachem AG, Switzerland.
  • Peptide corresponding to the 10 utmost C-terminal amino acids of human Hsp90 was obtained from GL Biochem Ltd, Shanghai, China.
  • EZ-link Plus activated peroxidase kit was obtained from Pierce Thermo Scientific Inc.
  • Peptide coupling to activated horse-radish peroxidase (C90-HRP) was performed according to manufacturer ' s protocol.
  • EST clones encoding human Tom34 and Tom70 proteins were obtained from I.M.A.G.E. consortium distributors Source Bioscience, Cambridge, UK. Open reading frame of the Tom34 and Tom70 was PCR amplified and sub-cloned into pGEX-6 plasmid (GE Healthcare, Uppsala, Sweden).
  • Plasmid was further transformed into BL21 E.coli strain and protein expression was induced by addition of 1 mM IPTG for 2 h. Proteins were purified using Gluthatione Sepharose, GE Healthcare, Uppsala, Sweden. For dot-blot experiments (e.g. in Example 2 below), 1 ⁇ g of protein was applied on nitrocellulose membrane (Whatman, Madestone, UK), followed by incubation of membrane in TBS-T buffer supplemented with 5 % milk powder for 30 min, 25 °C. Indicated amounts of Pyrimido[1 ,2-a]benzimidazol-4(1 H)-one (GMP-1) or its derivatives were added from DMSO stock solution and incubation continued for another 30 min.
  • GMP-1 Pyrimido[1 ,2-a]benzimidazol-4(1 H)-one
  • C90-HRP 1 :200 times dilution was added to the membrane for 1 h 25 °C.
  • the membrane was subsequently washed 3 times with TBS-T for 10 min, 25 °C and signals were detected using SuperSignal West Pico enhanced chemiluminescence system (ThermoScientific, Rockford IL, USA). Analysis and quantification was performed using a digital imaging camera (Bio-Rad) with QuantityOne software.
  • SH-SY5Y human neuroblastoma cells were obtained from the American Tissue Culture Collection, ATCC and maintained in DMEM supplemented with 10% FBS and 1 % penicillin-streptomycin (Gibco/lnvitrogen, Carlsbad, CA, USA). Cells were cultured in 5% CO2 -95% air at 37 °C. Sub-cellular fractionation was performed as described in (Pavlov PF et al, 201 1). For viability assays (e.g. in Example 5 below), cellular toxicity was assessed with MTT Cell Proliferation Kit I, Roche Applied Science, Indianapolis, IN, USA according the manufacturer ' s protocol. Indicated amounts of GMP-1 or its derivatives were added from DMSO stock solution directly to the culture media and incubated for 12 h.
  • Brain mitochondria were isolated (e.g. for use in western blot analysis, such as in Example 16 below) using differential centrifugation according previously published protocol (Devi, L, Prabhu, B.M., Galati, D. F., Avadhani, N. G. and Anandatheerthavarada, H. K., J Neurosci., 26, 9057-9068 (2006)).
  • Antibodies used in the studies were: 22c11 (MAB348) from Millipore, Temecula, CA, USA, Tom40 (Sc-1 1414) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-APP and amyloid beta (6E10) from Covance, USA, CD11 b (ab75476) and GFAP (ab7260) were from Abeam. Immunoprecipitation was performed from SH-SY5Y cells (0.2 mg of protein) treated with DMSO or 50 ⁇ of GMP-1 for 12 h. Cells were scraped from the surface in the presence of TBS with 0.2 % Triton X-100 and protease inhibitor cocktail Roche Applied Science, Indianapolis, IN, USA.
  • compound absorbance spectra were measured with NanoDrop ND-100 spectrophotometer (Saveen).
  • compound 4 also referred to herein as GMP-1
  • GMP-1 was used (0.5 mM in 0.1 M MOPS-KOH pH 7.2).
  • MOPS-KOH buffers of various pH from 5.0 to 8.0 were used.
  • Compound 4 also referred to herein as GMP-1 , at equimolar amounts in 0.1 M MOPS-KOH pH 7.5: CuCI 2 , ZnS0 4 , AlC , NiS0 4 , MnCI 2 , CoCI 2 , FeCI 2 , FeC , Pb(N03) 2 , CaCI 2 , MgCI 2 and absorbance spectra without and with salts were compared.
  • proteolysis of ⁇ -casein with collagenase was performed as following: 0.02 mg/ml of collagenase was incubated with 0.1 % DMSO or with 50 ⁇ of EDTA, 50 ⁇ compound or 50 ⁇ of clioquinol for 30 min, 4 °C.
  • ⁇ -casein was added from stock solution of 10 mg/ml to final concentration of 1 mg/ml and incubated for 10 min, 4°C. 2 x SDS sample buffer was added and immediately boiled for 5 min and loaded on SDS-PAGE. Gels were subsequently stained with Coumassie Brilliant Blue R-250 and photographed.
  • Cytochrome oxidase activity was measured (e.g. in Example 8 and 19 below) by decrease in absorbance of ferrocytochrome c at 550 nm.
  • Isolated mouse brain mitochondria (0.5 mg/ml) in the buffer containing 10 mM Tris-HCI, pH 7.0, 0.5 M sucrose, 0,05 % Triton X- 100 were incubated with 0.1 % DMSO alone, 50 ⁇ of clioquinol or 50 ⁇ of GMP-1 for 2 h, 4°C. After addition of ferrocytochrome c ratios of decrease in 550 nm absorbance were immediately measured. Experiments were performed in triplicate.
  • ⁇ 42 monomer was isolated by size exclusion chromatography over a Superdex 75 column (GE Healthcare) in 20 mM sodium phosphate, 200 ⁇ EDTA, 0.02% NaN 3 at pH 8 or 20 mM Tris-HCI pH 7.5 and kept on ice. Every sample was supplemented with 10 ⁇ ThT from a 1 mM stock solution. 5 ⁇ of ⁇ 4 2 was incubated with 5 ⁇ of CuCI 2 for 4 h at 25°C followed by addition of 0.01 % of DMSO alone, 5 ⁇ of clioquinol alone or 5 ⁇ of compound. Fluorescence was measured every 5 min during 16 h. Standard deviation of four measurements shown.
  • fly lines containing single and double copies of a signal-peptide- ⁇ transgene were generated as described (Crowther, D. C. et al., Neuroscience, 132, 123-135 (2005)).
  • the fly line expressing in the neurons dimer ⁇ 4 2 peptide connected via a linker of 12 amino acids was generated as described (Speretta, E. et al., 287, 20748-20754 (2012)).
  • fly assays For fly assays (e.g. in Examples 1 1 and 12 below), flies were maintained on the standard food containing 1 % Agar, 8 % Brewer ' s yeast, 8 % fructose, 5 % potato dry powder, 0.05 % Nipagin, 0.1 % ascorbate. Indicated amounts of compound were added directly to the food during solidification.
  • the fly mobility assay represents the percentage of flies that able to cross the line at 8 cm from the bottom of test tube in 10 seconds.
  • the survival assay in flies expressing dimer ⁇ 4 2 in the neurons was calculated as percentage of adult flies carrying the transgene, plain wings phenotype, to the total number of hatched flies with plain + curly wings phenotype.
  • Transgenic flies expressing the mutant form of human FUS protein were generated as described by (Lanson, N. A. Jr., et ai, Hum Mol Genet, 20, 2510-2523 (201 1)). Survival analysis of transgenic drosophila flies expressing the mutant form of human FUS protein (e.g. in Example 20 below) was performed in the presence of various amounts of Compound 4, also referred to herein as GMP-1.
  • mice Experiments with 5xFAD transgenic mice were performed at QPS CRO facility in Graz, Austria. Four groups of 15 animals were used: tg mice having 0.5 % DMSO in their drinking water ad libitum as placebo; tg mice that received 16.7 mg/kg of compound in the drinking water from 3 weeks of age until the end of experiment at 6 months of age; tg mice that received 16.7 mg/kg of compound in the drinking water at the age of 5.5 weeks during 2 weeks; non-tg littermates receiving 0.5 % DMSO in the drinking water.
  • Open Field test e.g. in Example 13 below
  • a Plexiglas Open Field 48x48 cm; TSE-System®
  • the infrared photo beams were placed in a 1.4 cm distance around the box. Each test session lasted for 5 minutes to check the mouse's behaviour in the new surroundings. Testing was performed under standard room lighting conditions during the light phase of the circadian cycle.
  • the Contextual Fear Conditioning test (e.g. in Example 14 below) was conducted in an automated box provided by TSE-Systems, Germany. Mice were trained and tested on 2 consecutive days. On the training day, mice received a foot shock (0.5 mA, 2 s) 5 seconds after being placed into the conditioning chamber. 30 seconds afterwards they were returned to their home cage again. 24 hours after training, mice were tested by being returned to the conditioning chamber for 5 minutes without any shock, and freezing behaviour was recorded by the automated system and evaluated separately every minute. Freezing is defined as lack of movement except that required for respiration and is expressed as freezing time in percent of the testing time.
  • Histological examination was performed with sagittal cryosections (10 ⁇ thickness) which have been prepared from fixed frozen hemibrains.
  • the right hemisphere of each mouse was systematically and uniformly sectioned at 12 mediolateral levels (collecting 10 sections per level and discarding the next 20 sections) on a Leica CM 3050S cryotome. Collection of sections started with a random section at approximately 0.2 mm lateral from midline and extended to approximately 3.6 mm lateral (based on the Mouse Brain Atlas). Sections were stored at -20 °C until used in histological stainings. In order to analyze different features of histopathology the following targets were chosen for immunofluorescent labeling and quantitative analysis (e.g.
  • astroglia GFAP
  • activated microglia CD1 1 b
  • ⁇ -amyloid 6E10
  • All measurements except region size are threshold based, thus objects above certain intensity and above a certain size are automatically detected by ImageProPlus software (v6.2).
  • the measurements are done within an area of interest (AOI), which is manually delineated for each slice and each brain region. Using this AOI, the size of the specific brain area is determined. Values of five slices per animal deriving from five different medio-sagittal levels were averaged to an individual mean; group values were calculated using the individual means. Data were tested for normality using a Kolmogorov Smirnov test; differences between groups were calculated by one-way ANOVA followed by a Newman Keuls post hoc test, the alpha-error set to 0.05.
  • example compounds described herein may be named and/or represented as tautomeric forms of compounds of formula I (which compounds may be renamed and/or redrawn in the corresponding tautomeric form as required for compounds of formula I).
  • FIG. 2B shows immunostaining results of fractionated SH-SY5Y cells with 22c1 1 antibodies, as performed in accordance with the general experimental procedures described above. Signal quantification in the mitochondrial and light membrane fractions treated either with DMSO only or with Compound 4, also referred to herein as GMP-1 , from three independent experiments is presented.
  • Figure 3A schematically presents the putative copper/zinc binding site on a compound of formula I as described herein.
  • Figure 3C represents pH-dependent binding of Cu 2 7Zn 2+ to Compound 4, also referred to herein as GMP-1 , calculated as percentage of light absorbance at 330 nm. It was noted that binding is sharply decreased at pH ⁇ 6.5 with 50 % binding at pH 6.3.
  • Example 7 An SDS-PAGE analysis of ⁇ -casein cleavage with collagenase in the absence or presence of various metal chelators was performed in accordance with the general experimental procedures described herein, as follows:
  • Lane 1 ⁇ -casein alone, lanes 2-5 ⁇ -casein cleavage with collagenase;
  • Lane 2 no chelator addition
  • lane 3 collagenase preincubated with 50 ⁇ EDTA for 10 min
  • lane 4 collagenase preincubated with 50 ⁇ of Compound 4, also referred to herein as GMP-1 , for 10 min
  • lane 5 collagenase preincubated with 50 ⁇ clioquinol for 10 min.
  • cytochrome oxidase activity was measured in isolated mouse brain mitochondria incubated with DMSO only or with 50 ⁇ clioquinol and 50 ⁇ of Compound 4, also referred to herein as GMP-1.
  • Figure 4C represents time course of thioflavin T fluorescence measurement of ⁇ 4 2 (3 ⁇ ) fibrillization in the presence of Cu2+ alone (5 ⁇ ) or together with clioquinol (5 ⁇ ) or Compound 4, also referred to herein as GMP-1 (5 ⁇ ), as analysed in accordance with the general experimental procedures described herein above.
  • ⁇ 4 2 and Compound 4, also referred to herein as GMP-1 were added to the culture medium of differentiated SH-SY5Y cells and toxicity was monitored after an incubation period of 48 h using an MTT assay.
  • Example 12 Figure 6B represents results of a viability assay using flies expressing in the neurons dimer ⁇ 4 2 peptide connected via linker of 12 amino acids (seesperetta, E. et al., J Biol Chem., 287, 20748-20754 (2012)), which is more toxic variant of ⁇ 4 2, in accordance with the general experimental procedures described herein above. These flies exhibit temperature-dependent survival deficit.
  • Example 14 Figure 7B represents the results of contextual fear conditioning test using 6 months old transgenic mice and control non-transgenic mice, following the general experimental procedures described herein above.
  • Transgenic mice were treated with DMSO only or with 16.7 mg/kg of Compound 4, also referred to herein as GMP-1 , in the drinking water for 5 months or for 2 weeks prior the test. 15 animals in each group were used in the study.
  • Compound 4 also referred to herein as GMP-1
  • FIG. 8A represents immunohistochemical results of brain cortex and hippocampus sections stained with antibodies directed to ⁇ 4 2 (6E10), in accordance with the general experimental procedures described herein above, as follows:
  • Graph A represents the ⁇ plaque area of tg animals treated with Compound 4, also referred to herein as GMP-1 , for 2 weeks prior the test;
  • Graph B represents the ⁇ plaque area of tg animals treated with DMSO only
  • Graph C represents the ⁇ plaque area of tg animals treated with Compound 4, also referred to herein as GMP-1 , for 5 month prior the test;
  • Graph D represents the ⁇ plaque area of non-tg animals. Four animals in each group were analysed.
  • Figure 8B provides the results of a western blot analysis of total brain extract or purified mitochondria with 6E10 antibody, in accordance with the general experimental procedures described herein.
  • Tg mice were treated either with DMSO only or with Compound 4, also referred to herein as GMP-1 , for 5 months prior to analysis. Six animals in each group were used.
  • Example 17
  • Figure 9A represents immunohistochemical results of brain cortex and hippocampus sections stained with antibodies directed GFAP protein, a marker for astrocytosis, in accordance with the general experimental procedures described herein above, as follows:
  • Graph A represents the ⁇ plaque area of tg animals treated with Compound 4, also referred to herein as GMP-1 , for 2 weeks prior the test;
  • Graph B represents the ⁇ plaque area of tg animals treated with DMSO only
  • Graph C represents the ⁇ plaque area of tg animals treated with Compound 4, also referred to herein as GMP-1 , for 5 months prior the test;
  • Graph D represents the ⁇ plaque area of non-tg animals. Four animals in each group were analysed.
  • Figure 9B represents immunohistochemical results of brain cortex and hippocampus sections stained with antibodies directed CD11 b protein, a marker for microglia activation, in accordance with the general experimental procedures described herein, as follows:
  • Graph A represents the ⁇ plaque area of tg animals treated with Compound 4, also referred to herein as GMP-1 , for 2 weeks prior the test;
  • Graph B represents the ⁇ plaque area of tg animals treated with DMSO only
  • Graph C represents the ⁇ plaque area of tg animals treated with Compound 4, also referred to herein as GMP-1 , for 5 months prior to the test;
  • Graph D represents the ⁇ plaque area of non-tg animals.
  • cytochrome oxidase activity in purified brain mitochondria from non-tg mice and 5XFAD mice was performed: 5xFAD mice treated with DMSO only or 5xFAD mice treated with 16.7 mg/kg of Compound 4, also referred to herein as GMP-1 , in the drinking water for 5 months. Six animals in each group were analysed.

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Abstract

L'invention concerne un composé de formule I (I) ou un tautomère et/ou un sel de qualité pharmaceutique de celui-ci, pour utilisation dans le traitement ou la prévention d'un trouble neurodégénératif, formule dans laquelle X, Y, Z, R1, R2 et n sont tels que définis dans la description.
PCT/GB2017/050866 2016-03-29 2017-03-28 Pyrimidobenzimidazoles pour utilisation dans le traitement et la prévention de troubles neurodégénératifs WO2017168137A1 (fr)

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CN108690027A (zh) * 2018-06-15 2018-10-23 广州大学 一种2-二氟亚甲基取代嘧啶并[1,2-a]苯并咪唑化合物及其制备和应用
WO2020227576A1 (fr) 2019-05-09 2020-11-12 Genentech, Inc. Synthèse régio-sélective d'imidazo[1,2-a]pyrimidines
US20210128561A1 (en) * 2019-10-31 2021-05-06 Rheinische-Friedrich-Wilhelms-Universität Bonn MRGX Receptor Antagonists
WO2022111473A1 (fr) * 2020-11-26 2022-06-02 Bioardis Llc Composés utilisés en tant qu'antagonistes de mrgprx2

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108250202A (zh) * 2018-01-15 2018-07-06 陕西师范大学 2,3-二取代苯并咪唑并[1,2-a]嘧啶类化合物及其制备方法和应用
CN108690027A (zh) * 2018-06-15 2018-10-23 广州大学 一种2-二氟亚甲基取代嘧啶并[1,2-a]苯并咪唑化合物及其制备和应用
WO2020227576A1 (fr) 2019-05-09 2020-11-12 Genentech, Inc. Synthèse régio-sélective d'imidazo[1,2-a]pyrimidines
US20210128561A1 (en) * 2019-10-31 2021-05-06 Rheinische-Friedrich-Wilhelms-Universität Bonn MRGX Receptor Antagonists
WO2022111473A1 (fr) * 2020-11-26 2022-06-02 Bioardis Llc Composés utilisés en tant qu'antagonistes de mrgprx2

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