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WO2015069752A1 - Acetylcholine binding protein ligands, cooperative nachr modulators and methods for making and using - Google Patents

Acetylcholine binding protein ligands, cooperative nachr modulators and methods for making and using Download PDF

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Publication number
WO2015069752A1
WO2015069752A1 PCT/US2014/064104 US2014064104W WO2015069752A1 WO 2015069752 A1 WO2015069752 A1 WO 2015069752A1 US 2014064104 W US2014064104 W US 2014064104W WO 2015069752 A1 WO2015069752 A1 WO 2015069752A1
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Prior art keywords
substituted
optionally
unsubstituted
group
nachr
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PCT/US2014/064104
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French (fr)
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WO2015069752A9 (en
Inventor
Palmer Taylor
Katarzyna Kaczanowska
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The Regents Of The University Of California
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Publication of WO2015069752A1 publication Critical patent/WO2015069752A1/en
Publication of WO2015069752A9 publication Critical patent/WO2015069752A9/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic 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
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic 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
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/53751,4-Oxazines, e.g. morpholine
    • A61K31/53771,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/14Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16Anti-Parkinson drugs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/18Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D239/00Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/32One oxygen, sulfur or nitrogen atom
    • C07D239/42One nitrogen atom
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D239/00Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/48Two nitrogen atoms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D401/00Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D401/00Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D401/00Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D405/00Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D405/00Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D409/00Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • This invention relates to a family of compounds that interact with the
  • acetylcholine binding protein and the nicotinic acetylcholine receptor (nAChR) at the subunit interfaces in a cooperative manner. Either through their pre-synaptic or post- 20 synaptic actions, they modulate the release of acetylcholine or importantly other
  • neurotransmitters include the excitatory amino acids such as glutamate or aspartate; the inhibitor amino acids, such as ⁇ -aminobutyric acid (GABA) and glycine;
  • bigenic amines such as norepinephrine, epinephrine, dopamine and serotonin.
  • the invention provides
  • compositions responsive to modulating, decreasing or increasing the activity of an nAChR in the CNS or brain do not resemble acetylcholine structurally, but rather display an unusual cooperative binding interaction involving multiple sites on this pentameric protein. If the pentameric protein is composed of identical subunits, exemplary compounds of the invention can facilitate or inhibit the binding of acetylcholine. If the subunits are not identical, exemplary compounds of the invention can affect acetylcholine binding and stimulation in an allosteric manner.
  • the invention provides compositions and methods for treating, ameliorating, preventing or lessening the symptoms of an tobacco or nicotine-related addiction, e.g., thereby promoting smoking cessation, or a substance abuse, by, e.g., preventing the abstinence syndrome and addiction in two ways; (a) serving as a partial agonist alternative to nicotine; and/or, (b) ameliorating the symptoms of withdrawal and abstinence through their actions on the dopamine and other neurotransmitter systems.
  • the invention provides compositions and methods for treating, ameliorating, preventing or lessening the symptoms of disorders of development of the central nervous system, such as autism, schizophrenia, and other psychosis.
  • disorders of aging such as Alzheimer's diseases Parkinson's disease or psychoses.
  • the invention provides compositions and methods for treating, ameliorating, preventing or lessening the symptoms of disorders of the peripheral nervous system with its sensory afferent and efferent pathways, including treating, ameliorating, preventing or lessening acute and chronic pain, and providing pain relief, including allodynia, including movement, thermal or mechanical allodynia.
  • the invention provides compositions and methods for treating, ameliorating, preventing or lessening the symptoms of any condition responsive to an increase in levels or activity of dopamine in the CNS or brain, or any disease or condition responsive to the modulation of, or a decrease or an increase in the activity of nAChR.
  • AChBP acetylcholine binding protein
  • compounds of the invention also can modulate GABA, glycine and 5-hydroxytryptamine receptors in the CNS.
  • Nicotinic acetylcholine receptors are pentamers, which means that they are formed by five units that can be identical (or homomeric) or have different structures (or be heteromeric). Some proteins that are composed of more than one unit, and therefore have multiple binding sites, exhibit so called
  • nAChR neuropeptide
  • nicotine increases the release of dopamine in the brain, a neurotransmitter that is responsible for feelings of pleasure. In theory, without the nicotine-induced elevation of dopamine levels, tobacco would not produce this type of reward.
  • nAChR nicotinic acetylcholine receptor
  • AChBP acetylcholine binding protein
  • the invention provides compounds that
  • nAChR nicotinic acetylcholine receptor
  • the invention provides methods for formulating or using these compounds to increase or stimulate an nAChR functional response, e.g., to increase dopamine levels or activity.
  • the invention provides compounds,
  • compositions or formulations comprising:
  • Rl, R2, R3 and R4 are independently selected from the group consisting of: a hydrogen, an aryl (wherein optionally the aryl is any 5-or 6-membered ring, or is selected from the group consisting of: a heteroaryl, an aryl halide, a heteroaryl cycloalkyl, a phenyl, a naphthyl, a thienyl, an indolyl, a thiophene, or a isoxasole), an unsubstituted amino or a substituted amino (NRR'), a halo, a hydroxy (-OH), a substituted or an unsubstituted hydroxy (-OR), a phenoxy, a thiol (-SH), a substituted or an unsubstituted thiol (-SR), a cyano (-CN), a formyl (-CHO), a substituted or unsubstituted alkyl (wherein
  • a methyl-aryl substituent a benzylic substituent, an alkenyl, an alkynyl, a cycloalkyl
  • the cycloalkyl is selected from the group consisting of: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl, - cycloheptyl, and - cyclooctyl
  • a cycloalkenyl a substituted alkyl, a substituted alkenyl, a substituted alkynyl, a substituted cycloalkyl, a substituted cycloalkenyl, an aryl, a heteroaryl silyl, a heterosilyl and a heterocyclic group
  • the heterocyclic group is selected from the group consisting of: a saturated heterocyclic and/or a nonsaturated heterocyclic, and optionally the saturated heterocyclic and/or a non-cyclic and a
  • R2 is a substituted amino (NRR', or N-R2-R3) , or
  • Rl is an amino group
  • R2 is a substituted amino (NRR', -R2-R3)
  • R3 is a hydrogen
  • R4 is an aryl group
  • R2 and R3 of Formula II, or Rl and R2 of formula III are independently selected the group consisting of:
  • a hydrogen an aryl (wherein optionally the aryl is any 5-or 6-membered ring, or is selected from the group consisting of: a heteroaryl, an aryl halide, a heteroaryl cycloalkyl, a phenyl, a naphthyl, a thienyl, an indolyl, a thiophene, or a isoxasole), an unsubstituted amino or a substituted amino (NRR'), a halo, a hydroxy (-OH), a substituted or an unsubstituted hydroxy (-OR), a phenoxy, a thiol (-SH), a substituted or an unsubstituted thiol (-SR), a cyano (-CN), a formyl (-CHO), a substituted or unsubstituted alkyl (wherein optionally the alkyl is selected from the group consisting of: - methyl, -
  • a methyl-aryl substituent a benzylic substituent, an alkenyl, an alkynyl, a cycloalkyl
  • the cycloalkyl is selected from the group consisting of: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl, - cycloheptyl, and - cyclooctyl
  • a cycloalkenyl a substituted alkyl, a substituted alkenyl, a substituted alkynyl, a substituted cycloalkyl, a substituted cycloalkenyl, an aryl, a heteroaryl silyl, a heterosilyl and a heterocyclic group
  • the heterocyclic group is selected from the group consisting of: a saturated heterocyclic and/or a nonsaturated heterocyclic, and optionally the saturated heterocyclic and/or a non-cyclic and a
  • X, Y and Z are independently selected from the group consisting of: a C and an N
  • Rl, R2, R3, R4 and R5 of Formula IV are independently selected from the group consisting of:
  • a hydrogen an aryl (wherein optionally the aryl is any 5-or 6-membered ring, or is selected from the group consisting of: a heteroaryl, an aryl halide, a heteroaryl cycloalkyl, a phenyl, a naphthyl, a thienyl, an indolyl, a thiophene, or a isoxasole), an unsubstituted amino or a substituted amino (NRR'), a halo, a hydroxy (-OH), a substituted or an unsubstituted hydroxy (-OR), a phenoxy, a thiol (-SH), a substituted or an unsubstituted thiol (-SR), a cyano (-CN), a formyl (-CHO), a substituted or unsubstituted alkyl (wherein optionally the alkyl is selected from the group consisting of: - methyl, -
  • a halogen a methyl-aryl substituent, a benzylic substituent, an alkenyl, an alkynyl, a cycloalkyl
  • the cycloalkyl is selected from the group consisting of: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl, - cycloheptyl, and - cyclooctyl
  • a cycloalkenyl a substituted alkenyl, a substituted alkynyl, a substituted cycloalkyl, a substituted cycloalkenyl, a heteroaryl silyl, a heterosilyl and a heterocyclic group
  • the heterocyclic group is selected from the group consisting of: a saturated heterocyclic and/or a nonsaturated heterocyclic, and optionally the saturated heterocyclic and/or a nonsaturated heterocyclic is
  • X, Y and Z are independently selected from the group consisting of: a C an N, an O and an S,
  • Rl, R2, R3 and R4, of Formula V are independently selected from the group consisting of: a hydrogen, an aryl (wherein optionally the aryl is any 5-or 6-membered ring, or is selected from the group consisting of: a heteroaryl, an aryl halide, a heteroaryl cycloalkyl, a phenyl, a naphthyl, a thienyl, an indolyl, a thiophene, or a isoxasole), an unsubstituted amino or a substituted amino (NRR'), a halo, a hydroxy (-OH), a substituted or an unsubstituted hydroxy (-OR), a phenoxy, a thiol (-SH), a substituted or an unsubstituted thiol (-SR), a cyano (-CN), a formyl (-CHO), a substituted or unsubstituted alkyl (
  • a halogen a methyl-aryl substituent, a benzylic substituent, an alkenyl, an alkynyl, a cycloalkyl
  • the cycloalkyl is selected from the group consisting of: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl, - cycloheptyl, and - cyclooctyl
  • a cycloalkenyl a substituted alkyl, a substituted alkenyl, a substituted alkynyl, a substituted cycloalkyl, a substituted cycloalkenyl, an aryl, a heteroaryl silyl, a heterosilyl and a heterocyclic group
  • the heterocyclic group is selected from the group consisting of: a saturated heterocyclic and/or a nonsaturated heterocyclic, and optionally the saturated heterocyclic
  • nAChR nicotinic acetylcholine receptor
  • acetylcholine ligand- acetylcholine receptor response has a positive or a negative cooperativity in an acetylcholine ligand- acetylcholine receptor response, or in ligand occupation of the acetylcholine binding protein (AChBP), or
  • nAChR nicotinic acetylcholine receptor
  • the invention provides products of manufacture or a device capable of injecting, causing inhalation of, adsorption of, or otherwise designed for administering for either enteral or parenteral administration: a compound, composition or formulation of the invention to an individual in need thereof, wherein optionally the product of manufacture or a device comprises: a compound, composition or formulation of the invention.
  • the compound, composition or formulation of the invention is formulated for administration in vivo; or for enteral or parenteral administration, or for oral, ophthalmic, topical, oral, intravenous (IV), intramuscular (IM), intrathecal, subcutaneous (SC), intracerebral, epidural, intracranial or rectal administration, or by inhalation.
  • IV intravenous
  • IM intramuscular
  • SC subcutaneous
  • intracerebral epidural
  • intracranial or rectal administration or by inhalation.
  • the compound, composition or formulation is formulated as: a particle, a nanoparticle, a liposome, a tablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, a suspension, a syrup, an emulsion, a lotion, an ointment, an aerosol, a spray, a lozenge, an ophthalmic preparation, an aqueous or a sterile or an injectable solution, a patch (optionally a transdermal patch or a medicated adhesive patch), or an implant.
  • the invention provides pharmaceutical compositions or formulations comprising a compound, composition or formulation of the invention, wherein optionally the pharmaceutical composition or formulation further comprises a pharmaceutically acceptable excipient.
  • the invention provides products of manufacture or a device, comprising a compound, composition or formulation of the invention, or a pharmaceutical composition or formulation of the invention, wherein optionally the product of manufacture or device is a medical device or an implant, wherein optionally the product of manufacture or device is designed to be capable of injecting, causing inhalation of, adsorption of, or otherwise administering for either enteral or parenteral administration a compound, composition or formulation of the invention, or a pharmaceutical composition or formulation of the invention.
  • the invention provides a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump, comprising a compound, composition or formulation of the invention, or a pharmaceutical composition or formulation of the invention.
  • the invention provides methods for
  • nAChR nicotinic acetylcholine receptor
  • nAChR nicotinic acetylcholine receptor
  • acetylcholine ligand- acetylcholine receptor response having a positive or a negative cooperativity in an acetylcholine ligand- acetylcholine receptor response, or in ligand occupation of the acetylcholine binding protein (AChBP), or
  • nAChR nicotinic acetylcholine receptor
  • a device comprising contacting the AChR with a compound, composition or formulation of the invention, or administering to an individual in need thereof a product of manufacture or a device of the invention, or a or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of the invention, thereby
  • nAChR nicotinic acetylcholine receptor
  • nAChR nicotinic acetylcholine receptor
  • AChBP acetylcholine binding protein
  • nAChR nicotinic acetylcholine receptor
  • the contacting is in vitro, ex vivo or in vivo.
  • the invention provides methods for increasing, modulating or stimulating the release of or activity of a neurotransmitter or a
  • nAChR nicotinic acetylcholine receptor
  • the neurotransmitter or a neuromodulator comprises a glutamate, a gamma aminobutyric acid (GABA), a glycine, a serotonin, a peptide or a neuropeptide (optionally a galanin, an enkephalin, an acetylcholine), a norepinephrine, or a biogenic amine (optionally a dopamine, a noradrenaline, an adrenaline, or a
  • GABA gamma aminobutyric acid
  • a pump comprising: administering to an individual in need thereof a compound, composition or formulation of the invention, or a pharmaceutical composition or formulation of the invention, or administering to an individual in need thereof a product of manufacture or a device of the invention, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of the invention,
  • nAChR nicotinic acetylcholine receptor
  • the contacting is in vitro, ex vivo or in vivo.
  • the invention provides methods for treating, ameliorating, preventing or lessening the symptoms of diseases or conditions that are responsive to modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or are responsive to modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain, wherein optionally the neurotransmitter or a neuromodulator comprises a glutamate, a gamma aminobutyric acid (GABA), a glycine, a serotonin, a peptide or a neuropeptide (optionally a galanin, an enkephalin, an acetylcholine), a norepinephrine, or a biogenic amine (optionally a dopamine, a noradrenaline, an adrenaline, or a catecholamine), comprising:
  • a pump comprising: administering to an individual in need thereof a compound, composition or formulation of the invention, or a pharmaceutical composition or formulation of the invention, or administering to an individual in need thereof a product of manufacture or a device of the invention, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of the invention,
  • nAChR nicotinic acetylcholine receptor
  • the contacting is in vitro, ex vivo or in vivo.
  • the invention provides methods for treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse, optionally an addiction or substance abuse involving use of tobacco or nicotine-related products, involving modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or responsive to modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain,
  • nAChR nicotinic acetylcholine receptor
  • the neurotransmitter or a neuromodulator comprises a glutamate, a gamma aminobutyric acid (GABA), a glycine, a serotonin, a peptide or a neuropeptide (optionally a galanin, an enkephalin, an acetylcholine), a norepinephrine, or a biogenic amine (optionally a dopamine, a noradrenaline, an adrenaline, or a catecholamine), or for treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse involving tobacco or nicotine use, cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, or 3,4-methylenedioxy-N- methylamphetamine (MDMA), comprising:
  • a pharmaceutical composition or formulation of the invention a product of manufacture or a device of the invention, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of the invention,
  • an addiction or substance abuse optionally an addiction or substance abuse involving use of tobacco or nicotine-related products, involving modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or responsive to modulating, decreasing or increasing the activity of a nicotinic
  • acetylcholine receptor in the CNS or brain, or treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse involving tobacco or nicotine use, cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, or 3,4-methylenedioxy-N-methylamphetamine (MDMA),
  • the contacting is in vitro, ex vivo or in vivo.
  • the invention provides methods for treating, ameliorating, preventing or lessening the symptoms of a disease or condition responsive to an increase in levels or activity of a neurotransmitter or a neuromodulator in the peripheral nervous system (PNS), the central nervous system (CNS) or brain, or a disease or condition responsive to the modulation of, or a decrease or an increase in the activity of a nicotinic acetylcholine receptor (nAChR), or treating, ameliorating, preventing or lessening the symptoms of a dementia, Parkinson's disease, pain or chronic pain, an allodynia, a psychosis, autism, or a schizophrenia,
  • PNS peripheral nervous system
  • CNS central nervous system
  • nAChR nicotinic acetylcholine receptor
  • the neurotransmitter or a neuromodulator comprises a glutamate, a gamma aminobutyric acid (GABA), a glycine, a serotonin, a peptide or a neuropeptide (optionally a galanin, an enkephalin, an acetylcholine), a norepinephrine, or a biogenic amine (optionally a dopamine, a noradrenaline, an adrenaline, or a
  • GABA gamma aminobutyric acid
  • a pharmaceutical composition or formulation of the invention comprising: administering to an individual in need thereof a compound, composition or formulation of the invention, or administering or applying to an individual in need thereof: a pharmaceutical composition or formulation of the invention; a product of manufacture or a device of the invention, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of the invention,
  • a disease or condition responsive to an increase in levels or activity of a neurotransmitter or a neuromodulator in the peripheral nervous system (P S), the central nervous system (CNS) or brain, or a disease or condition responsive to the modulation of, or a decrease or an increase in the activity of a nicotinic acetylcholine receptor (nAChR), or treating, ameliorating, preventing or lessening the symptoms of a dementia, Parkinson's disease, pain or chronic pain, an allodynia, a psychosis, autism, or a schizophrenia,
  • P S peripheral nervous system
  • CNS central nervous system
  • nAChR nicotinic acetylcholine receptor
  • the contacting is in vitro, ex vivo or in vivo.
  • kits comprising a compound, composition or formulation of the invention, a product of manufacture or a device of the invention, and/or optionally comprising ingredients and/or instructions for practicing a method of the invention.
  • the invention provides kits comprising a compound, composition or formulation of the invention, a product of manufacture or a device of the invention, and/or optionally comprising ingredients and/or instructions for practicing a method of the invention.
  • kits comprising a compound, composition or formulation of the invention; a pharmaceutical composition or formulation of the invention; a product of manufacture or a device of the invention, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi- chambered pump of the invention, and/or optionally comprising ingredients and/or instructions for practicing a method of the invention.
  • the invention provides uses of a compound, composition or formulation of the invention, in the manufacture of a medicament.
  • the invention provides uses of a compound, composition or formulation of the invention, in the manufacture of a medicament for:
  • nAChR nicotinic acetylcholine receptor
  • nAChR nicotinic acetylcholine receptor
  • nAChR nicotinic acetylcholine receptor
  • neurotransmitter or a neuromodulator in the central nervous system (CNS) or the brain or modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain,
  • nAChR nicotinic acetylcholine receptor
  • nAChR nicotinic acetylcholine receptor
  • nAChR nicotinic acetylcholine receptor
  • PNS peripheral nervous system
  • CNS nicotinic acetylcholine receptor
  • the invention provides therapeutic combinations comprising: a compound, composition or formulation of the invention: a pharmaceutical composition or formulation of the invention; a product of manufacture or a device of the invention, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi- chambered pump of the invention.
  • Figure 1A in table form, and Figure IB and Figure 1C in graphic form, summarizes Ki and slope (Hill coefficient, nn) data from radioligand binding
  • Figure 2A schematically illustrates three exemplary synthetic pathways of the invention to generate exemplary 2,4,6-substituted pyrimidine compounds of the invention; as discussed in detail in Example 1, below.
  • Fig. 2B schematically illustrates exemplary compounds of the invention that were synthesized and screened; as discussed in detail in Example 1, below.
  • Figure 2C graphically illustrates the results of quick screen results of the so-called "KK-169 analog" exemplary compounds of the invention, including exemplary compounds of the invention KK 301-A, KK 301-B, KK 302, KK 303, KK 304-A, KK 304-B and KK 305 (see Figure 4, for structures); as discussed in detail in Example 1, below.
  • Figure 3 A schematically illustrates an exemplary compound of the invention, and graphically illustrates the results of an ⁇ 4 ⁇ 2 nAChR agonist screening of a collection of 80 compounds using cell-based medium throughput fluorescence assays, which identified one exemplary compound of the invention, the so-called "KK-253B", that acted as a ⁇ 4 ⁇ 2 nAChR agonist and did not activate a7 nAChR; as discussed in detail in Example 1, below.
  • Figure 3B schematically illustrates exemplary compounds of the invention identified using cell-based assay as ⁇ 4 ⁇ 2 nAChR antagonists; as discussed in detail in Example 1, below.
  • Figure 5 graphically illustrates data from titration curves using radioligand binding assay for the 4,6 substituted 2-aminopyrimidines showing the range of potencies and Hill coefficients for ligand binding:
  • Fig. 5A graphically illustrates data showing that if the binding of ligand at one site lowers the affinity for ligand at another site on an adjacent subunit, the protein exhibits negative cooperativity or induced site heterogeneity, n H ⁇ 1;
  • Fig. 5C graphically illustrates data showing that if the binding of ligand at one site increases the affinity for ligand at another site, the macromolecule exhibits positive cooperativity (nn >1); as discussed in detail in Example 1, below.
  • FIG. 6A schematically illustrates an exemplary scintillation proximity screening scheme of the invention comprising screening of a compound library by a radioligand binding assay against AChBPs; this screen assay resulted in the identification of several additional exemplary compounds of this invention, more so-called “lead structures", with low micromolar affinities; and from these exemplary compounds, or “leads", approximately 40 exemplary compounds, or analogs, were identified, synthesized and screened, as illustrated in Figure 6B.
  • Figure 6B graphically illustrates, and Figure 6C in table form illustrates, radioligand screening results of the pyrimidine series against Lymnea AChBP, where the compounds marked in green (or the so-called "AC" fraction, the middle of the 3 fractions) also have low micromolar Kd values for Aplysia AChBP; as discussed in detail in Example 1, below.
  • Figure 7 schematically illustrates the crystal structure of Ls AChBP-ligand complex: Fig. 7A schematically illustrates a side image, and Fig. 7B
  • FIG. 7C schematically illustrates ligands having n H ⁇ l for both
  • Fig. 7D schematically illustrates n H ⁇ l and n H >l images superimposed; as discussed in detail in Example 1, below.
  • Figure 8 graphically illustrates data from representative titration profiles for 4,6-substituted 2-aminopyrimidine (exemplary compounds of the invention, see Table 4 to associate numbering with structure) competition with 3 H- epibatidine binding showing a range of dissociation constants (K d ) and Hill coefficients (n H ) for ligand binding to Zs-AChBP; as discussed in detail in Example 2, below.
  • Figure 9 shows X-ray crystal structures of exemplary compounds of the invention (ligands) 32 and 33 (negative cooperativity, n H ⁇ l) and exemplary compound 15 (positive cooperativity, n H >l), in complexes with Zs-AChBP:
  • FIG. 9(C) illustrates overlay of exemplary compound 32 (blue) and exemplary compound 33 (yellow) crystal structures; and, Fig 9(D): illustrates a superimposition of exemplary compound 15 (yellow) and exemplary compound 10 33 (blue) crystal structures; as discussed in detail in Example 2, below.
  • Figure 10 shows the superimposition of Zs-AChBP X-ray crystal structures in complex with exemplary compound 33 (Fig. 10A) and exemplary compound 15 (Fig. 10B) with nicotine; as discussed in detail in Example 2, below.
  • Figure 11 shows the global differences in X-ray crystal structures of Ls-
  • FIG. 1 1(A) schematically illustrates a top (apical) view on superimposed (UCSF chimera) Apo pentamer (in blue) and with bound exemplary compound 15 (in red), dashed lines (blue and red respectively) indicate 0 most significant differences in quaternary structures quantified by measuring distances between T13 backbone alpha carbon of distant subunits;
  • Fig. 1 1(B) schematically illustrates a superimposition (PyMOL) of Zs-AChBP Apo, chain D (in blue) and exemplary compound 15 complex, chain D (in red);
  • Figure 12 shows a comparison of Zs-AChBP quaternary changes
  • Fig. 12(A) schematically illustrates an overlay of Ls- AChBP crystal structure in its Apo form
  • Fig. 12(B) illustrates a chart representing 'bloom'
  • x axis delta distance between C a observed in Zs-AChBP - ligand complexes when compared with Zs-AChBP Apo form
  • y axis relative distance from the protein vestibule
  • Fig. 12(A) schematically illustrates an overlay of Ls- AChBP crystal structure in its Apo form
  • Fig. 12(B) illustrates a chart representing 'bloom'
  • x axis delta distance between C a observed in Zs-AChBP - ligand complexes when compared with Zs-AChBP Apo form
  • y axis relative distance from the protein vestibule
  • Fig. 12(A) schematically illustrates an overlay of Ls- AChBP crystal structure in its Apo form
  • Fig. 12(B) illustrates a chart representing
  • FIG. 12(C) illustrates a chart representing 'twist' of the pentameric structure
  • x axis delta dihedral angle between C a observed in Zs-AChBP - ligand complexes when compared with Zs-AChBP Apo form; as discussed in detail in Example 2, below.
  • Figure 14 illustrates Table 6, showing data of competition between exemplary compounds of the invention (numbering corresponds to Table 4 compound numbers) as substituted 2-aminopyrimidines against 3 H-epibatidine binding to Zs-AChBP; as discussed in detail in Example 2, below.
  • Figure 15 schematically illustrates an overlay of exemplary compound 15 (yellow) and exemplary compound 32 (blue) crystal structures;
  • Fig. 15(B) schematically illustrates superimposition of exemplary compound 15
  • Figure 16 illustrates a screening assay demonstrating activity of exemplary compounds of the invention (so-called compounds 17, KK-311-D and 171 A, see Table 4 for structures), using activation of a7-nAChr CNiFERS with l-(5-chloro- 2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl);
  • Fig. 16A and Fig. 16B graphically illustrates the results at 50 uM and 10 uM concentrations
  • Fig. 15C graphically illustrates these results; as discussed in detail in Example 2, below.
  • Figure 17 schematically illustrates three (1, 2, 3) exemplary synthetic pathways of the invention to generate exemplary 2,4,6-substituted pyrimidine compounds of the invention, including the so-called Formula I genus structure; as discussed in detail in Example 2, below.
  • Figure 18 schematically illustrates exemplary synthetic pathways of the invention to generate exemplary 2,4,5 -substituted pyrimidine compounds of the invention, including the so-called Formula I genus structure; as discussed in detail in Example 2, below.
  • Like reference symbols in the various drawings indicate like elements.
  • the invention provides compounds and
  • compositions that are selective ligands to acetylcholine binding protein (AChBP) and have unique properties.
  • AChBP acetylcholine binding protein
  • these AChBP ligands have both negative as well as positive
  • This invention for the first time describes cooperative binding activity by the acetylcholine binding protein (AChBP), compounds and compositions that can modulate this cooperative binding activity. Since AChBP only consists of the extracellular domain of the nAChR, compounds of the invention uniquely show that cooperativity, be it negative or positive, can occur in a circumferential
  • this invention widens the structural base for achieving selectivity with the nAChR.
  • the structures of exemplary compounds of the invention depart substantially from structures of the classical agonists and antagonists of the nAChR.
  • the invention establishes a previously-unknown level of conformational communication of AChBP subunits upon ligand binding. Interaction of a ligand in the first binding pocket causes structural changes between subunits resulting in a transition between different affinity states. In case of negative cooperativity, the unoccupied site in the pentamer becomes structurally restrained, leading to reduced affinity for binding of the second ligand. By translating the phenomena to nicotinic receptors, ligands
  • receptors e.g., nicotinic and ligand gated ion channel
  • compositions and formulations of the present disclosure are provided in alternative embodiments.
  • inventions act as ligands that bind cooperatively to the acetylcholine binding
  • compositions and methods are described in detail below.
  • compositions and formulations of the invention have relatively high affinities, or low dissociation constants - particularly for the negatively cooperative ligands.
  • compositions and formulations of the invention cause
  • conformational changes in the acetylcholine binding protein that are seen globally in the protein; these conformational changes are unique to the 2-aminopyrimidines that are not seen in the classical agonists (for example, nicotine or epibatidine) or antagonists (for example, benzylidene anabaseine,
  • compositions and formulations of the invention can stimulate the alpha-7 nicotinic receptor, thus acting as drugs or pharmaceutically active reagents.
  • the invention provides exemplary compounds of the invention as "congeneric structures" that exhibit cooperativity, where many members of the family show Hill slopes greater than 1.0 or considerably less than 1.0. The latter is not due to heterogeneity of the binding protein template, since AChBP has five identical sites as examined crystallographically and by conventional ligand binding. These cooperative interactions appear not to require a direct connection with the channel gating area to elicit subunit interactions and cooperativity. Rather, cooperativity can be confined to the extra-cellular domain and is mediated circumferentially around the cylindrical pentamer. This unique feature of compounds of the invention, these AChBP ligands, makes them effective as medications with distinct pharmacological profiles.
  • Figure 4 illustrates generic structures of the invention in cooperative series 1; structure of most potent ligands, 4,6-substituted 2-amino pyrimidines 2.
  • the invention provides compounds that
  • nAChR nicotinic acetylcholine receptor
  • exemplary compounds of the invention can stimulate a nAChR functional response.
  • the invention provides two
  • types (or classes or genuses) of exemplary compounds one class (or type or
  • nAChR binding proteins binds in a cooperative manner to nAChR binding proteins, and a second exemplary class (or type or genus) activates a functional response of an nAChR by its binding.
  • both classes of compounds of the invention can increase the release of dopamine in the CNS or brain, or increase the activity of nAChR in the CNS or brain.
  • the invention provides compounds, compositions and methods for increasing or stimulating the release of or activity of dopamine in the central nervous system (CNS), including the brain, or increasing the activity of nAChR in the CNS or brain, and compounds, compositions and methods for treating, ameliorating, preventing or lessening the symptoms of diseases or conditions that are responsive to an increase in levels or activity of dopamine in the CNS or brain, or responsive to an increase in the activity of nAChR in the CNS or brain.
  • CNS central nervous system
  • the invention provides compounds, compositions and methods for treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse involving increasing levels or activity of dopamine in the CNS or brain, or responsive to an increase in the activity of nAChR in the CNS or brain, for example, for treating, ameliorating, preventing or lessening the symptoms of an addiction or a substance abuse, e.g., an addiction or a substance abuse involving cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, such as 3,4-methylenedioxy-N-methylamphetamine
  • the invention provides compounds, compositions and methods for treating, ameliorating, preventing or lessening the symptoms of a disease or condition responsive to an increase in levels or activity of dopamine in the CNS or brain, or responsive to an increase in the activity of nAChR in the CNS or brain, for example, dementia, Parkinson's disease, pain or chronic pain, allodynia, autism, psychosis or schizophrenia.
  • the invention also provides bioisosteres of compounds of the invention, e.g., compounds having a structure as set forth in Table 1, Table 2, Table 3 or Table 4.
  • bioisosteres of the invention are compounds of the invention comprising one or more substituent and/or group replacements with a substituent and/or group having substantially similar physical or chemical properties which produce substantially similar
  • the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of a
  • bioisosteres of compounds of the disclosure are provided.
  • bioisosteres of compounds of the disclosure are provided.
  • inventions are made by replacing one or more hydrogen atom(s) with one or more fluorine atom(s), e.g., at a site of metabolic oxidation; this may prevent
  • the molecule may have a blocked pathway for metabolism
  • the invention provides compounds and compositions, including formulations and pharmaceutical compositions, for use in in vivo, in vitro or ex vivo methods, e.g., for:
  • an addiction or substance abuse e.g., an addiction or substance abuse involving cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, or 3,4- methylenedioxy-N-methylamphetamine (MDMA)
  • an addiction or substance abuse e.g., an addiction or substance abuse involving cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, or 3,4- methylenedioxy-N-methylamphetamine (MDMA)
  • a dementia for treating, ameliorating, preventing or lessening the symptoms of a dementia, Parkinson's disease, pain or chronic pain, allodynia, autism, psychosis or schizophrenia.
  • compositions of the invention can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally.
  • pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, capsules, suspensions, taken orally, suppositories and salves, lotions and the like.
  • Pharmaceutical formulations of this invention may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc.
  • the pharmaceutical compounds can be delivered by
  • transdermally by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • compositions of the invention are delivered orally, e.g., as pharmaceutical formulations for oral administration, and can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages.
  • Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
  • Oral carriers can be elixirs, syrups, capsules, tablets, pills, geltabs and the like.
  • compositions for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
  • suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose,
  • liquid carriers are used to manufacture or formulate compounds of this invention, or a composition used to practice the methods of this invention, including carriers for preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compounds.
  • the active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • the liquid carrier can comprise other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators.
  • solid carriers are used to manufacture or formulate compounds of this invention, or a composition used to practice the methods of this invention, including solid carriers comprising substances such as lactose, starch, glucose, methyl-cellulose, magnesium stearate, dicalcium phosphate, mannitol and the like.
  • a solid carrier can further include one or more substances acting as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material.
  • the carrier can be a finely divided solid which is in admixture with the finely divided active compound.
  • the active compound is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methylcellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • compounds and pharmaceutical compositions of the invention are formulated as and/or delivered as patches, e.g., a transdermal patch or a medicated adhesive patch that is placed on the skin or mucous membrane to deliver a specific dose of drug or medication (e.g., compounds and pharmaceutical compositions of the invention) through the skin and into the bloodstream.
  • a transdermal drug delivery route over other types of medication delivery such as oral, topical, intravenous, intramuscular, etc. can be that the patch provides a controlled release of the drug or medication into the patient, optionally through either a porous membrane covering a reservoir of medication or through body heat melting thin layers of medication embedded in the adhesive.
  • a patch is a single-layer drug-in-adhesive patch; in this exemplary embodiment, the adhesive layer also contains the drug or medication (e.g., compounds and pharmaceutical compositions of the invention).
  • the adhesive layer can not only serves to adhere the various layers together, along with the entire system to the skin, but also can be responsible for the releasing of the drug or medication.
  • the adhesive layer can be surrounded by a temporary liner and a backing.
  • a patch is a multi-layer drug-in- adhesive patch, which is similar to the single-layer system, but it adds another layer of drug-in-adhesive, optionally separated by a membrane.
  • One of the layers can be for immediate release of a drug or medication (e.g., compounds and pharmaceutical compositions of the invention) and other layer is for control release of the same and/or different drug or medication from the reservoir.
  • This patch also can have a temporary liner-layer and a permanent backing.
  • drug release depends on membrane permeability and diffusion of drug molecules.
  • a patch is a reservoir transdermal system, which has a separate drug layer; the drug layer can be a liquid or gel compartment comprising a drug solution or a suspension separated by the adhesive layer.
  • the drug reservoir can be totally encapsulated in a shallow compartment molded from a drug-impermeable metallic plastic laminate, optionally with a rate-controlling membrane made of a polymer (e.g., a vinyl acetate) on one surface.
  • This patch also can be backed by a backing layer.
  • the rate of release can be designed to be zero order.
  • a patch is a matrix system, or so-called “monolithic device", which comprises a drug layer of a solid or a semisolid matrix comprising a drug solution or a suspension (e.g., comprising compounds and pharmaceutical compositions of the invention).
  • the adhesive layer in this patch can surround the drug layer, optionally partially overlaying it.
  • compounds and pharmaceutical compositions of the invention are formulated as and/or delivered as or in so-called "thin-film” or dissolving film delivery systems. These can be used to administer a drug solution or a suspension (e.g., comprising compounds and pharmaceutical compositions of the invention) via absorption in the mouth (e.g., buccally or sublingually) and/or via the small intestines or otherwise enterically.
  • a film can be prepared using a hydrophilic polymer that rapidly dissolves on a mucous membrane, e.g., in the tongue or buccal cavity or esophagus or intestine, thus delivering the drug to the systemic circulation via dissolution when contact with liquid (e.g., a bodily fluid) is made.
  • thin-film drug delivery is used as an alternative to or with another delivery modality, e.g., tablets, capsules, liquids and the like. They can be similar in size, shape and thickness to a postage stamp, and can be designed for oral administration, with the user placing the strip on or under the tongue (sublingual) or along the inside of the cheek (buccal). As the strip dissolves, the drug can enter the blood stream enterically, buccally or sublingually.
  • thin-films are made of combination of microcrystalline cellulose and maltodextrin, and can also include plasticizers, phthalate, glycols.
  • concentrations of therapeutically active compound in a formulation can be from between about 0.1% to about 100%, e.g., having at least about 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or more, by weight.
  • therapeutic formulations are prepared by any method well known in the art, e.g., as described by Brunton et al, eds., Goodman and Gilman's: The Pharmacological Bases of Therapeutics , 12th ed., McGraw-Hill, 201 1; Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; Avis et al, eds., Pharmaceutical Dosage Forms: Parenteral Medications, published by Marcel Dekker, Inc., N.Y., 1993; Lieberman et al, eds., Pharmaceutical Dosage Forms: Tablets, published by Marcel Dekker, Inc., N.Y., 1990; and Lieberman et al., eds., Pharmaceutical Dosage Forms: Disperse Systems, published by Marcel Dekker, Inc., N.Y., 1990.
  • therapeutic formulations are delivered by any effective means appropriated for a particular treatment.
  • the suitable means include oral, rectal, vaginal, nasal, pulmonary administration, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) infusion into the bloodstream.
  • parenteral administration antitumor agents of the present invention may be formulated in a variety of ways.
  • Aqueous solutions of the modulators can be encapsulated in polymeric beads, liposomes, nanoparticles or other injectable depot formulations known to those of skill in the art.
  • compounds of the invention are administered encapsulated in liposomes.
  • compositions are present both in an aqueous layer and in a lipidic layer, e.g., a liposomic suspension.
  • a hydrophobic layer comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such a diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like.
  • an exemplary dosage may be about 30 mg/kg administered e.g., by intravenous therapy, e.g., over between about 15 to 30 minutes, or by intramuscular injection or subcutaneous injection, e.g., repeated later in intervals, e.g., at about 60 minutes later.
  • intravenous therapy e.g., over between about 15 to 30 minutes
  • intramuscular injection or subcutaneous injection e.g., repeated later in intervals, e.g., at about 60 minutes later.
  • an exemplary dosage and administration is as a 500 mg/h continuous IV infusion.
  • an exemplary dosage and administration is as a 500 mg/h continuous IV infusion.
  • an exemplary dosage and administration is as a 500 mg/h continuous IV infusion.
  • administration is at between about 20 to 50 mg/kg, optionally followed by a maintenance infusion at between about 5 to 10 mg/kg/h.
  • an exemplary dosage and administration is based on long term, low dosage administration, for example, by a slow release pharmaceutical vehicle, or by slow release from an implant.
  • compositions of the invention are formulated in a buffer, in a saline solution, in a powder, an emulsion, in a vesicle, in a liposome, in a
  • compositions can be formulated in any way and can be applied in a variety of concentrations and forms depending on the desired in vivo, in vitro or ex vivo conditions, a desired in vivo, in vitro or ex vivo method of administration and the like. Details on techniques for in vivo, in vitro or ex vivo formulations and administrations are well described in the scientific and patent literature. Formulations and/or carriers used to practice this invention can be in forms such as tablets, pills, powders, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for in vivo, in vitro or ex vivo
  • the compounds (e.g., formulations) of the invention can comprise a solution of compounds of the invention, including stereoisomers, derivatives and analogs thereof, disposed in or dissolved in a pharmaceutically acceptable carrier, e.g., acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride.
  • a pharmaceutically acceptable carrier e.g., acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride.
  • sterile fixed oils can be employed as a solvent or suspending medium.
  • any fixed oil can be employed including synthetic mono- or diglycerides, or fatty acids such as oleic acid.
  • solutions and formulations used to practice the invention are sterile and can be manufactured to be generally free of undesirable matter. In one embodiment, these solutions and formulations are sterilized by conventional, well known sterilization techniques.
  • solutions and formulations used to practice the invention can comprise auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of active agent in these formulations can vary widely, and can be selected primarily based on fluid volumes, viscosities and the like, in accordance with the particular mode of in vivo, in vitro or ex vivo administration selected and the desired results.
  • compositions and formulations of the invention can be delivered by the use of liposomes.
  • liposomes particularly where the liposome surface carries ligands specific for target cells or organs, or are otherwise preferentially directed to a specific tissue or organ type, one can focus the delivery of the active agent into a target cells in an in vivo, in vitro or ex vivo application.
  • compositions and formulations of the invention can be directly administered, e.g., under sterile conditions, to an individual (e.g., a patient) to be treated.
  • the modulators can be administered alone or as the active ingredient of a pharmaceutical composition.
  • Compositions and formulations of this invention can be combined with or used in association with other therapeutic agents. For example, an individual may be treated concurrently with conventional therapeutic agents.
  • a compound, a formulation or mixture of compounds of the invention is/are administered parenterally in an appropriate co-solvent to enable distribution from the site of IM, SC or IV injection, to prevent post- injection precipitation by virtue of a change in pH, for example, as described in J. Pharm. Pharmacol: 62:873-82 (2010); Adv. Drug Delivery Rev. 59:603-07 (2007), and to ensure "solubilization" conditions at the injection site, e.g., as described in J. Pharm. Pharmacol 62: 1607-21; Anesth Analg 79: 933-39 (1994); J. Pharm. Pharmacol 65 1429-39 (2013).
  • the compounds of the invention can be administered at low pH (e.g., between about pH 4 to 6) or high pH (e.g., between about pH 8 to 11).
  • these alternative embodiments involve: Low pH solutions adjusted with acetic acid; High pH solutions adjusted with a 2 C0 3 (pH 10- 11); Co-solvent formulation at neutral pH to include propylene glycol (up to 50%), polyethylene glycol, 2-hydroxypropyl ⁇ -cyclodextrin and combinations and congeners thereof; and/or, micellular dispersions with surface active agents.
  • oral (p.o.) preparations encompass tablets and capsules, including syrups emulsions and suspensions to insure distribution throughout the gastrointestinal (GI) tract.
  • GI gastrointestinal
  • the invention also provides nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising compounds and compositions used to practice the methods of this invention.
  • the invention provides nanoparticles, nanolipoparticles, vesicles and liposomal membranes for low dosage and/or slow release of a compound of the invention.
  • the invention provides multilayered liposomes comprising compounds used to practice this invention, e.g., as described in Park, et al, U.S. Pat. Pub. No. 20070082042.
  • the multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice this invention.
  • Liposomes can be made using any method, e.g., as described in Park, et al, U.S. Pat. Pub. No. 20070042031, including method of producing a liposome by encapsulating an active agent (e.g., a compound of the invention), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution.
  • an active agent e.g., a compound of the invention
  • liposome compositions used to practice this invention comprise a substituted ammonium and/or polyanions, e.g., for targeting delivery of a compound (e.g., e.g., a compound of the invention) used to practice this invention to a desired cell type or organ, e.g., brain, as described e.g., in U.S. Pat. Pub. No.
  • the invention also provides nanoparticles comprising compounds (e.g., a compound of the invention) used to practice this invention in the form of active agent- containing nanoparticles (e.g., a secondary nanoparticle), as described, e.g., in U.S. Pat. Pub. No. 20070077286.
  • the invention provides nanoparticles comprising a fat-soluble active agent of this invention or a fat-solubilized water-soluble active agent to act with a bivalent or trivalent metal salt.
  • solid lipid suspensions can be used to formulate and to deliver compositions used to practice this invention to mammalian cells in vivo, in vitro or ex vivo, as described, e.g., in U.S. Pat. Pub. No. 20050136121.
  • compositions and formulations of the invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions or formulations of the invention are administered to a subject that is responsive to modulating, decreasing or increasing levels or activity of a dopamine in the CNS or brain, or is responsive to modulating, decreasing or increasing the activity of an nAChR in the CNS or brain (a "therapeutically effective amount").
  • the pharmaceutical compositions and formulations of the invention also can be administered as a preventative agent, e.g., prophylactically.
  • the amount of pharmaceutical composition adequate to accomplish this is defined as a "therapeutically effective dose.”
  • the dosage schedule and amounts effective for this use, i.e., the "dosing regimen,” will depend upon a variety of factors, including the stage of the exposure, the severity of the exposure, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
  • the dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51 :337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84: 1144-1 146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; the latest Remington's, supra).
  • pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:
  • the invention also provides products of manufacture and kits for
  • the invention provides products of manufacture and kits comprising compounds, compositions and formulations of this invention, and comprising all the components needed to practice a method of the invention.
  • kits comprising compounds, compositions and formulations of this invention, and comprising compositions and/or instructions for practicing methods of the invention.
  • the invention provides kits comprising: a composition used to practice a method of any of the invention, optionally comprising instructions for use thereof.
  • the invention provides pumps, devices, subcutaneous infusion devices, continuous subcutaneous infusion device, infusion pens, needles, reservoirs, ampoules, vials, syringes, cartridges, disposable pen or jet injectors, prefilled pens or syringes or cartridges, cartridge or disposable pen or jet injectors, two chambered or multi-chambered pumps, syringes, cartridges or pens or jet injectors comprising a composition, composition or a formulation of the invention.
  • the injector is an auto injector, e.g., a SMART JECT® autoinjector (Janssen Research and Development LLC); or a MOLLY®, or DAI®, or DAI-RNS® autoinjector (SHL Group, Deerfield Beach, FL).
  • the injector is a hypodermic or a piston syringe.
  • Example 1 Exemplary compositions, formulations and combinations of the invention
  • This example describes exemplary compositions, formulations and combinations of the invention, and methods for making them.
  • the inventors used in situ click chemistry (see e.g., Kolb (2001)
  • nn ⁇ l the affinity for ligand at another site
  • the protein exhibits negative cooperativity, nn ⁇ l . It may also mean that two different independent binding sites with different affinities participate in ligand binding (see, e.g., KRIJSEK, Physiol. Res., 2004). Cooperative binding to AChBP can lead to ligands with unique pharmacological profiles.
  • nAChR agonists and antagonists To identify and characterize nAChR agonists and antagonists, we used cell4oased medium throughput fluorescence assays. One compound, KK-253B, passed the initial quick screen as a ⁇ 4 ⁇ 2 nAChR agonist and did not activate a7 nAChR. Additionally we found series of compounds that act as ⁇ 4 ⁇ 2 nAChR antagonists, as illustrated in Figure 3. For performing in situ click chemistry to identify potential dual-site binders, azides and alkynes were used as complementary reactants in triazole formation directed by the protein itself. Soluble forms of ⁇ 4 ⁇ 2 nAChR subtypes were used in these experiments. Ligands identified by mass spectrometry can be re-synthesized and tested to confirm activity.
  • Figure 7 schematically illustrates the crystal structure of Ls AChBP-ligand complex: Fig. 7(A) Side and Fig. 7(B) bottom side view showing molecules in five binding sites.
  • Figure 8 graphically illustrates representative titration profiles for 4,6-substituted
  • 2-aminopyrimidine (exemplary compounds of the invention, see Table 4, to associate numbering with structure) competition with 3 H-epibatidine binding showing a range of dissociation constants (3 ⁇ 4) and Hill coefficients (nn) for ligand binding to Zs-AChBP.
  • Figure 9 graphically illustrates global differences in X-ray crystal structures of Ls- AChBP bound cooperative ligands in comparison with crystal structure of Zs-AChBP in its Apo form.
  • Figure 14 summarizes data of X-ray crystal structures of exemplary compounds of the invention (ligands) 32 and 33 (negative cooperativity, n H ⁇ l) and exemplary compound 15 (positive cooperativity, n H >l), in complexes with Zs-AChBP:
  • Fig 9(A) illustrates radial view of Ls -AChBP pentameric structure in complex with exemplary compound of the invention 32;
  • Fig 9(B) illustrates an expanded radial view of exemplary compound 32 in binding site, including ligand electron density;
  • Fig. 9(C) illustrates overlay of exemplary compound 32 (blue) and exemplary compound 33
  • Fig 9(D) illustrates a superimposition of exemplary compound 15 (yellow) and exemplary compound 33 (blue) crystal structures.
  • Figure 17 schematically illustrates three (1, 2, 3) exemplary synthetic
  • Figure 18 schematically illustrates exemplary synthetic pathways of the invention to generate exemplary 2,4,5 -substituted pyrimidine compounds of the invention, including the so-called Formula I genus structure.
  • compositions including 4,6- disubstituted 2-aminopyrimidines, formulations and combinations of the
  • Both sets of molecules bind at the agonist-antagonist site, as expected from their competition with epibatidine (or, (lR,2R,45)-(+)-6-(6-chloro-3-pyridyl)-7-azabicyclo[2.2.1]heptane).
  • An analysis of AChBP quaternary structure shows that cooperative ligand binding is associated with a blooming or flare conformation, a structural change not observed with the classical, non- cooperative, nicotinic ligands.
  • Exemplary compounds of the invention behaved as positively and negatively cooperative ligands and exhibited unique features in the detailed binding determinants and poses of the complexes.
  • Nicotinic acetylcholine receptors function as allosteric pentamers of identical or homologous transmembrane spanning subunits. Ligand binding at two or more of the five inter-subunit sites, located radially in the extracellular domain, drives a conformational change that results in the opening of a centrosymmetric transmembrane channel, internally constructed amongst the five subunits (See Fig. l2A) (1-4). Up to five potential agonist-competitive antagonist sites on the pentamer are found at the outer perimeter of the subunit interfaces. Amino acid side chain determinants on both subunit interfaces dictate selectivity amongst the many subtypes of nAChRs.
  • nAChRs are hetero-oligomeric, where the sites of ligand occupation are not identical (1-4). This arrangement arises when a common a-subunit pairs with one or more non-identical subunit partners, termed non a-subunits (7-8). Non-identity of the subunit interface complementary to the a subunit may also give rise to heterogeneity in binding constants typically seen for antagonists and mask partially the degree of agonist cooperativity. An exception to this is the a7-neuronal nAChR composed of five identical subunits and exhibiting a high degree of cooperativity for agonist activation (9).
  • acetylcholine binding protein was characterized from mollusks (15-
  • AChBP exhibits a similar profile of ligand selectivity toward the classical nicotinic agonists and antagonists of quaternary amine, tertiary and secondary amine (alkaloid), imine, and peptide origin that bind nicotinic receptors (18-25). If looked at solely on the basis of ligand binding capacities, AChBP could be considered as a distinct subtype of nAChR.
  • Ligand 15 has a clear electron density for the bi-aryl ring of the molecule, but a poor density of the alkyl chain possibly arising from multiple flexible conformations of the C loop. Several residues in F the loop (T155-E163) gave unresolved electron densities and were consequently excluded from the models. Additionally, in complex with ligand 15, residues 185-189 in the C loop encompassing the vicinal Cys-Cys bond were not seen in nine often subunits of the dimer of pentamers.
  • Loop closure in the presence of bound ligands is 8.4 A for 32 and 33 structures and 8.2 A for 15.
  • the ligands contact amino acids from both the principal face with residues from loops A (Y89), B (W143), and C (Y185 and Y192), and complementary face (loops D: W53, E: LI 12 and Ml 14, F: Y164). Parallel displaced ⁇ -stacking interactions with W143 are present in all structures.
  • the ring rotation results in its parallel alignment with Y192 side chain (2.9 A and greater).
  • Morpholine (32) or 4-methyl-piperidine (33) substituents appear to associate with Y89 and Y185. Nitrogen atoms of these rings are positioned to stack with all 7 atoms of the Y185 ring with distances ranging between 3.6-4.3 A. Interactions of these substituents on the complementary subunit face are predominantly hydrophobic.
  • the altered position of the indole of W53 in 32 and 33, compared with 15, is associated with a change in side chain orientation of neighboring residues Ml 14 and Q55.
  • Phenyl rings substituted at the pyrimidine 6-position interact mainly with W143, Y192 and CI 88.
  • fluorines in trifluoromethyl substituent are in the vicinity of T 144 side chain and interact with multiple loop F residues, including LI 12 and Ml 14 as well as neighboring water molecules, with interaction extending to Ml 14 and R104 side chains.
  • the flexible aliphatic chain of 15 at the 4 position in the pyrimidine does not yield discernable electron densities except for chain D. This likely reflects multiple conformations of the flexible C loop not constrained by the symmetry related molecule in the crystal structure.
  • Rotation of the pyrimidine ring and presence of the alkyl chain in 15 brings the ligand in close contact with Y 185 side chain ( ⁇ 3.0 A to N4 of the ligand) and causes the tyrosine ring to rotate toward the gorge interface presumably to avoid a clash with the alkyl chain of the ligand.
  • the indole of W53 on the complementary face rotates towards the subunit interface and is in contact with ligand N4 (3.5 A).
  • the phenyl ring in ligand 15 has a similar position as the aromatic ring in complexes 32 or 33, but its contacts are altered by methoxy- substituent interacting with T144 and with LI 02, LI 12 and Ml 14 in the complementary face.
  • Major differences for 15, when compared to complexes of ligands showing negative cooperativity, are seen in Y185, Y164, Ml 14 and W53 side chains conformations.
  • the indole side chain in W53 changes its rotameric position.
  • Ml 14 is brought in contact with the 6- substituted trifluoromethyl phenyl group of the ligand.
  • the position of the 32/33 phenyl ring is similar to the pyridine ring in nicotine.
  • the trifluoromethyl group forces LI 12 to change its rotameric position.
  • Loop C in ligand 15 shows significant variation, reflected in the Y185 side chain conformation (Fig. 3B).
  • Residues W53 and Ml 14 in the 32/33 complexes, that represent most significant departures from nicotine, in the 15 X-ray structure have orientations similar to those in the nicotine complex.
  • the indole ring of W53 side chain has more extensive contacts with the ligand 15 than with nicotine, through its substituent at the position 4 in the pyrimidine ring.
  • the rotational state of Y164 on complementary face changes as well.
  • AChBP subunits accompanying ligand binding We report a series of selective AChBP ligands exhibiting negative as well as positive cooperativity, a type of allosteric behavior in which binding interactions in an oligomeric protein take place between distant subunit interfaces. Selected ligands showing marked differences in cooperativity were used to obtain three atomic resolution, X-ray crystal structures of ligands with different Hill slope values in their complexes with Zs-AChBP. The lower affinity ligands showing positive cooperativity, along with the high affinity of the negatively cooperative ligands, serve to achieve full occupation of ligand in the crystal structures.
  • the 2-aminopyrimidines may be considered as electron-rich ring systems capable of ring stacking with the side chains of Y192 and W143.
  • the pyrimidine ring nitrogens are not as exposed as the bicyclic ring in epibatidine and the pyrrolidine nitrogen in nicotine affording a proper directionality for hydrogen bonding.
  • the global conformational changes primarily manifested by blooming appear characteristic of the substituted 2-aminopyrimidines and are not seen with carbamylcholine and nicotine (Fig. 4 and Fig. S2). To date, a clear state change has only been documented for the substituted 2-aminopyrimidines.
  • AChBP quaternary structure add another dimension to considering subunit interactions.
  • the "blooming" and torsional conformational changes noted with AChBP show analogies with those observed in GLIC (13, 27) in relation with correspondences of protein sequence (Fig. 4, A, B & C and Fig. S2).
  • the amino terminal helix, regions between residues 58-70 and between 106-110 all show increased distances between diametrically opposed subunits.
  • the sharper negative peaks around residues 156 and 185 likely involve local perturbations of the C and F loops proximal to the ligand binding site resulting in local compaction of the structure, measured in Ca distances.
  • the comparison of the dihedral bond angles should reflect a torsional or twist motion as seen between the two states of GLIC (13, 27). Changes in dihedral angle positions are small (Fig. S2, C), but involve the same set of residues. Hence, when the binding of the 2- aminopyrimidines are compared with the pH dependent conformations of GLIC, the dominant common change is found with the Ca distances between diametric subunits (blooming) (Fig. S2 B), rather than the dihedral angles for torsional movement (twist) (Fig. S2 C).
  • interfacial binding sites residing under the C loop of AChBP and the nAChR appear surprisingly accommodating for the binding of ligands of different structure.
  • Stornaiaolo and colleagues have reported on large planar, aromatic molecules binding under the C loop in a stacked sandwich fashion, and extending the C loop (35).
  • benzodiazepines (36-37) and other sedative agents (38) that act in this manner with the GABA receptor (39-40). Accordingly, new dimensions for achieving pharmacologic selectivity for particular nAChR subtypes may result with the cooperative nAChR ligands possessing electron-rich substituted 2-aminopyrimidines.
  • Splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), pentet (p), doublet of doublets (dd), doublet of doublet of doublets (ddd), doublet of triplets (dt), triplet of doublets (td) and broad (b); the value of chemical shifts ( ⁇ ) are given in ppm and coupling constants (J) in hertz (Hz).
  • Routine MS spectra were acquired in the positive ion mode using Agilent G2446ATM Lc/MSD Trap XCTTM coupled to an Agilent 1100TM HPLC.
  • Representative compounds were characterized by high-resolution mass spectrometry (HR-MS) by using an Agilent 6230 ESI-TOFMS. Analytical and preparative TLC was performed on aluminum-backed plates (EMD Chemicals, San Diego, CA) and visualized by exposure to UV light.
  • HR-MS high-resolution mass spectrometry
  • Scheme SI Synthetic pathway leading to 4,6-substituted 2-aminopyrimidines 1-38.
  • boronic acid (0.30 mmol) was added to a solution of appropriate chloropyrimidine (0.15 mmol) in N,N-dimethylacetamide (1.5 mL).
  • Potassium carbonate 2M (0.2mL) was added following the addition of 1 , 1 '-Bis(diplienylpliosphino)ferrocene- palladium(II)dichloridedichlorometha.ne complex (0.0112 mg, 0.015 mmol).
  • the resulting mixture was stirred in a capped glass vial at 140 °C for 2h. The solvents were removed under reduced pressure, brine was added and the mixture was extracted with ethyl acetate (3 x 20 mL).
  • CDCI 3 ⁇ 1.35 (m, 2 H), 1.41 (s, 9 H), 1.46 (m, 2 H), 1.57 (m, 2 H), 3.08 (m, 2 H), 3.28 (bs, 2 H), 5.09 (bs, 2 H), 6.13 (s, 1 H), 7.64 (m, 2 H), 7.96 (m, 2 H).
  • ESI- MS [Ci 9 H 2 4F 3 50 2 + H] + 412 (100). (m,
  • N 4 ,iV 4 -dibenzyl-6-(4-(trifluoromethyl) phenyl)pyrimidine-2,4-diamine AC-19-22).
  • 5-Bromo-2-chloro-4-(4-methylpiperidin-l- yl)pyrimidine AC-2-P: A solution 4- methylpiperidine (0.362 g, 3.65 mmol) and DIPEA (0.567 g, 4.38 mmol) in 10 ml THF was added dropwise to an ice-cold solution of 5-bromo-2,4-dichloropyrimidine (1.0 g, 4.38 mmol) in 10 ml dry THF. The solution was warmed up to room temperature and stirred overnight. Evaporation of the solvent provided with a crude product that was purified by
  • Figure 8 Representative titration profiles for 4,6-substituted 2-aminopyrimidine competition (using exemplary compounds of the invention 1, 15, 30 and 32, see Table 4, above) with 3 H-epibatidine binding showing a range of dissociation constants (3 ⁇ 4) and Hill coefficients (nn) for ligand binding to Zs-AChBP. Measurements were carried out by a scintillation proximity assay and are reported as an average of at least three individual experiments ( ⁇ S.D.).
  • Figure 9 shows X-ray crystal structures of exemplary compounds of the invention (ligands) 32 and 33 (negative cooperativity, n H ⁇ l) and 15 (positive cooperativity, n H >l), in complexes with Zs-AChBP (compound numbering corresponds to Table 4).
  • Fig 9(A) illustrates radial view of Zs-AChBP pentameric structure in complex with exemplary compound of the invention 32 (compound numbering corresponds to Table 4). Full occupation of the 10 binding sites in the unit crystal of a dimer of pentamers was evident. A principal, C loop containing, and complementary face are shown in grey and purple.
  • Fig 9(B) illustrates an expanded radial view of exemplary compound 32 in binding site, including ligand electron density.
  • Fig 9(C) illustrates an overlay of exemplary compound 32 (blue) and exemplary compound 33 (yellow) crystal structures. Side chain carbons for exemplary compound 32 are in turquoise. The overlay shows little or no variance in ligand pose or side chain positions.
  • exemplary compound 33 (yellow) and exemplary compound 33 (blue) crystal structures. Side chain carbons for exemplary compound 33 are in turquoise.
  • the positively exemplary compound 15 and negatively exemplary compounds 32/33 exemplary compounds of the invention are in turquoise.
  • Figure 10 shows the superimposition of Zs-AChBP X-ray crystal structures in complex with exemplary compound 33 (Fig. 10A) and exemplary compound 15 (Fig. 10B) with nicotine (PDB code: 1UW6, 18) (in orange).
  • exemplary compound 33 and exemplary compound 15 carbons are shown in yellow, nicotine in orange.
  • the protein side chains are shown in grey for exemplary compounds 33 and 15 and pink for nicotine.
  • Both ligands show distinctly different positions from the pyrrolidine and pyridine rings of nicotine, as well as the side chain positions of residues in the C loop in the principal subunit (Y89, Y185) and the complementary (W53 and Y164) subunit face.
  • Figure 11 shows the global differences in X-ray crystal structures of Zs-AChBP bound cooperative ligands in comparison with crystal structure of Zs-AChBP in its Apo form (PDB code: 1UX2) and GLIC (4NPP):
  • Fig. 1 1(A) schematically illustrates a top (apical) view on superimposed (UCSF chimera) Apo pentamer (in blue) and with bound exemplary compound 15 (in red), dashed lines (blue and red respectively) indicate most significant differences in quaternary structures quantified by measuring distances between T13 backbone alpha carbon of distant subunits;
  • Fig. 1 1(A) schematically illustrates a top (apical) view on superimposed (UCSF chimera) Apo pentamer (in blue) and with bound exemplary compound 15 (in red), dashed lines (blue and red respectively) indicate most significant differences in quaternary structures quantified by measuring distances between T13 backbone alpha carbon of distant subunits;
  • FIG. 1 1(B) schematically illustrates a superimposition (PyMOL) of Zs-AChBP Apo, chain D (in blue) and exemplary compound 15 complex, chain D (in red).
  • Major differences in the quaternary structures of the AChBP are marked with dashed rectangles (RMS value of approximately 0.5 or greater).
  • the Ls -AChBP was expressed and purified as previously reported (1, 2). Briefly,
  • AChBP was expressed with an amino-terminal FLAG epitope tag and secreted from stably transfected HEK293S cells lacking the N-acetylglucosaminyltransferase I (GnTI-) gene.
  • the protein was purified using FLAG-antibody resin and eluted with FLAG peptide (Sigma) and was further characterized by size-exclusion chromatography (SUPEROSE 6 10/300 GLTM column (GE Healthcare) in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl,
  • AChBP pentamers were then concentrated in a YM50TM Centricon ultrafiltration unit (Millipore) removing monomeric subunits and trace contaminants. Protein concentrations were determined by UV absorbance at 280 nm (NANODROP (NanoDrop) 2000cTM spectrophotometer, ThermoScientific) and confirmed by Bradford assay.
  • NANODROP NanoDrop 2000cTM spectrophotometer
  • a scintillation proximity assay was employed to measure ligand binding to AChBP using [ 3 H]-epibatidine (5 nM, GE Healthcare) as the labeled ligand,
  • polyvinyltoluene anti-mouse SPA scintillation beads (0.17 mg/mL final concentration, GE Healthcare), monoclonal anti-FLAG M2 antibody from mouse 1 :8000 dilution
  • Ligand - Ls -AChBP complexes was formed by
  • Table 7 Data collection and refinement statistics.
  • Fig. 12(A) schematically illustrates an overlay of Zs-AChBP crystal structure in its Apo form (PDB code: 1UX2, shown in blue) and in complex with ligand 15 (in red), with GLIC X-ray crystal structure at pH 7.5 (PDB code: 4NPQ, shown in grey); X- ray structure of Zs-AChBP complex with nicotine and carbamylcholine (carbachol) used as controls (PDB codes: 1UW6 and 1UV6 respectively); stars represents position of the
  • Fig. 12(B) illustrates a chart representing 'bloom'; x axis: delta distance between C a observed in Zs-AChBP - ligand complexes when compared with Zs-AChBP Apo form (GLIC: X-ray structure obtained at pH 7.5 compared to the structure at pH 4); y axis: relative distance from the protein vestibule.
  • Fig. 12(C) illustrates a chart representing 'twist' of the pentameric structure; x axis: delta
  • reference points were defined by average coordinates of residue 194, average 5 coordinates of residue 12 and coordinates of residue 194 from corresponding subunit.
  • For GLIC reference points were defined by coordinates of residue 284 from corresponding subunit, average coordinates of residue 284 and average coordinates of residue 22.
  • Figure 14 Table 6, illustrated as Figure 14: shows competition between exemplary compounds of the invention (numbering corresponds to Table 4 compound
  • Figure 15 schematically illustrates an overlay of exemplary compound
  • Figure 16 illustrates a screening assay demonstrating activity of exemplary compounds of the invention (so-called compounds 17, KK-311-D and 171A, see
  • Fig. 16A and Fig. 16B compounds was screened at 50 uM and 10 uM, respectively, the nicotine agonist at 40 uM, and methyllycaconitine (or MLA) agonist at 400 uM; nicotine and MLA used a positive controls.
  • Compound 171 A (see Table 4) gave approximately 90% activation compared to nicotine.
  • Hibbs RE et al. (2009) Structural determinants for interaction of partial agonists with acetylcholine binding protein and neuronal alpha7 nicotinic acetylcholine receptor. £M5O J28(19):3040-3051.
  • Brams M, et al. (201 1) Crystal structures of a cysteine-modified mutant in loop D of acetylcholine-binding protein. J Biol Chem 286(6):4420-4428. Shahsavar A, et al. (2012) Crystal structure of Lymnaea stagnalis AChBP complexed with the potent nAChR antagonist ⁇ suggests a unique mode of antagonism. PLoS One 7(8):e40757.
  • Nicotine binding to brain receptors requires a strong cation-pi interaction.
  • Alpha-conotoxin OmIA is a potent ligand for the acetylcholine-binding protein as well as alpha3beta2 and alpha7 nicotinic acetylcholine receptors. J Biol Chem 281(34):24678-24686.
  • GABAA gamma-aminobutyric acid type-A

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Abstract

The invention provides compositions and methods a) for increasing or stimulating the release of other transmitters (excitatory amino acids, inhibitor amino acids and biogenic amines) in the central nervous system (CNS) or the brain, or modulating, decreasing or increasing the activity of an AChR in the CNS or brain, b) for treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse involving modulating, decreasing or increasing levels or activity of dopamine in the CNS or brain, c) for treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse involving e.g., drugs, narcotics or tobacco use, and d) for treating, ameliorating, preventing or lessening the symptoms of a disease or condition i.e., dementia, Parkinson's disease, pain or chronic pain, an allodynia, autism, psychosis or schizophrenia, responsive to the modulation of, or a decrease or an increase in the activity of nAChR.

Description

ACETYLCHOLINE BINDING PROTEIN LIGANDS, COOPERATIVE NACHR MODULATORS AND
METHODS FOR MAKING AND USING
5
RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. (USSN) 61/900,253, filed November 05, 2013. The
aforementioned application is expressly incorporated herein by reference in its entirety 10 and for all purposes.
GOVERNMENT RIGHTS
This invention was made with government support under grant GM 18360- 40, awarded by the National Institutes of Health (NIH). The government has
15 certain rights in the invention.
TECHNICAL FIELD
This invention relates to a family of compounds that interact with the
acetylcholine binding protein and the nicotinic acetylcholine receptor (nAChR) at the subunit interfaces in a cooperative manner. Either through their pre-synaptic or post- 20 synaptic actions, they modulate the release of acetylcholine or importantly other
neurotransmitters. These include the excitatory amino acids such as glutamate or aspartate; the inhibitor amino acids, such as γ-aminobutyric acid (GABA) and glycine;
and bigenic amines such as norepinephrine, epinephrine, dopamine and serotonin.
In particular, in alternative embodiments, the invention provides
25 compositions and methods for stimulating or inhibiting the release of or activity
these neurotransmitter or other neurohormones in the central nervous system
(CNS) or the brain, and the peripheral autonomic and somatic motor system in the periphery. In alternative embodiments, this action is mediated through various
subtypes of nAChR initiating depolarization or ion fluxes to stimulate other
30 neurotransmitter receptor systems. In alternative embodiments, the invention
provides compositions and methods for modulating, decreasing or increasing
levels of activity of dopamine in the CNS or brain, or methods for administering
compositions responsive to modulating, decreasing or increasing the activity of an nAChR in the CNS or brain. In alternative embodiments, compositions of the invention do not resemble acetylcholine structurally, but rather display an unusual cooperative binding interaction involving multiple sites on this pentameric protein. If the pentameric protein is composed of identical subunits, exemplary compounds of the invention can facilitate or inhibit the binding of acetylcholine. If the subunits are not identical, exemplary compounds of the invention can affect acetylcholine binding and stimulation in an allosteric manner.
In alternative embodiments, the invention provides compositions and methods for treating, ameliorating, preventing or lessening the symptoms of an tobacco or nicotine-related addiction, e.g., thereby promoting smoking cessation, or a substance abuse, by, e.g., preventing the abstinence syndrome and addiction in two ways; (a) serving as a partial agonist alternative to nicotine; and/or, (b) ameliorating the symptoms of withdrawal and abstinence through their actions on the dopamine and other neurotransmitter systems.
In alternative embodiments, the invention provides compositions and methods for treating, ameliorating, preventing or lessening the symptoms of disorders of development of the central nervous system, such as autism, schizophrenia, and other psychosis. A similar situation applies to disorders of aging, such as Alzheimer's diseases Parkinson's disease or psychoses.
In alternative embodiments, the invention provides compositions and methods for treating, ameliorating, preventing or lessening the symptoms of disorders of the peripheral nervous system with its sensory afferent and efferent pathways, including treating, ameliorating, preventing or lessening acute and chronic pain, and providing pain relief, including allodynia, including movement, thermal or mechanical allodynia.
In alternative embodiments, the invention provides compositions and methods for treating, ameliorating, preventing or lessening the symptoms of any condition responsive to an increase in levels or activity of dopamine in the CNS or brain, or any disease or condition responsive to the modulation of, or a decrease or an increase in the activity of nAChR. Since the acetylcholine binding protein (AChBP), from which the structure of the ligand complexes is deduced, is homologous to other pentameric ligand-gated ion channels, compounds of the invention also can modulate GABA, glycine and 5-hydroxytryptamine receptors in the CNS. BACKGROUND
Nicotinic acetylcholine receptors (nAChRs) are pentamers, which means that they are formed by five units that can be identical (or homomeric) or have different structures (or be heteromeric). Some proteins that are composed of more than one unit, and therefore have multiple binding sites, exhibit so called
cooperative binding. This means that when one molecule binds to one binding site at a subunit interface, the protein changes its shape (conformation) in such way, that it affects binding at other interfaces. Such ligands for nAChR proteins
haven't been described in the literature
Many different types of nAChR exist in the body, located in different
tissues and having different functions, but all closely related in structure. For
example, it has been hypothesized that agents which increase the activity of the alpha4beta2 nAChR subtype can produce an increase in dopamine levels,
counteracting the low dopamine levels that can occur, for example, in the absence of nicotine during smoking cessation attempts.
When a person inhales cigarette smoke, the nicotine in the smoke is
absorbed into the bloodstream and gains access to the brain within seconds. By activation of certain receptors, including nAChRs, nicotine increases the release of dopamine in the brain, a neurotransmitter that is responsible for feelings of pleasure. In theory, without the nicotine-induced elevation of dopamine levels, tobacco would not produce this type of reward.
The nicotinic acetylcholine receptor (nAChR) and the acetylcholine binding protein (AChBP) are pentameric oligomers in which binding sites for nicotinic agonists and competitive antagonists are found at selected subunit interfaces. The nAChR spontaneously exists in multiple conformations associated with its activation and desensitization steps, and conformations are selectively stabilized by binding of agonists and antagonists. In the nAChR, agonist binding and the associated conformational changes accompanying activation and desensitization are cooperative. AChBP, which lacks the transmembrane spanning and cytoplasmic domains, serves as a homology model of the extracellular domain of the nAChRs. nAChR structure is known at atomic resolution. SUMMARY
In alternative embodiments, the invention provides compounds that
exhibit cooperative binding to nicotinic acetylcholine receptor (nAChR) protein and stimulate an nAChR functional response. In alternative embodiments, the invention provides methods for formulating or using these compounds to increase or stimulate an nAChR functional response, e.g., to increase dopamine levels or activity.
In alternative embodiments, the invention provides compounds,
compositions or formulations comprising:
a) (i) a compound having a formula as set forth in Formula I;
Figure imgf000006_0001
Formula I
wherein Rl, R2, R3 and R4 are independently selected from the group consisting of: a hydrogen, an aryl (wherein optionally the aryl is any 5-or 6-membered ring, or is selected from the group consisting of: a heteroaryl, an aryl halide, a heteroaryl cycloalkyl, a phenyl, a naphthyl, a thienyl, an indolyl, a thiophene, or a isoxasole), an unsubstituted amino or a substituted amino (NRR'), a halo, a hydroxy (-OH), a substituted or an unsubstituted hydroxy (-OR), a phenoxy, a thiol (-SH), a substituted or an unsubstituted thiol (-SR), a cyano (-CN), a formyl (-CHO), a substituted or unsubstituted alkyl (wherein optionally the alkyl is selected from the group consisting of: - methyl, -ethyl, -propyl, -butyl, -i-propyl, and- i-butyl), a haloalkyl, a substituted or unsubstituted alkene, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkyne, a heteroalkyl, a heteroalkenyl, a heteroalkynyl, a substituted or unsubstituted aryl, a nitro (— N02), an alkoxy, a haloalkoxy, a thioalkoxy, a substituted or unsubstituted alkanoyl, a haloalkanoyl and a carbonyloxy group,
a methyl-aryl substituent, a benzylic substituent, an alkenyl, an alkynyl, a cycloalkyl (wherein optionally the cycloalkyl is selected from the group consisting of: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl, - cycloheptyl, and - cyclooctyl), a cycloalkenyl, a substituted alkyl, a substituted alkenyl, a substituted alkynyl, a substituted cycloalkyl, a substituted cycloalkenyl, an aryl, a heteroaryl silyl, a heterosilyl and a heterocyclic group (wherein optionally the heterocyclic group is selected from the group consisting of: a saturated heterocyclic and/or a nonsaturated heterocyclic, and optionally the saturated heterocyclic and/or a nonsaturated heterocyclic is selected from the group consisting of: -aziridine, -oxirane- thiirane, -azirine, - oxirene, -thiirene, -azetidine, -oxetane, -thietane, -azete, -oxete, -thiete, - pyrrolidine, -oxolane, -thiolane, -pyrrole, -furan, -thiophene, -piperidine, - oxane, -thiane, -pyridine, -pyran, -thiopyran, -azepane, -oxepane, -thiepane, - azepine, -oxepine, -thiepine, -azocane, and -azocine),
a carboxy (COOH), a carboxy derivative, a carboxylic halide (COX), an anhydride (COOCOR), an amide (CONRR'), an ester (COOR), a ketone (COR), an aldehyde (CHO) and a cyano (CN), or
an amidine, an N-substituted or unsubstituted amidines (— C(NR)NR'R") and a carbamidate (— CNOR),
wherein optionally in Formula I, R2 is a substituted amino (NRR', or N-R2-R3) , or
N
^ :' ^ * Formula II wherein optionally in Formula I, Rl is an amino group, R2 is a substituted amino (NRR', -R2-R3), R3 is a hydrogen and R4 is an aryl group, or
Figure imgf000007_0001
Formula III,
wherein R2 and R3 of Formula II, or Rl and R2 of formula III, are independently selected the group consisting of:
a hydrogen, an aryl (wherein optionally the aryl is any 5-or 6-membered ring, or is selected from the group consisting of: a heteroaryl, an aryl halide, a heteroaryl cycloalkyl, a phenyl, a naphthyl, a thienyl, an indolyl, a thiophene, or a isoxasole), an unsubstituted amino or a substituted amino (NRR'), a halo, a hydroxy (-OH), a substituted or an unsubstituted hydroxy (-OR), a phenoxy, a thiol (-SH), a substituted or an unsubstituted thiol (-SR), a cyano (-CN), a formyl (-CHO), a substituted or unsubstituted alkyl (wherein optionally the alkyl is selected from the group consisting of: - methyl, -ethyl, -propyl, -butyl, -i-propyl, and- i-butyl), a haloalkyl, a substituted or unsubstituted alkene, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkyne, a heteroalkyl, a heteroalkenyl, a heteroalkynyl, a substituted or unsubstituted aryl, a nitro (— N02), an alkoxy, a haloalkoxy, a thioalkoxy, a substituted or unsubstituted alkanoyl, a haloalkanoyl and a carbonyloxy group,
a methyl-aryl substituent, a benzylic substituent, an alkenyl, an alkynyl, a cycloalkyl (wherein optionally the cycloalkyl is selected from the group consisting of: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl, - cycloheptyl, and - cyclooctyl), a cycloalkenyl, a substituted alkyl, a substituted alkenyl, a substituted alkynyl, a substituted cycloalkyl, a substituted cycloalkenyl, an aryl, a heteroaryl silyl, a heterosilyl and a heterocyclic group (wherein optionally the heterocyclic group is selected from the group consisting of: a saturated heterocyclic and/or a nonsaturated heterocyclic, and optionally the saturated heterocyclic and/or a nonsaturated heterocyclic is selected from the group consisting of: -aziridine, -oxirane- thiirane, -azirine, - oxirene, -thiirene, -azetidine, -oxetane, -thietane, -azete, -oxete, -thiete, - pyrrolidine, -oxolane, -thiolane, -pyrrole, -furan, -thiophene, -piperidine, - oxane, -thiane, -pyridine, -pyran, -thiopyran, -azepane, -oxepane, -thiepane, - azepine, -oxepine, -thiepine, -azocane, and -azocine),
a carboxy (COOH), a carboxy derivative, a carboxylic halide (COX), an anhydride (COOCOR), an amide (CONRR'), an ester (COOR), a ketone (COR), an aldehyde (CHO) and a cyano (CN), or an amidine, an N-substituted or unsubstituted amidines (- C(NR)NR'R") and a carbamidate (-CNOR);
(ii) a compound having a formula as set forth in Formula III, wherein the aryl group of Formula III is a compound having a structure of Formula IV, wherein R6 of Formula IV is the compound of Formula I, Formula II, or Formula III, or, the R4 group of Formula I is a compound of Formula IV, or the Ar group of Formula III is a compound of Formula IV:
Figure imgf000009_0001
Formula IV,
wherein in Formula IV:
X, Y and Z, are independently selected from the group consisting of: a C and an N, Rl, R2, R3, R4 and R5 of Formula IV are independently selected from the group consisting of:
a hydrogen, an aryl (wherein optionally the aryl is any 5-or 6-membered ring, or is selected from the group consisting of: a heteroaryl, an aryl halide, a heteroaryl cycloalkyl, a phenyl, a naphthyl, a thienyl, an indolyl, a thiophene, or a isoxasole), an unsubstituted amino or a substituted amino (NRR'), a halo, a hydroxy (-OH), a substituted or an unsubstituted hydroxy (-OR), a phenoxy, a thiol (-SH), a substituted or an unsubstituted thiol (-SR), a cyano (-CN), a formyl (-CHO), a substituted or unsubstituted alkyl (wherein optionally the alkyl is selected from the group consisting of: - methyl, -ethyl, -propyl, -butyl, -i-propyl, and- i-butyl), a haloalkyl, a substituted or unsubstituted alkene, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkyne, a heteroalkyl, a heteroalkenyl, a heteroalkynyl, a substituted or unsubstituted aryl, a nitro (— N02), an alkoxy, a haloalkoxy, a thioalkoxy, a substituted or unsubstituted alkanoyl, a haloalkanoyl and a carbonyloxy group
a halogen, a methyl-aryl substituent, a benzylic substituent, an alkenyl, an alkynyl, a cycloalkyl (wherein optionally the cycloalkyl is selected from the group consisting of: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl, - cycloheptyl, and - cyclooctyl), a cycloalkenyl, a substituted alkenyl, a substituted alkynyl, a substituted cycloalkyl, a substituted cycloalkenyl, a heteroaryl silyl, a heterosilyl and a heterocyclic group (wherein optionally the heterocyclic group is selected from the group consisting of: a saturated heterocyclic and/or a nonsaturated heterocyclic, and optionally the saturated heterocyclic and/or a nonsaturated heterocyclic is selected from the group consisting of: -aziridine, -oxirane- thiirane, -azirine, -oxirene, -thiirene, - azetidine, -oxetane, -thietane, -azete, -oxete, -thiete, -pyrrolidine, -oxolane, - thiolane, -pyrrole, -furan, -thiophene, -piperidine, -oxane, -thiane, -pyridine, - pyran, -thiopyran, -azepane, -oxepane, -thiepane, -azepine, -oxepine, -thiepine, -azocane, and -azocine),
a carboxy (COOH), a carboxy derivative, a carboxylic halide (COX), an anhydride (COOCOR), an amide (CONRR'), an ester (COOR), a ketone (COR), an aldehyde (CHO) and a cyano (CN), or
an amidine, an N-substituted or unsubstituted amidines (— C(NR)NR'R") and a carbamidate (— CNOR);
(iii) a compound having a formula as set forth in Formula III, wherein the aryl group of Formula III is a compound having a structure of Formula V, wherein R5 of Formula V is the compound of Formula I, Formula II, or Formula III, or, the R4 group of Formula I is a compound of Formula V, or the Ar group of Formula III is a compound of Formula V:
Figure imgf000010_0001
Formula V,
wherein in Formula V:
wherein X, Y and Z, are independently selected from the group consisting of: a C an N, an O and an S,
Rl, R2, R3 and R4, of Formula V are independently selected from the group consisting of: a hydrogen, an aryl (wherein optionally the aryl is any 5-or 6-membered ring, or is selected from the group consisting of: a heteroaryl, an aryl halide, a heteroaryl cycloalkyl, a phenyl, a naphthyl, a thienyl, an indolyl, a thiophene, or a isoxasole), an unsubstituted amino or a substituted amino (NRR'), a halo, a hydroxy (-OH), a substituted or an unsubstituted hydroxy (-OR), a phenoxy, a thiol (-SH), a substituted or an unsubstituted thiol (-SR), a cyano (-CN), a formyl (-CHO), a substituted or unsubstituted alkyl (wherein optionally the alkyl is selected from the group consisting of: - methyl, -ethyl, -propyl, -butyl, -i-propyl, and- i-butyl), a haloalkyl, a substituted or unsubstituted alkene, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkyne, a heteroalkyl, a heteroalkenyl, a heteroalkynyl, a substituted or unsubstituted aryl, a nitro (— N02), an alkoxy, a haloalkoxy, a thioalkoxy, a substituted or unsubstituted alkanoyl, a haloalkanoyl and a carbonyloxy group
a halogen, a methyl-aryl substituent, a benzylic substituent, an alkenyl, an alkynyl, a cycloalkyl (wherein optionally the cycloalkyl is selected from the group consisting of: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl, - cycloheptyl, and - cyclooctyl), a cycloalkenyl, a substituted alkyl, a substituted alkenyl, a substituted alkynyl, a substituted cycloalkyl, a substituted cycloalkenyl, an aryl, a heteroaryl silyl, a heterosilyl and a heterocyclic group (wherein optionally the heterocyclic group is selected from the group consisting of: a saturated heterocyclic and/or a nonsaturated heterocyclic, and optionally the saturated heterocyclic and/or a nonsaturated heterocyclic is selected from the group consisting of: -aziridine, -oxirane- thiirane, -azirine, - oxirene, -thiirene, -azetidine, -oxetane, -thietane, -azete, -oxete, -thiete, - pyrrolidine, -oxolane, -thiolane, -pyrrole, -furan, -thiophene, -piperidine, - oxane, -thiane, -pyridine, -pyran, -thiopyran, -azepane, -oxepane, -thiepane, - azepine, -oxepine, -thiepine, -azocane, and -azocine),
a carboxy (COOH), a carboxy derivative, a carboxylic halide (COX), an anhydride (COOCOR), an amide (CONRR'), an ester (COOR), a ketone (COR), an aldehyde (CHO) and a cyano (CN), or
an amidine, an N-substituted or unsubstituted amidines (— C(NR)NR'R") and a carbamidate (— CNOR); wherein the compound, composition or formulation of any of (i), (ii) or (iii): exhibits a cooperative binding to a nicotinic acetylcholine receptor (nAChR) protein and modulates, inhibits or stimulates an AChR functional response, or
has a positive or a negative cooperativity in an acetylcholine ligand- acetylcholine receptor response, or in ligand occupation of the acetylcholine binding protein (AChBP), or
can modulate, inhibit or increase the magnitude and rapidity of a functional response of a nicotinic acetylcholine receptor (nAChR);
(b) a compound as set forth in Figure 2A, 2B, 3A, 3B, Figure 4, or Table 4; wherein optionally the compound is selected from the group consisting of:
Figure imgf000012_0001
-(4-Methoxyphenyl)-Ni-octylpyrimidine-2,4-diamine,
Figure imgf000012_0002
-Morpholino-6-(4-(trifluoromethyl)phenyl)pyrimidin-2-amine,
Figure imgf000012_0003
-(4-Methylpiperidin- l-yl)-6-(4-(trifluoromethyl)phenyl)pyrimidin-2-
Figure imgf000012_0004
Ethyl 2-amino-6-phenylpyrimidine-4-carboxylate,
Figure imgf000013_0001
6-(4-Methoxyphenyl)-N4,N4-bis( yridin-2-ylmethyl)pyrimidine-2,4-diamine; and a combination thereof;
(c) an enantiomer, a stereoisomer, an analog or a bioisostere of any of (a) or (b), or
(d) a salt of, or a pharmaceutically acceptable salt of, any of (a), (b) or (c).
In alternative embodiments, the invention provides products of manufacture or a device capable of injecting, causing inhalation of, adsorption of, or otherwise designed for administering for either enteral or parenteral administration: a compound, composition or formulation of the invention to an individual in need thereof, wherein optionally the product of manufacture or a device comprises: a compound, composition or formulation of the invention.
In alternative embodiments, the compound, composition or formulation of the invention is formulated for administration in vivo; or for enteral or parenteral administration, or for oral, ophthalmic, topical, oral, intravenous (IV), intramuscular (IM), intrathecal, subcutaneous (SC), intracerebral, epidural, intracranial or rectal administration, or by inhalation. In alternative embodiments, the compound, composition or formulation is formulated as: a particle, a nanoparticle, a liposome, a tablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, a suspension, a syrup, an emulsion, a lotion, an ointment, an aerosol, a spray, a lozenge, an ophthalmic preparation, an aqueous or a sterile or an injectable solution, a patch (optionally a transdermal patch or a medicated adhesive patch), or an implant.
In alternative embodiments, the invention provides pharmaceutical compositions or formulations comprising a compound, composition or formulation of the invention, wherein optionally the pharmaceutical composition or formulation further comprises a pharmaceutically acceptable excipient.
In alternative embodiments, the invention provides products of manufacture or a device, comprising a compound, composition or formulation of the invention, or a pharmaceutical composition or formulation of the invention, wherein optionally the product of manufacture or device is a medical device or an implant, wherein optionally the product of manufacture or device is designed to be capable of injecting, causing inhalation of, adsorption of, or otherwise administering for either enteral or parenteral administration a compound, composition or formulation of the invention, or a pharmaceutical composition or formulation of the invention.
In alternative embodiments, the invention provides a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump, comprising a compound, composition or formulation of the invention, or a pharmaceutical composition or formulation of the invention.
In alternative embodiments, the invention provides methods for
modulating, inhibiting or stimulating a nicotinic acetylcholine receptor (nAChR) functional response,
modulating, inhibiting or stimulating an nAChR response by cooperative binding to a nicotinic acetylcholine receptor (nAChR) protein,
having a positive or a negative cooperativity in an acetylcholine ligand- acetylcholine receptor response, or in ligand occupation of the acetylcholine binding protein (AChBP), or
modulating, inhibiting or stimulating the magnitude and rapidity of a functional response of a nicotinic acetylcholine receptor (nAChR),
comprising contacting the AChR with a compound, composition or formulation of the invention, or administering to an individual in need thereof a product of manufacture or a device of the invention, or a or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of the invention, thereby
modulating, inhibiting or stimulating a nicotinic acetylcholine receptor (nAChR) functional response,
modulating, inhibiting or stimulating an nAChR response by cooperative binding to a nicotinic acetylcholine receptor (nAChR) protein, having a positive or a negative cooperativity in an acetylcholine ligand- acetylcholine receptor response, or in ligand occupation of the acetylcholine binding protein (AChBP), or
modulating, inhibiting or stimulating the magnitude and rapidity of a functional response of a nicotinic acetylcholine receptor (nAChR),
wherein optionally the contacting is in vitro, ex vivo or in vivo.
In alternative embodiments, the invention provides methods for increasing, modulating or stimulating the release of or activity of a neurotransmitter or a
neuromodulator in the central nervous system (CNS) or the brain, or modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain,
wherein optionally the neurotransmitter or a neuromodulator comprises a glutamate, a gamma aminobutyric acid (GABA), a glycine, a serotonin, a peptide or a neuropeptide (optionally a galanin, an enkephalin, an acetylcholine), a norepinephrine, or a biogenic amine (optionally a dopamine, a noradrenaline, an adrenaline, or a
catecholamine),
comprising: administering to an individual in need thereof a compound, composition or formulation of the invention, or a pharmaceutical composition or formulation of the invention, or administering to an individual in need thereof a product of manufacture or a device of the invention, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of the invention,
thereby increasing, modulating or stimulating the release of or activity of a neurotransmitter or a neuromodulator in the central nervous system (CNS) or the brain, or modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain
wherein optionally the contacting is in vitro, ex vivo or in vivo.
In alternative embodiments, the invention provides methods for treating, ameliorating, preventing or lessening the symptoms of diseases or conditions that are responsive to modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or are responsive to modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain, wherein optionally the neurotransmitter or a neuromodulator comprises a glutamate, a gamma aminobutyric acid (GABA), a glycine, a serotonin, a peptide or a neuropeptide (optionally a galanin, an enkephalin, an acetylcholine), a norepinephrine, or a biogenic amine (optionally a dopamine, a noradrenaline, an adrenaline, or a catecholamine), comprising:
comprising: administering to an individual in need thereof a compound, composition or formulation of the invention, or a pharmaceutical composition or formulation of the invention, or administering to an individual in need thereof a product of manufacture or a device of the invention, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of the invention,
thereby treating, ameliorating, preventing or lessening the symptoms of diseases or conditions that are responsive to modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or are responsive to modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain,
wherein optionally the contacting is in vitro, ex vivo or in vivo.
In alternative embodiments, the invention provides methods for treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse, optionally an addiction or substance abuse involving use of tobacco or nicotine-related products, involving modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or responsive to modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain,
wherein optionally the neurotransmitter or a neuromodulator comprises a glutamate, a gamma aminobutyric acid (GABA), a glycine, a serotonin, a peptide or a neuropeptide (optionally a galanin, an enkephalin, an acetylcholine), a norepinephrine, or a biogenic amine (optionally a dopamine, a noradrenaline, an adrenaline, or a catecholamine), or for treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse involving tobacco or nicotine use, cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, or 3,4-methylenedioxy-N- methylamphetamine (MDMA), comprising:
administering to an individual in need thereof a compound, composition or formulation of the invention, or administering or applying to an individual in need thereof: a pharmaceutical composition or formulation of the invention; a product of manufacture or a device of the invention, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of the invention,
thereby treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse, optionally an addiction or substance abuse involving use of tobacco or nicotine-related products, involving modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or responsive to modulating, decreasing or increasing the activity of a nicotinic
acetylcholine receptor (nAChR) in the CNS or brain, or treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse involving tobacco or nicotine use, cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, or 3,4-methylenedioxy-N-methylamphetamine (MDMA),
wherein optionally the contacting is in vitro, ex vivo or in vivo.
In alternative embodiments, the invention provides methods for treating, ameliorating, preventing or lessening the symptoms of a disease or condition responsive to an increase in levels or activity of a neurotransmitter or a neuromodulator in the peripheral nervous system (PNS), the central nervous system (CNS) or brain, or a disease or condition responsive to the modulation of, or a decrease or an increase in the activity of a nicotinic acetylcholine receptor (nAChR), or treating, ameliorating, preventing or lessening the symptoms of a dementia, Parkinson's disease, pain or chronic pain, an allodynia, a psychosis, autism, or a schizophrenia,
wherein optionally the neurotransmitter or a neuromodulator comprises a glutamate, a gamma aminobutyric acid (GABA), a glycine, a serotonin, a peptide or a neuropeptide (optionally a galanin, an enkephalin, an acetylcholine), a norepinephrine, or a biogenic amine (optionally a dopamine, a noradrenaline, an adrenaline, or a
catecholamine),
comprising: administering to an individual in need thereof a compound, composition or formulation of the invention, or administering or applying to an individual in need thereof: a pharmaceutical composition or formulation of the invention; a product of manufacture or a device of the invention, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of the invention,
thereby: treating, ameliorating, preventing or lessening the symptoms of a disease or condition responsive to an increase in levels or activity of a neurotransmitter or a neuromodulator in the peripheral nervous system (P S), the central nervous system (CNS) or brain, or a disease or condition responsive to the modulation of, or a decrease or an increase in the activity of a nicotinic acetylcholine receptor (nAChR), or treating, ameliorating, preventing or lessening the symptoms of a dementia, Parkinson's disease, pain or chronic pain, an allodynia, a psychosis, autism, or a schizophrenia,
wherein optionally the contacting is in vitro, ex vivo or in vivo.
In alternative embodiments, the invention provides kits comprising a compound, composition or formulation of the invention, a product of manufacture or a device of the invention, and/or optionally comprising ingredients and/or instructions for practicing a method of the invention. In alternative embodiments, the invention provides kits comprising a compound, composition or formulation of the invention, a product of manufacture or a device of the invention, and/or optionally comprising ingredients and/or instructions for practicing a method of the invention.
In alternative embodiments, the invention provides kits comprising a compound, composition or formulation of the invention; a pharmaceutical composition or formulation of the invention; a product of manufacture or a device of the invention, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi- chambered pump of the invention, and/or optionally comprising ingredients and/or instructions for practicing a method of the invention.
In alternative embodiments, the invention provides uses of a compound, composition or formulation of the invention, in the manufacture of a medicament.
In alternative embodiments, the invention provides uses of a compound, composition or formulation of the invention, in the manufacture of a medicament for:
modulating, inhibiting or stimulating a nicotinic acetylcholine receptor (nAChR) functional response,
modulating, inhibiting or stimulating an nAChR response by cooperative binding to a nicotinic acetylcholine receptor (nAChR) protein,
having a positive or a negative cooperativity in an acetylcholine ligand- acetylcholine receptor response, or in ligand occupation of the acetylcholine binding protein (AChBP),
modulating, inhibiting or stimulating the magnitude and rapidity of a functional response of a nicotinic acetylcholine receptor (nAChR),
increasing, modulating or stimulating the release of or activity of a
neurotransmitter or a neuromodulator in the central nervous system (CNS) or the brain, or modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain,
treating, ameliorating, preventing or lessening the symptoms of diseases or conditions that are responsive to modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or are responsive to modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain,
treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse, optionally an addiction or substance abuse involving use of tobacco or nicotine-related products, involving modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or responsive to modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain, or
treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse involving tobacco or nicotine use, cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, or 3,4-methylenedioxy-N- methylamphetamine (MDMA),
treating, ameliorating, preventing or lessening the symptoms of a disease or condition responsive to an increase in levels or activity of a neurotransmitter or a neuromodulator in the peripheral nervous system (PNS), the central nervous system
(CNS) or brain, or a disease or condition responsive to the modulation of, or a decrease or an increase in the activity of a nicotinic acetylcholine receptor (nAChR), or
treating, ameliorating, preventing or lessening the symptoms of a dementia, Parkinson's disease, pain or chronic pain, an allodynia, a psychosis, autism, or a schizophrenia.
In alternative embodiments, the invention provides therapeutic combinations comprising: a compound, composition or formulation of the invention: a pharmaceutical composition or formulation of the invention; a product of manufacture or a device of the invention, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi- chambered pump of the invention. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby
expressly incorporated by reference for all purposes.
DESCRIPTION OF DRAWINGS
The drawings set forth herein are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figure 1A in table form, and Figure IB and Figure 1C in graphic form, summarizes Ki and slope (Hill coefficient, nn) data from radioligand binding
assays, N=3, which involved a screening of a collection of 80 compounds by a radioligand binding assay against AChBPs; these assays resulted in the identification of several lead structures with low micromolar affinities: including the exemplary compounds KK-141G, KK-169, as illustrated in Figure IB; and, the exemplary compounds KK-141G, KK-191G, as illustrated in Figure 1C) and unusually high Hill coefficient (nH) values, as illustrated in Figure 1A; as discussed in detail in Example 1, below.
Figure 2A schematically illustrates three exemplary synthetic pathways of the invention to generate exemplary 2,4,6-substituted pyrimidine compounds of the invention; as discussed in detail in Example 1, below.
Fig. 2B schematically illustrates exemplary compounds of the invention that were synthesized and screened; as discussed in detail in Example 1, below.
Figure 2C graphically illustrates the results of quick screen results of the so-called "KK-169 analog" exemplary compounds of the invention, including exemplary compounds of the invention KK 301-A, KK 301-B, KK 302, KK 303, KK 304-A, KK 304-B and KK 305 (see Figure 4, for structures); as discussed in detail in Example 1, below.
Figure 3 A schematically illustrates an exemplary compound of the invention, and graphically illustrates the results of an α4β2 nAChR agonist screening of a collection of 80 compounds using cell-based medium throughput fluorescence assays, which identified one exemplary compound of the invention, the so-called "KK-253B", that acted as a α4β2 nAChR agonist and did not activate a7 nAChR; as discussed in detail in Example 1, below.
Figure 3B schematically illustrates exemplary compounds of the invention identified using cell-based assay as α4β2 nAChR antagonists; as discussed in detail in Example 1, below.
Figure 4 schematically illustrates exemplary compounds of the invention, the so-called Formula I and Formula II genus structures, which we classified in "cooperative series 1", and include structures of potent ligands of this invention, including 4,6-substituted 2-amino pyrimidines 2; and these exemplary 4,6- substituted 2-amino pyrimidines 1 can be divided in three groups when based on Hill coefficient values, ligands with nH < 1, nH = 1 and nH >1, as illustrated in Figure 5; as discussed in detail in Example 1, below.
Figure 5 graphically illustrates data from titration curves using radioligand binding assay for the 4,6 substituted 2-aminopyrimidines showing the range of potencies and Hill coefficients for ligand binding: Fig. 5A, graphically illustrates data showing that if the binding of ligand at one site lowers the affinity for ligand at another site on an adjacent subunit, the protein exhibits negative cooperativity or induced site heterogeneity, nH < 1; Fig. 5B, graphically illustrates data showing that nicotine and all classical agonists or antagonists are examples of a non- cooperative system for AChBP with nH = 1 ; and, Fig. 5C, graphically illustrates data showing that if the binding of ligand at one site increases the affinity for ligand at another site, the macromolecule exhibits positive cooperativity (nn >1); as discussed in detail in Example 1, below.
Figure 6A schematically illustrates an exemplary scintillation proximity screening scheme of the invention comprising screening of a compound library by a radioligand binding assay against AChBPs; this screen assay resulted in the identification of several additional exemplary compounds of this invention, more so-called "lead structures", with low micromolar affinities; and from these exemplary compounds, or "leads", approximately 40 exemplary compounds, or analogs, were identified, synthesized and screened, as illustrated in Figure 6B.
Figure 6B graphically illustrates, and Figure 6C in table form illustrates, radioligand screening results of the pyrimidine series against Lymnea AChBP, where the compounds marked in green (or the so-called "AC" fraction, the middle of the 3 fractions) also have low micromolar Kd values for Aplysia AChBP; as discussed in detail in Example 1, below.
Figure 7 schematically illustrates the crystal structure of Ls AChBP-ligand complex: Fig. 7A schematically illustrates a side image, and Fig. 7B
schematically illustrates a bottom side view image showing molecules in five binding sites; and, Fig. 7C schematically illustrates ligands having nH<l for both, and Fig. 7D schematically illustrates nH <l and nH >l images superimposed; as discussed in detail in Example 1, below.
Figure 8 graphically illustrates data from representative titration profiles for 4,6-substituted 2-aminopyrimidine (exemplary compounds of the invention, see Table 4 to associate numbering with structure) competition with 3H- epibatidine binding showing a range of dissociation constants (Kd) and Hill coefficients (nH) for ligand binding to Zs-AChBP; as discussed in detail in Example 2, below. Figure 9 shows X-ray crystal structures of exemplary compounds of the invention (ligands) 32 and 33 (negative cooperativity, nH<l) and exemplary compound 15 (positive cooperativity, nH>l), in complexes with Zs-AChBP: Fig 9(A): illustrates radial view of Zs-AChBP pentameric structure in complex with 5 exemplary compound of the invention 32; Fig 9(B): illustrates an expanded radial view of exemplary compound 32 in binding site, including ligand electron density; Fig. 9(C) illustrates overlay of exemplary compound 32 (blue) and exemplary compound 33 (yellow) crystal structures; and, Fig 9(D): illustrates a superimposition of exemplary compound 15 (yellow) and exemplary compound 10 33 (blue) crystal structures; as discussed in detail in Example 2, below.
Figure 10 shows the superimposition of Zs-AChBP X-ray crystal structures in complex with exemplary compound 33 (Fig. 10A) and exemplary compound 15 (Fig. 10B) with nicotine; as discussed in detail in Example 2, below.
15 Figure 11 shows the global differences in X-ray crystal structures of Ls-
AChBP bound cooperative ligands in comparison with crystal structure of Ls- AChBP in its Apo form: Fig. 1 1(A) schematically illustrates a top (apical) view on superimposed (UCSF chimera) Apo pentamer (in blue) and with bound exemplary compound 15 (in red), dashed lines (blue and red respectively) indicate 0 most significant differences in quaternary structures quantified by measuring distances between T13 backbone alpha carbon of distant subunits; Fig. 1 1(B) schematically illustrates a superimposition (PyMOL) of Zs-AChBP Apo, chain D (in blue) and exemplary compound 15 complex, chain D (in red); Fig. 1 1(C) illustrates a chart of differences of Ca distances (n=5) of diametrical subunits 5 observed in cooperative ligands relative to Apo in comparison with GLIC; Fig.
1 1(D) illustrates a plot of differences of Ca dihedral angles (n=5) observed in cooperative ligands relative to Apo in comparison with GLIC; as discussed in detail in Example 2, below.
Figure 12 shows a comparison of Zs-AChBP quaternary changes with
30 changes observed for GLIC: Fig. 12(A) schematically illustrates an overlay of Ls- AChBP crystal structure in its Apo form; Fig. 12(B) illustrates a chart representing 'bloom'; x axis: delta distance between Ca observed in Zs-AChBP - ligand complexes when compared with Zs-AChBP Apo form, y axis: relative distance from the protein vestibule; and, Fig. 12(C) illustrates a chart representing 'twist' of the pentameric structure; x axis: delta dihedral angle between Ca observed in Zs-AChBP - ligand complexes when compared with Zs-AChBP Apo form; as discussed in detail in Example 2, below.
Figure 13 graphically illustrates the comparison of delta distance ± SD
('bloom') observed in the Zs-AChBP complexes binding pocket; nicotine X-ray structure used as a control; exemplary compounds of the invention 15, 32, and 33 are used (numbering corresponds to Table 4 structure numbers).
Figure 14 illustrates Table 6, showing data of competition between exemplary compounds of the invention (numbering corresponds to Table 4 compound numbers) as substituted 2-aminopyrimidines against 3H-epibatidine binding to Zs-AChBP; as discussed in detail in Example 2, below.
Figure 15: Fig. 15(A) schematically illustrates an overlay of exemplary compound 15 (yellow) and exemplary compound 32 (blue) crystal structures; Fig. 15(B) schematically illustrates superimposition of exemplary compound 15
(yellow) and exemplary compound 33 (blue) crystal structures (numbering corresponds to Table 4 compound numbers); as discussed in detail in Example 2, below.
Figure 16 illustrates a screening assay demonstrating activity of exemplary compounds of the invention (so-called compounds 17, KK-311-D and 171 A, see Table 4 for structures), using activation of a7-nAChr CNiFERS with l-(5-chloro- 2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl); Fig. 16A and Fig. 16B graphically illustrates the results at 50 uM and 10 uM concentrations,
respectively, and Fig. 15C graphically illustrates these results; as discussed in detail in Example 2, below.
Figure 17 schematically illustrates three (1, 2, 3) exemplary synthetic pathways of the invention to generate exemplary 2,4,6-substituted pyrimidine compounds of the invention, including the so-called Formula I genus structure; as discussed in detail in Example 2, below.
Figure 18 schematically illustrates exemplary synthetic pathways of the invention to generate exemplary 2,4,5 -substituted pyrimidine compounds of the invention, including the so-called Formula I genus structure; as discussed in detail in Example 2, below. Like reference symbols in the various drawings indicate like elements.
Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention.
DETAILED DESCRIPTION
In alternative embodiments, the invention provides compounds and
compositions that are selective ligands to acetylcholine binding protein (AChBP) and have unique properties. Exemplary compounds and compositions of the
invention, these AChBP ligands, have both negative as well as positive
cooperativity, which is a type of allostery in which binding at one subunit
interface in an oligomeric protein affects the affinity of a distant interface. This unique feature of these exemplary compounds and compositions of the invention make them useful as medications with distinct pharmacological profiles.
This invention for the first time describes cooperative binding activity by the acetylcholine binding protein (AChBP), compounds and compositions that can modulate this cooperative binding activity. Since AChBP only consists of the extracellular domain of the nAChR, compounds of the invention uniquely show that cooperativity, be it negative or positive, can occur in a circumferential
fashion in the extracellular domain. Studies with the nAChR indicated that
cooperativity is mediated through the transmembrane domains, but this is no
longer the case. Hence, this invention widens the structural base for achieving selectivity with the nAChR. Moreover, the structures of exemplary compounds of the invention depart substantially from structures of the classical agonists and antagonists of the nAChR. Hence, the structural differences of exemplary
compounds of the invention allows them to be subtype selective.
The invention establishes a previously-unknown level of conformational communication of AChBP subunits upon ligand binding. Interaction of a ligand in the first binding pocket causes structural changes between subunits resulting in a transition between different affinity states. In case of negative cooperativity, the unoccupied site in the pentamer becomes structurally restrained, leading to reduced affinity for binding of the second ligand. By translating the phenomena to nicotinic receptors, ligands
showing different cooperativity profiles may give distinct functional responses dependent on receptor sites occupations at different concentrations. A difference in signaling of this sort may be very important to the magnitude and rapidity of functional responses of receptors (e.g., nicotinic and ligand gated ion channel) in the CNS and brain.
In alternative embodiments, compositions and formulations of the
invention act as ligands that bind cooperatively to the acetylcholine binding
protein with positive and negative cooperativity in the same family of structures the 2-aminopyrimidines. In alternative embodiments, compositions and
formulations of the invention have relatively high affinities, or low dissociation constants - particularly for the negatively cooperative ligands. In alternative embodiments, compositions and formulations of the invention cause
conformational changes in the acetylcholine binding protein that are seen globally in the protein; these conformational changes are unique to the 2-aminopyrimidines that are not seen in the classical agonists (for example, nicotine or epibatidine) or antagonists (for example, benzylidene anabaseine,
methyllycaconitine (MLA) etc.). In alternative embodiments, exemplary
compositions and formulations of the invention can stimulate the alpha-7 nicotinic receptor, thus acting as drugs or pharmaceutically active reagents.
Screening of a collection of 80 compounds by a radioligand binding assay against AChBPs resulted in the identification of exemplary compounds of the invention, so-called "lead structures", with low micromolar affinities (e.g., KK- 141G, KK-169, as illustrated in Figure IB) and unusually high Hill coefficient
(nH) values, as illustrated in Figure 1A. Following this finding approximately 50 analogs were synthesized and screened providing information about structure- activity relationships.
Until this discovery, ligands associating with the AChBP ligands exhibit binding as predicted for an equivalent set of five sites in the pentameric molecule, a situation also seen with classical natural product agonists and antagonists binding to the AChBP. In alternative embodiments, the invention provides exemplary compounds of the invention as "congeneric structures" that exhibit cooperativity, where many members of the family show Hill slopes greater than 1.0 or considerably less than 1.0. The latter is not due to heterogeneity of the binding protein template, since AChBP has five identical sites as examined crystallographically and by conventional ligand binding. These cooperative interactions appear not to require a direct connection with the channel gating area to elicit subunit interactions and cooperativity. Rather, cooperativity can be confined to the extra-cellular domain and is mediated circumferentially around the cylindrical pentamer. This unique feature of compounds of the invention, these AChBP ligands, makes them effective as medications with distinct pharmacological profiles.
Figure 4 illustrates generic structures of the invention in cooperative series 1; structure of most potent ligands, 4,6-substituted 2-amino pyrimidines 2. We observed that the series of 4,6-substituted 2-amino pyrimidines 1 (Figure 4) can be divided in three groups when based on Hill coefficient values, ligands with nH < 1, nH = 1 and nH >1 (Figure 5):
(1) Nicotine and all classical agonists or antagonists are examples of a non- cooperative system for AChBP with nH = 1 (see Fig. 5B).
(2) If the binding of ligand at one site increases the affinity for ligand at another site, the macromolecule exhibits positive cooperativity (nn >1) (see Fig. 5C).
(3) Conversely, if the binding of ligand at one site lowers the affinity for ligand at another site on an adjacent subunit, the protein exhibits negative cooperativity or induced site heterogeneity, nH < 1 (see Fig. 5A).
Hence, partial occupation of sites in the pentamer may diminish occupation of remaining sites through changes in conformation. Through the structural analysis, we can delineate the structural features of the ligands inducing this new mode of association with the extracellular domain of the receptor. Our approach of finding a series of approximately 50 congeneric ligands where individual members, distinguished by positive and negative cooperativity, sheds light on subunit interactions in this homopentamer. For example, cooperative interactions appear to link in a circumferential fashion around the perimeter of the extracellular domain and do not necessarily require a direct connection with the channel gate area to elicit subunit interactions. Figure 5 graphically illustrates data from titration curves using radioligand binding assay for the 4,6 substituted 2-aminopyrimidines showing the range of potencies and Hill coefficients for ligand binding. The black curves are nicotine nn=l.
Observed cooperativity is not due to heterogeneity of the binding protein template, since AChBP has five identical sites as examined crystallographically and by
conventional ligand binding. We solved structures at high resolution for three cooperative ligands in complexes with Ls ACHBP (nH < 1 : KK-31 1A and KK-321C; nH > 1 : KK- 325B). The ligands with the steep Hill slopes require higher concentrations to occupy the first site, but subsequent occupations of four remaining sites are facilitated. By contrast, ligands exhibiting the shallow slopes occupy the first site at lower concentrations, but each subsequent occupation is more difficult statistically.
Having examined the binding poses of various ligands showing defined stoichiometries, we can define four distinctive means of stabilization of ligands at the orthosteric site:
(1) quaternary amines, stabilized through cation-π interactions,
(2) secondary and tertiary amines and imines stabilized through hydrogen bond donation from the protonated nitrogen to a backbone carbonyl of a tryptophan on the principal subunit,
(3) peptides that conform to the dimensions of the C loop or a space bounded by its radial surface, and
(4) this new set of ligands showing diverse cooperativity profiles. Because amino nitrogens do not protonate at physiological pH values, the
2-aminopymidine ring system and nitrogens immediately attached to it are
uncharged, and it may be that this electron rich ring system contributes to the
specificity of these compounds.
In alternative embodiments, the invention provides compounds that
exhibit cooperative binding to nicotinic acetylcholine receptor (nAChR) protein.
By testing exemplary compounds of the invention using cell-based assays, it was demonstrated that exemplary compounds of the invention can stimulate a nAChR functional response. In alternative embodiments, the invention provides two
types (or classes or genuses) of exemplary compounds: one class (or type or
genus) binds in a cooperative manner to nAChR binding proteins, and a second exemplary class (or type or genus) activates a functional response of an nAChR by its binding.
In alternative embodiments, both classes of compounds of the invention can increase the release of dopamine in the CNS or brain, or increase the activity of nAChR in the CNS or brain. Thus, in alternative embodiments, the invention provides compounds, compositions and methods for increasing or stimulating the release of or activity of dopamine in the central nervous system (CNS), including the brain, or increasing the activity of nAChR in the CNS or brain, and compounds, compositions and methods for treating, ameliorating, preventing or lessening the symptoms of diseases or conditions that are responsive to an increase in levels or activity of dopamine in the CNS or brain, or responsive to an increase in the activity of nAChR in the CNS or brain. In alternative
embodiments, the invention provides compounds, compositions and methods for treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse involving increasing levels or activity of dopamine in the CNS or brain, or responsive to an increase in the activity of nAChR in the CNS or brain, for example, for treating, ameliorating, preventing or lessening the symptoms of an addiction or a substance abuse, e.g., an addiction or a substance abuse involving cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, such as 3,4-methylenedioxy-N-methylamphetamine
(MDMA).
In alternative embodiments, the invention provides compounds, compositions and methods for treating, ameliorating, preventing or lessening the symptoms of a disease or condition responsive to an increase in levels or activity of dopamine in the CNS or brain, or responsive to an increase in the activity of nAChR in the CNS or brain, for example, dementia, Parkinson's disease, pain or chronic pain, allodynia, autism, psychosis or schizophrenia.
Bioisosteres of Compounds of the Invention
In alternative embodiments, the invention also provides bioisosteres of compounds of the invention, e.g., compounds having a structure as set forth in Table 1, Table 2, Table 3 or Table 4. In alternative embodiments, bioisosteres of the invention are compounds of the invention comprising one or more substituent and/or group replacements with a substituent and/or group having substantially similar physical or chemical properties which produce substantially similar
biological properties to a compound of the invention, or stereoisomer, racemer or isomer thereof. In one embodiment, the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of a
compound without making significant changes in chemical structures.
For example, in one embodiment, bioisosteres of compounds of the
invention are made by replacing one or more hydrogen atom(s) with one or more fluorine atom(s), e.g., at a site of metabolic oxidation; this may prevent
metabolism (catabolism) from taking place. Because the fluorine atom is similar in size to the hydrogen atom the overall topology of the molecule is not
significantly affected, leaving the desired biological activity unaffected.
However, with a blocked pathway for metabolism, the molecule may have a
longer half-life or be less toxic, and the like. Formulations and pharmaceutical compositions
In alternative embodiments, the invention provides compounds and compositions, including formulations and pharmaceutical compositions, for use in in vivo, in vitro or ex vivo methods, e.g., for:
treating, ameliorating, preventing or lessening the symptoms of diseases or conditions that are responsive to modulating, decreasing or increasing levels or activity of a dopamine in the CNS or brain, or are responsive to modulating, decreasing or increasing the activity of an AChR in the CNS or brain,
treating, ameliorating, preventing or lessening the symptoms of an addiction or a substance abuse involving modulating, decreasing or increasing levels or activity of dopamine in the CNS or brain, or responsive to modulating, decreasing or increasing the activity of an AChR in the CNS or brain,
treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse, e.g., an addiction or substance abuse involving cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, or 3,4- methylenedioxy-N-methylamphetamine (MDMA),
treating, ameliorating, preventing or lessening the symptoms of a disease or condition responsive to an increase in levels or activity of dopamine in the CNS or brain, or a disease or condition responsive to the modulation of, or a decrease or an increase in the activity of nAChR; or,
for treating, ameliorating, preventing or lessening the symptoms of a dementia, Parkinson's disease, pain or chronic pain, allodynia, autism, psychosis or schizophrenia.
In alternative embodiments, the pharmaceutical compositions of the invention can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. In alternative embodiments, pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, capsules, suspensions, taken orally, suppositories and salves, lotions and the like. Pharmaceutical formulations of this invention may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, geltabs, on patches, in a thin-film or dissolving film, in implants, etc. In practicing this invention, the pharmaceutical compounds can be delivered by
transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
In alternative embodiment, compositions of the invention are delivered orally, e.g., as pharmaceutical formulations for oral administration, and can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Oral carriers can be elixirs, syrups, capsules, tablets, pills, geltabs and the like. Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, dextrins, or a salt thereof, such as sodium alginate. In alternative embodiments, liquid carriers are used to manufacture or formulate compounds of this invention, or a composition used to practice the methods of this invention, including carriers for preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compounds. The active ingredient (e.g., a composition of this invention) can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can comprise other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators.
In alternative embodiments, solid carriers are used to manufacture or formulate compounds of this invention, or a composition used to practice the methods of this invention, including solid carriers comprising substances such as lactose, starch, glucose, methyl-cellulose, magnesium stearate, dicalcium phosphate, mannitol and the like. A solid carrier can further include one or more substances acting as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material. In powders, the carrier can be a finely divided solid which is in admixture with the finely divided active compound. In tablets, the active compound is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methylcellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
In alternative embodiments, compounds and pharmaceutical compositions of the invention are formulated as and/or delivered as patches, e.g., a transdermal patch or a medicated adhesive patch that is placed on the skin or mucous membrane to deliver a specific dose of drug or medication (e.g., compounds and pharmaceutical compositions of the invention) through the skin and into the bloodstream. An advantage of a transdermal drug delivery route over other types of medication delivery such as oral, topical, intravenous, intramuscular, etc. can be that the patch provides a controlled release of the drug or medication into the patient, optionally through either a porous membrane covering a reservoir of medication or through body heat melting thin layers of medication embedded in the adhesive.
In alternative embodiments, a patch is a single-layer drug-in-adhesive patch; in this exemplary embodiment, the adhesive layer also contains the drug or medication (e.g., compounds and pharmaceutical compositions of the invention). In this type of patch the adhesive layer can not only serves to adhere the various layers together, along with the entire system to the skin, but also can be responsible for the releasing of the drug or medication. The adhesive layer can be surrounded by a temporary liner and a backing.
In alternative embodiments, a patch is a multi-layer drug-in- adhesive patch, which is similar to the single-layer system, but it adds another layer of drug-in-adhesive, optionally separated by a membrane. One of the layers can be for immediate release of a drug or medication (e.g., compounds and pharmaceutical compositions of the invention) and other layer is for control release of the same and/or different drug or medication from the reservoir. This patch also can have a temporary liner-layer and a permanent backing. In alternative embodiments, drug release depends on membrane permeability and diffusion of drug molecules.
In alternative embodiments, a patch is a reservoir transdermal system, which has a separate drug layer; the drug layer can be a liquid or gel compartment comprising a drug solution or a suspension separated by the adhesive layer. The drug reservoir can be totally encapsulated in a shallow compartment molded from a drug-impermeable metallic plastic laminate, optionally with a rate-controlling membrane made of a polymer (e.g., a vinyl acetate) on one surface. This patch also can be backed by a backing layer. In a reservoir transdermal system the rate of release can be designed to be zero order. In alternative embodiments, a patch is a matrix system, or so-called "monolithic device", which comprises a drug layer of a solid or a semisolid matrix comprising a drug solution or a suspension (e.g., comprising compounds and pharmaceutical compositions of the invention). The adhesive layer in this patch can surround the drug layer, optionally partially overlaying it.
In alternative embodiments, compounds and pharmaceutical compositions of the invention are formulated as and/or delivered as or in so-called "thin-film" or dissolving film delivery systems. These can be used to administer a drug solution or a suspension (e.g., comprising compounds and pharmaceutical compositions of the invention) via absorption in the mouth (e.g., buccally or sublingually) and/or via the small intestines or otherwise enterically. A film can be prepared using a hydrophilic polymer that rapidly dissolves on a mucous membrane, e.g., in the tongue or buccal cavity or esophagus or intestine, thus delivering the drug to the systemic circulation via dissolution when contact with liquid (e.g., a bodily fluid) is made.
In alternative embodiments, thin-film drug delivery is used as an alternative to or with another delivery modality, e.g., tablets, capsules, liquids and the like. They can be similar in size, shape and thickness to a postage stamp, and can be designed for oral administration, with the user placing the strip on or under the tongue (sublingual) or along the inside of the cheek (buccal). As the strip dissolves, the drug can enter the blood stream enterically, buccally or sublingually. In alternative embodiments, thin-films are made of combination of microcrystalline cellulose and maltodextrin, and can also include plasticizers, phthalate, glycols.
In alternative embodiments, concentrations of therapeutically active compound in a formulation can be from between about 0.1% to about 100%, e.g., having at least about 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or more, by weight.
In alternative embodiments, therapeutic formulations are prepared by any method well known in the art, e.g., as described by Brunton et al, eds., Goodman and Gilman's: The Pharmacological Bases of Therapeutics , 12th ed., McGraw-Hill, 201 1; Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; Avis et al, eds., Pharmaceutical Dosage Forms: Parenteral Medications, published by Marcel Dekker, Inc., N.Y., 1993; Lieberman et al, eds., Pharmaceutical Dosage Forms: Tablets, published by Marcel Dekker, Inc., N.Y., 1990; and Lieberman et al., eds., Pharmaceutical Dosage Forms: Disperse Systems, published by Marcel Dekker, Inc., N.Y., 1990.
In alternative embodiments, therapeutic formulations are delivered by any effective means appropriated for a particular treatment. For example, depending on the specific antitumor agent to be administered, the suitable means include oral, rectal, vaginal, nasal, pulmonary administration, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) infusion into the bloodstream. For parenteral administration, antitumor agents of the present invention may be formulated in a variety of ways. Aqueous solutions of the modulators can be encapsulated in polymeric beads, liposomes, nanoparticles or other injectable depot formulations known to those of skill in the art. In alternative embodiments, compounds of the invention are administered encapsulated in liposomes. In alternative embodiments, depending upon solubility, compositions are present both in an aqueous layer and in a lipidic layer, e.g., a liposomic suspension. In alternative embodiments, a hydrophobic layer comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such a diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. For example, in adults, an exemplary dosage may be about 30 mg/kg administered e.g., by intravenous therapy, e.g., over between about 15 to 30 minutes, or by intramuscular injection or subcutaneous injection, e.g., repeated later in intervals, e.g., at about 60 minutes later. In alternative
embodiments, an exemplary dosage and administration is as a 500 mg/h continuous IV infusion. In alternative embodiments, for children, an exemplary dosage and
administration is at between about 20 to 50 mg/kg, optionally followed by a maintenance infusion at between about 5 to 10 mg/kg/h.
In alternative embodiments, an exemplary dosage and administration is based on long term, low dosage administration, for example, by a slow release pharmaceutical vehicle, or by slow release from an implant.
Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co., Easton PA ("Remington's"). For example, in alternative embodiments, these compositions of the invention are formulated in a buffer, in a saline solution, in a powder, an emulsion, in a vesicle, in a liposome, in a
nanoparticle, in a nanolipoparticle and the like. In alternative embodiments, the compositions can be formulated in any way and can be applied in a variety of concentrations and forms depending on the desired in vivo, in vitro or ex vivo conditions, a desired in vivo, in vitro or ex vivo method of administration and the like. Details on techniques for in vivo, in vitro or ex vivo formulations and administrations are well described in the scientific and patent literature. Formulations and/or carriers used to practice this invention can be in forms such as tablets, pills, powders, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for in vivo, in vitro or ex vivo
applications.
In practicing this invention, the compounds (e.g., formulations) of the invention can comprise a solution of compounds of the invention, including stereoisomers, derivatives and analogs thereof, disposed in or dissolved in a pharmaceutically acceptable carrier, e.g., acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any fixed oil can be employed including synthetic mono- or diglycerides, or fatty acids such as oleic acid. In one embodiment, solutions and formulations used to practice the invention are sterile and can be manufactured to be generally free of undesirable matter. In one embodiment, these solutions and formulations are sterilized by conventional, well known sterilization techniques.
The solutions and formulations used to practice the invention can comprise auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and can be selected primarily based on fluid volumes, viscosities and the like, in accordance with the particular mode of in vivo, in vitro or ex vivo administration selected and the desired results.
The compositions and formulations of the invention can be delivered by the use of liposomes. In alternative embodiments, by using liposomes, particularly where the liposome surface carries ligands specific for target cells or organs, or are otherwise preferentially directed to a specific tissue or organ type, one can focus the delivery of the active agent into a target cells in an in vivo, in vitro or ex vivo application.
The compositions and formulations of the invention can be directly administered, e.g., under sterile conditions, to an individual (e.g., a patient) to be treated. The modulators can be administered alone or as the active ingredient of a pharmaceutical composition. Compositions and formulations of this invention can be combined with or used in association with other therapeutic agents. For example, an individual may be treated concurrently with conventional therapeutic agents.
In alternative embodiments, a compound, a formulation or mixture of compounds of the invention is/are administered parenterally in an appropriate co-solvent to enable distribution from the site of IM, SC or IV injection, to prevent post- injection precipitation by virtue of a change in pH, for example, as described in J. Pharm. Pharmacol: 62:873-82 (2010); Adv. Drug Delivery Rev. 59:603-07 (2007), and to ensure "solubilization" conditions at the injection site, e.g., as described in J. Pharm. Pharmacol 62: 1607-21; Anesth Analg 79: 933-39 (1994); J. Pharm. Pharmacol 65 1429-39 (2013). Although these compounds with two ionization equilibria can form zwitterions, the predominant species is uncharged at physiologic pH. Similar to certain parenteral anesthetics, the compounds of the invention can be administered at low pH (e.g., between about pH 4 to 6) or high pH (e.g., between about pH 8 to 11). Hence these alternative embodiments involve: Low pH solutions adjusted with acetic acid; High pH solutions adjusted with a2C03 (pH 10- 11); Co-solvent formulation at neutral pH to include propylene glycol (up to 50%), polyethylene glycol, 2-hydroxypropyl β-cyclodextrin and combinations and congeners thereof; and/or, micellular dispersions with surface active agents.
These approaches can insure more rapid systemic absorption from the sites of administration. The above references document enhanced rates of absorption using these procedures for drugs of similar solubility: ketamine, etomidate, thiopental, diclofenac, aripiprazole, carbamazepine.
In alternative embodiments, oral (p.o.) preparations encompass tablets and capsules, including syrups emulsions and suspensions to insure distribution throughout the gastrointestinal (GI) tract. Nanoparticles, Nanolipoparticles and Liposomes
The invention also provides nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising compounds and compositions used to practice the methods of this invention. For example, in alternative embodiments, the invention provides nanoparticles, nanolipoparticles, vesicles and liposomal membranes for low dosage and/or slow release of a compound of the invention.
The invention provides multilayered liposomes comprising compounds used to practice this invention, e.g., as described in Park, et al, U.S. Pat. Pub. No. 20070082042. The multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice this invention.
Liposomes can be made using any method, e.g., as described in Park, et al, U.S. Pat. Pub. No. 20070042031, including method of producing a liposome by encapsulating an active agent (e.g., a compound of the invention), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution.
. In one embodiment, liposome compositions used to practice this invention comprise a substituted ammonium and/or polyanions, e.g., for targeting delivery of a compound (e.g., e.g., a compound of the invention) used to practice this invention to a desired cell type or organ, e.g., brain, as described e.g., in U.S. Pat. Pub. No.
20070110798.
The invention also provides nanoparticles comprising compounds (e.g., a compound of the invention) used to practice this invention in the form of active agent- containing nanoparticles (e.g., a secondary nanoparticle), as described, e.g., in U.S. Pat. Pub. No. 20070077286. In one embodiment, the invention provides nanoparticles comprising a fat-soluble active agent of this invention or a fat-solubilized water-soluble active agent to act with a bivalent or trivalent metal salt. In one embodiment, solid lipid suspensions can be used to formulate and to deliver compositions used to practice this invention to mammalian cells in vivo, in vitro or ex vivo, as described, e.g., in U.S. Pat. Pub. No. 20050136121.
Dosaging
The pharmaceutical compositions and formulations of the invention can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions or formulations of the invention are administered to a subject that is responsive to modulating, decreasing or increasing levels or activity of a dopamine in the CNS or brain, or is responsive to modulating, decreasing or increasing the activity of an nAChR in the CNS or brain (a "therapeutically effective amount"). The pharmaceutical compositions and formulations of the invention also can be administered as a preventative agent, e.g., prophylactically.
The amount of pharmaceutical composition adequate to accomplish this is defined as a "therapeutically effective dose." The dosage schedule and amounts effective for this use, i.e., the "dosing regimen," will depend upon a variety of factors, including the stage of the exposure, the severity of the exposure, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51 :337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84: 1144-1 146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; the latest Remington's, supra). The state of the art allows the clinician to determine the dosage regimen for each individual patient and particular active agent. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate. Products of Manufacture, Kits
The invention also provides products of manufacture and kits for
practicing the methods of this invention, and comprising compounds, compositions and formulations of this invention, including bioisostere compounds of the invention. In alternative embodiments, the invention provides products of manufacture and kits comprising compounds, compositions and formulations of this invention, and comprising all the components needed to practice a method of the invention.
The invention provides kits comprising compounds, compositions and formulations of this invention, and comprising compositions and/or instructions for practicing methods of the invention. In alternative embodiments, the invention provides kits comprising: a composition used to practice a method of any of the invention, optionally comprising instructions for use thereof.
In alternative embodiments, the invention provides pumps, devices, subcutaneous infusion devices, continuous subcutaneous infusion device, infusion pens, needles, reservoirs, ampoules, vials, syringes, cartridges, disposable pen or jet injectors, prefilled pens or syringes or cartridges, cartridge or disposable pen or jet injectors, two chambered or multi-chambered pumps, syringes, cartridges or pens or jet injectors comprising a composition, composition or a formulation of the invention. In alternative embodiments, the injector is an auto injector, e.g., a SMART JECT® autoinjector (Janssen Research and Development LLC); or a MOLLY®, or DAI®, or DAI-RNS® autoinjector (SHL Group, Deerfield Beach, FL). In alternative embodiments, the injector is a hypodermic or a piston syringe.
The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples. EXAMPLES
Example 1 : Exemplary compositions, formulations and combinations of the invention
This example describes exemplary compositions, formulations and combinations of the invention, and methods for making them.
The inventors used in situ click chemistry (see e.g., Kolb (2001)
Angewandte Chemie International Edition 40 (1 1): 2004-2021), which uses the target "alpha4beta2 nAChR protein" to assemble its own molecular modulator. An "anchor molecule" having a linker that does not react with the protein but is able to react with other molecules that bind nearby on the protein surface was discovered. In this assay, these anchor molecules were incubated in the presence of the protein with collection of such small molecules. The formation of a linked molecule, which can only occur if both portions bind to the protein, creates a "biligand" that binds more tightly than either piece alone. The occurrence of a successful target-templated reaction is indicated by very sensitive mass spectrometry methods, identifying the molecule formed and allowing us to make it in quantity.
Initially, a collection of more than 80 novel compounds were screened using various assays, including radioligand binding assays for three AChBP as well as two cell based assays, a7- and α4β2- nAChR CNiFERs (see e.g., Yamauchi JG et al, PLoS ONE, 2011).
Three substituted pyrimidines were found to bind to binding proteins with low micro molar affinity, as illustrated in Figure 1. Interestingly all three molecules showed average Hill Slope value different than 1.0. The Hill coefficient (nn) provides a quantitative method for characterizing binding cooperativity (see e.g., Briggs, Biophys. Chem., 1983). In a non-cooperative system such as nicotine, n = 1 at all ligand concentrations (Figure 1). In contrast, compounds KK-169, KK-141G and KK-191am3 show values different than 1.0, which indicate cooperative binding. Cooperative binding is a special case of allostery that requires multiple binding sites, since cooperativity results from the interactions between them. In many proteins (such as hemoglobin) the binding of the first ligand to the protein can change the affinity for the second ligand (see e.g., Miele, J. Mol. Biol. 2012).
Summary of the AChBPs activities of the originally identified lead structures: the table of Figure 1A summarizes ¾ values (μΜ) and Hill coefficient, nn obtained by radioligand proximity assay against Ls- and Ac- AChBP, which involved a screening of a collection of 80 compounds by a radioligand binding assay against AChBPs; these assays resulted in the identification of several lead structures with low micromolar affinities (e.g., the exemplary KK-141G, KK-169, KK-191am3) and unusually high Hill coefficient (nH) values. If the binding of ligand at one site increases the affinity for ligand at another site, the macromolecule exhibits positive cooperativity (nn >1).
Conversely, if the binding of ligand at one site lowers the affinity for ligand at another site, the protein exhibits negative cooperativity, nn<l . It may also mean that two different independent binding sites with different affinities participate in ligand binding (see, e.g., KRIJSEK, Physiol. Res., 2004). Cooperative binding to AChBP can lead to ligands with unique pharmacological profiles.
Based on the initial screen we synthesized additional analogs of KK-169. As outlined in Figure 2, entry (A), three different synthetic pathways were explored to increase flexibility of pyrimidine ring substitution. All of them employed inexpensive, commercially available starting materials. One exemplary pathway, route or scheme of the invention, as illustrated in Figure 2, entry A, path 1, was to react 2,4,6-trichloropyrimidine first with a boronic acid followed by reaction with an amine. Remaining, unreacted chlorine in the position 2 was then substituted by number of different nucleophiles. An exemplary, alternative route or scheme of the invention, as illustrated in Figure 2, entry A, path 2, started with 4,6-dichloro-2amniopyrmididine that after substitution with an amine and palladium coupling reactions led to a substituted 2-aminopyrimidine. Another exemplary scheme of the invention, an aldol condensation of aromatic aldehydes with pyruvate, as illustrated in Figure 2, entry A, path 3, gave unsaturated intermediates that was reacted with guanidine to afford substituted 2- amniopyrimidines having carbonyl group in position 4.
These exemplary pathways of the invention complement each other and open an access to high number of analogs with various substituents in three positions in pyrimidine ring. Synthesized compounds (see Figure 2, entry B), analogs of KK-169 were tested and one of them, KK-304B showed strong binding to Ls AChBP, as illustrated in Figure 2, entry C.
To identify and characterize nAChR agonists and antagonists, we used cell4oased medium throughput fluorescence assays. One compound, KK-253B, passed the initial quick screen as a α4β2 nAChR agonist and did not activate a7 nAChR. Additionally we found series of compounds that act as α4β2 nAChR antagonists, as illustrated in Figure 3. For performing in situ click chemistry to identify potential dual-site binders, azides and alkynes were used as complementary reactants in triazole formation directed by the protein itself. Soluble forms of α4β2 nAChR subtypes were used in these experiments. Ligands identified by mass spectrometry can be re-synthesized and tested to confirm activity.
Screening of a collection of 80 compounds using cell-based medium throughput fluorescence assays identified one compound, KK-253B (see Figure 3(A)), that acted as a α4β2 nAChR agonist and did not activate a7 nAChR.3
In a separate series of experiments, screening of the compound library by a radioligand binding assay against AChBPs resulted in the identification of several additional exemplary compounds of this invention, more so-called "lead structures", with low micromolar affinities. Following initial leads,
approximately 40 compounds, or analogs, were synthesized and screened, as illustrated in Figure 6(A), illustrating the initial screen using scintillation proximity assay, and Figure 6(B) and 6(c), showing radioligand screening results of the pyrimidine series against Lymnea AChBP, where the compounds marked in green also have low micromolar Kd values for Aplysia AChBP; thus, giving information about structure-activity relationships.
Having examined the binding poses of various ligands showing defined stoichiometries,6 we demonstrated and identified four distinctive means of stabilization of ligands at the orthosteric site:
(1) Quaternary amines, stabilized through cation-p interactions,
(2) secondary and tertiary amines and imines stabilized through hydrogen bond donation from the protonated nitrogen to a backbone carbonyl of a tryptophan on the principal subunit,
(3) peptides that conform to the dimensions of the C loop or a space bounded by its radial surface, and
(4) this new set of ligands showing this diverse cooperativity, as illustrated in (Figure 7).
Through the structural analysis, we delineated the structural features of the ligands inducing this new mode of association with the extracellular domain of the receptor. Figure 7 schematically illustrates the crystal structure of Ls AChBP-ligand complex: Fig. 7(A) Side and Fig. 7(B) bottom side view showing molecules in five binding sites. Ligands Fig. 7(C) nH<l for both Fig. 7(D) nH <l and nH >l
superimposed.
Figure 8 graphically illustrates representative titration profiles for 4,6-substituted
2-aminopyrimidine (exemplary compounds of the invention, see Table 4, to associate numbering with structure) competition with 3H-epibatidine binding showing a range of dissociation constants (¾) and Hill coefficients (nn) for ligand binding to Zs-AChBP.
Figure 9 graphically illustrates global differences in X-ray crystal structures of Ls- AChBP bound cooperative ligands in comparison with crystal structure of Zs-AChBP in its Apo form.
Figure 14, as Table 6, summarizes data of X-ray crystal structures of exemplary compounds of the invention (ligands) 32 and 33 (negative cooperativity, nH<l) and exemplary compound 15 (positive cooperativity, nH>l), in complexes with Zs-AChBP: Fig 9(A): illustrates radial view of Ls -AChBP pentameric structure in complex with exemplary compound of the invention 32; Fig 9(B): illustrates an expanded radial view of exemplary compound 32 in binding site, including ligand electron density; Fig. 9(C) illustrates overlay of exemplary compound 32 (blue) and exemplary compound 33
(yellow) crystal structures; and, Fig 9(D): illustrates a superimposition of exemplary compound 15 (yellow) and exemplary compound 33 (blue) crystal structures.
Figure 17 schematically illustrates three (1, 2, 3) exemplary synthetic
pathways of the invention to generate exemplary 2,4,6-substituted pyrimidine compounds of the invention, including the so-called Formula I genus structure.
Reagents and conditions for the protocols of Figure 17: (a) Boronic acid,
palladium catalyst; (b) amine; (c) nucleophile; (d) Amine; (e) (i) KOH, MeOH (ii) AcCl, EtOH, reflux (f) urea or guanidine or amidine.
Figure 16C graphically illustrates data of the activation of a7-nAChR CNiFERs after 30 min cell incubation with PNU- 120596. Concentration dependent response of a7- nAChR CNiFERs to Ac-171A (EC50 = 4.2 μΜ). Nicotine used as a control. Assay details were as described in: Yamauchi JG, Nemecz A, Nguyen QT, Muller A, Schroeder LF, Talley TT, Lindstrom J, Kleinfeld D, Taylor P. Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors, PLoS One. 2011,
6(l):el6519. Figure 18 schematically illustrates exemplary synthetic pathways of the invention to generate exemplary 2,4,5 -substituted pyrimidine compounds of the invention, including the so-called Formula I genus structure. Reagents and
conditions for the protocols of Figure 18: Reagents and conditions: (a) amine; (b) boronic acid, palladium catalyst; (c) KCN; (d) NaOH or KOtBu; (e) amine,
coupling reagent; (f) a 3; (g) alkyne, Cu (I); (h) H2.
The following table summarizes data of exemplary 4,6-substituted 2- aminopyrimidines in vitro screening data obtained with radioligand binding assay (Ka and Hill slope for Ls-, Ac- and Y55 W-AchBPs) and cell based assays (EC50 for α7-, α4β2- nAChRs and 5HT3A receptor):
Figure imgf000045_0001
References - Example 1
(1) (i) Rahman, S. Prog. Mol. Biol. Transl. Sci. 2011, 98, 349. (ii) Caponnetto, P.; Russo, C; Polosa, R. Curr. Opin. Pharmacol. 2012, 12, 229.
(2) De Biasi, M.; Dani, J. A. Annu. Rev. Neurosci. 201 1, 34, 105.
(3) Yamauchi, J. G.; Nemecz, A.; Nguyen, Q. T.; Muller, A.; Schroeder, L. F.;
Talley, T. T.; Lindstrom, J.; Kleinfeld, D.; Taylor; P. PLoS ONE 2011, 6, 16519.
(4) Monod, J.; Wyman, J.; Changeoux, J.-P. J. Mol. Biol. 1965, 12, 88-1 18.
(5) Allosteric Interactions and Biological Regulation (Part I), J. Mol. Biol.
2013, 425, 1391-1592.
(6) Miller, M. T.; Hansen, S. B.; Mcintosh, J. M.; Olivera, B. M.; Taylor, P.
FASEB J. 2006, 20, A244. Example 2: Structural Basis for Cooperative Interactions of Substituted 2- Aminopyrimidines with the Acetylcholine Binding Protein
This example describes exemplary compositions, including 4,6- disubstituted 2-aminopyrimidines, formulations and combinations of the
invention, and methods for making them.
We have identified unique cooperative binding behavior of exemplary 4,6- disubstituted 2-aminopyrimidine compounds of the invention to a Lymnaea AChBP, with different molecular variants exhibiting positive, nn > 1.0, and negative cooperativity, nn < 1.0. Therefore, for a distinctive set of ligands, the extracellular domain of a nAChR surrogate suffices to accommodate cooperative interactions. X-ray crystal structures of AChBP complexes with examples of each allowed the identification of structural features in the ligands that confer differences in cooperative behavior. Both sets of molecules bind at the agonist-antagonist site, as expected from their competition with epibatidine (or, (lR,2R,45)-(+)-6-(6-chloro-3-pyridyl)-7-azabicyclo[2.2.1]heptane). An analysis of AChBP quaternary structure shows that cooperative ligand binding is associated with a blooming or flare conformation, a structural change not observed with the classical, non- cooperative, nicotinic ligands. Exemplary compounds of the invention behaved as positively and negatively cooperative ligands and exhibited unique features in the detailed binding determinants and poses of the complexes.
Heretofore, ligand recognition at each subunit interface of the AChBP has been found to be independent of the other interfaces, representing a disconnection between the properties of the AChBP and the full receptor that it is intended to model. These results comprise the first examples of cooperative binding with the extracellular domain, providing insights into the structural basis for interactions between subunits. Within a single series of congeneric molecules, both positively and negatively cooperative behaviors towards AChBP are manifest. Hence, a distinct mode of binding to the agonist- competitive antagonist site is established in the AChBP protein.
Nicotinic acetylcholine receptors (nAChRs) function as allosteric pentamers of identical or homologous transmembrane spanning subunits. Ligand binding at two or more of the five inter-subunit sites, located radially in the extracellular domain, drives a conformational change that results in the opening of a centrosymmetric transmembrane channel, internally constructed amongst the five subunits (See Fig. l2A) (1-4). Up to five potential agonist-competitive antagonist sites on the pentamer are found at the outer perimeter of the subunit interfaces. Amino acid side chain determinants on both subunit interfaces dictate selectivity amongst the many subtypes of nAChRs. The interconversion between resting, active and desensitized states occurs in the absence of ligands, and partial occupation of the binding sites suffices for agonist activation of the receptor and its antagonism (5-7). Cooperativity of agonist association and its coupling to channel gating likely play important roles in the dynamics of nicotinic responses and in sharpening the concentration and temporal windows for activation.
As revealed in functional studies, most nAChRs are hetero-oligomeric, where the sites of ligand occupation are not identical (1-4). This arrangement arises when a common a-subunit pairs with one or more non-identical subunit partners, termed non a-subunits (7-8). Non-identity of the subunit interface complementary to the a subunit may also give rise to heterogeneity in binding constants typically seen for antagonists and mask partially the degree of agonist cooperativity. An exception to this is the a7-neuronal nAChR composed of five identical subunits and exhibiting a high degree of cooperativity for agonist activation (9). Recently, sequence alignments identified genes coding for pentameric ligand-gated ion channels in prokaryotes led to the resolution of the first structure by X-ray crystallography on 3D crystals of a pentameric receptor protein from Erminia chrysanthemi (ELIC) (10) and Gloeobacter violaceus (GLIC) (11-12) and provided high resolution structures of the two end-point states of the cooperative gating mechanism in the same pentameric, ligand-gated ion channel (GLIC) (13). Recently, the first structure of a eukaryotic member of the family, the anionic glutamate receptor from Caenorhabditis elegans (GluCl), was solved at atomic resolution (14) revealing remarkable identity of 3D structure with GLIC.
The acetylcholine binding protein (AChBP) was characterized from mollusks (15-
17) and consists of only a homologous extracellular domain of the nAChR. Assembled as a homomeric pentamer, AChBP exhibits a similar profile of ligand selectivity toward the classical nicotinic agonists and antagonists of quaternary amine, tertiary and secondary amine (alkaloid), imine, and peptide origin that bind nicotinic receptors (18-25). If looked at solely on the basis of ligand binding capacities, AChBP could be considered as a distinct subtype of nAChR. While its homomeric composition and ligand selectivity best resemble the a 7-subtype of nAChR, when the concentration dependence of ligand occupation has been examined, no evidence of cooperativity emerged (21). This suggests that the cooperative behavior for both activation and desensitization of receptors, seen for the classical nicotinic agonists with nAChRs, might arise from a cooperative torsional motion driven by the transmembrane spanning domain of the receptor (26).
We demonstrate here a set of ligands, all exemplary compounds of this invention, that bind to the AChBP in a cooperative fashion, whereby binding to a single subunit affects the binding energy at identical interfaces in the pentamer. Hence, interactions within the extracellular domain of this family of homologous pentameric proteins establish a circumferential linkage between subunit interfaces which results in cooperative behavior.
Results
Association of Substituted 2-Aminopyrimidines with AChBP Reveals Positive and Negative Cooperativity.
After preliminary assessment of other scaffolds, we focused our attention on 4,6- disubstituted 2-aminopyrimidines as being representative of drug-like molecules having a propensity to associate with neurological signaling receptors. These exemplary compounds showed selectivity for binding to Lymnaea stagnalis (Ls)-AOaS? when characterized in a radioligand competition assay. These exemplary compounds of the invention could be divided in three groups of binding profiles based on Hill coefficients: ligands with nH < 1, nH approximately 1, and nH >1; see Figure 8.
Nicotine competition with epibatidine served as our positive control and is an example of a non-cooperative ligand for AChBP with nn = 1. If the binding of ligand at one site increases the affinity for ligand at a corresponding homologous sites on the pentamer, the ligand exhibits positive cooperativity (nn >1) with AChBP. Conversely, if the binding of ligand at one site diminishes the affinity for ligand at another site, the protein exhibits negative cooperativity or site heterogeneity, nn < 1. Hence, partial occupation of sites in the pentamer diminishes allosterically with occupation of the remaining sites.
Overall, a broad range of affinities are revealed in this series of congeneric compounds with apparent ¾ values of 0.2 nM to > 10 μΜ (Table 1). Many compounds from the series showed Hill slope values close to one (Tablel ; 28, 37). However, compounds with large Hill coefficients were identified, and were found to exhibit lower, but still respectable, affinities (Fig. 1; 1, 15). In contrast, some ligands with shallow titration curves bound with exceptionally high affinity (Fig. 1 ; 30, 32). There was significant tolerance to substitution at position 4 of the pyrimidine ring, accepting lipophilic alkyl (Table 1 ; 12-15) and aryl (Table 1 ; 28-30), as well as more polar groups (Table 1; 27, 35). These substituents strongly influence the range of ligand ¾ and Hill coefficients. A few heterocyclic aromatic rings substituted at 6-position were tested (Table 1; 9-11), but no aryl group was found to give superior activity compared with substituted phenyl ring.
Crystal Structures of Zs-AChBP with Substituted 2-Aminopyrimidines Shows Distinctive Tertiary Structural Changes at the Binding Interface. To gain structural insight, crystallization of several complexes was attempted. Crystal structures with Zs-AChBP in complex with ligands showing negative (32, Fig. 2A and B, 33 Fig. 2C; chains A and B) and positive (15, Fig. 2D; chains D and E) cooperativity were refined to 3.0, 2.1 and 2.7 A respectively. For statistics on data collections, see SI Appendix. The Zs-AChBP complex with bound cooperative ligands reflected full occupation of the pentamer and a well resolved electron density of all 10 subunits (Fig. 2A and B). Ligand 15 has a clear electron density for the bi-aryl ring of the molecule, but a poor density of the alkyl chain possibly arising from multiple flexible conformations of the C loop. Several residues in F the loop (T155-E163) gave unresolved electron densities and were consequently excluded from the models. Additionally, in complex with ligand 15, residues 185-189 in the C loop encompassing the vicinal Cys-Cys bond were not seen in nine often subunits of the dimer of pentamers.
The structure of Zs-AChBP in complex with 33 superimposed on 32 with r.m.s. deviation of 0.38 A for 1,212 Ca atoms (Fig. 2C) and on 15 with r.m.s. deviation of 0.34 A for 1, 132 Ca atoms (Fig. 2D). Binding orientations show high degree of similarity at all five binding sites in each pentamer and a similar orientation of the ligands. All three compounds bind underneath a closed C loop at the interface between two subunits. Loop closure in the presence of bound ligands, as measured from the backbone carbonyl of W143 in the A loop to the γ-sulfur atom of the first vicinal Cys disulfide linked residue in the C loop (C187 in Zs-AChBP), is 8.4 A for 32 and 33 structures and 8.2 A for 15. The ligands contact amino acids from both the principal face with residues from loops A (Y89), B (W143), and C (Y185 and Y192), and complementary face (loops D: W53, E: LI 12 and Ml 14, F: Y164). Parallel displaced π-stacking interactions with W143 are present in all structures. Importantly, the pyrimidine ring of 15, the ligand showing positive cooperativity, rotates by -26-36° compared to 32 and 33 showing negative cooperativity (Fig. S5). The ring rotation results in its parallel alignment with Y192 side chain (2.9 A and greater). The only potentially protonable nitrogen in the ligands at physiological pH, is in the pyrimidine ring at the position Nl (pKa~6.7-6.8; Marvin Sketch 5.12.3). It resides within hydrogen bonding distance of the carbonyl backbone oxygen of W143 (2.7-2.9 A) and as close as 3.8-3.9 A to W143 side chain aromatic ring. Other interactions in the complexes come from polar contacts formed by ligands N2 atoms with the hydroxyl group of Y89 side chain from the B-loop (2.9 A in 32 and 33; 2.7 A in 15) and carbonyl oxygen of S142 (2.6-2.8) A for all three ligands.
Morpholine (32) or 4-methyl-piperidine (33) substituents appear to associate with Y89 and Y185. Nitrogen atoms of these rings are positioned to stack with all 7 atoms of the Y185 ring with distances ranging between 3.6-4.3 A. Interactions of these substituents on the complementary subunit face are predominantly hydrophobic. The altered position of the indole of W53 in 32 and 33, compared with 15, is associated with a change in side chain orientation of neighboring residues Ml 14 and Q55. Phenyl rings substituted at the pyrimidine 6-position interact mainly with W143, Y192 and CI 88. Additionally, fluorines in trifluoromethyl substituent are in the vicinity of T 144 side chain and interact with multiple loop F residues, including LI 12 and Ml 14 as well as neighboring water molecules, with interaction extending to Ml 14 and R104 side chains.
The flexible aliphatic chain of 15 at the 4 position in the pyrimidine does not yield discernable electron densities except for chain D. This likely reflects multiple conformations of the flexible C loop not constrained by the symmetry related molecule in the crystal structure. Rotation of the pyrimidine ring and presence of the alkyl chain in 15 brings the ligand in close contact with Y 185 side chain (~3.0 A to N4 of the ligand) and causes the tyrosine ring to rotate toward the gorge interface presumably to avoid a clash with the alkyl chain of the ligand. Also, the indole of W53 on the complementary face rotates towards the subunit interface and is in contact with ligand N4 (3.5 A). The phenyl ring in ligand 15 has a similar position as the aromatic ring in complexes 32 or 33, but its contacts are altered by methoxy- substituent interacting with T144 and with LI 02, LI 12 and Ml 14 in the complementary face. Major differences for 15, when compared to complexes of ligands showing negative cooperativity, are seen in Y185, Y164, Ml 14 and W53 side chains conformations.
The interactions of 32/33 are compared with nicotine the in Zs-AChBP binding pocket (Protein Data Bank, PDB ID code 1UW6) (18) in Figure 3A. The pyrrolidine ring in nicotine only partially overlaps with pyrimidine ring of cooperative ligands, and nitrogens of these substituents are well aligned. In contrast to nicotine, the side chain of Y89 shifts to bring its hydroxyl group in hydrogen bonding distance of the pyrimidine nitrogens in the 32, 33 and 15 crystal structures. Striking differences in side chain positions in nicotine complex compared to 32 and 33 are seen on the complementary face of the binding site that forms interactions through residues that are only partially conserved in AChBPs from different species. To accommodate the morpholine or methylpiperidine substituents in the 32/33 complex, the indole side chain in W53 changes its rotameric position. As a consequence Ml 14 is brought in contact with the 6- substituted trifluoromethyl phenyl group of the ligand. The position of the 32/33 phenyl ring is similar to the pyridine ring in nicotine. The trifluoromethyl group forces LI 12 to change its rotameric position. The presence of fluorines, however, affords additional hydrophobic stabilization with multiple residues on complementary subunit.
Compared with the nicotine-AChBP complex, Loop C in ligand 15 (Chain D) shows significant variation, reflected in the Y185 side chain conformation (Fig. 3B). Residues W53 and Ml 14 in the 32/33 complexes, that represent most significant departures from nicotine, in the 15 X-ray structure have orientations similar to those in the nicotine complex. However, the indole ring of W53 side chain has more extensive contacts with the ligand 15 than with nicotine, through its substituent at the position 4 in the pyrimidine ring. To avoid steric occlusion with Y 185 in the complex, the rotational state of Y164 on complementary face changes as well.
Quaternary Structural Changes of Zs-AChBP.
Superimposing Zs-AChBP in the Apo form with the complexes of substituted 2- aminopyrimidines showing cooperativity (ligands 15, 32, 33) also revealed a major change in quaternary structure (Fig. 4, Panel A). Distances between Ca for T 13 in the apical -helices of diametrically opposed subunits showed 7 A greater trans-subunit spans than for the Apo reference structures or the nicotine complex. By superimposing a single chain in each pentamer (Fig. 4B), differences in tertiary structure could be observed for six regions (r.m.s.d. approximately 5 or greater, Swiss-PdbViewer): residues 8-14 (N-terminal a-helix), loop 22-24, 43-44 (loop connecting βΐ and β2), 61-69 (η ΐ and its continuation), 156-162 (region in β8 N-terminal to loop F), 182-185 (part of β9 and loop C). Based on a similar analysis for nicotine and carbamylcholine Zs-AChBP crystal 5 structures (SI Appendix Fig. S2), differences observed for four regions appeared to be unique for cooperative ligand complexes (regions 8-14, 22-24, 42-43 and 61-69). To quantify observed variations, two types of calculations were performed using 1UX2 as a reference structure (for detailed description of calculations see SI Appendix).
Differences in distances between every a carbon (Ca) of diametrically opposed
10 subunits were calculated relative to the reference structure (Fig. 4C). Additionally
differences in dihedral angles for every Ca residue were calculated (using three reference points) relative the reference structure (Fig. 4D). Values were compared to those obtained for the nicotine complex and the open and closed states of prokaryotic GLIC protein (13). For the a -carbon distances (Fig. 4C), significant differences compared to nicotine
15 complex were observed for residues 1-120 with largest differences emerging in the N- terminal helix. Interestingly these differences showed similar patterns in linear sequence to those observed for GLIC (13) and described as a blooming motion of the protein ECD. Region 155-162 in complex 15 shows a significant distance reduction compared to nicotine. This is a highly flexible part of the protein, as is the case for complexes 32 and
20 33, where this region is omitted due to limiting densities in the crystal structure. Although short sequence regions in the crystal with positively cooperative ligand 15 diverge from crystals of 32/33 with negative cooperativity (e.g. residues 182-189), owing to differences between ligands 32 and 33 themselves, interpretation of the fine structural differences between 15 and 32, 33 will require additional study. Based on the rearrangements
25 observed at the interface of the orthosteric agonist/antagonist pocket (SI Appendix, Fig.
S3) the greatest deviations from nicotine (up to ~4 A) are evident for Y185 and C187-8 in the crystal structure of the 32 and 33 complexes.
Relative differences of dihedral angles show small torsional or twist motions seen for residues 1-23 with as much as a five degree shift for Rl 1. Also residues 157-160
30 exhibit a similar tendency with changes up to eight degrees. Data for 32 and 33 overlay very well and do not differ much from 15 across the entire sequence, especially for above mentioned regions with greatest torsional motions that are not observed for nicotine. However, the patterns of dihedral angle changes are far smaller than seen in the GLIC structure and AChR models (13, 26-27).
An alternative presentation of the data is shown in SI Appendix Fig. S2 were all values were projected on to a centrosymmetric axis in the pentamer and reflect relative distances from a reference point at the transmembrane region interface. The blooming amplitude develops from cytoplasmic towards the extracellular, apical region resulting in increased intersubunit distances between diametrically opposed, non-adjacent subunits. Accordingly, the blooming quaternary structure is evident for all three members of the 2- aminopyrimidine series and emphasizes the importance of the conformational differences in quaternary structure associated with the state changes. These sequence patterns of quaternary rearrangements reflected in distances between diametric subunits are in close correspondence in residue positions with those found in the open and closed channel forms of GLIC (13).
Discussion
Our studies establish a previously unknown level of conformational
communication between AChBP subunits accompanying ligand binding. We report a series of selective AChBP ligands exhibiting negative as well as positive cooperativity, a type of allosteric behavior in which binding interactions in an oligomeric protein take place between distant subunit interfaces. Selected ligands showing marked differences in cooperativity were used to obtain three atomic resolution, X-ray crystal structures of ligands with different Hill slope values in their complexes with Zs-AChBP. The lower affinity ligands showing positive cooperativity, along with the high affinity of the negatively cooperative ligands, serve to achieve full occupation of ligand in the crystal structures. These cooperative interactions occur in the absence of the transmembrane, channel gating domain required of the nAChR to elicit subunit interactions and cooperativity. Rather cooperativity in AChBP is confined to the extracellular domain of the nAChR reference structure and is mediated circumferentially around the cylindrical pentamer.
The differences in side chain positions at the subunit interface (W53, Y164, Yl 85), that distinguish between positively and negatively cooperative ligands also diverge from the nicotine side chain positions. Nicotine also adds Y89 to the perturbed side chains. Changes in quaternary structure, noted, in reference to the gating transition of prokaryotic GLIC, as blooming (13, 27), are not seen for nicotine and carbamylcholine, where the Apo form of the receptor is used as a reference state. However, it should be recognized that the apo-receptor contains HEPES buffer occupying the reference site (16). Hence, at this stage, a nAChBP unoccupied by ligand is not available in a crystallized form.
Nevertheless, we should ask what distinguishes the substituted 2- aminopyrimidines from the conventional ligands hitherto fore characterized. They may fit into three categories: (a) Quaternary amines stabilized by a cation -π interaction between the cation and a nest of surrounding aromatic side chains. Such interactions are evident in the crystal structures of acetylcholine, carbamylcholine and other quaternary complexes (16, 18) and the energetics have been established through mutagenesis studies modifying the polarizability, and electronegativity of the aromatic side chains in the binding pocket (28-29); (b) Secondary and tertiary amines (19-21) and imines (21), which, when in a protonated state, hydrogen bond to the backbone carbonyl of W 143. The protonated dihydropyridine-bound state for the benzylidene anabaseines has been demonstrated by difference spectroscopy (30); (c) Peptides, such as the a-conotoxins and a-neurotoxins, stabilized by multiple interactions on the C loop and interfacial residues on both the principal and complementary faces (31-34). The binding interactions of the 2- aminopyrimidines do not appear to follow the above patterns and therefore these structures constitute a distinct fourth group of ligands. The nitrogens in the 2- aminopyrimidine ring are not very basic, suggesting a far higher energy requirement for the ligand to bind in a protonated state than that found for secondary and tertiary amines and the imines. Hence, the 2-aminopyrimidines may be considered as electron-rich ring systems capable of ring stacking with the side chains of Y192 and W143. The pyrimidine ring nitrogens are not as exposed as the bicyclic ring in epibatidine and the pyrrolidine nitrogen in nicotine affording a proper directionality for hydrogen bonding. Thus, the global conformational changes primarily manifested by blooming appear characteristic of the substituted 2-aminopyrimidines and are not seen with carbamylcholine and nicotine (Fig. 4 and Fig. S2). To date, a clear state change has only been documented for the substituted 2-aminopyrimidines.
The changes in AChBP quaternary structure add another dimension to considering subunit interactions. The "blooming" and torsional conformational changes noted with AChBP show analogies with those observed in GLIC (13, 27) in relation with correspondences of protein sequence (Fig. 4, A, B & C and Fig. S2). The amino terminal helix, regions between residues 58-70 and between 106-110 all show increased distances between diametrically opposed subunits. The sharper negative peaks around residues 156 and 185 likely involve local perturbations of the C and F loops proximal to the ligand binding site resulting in local compaction of the structure, measured in Ca distances. The comparison of the dihedral bond angles should reflect a torsional or twist motion as seen between the two states of GLIC (13, 27). Changes in dihedral angle positions are small (Fig. S2, C), but involve the same set of residues. Hence, when the binding of the 2- aminopyrimidines are compared with the pH dependent conformations of GLIC, the dominant common change is found with the Ca distances between diametric subunits (blooming) (Fig. S2 B), rather than the dihedral angles for torsional movement (twist) (Fig. S2 C).
The interfacial binding sites residing under the C loop of AChBP and the nAChR appear surprisingly accommodating for the binding of ligands of different structure. For example, Stornaiaolo and colleagues have reported on large planar, aromatic molecules binding under the C loop in a stacked sandwich fashion, and extending the C loop (35).
The cooperative ligands binding to AChBP add a new dimension to ligands interaction with the extracellular domain of the pentameric ligand-gated ion channels. We are currently examining the structural determinants of selectivity of this ligand family with other AChBP 's and the homomeric a l nAChR. With homomeric nAChRs, selectivity for the primary agonist could be altered through partial site occupation by the 2-aminopyrimidines showing negative cooperativity. In the case of the predominant heteromeric receptors where the binding interfaces will differ, such ligands may possibly serve as positive or negative allosteric modulators at sites distinct from those occupied by agonist and competitive antagonist (36). Such appears to be the case for the
benzodiazepines (36-37) and other sedative agents (38) that act in this manner with the GABA receptor (39-40). Accordingly, new dimensions for achieving pharmacologic selectivity for particular nAChR subtypes may result with the cooperative nAChR ligands possessing electron-rich substituted 2-aminopyrimidines.
Materials and Methods
Chemistry: Reagents and solvents were purchased from commercial sources and used without further purification. Unless otherwise stated, the reactions were performed under ambient conditions without attempts to exclude air or moisture other than capping the reaction vessels. NMR spectra were obtained on Bruker AMX-400™ and Briiker Avance III 600™ MHz instruments and referenced to the signals of residual 54protium in the NMR solvent. Splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), pentet (p), doublet of doublets (dd), doublet of doublet of doublets (ddd), doublet of triplets (dt), triplet of doublets (td) and broad (b); the value of chemical shifts (δ) are given in ppm and coupling constants (J) in hertz (Hz). Routine MS spectra were acquired in the positive ion mode using Agilent G2446A™ Lc/MSD Trap XCT™ coupled to an Agilent 1100™ HPLC. Representative compounds were characterized by high-resolution mass spectrometry (HR-MS) by using an Agilent 6230 ESI-TOFMS. Analytical and preparative TLC was performed on aluminum-backed plates (EMD Chemicals, San Diego, CA) and visualized by exposure to UV light.
Synthetic procedures: A straightforward synthetic pathway allowed access to multiple 2-aminopyrimidine analogs varying in substitution at positions 4 and 6 in the pyrimidine ring (Scheme SI). Commercially available 2-amino-4,6-dichloropyrimidine A was reacted with appropriate amines to afford intermediates B1-B24. In the subsequent step, compounds B were subjected to a coupling reaction with various boronic acids using tetrakis (triphenylphosphine) palladium(O) as a catalyst to give products 1-38 in good to excellent yields. If tert-butyloxycarbonyl (boc) protected the amine functional group in the described approach, free amines were generated by subsequent acidic cleavage with 10% trifluoroacetic acid solution in dichloromethane. This synthetic route enabled us to synthesize approximately 40 analogs of the lead with separate modifications at the 4 and 6 positions.
Boronic acid, Pd(PPh3)4
Na2CO3- 10H2O
Toluene/EtO H/H20, 95 °C, 24 h
Figure imgf000056_0001
Scheme SI. Synthetic pathway leading to 4,6-substituted 2-aminopyrimidines 1-38.
General procedure for synthesis of 4-chloro-6-aminopyrimidin-2-amines B1-B24:
An appropriate amine (2.20 mmol) was added to a solution of 4,6- dichloropyrimidin-2 -amine (0.328 g, 2.00 mmol) and N,N-diisopropylethylamine (0.383 mL, 2.20 mmol) in DMF (3 mL) and the solution was stirred at 80 °C for 2-6 hours (reaction monitored by TLC). The solvents were removed under reduced pressure, brine was added and the mixture was extracted with ethyl acetate (3 x 20 mL). The organic layers were washed with water and saturated sodium chloride solution, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated on a rotary evaporator, and the title compound was isolated by column chromatography on silica gel or by crystallization.
General procedure for synthesis of 6-aryl-2.4-diaminopyrimidine 1-38:
Appropriate bornonic acid (0.60 mmol) was added to a solution of appropriate chloropyrimidine (0.50 mmol) in toluene/ethanol (1 : 1, 4 mL). Sodium carbonate decahydrate (0.286 g, 1.0 mmol) was added following the addition of
tetrakis(triphenylphosphine)palladium(0) (57.8 mg, 0.050 mmol). The resulting mixture was stirred in a capped glass vial at 95 °C for 2-4 h. After cooling the mixture to room temperature, water was added and the mixture was extracted with ethyl acetate (3 x 20 mL). The organic layers were washed with brine, dried over anhydrous magnesium sulphate, and filtered. The filtrate was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel. Boc -protected pyrimidines (0.05 mmol) was dissolved in 10% TFA/DCM (1 mL) and the mixture was left for 1 hour at room temperature. The solvents were removed and the deprotected product was used without further purification.
General procedure for synthesis of 6-aryl-2,4-diaminopyrimidine AC-171BJ71C:
Appropriate boronic acid (0.30 mmol) was added to a solution of appropriate chloropyrimidine (0.15 mmol) in N,N-dimethylacetamide (1.5 mL). Potassium carbonate 2M (0.2mL) was added following the addition of 1 , 1 '-Bis(diplienylpliosphino)ferrocene- palladium(II)dichloridedichlorometha.ne complex (0.0112 mg, 0.015 mmol). The resulting mixture was stirred in a capped glass vial at 140 °C for 2h. The solvents were removed under reduced pressure, brine was added and the mixture was extracted with ethyl acetate (3 x 20 mL). The organic layers were washed with brine, dried over anhydrous magnesium sulphate, and filtered. The filtrate was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel. Table 4. Structures of Exemplary Compounds of the Invention; Analytical Details for Synthesized Exemplary Compounds of the Invention; Exemplary Synthetic Protocols:
EXEMPLARY COMPOUND OF THE
EXEMPLARY SYNTHETIC PROTOCOL INVENTION
6-( hloro-A4- cycloheptylpyrimidine-2,4- diamine (B-1). Purification: column chromatography on silica gel using
hexane/ethyl acetate (4: 1 and 1 : 1) as the eluent. White solid, 80% yield. ¾ NMR (400 MHz,
Figure imgf000058_0001
CDCI3) £ 1.41-1.68 (m, 10 H), 1.94 (m, 2 H),
3.64 (bs, 1 H), 4.83 (s, 3 H), 5.72 (s, 1 H). ESI- MS: [CiiH17ClN4 + H]+ 241 (100).
6-Chloro-7V4-cyclopropylpyrimidine-2,4- diamine (B-2). Purification: column chromatography on silica gel using
hexane/ethyl acetate (4: 1 and 1 : 1) as the eluent. White solid, 75% yield. ¾ NMR (400 MHz, CDCI3) δ 0.56 (m, 2 H), 0.81 (m, 2 H), 2.48
CI (m, 1 H), 4.92 (bs, 2 H), 5.33 (s, 1 H), 6.13 (s,
Figure imgf000058_0002
1 H). 13C NMR (150 MHz, CDCI3) δ 7.6
(CH2), 23.2 (CH), 92.7 (CH), 141.8 (C), 162.0 (C), 165.6 (C). ESI-MS: [C7H9C1N4 + H]+ 185 (100).
6-Chloro- /V4-heptylpyrimidine-2,4-diamine (B-3). Purification: column chromatography on silica gel using hexane/ethyl acetate (4: 1 and 1 : 1) as the eluent. White solid, 70% yield. XH NMR (400 MHz, CDC13) δ 0.85 (t, J= 6.8 Hz,
Figure imgf000058_0003
3 H), 1.27 (m, 8 H), 1.53 (m, 2 H), 3.17 (bs, 2
H), 5.19 (s, 3 H), 5.74 (s, 1 H). 13C NMR (100 MHz, CDCI3) δ 14.0 (CH3), 22.5 (CH2), 26.8 (CH2), 28.9 (CH2), 29.2 (CH2), 31.7 (CH2), 41.5 (CH2), 162.4 (C), 164.2 (C). ESI-MS: [CiiH19ClN3 + H]+ 243 (100).
6-Chloro-iV?-octylpyrimidine-2,4-diamine (B-4). Purification: column chromatography on silica gel using hexane/ethyl acetate (6: 1) as the eluent. Pale solid, 0.425 g, 83% yield. ¾ NMR (600 MHz, CDC13) δ 0.86 (t, J= 7.0 Hz, 3 H), 1.21-1.38 (m, 10 H), 1.55 (m, 2 H), 3.20 (bs, 2 H), 5.01 (bs, 2 H), 5.75 (s, 1 H). 13C
Figure imgf000059_0001
NMR (150 MHz, CDC13) £ 14.0 (CH3), 22.6
(CH2), 26.8 (CH2), 29.1 (CH2), 29.2 (CH2), 29.3 (CH2), 31.7 (CH2), 41.6 (CH2), 120.0 (C), 162.4 (C), 164.3 (C). ESI-MS: [Ci2H21ClN4 + H]+ 257 (100).
teri-Butyl 5-(2-amino-6-chloropyrimidin-4- ylamino)pentylcarbamate (B-5). Purification: column chromatography on silica gel using hexane/ethyl acetate (4: 1) and ethyl acetate/methanol (10: 1) as the eluent. Colorless oil, 0.664 g, 99% yield. ¾ NMR (600 MHz,
Figure imgf000059_0002
CDCI3) £ 1.39 (m, 2 H), 1.45 (s, 9 H), 1.52 (m,
2 H), 1.60 (m, 2 H), 3.13 (m, 2 H), 3.23 (m, 2 H), 5.00 (bs, 3 H), 5.78 (s, 1 H), 8.02 (s, 1 H). ESI-MS: [Ci4H24ClN502 + H]+ 330 (100). tert-Butyl 4-(2-amino-6-chloropyrimidin-4- ylamino)butylcarbamate (B-6). Purification: column chromatography on silica gel using hexane/ethyl acetate (4: 1) and ethyl acetate/methanol (10: 1) as the eluent. Yellow
Figure imgf000059_0003
oil, 0.650 g, 99% yield. ¾ NMR (600 MHz, CDCI3) £ 1.42 (s, 9 H), 1.52 (m, 2 H), 1.59 (m,
2 H), 3.15 (m, 2 H), 3.29 (m, 2 H), 4.96 (bs, 2 H), 5.09 (s, 1 H), 5.76 (s, 1 H), 7.99 (s, 1 H). ESI-MS: [Ci3H22ClN502 + H]+ 316 (100). tert-Butyl 3-(2-amino-6-chloropyrimidin-4- ylamino)propylcarbamate (B-7). Purification: column chromatography on silica gel using hexane/ethyl acetate (4: 1) and ethyl acetate/methanol (10: 1) as the eluent. Yellow oil, 0.640 g, 99% yield. ¾ NMR (600 MHz,
Figure imgf000060_0001
CDCI3) £ 1.43 (s, 9 H), 1.68 (m, 2 H), 3.17 (m,
2 H), 3.35 (m, 2 H), 5.04 (bs, 2 H), 5.46 (s, 1 H), 5.78 (s, 1 H). ESI-MS: [Ci2H2oClN502 + H]+ 302 (100).
4-(2-Amino-6-chloropyrimidin-4- ylamino)butanoic acid (B-8). Purification: column chromatography on silica gel using ethyl acetate/methanol (10: 1 and 7: 1) as the eluent. White solid, 0.150 g, 34% yield. XH NMR (600 MHz, DMSO-D6) δ 1.69 (m, 2 H),
Figure imgf000060_0002
2.24 (t, J= 7.4 Hz, 2 H), 3.21 (m, 2 H), 5.71 (s,
1 H), 6.36 (s, 2 H), 7.12 (s, 1 H). ESI-MS: [C8HnClN402 + H]+ 231 (100).
6-Chloro-7V4-(tetrahydro-2H-pyran-4- yl)pyrimidine-2,4-diamine (B-9).
Purification: column chromatography on silica gel using hexane/ethyl acetate (2: 1) as the eluent. White solid, 0.330 g, 72% yield. XH
Figure imgf000060_0003
NMR (600 MHz, MeOD) δ 1.44 (m, 2 H),
1.85 (m, 2 H), 3.42 (td, J= 11.7, 2.0 Hz, 2 H), 3.87 (dt, J= 11.2, 3.0 Hz, 2 H), 5.74 (s, 1 H). ESI-MS: [C9H13C1N40 + H]+ 229 (100).
6-Chloro-7V4-(2-morpholinoethyl) pyrimidine-2,4-diamine (B-10). Purification: column chromatography on silica gel using ethyl acetate/methanol (10: 1) as the eluent. White solid, 0.316 g, 78% yield. XH NMR (600 MHz, MeOD) δ 2.44 (m, 4 H), 2.48 (t, J= 6.6
Figure imgf000061_0001
Hz, 2 H), 3.40 (s, 2 H), 3.63 (t, J= 4.6 Hz, 4 H), 5.78 (s, 1 H). ESI-MS: [Ci0H16ClN5O + H]+ 258 (100).
6-Chloro-7V4-(4-methylpiperazin-l- yl)pyrimidine-2,4-diamine (B-l 1).
Purification: column chromatography on silica gel using ethyl acetate/methanol (10: 1) as the eluent. Pale solid, 0.267 g, 55% yield. ¾ NMR (600 MHz, MeOD) £2.31 (s, 3 H), 2.45 (m, 4
Figure imgf000061_0002
H), 3.62 (m, 4 H), 6.09 (s, 1 H). 13C NMR (150
MHz, MeOD) £44.6 (CH2), 46.1 (CH3), 55.5 (CH2), 92.7 (CH), 160.9 (C), 164.1 (C), 165.0 (C). ESI-MS: [C9Hi5ClN6 + H]+ 243 (100).
4-(2-(2-Amino-6-chloropyrimidin-4- ylamino)ethyl)phenol (B-12). Purification: column chromatography on silica gel using hexane/ethyl acetate (1 : 1) as the eluent. Orange oil, 0.330 g, 62% yield. ¾ NMR (600 MHz, MeOD) δ 2.65 (m, 2 H), 3.39 bs, 2 H), 5.70 (s,
Figure imgf000061_0003
1 H), 6.63 (m, 2 H), 6.93 (m, 2 H).13C NMR
(150 MHz, MeOD) δ 35.6 (CH2), 116.2 (CH), 130.7 (CH), 156.8 (C), 165.7 (C). ESI-MS: [Ci2Hi3ClN40 + H]+ 265 (100).
Figure imgf000062_0001
4-Chloro-6-morpholinopyrimidin-2-amine (B-16). Purification: column chromatography on silica gel using hexane/ethyl acetate (3: 1 and 1: 1) as the eluent. White solid, 0.334 g, 78% yield. ESI-MS: [C8HiiClN4 + H]+215 (100). ¾ NMR (600 MHz, CDC13) δ 3.53 (m, 4 H), 3.72 (m, 4 H), 4.96 (bs, 2 H), 5.92 (m, 1
Figure imgf000063_0001
H). 13C NMR (150 MHz, CDC13) δ 44.3
(CH2), 66.4 (CH2), 92.3 (CH), 160.5 (C), 162.1 (C), 163.8 (C). ). ESI-MS: [C8HiiClN40 + H]+ 215 (100).
4-Chloro-6-(4-methylpiperidin-l- yl)pyrimidin-2-amine (B-17). Purification: column chromatography on silica gel using hexane/ethyl acetate (3: 1 and 1 : 1) as the eluent. White solid, 0.407 g, 90% yield. XH NMR (600 MHz, CDC13) £0.95 (d, J= 6.6 Hz, 3 H), 1.12 (m, 2 H), 1.63 (m, 1 H), 1.69 (m, 2 H), 2.82 (td, J= 12.8, 2.0 Hz, 2 H), 4.26 (bs, 2 H), 4.99
Figure imgf000063_0002
(bs, 2 H), 5.95 (s, 1 H). 13C NMR (150 MHz,
MeOD) £21.7 (CH3), 31.1 (CH), 33.7 (CH2), 44.5 (CH2), 92.2 (CH), 160.1 (C), 162.3 (C), 163.3 (C). ESI-MS: [Ci0H15ClN4 + H]+227 (100).
teri-Butyl 4-(2-amino-6-chloropyrimidin-4- yl)piperazine-l-carboxylate (B-18).
Purification: column chromatography on silica gel using hexane/ethyl acetate (6: 1) as the eluent. Orange solid, 0.511 g, 82% yield. XH
Figure imgf000063_0003
NMR (600 MHz, MeOD) δ 1.41 (s, 9 H), 3.40
(m, 4 H), 3.55 (m, 4 H). 6.05 (s, 1 H). 13C NMR (150 MHz, MeOD) 28.6 (CH3), 44.8
(CH2), 81.6 (C), 92.8 (CH), 156.4 (C), 160.9 (C), 165.1 (C). ESI-MS: [Ci3H2oClN502 + H]+ 314 (100).
4-Chloro-6-(4-(2-morpholinoethyl) piperazin-l-yl)pyrimidin-2-amine (B-19).
Purification: crystallization from ethanol. Orange crystals, 0.456 g, 70% yield. XH NMR (600 MHz, MeOD) δ 2.48 (m, 8 H), 2.51 (bs, 4 H), 3.56 (m, 4 H), 3.63 (m, 4 H), 6.03 (s, 1 H). 13C NMR (150 MHz, MeOD) δ 44.8
Figure imgf000064_0001
(CH2), 54.2 (CH2), 55.1 (CH2), 56.0 (CH2),
56.8 (CH2), 67.6 (CH2), 92.6 (CH), 160.8 (C), 164.1 (C), 165.0 (C). ESI-MS: [Ci4H23ClN60 H H]+ 327 (100).
4-Chloro-6-(4-(4-fluorophenyl)piperazin-l- yl)pyrimidin-2-amine (B-20). Purification: column chromatography on silica gel using hexane/ethyl acetate (6: 1) as the eluent. Orange solid, 0.460 g, 75% yield. XH NMR (600 MHz, MeOD) δ 3.10 (m, 4 H), 3.75 (m, 4 H), 6.14 (s, 1 H), 6.97 (m, 2 H), 6.98 (m, 2 H). 13C NMR (150 MHz, MeOD) £45.1 (CH2), 51.3
Figure imgf000064_0002
(CH2), 92.7 (CH), 116.4 (d, 2JC-F = 22.0 Hz, CH), 119.7 (d, 3JC-F = 1.1 Hz, CH), 149.4 (C), 159.7 (C), 160.8 (C), 165.0 (C). ESI-MS: [Ci4H15ClFN5 + H]+ 308 (100).
7V4-cycloheptyl-6-(4-(trifluoromethyl) phenyl)pyrimidine-2,4-diamine (1).
Synthesized using B-l as the starting material.
Figure imgf000064_0003
Purification: column chromatography on silica gel using hexane/ethyl acetate (2: 1) as the eluent. Pale solid, 0.110 g, 62% yield. ¾ NMR (400 MHz, CDC13) δ 1.48-1.72 (m, 10 H), 2.00 (m, 2 H), 3.82 (bs, 1 H), 5.05 (bs, 2 H), 6.08 (s, 1 H), 7.67 (m, 2 H), 7.96 (m, 2 H). ESI-MS: [C18H21F3N4 + H]+ 351 (100).
7V4-ycloheptyl-6-(pyridin-4-yl)pyrimidine- 2,4-diamine (2). Synthesized using B-l as the starting material. Purification: column chromatography on silica gel using
hexane/ethyl acetate (2: 1 and 1 : 1) as the eluent. White solid, 0.10 mg, 71% yield. XH NMR (600 MHz, CDCI3) £ 1.46-1.72 (m, 10 H), 1.96-2.06 (m, 2 H), 4.94 (bs, 3 H), 6.14 (s, 1
Figure imgf000065_0001
H), 7.76 (m, 2 H), 8.69 (m, 2 H). 13C NMR
(150 MHz, CDCI3) £24.0 (CH2), 28.1 (CH2), 35.0 (CH2), 121.0 (CH), 145.8 (C), 150.2 (CH), 163.2 (C), 163.3 (C). ESI-MS: [d6H21N5 + H]+284 (100).
7V4-cyclopropyl-6-(4-(trifluoromethyl) phenyl)pyrimidine-2,4-diamine (3).
Synthesized using B-2 as the starting material. Purification: column chromatography on silica gel using hexane/ethyl acetate (2: 1 and 1 : 1) as the eluent. Pale solid, 75.0 mg, 51% yield. XH NMR (600 MHz, CDC13) δ 0.61 (m, 2 H), 0.83 (m, 2 H), 2.59 (s, 1 H), 4.87 (bs, 2 H), 5.30 (s,
Figure imgf000065_0002
1 H), 6.51 (s, 1 H), 7.69 (d, J= 7.5 Hz, 2 H),
8.03 (d, J= 7.9 Hz, 2 H). ljC NMR (150 MHz, CDCI3) δ 7.7 (CH2), 23.4 (CH), 125.5 (q, 3Jc-F = 3.4 Hz, CH), 127.3 (CH), 131.5 (q, 2JC-F = 32.1 Hz, C), 141.8 (C), 162.9 (C), 165.7 (C),
176.6 (C). ESI-MS: [C^H^F^ + H]+ 295 (100).
-heptyl-6-(4-(trifluoromethyl)
phenyl)pyrimidine-2,4-diamine (4).
Synthesized using B-3 as the starting material. Purification: column chromatography on silica gel using hexane/ethyl acetate (2: 1 and 1 : 1) as the eluent. Pale solid, 0.109 mg, 62% yield. ¾ NMR (600 MHz, CDC13) δ 0.88 (t, J= 6.8 Hz, 3 H), 1.21-1.38 (m, 8 H), 1.58 (m, 2 H), 3.33 (bs, 2 H), 5.48 (s, 1 H), 6.00 (s, 1 H), 7.52 (m, 2 H), 7.90 (bs, 2 H). 13C NMR (150 MHz,
Figure imgf000066_0001
CDC13) £ 14.0 (CH3), 22.5 (CH2), 26.8 (CH2), 28.9 (CH2), 29.1 (CH2), 31.7 (CH2), 124.1 (CH), 125.7 (q, 3JC-F = 3.4 Hz, CH), 127.1 (CH), 132.4 (q, 2JC-F = 33.3 Hz, C), 163.3 (C), 164.3 (C). ESI-MS: [CisH^F^ + H]+ 353 (100).
6-(3,5-Bis(trifluoromethyl)phenyl)-7V4- heptylpyrimidine-2,4-diamine (5).
Synthesized using B-3 as the starting material. Purification: column chromatography on silica gel using hexane/ethyl acetate (6: 1) as the eluent. Yellow oil, 0.210 g, 99% yield. XH NMR (600 MHz, CDC13) δ 0.87 (t, J= 7.0 Hz, 3 H), 1.22-1.47 (m, 8 H), 1.60 (q, J= 7.3 Hz, 2
Figure imgf000066_0002
H), 3.33 (bs, 2 H), 4.94 (bs, 2 H), 6.17 (s, 1 H),
7.90 (s, 1 H), 8.36 (s, 2 H). 13C NMR (150 MHz, CDCI3) £ 14.0 (CH3), 22.6 (CH2), 26.9 (CH2), 29.0 (CH2), 29.4 (CH2), 31.7 (CH2), 41.3 (CH2), 122.4 (C), 123.1 (m, CH), 124.2 (C), 126.9 (m, CH), 131.8 (m, C), 140.6 (C),
163.3 (C), 164.4 (C). ESI-MS: [Ci9H22F6N4 + H]+ 421 (100).
6-(2-Fluoro-4-(trifluoromethyl)phenyl)-7V4- heptylpyrimidine-2,4-diamine (6).
Synthesized using B-3 as the starting material. Purification: column chromatography on silica gel using hexane/ethyl acetate (6: 1) as the eluent. Yellow oil, 0.135 g, 73% yield. XH NMR (600 MHz, CDC13) δ 0.87 (t, J = 6.6 Hz, 3 H), 1.22-1.38 (m, 8 H), 1.58 (q, J= 7.3 Hz, 2
Figure imgf000067_0001
H), 3.28 (bs, 2 H) 4.90 (bs, 1H), 4.93 (bs, 2 H), 6.26 (s, 1 H), 7.37 (m, 1 H), 7.46 (m, 1 H), 8.08 (m, 1 H). ESI-MS: [Ci8H22F4N4 + H]+ 371 (100).
6-(2,4-Difluorophenyl)-/V4-heptylpyrimidine- 2,4-diamine (7). Synthesized using B-3 as the starting material. Purification: column chromatography on silica gel using
hexane/ethyl acetate (6: 1) as the eluent. Yellow oil, 0.132 g, 82% yield. ¾ NMR (600 MHz, CDC13) δ 0.87 (t, J = 7.0 Hz, 3 H), 1.23-1.37 (m, 8 H), 1.57 (m, 2 H), 3.27 (bs, 2 H), 4.85 (bs, 3 H), 6.21 (d, J= 1.5 Hz, 1 H), 6.84 (ddd, J = 1 1.3, 8.8, 2.5 Hz, 1 H), 6.93 (ddd, J= 8.4,
Figure imgf000067_0002
8.4, 2.4 Hz, 1 H), 7.97 (ddd, J= 10.3, 8.6, 8.6 Hz, 1 H). 13C NMR (150 MHz, CDC13) δ 14.0 (CH3), 22.6 (CH2), 26.9 (CH2), 29.0 (CH2), 29.4 (CH2), 31.7 (CH2), 41.4 (CH2), 104.3 (dd, Jc-F = 25.5, 25.3 Hz, CH), 1 11.6 (dd, Jc-F = 20.9, 3.3 Hz, CH), 131.7 6 (dd, Jc-F = 9.0, 4.4 Hz, CH), 161.0 (dd, Jc-F = 255.0, 12.1 Hz, C),
162.93 (C), 163.3 (dd, Jc-F = 251.6, 12.1 Hz, C). ESI-MS: [Ci7H22F2N4 + H]+ 321 (100).
-heptyl-6-(3,4,5-trifluorophenyl) pyrimidine-2,4-diamine (8). Synthesized using B-3 as the starting material. Purification: column chromatography on silica gel using hexane/ethyl acetate (6: 1) as the eluent. Yellow oil, 0.169 g, 99% yield. ¾ NMR (600 MHz, CDC13) δ 0.87 (t, J= 6.6 Hz, 3 H), 1.23-1.39
Figure imgf000068_0001
(m, 8 H), 1.59 (m, 2 H), 3.29 (bs, 2 H), 4.85 (bs, 3 H), 6.03 (s, 1 H), 7.55 (m, 2 H). ESI-MS: [Ci7H21F3N4 + H]+ 339 (100).
N/-heptyl-6-(pyridin-3-yl)pyrimidine-2,4- diamine (9). Synthesized using B-3 as the starting material. Purification: column chromatography on silica gel using
hexane/ethyl acetate (2: 1 and 1 : 1) as the eluent. Pale solid, 0.10 g, 70% yield. ¾ NMR (600 MHz, CDCI3) £0.86 (t, J= 6.9 Hz, 3 H), 1.21- 1.37 (m, 8 H), 1.58 (m, 2 H), 3.29 (bs, 2 H), 5.06 (bs, 3 H), 6.13 (s, 1 H), 7.35 (dd, J= 7.9,
Figure imgf000068_0002
4.8 Hz, 1 H), 8.21 (m, 1 H), 8.63 (m, 1 H), 9.08
(s, 1 H). 13C NMR (150 MHz, CDC13) δ 14.0 (CH3), 22.5 (CH2), 26.9 (CH2), 28.9 (CH2), 29.4 (CH2), 31.7 (CH2), 41.4 (CH2), 123.5 (CH), 134.1 (C), 134.7 (CH), 147.8 (CH), 150.1 (CH), 163.0 (C), 164.2 (C). ESI-MS: [Ci6H23N5 + H]+ 286 (100).
Figure imgf000069_0001
5.11 (bs, 2 H), 6.13 (s, 1 H), 7.65 (m, 2 H),
7.98 (m, 2 H). 13C NMR (150 MHz, CDC13) δ 14.0 (CH3), 22.6 (CH2), 26.9 (CH2), 29.2 (CH2), 29.2 (CH2), 29.4 (CH2), 31.8 (CH2), 41.4 (CH2), 125.4 (q, 3JC-F = 3.3 Hz, CH), 127.1 (CH), 131.3 (q, 2JC-F = 31.9 Hz, C), 142.0 (C), 163.3 (C), 164.3 (C). ESI-MS: [Ci9H25F3 4 + H]+ 367 (100).
6-(2-Fluoro-4-(trifluoromethyl)phenyl)-7V4- octylpyrimidine-2,4-diamine (13).
Synthesized using B-4 as the starting material. Purification: column chromatography on silica gel using hexane/ethyl acetate (6: 1) as the eluent. Pale solid, 0.133 g, 69% yield. ¾ NMR (600 MHz, CDC13) δ 0.97 (t, J= 7.0 Hz, 3 H),
Figure imgf000070_0001
1.38 (m, 8 H), 1.46 (m, 2 H), 1.68 (m, 2 H), 3.38 (bs, 2 H), 5.17 (bs, 2 H), 6.36 (s, 1 H), 7.48 (m, 1 H), 7.58 (m, 1 H), 8.18 (m, 1 H). ESI-MS: [C19H24F4N4 + H]+ 385 (100).
6-(4-Chlorophenyl)-/V4-octylpyrimidine-2,4- diamine (14). Synthesized using B-4 as the starting material. Purification: column chromatography on silica gel using hexane/ethyl acetate (3: 1) as the eluent. Yellow solid, 0.155 g, 93% yield. XH NMR (600 MHz, CDCI3) δ 0.88 (m, 3 H), 1.22-1.39 (m, 10 H), 1.58 (m, 2 H), 3.28 (bs, 2 H), 4.99 (bs, 2 H),
Figure imgf000070_0002
6.10 (d, J= 1.8 Hz, 1 H), 7.38 (dd, J= 8.5, 1.7 Hz, 2 H), 7.84 (m, 2 H). XH NMR (150 MHz, CDCI3) δ 14.0 (CH3), 22.6 (CH2), 26.9 (CH2), 29.2 (CH2), 29.2 (CH2), 29.4 (CH2), 31.7 (CH2), 41.2 (CH2), 128.1 (CH), 128.6 (CH), 135.6 (C), 136.9 (C), 163.1 (C), 164.3 (C).
ESI-MS: [Ci8H25ClN4 + H]+ 333 (100).
6-(4-Methoxyphenyl)-/V4-octylpyrimidine-2,4- diamine (15). Synthesized using B-4 as the starting material. Purification: column chromatography on silica gel using
hexane/ethyl acetate (1 : 1 and 1:2) as the eluent. Brown oil, 0.164 g, 99% yield. XH NMR (600 MHz, CDC13) δ 0.87 (t, J= 6.9 Hz, 3 H), 1.24- 1.41 (m, 10 H), 1.58 (m, 2 H), 3.27 (m, 2 H), 3.83 (s, 3 H), 4.83 (bs, 1 H), 4.89 (s, 2 H), 6.08 (s, 1 H), 6.93 (m, 2 H), 7.86 (m, 2 H). 'H NMR
Figure imgf000071_0001
(150 MHz, CDCI3) £ 14.0 (CH3), 22.6 (CH2), 26.9 (CH2), 29.2 (CH2), 29.3 (CH2), 29.5 (CH2), 31.8 (CH2), 41.5 (CH2), 55.3 (CH3), 113.8 (CH), 128.2 (CH), 130.9 (C), 161.0 (C), 163.0 (C), 164.3 (C). HR-ESI APCI-MS: calculated for [Ci9H28N40 + H]+ 329.2336, found 329.2339.
teri-Butyl 5-(2-amino-6-(4-(trifluoromethyl) phenyl)pyrimidin-4-ylamino)
pentylcarbamate (16). Synthesized using B-5 as the starting material. Purification: column chromatography on silica gel using hexane hexane/ethyl acetate (4: 1) and ethyl acetate/methanol (10: 1) as the eluent. Yellow oil, 0.190 g, 86% yield. ¾ NMR (600 MHz,
Figure imgf000071_0002
CDCI3) δ 1.35 (m, 2 H), 1.41 (s, 9 H), 1.46 (m, 2 H), 1.57 (m, 2 H), 3.08 (m, 2 H), 3.28 (bs, 2 H), 5.09 (bs, 2 H), 6.13 (s, 1 H), 7.64 (m, 2 H), 7.96 (m, 2 H). 13C NMR (150 MHz, CDC13) δ 23.9 (CH2), 28.4 (CH3), 28.9 (CH2), 29.7 (CH2), 40.2 (CH2), 125.3 (q, "JC-F = 3.3 Hz,
CH), 127.1 (CH), 131.3 (q, 2JC-F = 34.1 Hz, C), 141.9 (C), 163.2 (C), 164.3 (C). ESI-MS: [C2iH28F3 502 + H]+440 (100).
teri-Butyl 4-(2-amino-6-(4- (trifluoromethyl)phenyl)pyrimidin-4- ylamino)butylcarbamate (17). Synthesized using B-6 as the starting material. Purification: column chromatography on silica gel using hexane/ethyl acetate (4: 1) and ethyl acetate/methanol (10: 1) as the eluent. Brown oil, 0.134 g, 63% yield. ¾ NMR (600 MHz,
Figure imgf000072_0001
CDC13) δ 1.42 (s, 9 H), 1.54 (m, 2 H), 1.60 (m, 2 H), 3.15 (m, 2 H), 3.35 (bs, 2 H), 5.16 (bs, 2 H), 5.42 (bs, 1 H), 6.15 (s, 1 H), 7.61 (d, J= 7.0 Hz, 2 H), 7.90 (bs, 2 H). ESI-MS:
[C2oH26F3N502 + H]+426 (100).
teri-Butyl 3-(2-amino-6-(4- (trifluoromethyl)phenyl)pyrimidin-4- ylamino)propylcarbamate (18). Synthesized using B-7 as the starting material. Purification: column chromatography on silica gel using hexane/ethyl acetate (4: 1) and ethyl acetate/methanol (10: 1) as the eluent. Yellow oil, 0.150 g, 73% yield. ¾ NMR (600 MHz,
Figure imgf000072_0002
CDCI3) δ 1.42 (s, 1 H), 1.43 (s, 9 H), 1.68 (m, 2 H), 3.17 (m, 2 H), 3.40 (m, 2 H), 5.10 (bs, 2 H), 5.65 (bs, 1 H), 6.14 (s, 1 H), 7.62 (d, J= 7.0 Hz, 2 H), 7.93 (d, J = 7.5 Hz, 2 H). ESI- MS: [Ci9H24F3 502 + H]+412 (100). (m,
1 312
Figure imgf000073_0001
4-(2-Amino-6-(4-(trifluoromethyl)
phenyl)pyrimidin-4-ylamino)butanoic acid (24). Purification: column chromatography on silica gel using DCM/methanol (10: 1 and 7: 1) as the eluent. White solid, 17.0 mg, 10% yield. XH NMR (600 MHz, DMSO-D6) £ 1.74 (p, J =
Figure imgf000074_0001
7.0 Hz 2 H), 2.28 (t, J= 7.5 Hz, 2 H), 3.27 (bs,
2 H), 6.07 (s, 2 H), 6.31 (s, 1 H), 7.79 (d, J = 7.9 Hz, 2 H), 8.09 (m, 2 H). ESI-MS:
[Ci5H15F3 402 + H]+ 341 (100).
7V*-(tetrahydro-2H-pyran-4-yl)-6-(4- (trifluoromethyl)phenyl)pyrimidine-2,4- diamine (25). Purification: column chromatography on silica gel using hexane/ethyl acetate (1 :2) as the eluent. White solid, 0.10 g, 59% yield. XH NMR (600 MHz, CDC13) δ 1.51 (m, 2 H), 2.00 (m, 2 H), 3.52 mt, 2 H), 3.98 (m, 3 H), 4.79 (s, 1 H), 4.97 (bs,
Figure imgf000074_0002
2 H), 6.14 (s, 1 H), 7.66 (d, J= 8.1 Hz, 2 H),
7.96 (d, J= 8.1 Hz, 2 H). 13C NMR (150 MHz, CDCI3) £33.3 (CH2), 46.9 (CH), 66.7 (CH2), 125.4 (CH), 127.1 (CH), 141.8 (C), 163.3 (C). ESI-MS: [Ci6H17F3N40 + H]+ 339 (100).
7V*-(2-morpholinoethyl)-6-(4- (trifluoromethyl)phenyl)pyrimidine-2,4- diamine (26). Purification: column chromatography on silica gel using hexane/ethyl acetate (1 : 1) as the eluent. Yellow oil, 0.162 mg, 88% yield. XH NMR (600 MHz,
Figure imgf000074_0003
DMSO-D6) £2.39 (bs, 4 H), 2.45 (t, J= 6.6 Hz, 2 H), 3.41 (bs, 2 H), 3.57 (m, 4H), 6.16 (s, 1 H), 6.36 (s, 1 H), 7.77 (m, 2 H), 8.10 (bs, 2H). ljC NMR (150 MHz, DMSO-D6) £53.3
(CH2), 57.4 (CH2), 66.2 (CH2), 121.5 (C), 123.3 (C), 125.1 (C), 125.2 (q, 3JC-F = 3.3 Hz, CH), 126.9 (CH), 129.4 (q, 2JC-F = 30.8 Hz, C), 142.3 (C), 163.6 (C), 164.1 (C). ESI-MS: [Ci7H2oF3 50 + H]+ 368 (100).
7V4-(4-methylpiperazin-l-yl)-6-(4- (trifluoromethyl)phenyl)pyrimidine-2,4- diamine (27). Purification: column
chromatography on silica gel using ethyl acetate/methanol (9: 1) as the eluent. Pale solid, 0.128 g, 73% yield. XH NMR (600 MHz, MeOD) £ 2.23 (s, 3 H), 2.40 (m, 4 H), 3.63 (m, 4 H), 6.43 (s, 1 H), 7.63 (m, 2 H), 7.96 (m,
Figure imgf000075_0001
2 H). 13C NMR (150 MHz, MeOD) £44.6
(CH2), 46.1 (CH3), 55.6 (CH2), 92.0 (CH), 126.4 (q, 3JC-F = 3.3 Hz, CH), 128.7 (CH), 132.3 (q, 2JC-F = 31.9 Hz, C), 143.9 (C), 164.4 (C), 164.9 (C), 165.2 (C). ESI-MS:
[Ci6H19F3N6 + H]+ 353 (100).
4-(2-(2-Amino-6-(4-(trifluoromethyl) phenyl)pyrimidin-4-ylamino)ethyl)phenol (28). Purification: column chromatography on silica gel using hexane/ethyl acetate (1: 1) as the eluent. Yellow oil, 0.155 mg, 82% yield. XH NMR (600 MHz, MeOD) £2.84 (t, J= 7.15 Hz, 2 H), 3.71 (t, J= 7.15 Hz, 2 H), 6.35 (s, 1
Figure imgf000075_0002
H), 6.71 (m, 2 H), 7.07 (m, 2 H), 7.90 (m, 4 H). 13C NMR (150 MHz, MeOD) £35.1 (CH2), 43.9 (CH2), 97.7 (CH), 116.3 (CH), 127.4 (q, 3JC-F = 3.3 Hz, CH), 128.7 (CH), 130.8 (CH),
Figure imgf000076_0001
Figure imgf000077_0001
325.1271, found 325.1277. 4-(4-Methylpiperidin-l-yl)-6-(4- (trifluoromethyl)phenyl)pyrimidin-2-amine (33). Purification: column chromatography on silica gel using hexane/ethyl acetate (2: 1 and 1 : 1) as the eluent. White solid, 0.120 g, 71% yield. XH NMR (600 MHz, CDC13) δ 0.97 (d, J = 6.4 Hz, 3 H), 1.17 (td, J= 12.3, 4.0 Hz, 2 H), 1.66 (m, 1 H), 1.72 (m, 2 H), 2.87 (td, J= 12.8, 2.0 Hz, 2 H), 4.41 (m, 2 H), 4.93 (bs, 2 H), 6.36 (s, 1 H), 7.67 (d, J = 8.1 Hz, 2 H), 8.00 (d, J = 8.1 Hz, 2 H). ¾ NMR (600 MHz, CDC13)
Figure imgf000078_0001
δ 21.8 (CH3), 31.2 (CH), 33.8 (CH2), 44.5 (CH2), 91.1 (CH), 124.1 (q, 7JC-F = 271.8 Hz, C), 125.3 (q, 3JC-F = 3.3 Hz, CH), 127.2 (CH), 131.2 (q, 2JC-F = 31.9 Hz, C), 142.6 (C), 163.2 (C), 163.5 (C). HR-ESI/APCI-MS: calculated for [Ci7H19F3N4 + H]+ 337.1635, found 337.1637.
4-(2-Amino-6-(4-methylpiperidin-l- yl)pyrimidin-4-yl)phenol (34). Purification: column chromatography on silica gel using ethyl acetate/methanol (10: 1) as the eluent. White solid, 0.142 g, 99% yield. XH NMR (600 MHz, DMSO-D6) £0.90 (d, J= 6.2 Hz, 3 H), 1.03 (td, J= 3.7, 12.1 Hz, 2 H), 1.60 (m, 1 H), 1.63 (m, 2 H), 2.77 (m, 2 H), 4.40 (m, 2 H),
Figure imgf000078_0002
5.89 (s, 2 H), 6.42 (s, 1 H), 6.78 (m, 2 H), 7.87
(m, 2 H). 9.67 (s, 1 H). ¾ NMR (600 MHz DMSO-D6) δ 21.8 (CH3), 30.7 (CH), 33.5 (CH2), 43.8 (CH2), 87.5 (CH), 114.9 (CH),
128.0 (CH), 129.2 (C), 158.8 (C), 162.9 (C),
163.1 (C), 163.3 (C). ESI-MS: [Ci6H20N4O +
Figure imgf000079_0001
= 3.3 Hz, CH), 128.7 (CH), 132.2 (q, 2JC-F =
31.9 Hz, C), 143.9 (C), 164.3 (C), 164.9 (C), 165.1 (C). ESI-MS: [C2iH27F3 60 + H]+ 437 (100).
4-(4-(4-Fluorophenyl)piperazin-l-yl)-6-(4- (trifluoromethyl)phenyl)pyrimidin-2-amine (38). Purification: column chromatography on silica gel using hexane/ethyl acetate (2: 1 and 1 : 1) as the eluent. Pale solid, 0.207 mg, 99% yield. 'H NMR (600 MHz, MeOD) δ 3.15 (m, 4 H), 3.85 (m, 4 H), 6.57 (s, 1 H), 6.99 (m, 4 H), 7.73 (m, 2 H), 8.06 (m, 2 H). 13C NMR (150 MHz, MeOD) £45.1 (CH2), 51.5 (CH2),
Figure imgf000080_0001
92.1 (CH), 116.4 (d, 2JC F = 23A HZ, CH),
119.7 (d, 3Jc-F = l.l Hz, CH), 126.4 (q, 3JC-F = 4.0 Hz, CH), 128.7 (CH), 132.4 (q, 2JC F = 31.9 Hz, C), 143.8 (C), 149.5 (C), 158.2 (C), 159.7 (C), 164.3 (C), 164.8 (C), 165.1 (C). ESI-MS: [C2iH19F4N5 + H]+ 418 (100).
6-Chloro-N4,N4-bis(pyridin-2-ylmethyl) pyrimidine-2,4-diamine (KK-310).
Purification: column chromatography on silica gel using hexane/ethyl acetate (4: 1 and 1 : 1) as the eluent. White solid, 80% yield. XH NMR (400 MHz, CDC13) δ 1.41-1.68 (m, 10 H), 1.94
Figure imgf000080_0002
(m, 2 H), 3.64 (bs, 1 H), 4.83 (s, 3 H), 5.72 (s,
1 H). HR-ESI/APCI-MS: calculated for
[Ci6H15ClN6 + H]+ 327.1 124, found 327.11 19.
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
2.97 (s, 6 H), 4.72 (s, 2 H), 4.97 (bs, 4 H),
6.24 (s, 1 H), 6.67 (m, 2 H), 7.15 (ddd, J= 7.3, 4.9, 0.8 Hz, 2 H), 7.60 (m, 2 H), 7.73 (m, 2 H), 8.54 (m, 2 H). ESI-MS: [C24H25 7+ H]+ 412 (100).
4-(2-Amino-6-(bis(pyridin-2- ylmethyl)amino)pyrimidin-4-yl)phenol (AC- 171C). Purification: column chromatography on silica gel using ethyl acetate/methanol (7:3) as the eluent. Colorless solid, 46% yield. XH NMR (600 MHz, CDC13) £4.97 (bs, 4 H), 4.16 (s, 2 H), 6.16 (s, 1 H), 6.75 (m, 2 H), 7.18 (ddd,
Figure imgf000085_0001
J= 7.5, 4.8, 0.8 Hz, 2 H), 7.59 (m, 2 H), 7.62 (m, 2 H), 8.54 (m, 2 H). ESI-MS: [C22H2oN60+ H]+ 385 (100).
Nt,iV -bis(pyridin-3-ylmethyl)-6-(4- (trifluoromethyl)phenyl)pyrimidine-2,4- diamine (AC -20-2). Purification: column chromatography on silica gel using ethyl acetate/methanol (9: 1) as the eluent. Pale solid, 77% yield. ¾ NMR (600 MHz, CDC13) £4.79 (bs, 4 H), 4.99 (s, 2 H), 6.29 (s, 1 H), 7.27 (m,
Figure imgf000085_0002
2H), 7.57 (d, J= 7.7 Hz, 2 H), 7.65 (d, J= 8.4 Hz, 2 H), 7.90 (d, J= 7.9 Hz, 2 H), 8.55 (m, 4 H). ESI-MS: [C23H19F3N6+ H]+ 437 (100). 7V4,iV4-bis(pyridin-4-ylmethyl)-6-(4- (trifluoromethyl)phenyl)pyrimidine-2,4- diamine (AC-18-2).
Figure imgf000086_0001
N4,iV4-dibenzyl-6-(4-(trifluoromethyl) phenyl)pyrimidine-2,4-diamine (AC-19-2).
Purification: column chromatography on silica gel using hexane/ethyl acetate (9: 1) as the eluent. White solid, 77% yield. XH NMR (600 MHz, CDC13) £4.78 (bs, 4 H), 4.94 (s, 2 H), 6.30 (s, 1 H), 7.24 (m, 4H), 7.28 (m, 2 H), 7.33
Figure imgf000086_0002
(m, 4 H), 7.62 (d, J= 8.4 Hz, 2 H), 7.89 (d, J= 7.9 Hz, 2 H). ESI-MS: [C25H21F3N4+ H]+ 434 (100).
5-Bromo-2-chloro-4-(4-methylpiperidin-l- yl)pyrimidine (AC-2-P): A solution 4- methylpiperidine (0.362 g, 3.65 mmol) and DIPEA (0.567 g, 4.38 mmol) in 10 ml THF was added dropwise to an ice-cold solution of 5-bromo-2,4-dichloropyrimidine (1.0 g, 4.38 mmol) in 10 ml dry THF. The solution was warmed up to room temperature and stirred overnight. Evaporation of the solvent provided
Figure imgf000086_0003
with a crude product that was purified by
column chromatography on silica gel using 9: 1 hexanes/ethyl acetate as the eluent to yield 1.27 g AC-2-P, white solid, 89% yield. ESI-MS: [Ci0H13BrClN3+ H]+ 290 (100). 2-Chloro-4-(4-methylpiperidin-l-yl)-5-(4-
(trifluoromethyl)phenyl)pyrimidine (AC-5-
P). Synthesized as reported for AC- 17 IB.
Purification: column chromatography on silica gel using hexane/ethyl acetate (4:1) as the
Figure imgf000087_0001
eluent. Pale solid, 45% yield. ESI-MS:
[C17H17CIF3N3+ H]+ 356.
2-Azido-4-(4-methylpiperidin-l-yl)-5-(4-
(trifluoromethyl)phenyl)pyrimidine (AC-9-
P). Sodium azide (0.040 g, 0.61 mmol) was added to a solution of AC-5-P (0.110 g, 0.31 mmol) in DMF (4 mL) and the reaction
*'* Η mixture was stirred at 120 °C for 3 h. Water was added (20 mL) and the mixture was
,.··;·:·-% ,.·-Λχ extracted with DCM (3 x 10 mL). The organic ::: ··": § layers were dried over anhydrous magnesium sulphate, filtered and the solvents were removed under reduced pressure. The product, yellow oil (0.11 g, 99% yield), was used without further purification.
ESI-MS: [Ci7H17F3N6+ H]+ 363
4-(4-Methylpiperidin-l-yl)-5-(4-(trifluoro methyl)phenyl)pyrimidin-2-amine (KK- 337). Palladium on charcoal (lOwt. %) was added to nitrogen purged flask loaded with a solution of AC-9-P (0.036 g, 0.1 mmol) in methanol (2 mL). The mixture was purged with gaseous hydrogen and stirred overnight under 1
Figure imgf000087_0002
atm hydrogen pressure. The mixture was
filtered using celite, concentrated and purified with dichloromethane/methanol gradient to afford 0.020 g (59%) of the product (white solid). ESI-MS: [Ci7H19F3 4+ H]+ 337
4-(4-Methylpiperidin-l-yl)-2-(4-((2- (piperidin-l-yl)ethoxy)methyl)-lH-l,2,3- triazol-l-yl)-5-(4-(trifluoromethyl) phenyl)pyrimidine (AC-13). 1 M aqueous solution of copper (II) sulphate (0.10 rnL) and 1 M aqueous solution of sodium ascorbate (0.20 mL) were added to a solution of azide AC-9-P (0.041 g, 0.1 1 mmol) and NN- dimethylprop-2-yn- 1 -amine (0.010 g, 0.12 mmol) in methanol (5 mL) and the mixture was stirred at room temperature for 16 hours. The solvents were removed under reduced pressure, water was added (10 mL) and the mixture was extracted with dichloromethane (3 x 10 mL).
Figure imgf000088_0001
The combined organic phases were dried over anhydrous magnesium sulphate and concentrated. The product (pale solid, 0.033 g, 57%) was isolated by column chromatography on silica with dichloromethane/methanol gradient as eluent. ESI-MS: [C27H34F3N7O + H]+ 531
2,4-Dichloro-6-(4-(trifluoro methyl) phenyl)pyrimidine (KK-331).
4-Trifluoromethyl phenylboronic acid (0.19 g, 1.0 mmol) was added to a solution of 2,4,6- trichloropyrimidine (0.18 g, 1.0 mmol) in
Figure imgf000088_0002
THF/H2O (9: 1) (10 mL). Sodium carbonate decahydrate (0.43 g, 1.5 mmol) was added following the addition of palladium(II) acetate (5% mol) and triphenylphosphane (10% mol).
The resulting mixture was stirred in a capped glass vial at 60 °C for 16h. The solvents were removed under reduced pressure, brine was added and the mixture was extracted with hexanes (3 x 20 mL). The organic layers were washed with brine, dried over anhydrous magnesium sulphate, and filtered. The filtrate was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel using
hexane/ethyl acetate (20: 1) as eluent to afford 0.20 g (68%) of the product as white solid. ESI-MS: [C11H5CI2F3N2 + H]+ 293
2-Chloro-7V-(2-chloroethyl)-6-(4- (trifluoromethyl) phenyl)pyrimidin-4-amine (KK-309). Synthesized as reported for AC-2-P using KK-331 as the starting material.
Purification: column chromatography on silica gel using hexane/ethyl acetate (4: 1) as eluent.
Figure imgf000089_0001
Pale solid, 50% yield. ESI-MS: [CisHioClzFs s + H]+ 336
Ethyl 2-amino-6-phenylpyrimidine-4- carboxylate (KK-302). Guanidine
hydrochloride (0.029g, 0.30 mmol) was added to a solution of ethyl (£)-2-oxo-4-phenylbut-3- enoate (0.061 g, 0.30 mmol) in DMF (3 mL) and the mixture was heated in a sealed vial by
Figure imgf000089_0002
microwave irradiation at 180 °C for 80 minutes. The solvents were removed and the product (brown solid, 0.014 g, 19%) was isolated by column chromatography on silica gel with dichloromethane/methanol gradient. ESI-MS: [Ci3H13N302 + H]+ 244
Table 5 Binding of exemplary compounds of the invention to s-AChBP
Figure imgf000090_0001
Table 6 Binding of exemplary compounds of the invention to s-AChBP
Figure imgf000090_0002
Figure imgf000091_0001
Figure 8. Representative titration profiles for 4,6-substituted 2-aminopyrimidine competition (using exemplary compounds of the invention 1, 15, 30 and 32, see Table 4, above) with 3H-epibatidine binding showing a range of dissociation constants (¾) and Hill coefficients (nn) for ligand binding to Zs-AChBP. Measurements were carried out by a scintillation proximity assay and are reported as an average of at least three individual experiments (± S.D.).
Figure 9 shows X-ray crystal structures of exemplary compounds of the invention (ligands) 32 and 33 (negative cooperativity, nH<l) and 15 (positive cooperativity, nH>l), in complexes with Zs-AChBP (compound numbering corresponds to Table 4). Fig 9(A): illustrates radial view of Zs-AChBP pentameric structure in complex with exemplary compound of the invention 32 (compound numbering corresponds to Table 4). Full occupation of the 10 binding sites in the unit crystal of a dimer of pentamers was evident. A principal, C loop containing, and complementary face are shown in grey and purple. Fig 9(B): illustrates an expanded radial view of exemplary compound 32 in binding site, including ligand electron density. Side chain carbons of the principal and complementary subunits are shown in grey and purple, respectively. Ligand carbons are in yellow and fluorines in turquoise. Fig 9(C): illustrates an overlay of exemplary compound 32 (blue) and exemplary compound 33 (yellow) crystal structures. Side chain carbons for exemplary compound 32 are in turquoise. The overlay shows little or no variance in ligand pose or side chain positions. Fig 9(D): illustrates a superimposition of exemplary compound 15
(yellow) and exemplary compound 33 (blue) crystal structures. Side chain carbons for exemplary compound 33 are in turquoise. The positively exemplary compound 15 and negatively exemplary compounds 32/33 exemplary compounds of the invention
(cooperative ligands) show a similar positioning of the 4-substituted phenyl rings, but distinct poses or positions for the 2-aminopyrimidine ring and the substituents at position 4 of the pyrimidine ring. Marked changes in the orientation of the side chains of Y185, W53 and Y164 are evident for the positively and negatively cooperative ligands.
Figure 10 shows the superimposition of Zs-AChBP X-ray crystal structures in complex with exemplary compound 33 (Fig. 10A) and exemplary compound 15 (Fig. 10B) with nicotine (PDB code: 1UW6, 18) (in orange). Exemplary compound 33 and exemplary compound 15 carbons are shown in yellow, nicotine in orange. The protein side chains are shown in grey for exemplary compounds 33 and 15 and pink for nicotine. Both ligands show distinctly different positions from the pyrrolidine and pyridine rings of nicotine, as well as the side chain positions of residues in the C loop in the principal subunit (Y89, Y185) and the complementary (W53 and Y164) subunit face.
Figure 11 shows the global differences in X-ray crystal structures of Zs-AChBP bound cooperative ligands in comparison with crystal structure of Zs-AChBP in its Apo form (PDB code: 1UX2) and GLIC (4NPP): Fig. 1 1(A) schematically illustrates a top (apical) view on superimposed (UCSF chimera) Apo pentamer (in blue) and with bound exemplary compound 15 (in red), dashed lines (blue and red respectively) indicate most significant differences in quaternary structures quantified by measuring distances between T13 backbone alpha carbon of distant subunits; Fig. 1 1(B) schematically illustrates a superimposition (PyMOL) of Zs-AChBP Apo, chain D (in blue) and exemplary compound 15 complex, chain D (in red). Major differences in the quaternary structures of the AChBP are marked with dashed rectangles (RMS value of approximately 0.5 or greater). (C) Chart of differences of Ca distances (n=5) of diametrical subunits observed in cooperative ligands relative to Apo in comparison with GLIC (GLIC, closed form, is used as a reference). Nicotine used as a control. Observed differences reflect blooming profile of the protein complexes. (D) Plot of differences of Ca dihedral angles (n=5) observed in cooperative ligands relative to Apo in comparison with GLIC (GLIC closed form used as a reference). Nicotine used as a control. Observed differences reflect twisting profile of the protein complex.
PROTEIN PRODUCTION AND PURIFICATION.
The Ls -AChBP was expressed and purified as previously reported (1, 2). Briefly,
AChBP was expressed with an amino-terminal FLAG epitope tag and secreted from stably transfected HEK293S cells lacking the N-acetylglucosaminyltransferase I (GnTI-) gene. The protein was purified using FLAG-antibody resin and eluted with FLAG peptide (Sigma) and was further characterized by size-exclusion chromatography (SUPEROSE 6 10/300 GL™ column (GE Healthcare) in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl,
0.02% NaNs). Purified AChBP pentamers were then concentrated in a YM50™ Centricon ultrafiltration unit (Millipore) removing monomeric subunits and trace contaminants. Protein concentrations were determined by UV absorbance at 280 nm (NANODROP (NanoDrop) 2000c™ spectrophotometer, ThermoScientific) and confirmed by Bradford assay.
Assay of Ligand Binding to Ls -AChBP.
A scintillation proximity assay was employed to measure ligand binding to AChBP using [3H]-epibatidine (5 nM, GE Healthcare) as the labeled ligand,
polyvinyltoluene anti-mouse SPA scintillation beads (0.17 mg/mL final concentration, GE Healthcare), monoclonal anti-FLAG M2 antibody from mouse 1 :8000 dilution
(Sigma), and 0.50 nM AChBP in binding sites (2). Initial screens required dissociation of 50% of the bound epibatidine by 10 μΜ of the candidate competitive ligand. Full concentration curves were then generated for these higher affinity ligands. Nonspecific binding was determined in parallel by adding a saturating concentration (12.5 μΜ) of MLA (Tocris). The resulting mixtures were allowed to equilibrate at room temperature for a minimum of 1 h and measured on a 1450 MicroBeta TRILUX (TriLux)™ liquid scintillation counter (Wallac). Dissociation constants and Hill coefficients were determined from the competition profiles, concentration of epibatidine and its Ka (0.30 nM) (GRAPHPAD PRISM 6™). All measurements were completed in triplicate. Nicotine dissociation of labeled epibatidine was employed as a frame of reference. CRYSTALLOGRAPHY.
Complex Formation and Crystallization
Complexes of multiple substituted 2-aminopyrimidines were prepared for crystallization as described (2). Ligand - Ls -AChBP complexes was formed by
5 combining 2 of a solution of a compound (10 mM dissolved in DMSO) with 48 of the protein at a concentration of 5 mg/mL to achieve a stoichiometric excess of ligand to binding sites. Co-crystals were obtained by the vapor diffusion hanging drop method. Concentrated protein complex was mixed in a 1 μL/l μL ratio with selected Hampton CRYSTAL SCREEN CRYO™ solutions. Crystals of final size 0.3 mm x 0.3 mm x 0.2 10 mm were obtained using Reagent 3 (0.26 M ammonium phosphate monobasic, 35% v/v glycerol) and flash-cooled directly in liquid nitrogen. Final crystallization buffer mixture was at pH 5.
Data Collection, Phasing, and Model Refinement.
A full set of X-ray diffraction data was collected at 100 K at the Advanced Light
15 Source Synchrotron in Berkeley, California (BL 8.2.2). Data were processed using the HKL2000 program. Ligand - Zs-AChBP complex structures were solved by molecular replacement method with Phaser-MR using an ensemble of AChBP structures (PDB entry 1UX2) as the search model. The electron density maps were fitted with COOT, and structure refinement used the program Phenix-1.8.4. Refinement statistics are listed in
20 Table S2. Atomic coordinates and structure factors of the complex have been deposited in the Protein Data Bank (PDB entries 4QAA, 4QAB and 4QAC). The structural figures were generated using PyMOL.
Refinement Statistics.
Table 7: Data collection and refinement statistics.
Figure imgf000094_0001
Cell dimensions a, b, c (A) 83.41, 129.93, 122.75 j 122.64, 134.02, 147.02 j 240.06, 75.50, 149.76 j α,β,γ (°) 90.00, 106.21, 90.00 \ 90.00, 90.00, 90.00 j 90.00, 117.96,90.00
Resolution (A) 50.00-2.17 (2.95-2.83) j 50.00-3.00(3.05-3.00) j 50.00-1.91 (2.15-2.10) j
Rsym ΟΓ Rmerge 15.3 (83.6) 11.3 (50.6) I 10.1 (78.4)
Τΐσ{Γ) 11.3 (4.3) 41.0(8.1) I 17.7 (2.2)
Completeness (%) 99.8 (100) 100 (100) j 96.6 (97.3)
Redundancy 7.2 (7.7) 14.5 (14.7) j 7.4 (7.5)
Refinement
Resolution (A) 49.65-2.70 49.52-2.98 j 49.8-2.10
No. reflections 68,911 49,465 I 135,865
Rwork / Rfree 19.3/26.2 15.0/23.3 I 20.0/24.7
Ramachandran plot,
residues in most 95.0 94.8 1 98.9 favored regions (%)
No. molecules
Protein (all atoms) 17,293 17,236 1 17,777
Ligand 10 lo 1 10
Water 161 25 1 607
B-factors (A2)
Protein (main/side
28.2/31.7 25.4/32.2 6.0 chain) 1 22.5/2
Ligand 38.2 24.7 1 21.1 Water 21.6 19.3 23.7
Wilson B-factor 50.6 52.0 32.6
R.m.s. deviations
Bond lengths (A) 0.008 0.009 0.009
Bond angles (°) 1.2 1.2 1.2
Values in parentheses are for highest-resolution shell.
Crystallographic data analysis.
All calculations were performed with Sigma Plot using X-ray crystal structure 5 coordinates for Ca atoms of 1 pentamer (Zs-AChBP: residues 0-204). For Zs-AChBP crystal structures, its Apo form (PDB code: 1UX2) was used as a reference. For GLIC, crystal structure obtained at pH 4 (PDB code: 4NPP) was used as a reference. For residues 233-253 alternative conformation 'B' was used for calculations.
Figure 12: Comparison of Zs-AChBP quaternary changes with changes observed
10 for GLIC; Fig. 12(A) schematically illustrates an overlay of Zs-AChBP crystal structure in its Apo form (PDB code: 1UX2, shown in blue) and in complex with ligand 15 (in red), with GLIC X-ray crystal structure at pH 7.5 (PDB code: 4NPQ, shown in grey); X- ray structure of Zs-AChBP complex with nicotine and carbamylcholine (carbachol) used as controls (PDB codes: 1UW6 and 1UV6 respectively); stars represents position of the
15 reference residues on protein axis; Fig. 12(B) illustrates a chart representing 'bloom'; x axis: delta distance between Ca observed in Zs-AChBP - ligand complexes when compared with Zs-AChBP Apo form (GLIC: X-ray structure obtained at pH 7.5 compared to the structure at pH 4); y axis: relative distance from the protein vestibule. Fig. 12(C) illustrates a chart representing 'twist' of the pentameric structure; x axis: delta
20 dihedral angle between Ca observed in Zs-AChBP - ligand complexes when compared with Zs-AChBP Apo form (GLIC: X-ray structure obtained at pH 7.5 compared to the structure at pH 4); y axis: relative distance from the reference residue. Figure 13 graphically illustrates the comparison of delta distance ± SD ('bloom') observed in the Zs-AChBP complexes binding pocket; nicotine X-ray structure used as a control; exemplary compounds of the invention 15, 32, and 33 are used (numbering corresponds to Table 4 structure numbers). Since in Zs-AChBP complex with exemplary compound of the invention (ligand) 15 some residues in C-loop had no defined densities in four chains of the pentamer, values for residues 185, 187 and 188 could not be generated.
3D Distances Calculations (Fig. 12, Panels B and C and Fig. 13)
Distances (AB) between diametrical Ca (A and B) were calculated applying the equation:
Figure imgf000097_0001
Final value for every residue was an average of 5 distances (with exception for regions where specific residue wasn't present in all chains of a pentamer). Delta distance was obtained by subtracting values obtained for Apo from distances acquired for analyzed structures.
Calculations of Residues Relative Positions (Fig. 12, Panels B and C).
Structure alignment (UCSF Chimera) of Zs-AChBP with GLIC X-ray crystal structures superimposed terminal residue 205 of Zs-AChBP complex with residue 194 of GLIC, which is located in the protein vestibule region. Projections of residues position (obtained by averaging coordinates from all five chains for every residue; whenever a residue was missing in one or more chains in a pentamer, it was omitted in this analysis) taper toward an axis passing through the center of the pentagon. Relative distances from the vestibule were obtained by calculating distances between points defined by average of five coordinates for every residue and a point defined by average coordinates for the reference residue (residue 205 for Zs-AChBP and residue 194 for GLIC). Distances obtained for residues in GLIC transmembrane domain are presented as negative values (Fig. S2, Panels B and C).
Dihedral Angles Calculations (Fig. 12, Panel C). Dihedral angles (a, Fig. S4) for every Ca atom (¾) were calculated independently for every chain (X;) and an average was calculated (n=5; with exception for regions where specific residue wasn't present in all chains of a pentamer). In Zs-AChBP structures reference points were defined by average coordinates of residue 194, average 5 coordinates of residue 12 and coordinates of residue 194 from corresponding subunit. For GLIC reference points were defined by coordinates of residue 284 from corresponding subunit, average coordinates of residue 284 and average coordinates of residue 22.
Figure 14: Table 6, illustrated as Figure 14: shows competition between exemplary compounds of the invention (numbering corresponds to Table 4 compound
10 numbers) as substituted 2-aminopyrimidines against 3H-epibatidine binding to Zs-AChBP (ligand affinity constants (¾) and Hill coefficients (nn) for Zs-AChBP are reported as means from at least three individual experiments (±SD). *Ligands with solved crystal structure in complex with Zs-AChBP).
Figure 15: Fig. 15(A) schematically illustrates an overlay of exemplary compound
15 15 (yellow) and exemplary compound 32 (blue) crystal structures. Side chain carbons of the principal and complementary subunits in exemplary compound 15 are shown in grey and purple, respectively. Side chain carbons for exemplary compound 32 are in turquoise; Fig. 15(B) schematically illustrates superimposition of exemplary compound 15 (yellow) and exemplary compound 33 (blue) crystal structures. Side chain carbons for exemplary 0 compound 33 are in turquoise. The overlays show rotation of the pyrimidine ring in
ligand exemplary compound 15 relative to exemplary compounds 32/33 (numbering corresponds to Table 4 compound numbers).
Figure 16 illustrates a screening assay demonstrating activity of exemplary compounds of the invention (so-called compounds 17, KK-311-D and 171A, see
5 Table 4 for structures), using activation of a7-nAChr CNiFERS with l-(5-chloro- 2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea (also called PNU-120596, a drug that acts as a potent and selective positive allosteric modulator for the a7 subtype of neural nicotinic acetylcholine receptors), after a 30 minute (min) cell incubation; Fig. 16A and Fig. 16B graphically illustrates the results at 50 uM and
30 10 uM concentrations, respectively, and Fig. 16C graphically illustrates these
results. For Fig. 16A and Fig. 16B, compounds was screened at 50 uM and 10 uM, respectively, the nicotine agonist at 40 uM, and methyllycaconitine (or MLA) agonist at 400 uM; nicotine and MLA used a positive controls. Compound 171 A (see Table 4) gave approximately 90% activation compared to nicotine. Fig. 16C graphically illustrates a concentration dependent response of a7-nAChr CNiFERS to the exemplary compound 171 A, EC50 = 2.6 uM. Nicotine used a positive
control. Assay done as described by Yamauchi, et al. (201 1) PLOS vol.
6(l):cl6519.
References
I. Changeux JP (2013) 50 years of allosteric interactions: the twists and turns of the models. Nat Rev Mol Cell Biol 14(12):819-829.
2. Karlin A (2002) Emerging structure of the nicotinic acetylcholine receptors. Nat Rev Neurosci 3(2): 102-114.
3. Thompson A J, Lester HA, Lummis SC (2010) The structural basis of function in Cys-loop receptors. Q Rev Biophys 43(4):449-499.
4. Corringer PJ, et al. (2012) Structure and pharmacology of pentameric receptor channels: from bacteria to brain. Structure 20(6):941-956.
5. Heidmann T, Changeux JP (1979) Fast kinetic studies on the interaction of a fluorescent agonist with the membrane-bound acetylcholine receptor from Torpedo marmorata. Eur J Biochem 94(l):255-279.
6. Neubig RR, Cohen JB (1979) Equilibrium binding of [3H]tubocurarine and
[3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions. Biochemistry 18(24):5464-5475.
7. Sine SM, Taylor P (1980) The relationship between agonist occupation and the permeability response of the cholinergic receptor revealed by bound cobra alpha- toxin. J Biol Chem 255(21): 10144-10156.
8. Sine SM, Taylor P (1981) Relationship between reversible antagonist occupancy and the functional capacity of the acetylcholine receptor. J Biol Chem
256(13):6692-6699.
9. Bertrand D, et al. (2008) Positive allosteric modulation of the alpha7 nicotinic acetylcholine receptor: ligand interactions with distinct binding sites and evidence for a prominent role of the M2-M3 segment. Mol Pharmacol 74(5): 1407-1416.
10. Hilf RJ, Dutzler R (2008) X-ray structure of a prokaryotic pentameric ligand- gated ion channel. Nature 452(7185):375-379.
I I . Bocquet N, et al. (2009) X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation. Nature 457(7225): 11 1-114.
Hilf RJ, Dutzler R (2009) Structure of a potentially open state of a proton- activated pentameric ligand-gated ion channel. Nature 457(7225): 115-118.
Sauguet L, et al. (2014) Crystal structures of a pentameric ligand-gated ion channel provide a mechanism for activation. Proc Natl Acad Sci USA
1 11(3):966-971.
Hibbs RE, Gouaux E (201 1) Principles of activation and permeation in an anion- selective Cys-loop receptor. Nature 474(7349):54-60.
Smit AB, et al. (2001) A glia-derived acetylcholine-binding protein that modulates synaptic transmission. Nature 41 1(6835):261-268.
Brejc K, et al. (2001) Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 41 1(6835):269-276.
Hansen SB, et al. (2002) Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein. J Biol Chem 277(44):41299-41302.
Celie PH, et al. (2004) Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41(6):907- 914.
Hansen SB, et al. (2005) Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations. EMBO J24(20):3635-3646.
Hibbs RE, et al. (2009) Structural determinants for interaction of partial agonists with acetylcholine binding protein and neuronal alpha7 nicotinic acetylcholine receptor. £M5O J28(19):3040-3051.
Rucktooa P, et al. (2012) Structural characterization of binding mode of smoking cessation drugs to nicotinic acetylcholine receptors through study of ligand complexes with acetylcholine-binding protein. J Biol Chem 287(28):23283-23293. Hansen SB, Talley TT, Radic Z, Taylor P (2004) Structural and ligand recognition characteristics of an acetylcholine-binding protein from Aplysia californica. J Biol Chem 279(23):24197-24202.
Brams M, et al. (201 1) Crystal structures of a cysteine-modified mutant in loop D of acetylcholine-binding protein. J Biol Chem 286(6):4420-4428. Shahsavar A, et al. (2012) Crystal structure of Lymnaea stagnalis AChBP complexed with the potent nAChR antagonist ϋΗβΕ suggests a unique mode of antagonism. PLoS One 7(8):e40757.
Ulens C, et al. (2009) Use of acetylcholine binding protein in the search for novel alpha7 nicotinic receptor ligands. In silico docking, pharmacological screening, and X-ray analysis. J Med Chem 52(8):2372-2383.
Taly A, et al. (2005) Normal mode analysis suggests a quaternary twist model for the nicotinic receptor gating mechanism. Biophys J 88(6):3954-3965.
Calimet N, et al. (2013) A gating mechanism of pentameric ligand-gated ion channels. Proc Natl Acad Sci USA 1 10(42):E3987-3996.
Dougherty DA (2008) Cys-loop neuroreceptors: structure to the rescue? Chem Rev 108(5): 1642-1653.
Xiu X, Puskar NL, Shanata JA, Lester HA, Dougherty DA (2009) Nicotine binding to brain receptors requires a strong cation-pi interaction. Nature
458(7237):534-537.
Talley TT, et al. (2006) Spectroscopic analysis of benzylidene anabaseine complexes with acetylcholine binding proteins as models for ligand-nicotinic receptor interactions. Biochemistry 45(29):8894-8902.
Bourne Y, Talley TT, Hansen SB, Taylor P, Marchot P (2005) Crystal structure of a Cbtx-AChBP complex reveals essential interactions between snake alpha- neurotoxins and nicotinic receptors. EMBO J 24(8): 1512-1522.
Sine SM, Huang S, Li SX, daCosta CJ, Chen L (2013) Inter-residue coupling contributes to high-affinity subtype-selective binding of alpha-bungarotoxin to nicotinic receptors. Biochem J454(2):311-321.
Talley TT, et al. (2006) Alpha-conotoxin OmIA is a potent ligand for the acetylcholine-binding protein as well as alpha3beta2 and alpha7 nicotinic acetylcholine receptors. J Biol Chem 281(34):24678-24686.
Ulens C, et al. (2006) Structural determinants of selective alpha-conotoxin binding to a nicotinic acetylcholine receptor homolog AChBP. Proc Natl Acad Sci USA 103(10):3615-3620.27.
Stornaiuolo M, et al. (2013) Assembly of a pi-pi stack of ligands in the binding site of an acetylcholine-binding protein. Nat Commun 4: 1875. 36. Richter L, et al. (2012) Diazepam-bound GABAA receptor models identify new benzodiazepine binding-site ligands. Nat Chem Biol 8(5):455-464.
37. Morlock EV, Czajkowski C (2011) Different residues in the GABAA receptor benzodiazepine binding pocket mediate benzodiazepine efficacy and binding. Mol Pharmacol 80(1): 14-22.
38. Hanson SM, Morlock EV, Satyshur KA, Czajkowski C (2008) Structural
requirements for eszopiclone and Zolpidem binding to the gamma-aminobutyric acid type-A (GABAA) receptor are different. J Med Chem 51(22):7243-7252.
39. Changeux JP (2013) The concept of allosteric modulation: an overview. Drug Discov Today Technol 10(2):e223-228.
40. Smith GB, Olsen RW (2000) Deduction of amino acid residues in the GABA(A) receptor alpha subunits photoaffinity labeled with the benzodiazepine
flunitrazepam. Neuropharmacology 39(l):55-64. 1. Talley TT, et al. (2006) Spectroscopic analysis of benzylidene anabaseine complexes with acetylcholine binding proteins as models for ligand-nicotinic receptor interactions. Biochemistry 45(29):8894-8902.
2. Nemecz A, Taylor P (2011) Creating an alpha7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: crystallographic and ligand selectivity analyses. J Biol Chem 286(49):42555-42565.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A compound, a composition or a formulation comprising:
(a)
(i) a compound having a formula as set forth in Formula I;
Figure imgf000103_0001
Formula I
wherein Rl, R2, R3 and R4 are independently selected from the group consisting of: a hydrogen, an aryl (wherein optionally the aryl is any 5-or 6-membered ring, or is selected from the group consisting of: a heteroaryl, an aryl halide, a heteroaryl cycloalkyl, a phenyl, a naphthyl, a thienyl, an indolyl, a thiophene, or a isoxasole), an unsubstituted amino or a substituted amino (NRR'), a halo, a hydroxy (-OH), a substituted or an unsubstituted hydroxy (-OR), a phenoxy, a thiol (-SH), a substituted or an unsubstituted thiol (-SR), a cyano (-CN), a formyl (-CHO), a substituted or unsubstituted alkyl (wherein optionally the alkyl is selected from the group consisting of: - methyl, -ethyl, -propyl, -butyl, -i-propyl, and- i-butyl), a haloalkyl, a substituted or unsubstituted alkene, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkyne, a heteroalkyl, a heteroalkenyl, a heteroalkynyl, a substituted or unsubstituted aryl, a nitro (— N02), an alkoxy, a haloalkoxy, a thioalkoxy, a substituted or unsubstituted alkanoyl, a haloalkanoyl and a carbonyloxy group,
a methyl-aryl substituent, a benzylic substituent, an alkenyl, an alkynyl, a cycloalkyl (wherein optionally the cycloalkyl is selected from the group consisting of: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl, - cycloheptyl, and - cyclooctyl), a cycloalkenyl, a substituted alkyl, a substituted alkenyl, a substituted alkynyl, a substituted cycloalkyl, a substituted cycloalkenyl, an aryl, a heteroaryl silyl, a heterosilyl and a heterocyclic group (wherein optionally the heterocyclic group is selected from the group consisting of: a saturated heterocyclic and/or a nonsaturated heterocyclic, and optionally the saturated heterocyclic and/or a nonsaturated heterocyclic is selected from the group consisting of: -aziridine, -oxirane- thiirane, -azirine, - oxirene, -thiirene, -azetidine, -oxetane, -thietane, -azete, -oxete, -thiete, - pyrrolidine, -oxolane, -thiolane, -pyrrole, -furan, -thiophene, -piperidine, - oxane, -thiane, -pyridine, -pyran, -thiopyran, -azepane, -oxepane, -thiepane, - azepine, -oxepine, -thiepine, -azocane, and -azocine),
a carboxy (COOH), a carboxy derivative, a carboxylic halide (COX), an anhydride (COOCOR), an amide (CONRR'), an ester (COOR), a ketone (COR), an aldehyde (CHO) and a cyano (CN), or
an amidine, an N-substituted or unsubstituted amidines (— C(NR)NR'R") and a carbamidate (— CNOR),
wherein optionally in Formula I, R2 is a substituted amino (NRR', or N-R2-R3) group, or
Figure imgf000104_0001
Formula II wherein optionally in Formula I, Rl is an amino group, R2 is a substituted amino (NRR', is a hydrogen and R4 is an aryl group, or
Figure imgf000104_0002
Formula III,
wherein
R2 and R3 of Formula II, or Rl and R2 of formula III, are independently selected from the group consisting of:
a hydrogen, an aryl (wherein optionally the aryl is any 5-or 6-membered ring, or is selected from the group consisting of: a heteroaryl, an aryl halide, a heteroaryl cycloalkyl, a phenyl, a naphthyl, a thienyl, an indolyl, a thiophene, or a isoxasole), an unsubstituted amino or a substituted amino (NRR'), a halo, a hydroxy (-OH), a substituted or an unsubstituted hydroxy (-OR), a phenoxy, a thiol (-SH), a substituted or an unsubstituted thiol (-SR), a cyano (-CN), a formyl (-CHO), a substituted or unsubstituted alkyl (wherein optionally the alkyl is selected from the group consisting of: - methyl, -ethyl, -propyl, -butyl, -i-propyl, and- i-butyl), a haloalkyl, a substituted or unsubstituted alkene, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkyne, a heteroalkyl, a heteroalkenyl, a heteroalkynyl, a substituted or unsubstituted aryl, a nitro (— N02), an alkoxy, a haloalkoxy, a thioalkoxy, a substituted or unsubstituted alkanoyl, a haloalkanoyl and a carbonyloxy group,
a methyl-aryl substituent, a benzylic substituent, an alkenyl, an alkynyl, a cycloalkyl (wherein optionally the cycloalkyl is selected from the group consisting of: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl, - cycloheptyl, and - cyclooctyl), a cycloalkenyl, a substituted alkyl, a substituted alkenyl, a substituted alkynyl, a substituted cycloalkyl, a substituted cycloalkenyl, an aryl, a heteroaryl silyl, a heterosilyl and a heterocyclic group (wherein optionally the heterocyclic group is selected from the group consisting of: a saturated heterocyclic and/or a nonsaturated heterocyclic, and optionally the saturated heterocyclic and/or a nonsaturated heterocyclic is selected from the group consisting of: -aziridine, -oxirane- thiirane, -azirine, - oxirene, -thiirene, -azetidine, -oxetane, -thietane, -azete, -oxete, -thiete, - pyrrolidine, -oxolane, -thiolane, -pyrrole, -furan, -thiophene, -piperidine, - oxane, -thiane, -pyridine, -pyran, -thiopyran, -azepane, -oxepane, -thiepane, - azepine, -oxepine, -thiepine, -azocane, and -azocine),
a carboxy (COOH), a carboxy derivative, a carboxylic halide (COX), an anhydride (COOCOR), an amide (CONRR'), an ester (COOR), a ketone (COR), an aldehyde (CHO) and a cyano (CN), or
an amidine, an N-substituted or unsubstituted amidines (- C(NR)NR'R") and a carbamidate (-CNOR);
(ii) a compound having a formula as set forth in Formula III, wherein the aryl group of Formula III is a compound having a structure of Formula IV, wherein R6 of Formula IV is the compound of Formula I, Formula II, or Formula III, or, the R4 group of Formula I is a compound of Formula IV, or the Ar group of Formula III is a compound of
Figure imgf000106_0001
Formula IV,
wherein in Formula IV:
X, Y and Z, are independently selected from the group consisting of: a C and an N, Rl, R2, R3, R4 and R5 of Formula IV are independently selected from the group consisting of:
a hydrogen, an aryl (wherein optionally the aryl is any 5-or 6-membered ring, or is selected from the group consisting of: a heteroaryl, an aryl halide, a heteroaryl cycloalkyl, a phenyl, a naphthyl, a thienyl, an indolyl, a thiophene, or a isoxasole), an unsubstituted amino or a substituted amino (NRR'), a halo, a hydroxy (-OH), a substituted or an unsubstituted hydroxy (-OR), a phenoxy, a thiol (-SH), a substituted or an unsubstituted thiol (-SR), a cyano (-CN), a formyl (-CHO), a substituted or unsubstituted alkyl (wherein optionally the alkyl is selected from the group consisting of: - methyl, -ethyl, -propyl, -butyl, -i-propyl, and- i-butyl), a haloalkyl, a substituted or unsubstituted alkene, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkyne, a heteroalkyl, a heteroalkenyl, a heteroalkynyl, a substituted or unsubstituted aryl, a nitro (— N02), an alkoxy, a haloalkoxy, a thioalkoxy, a substituted or unsubstituted alkanoyl, a haloalkanoyl and a carbonyloxy group
a halogen, a methyl-aryl substituent, a benzylic substituent, an alkenyl, an alkynyl, a cycloalkyl (wherein optionally the cycloalkyl is selected from the group consisting of: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl, - cycloheptyl, and - cyclooctyl), a cycloalkenyl, a substituted alkenyl, a substituted alkynyl, a substituted cycloalkyl, a substituted cycloalkenyl, a heteroaryl silyl, a heterosilyl and a heterocyclic group (wherein optionally the heterocyclic group is selected from the group consisting of: a saturated heterocyclic and/or a nonsaturated heterocyclic, and optionally the saturated heterocyclic and/or a nonsaturated heterocyclic is selected from the group consisting of: -aziridine, -oxirane- thiirane, -azirine, -oxirene, -thiirene, - azetidine, -oxetane, -thietane, -azete, -oxete, -thiete, -pyrrolidine, -oxolane, - thiolane, -pyrrole, -furan, -thiophene, -piperidine, -oxane, -thiane, -pyridine, - pyran, -thiopyran, -azepane, -oxepane, -thiepane, -azepine, -oxepine, -thiepine, -azocane, and -azocine),
a carboxy (COOH), a carboxy derivative, a carboxylic halide (COX), an anhydride (COOCOR), an amide (CONRR'), an ester (COOR), a ketone (COR), an aldehyde (CHO) and a cyano (CN), or
an amidine, an N-substituted or unsubstituted amidines (— C(NR)NR'R") and a carbamidate (— CNOR);
(iii) a compound having a formula as set forth in Formula III, wherein the aryl group of Formula III is a compound having a structure of Formula V, wherein R5 of Formula V is the compound of Formula I, Formula II, or Formula III, or, the R4 group of Formula I is a compound of Formula V, or the Ar group of Formula III is a compound of Formula V:
Figure imgf000107_0001
Formula V,
wherein in Formula V:
wherein X, Y and Z, are independently selected from the group consisting of: a C an N, an O and an S,
Rl, R2, R3 and R4, of Formula V are independently selected from the group consisting of:
a hydrogen, an aryl (wherein optionally the aryl is any 5-or 6-membered ring, or is selected from the group consisting of: a heteroaryl, an aryl halide, a heteroaryl cycloalkyl, a phenyl, a naphthyl, a thienyl, an indolyl, a thiophene, or a isoxasole), an unsubstituted amino or a substituted amino (NRR'), a halo, a hydroxy (-OH), a substituted or an unsubstituted hydroxy (-OR), a phenoxy, a thiol (-SH), a substituted or an unsubstituted thiol (-SR), a cyano (-CN), a formyl (-CHO), a substituted or unsubstituted alkyl (wherein optionally the alkyl is selected from the group consisting of: - methyl, -ethyl, -propyl, -butyl, -i-propyl, and- i-butyl), a haloalkyl, a substituted or unsubstituted alkene, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted alkyne, a heteroalkyl, a heteroalkenyl, a heteroalkynyl, a substituted or unsubstituted aryl, a nitro (— N02), an alkoxy, a haloalkoxy, a thioalkoxy, a substituted or unsubstituted alkanoyl, a haloalkanoyl and a carbonyloxy group
a halogen, a methyl-aryl substituent, a benzylic substituent, an alkenyl, an alkynyl, a cycloalkyl (wherein optionally the cycloalkyl is selected from the group consisting of: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl, - cycloheptyl, and - cyclooctyl), a cycloalkenyl, a substituted alkyl, a substituted alkenyl, a substituted alkynyl, a substituted cycloalkyl, a substituted cycloalkenyl, an aryl, a heteroaryl silyl, a heterosilyl and a heterocyclic group (wherein optionally the heterocyclic group is selected from the group consisting of: a saturated heterocyclic and/or a nonsaturated heterocyclic, and optionally the saturated heterocyclic and/or a nonsaturated heterocyclic is selected from the group consisting of: -aziridine, -oxirane- thiirane, -azirine, - oxirene, -thiirene, -azetidine, -oxetane, -thietane, -azete, -oxete, -thiete, - pyrrolidine, -oxolane, -thiolane, -pyrrole, -furan, -thiophene, -piperidine, - oxane, -thiane, -pyridine, -pyran, -thiopyran, -azepane, -oxepane, -thiepane, - azepine, -oxepine, -thiepine, -azocane, and -azocine),
a carboxy (COOH), a carboxy derivative, a carboxylic halide (COX), an anhydride (COOCOR), an amide (CONRR'), an ester (COOR), a ketone (COR), an aldehyde (CHO) and a cyano (CN), or
an amidine, an N-substituted or unsubstituted amidines (— C(NR)NR'R") and a carbamidate (— CNOR);
wherein the compound, composition or formulation of any of (i), (ii) or (iii): exhibits a cooperative binding to a nicotinic acetylcholine receptor (nAChR) protein and modulates, inhibits or stimulates an AChR functional response, or
has a positive or a negative cooperativity in an acetylcholine ligand- acetylcholine receptor response, or in ligand occupation of the acetylcholine binding protein (AChBP), or can modulate, inhibit or increase the magnitude and rapidity of a functional response of a nicotinic acetylcholine receptor (nAChR);
(b) a compound as set forth in Figure 2A, 2B, 3A, 3B, Figure 4, or Table 4;
wherein optionally the compound is selected from the group consisting of:
Figure imgf000109_0001
-(4-Methoxyphenyl)-N4-octylpyrimidine-2,4-diamine,
Figure imgf000109_0002
-Morpholino-6-(4-(trifluoromethyl)phenyl)pyrimidin-2-amine,
Figure imgf000109_0003
-(4-Methylpiperidin- l-yl)-6-(4-(trifluoromethyl)phenyl)pyrimidin-2-
Figure imgf000109_0004
Ethyl 2-amino-6-phenylpyrimidine-4-carboxylate,
6-(4-Methoxyphenyl)-N4,N4-bis(pyridin-2-ylmethyl)pyrimidine-2,4-diamine; and a combination thereof;
(c) an enantiomer, a stereoisomer, an analog or a bioisostere of any of (a) or (b), or
(d) a salt of, or a pharmaceutically acceptable salt of, any of (a), (b) or (c).
2. The compound, composition or formulation of claim 1, wherein the compound, composition or formulation is formulated for administration in vivo; or for enteral or parenteral administration, or for ophthalmic, topical, oral, intravenous (IV), intramuscular (IM), intrathecal, subcutaneous (SC), intracerebral, epidural, intracranial or rectal administration, or by inhalation.
3. The compound, composition or formulation of claim 1 or claim 2, wherein the compound, composition or formulation is formulated as: a particle, a nanoparticle, a liposome, a tablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, a suspension, a syrup, an emulsion, a lotion, an ointment, an aerosol, a spray, a lozenge, an ophthalmic preparation, an aqueous or a sterile or an injectable solution, a patch (optionally a transdermal patch or a medicated adhesive patch), or an implant.
4. A pharmaceutical composition or formulation comprising a compound, composition or formulation of any of claims 1 to 3, wherein optionally the pharmaceutical composition or formulation further comprises a pharmaceutically acceptable excipient.
5. A product of manufacture or a device, comprising a compound, composition or formulation of any of claims 1 to 3, or a pharmaceutical composition or formulation of claim 4,
wherein optionally the product of manufacture or device is a medical device or an implant,
wherein optionally the product of manufacture or device is designed to be capable of injecting, causing inhalation of, adsorption of, or otherwise administering for either enteral or parenteral administration a compound, composition or formulation of claims 1 to 3, or a pharmaceutical composition or formulation of claim 4.
6. A pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi- chambered pump, comprising a compound, composition or formulation of any of claims 1 to 3, or a pharmaceutical composition or formulation of claim 4.
7. A method for:
modulating, inhibiting or stimulating a nicotinic acetylcholine receptor (nAChR) functional response,
modulating, inhibiting or stimulating an nAChR response by cooperative binding to a nicotinic acetylcholine receptor (nAChR) protein,
having a positive or a negative cooperativity in an acetylcholine ligand- acetylcholine receptor response, or in ligand occupation of the acetylcholine binding protein (AChBP), or
modulating, inhibiting or stimulating the magnitude and rapidity of a functional response of a nicotinic acetylcholine receptor (nAChR),
comprising contacting the nAChR with a compound, composition or formulation of any of claims 1 to 3, or administering or applying to an individual in need thereof: a pharmaceutical composition or formulation of claim 4; a product of manufacture or a device of claim 5, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump, of claim 6,
thereby
modulating, inhibiting or stimulating a nicotinic acetylcholine receptor (nAChR) functional response,
modulating, inhibiting or stimulating an nAChR response by cooperative binding to a nicotinic acetylcholine receptor (nAChR) protein,
having a positive or a negative cooperativity in an acetylcholine ligand- acetylcholine receptor response, or in ligand occupation of the acetylcholine binding protein (AChBP), or
modulating, inhibiting or stimulating the magnitude and rapidity of a functional response of a nicotinic acetylcholine receptor (nAChR),
wherein optionally the contacting is in vitro, ex vivo or in vivo.
8. A method for increasing, modulating or stimulating the release of or activity of a neurotransmitter or a neuromodulator in the central nervous system (CNS) or the brain, or modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain,
wherein optionally the neurotransmitter or a neuromodulator comprises a glutamate, a gamma aminobutyric acid (GABA), a glycine, a serotonin, a peptide or a neuropeptide (optionally a galanin, an enkephalin, an acetylcholine), a norepinephrine, or a biogenic amine (optionally a dopamine, a noradrenaline, an adrenaline, or a
catecholamine),
comprising:
administering to an individual in need thereof a compound, composition or formulation of any of claims 1 to 3, or a pharmaceutical composition or formulation of claim 4, or administering to an individual in need thereof a product of manufacture or a device of claim 5, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of claim 6,
thereby increasing, modulating or stimulating the release of or activity of a neurotransmitter or a neuromodulator in the central nervous system (CNS) or the brain, or modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain
wherein optionally the contacting is in vitro, ex vivo or in vivo.
9. A method for treating, ameliorating, preventing or lessening the symptoms of diseases or conditions that are responsive to modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or are responsive to modulating, decreasing or increasing the activity of a nicotinic
acetylcholine receptor (nAChR) in the CNS or brain,
wherein optionally the neurotransmitter or a neuromodulator comprises a glutamate, a gamma aminobutyric acid (GABA), a glycine, a serotonin, a peptide or a neuropeptide (optionally a galanin, an enkephalin, an acetylcholine), a norepinephrine, or a biogenic amine (optionally a dopamine, a noradrenaline, an adrenaline, or a
catecholamine),
comprising:
administering to an individual in need thereof a compound, composition or formulation of any of claims 1 to 3, or administering or applying to an individual in need thereof: a pharmaceutical composition or formulation of claim 4; a product of manufacture or a device of claim 5, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of claim 6,
thereby treating, ameliorating, preventing or lessening the symptoms of diseases or conditions that are responsive to modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or are responsive to modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain,
wherein optionally the contacting is in vitro, ex vivo or in vivo.
10. A method for:
treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse, optionally an addiction or substance abuse involving use of tobacco or nicotine-related products, involving modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or responsive to modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain,
wherein optionally the neurotransmitter or a neuromodulator comprises a glutamate, a gamma aminobutyric acid (GABA), a glycine, a serotonin, a peptide or a neuropeptide (optionally a galanin, an enkephalin, an acetylcholine), a norepinephrine, or a biogenic amine (optionally a dopamine, a noradrenaline, an adrenaline, or a
catecholamine), or
for treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse involving tobacco or nicotine use, cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, or 3,4-methylenedioxy-N- methylamphetamine (MDMA), comprising:
administering to an individual in need thereof a compound, composition or formulation of any of claims 1 to 3, or administering or applying to an individual in need thereof: a pharmaceutical composition or formulation of claim 4; a product of manufacture or a device of claim 5, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of claim 6,
thereby: treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse, optionally an addiction or substance abuse involving use of tobacco or nicotine-related products, involving modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or responsive to modulating, decreasing or increasing the activity of a nicotinic
acetylcholine receptor (nAChR) in the CNS or brain, or
treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse involving tobacco or nicotine use, cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, or 3,4-methylenedioxy-N- methylamphetamine (MDMA),
wherein optionally the contacting is in vitro, ex vivo or in vivo.
1 1. A method for:
treating, ameliorating, preventing or lessening the symptoms of a disease or condition responsive to an increase in levels or activity of a neurotransmitter or a neuromodulator in the peripheral nervous system (PNS), the central nervous system (CNS) or brain, or a disease or condition responsive to the modulation of, or a decrease or an increase in the activity of a nicotinic acetylcholine receptor (nAChR), or
treating, ameliorating, preventing or lessening the symptoms of a dementia, Parkinson's disease, pain or chronic pain, an allodynia, a psychosis, autism, or a schizophrenia, wherein optionally the neurotransmitter or a neuromodulator comprises a glutamate, a gamma aminobutyric acid (GABA), a glycine, a serotonin, a peptide or a neuropeptide (optionally a galanin, an enkephalin, an acetylcholine), a norepinephrine, or a biogenic amine (optionally a dopamine, a noradrenaline, an adrenaline, or a
catecholamine),
comprising: administering to an individual in need thereof a compound, composition or formulation of any of claims 1 to 3, or administering or applying to an individual in need thereof: a pharmaceutical composition or formulation of claim 4; a product of manufacture or a device of claim 5, or a pump, a patch, a device, a
subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of claim 6,
thereby
treating, ameliorating, preventing or lessening the symptoms of a disease or condition responsive to an increase in levels or activity of a neurotransmitter or a neuromodulator in the peripheral nervous system (P S), the central nervous system (CNS) or brain, or a disease or condition responsive to the modulation of, or a decrease or an increase in the activity of a nicotinic acetylcholine receptor (nAChR), or
treating, ameliorating, preventing or lessening the symptoms of a dementia,
Parkinson's disease, pain or chronic pain, an allodynia, a psychosis, autism, or a schizophrenia,
wherein optionally the contacting is in vitro, ex vivo or in vivo.
12. A kit comprising a compound, composition or formulation of any of claims 1 to 3, a pharmaceutical composition or formulation of claim 4; a product of manufacture or a device of claim 5, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of claim 6, and/or optionally comprising ingredients and/or instructions for practicing a method of any of claims 7 to 1 1.
13. Use of a compound, composition or formulation of claim 1, in the manufacture of a medicament.
14. Use of a compound, composition or formulation of claim 1, in the manufacture of a medicament for:
modulating, inhibiting or stimulating a nicotinic acetylcholine receptor (nAChR) functional response,
modulating, inhibiting or stimulating an nAChR response by cooperative binding to a nicotinic acetylcholine receptor (nAChR) protein,
having a positive or a negative cooperativity in an acetylcholine ligand- acetylcholine receptor response, or in ligand occupation of the acetylcholine binding protein (AChBP),
modulating, inhibiting or stimulating the magnitude and rapidity of a functional response of a nicotinic acetylcholine receptor (nAChR),
increasing, modulating or stimulating the release of or activity of a
neurotransmitter or a neuromodulator in the central nervous system (CNS) or the brain, or modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain,
treating, ameliorating, preventing or lessening the symptoms of diseases or conditions that are responsive to modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or are responsive to modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain,
treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse, optionally an addiction or substance abuse involving use of tobacco or nicotine-related products, involving modulating, decreasing or increasing levels or activity of a neurotransmitter or a neuromodulator in the CNS or brain, or responsive to modulating, decreasing or increasing the activity of a nicotinic acetylcholine receptor (nAChR) in the CNS or brain, or
treating, ameliorating, preventing or lessening the symptoms of an addiction or substance abuse involving tobacco or nicotine use, cigarette smoking, methylphenidate, cocaine, or amphetamines or methamphetamines, or 3,4-methylenedioxy-N- methylamphetamine (MDMA), treating, ameliorating, preventing or lessening the symptoms of a disease or condition responsive to an increase in levels or activity of a neurotransmitter or a neuromodulator in the peripheral nervous system (PNS), the central nervous system (CNS) or brain, or a disease or condition responsive to the modulation of, or a decrease or an increase in the activity of a nicotinic acetylcholine receptor (nAChR), or
treating, ameliorating, preventing or lessening the symptoms of a dementia, Parkinson's disease, pain or chronic pain, an allodynia, a psychosis, autism, or a schizophrenia.
15. A therapeutic combination comprising: a compound, composition or formulation of any of claims 1 to 3, a pharmaceutical composition or formulation of claim 4; a product of manufacture or a device of claim 5, or a pump, a patch, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, a pen, an infusion pen, a needle, a reservoir, an ampoules, a vial, a syringe, a cartridge, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of claim 6.
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106674131A (en) * 2016-12-14 2017-05-17 华南理工大学 Method for compounding polysubstituted pyrimidine heterocyclic compound
CN106950383A (en) * 2017-03-23 2017-07-14 南京农业大学 Purposes of the spider acetylcholine associated proteins in ligand-gated ion channel ligand screening
US9975886B1 (en) 2017-01-23 2018-05-22 Cadent Therapeutics, Inc. Potassium channel modulators
CN110023331A (en) * 2016-07-07 2019-07-16 霍华休斯医学研究院 The ion channel and application method of modified ligand gate
US10774064B2 (en) 2016-06-02 2020-09-15 Cadent Therapeutics, Inc. Potassium channel modulators
US10793551B2 (en) 2017-10-19 2020-10-06 Effector Therapeutics Inc. Benzimidazole-indole inhibitors of Mnk1 and Mnk2
WO2020210521A2 (en) 2019-04-12 2020-10-15 The Regents Of The University Of California Compositions and methods for increasing muscle mass and oxidative metabolism
US11993586B2 (en) 2018-10-22 2024-05-28 Novartis Ag Crystalline forms of potassium channel modulators
US11993580B1 (en) 2022-12-02 2024-05-28 Neumora Therapeutics, Inc. Methods of treating neurological disorders

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010120854A1 (en) * 2009-04-15 2010-10-21 Glaxosmithkline Llc Chemical compounds
US20110230485A1 (en) * 2008-10-09 2011-09-22 Neurosearch A/S 6-phenyl-pyrimidin-4-yl-(phenylamine or phenoxy) derivatives useful as modulators of nicotinic acetylcholine receptors
US20130158258A1 (en) * 2007-02-14 2013-06-20 Janssen Pharmaceutica Nv 2-aminopyrimidine modulators of the histamine h4 receptor
US20130158057A1 (en) * 2011-11-29 2013-06-20 Genentech, Inc. Aminopyrimidine derivatives as lrrk2 modulators

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130158258A1 (en) * 2007-02-14 2013-06-20 Janssen Pharmaceutica Nv 2-aminopyrimidine modulators of the histamine h4 receptor
US20110230485A1 (en) * 2008-10-09 2011-09-22 Neurosearch A/S 6-phenyl-pyrimidin-4-yl-(phenylamine or phenoxy) derivatives useful as modulators of nicotinic acetylcholine receptors
WO2010120854A1 (en) * 2009-04-15 2010-10-21 Glaxosmithkline Llc Chemical compounds
US20130158057A1 (en) * 2011-11-29 2013-06-20 Genentech, Inc. Aminopyrimidine derivatives as lrrk2 modulators

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KACZANOWSKA, K ET AL.: "Structural basis for cooperative interactions of substituted 2-aminopyrimidines with the acetylcholine binding protein", PNAS, vol. 111, no. 29, 22 July 2014 (2014-07-22), pages 10749 - 10754 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10774064B2 (en) 2016-06-02 2020-09-15 Cadent Therapeutics, Inc. Potassium channel modulators
CN110023331A (en) * 2016-07-07 2019-07-16 霍华休斯医学研究院 The ion channel and application method of modified ligand gate
CN106674131A (en) * 2016-12-14 2017-05-17 华南理工大学 Method for compounding polysubstituted pyrimidine heterocyclic compound
CN106674131B (en) * 2016-12-14 2019-08-20 华南理工大学 A method of synthesis polysubstituted pyrimidine heterocyclic compound
US10717728B2 (en) 2017-01-23 2020-07-21 Cadent Therapeutics, Inc. Potassium channel modulators
US9975886B1 (en) 2017-01-23 2018-05-22 Cadent Therapeutics, Inc. Potassium channel modulators
US10351553B2 (en) 2017-01-23 2019-07-16 Cadent Therapeutics, Inc. Potassium channel modulators
CN106950383B (en) * 2017-03-23 2018-09-21 南京农业大学 Purposes of the spider acetylcholine binding protein in ligand-gated ion channel ligand screening
CN106950383A (en) * 2017-03-23 2017-07-14 南京农业大学 Purposes of the spider acetylcholine associated proteins in ligand-gated ion channel ligand screening
US10793551B2 (en) 2017-10-19 2020-10-06 Effector Therapeutics Inc. Benzimidazole-indole inhibitors of Mnk1 and Mnk2
US11993586B2 (en) 2018-10-22 2024-05-28 Novartis Ag Crystalline forms of potassium channel modulators
WO2020210521A2 (en) 2019-04-12 2020-10-15 The Regents Of The University Of California Compositions and methods for increasing muscle mass and oxidative metabolism
EP3952849A4 (en) * 2019-04-12 2023-03-01 The Regents Of The University Of California Compositions and methods for increasing muscle mass and oxidative metabolism
JP7566775B2 (en) 2019-04-12 2024-10-15 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Compositions and methods for increasing muscle mass and oxidative metabolism
US11993580B1 (en) 2022-12-02 2024-05-28 Neumora Therapeutics, Inc. Methods of treating neurological disorders

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