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WO2016191366A1 - Modulateurs pour les sous-unités α2 et α4 de récepteur nicotinique de l'acétylcholine - Google Patents

Modulateurs pour les sous-unités α2 et α4 de récepteur nicotinique de l'acétylcholine Download PDF

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WO2016191366A1
WO2016191366A1 PCT/US2016/033774 US2016033774W WO2016191366A1 WO 2016191366 A1 WO2016191366 A1 WO 2016191366A1 US 2016033774 W US2016033774 W US 2016033774W WO 2016191366 A1 WO2016191366 A1 WO 2016191366A1
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pbtc
thiophene
nachrs
benzo
synthetic example
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PCT/US2016/033774
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English (en)
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Theodore M. Kamenecka
Paul Kenny
Jon M. Lindstrom
Jingyi Wang
Zhuang JIN
Christelle DOEBELIN
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The Scripps Research Institute
Icahn School Of Medicine At Mont Sinai
The Trustees Of The University Of Pennsylvania
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Publication of WO2016191366A1 publication Critical patent/WO2016191366A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • Nicotinic acetylcholine receptors are critical for nicotine addiction and important for several neuropsychiatric disorders (De Biasi and Dani, 201 1 ; Lewis and Picciotto, 2013; Picciotto, 2015). They are ligand-gated ion channels formed from five homologous subunits whose subtypes are defined by their subunit composition. There are twelve neuronal types of subunits: 2-10 and ⁇ 2-4. Homomeric nAChRs like ct7 assemble from only a7 subunits, while heteromeric nAChRs usually require both a and ⁇ subunits (Hurst et al., 2013; Zoli et al., 2014).
  • Both homomeric and heteromeric nAChRs form orthosteric agonist binding sites at interfaces between subunits in the extracellular domain.
  • various ligands have been identified which activate, inhibit, or potentiate activation of nAChRs from allosteric sites other than the agonist binding sites (Williams et al., 201 1 ; Hurst et al., 2013; Grupe et al., 2015).
  • PAMs positive allosteric modulators
  • NAMs negative allosteric modulators
  • allosteric agonists Williams et al., 201 1 ; Gill et al., 201 1 ; Gill-Thind et al., 2015).
  • nAChRs these drugs bind to various places in nAChRs, including the extracellular domain, transmembrane domain, and the extracellular C-terminus (i.e., C- tail) (Grupe et al., 2015; Williams et al., 201 1).
  • PAMs enhance nAChR function in an activity-dependent manner, potentially modulating the endogenous pattern of signaling rather than constantly activating or desensitizing nAChRs.
  • PAMs also increase the potential for subtype specificity. This is because diversity of PAM binding sites in nAChRs provides better chances to develop selective therapeutics than does targeting the relatively similar ACh binding sites.
  • Type I PAMs increase peak responses.
  • Type II PAMs not only increase peak responses but also the duration of channel opening by delaying desensitization. This makes type II PAMs especially efficacious. In some cases, they can act as allosteric agonists (Gill et al., 201 1). Understanding the pharmacology and potentiation mechanism of PAMs should facilitate design of more potent and selective PAMs. There is no direct correlation between where a PAM binds and which type of PAM it is (Williams et al., 201 1).
  • PAMs bind in the transmembrane domain near the gate for the cation channel whose opening they influence (Williams et al., 201 1). These transmembrane PAMs can be either type I or type II. Here we describe a novel type II PAM, Br-PBTC, which binds at the C-tail of the 4 subunit.
  • the invention provides, in various embodiments, a compound of formula (I)
  • ring bearing R 1 comprises 0 or 1 nitrogen atom therewithin
  • R 1 is halo, cyano, (C
  • NR 2 or phenyl optionally substituted with 1 or 2 OR groups, wherein R is H or (C
  • -C4)alkyl, or two R 1 groups together form a methylenedioxy; m 0, 1, 2, or 3;
  • R 2 is H, (Ci-Gt)alkyl, or phenyl, wherein the phenyl is optionally substituted with 1 -2
  • R 3 is H, (Ci-C6)alkyl, (C r C6)acyl, or benzyl, wherein the benzyl is optionally substituted with 1 -2 R 4 ;
  • R 4 is halo, (C C 4 )alkyl, (C,-C 4 )alkoxy, or NR 2 ;
  • R 5 is independently at each occurrence H or (C] -C 4 )alkyl
  • n 2, 3, or 4;
  • the invention can further provide a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable excipient.
  • the invention can further provide a method of allosterically modulating an a5-nicotinic receptor, comprising contacting the receptor with an effective amount or concentration of the invention.
  • the allosteric modulation can be a positive allosteric modulation.
  • the invention can provide a method of treatment of nicotine addiction in a patient afflicted therewith, comprising administering to the patient an effective dose of a compound of the invention. Because 4 ⁇ 2 nicotinic receptors are lost in Alzheimer's disease and the weak positive allosteric modulator for all a subunits galantamine has been found useful for symptomatic therapy (Samochocki et al., 2003) it is possible that the more potent and efficacious positive allosteric modulators of the invention might be more useful and less liable to side effects because they are more specific and effect only a4 subunits.
  • FIGURE 1 Chemical structure and nAChR subtype-selectivity of the PAM Br- PBTC.
  • A Structural comparision of Br-PBTC and 17P-estradiol.
  • B
  • Concentration/response curves of Br-PBTC for potentiating activation of nAChR subtypes expressed in HEK cell lines were pre-applied for 15 minutes before acute application of ACh at EC 2 o concentrations (i.e., ⁇ 4 ⁇ 2, 0.4 ⁇ ; ⁇ 4 ⁇ 4, 1 ⁇ ; ⁇ 3 ⁇ 2, 4 ⁇ ; ⁇ 3 ⁇ 4, 5 ⁇ ; 2 ⁇ 2, 0.4 ⁇ ; ⁇ 2 ⁇ 4, 0.8 ⁇ ).
  • Potentiation effects were calculated by increased peak responses by Br-PBTC relative to responses evoked by ACh.
  • FIGURE 2 Schematic illustration of human nAChR a3 and 4 subunit chimeras.
  • the a3 sequences are grey and the a4 sequences are black.
  • a4 AAC is an cc4 subunit with its last four amino acids replaced with alanine-alanine-cysteine. These were chosen because this mutation inhibits the PAM effect of ⁇ -estradiol (Paradiso et al., 201 1 ).
  • This modified C- tail is annotated as a grey squiggly line.
  • FIGURE 3 Summary of potentiation effects of Br-PBTC on ⁇ 3/ ⁇ 4 nAChR chimeras expressed in oocytes.
  • Br-PBTC (3 ⁇ ) was co-applied with EC3 0 -40 ACh to each oocyte. Each data point was collected from more than four oocytes.
  • A Bar graph comparison of the PAM effects of Br-PBTC.
  • B Representative response kinetics for wild type ⁇ 3 ⁇ 2, ⁇ 4 ⁇ 2, ⁇ 4 ⁇ ⁇ 2 and ⁇ 3 (1 - 440) / ⁇ 4 (561 - 594) ⁇ 2 nAChRs.
  • FIGURE 4 Br-PBTC potentiates activation of both stiochiometries of ⁇ 4 ⁇ 2 nAChRs.
  • Concatameric nAChRs of defined stoichiometries were expressed in HEK cell lines.
  • ACh indicates ACh binding sites at subunit interfaces.
  • PAM indicates PAM binding sites near 4 C-tails.
  • B Concentration/response curves for Br-PBTC potentiation of EC40-50 ACh.
  • FIGURE 5 Br-PBTC PAM effect increases with the number of a4 subunits in a nAChR. Each data point was collected from more than five oocytes.
  • A Illustration of nAChRs constructs used that contain different numbers of a4 subunits.
  • B Potentiation by Br-PBTC (3 ⁇ ) increases with the number of 4 subunits in a nAChR. Br-PBTC was co- applied with 100 or 3000 ⁇ ACh.
  • FIGURE 6 Br-PBTC reactivates short-term desensitized nAChRs expressed in oocytes.
  • ACh 1000 ⁇ was applied to oocytes for 6 minutes before its co-application with Br-PBTC (3 ⁇ ). Each data point was collected from more than five oocytes.
  • A Br-PBTC requires two or more a4 subunits to reactivate short-term desensitized nAChRs. The efficacy of reactivation increases with more a4 subunits in a nAChR.
  • B Response kinetics from representative oocytes.
  • FIGURE 7 Br-PBTC reactivates short-term desensitized ( ⁇ 4 ⁇ 2) 2 ⁇ 4 and ( ⁇ 4 ⁇ 2) 2 ⁇ 2 nAChRs expressed in HEK cells. Saturating concentrations of agonists were added to desensitize nAChRs. Because of the low ACh affinity site at the ⁇ 4/ ⁇ 4 interface, higher concentrations of agonists were used for ( ⁇ 4 ⁇ 2) 2 ⁇ 4 than ( ⁇ 4 ⁇ 2) 2 ⁇ 2. Br-PBTC (3 ⁇ ) and ⁇ (1 ⁇ ) were added separately or together to nAChRs 6 minutes after addition of agonist. The antagonist ⁇ prevents activation.
  • A ACh (300 ⁇ ) and nicotine ( 100 ⁇ ) desensitized ( ⁇ 4 ⁇ 2) 2 ⁇ 4 nAChRs.
  • B ACh (100 ⁇ ) and nicotine (10 ⁇ ) desensitized ( ⁇ 4 ⁇ 2) 2 ⁇ 2 nAChRs.
  • FIGURE 8 Br-PBTC reactivates long-term desensitized nAChRs expressed in HEK cells.
  • FIGURE 9 Effect of Br-PBTC and conotoxin Mil on ( ⁇ 4 ⁇ 2)( ⁇ 6 ⁇ 2) ⁇ 3 nAChRs expressed in oocytes.
  • A Illustration of expressing ( ⁇ 4 ⁇ 2)( ⁇ 6 ⁇ 2) ⁇ 3 from a pentameric concatamer. These nAChRs have only one C-tail PAM site for Br-PBTC.
  • B Br-PBTC potentiated activation of ( ⁇ 4 ⁇ 2)( ⁇ 6 ⁇ 2) ⁇ 3 by ACh, but both activation and potentiation were blocked by the a6-selective antagonist, conotoxin Mil (50 nM). Responses to ACh (3 ⁇ ) are shown in black and responses to ACh with Br-PBTC (3 ⁇ ) are shown in grey.
  • FIGURE 10 Proposed potentiation mechanism for C-tail PAMs.
  • A States of nAChRs bound with an agonist or antagonist. Upon agonist binding, nAChRs go through various conformation changes from the resting state (R) to the open state (O), and non- conductive short-term (D s ) or long-term (D L ) desensitized states. When an antagonist binds to nAChRs, nAChRs go into an inactive state (I) or is held in a resting state that prevents further activation by agonists. nAChRs may pass through various transitional states, which are not displayed in the figure.
  • B Hypothetical PAM effects on probability of nAChR states.
  • FIGURE 11 A graph of data indicating that compound SRI 3521 reduced nicotine intake in a dose-dependent manner when administered by intraperitoneal injection 30 min prior to the l h nicotine self-administration session.
  • FIGURE 12 Protein backbone structure model of an ( ⁇ 4 ⁇ 2) 2 ⁇ 2 AChR showing docking of ACh to an ⁇ 4/ ⁇ 2 binding site and docking of the PAM Br-PBTC to the transmembrane region of an a4 subunit. The large cytoplasmic domains of the subunits are not shown. cc4 subunits are green, and ⁇ 2 subunits are yellow. To the right on top is an expanded view of the ACh binding site showing the a4 C loop closed over an ACh molecule bound between a4 and ⁇ 2 subunits.
  • an effective amount when used to describe therapy to an individual suffering from a disorder, refers to the quantity or concentration of a compound of the invention that is effective to inhibit or otherwise act on an a5-nicotinic receptor in the individual's tissues wherein an a5-nicotinic receptor involved in the disorder, such as nicotine addiction, wherein such inhibition or other action occurs to an extent sufficient to produce a beneficial therapeutic effect.
  • Treating” or “treatment” within the meaning herein refers to an alleviation of symptoms associated with a disorder or disease, or inhibition of further progression or worsening of those symptoms, or prevention or prophylaxis of the disease or disorder, or curing the disease or disorder.
  • an "effective amount” or a “therapeutically effective amount” of a compound of the invention refers to an amount of the compound that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents, or provides prophylaxis for, the disorder or condition.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of compounds of the invention are outweighed by the therapeutically beneficial effects.
  • phrases such as "under conditions suitable to provide” or “under conditions sufficient to yield” or the like, in the context of methods of synthesis, as used herein refers to reaction conditions, such as time, temperature, solvent, reactant concentrations, and the like, that are within ordinary skill for an experimenter to vary, that provide a useful quantity or yield of a reaction product. It is not necessary that the desired reaction product be the only reaction product or that the starting materials be entirely consumed, provided the desired reaction product can be isolated or otherwise further used.
  • chemically feasible is meant a bonding arrangement or a compound where the generally understood rules of organic structure are not violated; for example a structure within a definition of a claim that would contain in certain situations, e.g., a pentavalent carbon atom that would not exist in nature would be understood to not be within the claim.
  • the structures disclosed herein, in all of their embodiments are intended to include only “chemically feasible” structures, and any recited structures that are not chemically feasible, for example in a structure shown with variable atoms or groups, are not intended to be disclosed or claimed herein.
  • an "analog" of a chemical structure refers to a chemical structure that preserves substantial similarity with the parent structure, although it may not be readily derived synthetically from the parent structure.
  • a related chemical structure that is readily derived synthetically from a parent chemical structure is referred to as a "derivative.” All single enantiomer, diastereomeric, and racemic forms of a structure are intended, unless a particular stereochemistry or isomeric form is specifically indicated. In several instances though an individual stereoisomer is described among specifically claimed compounds, the stereochemical designation does not imply that alternate isomeric forms are less preferred, undesired, or not claimed.
  • Compounds used in the present invention can include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions, at any degree of enrichment. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these are all within the scope of the invention.
  • each individual integral number representing the number of carbon atoms is intended.
  • recitation of a (Ci-C4)alkyl group indicates that the alkyl group can be any of methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, or tert-butyl. It is understood that a specification of a number of carbon atoms must be an integer.
  • Alkyl groups include straight chain and branched carbon-based groups having from 1 to about 20 carbon atoms, and typically from 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms.
  • straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n- octyl groups.
  • alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups.
  • alkyl encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl.
  • alkoxy refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined above.
  • linear alkoxy groups include but are not limited to methoxy, ethoxy, n-propoxy, n-butoxy, n-pentyloxy, n-hexyloxy, and the like.
  • branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like.
  • haloalkyl group includes mono-halo alkyl groups, poly-halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by the same or differing halogen atoms, such as fluorine and/or chlorine atoms.
  • haloalkyl include trifluoromethyl, 1 , 1 -dichloroethyl, 1 ,2-dichloroethyl, l ,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and the like.
  • acyl group refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom.
  • the carbonyl carbon atom is also bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like.
  • amine includes primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like.
  • Amines include but are not limited to R-NH 2 , wherein R is a carbon-based moiety, for example, alkylamines, arylamines, alkylarylamines; R 2 NH wherein each R is independently selected carbon-based moiety, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected carbon- based moiety, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like.
  • the term "amine” as used herein also includes positively charged (cationic) forms such as amine salts and quaternarized amines.
  • amino group is a substituent group of the form -NH 2 , -NHR, -NR 2 , or -NR 3 + , wherein each R is an independently selected carbon-based group, and protonated forms of each, except for -NR 3 + , which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine.
  • An “amino group” within the meaning herein can be a primary, secondary, tertiary or quaternary amino group.
  • alkylamino includes a monoalkylamino, dialkylamino, and trialkylamino (trialkylammonium) group.
  • a “salt” as is well known in the art includes an organic compound such as a carboxylic acid, a sulfonic acid, or an amine, in ionic form, in combination with a counterion.
  • acids in their anionic form can form salts with cations such as metal cations, for example sodium, potassium, and the like; with ammonium salts such as ⁇ 3 ⁇ 4 + or the cations of various amines, including tetraalkyl ammonium salts such as tetramethylammonium, or other cations such as trimethylsulfonium, and the like.
  • a “pharmaceutically acceptable” or “pharmacologically acceptable” salt is a salt formed from an ion that has been approved for human consumption and is generally non-toxic, such as a chloride salt or a sodium salt.
  • Suitable pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids.
  • Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,
  • benzenesulfonic pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, ⁇ -hydroxybutyric, salicylic, galactaric and galacturonic acid.
  • pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates.
  • the compounds described herein can be prepared in a number of ways based on the teachings contained herein and synthetic procedures known in the art.
  • synthetic procedures known in the art.
  • all proposed reaction conditions including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be chosen to be the conditions standard for that reaction, unless otherwise indicated.
  • the functionality present on various portions of the molecule should be compatible with the reagents and reactions proposed.
  • Substituents not compatible with the reaction conditions will be apparent to one skilled in the art, and alternate methods are therefore indicated.
  • the starting materials for the examples are either commercially available or are readily prepared by standard methods from known materials.
  • the present invention further embraces isolated compounds of the invention.
  • isolated compound refers to a preparation of a compound of the invention, or a mixture of compounds the invention, wherein the isolated compound has been separated from the reagents used, and/or byproducts formed, in the synthesis of the compound or compounds. "Isolated” does not mean that the preparation is technically pure
  • an "isolated compound” refers to a preparation of a compound of the invention or a mixture of compounds of the invention, which contains the named compound or mixture of compounds of the invention in an amount of at least 10 percent by weight of the total weight.
  • the preparation contains the named compound or mixture of compounds in an amount of at least 50 percent by weight of the total weight; more preferably at least 80 percent by weight of the total weight; and most preferably at least 90 percent, at least 95 percent or at least 98 percent by weight of the total weight of the preparation.
  • the compounds of the invention and intermediates may be isolated from their reaction mixtures and purified by standard techniques such as filtration, liquid-liquid extraction, solid phase extraction, distillation, recrystallization or chromatography, including flash column chromatography, or HPLC.
  • Isolated optical isomer or “isolated enantiomer” means a compound which has been substantially purified from the corresponding optical isomer(s) of the same formula.
  • the isolated isomer is at least about 80%, more preferably at least 90%
  • enantiomeric purity is meant the percent of the predominant enantiomer in an enantiomeric mixture of optical isomers of a compound. A pure single enantiomer has an enantiomeric purity of 100%.
  • Isolated optical isomers may be purified from racemic mixtures by well-known chiral separation techniques. According to one such method, a racemic mixture of a compound of the invention, or a chiral intermediate thereof, is separated into 99% wt.% pure optical isomers by HPLC using a suitable chiral column, such as a member of the series of
  • DAICEL ® CHIRALPAK ® family of columns (Daicel Chemical Industries, Ltd., Tokyo, Japan). The column is operated according to the manufacturer's instructions.
  • PAMs Positive allosteric modulators
  • nAChR nicotinic acetylcholine receptors
  • Br-BPTC binds to the C-terminal extracellular sequences of a4 subunits, which is also a PAM site for steroid hormone estrogens such as 17- ⁇ estradiol.
  • Br-PBTC is much more potent than estrogens.
  • the non-steroid Br-PBTC only requires one a4 subunit to potentiate nAChR function, and its potentiation is stronger with more a4 subunits.
  • Br-BPTC potentiate activation of ( ⁇ 4 ⁇ 2)( ⁇ 6 ⁇ 2) ⁇ 3 but not ( 6 ⁇ 2)2 ⁇ 3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different cc6* nAChR subtypes.
  • Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short-term desensitized nAChRs that have at least two a4 subunits, but not those with only one.
  • Br-PBTC selectively affects a2 and a4 subunits
  • Br-PBTC Br-PBTC increased activation by EC2 0 ACh of a2- and a4-containing nAChRs by 1 19-560% (Table 1 ).
  • Br-PBTC potentiated activation of nAChRs more than ⁇ 4- ⁇ 3 ⁇ nAChRs.
  • Our ⁇ 2 ⁇ 4 and ⁇ 4 ⁇ 4 nAChR cell lines preferably express more of the ( ⁇ ) 2 ⁇ stoichiometry than the ( ⁇ )2( stoichiometry, while our 2 ⁇ 2 and ⁇ 4 ⁇ 2 lines express more of the ( ⁇ ) 2 stoichiometry (Wang et al., 2015).
  • the higher efficacy of Br-PBTC on 2 ⁇ 2 and ⁇ 4 ⁇ 2 nAChRs could result from Br-PBTC having greater effects on the ( ⁇ )2 ⁇ stoichiometry.
  • Br-PBTC 0 values for Br-PBTC ranged from 0.261 to 0.660 ⁇ (Table 1 ), equal to the most potent nAChR PAMs (Williams et al., 201 1 ; Grupe et al., 2015). At more than 3 ⁇ , Br-PBTC inhibited its own potentiation effect, perhaps because it behaved as an open channel blocker like some other nAChR PAMs and ACh itself (Weltzin and Schutle, 2010) (see Figure 1). Br-PBTC did not alter activation by ACh of ⁇ 3 ⁇ 2 or ⁇ 3 ⁇ 4 nAChRs (Fig. I B). Moreover, Br-PBTC did not activate any nAChR subtype by itself (data not shown). Therefore, Br-PBTC is an a2 and a4 nAChR subtype-selective PAM.
  • Br-PBTC binds to the extracellular C-terminal domain of a4 subunits Since Br-PBTC has no effect on a3* nAChRs, we expressed various chimeras of a3 and a4 subunits in Xenopus oocytes to identify the Br-PBTC binding site in the 4 subunit.
  • Figure 2 illustrates the chimeras of a3 and a4 that we used. Since Br-PBTC potentiates activation of ACh more strongly at intermediate agonist concentrations, we used ACh at EC30-40 to test PAM effects of Br-PBTC on 3 ⁇ 2, ⁇ 4 ⁇ 2 and their chimeras (Fig. 3 A).
  • Br- PBTC did not potentiate ⁇ 3 ⁇ 2 nAChRs expressed in oocytes.
  • Chimeras ⁇ 4 (1 "207) / ⁇ 3 (208"446) and a4 (l " 297) /a3 (298"446) which have the a3 cytoplasmic, M4, and C-tails, abolished potentiation by Br- PBTC.
  • Br-PBTC increases the sensitivity to ACh of the a4/a4, but not the ⁇ 4/ ⁇ 2 ACh site
  • Pentameric ⁇ 4 ⁇ 2 nAChRs assemble into two stoichiometries, ( 4 ⁇ 2) 2 ⁇ 4 and
  • nAChRs both have two 4/ ⁇ 2 ACh binding sites, but there is a third ⁇ 4/ ⁇ 4 ACh binding site in ( ⁇ 4 ⁇ 2) 2 4 (Harpsoe et al., 201 1 , Mazafarro et al., 201 1 ).
  • the site- selective agonist NS9283 binds only at the ⁇ 4/ ⁇ 4 site, increasing responses to low concentrations of ACh activating the ⁇ 4/ ⁇ 2 sites (Wang et al., 2015; Olsen et al., 2014;
  • PAMs increase the potency and/or efficacy of an agonist via promoting agonist activation.
  • Br-PBTC increased maximum efficacy of ACh on ( ⁇ 4 ⁇ 2) 2 ⁇ 4 nAChRs by 30 %.
  • Br-PBTC requires the three a4 subunits in ( ⁇ 4 ⁇ 2) 2 ⁇ 4 to affect agonist affinity
  • Br-PBTC increases agonist affinity to the low ACh affinity ⁇ 4/ ⁇ 4 site, but does not affect the high ACh affinity ⁇ 4/ ⁇ 2 sites.
  • ⁇ 2- ⁇ 4- ⁇ 2- ⁇ 4 concatamers with a3 subunits in oocytes to obtain ( ⁇ 4 ⁇ 2) 2 ⁇ 3, which has two ⁇ 4 ⁇ 2 binding sites like ( ⁇ 4 ⁇ 2) 2 ⁇ 2 and an additional low ACh affinity 3 ⁇ 4 site like
  • Br-PBTC increased the sensitivity of ( ⁇ 4 ⁇ 2) 2 ⁇ 4 to ACh by 37 fold, but changed the sensitivities of ( ⁇ 4 ⁇ 2) 2 ⁇ 3 and ( ⁇ 4 ⁇ 2) 2 ⁇ 2 very little (Table 2). This suggests that three a4 subunits are required for Br- PBTC to increase agonist sensitivity of nAChRs.
  • Br-PBTC potentiates nAChRs through a single a.4 subunit
  • nAChRs have similar numbers of agonist binding sites and agonist affinity. They all have at least one high ACh affinity ⁇ 4/ ⁇ 2 site. A low affinity ACh site can be formed at ⁇ 4/ ⁇ 4 and ⁇ 3/ ⁇ 4 interfaces (Wang et al., 2015; Harpsoe et al., 201 1 ; Mazzaferro et al., 201 1 ). ( ⁇ 3 ⁇ 4) 2 ⁇ 3 nAChRs showed lower ACh sensitivity than ( ⁇ 3 ⁇ 4) 2 ⁇ 4 nAChRs (Krashia et al., 2010).
  • the ⁇ 4/ ⁇ 4 site-selective agonist NS9283 also potentiated activation of ( ⁇ 4 ⁇ 2)( ⁇ 3 ⁇ 2) ⁇ 3 nAChRs (data not shown). Therefore, a low affinity ACh site is likely to be present at the ⁇ 3/ ⁇ 3 interface.
  • Br-PBTC can increase channel activation by a maximal concentration of ACh. This is similar to was observed with 4 ⁇ 2 nAChRs expressed in HEK cells (Fig. 4B). At higher concentrations of agonists, nAChRs desensitize more rapidly. The potentiation by Br-PBTC on 3000 ⁇ ACh could be due to increasing channel conductance, or increased open state probability, or destabilizing or slowing entry into the desensitized state.
  • Br-PBTC can reactivate both short-term and long-term desensitized nAChRs
  • nAChRs were all desensitized because application of ⁇ to these nAChRs showed no blockage of activation (black traces in Fig. 8, A and B).
  • Br-PBTC (4 ⁇ ) efficiently reactivated nicotine long-term desensitized ( 4 ⁇ 2) 2 ⁇ 4 nAChRs, but only weakly reactivated desensitized ( ⁇ 4 ⁇ 2) 2 ⁇ 2 nAChRs (Fig. 8, A and B).
  • the desensitized ( ⁇ 4 ⁇ 2) 2 ⁇ 2 could be less sensitive to reactivation by Br-PBTC.
  • Br-PBTC can equally reactivate both stoichiometrics of ⁇ 4 ⁇ 2 nAChRs after short-term desensitization by agonists, but reactivates long-term desensitized ( ⁇ 4 ⁇ 2) 2 ⁇ 4 nAChRs more efficaciously.
  • This potentiation of Br-PBTC is specific to agonist- desensitized nAChRs.
  • Br-PBTC could not reactivate antagonist-inactivated nAChRs (green traces in Fig. 8).
  • Br-PBTC did not potentiate activation of ( ⁇ 6 ⁇ 2) 2 ⁇ 3 expressed in oocytes (data not shown), but it increased ACh (3 ⁇ ) activation of ( ⁇ 6 ⁇ 2)( ⁇ 4 ⁇ 2) ⁇ 3 by 99.0 ⁇ 13.6% (representative kinetics shown in Fig. 9A). This is consistent with the finding in Figure 5 that only one a4 subunit is required for Br-PBTC potentiation.
  • One feature of ( ⁇ 6 ⁇ 2)( ⁇ 4 ⁇ 2) ⁇ 3 is that the competitive antagonist a-conotoxin Mil selectively blocks its activation from the ⁇ 6/ ⁇ 2 interface. This antagonist site is far away from the 4 C-tail where Br-PBTC acts.
  • a-Conotoxin Mil 50 nM completely blocked activation by ACh and potentiation by Br-PBTC (Fig. 9B).
  • activation is a cooperative event involving conformational change in the whole nAChR and antagonist inhibition of any one ACh site is sufficient to prevent activation (Unwin and Fujiyoshi, 2012; Fletcher and Steinbach, 1996).
  • Blockage by competitive antagonists also applies to potentiation of Br-PBTC on other a4* nAChRs.
  • the competitive antagonist ⁇ selective for ⁇ 2 nAChRs blocked activation of HEK cell lines expressing ( ⁇ 4 ⁇ 2) 2 ⁇ 2 and ( ⁇ 4 ⁇ 2) 2 ⁇ 4 nAChRs in the presence of Br-PBTC.
  • ⁇ also inhibited reactivation of both short-term and long-term desensitized nAChRs by Br-PBTC (grey traces in Figs. 7 and 8).
  • Figs. 1 and 3 submicromolar affinity (Figs. 1 and 3) (Paradiso et al., 2001).
  • the a4 C-tail can be engineered onto ⁇ 2 subunits and enabled estrogens to potentiate through this mutant ⁇ 2 subunit (Jin et al., 201 1 ).
  • a suitable PAM to bind the C-tail of ⁇ 2 and interact with the end of its M4 might produce a p2-selective effect. Perhaps in this way PAMs could be found that would be selective for any subunit.
  • These ligands might behave similarly to type II PAMs like Br-PBTC, but they might also be NAMs or allosteric agonists, depending on their structures. There is not clear guidance for how to design or select such ligands, but 17 ⁇ - estradiol and Br-PBTC illustrate examples of structurally different compounds with similar PAM properties but very different affinities. Suitable selection approaches using
  • stoichiometry-specific nAChR cell lines might allow discovery of PAMs, NAMs, and allosteric agonists for many nAChR subunits that will be useful tools for studying nAChRs and as drugs.
  • the C-tail PAM site is stereoselective. Neither the enantiomer of Br-PBTC nor estrogens potentiate a4* nAChRs (Paradiso et al., 2001 ). Stereoselectivity suggests that the PAM and the C-tail of a4 subunit are interacting with protein rather than membrane lipid. PAM bound to the short a4 C-tail must interact stereospecifically with a nearby region, probably on the same subunit, which is capable of influencing the channel gate. There are prolines at the extracellular end of M4 transmembrane domains. These prolines may contribute to a stereoselective site that interacts with PAM bound to the C-tail to mediate PAM effects.
  • ivermectin acts as a PAM on cc7 (Williams et al., 201 1) and is an allosteric agonist on glutamate gated chloride channels where its binding site has been localized in receptor crystals to near the C-terminal end of M4 (Hibbs and Gouaux, 201 1).
  • the C-tail PAMs could function through targeting a similar region of M4.
  • Br-PBTC is a better tool than estrogens to study the relationship between occupancy and potentiation of PAMs acting at the C-tail. That Br-PBTC potentiated linked a4 C-tail in concatamers also suggests that certain conformation of the C-tail is not required for potentiation from this site. The linker in ⁇ 4- ⁇ 2 concatamer might have prevented entrance of estrogens into the C-tail site.
  • nAChRs go into an inactive state (I) or are forced to remain in a resting state that prevents activation (Fig. 10A).
  • PAM binds to the C-tail of 4
  • Fig. 10B The increase of channel open probability only requires one C-tail site, and its extent is proportionate to the number of C-tail PAM sites in a nAChR (Fig. 10B).
  • nAChRs are the most prevalent subtypes in brain (Gaimarri et al., 2007). PAMs promoting activation of these nAChRs could be beneficial in improving cognition, movement, learning and memory, and reducing pain, or aggressive behaviors, thus beneficial for analgesia, Parkinson, or other dementia diseases (Srinivasan et al., 2014; Grupe et al., 2015; Lewis and Picciotto, 2013; Picciotto et al., 2015).
  • dFBr desformylflustrabromine
  • nAChRs form complex subtypes such as ( ⁇ 4 ⁇ 2) 2 ⁇ 2, ( ⁇ 4 ⁇ 2) 2 ⁇ 3, ( 6 ⁇ 2) 2 ⁇ 3 and ( ⁇ 6 ⁇ 2)( 4 ⁇ 2) ⁇ 3 (Wang et al., 2014).
  • the nAChR subtype expression pattern differs between brain areas (Wang et al., 2014; Zoli et al., 2014).
  • the a6-selective antagonist a-conotoxin Mil helps distinguish a6 and non- 6 nAChRs.
  • Br-PBTC selectively potentiates 6 ⁇ 4* nAChRs (Fig. 9). This differentiates them from ⁇ 6( ⁇ 4) nAChRs.
  • Br- PBTC can further distinguish ⁇ 4 ⁇ 6* from ⁇ 4( ⁇ 6) nAChRs.
  • the invention provides in various embodiments a compound of formula
  • ring bearing R 1 comprises 0 or 1 nitrogen atom therewithin
  • R 1 is halo, cyano, (Ci -C ⁇ alkyl, (Ci-C4)haloalkyl, (Ci-C4)alkoxy, (C] -C4)haloalkoxy,
  • R 2 is H, (Ci-C 4 )alkyl, or phenyl, wherein the phenyl is optionally substituted with 1 -2
  • R 3 is H, (Ci-C 6 )alkyl, (Ci-C 6 )acyl, or benzyl, wherein the benzyl is optionally substituted with 1-2 R 4 ;
  • R 4 is halo, (C r C 4 )alkyl, (Ci-C 4 )alkoxy, or NR 2 ;
  • R 5 is independently at each occurrence H or (C)-C 4 )alkyl
  • n 2, 3, or 4;
  • the compound can be any of the compounds noted in Table 3, below. Bioactivity data for the compounds of Table 3 are provided in Table 4, below.
  • the invention can further provide a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable excipient.
  • the invention provides a method of allosterically modulating an a5-nicotinic receptor, comprising contacting the receptor with an effective amount or concentration of the compound of the invention.
  • the invention provides a method of treatment of nicotine addiction in a patient afflicted therewith, comprising administering to the patient an effective dose of a compound of the invention.
  • Nicotinic acetylcholine receptors From basic science to therapeutics. Pharmacol. Ther. 137, 22-54
  • a binding element for 17 ⁇ - estradiol can be placed on any subunit of a nicotinic ⁇ 4 ⁇ 2 receptor. J. Neurosci. 31, 5045- 5054
  • the nicotinic a5 subunit can replace either an acetylcholine-binding or nonbinding subunit in the ⁇ 4 ⁇ 2* neuronal nicotinic receptor.
  • Nicotine acts as a pharmacological chaperone to up-regulate human ⁇ 4 ⁇ 2 acetylcholine receptors. Mol.
  • Galantamine is an allosterically potentiating ligand of neuronal nicotinic but not of muscarinic acetylcholine receptors. J. Pharmacol, and Exp. Ther. 305: 1024-1036.
  • nAChRs A therapeutic target for Parkinson's disease. Pharmacol. Res. 83, 20-29.
  • N.D refers to data not detected when the PAM effects are too small to obtain meaningful potency and efficacy values.
  • ACh concentration/response curves were determined on oocytes or HEK cell lines expressing defined stiochiometries. The maximum efficacy was defined as 100% for ACh without PAMs.
  • defined stoichiometrics were obtained by injecting ⁇ 2- ⁇ 4- ⁇ 2- ⁇ 4 concatamers with a free subunit. Each data point was collected from more than four oocytes or more than three wells of cells. "*" indicates data reported previously (Wang et al., 2015).
  • the ( ⁇ 4 ⁇ 2) 2 ⁇ 4 cell line exhibits a two component concentration/response curve due to a high sensitivity component reflecting its two ⁇ 4/ ⁇ 2 ACh binding sites and a low affinity component reflecting activation in combination with the low sensitivity ct4/a4 site.
  • concentration/response data for ( 4 ⁇ 2) 2 ⁇ 4 obtained from oocytes were too noisy to fit a biphasic curve, so was approximated with a monophasic curve.
  • Br-PBTC (SR-13523) was synthesized as described in the synthetic example ). A 10 mM stock of Br-PBTC was prepared in dimethyl sulfoxide. Dilutions of drugs were prepared daily in testing buffer before use. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
  • Benzothiophene 2-carboxylic acids were prepared from appropriately substituted benzaldehyde and methyl (or ethyl) mercaptoacetate, and followed by base hydrolysis. Then, the Benzothiophene 2-carboxylic acid was coupled with (R)-tert-butyl 3-aminopyrrolidine-l- carboxylate (or (R)-tert-butyl 3-aminopiperidine-l -carboxylate) under the assistance of HATU. The protecting group BOC was deprotected by TFA in DCM to give analogue with N-H, which can be further modified into analogue with N-R 5 .
  • 2,4-Dichloro-6-fluorobenzaldehyde (300.0 mg, 1.6 mmol), ethyl mercaptoacetate (204 uL, 1.9 mmol), Et 3 N (556 uL, 4.0 mmol) and CH 3 CN (10 mL) were added into a 50 ml round bottom flask, and stirred at 60 °C for overnight.
  • the CH 3 CN was removed in vacuo, and the residue was dissolved in ethyl acetate (30 ml) and water (10 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (2x).
  • Ethyl 4-bromobenzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 2-bromo-6-fluorobenzaldehyde and ethyl mercaptoacetate. ⁇
  • Ethyl 5-bromobenzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 5-bromo-2-fluorobenzaldehyde and ethyl mercaptoacetate. ⁇
  • Ethyl 6-chlorobenzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 4-chloro-2-fluorobenzaldehyde and ethyl mercaptoacetate. ⁇
  • Ethyl 7-chlorobenzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 3-chloro-2-fluorobenzaldehyde and ethyl mercaptoacetate.
  • Ethyl 6-bromobenzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 4-bromo-2-fluorobenzaldehyde and ethyl mercaptoacetate.
  • Ethyl 7-bromobenzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 3-bromo-2-fluorobenzaldehyde and ethyl mercaptoacetate.
  • Ethyl 4-(trifluoromethyl)benzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 2-fluoro-6-(trifluoromethyl)benzaldehyde and ethyl mercaptoacetate.
  • Ethyl 4-fluorobenzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 2,6-difluorobenzaldehyde and ethyl mercaptoacetate.
  • Ethyl benzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 2-fluorobenzaldehyde and ethyl mercaptoacetate.
  • Ethyl 6-(dimethylamino)benzo[b]thiophene-2-carboxylate was prepared by general procedure A in Synthetic Example 1 using 4-(dimethylamino)-2-nitrobenzaldehyde and ethyl mercaptoacetate.
  • Ethyl 7-chloro-6-fluorobenzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 3-chloro-2,4-difluorobenzaldehyde and ethyl
  • Ethyl 7-methoxybenzo[b]thiophene-2-carboxylate was prepared by general procedure A in Synthetic Example 1 using 3-methoxy-2-nitrobenzaldehyde and ethyl mercaptoacetate.
  • Ethyl thieno 2',3':4,5]benzo l,2-dl l,31dioxole-6-carboxylate Ethyl thieno[2',3':4,5]benzo[l ,2-d][ l,3]dioxole-6-carboxylate was prepared by general procedure A in Synthetic Example 1 using 6-nitrobenzo[d][ l ,3]dioxole-5-carbaldehyde and ethyl mercaptoacetate.
  • Ethyl 7-fluorobenzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 2,3-difluorobenzaldehyde and ethyl mercaptoacetate.
  • Ethyl 4-methoxybenzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 2-fJuoro-6-methoxybenzaldehyde and ethyl mercaptoacetate.
  • Ethyl 4,7-dichlorobenzo[b]thiophene-2-carboxylate was prepared by general procedure A in Synthetic Example 1 using 2,3,6-trichlorobenzaldehyde and ethyl mercaptoacetate, and final compound was mixed with ethyl 4,5-dichlorobenzo[b]thiophene-2-carboxylate.
  • Ethyl 4,7-dibromobenzo[b]thiophene-2-carboxylate was prepared by general procedure B in Synthetic Example 5 using 3,6-dibromo-2-fluorobenzaldehyde and ethyl mercaptoacetate.
  • Concatamers were formed by linking the C-terminus of one subunit to the N-terminus of the next. Synthesis of the tetrameric concatamer p2(AGS) 6 a4(AGS)i 2 p2(AGS)6( 4 (abbreviated as ⁇ 2- ⁇ 4- ⁇ 2- ⁇ 4) and the trimeric concatamer p2(QAP) n 4(QAP) n p2
  • the trimeric concatamer p2(QAP) n a4(QAP) n p2 (abbreviated as ⁇ 2- ⁇ 4- ⁇ 2) was synthesized through linking together ⁇ 2( ⁇ ) ⁇ ⁇ 4 with QAP linker and ⁇ 2.
  • ⁇ 2((3 ⁇ ) ⁇ ⁇ 4 was made similarly as ⁇ 3( ⁇ . ⁇ ) ⁇ ⁇ 6 which was describe (Ley et al, 2014).
  • a BspEI site was introduced at the end of mature peptide of ⁇ 2 using
  • the second (QAP) n linker was prepared from the p2(QAP) n 4 piece. We mutated Fspl site at the beginning of a4 sequence into BstBI site. The second (QAP) n linker with new restriction sites was cut out using Xmal site and BstBI enzymes. We introduced a BstBI restriction site at the beginning of mature peptide of ⁇ 2 using
  • GGCATGATCTTCGAAACGGATACAGAGGAG oligo allowed us to link together ⁇ 2 ⁇ ) ⁇ 4 dimer with Agel site, QAP linker with Xmal and BstBI ends, and ⁇ 2 subunits with BstBI restriction site at the beginning of mature peptide.
  • Resulting construct has been recloned into pBS SK(-) vector using EcoRI restriction enzyme. Resulting clone has been linearized with EcoRV for expression in oocytes.
  • the ⁇ 3 ( 40) / ⁇ 4 (56 94) were prepared from ligating three pieces of DNA: a 0.6 kb fragment from the Ncol to BstEII site of the (x3 subunit, a 1 kb fragment from the Hidlll to BstEII site of the a3 subunit, and a 3.1 kb fragment from the Ncol to Hidlll site of the a4 subunit in the pSP64 vector.
  • the ligation mixture was transformed into XL I O-Gold ultracompetent cells (Stratagene, La Jolla CA) and the right clone was chosen from a restriction enzyme digestion.
  • a C-tail mutant (noted as 4 AAC ) was obtained by mutating the last four amino acids of the (x4 subunit, alanine-glycine-methionine-isoleucine, to analine-analine-cysteine followed by a stop codon. Mutations were introduced using the PfuUltra high-fidelity DNA polymerase (Agilent, Santa Clara, CA), following the manufacturer's instructions. All mutations were confirmed by sequencing.
  • cRNA transcripts were prepared in vitro using mMessage mMachine kits (Ambion, Austin, TX). Concentrations of cDNAs and cRNAs were calculated by spectrophotometry.
  • HEK cells that express only one stiochiomety either ( ⁇ 4 ⁇ 2) 2 ⁇ 4 or ( ⁇ 4 ⁇ 2) 2 ⁇ 2, were obtained by transfecting a dimeric concatamer p2(QAP) n a4 cell line with a4 or ⁇ 2 subunits (Kuryatov et al., in preparation).
  • FLEXstation experiments For functional tests of nAChRs expressed in HEK cells, we used a FLEXstation (Molecular Devices, Sunnyvale, CA) bench-top scanning fluorometer as described by Kuryatov et al. (2005). To increase the expression level of ⁇ 2 ⁇ 3, ⁇ 3 ⁇ 2 and ( ⁇ 4 ⁇ 2) 2 ⁇ 2 nAChRs, the plates were incubated at 29 °C for 20 hours before being tested. A membrane potential fluorescent indicator kit (Molecular Devices, Sunnyvale, CA) was used according to the manufacturer's protocols. In PAM experiments, serial dilutions of Br-PBTC were manually added to the assay plate 15 min prior to addition of agonists during recording, unless otherwise noted.
  • I(x) I max [x" H /(x" H +EC 50 " H )J, where I(x) is the peak current measured at the drug concentration x, I max is the maximum current peak at the saturating concentration, EC 50 is the drug concentration required to achieve half of the maximum response, and nH ⁇ ' s the Hill coefficient.
  • Ooctye removal and injection Oocytes were removed surgically from Xenopus laevis and defolliculated as described (Gerzanich et al., 1997; Wang et al., 2015).
  • Oocyte injections were performed within 48 hours after surgery. Oocytes were injected with 20-40 ng of concatamer cRNA and free single subunit at 1 : 1 ratio. A total of 2- 20 ng cRNA were injected for free wild type or chimeric a and ⁇ subunits at 4: 1 ratio to force expression of the ( )3( ⁇ 2) 2 stoichiometry. Function was assayed 3-7 days after injection.
  • Electrophysiology Currents in oocytes were measured using the OpusXpress 6000A (Molecular Devices, Union City, CA), an automated two-electrode voltage clamp amplifier that enables recording up to eight oocytes in parallel (Wang et al., 2015). Oocytes were voltage-clamped at a holding potential of -50 mV. 200 ⁇ of drugs were delivered on top of oocytes for 4 seconds (s) through the sidewall of the bath to minimize disturbance to oocytes.
  • oocytes received a 30 s pre-wash and 223 s post-wash of ND96 solution (96 mM NaCl, 2 mM KC1, 1.8 mM CaCl 2 , 1 mM MgCl 2; 5 mM HEPES, pH 7.6) with 0.5 ⁇ atropine perfused through the bath at a rate of 3 ml/min, unless otherwise noted.
  • ND96 solution 96 mM NaCl, 2 mM KC1, 1.8 mM CaCl 2 , 1 mM MgCl 2; 5 mM HEPES, pH 7.6
  • Peak amplitudes of experimental responses were calculated relative to ACh responses to normalize the data and compensate for variable expression levels among oocytes.
  • PAM effect of Br-PBTC was calculated by increased responses with Br-PBTC relative to responses to ACh alone. Mean and standard error were calculated from normalized responses.
  • Table 4 provides biodata for the exemplary compounds of the invention.
  • rats weighing 250-300 g were housed in groups of 1 -23 per cage, in a temperature-controlled vivarium under a reversed 12-h light/dark cycle (lights off at 8 am). Food and water were provided ad libitum until behavioral training commences. During training, rats were food-restricted to maintain -85-90% of their free-feeding body weight. Behavioral testing occurred during the dark portion of the light/dark cycle between the hours of 9 am-1 pm, during the early portion of the dark phase of the cycle. All procedures were conducted in strict adherence with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute.
  • Rats were anesthetized by inhalation of 1 -3% isoflurane in oxygen and silastic catheters were inserted into the jugular veins.
  • the catheters consist of a 14 cm length of silastic tubing fitted to a guide cannula (Plastics One, Wallingford, CT), bent at a curved right angle and encased in dental acrylic.
  • the catheter tubing was passed subcutaneously from each animal's back to the right jugular vein, and 1 cm length of the catheter tip is inserted into the vein. After surgery, catheters are flushed daily with 0.1 mL of a heparinized (30 USP units/ml) sterile saline solution.
  • rats were mildly food restricted to 85-90%) of their free-feeding body weight and trained to press a lever in an operant chamber (Med Associates, St. Albans, VT) for food pellets (20 mg; TestDiet, Richmond, IN) under a fixed-ratio 5, time out 20 s (FR5TO20 s) schedule of reinforcement prior to catheter implantation.
  • FR5TO20 s time out 20 s
  • rats were permitted to acquire IV nicotine self-administration by autoshaping during 1 -h daily sessions, 7 days per week. Nicotine was delivered through the tubing into the IV catheter by a Razel syringe pump (Med Associates). Each nicotine self-administration session was performed using two retractable levers ( 1 active; 1 inactive).

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Abstract

La présente invention concerne des modulateurs allostériques positifs (PAM) de récepteurs nicotiniques de l'acétylcholine (nAChR) qui sont d'importants candidats thérapeutiques ainsi que des outils de recherche de valeur. Nous avons identifié un nouveau PAM de type II, (R)-7-bromo-N-(pipéridin-3-yl)benzo[b]thiophène-2-carboxamide (Br-PBTC), qui augmente l'activation et réactive des nAChRs désensibilisés. Ce composé augmente les réponses causées par l'acétylcholine des nAChR α2* et α4*, mais est sans effet sur les nAChR α3* et α6* ("*" indique la présence d'autres sous-unités de nAChR). Br-BPTC se lie aux séquences extracellulaires C-terminales des sous-unités a4, qui est également un site PAM pour les hormones stéroïdes oestrogènes telles que le 17-β estradiol. Br-PBTC est beaucoup plus puissant que les oestrogènes. Comme le 17-P-estradiol, le Br-PBTC non-stéroïde ne nécessite qu'un seul sous-unité α4 pour potentialiser la fonction de nAChR, et sa potentialisation est plus forte avec plus de sous-unités a4. Cette fonction permet à Br-BPTC de potentialiser l'activation de nAChR (α4β2)(α6β2)β3 mais pas (α6β2)2β3. L'invention concerne différents analogues bioactifs de Br-PBTC.
PCT/US2016/033774 2015-05-28 2016-05-23 Modulateurs pour les sous-unités α2 et α4 de récepteur nicotinique de l'acétylcholine WO2016191366A1 (fr)

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CN114907335A (zh) * 2022-02-25 2022-08-16 陕西维世诺新材料有限公司 2-（苯并噻吩-2-基）苯并[d]惡唑衍生物、制备方法及应用

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