HK1186469A - (2s,3r)-n-(2-((3-pyridinyl)methyl)-1-azabicyclo[2.2.2]oct-3-yl)benzofuran-2-carboxamide, novel salt forms, and methods of use thereof - Google Patents

Description

(2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, novel salt forms, and methods of use thereof

CROSS-REFERENCE TO PRIOR APPLICATIONS

The present invention claims U.S. provisional application No. 60/971,654 filed on 12/9/2007; U.S. provisional application No. 60/953,610 filed on 8/2/2007; U.S. provisional application No. 60/953,613 filed on 8/2/2007; and U.S. provisional application No. 60/953,614 filed on 2.8.2007, each of which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, novel salt forms thereof, processes for its preparation, novel intermediates, and methods of treating a wide variety of conditions and disorders, including those associated with dysfunction of the central and autonomic nervous systems.

Background

Neuronal Nicotinic Receptors (NNRs) characteristic of the Central Nervous System (CNS) have been shown to exist in several subtypes, the most common of which are the α 4 β 2 and α 7 subtypes. See, for example, Schmitt, Current med. chem.7:749(2000), which is hereby incorporated by reference into the present specification. Ligands that interact with the α 7NNR subtype have been proposed for use in the treatment of a variety of conditions and disorders. See Mazurov et al, curr. Med. chem.13:1567-1584(2006) and references cited therein, which are incorporated herein by reference for background understanding of the α 7 neuronal nicotinic receptor subtype. The most prominent of these conditions and disorders are cognitive impairment, schizophrenia, inflammation, angiogenesis, neuropathic pain and fibromyalgia.

In necropsy brain tissue from schizophrenic patients, hippocampal NNR numbers decreased. Moreover, there is an improved psychological effect in smoking schizophrenia patients relative to non-smoking schizophrenia patients. Nicotine ameliorates sensory gating deficits in animals and schizophrenic patients. Blockade of the α 7NNR subtype induces gating defects similar to those seen in schizophrenia. See, for example, Leonard et al, Schizophrania Bulletin22(3):431(1996), which is incorporated herein by reference. Biochemical, molecular and genetic studies of sensory processing in patients with defects in P50 auditory evoked potential gating suggest that the α 7NNR subtype may play a role in inhibitory neuronal pathways. See, for example, Freedman et al, Biological Psychiatry38(1):22(1995), which is incorporated by reference herein.

More recently, α 7NNR has been proposed as a mediator of angiogenesis, as described by Heeschen et al, j.clin.invest.100:527(2002), which is incorporated herein by reference. In these studies, inhibition of the α 7 subtype was shown to reduce inflammatory angiogenesis. Furthermore, α 7 NNRs have been proposed as targets for controlling neurogenesis and tumor growth (Utsugeisawa et al, Molecular Brain Research 106(1-2):88(2002) and U.S. patent application 2002/0016371, each of which is incorporated herein by reference). Finally, the role of the α 7 subtype in cognition (Levin and Rezvani, Current Drug Targets: CNS and Neurological Disorders1(4): 423(2002)), neuroprotection (O' Neill et al, Current Drug Targets: CNS and Neurological Disorders1(4): 399(2002) and Jeararasingam et al, Neuroscience 109(2):275(2002)) and neuropathic pain (Xiao et al, Proc. Nat. Acad. Sci. US (99) (12):8360(2002)) was recently recognized, each of which is incorporated herein by reference.

A variety of compounds have been reported to interact with α 7 NNRs and have been proposed as therapeutic agents on this basis. See, for example, PCT WO99/62505, PCT WO99/03859, PCT WO97/30998, PCT WO01/36417, PCT WO02/15662, PCT WO02/16355, PCT WO02/16356, PCT WO02/16357, PCT WO02/16358, PCT WO02/17358, Stevens et al, Psychopharm.136:320(1998), Dolle et al, J.Labelled Comp.Radiophorm.44: 785(2001) and Macor et al, bioorg.Med. chem.Lett.11:319(2001) and references cited therein, for background teachings on α 7NNR and proposed therapeutics, the above references being incorporated by reference into this specification. In these compounds, a common structural subject is the structure of substituted tertiary bicyclic amines (e.g., quinuclidine). Similar substituted quinuclidine compounds have also been reported to bind to muscarinic receptors. See, for example, U.S. Pat. No. 5,712,270 to Sabb, as well as PCTWO02/00652 and PCT WO02/051841, each of which is incorporated by reference herein for these compounds.

A limitation of some nicotinic compounds is that they are associated with a variety of undesirable side effects, such as those caused by stimulation of muscle and ganglionic receptors. There is a continuing need for compounds, compositions and methods for preventing or treating various conditions or disorders, such as CNS disorders, including alleviating the symptoms of these disorders, wherein the compounds exhibit nicotinic pharmacology with beneficial effects, i.e., affecting the CNS function, but without significant associated side effects. There remains a need for compounds, compositions and methods that affect CNS function without significantly affecting nicotinic receptor subtypes that may induce undesirable side effects such as significant activity at cardiovascular and skeletal muscle sites. The invention provides such compounds, compositions and methods.

Summary of The Invention

One aspect of the invention is (2S,3R) N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, or a pharmaceutically acceptable salt thereof Another aspect of the invention is (2S,3R) N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, or a pharmaceutically acceptable salt thereof, in substantially pure form Another aspect of the invention is (2S,3R) N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, or a (2S,3R) isomer that is substantially free of (2S,3S), (2R,3S), or (2R,3R A pharmaceutically acceptable salt thereof.

In addition, another aspect of the present invention is a stereoisomerically enriched (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide or a pharmaceutically acceptable salt thereof.

Another aspect of the invention is an acid salt of (2S,3R) N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, wherein the acid is selected from hydrochloric acid, sulfuric acid, phosphoric acid, maleic acid, p-toluenesulfonic acid, galactaric acid (mucic acid), D-mandelic acid, D-tartaric acid, methanesulfonic acid, R-and S-10-camphorsulfonic acid, ketoglutaric acid, or hippuric acid in one embodiment, the stoichiometry of (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide with acid is 2:1, 1:1, or 1:2 in one embodiment, the stoichiometry is 1: 1. One embodiment of the invention is (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide hydrochloride or a hydrate or solvate thereof, including a partial hydrate or a partial solvate. Another embodiment is (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide monohydrochloride or a hydrate or solvate thereof, including a partial hydrate or a partial solvate.

The invention also provides scalable syntheses of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide and novel intermediates.

The scope of the present invention includes all combinations of aspects, embodiments and preferences described in this specification.

Drawings

FIG. 1A1-1A4 is a schematic representation of the rat α 7 receptor pair (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide expressed in mammalian GH4C1 cells; the racemate, i.e., a mixture of (2S,3R), (2R,3S), (2R,3R), and (2S, 3S); individual stereoisomers; and acetylcholine (ACh) response.

FIG. 1B is a graph depicting the rat α 7 receptor pair expressed in mammalian GH4C1 cells versus (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide at effective plasma concentrations; the racemate, i.e., a mixture of (2S,3R), (2R,3S), (2R,3R), and (2S, 3S); and comparison of functional responses of individual stereoisomers.

FIG. 2A is a graph showing the response of human α 7 receptor expressed in Xenopus oocytes to (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide.

Figure 2B is a graph illustrating a control response at the human α 7 receptor following administration of the indicated concentrations of compound. Data were normalized to the net charge of the control 300 μ M ACh response obtained 5 minutes prior to the experimental agonist-induced response. Each dot represents the mean of the normalized responses of at least 4 oocytes. + -. SEM.

Figure 3 illustrates the evaluation of cognitive effects in an Object Recognition (OR) model demonstrating that (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide has a positive effect when administered intraperitoneally at 0.3 and 1mg/kg,. p < 0.5.

Figure 4 illustrates the evaluation of cognitive effects in the OR model, demonstrating that (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide has a positive effect when administered orally over a wide dose range (0.3-10mg/kg), p < 0.5.

FIG. 5 is a graph depicting the effect of intraperitoneally administered (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide in preventing cognitive deficits induced by MK-801 (also known as dezocyclopine, a commercially available uncompetitive antagonist of the NMDA receptor) in the OR task.

Fig. 6 illustrates that the mean time spent on object a 30 min, 6h OR 24 h after the last subacute administration (oral administration) by the vehicle-treated group in the OR task did not differ significantly with respect to the mean time spent on object B (p =0.17, p =0.35 and p =0.12, respectively). Alternatively, it took significantly more time (P < 0.05) for the subject to explore object B (a new object) than object a (a familiar object) 30 minutes, 2 hours, 6 hours, and 18 hours after the last subacute administration of 0.3mg/kg of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide. In addition, the cognitive index in animals treated with 0.3mg/kg of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide was significantly higher at 2 hours (75%) and 6 hours (71%) than the cognitive index of the group treated with vehicle at 30 minutes after the last dose (54%).

Fig. 7 illustrates the evaluation of cognitive effects in the Radial Arm Maze (RAM) model. (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide (0.1, 0.3, and 1.0mg/kg) was administered orally 30 minutes prior to the daily training period. During the second week of dosing, the improvement in performance of the task was evident in the group treated with 0.3mg/kg of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide.

Figure 8 illustrates a study of antipsychotic effect measured as hyperactivity induced by dopamine hyperstimulation, showing that (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide (0.3 and 1.0 mg/kg; subcutaneous administration) attenuates locomotor activity induced by apomorphine (1.0mg/kg) following subcutaneous administration in rats.

Figure 9 illustrates an antipsychotic evaluation, prepulse inhibition, showing that the defect induced by apomorphine was reversed by pretreatment with (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide following subcutaneous administration.

Fig. 10A illustrates the results of X-ray crystallographic analysis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide monohydrochloride, confirming the absolute stereochemistry of this material. The depicted compounds are partially hydrated hydrochlorides, as shown by the fully ordered chloride ion and the water molecules partially occupied in the asymmetric unit.

Figure 10B illustrates the results of X-ray crystallographic analysis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide monohydrochloride, confirming the absolute stereochemistry of this material, as described using the numbering scheme for reference. The view is looking down the crystallographic b-axis of the unit cell. Hydrogen bonds between molecules are indicated by dashed lines.

FIG. 11A is a graph showing the results of X-ray crystallography analysis of (2R,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide p-chlorobenzoate, confirming the absolute stereochemistry of this material.

Figure 11B illustrates the results of an X-ray crystallographic analysis of (2R,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide p-chlorobenzoate, establishing the absolute stereochemistry of that material, described using the numbering scheme for reference.

Figure 12 illustrates a complete chromatogram characterizing four stereoisomers of N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, where 2S,3R shows a peak at a retention time of 5.3 minutes, 2R,3S shows a peak at a retention time of 7.3 minutes, 2R,3R shows a peak at a retention time of 8.2 minutes, and 2S,3S shows a peak at a retention time of 12.4 minutes. As described in the specification, the mobile phase required for analysis to provide sufficient resolution formed a 60:40:0.2 composition of hexane: ethanol: di-n-butylamine at a flow rate of 1.0 ml/min, a column temperature of 20 ℃ and a UV detection wavelength of 270 nm.

FIG. 13 is an XRPD of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide monohydrochloride, illustrating the observed pattern (lighter) and the calculated pattern (darker). The two patterns are consistent in2 θ values, with small differences in intensity and peak width attributable to instrument resolution and preferred orientation effects. As described in this specification, other minor differences may be attributed to temperature variations due to observed data collected at room temperature and data calculated from the structure of 120K.

FIG. 14 is an XRPD of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide monotoluene sulfonate.

Detailed description of the invention

The specific compound (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, having affinity (. ltoreq.1 nM Ki values) and selectivity for the α 7NNR subtype, showed efficacy in animal models of cognition (cognitive enhancement) and psychosis (antipsychotic effects).

One aspect of the present invention is (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, or a pharmaceutically acceptable salt thereof, another aspect is (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, or a pharmaceutically acceptable salt thereof, in substantially pure form, another aspect is (2S,3R) N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, or a pharmaceutical thereof, that is substantially free of (2S,3S), (2R,3S), or (2R,3R) isomers A pharmaceutically acceptable salt.

In addition, another aspect is a stereoisomerically enriched (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide or a pharmaceutically acceptable salt thereof.

Another aspect of the invention is an acid salt of (2S,3R) N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, wherein the acid is selected from hydrochloric acid, sulfuric acid, phosphoric acid, maleic acid, p-toluenesulfonic acid, galactaric acid (mucic acid), D-mandelic acid, D-tartaric acid, methanesulfonic acid, R-and S-10-camphorsulfonic acid, ketoglutaric acid, or hippuric acid in one embodiment, the stoichiometry of (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide with acid is 2:1, 1:1, or 1:2 in one embodiment, the stoichiometry is 1: 1. One embodiment of the invention is (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide hydrochloride or a hydrate or solvate thereof, including a partial hydrate or a partial solvate. Another embodiment is (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide monohydrochloride or a hydrate or solvate thereof, including a partial hydrate or a partial solvate.

Another aspect of the invention is (2S,3R) - (2- ((3-pyridinyl) methyl) -3-amino-1-azabicyclo [2.2.2] octane.

Another aspect of the invention is a pharmaceutical composition comprising a compound of the invention and one or more pharmaceutically acceptable carriers.

Another aspect of the invention is a method of treating or preventing a central nervous system disorder, inflammation, pain, or neovascularization, comprising administering a compound of the invention. In one embodiment, the central nervous system disorder is characterized by an alteration in normal neurotransmitter release. In one embodiment, the central nervous system disorder is selected from mild cognitive impairment, age-related memory impairment, pre-senile dementia, early onset alzheimer's disease, senile dementia, dementia of the alzheimer's type, alzheimer's disease, Lewy Body (Body) dementia, micro-infarct dementia, AIDS-related dementia, HIV dementia, multiple cerebral infarctions, parkinsonism, parkinson's disease, pick's disease, progressive supranuclear palsy, huntington's chorea, tardive dyskinesia, hyperkinesia, mania, attention deficit disorder, attention deficit hyperactivity disorder, anxiety, depression, dyslexia, schizophrenia, cognitive dysfunction in schizophrenia, depression, obsessive-compulsive disorders, or tourette's syndrome. In one embodiment, the central nervous system disorder is selected from alzheimer's disease, mania, attention deficit disorder, attention deficit hyperactivity disorder, anxiety, dyslexia, schizophrenia, cognitive dysfunction in schizophrenia, depression, obsessive-compulsive disorders, or tourette's syndrome.

Another aspect of the invention includes the use of a compound of the invention in the manufacture of a medicament for the treatment or prevention of a central nervous system disorder, inflammation, pain, or neovascularization. In one embodiment, the central nervous system disorder is characterized by an alteration in normal neurotransmitter release. In one embodiment, the central nervous system disorder is selected from mild cognitive impairment, age-related memory impairment, pre-senile dementia, early onset alzheimer's disease, senile dementia, dementia of the alzheimer's type, alzheimer's disease, lewy body dementia, micro-infarct dementia, AIDS-related dementia, HIV dementia, multiple cerebral infarcts, parkinsonism, parkinson's disease, pick's disease, progressive supranuclear palsy, huntington's chorea, tardive dyskinesia, hyperkinesia, mania, attention deficit disorder, attention deficit hyperactivity disorder, anxiety, depression, dyslexia, schizophrenia, cognitive dysfunction in schizophrenia, depression, obsessive-compulsive disorders, or tourette's syndrome. In one embodiment, the central nervous system disorder is selected from alzheimer's disease, mania, attention deficit disorder, attention deficit hyperactivity disorder, anxiety, dyslexia, schizophrenia, cognitive dysfunction in schizophrenia, depression, obsessive-compulsive disorders, or tourette's syndrome.

Another aspect of the present invention is a compound of the present invention for use in the treatment or prevention of a central nervous system disorder, inflammation, pain, or neovascularization. In one embodiment, the central nervous system disorder is characterized by an alteration in normal neurotransmitter release. In one embodiment, the central nervous system disorder is selected from mild cognitive impairment, age-related memory impairment, pre-senile dementia, early onset alzheimer's disease, senile dementia, dementia of the alzheimer's type, alzheimer's disease, lewy body dementia, micro-infarct dementia, AIDS-related dementia, HIV dementia, multiple cerebral infarcts, parkinsonism, parkinson's disease, pick's disease, progressive supranuclear palsy, huntington's chorea, tardive dyskinesia, hyperkinesia, mania, attention deficit disorder, attention deficit hyperactivity disorder, anxiety, depression, dyslexia, schizophrenia, cognitive dysfunction in schizophrenia, depression, obsessive-compulsive disorders, or tourette's syndrome. In one embodiment, the central nervous system disorder is selected from alzheimer's disease, mania, attention deficit disorder, attention deficit hyperactivity disorder, anxiety, dyslexia, schizophrenia, cognitive dysfunction in schizophrenia, depression, obsessive-compulsive disorders, or tourette's syndrome.

In the above methods and uses, in one embodiment of the invention, the effective dose is about 1mg to 10mg per 24 hour period.

Another aspect of the invention is a process for the preparation of (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide or a pharmaceutically acceptable salt thereof by sequential kinetic resolution and stereoselective reductive amination of (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-one.

The scope of the present invention includes all combinations of aspects, embodiments and preferences described in this specification.

Commercial development of drug candidates involves a number of steps, including scale-up chemical synthesis and purification, finding the optimal salt form, and the like. In the formulation of pharmaceutical compositions, the drug substance is preferably in a form that can be conveniently handled and processed. Considerations include commercial viability and consistency of manufacture. In addition, in preparing a pharmaceutical composition, it is important to provide a reliable, reproducible and constant plasma concentration profile of the drug after administration to a patient.

Chemical stability, solid state stability and "shelf life" of the active ingredient are also very important factors. These drug substances and compositions containing them should preferably be capable of being effectively preserved for an appreciable period of time without exhibiting significant changes in the physicochemical characteristics of the active ingredient (e.g., its chemical composition, density, hygroscopicity and solubility). In addition, it is also important to be able to provide the drug in a form that is as chemically pure as possible. These features of the present invention are discussed in more detail below.

I. Compound (I)

The compound of the present invention is in the form of (2S,3R) -N (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide represented by the following compound a or a pharmaceutically acceptable salt of compound a.

Compound A

The racemic compound N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, the synthesis, and utility of medical treatment are described in U.S. patent No.6,953,855 to Mazurov et al, which is incorporated herein by reference.

Racemic N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide is a high affinity ligand for the α 7 subtype of neuronal nicotinic receptor (NNR.) racemic N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide contains two asymmetrically substituted carbon atoms therefore racemic N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide is present in the form of four stereoisomers, i.e. (S, S), (S, R), (R, r) and (R, S) are present. (S, R), i.e. (2S,3R), is Compound A.

It was previously believed that the major ones generated in the reported synthesis (including U.S. Pat. No.6,953,855)In stereoisomeric form in the form of 1-azabicyclo [2.2.2]The cis-relative configuration at the 2 and 3 positions of the octane (quinuclidine) ring is characteristic. In other words, the syn diastereomer (pair (2R,3R) and (2S,3S) of enantiomers) is believed to be the predominant form obtained when prepared according to the reported method. This determination of major cis synthesis is based on: (i) of the 2-and 3-position hydrogen nuclei of quinuclidines and of isolated diastereomeric (cis-and trans) intermediates¹H coupling constants compared to literature reported coupling constants; and (ii) by analogy to literature descriptions such as Warawa et al, J.Med.chem.18(6): 587-. Thus, it is expected that a cis configuration will be formed. As such, the results from biological assays using racemates are presumed to be due to the predominantly cis configuration.

It has now been found by means of X-ray diffraction analysis of crystalline salt forms and analogues that the major diastereomer produced in the initial synthesis is actually the trans diastereomer. In addition, it has been found that the two enantiomers with trans relative stereochemistry, namely (2S,3R) and (2R,3S), differ substantially from each other in their ability to interact with the α 7NNR subtype. The (2S,3R) configuration, compound a, has greater activity.

By further analysis, it has been found that compound a has a structure that makes it react with: i) each of the other three stereoisomers obtained separately, ii) a mixture of all four stereoisomers, i.e. a racemate; and iii) other distinctive pharmacological properties of the α 7NNR ligand reported in the literature.

(2S,3R) -N (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Oct-3-yl) benzofuran-2-carboxamide (Compound A) is a highly selective full agonist at the α 7NNR receptor with a significantly low EC₅₀(for activation) value and in EC₅₀And IC₅₀(for residual inhibition) with good distance difference between them, at wide therapeutically useful concentrationsFunctional agonism is provided within the scope.

An amplifiable, large-scale synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide

The specific synthetic steps differ in their feasibility to scale up. It was found that the reaction lacks scalability for a variety of reasons, including safety considerations, reagent expense, difficult work-up or purification, reaction kinetics (thermodynamics or kinetics), and reaction yield.

The synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide described in this specification has been used to generate kilogram quantities of material and the component reactions have been carried out on a kilogram scale in high yield.

Scalable synthesis employs dynamic resolution of the racemizable ketone (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octan-3-one) and stereoselective reduction (reductive amination) of the (R) - α -methylbenzylamine imine derivative of the resolved ketone. The synthetic sequences reported in this specification can be easily scaled up and dispense with chromatographic purification.

Preparation of a novel salt form of (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide

(2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide is an amorphous powder with very limited water solubility as the free base this free base is reacted with inorganic and organic acids to form certain acid addition salts that have physical and chemical properties that are advantageous for the preparation of pharmaceutical compositions, including but not limited to crystallinity, water solubility and stability.

Depending on the manner in which the salts described herein are formed, the salts may have a crystal structure that occludes the solvent present during salt formation. Thus, the salts may exist as hydrates and other solvates with different stoichiometries of solvent relative to (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide.

The method of preparing the salt form may vary. The preparation of a salt form of (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide generally comprises:

(i) mixing the free base or a solution of the free base, i.e. a solution of (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide in a suitable solvent with the neat acid or with a solution of the acid in a suitable solvent;

(iia) if necessary, cooling the resulting salt solution to cause precipitation; or

(iib) adding a suitable anti-solvent to cause precipitation; or

(iic) evaporating the first solvent and adding new solvent and repeating step (iia) or step (iib); and

(iii) the resulting salt was filtered and collected.

In one embodiment, (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide is stereoisomerically enriched in one embodiment enantiomeric and/or diastereomeric excess is 90% or greater in one embodiment enantiomeric and/or diastereomeric excess is 95% or greater in one embodiment enantiomeric and/or diastereomeric excess is 98% or greater in one embodiment enantiomeric and/or diastereomeric excess is 99% or greater in one embodiment enantiomeric and/or diastereomeric excess is 99.5% or greater in one embodiment.

The stoichiometry used, solvent mix, solute concentration and temperature can vary. Representative solvents that may be used for the preparation or recrystallization of the salt form include, but are not limited to, ethanol, methanol, isopropanol, acetone, ethyl acetate, and acetonitrile.

Examples of suitable pharmaceutically acceptable salts include inorganic acid addition salts such as chloride, bromide, sulfate, phosphate and nitrate; organic acid addition salts such as acetate, galactarate, propionate, succinate, lactate, glycolate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, p-toluenesulfonate and ascorbate; and salts with amino acids such as aspartate and glutamate. Salts may sometimes be hydrates or ethanol solvates. Representative salts are provided as described in U.S. patent No. 5,597,919 to Dull et al, U.S. patent No. 5,616,716 to Dull et al, and U.S. patent No. 5,663,356 to Ruecroft et al, each of which is incorporated herein by reference.

Salt screening of the free base (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide shows that, although many pharmaceutically acceptable salts of acids can be formed, only a few of these salts have acceptable properties for commercial manufacture. The salts of hydrochloric acid, phosphoric acid, maleic acid, and p-toluenesulfonic acid each exhibit additional desirable properties including high melting points, good water solubility, and low hygroscopicity. These characteristics in these salts are unexpected.

Pharmaceutical compositions

The pharmaceutical compositions of the present invention include the salts described herein in pure form or in the form of compositions wherein the compound is combined with any other pharmaceutically compatible product, which may be inert or physiologically active. The resulting pharmaceutical compositions can be used to prevent a condition or disorder in a subject predisposed to the condition or disorder, and/or to treat a subject suffering from the condition or disorder. The pharmaceutical compositions of the present specification include a compound of the present invention and/or a pharmaceutically acceptable salt thereof.

The mode of administration of the compounds may vary. The compositions are preferably administered orally (e.g., in liquid form in a solvent such as an aqueous or non-aqueous liquid, or in a solid carrier). Preferred compositions for oral administration include pills, tablets, capsules, caplets, syrups and solutions, including hard gelatin capsules and time release capsules. Standard excipients include binders, fillers, colorants, solubilizers, and the like. The compositions may be formulated in unit dosage form, or as multiple doses or sub-unit dosages. Preferred compositions are in liquid or semi-solid form. Compositions comprising a liquid pharmaceutically inert carrier such as water or other pharmaceutically compatible liquid or semi-solid may be used. The use of such liquids and semi-solids is well known to those skilled in the art.

The compositions may also be administered by injection, i.e., intravenously, intramuscularly, subcutaneously, intraperitoneally, intraarterially, intrathecally; and administration by the intracerebroventricular route. Intravenous administration is the preferred method of injection. Suitable carriers for injection are well known to those skilled in the art and include 5% dextrose solution, saline and phosphate buffered saline. The pharmaceutical product may also be administered as an infusion or injection (e.g., as a suspension or emulsion in a pharmaceutically acceptable liquid or liquid mixture).

The formulations may also be administered using other means, such as rectal administration. Formulations useful for rectal administration, such as suppositories, are well known to those skilled in the art. Pharmaceutical products may also be administered by inhalation (e.g., nasally in aerosol form or using a delivery article of the type described in U.S. patent No. 4,922,901 to Brooks et al, which is incorporated herein in its entirety); topical (e.g., in the form of a lotion); transdermal (e.g., using a transdermal patch) or iontophoretic administration; or by sublingual or buccal administration. Although it is possible to administer the compound in the form of a bulk active chemical, it is preferred to provide the pharmaceutical product in the form of a pharmaceutical composition or pharmaceutical formulation for efficient and efficacious administration.

Exemplary methods of administration of the compounds are well known to those skilled in the art. The usefulness of these formulations may vary depending on the particular composition used and the particular subject being treated. These formulations may contain a liquid carrier, which may be oily, aqueous, emulsified or contain certain solvents suitable for the mode of administration.

The composition may be administered to a warm-blooded animal (e.g. a mammal such as a mouse, rat, cat, rabbit, dog, pig, cow or monkey) periodically or at a gradual, continuous, constant or controlled rate, but is advantageously administered to a human. In addition, the number of days and times of daily administration of the pharmaceutical formulation may vary.

Other suitable methods for administration of the compounds of the present invention are described in Smith et al, U.S. patent No. 5,604,231, the contents of which are incorporated herein by reference.

In embodiments of the invention and as would be understood by one of skill in the art, the compounds of the invention may be administered in combination with other therapeutic compounds. For example, the compounds of the present invention may be used in combination with substances such as: other NNR ligands (such as valnemulin), antioxidants (such as free radical scavengers), antibacterial agents (such as penicillins), antiviral agents (such as nucleoside analogues, e.g. zidovudine and acyclovir), anticoagulants (such as warfarin), anti-inflammatory agents (such as NSAIDs), antipyretics, analgesics, narcotics (such as intraoperative narcotics), acetylcholinesterase inhibitors (such as donepezil and galantamine), antipsychotics (such as haloperidol, clozapine, olanzapine and quetiapine), immunosuppressive agents (such as cyclosporin and methotrexate), neuroprotective agents, steroids (such as steroid hormones), corticosteroids (such as dexamethasone, prednisone and hydrocortisone), vitamins, minerals, nutraceuticals, antidepressants (such as imipramine, fluoxetine, paroxetine, escitalopram, sertraline, venlafaxine and duloxetine), anxiolytics (such as alprazolam and buspirone), anticonvulsants (such as phenytoin and gabapentin), vasodilators (such as prazosin and sildenafil), mood-stabilizing drugs (such as valproate and aripiprazole), anti-cancer drugs (such as antiproliferatives), antihypertensives (such as atenolol, clonidine, amlodipine, verapamil and olmesartan), laxatives, stool softeners, diuretics (such as furosemide), antispasmodics (such as bicyline), anti-dyskinesias and anti-ulcers (such as esomeprazole).

The compounds of the present invention may be used alone or in combination with other therapeutic agents. Such combinations of pharmaceutically active agents may be administered together or separately, and when administered separately, administration may be simultaneous or sequential in any order. The amount of compound or agent and the relative timing of administration will be selected so as to achieve the desired therapeutic effect. The combined administration may be simultaneous administration in the form of: (1) a monolithic pharmaceutical composition comprising a plurality of compounds; or (2) separate pharmaceutical compositions, each comprising one of the compounds. Alternatively, the combination may be administered separately in a sequential manner in which one therapeutic agent is administered first and then the other therapeutic agent, or vice versa. The sequential administration may be spaced closer or further apart in time. The compounds of the present invention are useful in the treatment of a variety of disorders and conditions, and thus, the compounds of the present invention can be used in combination with a variety of other suitable therapeutic agents that can be used in the treatment or prevention of such disorders or conditions.

A suitable dose of a compound is an amount effective to prevent a patient suffering from a disorder from developing symptoms of the disorder or to treat some symptoms of the disorder. Reference to an "effective amount," "therapeutic amount," or "effective dose" is a quantity sufficient to elicit the desired pharmacological or therapeutic effect and thereby result in an effective prevention or treatment of the disorder.

When treating a CNS disorder, an effective amount of a compound is an amount sufficient to cross the blood-brain barrier of a subject in order to bind to the relevant receptor sites in the brain of the subject and modulate the activity of the relevant NNR subtypes (e.g., provide neurotransmitter secretion, thereby resulting in effective prevention or treatment of the disorder). An example of prevention of a disorder is represented by delaying the onset of symptoms of the disorder. Examples of treatment of a disorder are reduction of symptoms associated with the disorder or amelioration of recurrence of symptoms of the disorder. Preferably, the effective amount is sufficient to achieve the desired result, but insufficient to cause significant side effects.

The effective dose may vary depending on factors such as the condition of the patient, the severity of the symptoms of the disorder, and the mode of administration of the pharmaceutical composition. For human patients, an effective dose of a typical compound generally requires administration of an amount of the compound sufficient to modulate the activity of the relevant NNR, but insufficient to induce an effect on skeletal muscle and ganglia to any significant degree. The effective dose of the compound will, of course, vary from patient to patient, but generally will include amounts ranging from a starting point at which a CNS effect or other desired therapeutic effect occurs, but below the amount at which a muscular effect is observed.

The compounds described herein, when used in effective amounts according to the methods described herein, can provide a degree of prevention of progression of a CNS disorder or other disorder, amelioration of symptoms of a CNS disorder or other disorder, or amelioration of recurrence of a CNS disorder or other disorder to a certain degree. The effective amount of these compounds is generally below the threshold concentration required to elicit any significant side effects (e.g., those associated with skeletal muscle or ganglia). The compounds may be administered within the therapeutic window in which certain CNS disorders and other disorders are treated and certain side effects are avoided. Ideally, an effective dose of a compound described herein is sufficient to provide the desired effect for the disorder but insufficient (i.e., not at a high enough level) to provide the undesired side effects. Preferably, the compound is administered at a dose of 1/5, typically less than 1/10, that is effective in treating CNS or other disorders, but less than that amount required to elicit some side effects to any significant degree.

Most preferably, the effective dose is a very low concentration when the greatest effect is observed with minimal side effects. Typically, an effective dose of these compounds generally requires that the compound be administered in an amount of less than 5mg/kg of patient weight. Typically, the compounds of the invention are administered at less than about 1mg/kg of patient body weight, typically at less than about 100 μ g/kg of patient body weight, but often from about 10 μ g to less than 100 μ g/kg of patient body weight. The effective doses described above generally represent the amount administered as a single dose, or as an amount administered in one or more doses over a 24 hour period.

For human patients, an effective dose of a typical compound generally requires administration of the compound in an amount of at least about 1 mg/24 hours/patient, usually at least about 10 mg/24 hours/patient, and often at least about 100 mg/24 hours/patient. For human patients, an effective dose of a typical compound requires administration of an amount of the compound that generally does not exceed about 500 mg/24 hours/patient, usually does not exceed about 400 mg/24 hours/patient, and often does not exceed about 300 mg/24 hours/patient. In addition, the compositions can advantageously be administered in an effective dose such that the concentration of the compound in the plasma of the patient does not generally exceed 50ng/mL, usually does not exceed 30ng/mL, and often does not exceed 10 ng/mL. In one embodiment of the invention, the effective dose is about 1mg to 10mg over a 24 hour period.

Methods of use of pharmaceutical compositions

An "agonist" as used herein is a substance that stimulates its binding partner (usually the receptor). Stimuli are defined in the context of specific assays, or are clearly evident from literature discussions, which compare factors or substances accepted as "agonists" or "antagonists" of specific binding partners in substantially similar environments as would be understood by one skilled in the art. Stimulation may be defined in terms of an increase in a particular effect or function induced by the interaction of an agonist or partial agonist with a binding partner and may include allosteric effects.

As used herein, an "antagonist" is a substance that inhibits its binding partner (typically a receptor). Inhibition is defined in the context of a particular assay, or is clearly evident from literature discussions, which compare factors or substances accepted as "agonists" or "antagonists" of a particular binding partner under substantially similar circumstances as would be understood by one skilled in the art. Inhibition may be defined in terms of a reduction in a particular effect or function induced by the interaction of the antagonist with the binding partner and may include allosteric effects.

As used herein, a "partial agonist" or "partial antagonist" is a level of stimulation or inhibition that provides a non-full or incomplete agonistic or antagonistic effect, respectively, to its binding partner. It will be appreciated that inhibition, and thus inhibition, is inherently defined for any substance or class of substances that is to be defined as an agonist, antagonist or partial agonist.

"intrinsic activity" or "efficacy" as used herein relates to some measure of the biological effectiveness of the binding partner complex. With respect to receptor pharmacology, the context in which intrinsic activity or efficacy is defined should be based on the context of the binding partner (e.g., receptor/ligand) complex and the considerations of activity associated with a particular biological outcome. For example, in some cases, intrinsic activity may vary depending on the particular second messenger system involved. See Hoyer, d, and Boddeke, h., Trends pharmacol. sci14(7):270-5(1993), which are incorporated by reference into this specification for this teaching. The specific assessments of these contextual associations in what cases are associated and how they are associated in the context of the present invention will be readily apparent to those of skill in the art.

Receptor modulation as used herein includes agonism, partial agonism, antagonism, partial antagonism or inverse agonism of the receptor.

Neurotransmitters for which release is mediated by the compounds described herein, as used herein, include, but are not limited to, acetylcholine, dopamine, norepinephrine, 5-hydroxytryptamine and glutamate, and the compounds described herein act as modulators of the α 7 subtype of CNS NNR.

The term "prevention" or "preventing" as used herein includes any degree of reduction in the progression of a disease, disorder or condition, or any degree of delay in the onset of a disease, disorder or condition. The term includes providing a protective effect against a particular disease, disorder, or condition as well as including amelioration of recurrence of the disease, disorder, or condition. Thus, in another aspect, the invention provides methods of treating a patient having an NNR-or nAChR-mediated disorder or a subject at risk of developing or experiencing a relapse of a disorder mediated by NNR or nAChR. The compounds and pharmaceutical compositions of the invention may be used to achieve a beneficial therapeutic or prophylactic effect, for example, in a subject suffering from CNS dysfunction.

As described above, the free base and salt compounds of the invention modulate an α 7NNR subtype characteristic of the CNS, and can be used to prevent or treat conditions or disorders, including CNS conditions or disorders, in subjects suffering from or susceptible to such conditions or disorders by modulating an α 7 NNR. The compounds are capable of selectively binding to α 7NNR and expressing nicotinic pharmacology, e.g., acting as agonists, partial agonists, antagonists, as described. For example, the compounds of the present invention, when administered in an effective amount to a patient in need thereof, provide a degree of prevention of the progression of the CNS disorder, i.e., provide a protective effect, ameliorate the symptoms of the CNS disorder, or ameliorate the recurrence of the CNS disorder, or a combination thereof.

The compounds of the invention are useful in the treatment or prevention of other types of conditions and disorders for which other types of nicotinic compounds have been proposed or shown to be useful as therapeutic agents. See, for example, the literature cited above, and Williams et al, Drug News Perspec.7(4):205(1994), Arneric et al, CNS Drug Rev.1(1):1-26(1995), Arneric et al, exp.Opin.invest.Drugs5(1):79-100(1996), Bencherif et al, J.Pharmacol.exp.Ther.279:1413(1996), Lippiello et al, J.Pharmacol.exp.Ther.279:1422(1996), Damaj et al, J.Pharmacol.exp.Ther.291:390 (1999); chiari et al, Anesthesiology91:1447(1999), Lavand' homme and Eisenbach, Anesthesiology91:1455(1999), Holladay et al, J.Med.Chem.40(28):4169-94(1997), Bannon et al, Science279:77(1998), PCT WO94/08992, PCT WO96/31475, PCT WO96/40682, and U.S. Pat. No. 5,583,140 to Bencherif et al, U.S. Pat. No. 5,597,919 to Dull et al, U.S. Pat. No. 5,604,231 to Smith et al, and U.S. Pat. No. 5,852,041 to Cosford et al, the contents of which are incorporated herein by reference for these therapeutic teachings.

The compounds and pharmaceutical compositions thereof are useful for treating or preventing a variety of CNS disorders, including neurodegenerative disorders, neuropsychiatric disorders, neurological disorders, and addiction. The compounds and pharmaceutical compositions thereof are useful for treating or preventing age-related and other cognitive deficits and dysfunctions; attention disorders and dementias, including those caused by infectious agents or metabolic disorders; providing neuroprotection; treatment of convulsions and multiple cerebral infarction; treating mood disorders, obsessive-compulsive disorder and addictive behaviors; providing analgesia; control inflammation, such as that mediated by cytokines and nuclear factor κ B; treating inflammatory disorders; providing pain relief; treating metabolic disorders such as diabetes or metabolic syndrome; for the treatment of infections, as anti-infective agents for the treatment of bacterial, fungal and viral infections.

CNS disorders

Among the disorders, diseases, and conditions that may be treated or prevented using the compounds and pharmaceutical compositions of the present invention are: age-associated memory impairment (AAMI), Mild Cognitive Impairment (MCI), age-associated cognitive decline (ARCD), pre-senile dementia, early onset Alzheimer's disease, senile dementia, dementia of the Alzheimer's type, Alzheimer's disease, dementia without dementia (CIND), Lewy body dementia, HIV dementia, AIDS dementia complex, vascular dementia, Down's (Down) syndrome, head trauma, Traumatic Brain Injury (TBI), dementia pugilistica, Creutzfeld-Jacob disease and prion disease, stroke, ischemia, attention deficit disorder, attention deficit hyperactivity disorder, dyslexia, schizophrenia, schizophreniform disorder, schizoaffective disorder, cognitive dysfunction in schizophrenia, cognitive deficits in schizophrenia such as memory (including working memory), executive function, attention deficit hyperactivity disorder, dementia, schizophrenia, vascular dementia, Vigilance, information processing and learning, dementia associated with schizophrenia (whether mild, moderate or severe), parkinsonism, including parkinson's disease, postencephalitic parkinsonism, guam parkinsonism-dementia, frontotemporal dementia of parkinson's disease type (FTDP), Pick's disease, Niemann-Pick's disease, huntington's chorea, tardive dyskinesia, hyperkinesia, progressive supranuclear palsy, restless leg syndrome, multiple sclerosis, Amyotrophic Lateral Sclerosis (ALS), Motor Neuron Disease (MND), Multiple System Atrophy (MSA), corticobasal degeneration, Guillain-Barre syndrome (GBS), Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), epilepsy, autosomal dominant nocturnal frontal epilepsy, mania, anxiety, depression, premenstrual dysphoric disorder, panic disorder, bulimia, anorexia, narcolepsy, excessive daytime sleepiness, bipolar disorder, generalized anxiety disorder, obsessive-compulsive disorder, rage outbursts, oppositional defiant disorder, Tourette's (Tourette's) syndrome, autism, drug and alcohol addiction, tobacco addiction, obesity, cachexia, psoriasis, lupus, acute cholangitis, aphthous stomatitis, ulcers, asthma, ulcerative colitis, inflammatory bowel disease, crohn's disease, post-operative ileus, spasmodic dystonia, diarrhea, constipation, pouchitis, pancreatitis, viral pneumonia, arthritis, including rheumatoid arthritis and osteoarthritis, endotoxemia, sepsis, septic shock, central nervous syndrome, irritable bowel syndrome with symptoms of excessive eating, anorexia, bulimia, excessive daytime sleepiness, and other symptoms of the, Atherosclerosis, idiopathic pulmonary fibrosis, acute pain, chronic pain, neuropathy, urinary incontinence, diabetes, and neoplasia.

Cognitive impairment or dysfunction may be associated with mental diseases or conditions such as: schizophrenia and other psychoses, including, but not limited to, psychosis, schizophreniform disorder, schizoaffective disorder, delusional disorder, brief psychotic disorder, shared psychotic disorder and psychosis resulting from one or more general medical conditions, dementia, and other cognitive disorders, including, but not limited to, mild cognitive impairment, presenile dementia, Alzheimer's disease, senile dementia, dementia of the Alzheimer's type, age-related memory impairment, Lewy body dementia, vascular dementia, AIDS dementia complex, dyslexia, parkinsonism including Parkinson's disease, cognitive impairment and dementia of Parkinson's disease, cognitive impairment of multiple sclerosis, cognitive impairment resulting from traumatic brain injury, dementia resulting from other general medical conditions, anxiety disorders, including, but not limited to, panic disorder without agoraphobia, panic disorder with agoraphobia, agoraphobia without history of panic disorder, specific phobias, social phobias, obsessive-compulsive disorders, post-traumatic stress disorder, acute stress disorder, generalized anxiety disorder and generalized anxiety disorder caused by a general medical condition, mood disorders including, but not limited to, major depressive disorder, mood dysthymic disorder, bipolar depression, bipolar mania, bipolar I disorder, depression associated with mania, depression or mixed episodes, bipolar II disorder, circulatory disorder and mood disorder caused by a general medical condition, sleep disorders including, but not limited to, sleep disorders, primary insomnia, primary sleepiness, narcolepsy, deep sleep disorders, dreaminess disorders, sleep panic disorders and sleep disorders, mental retardation, learning disorders, motor skills disorders, communication disorders, pervasive development disorders, attention deficit and disruptive behavior disorders, attention deficit hyperactivity disorder, eating and eating disorders in infants, children or adults, spasticity disorders, excretory disorders, substance-related disorders including, but not limited to, substance dependence, substance abuse, substance intoxication, substance withdrawal, alcohol-related disorders, amphetamine or amphetamine-like related disorders, caffeine-related disorders, cannabis-related disorders, cocaine-related disorders, hallucinogen-related disorders, inhalant-related disorders, nicotine-related disorders, opioid-related disorders, phencyclidine or phencyclidine-like related disorders, and sedative, hypnotic or anxiolytic-related disorders, personality disorders including, but not limited to, obsessive-compulsive personality disorders and impulse-control disorders.

The symptoms of schizophrenia are generally divided into three categories: positive, negative and cognitive symptoms. Positive symptoms, which may sometimes be referred to as "psychotic" symptoms, include delusions and hallucinations. By "positive" is meant having a significant symptom. Negative symptoms include a flat mood or lack of expression, inability to start or complete an activity, short speech and lack of content, and a lack of pleasure or disinterest in the activity. "negative" refers to the absence of other certain features that are present in healthy subjects. Cognitive symptoms belong to the thought process. Cognitive symptoms include cognitive Deficits, such as memory including working memory, executive function, attention, alertness, information processing and learning, see, e.g., cognitive function in Schizophrenia: defaits, Functional sequences, and future Treatment, psychiatr. clin. n. am.26(2003)25-40 by Sharma et al, which is incorporated herein by reference. Schizophrenia also affects mood. Although many subjects suffering from schizophrenia become depressed, some subjects have significant mood swings and even a biphasic-like state.

The above conditions and disorders are discussed in further detail in, for example, the following documents: the American Psychiatric Association of Diagnostic and Statistical Manual of Mental Disorders, 4 th edition, Text review, Washington, DC, American Psychiatric Association, 2000; this reference is incorporated by reference into the present specification with respect to defining these conditions and disorders. See also the manual for a detailed description of symptoms and diagnostic features related to substance use, abuse and dependence.

Preferably, the treatment or prevention of the diseases, disorders, and conditions is performed without significant adverse side effects, including, for example, significant increases in blood pressure and heart rate, significant adverse effects on the gastrointestinal tract, and significant effects on skeletal muscle.

The compounds of the invention, when used in effective amounts, are believed to modulate the activity of α 7NNR without significant interaction with nicotinic subtypes characteristic of the human ganglia (as evidenced by a lack of ability to elicit nicotinic function in adrenal chromaffin tissue) or with nicotinic subtypes characteristic of skeletal muscle (as evidenced by a lack of ability to elicit nicotinic function in cell preparations expressing muscle-type nicotinic receptors). Thus, these compounds are believed to be capable of treating or preventing diseases, disorders, and conditions without eliciting significant side effects associated with activity at the ganglion and neuromuscular sites. Thus, administration of the compounds is believed to provide a therapeutic window within which treatment of certain diseases, disorders, and conditions is provided, and certain side effects are avoided. That is, an effective dose of the compound is considered sufficient to provide the desired effect for the disorder, disease, or condition, but is considered insufficient (i.e., not at a sufficiently high level) to provide undesirable side effects.

Accordingly, the present invention provides the use of a compound of the invention, or a pharmaceutically acceptable salt thereof, in therapy, such as the therapies described above.

In a further aspect of the invention there is provided the use of a compound of the invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a CNS disorder, such as a disorder, disease or condition as hereinbefore described.

Inflammation(s)

The nervous system (mainly via the vagus nerve) is known to modulate the magnitude of the innate immune response by inhibiting the release of macrophage Tumor Necrosis Factor (TNF). This physiological mechanism is termed The "cholinergic anti-inflammatory pathway" (see, e.g., Tracey, "The inflammation reflex," Nature420:853-9(2002), which is incorporated herein by reference). Excessive amounts of inflammatory factors and tumor necrosis factor synthesis lead to morbidity and even mortality in a variety of diseases. These diseases include, but are not limited to, endotoxemia, rheumatoid arthritis, osteoarthritis, psoriasis, asthma, atherosclerosis, idiopathic pulmonary fibrosis, and inflammatory bowel disease.

Inflammatory conditions that may be treated or prevented by administration of a compound described herein include, but are not limited to: chronic and acute inflammation, psoriasis, endotoxemia, gout, acute pseudogout, acute gouty arthritis, rheumatoid arthritis, osteoarthritis, polymyositis, dermatomyositis, ankylosing spondylitis, Still's disease, adult-onset Still's disease, allograft rejection, chronic transplant rejection, asthma, atherosclerosis, monocyte-macrophage dependent lung injury, idiopathic pulmonary fibrosis, atopic dermatitis, chronic obstructive pulmonary disease, adult respiratory distress syndrome, acute chest syndrome in sickle cell disease, inflammatory bowel disease, crohn's disease, ulcerative colitis, acute cholangitis, aphthous stomatitis, pouchitis, glomerulonephritis, lupus nephritis, thrombosis, and graft-versus-host reaction.

Inflammatory response associated with bacterial and/or viral infection

Many bacterial and/or viral infections (e.g., meningitis, hepatitis, and nephritis) are associated with side effects caused by the formation of toxins, the body's natural response to bacteria or viruses and/or toxins. As noted above, the body's response to infection often involves the production of large amounts of TNF and/or other cytokines. Over-expression of these cytokines can lead to severe injury such as septic shock (when the bacteria are septic), endotoxic shock, urosepsis and toxic shock syndrome.

Cytokine expression is mediated by NNRs and can be inhibited by administration of agonists or partial agonists of these receptors. Thus, the compounds described herein as agonists or partial agonists of these receptors are useful for minimizing the inflammatory response associated with bacterial infections as well as with viral and fungal infections. Examples of such bacterial infections include anthrax, botulism and sepsis. Some of these compounds also have antimicrobial properties.

These compounds may also serve as adjunctive therapeutic agents in combination with therapeutic agents currently used to treat bacterial, viral and fungal infections, such as antibiotics, antivirals and antifungals. Antitoxins can also be used to bind to toxins produced by infectious agents and allow the bound toxin to travel through the body without producing an inflammatory response. Examples of antitoxins are disclosed, for example, in U.S. patent No.6,310,043 to Bundle et al, which is incorporated herein by reference. Other agents effective against bacteria and other toxins may be effective and their therapeutic effects may be complemented by co-administration with the compounds described herein.

Pain (due to cold or dampness)

The compounds can be administered for the treatment and/or prevention of pain, including acute pain, neurological pain, inflammatory pain, neuropathic pain, and chronic pain. The analgesic activity of the compounds described in this specification can be demonstrated in models of persistent inflammatory and neuropathic pain, such as those described in U.S. patent application publication No. 20010056084a1 (Allgeier et al), which is incorporated herein by reference, where hyperalgesia is exhibited in the complete Freund's (Freund) adjuvant rat model of inflammatory pain and mechanical hyperalgesia is exhibited in the partial sciatic nerve ligation model of neuropathic pain in mice.

The analgesic effect is useful in the treatment of pain of various causes or etiology, particularly in the treatment of inflammatory pain and associated hyperalgesia, neuropathic pain and associated hyperalgesia, chronic pain (e.g., severe chronic pain, post-operative pain, and pain associated with various conditions including cancer, angina, renal or biliary colic, menstruation, migraine and gout). Inflammatory pain can be caused by different causes, including arthritis and rheumatoid diseases, synovitis and vasculitis. Neuropathic pain includes trigeminal or herpetic neuralgia, diabetic neuropathic pain, causalgia, lower back pain, and afferent nerve block syndromes such as brachial plexus avulsion.

Neovascularization

α 7NNR is associated with neovascularization. Inhibition of neovascularization, for example, by administering an antagonist of α 7NNR (or a partial agonist at certain doses), conditions characterized by undesirable neovascularization or angiogenesis can be treated or prevented. These conditions may include those characterized by inflammatory angiogenesis and/or angiogenesis induced by ischemia. Neovascularization associated with tumor growth can also be inhibited by administering a compound described herein that acts as an antagonist or partial agonist of α 7 NNR.

Specific antagonism of α 7NNR specific activity reduces the angiogenic response to inflammation, ischemia, and neoplasia. For guidance on suitable animal model systems for evaluating the compounds described herein, see, e.g., "A novel angiogenic therapy mediated by non-neurological nicotinic acetic chylene receptors," J.Clin.Invest.110(4):527-36(2002) "by Heeschen, C. et al, cells for α 7-specific inhibition of angiogenesis and angiogenic activity associated with human diseases (in vitro) and animal modeling, particularly for Lewis lung tumor models (in vivo, in mice, see, inter alia, pages 529 and 532-533), which are incorporated herein by reference.

Representative tumor types that can be treated using the compounds described herein include NSCLC, ovarian cancer, pancreatic cancer, breast cancer, colon cancer, rectal cancer, lung cancer, oropharyngeal cancer, hypopharynx cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, gall bladder cancer, bile duct cancer, small intestine cancer, urinary tract cancer, kidney cancer, bladder cancer, urothelial cancer, female genital tract cancer, cervical cancer, uterine cancer, ovarian cancer, choriocarcinoma, gestational trophoblastic disease, male genital tract cancer, prostate cancer, seminal vesicle cancer, testicular cancer, germ cell tumors, endocrine adenocarcinomas, thyroid cancer, adrenal cancer, pituitary cancer, skin cancer, hemangioma, melanoma, sarcoma, bone and soft tissue sarcomas, kaposi's sarcoma, brain tumors, neuroma, eye tumors, meningioma, astrocytoma, glioma, glioblastoma, retinoblastoma, neuroma, neuroblastoma, schwannoma, meningioma, solid tumors resulting from hematopoietic malignancies (such as leukemias, chloromas, plasmacytomas, and plaques and tumors of mycosis fungoides, and cutaneous T-cell lymphoma/leukemia), and solid tumors derived from lymphoma.

The compounds may also be used in combination with other forms of anti-cancer therapy, including co-administration with anti-neoplastic drugs such as cisplatin, doxorubicin, daunorubicin, and the like, and/or with anti-VEGF (vascular endothelial growth factor) agents, as is known in the art.

The compounds may be administered in a manner that targets them to the tumor site. For example, the compounds may be administered in microspheres, microparticles, or liposomes conjugated to various antibodies that direct the microparticles to the tumor. In addition, the compound may be present within microspheres, microparticles, or liposomes that have been appropriately sized to travel through arteries and veins, but enter and become immobilized within a capillary bed surrounding the tumor, and the compound is administered locally to the tumor. Such drug delivery devices are known in the art.

Other disorders

In addition to treating CNS disorders, inflammation, neovascularization, and pain, the compounds of the invention are useful for preventing or treating certain other conditions, diseases, and disorders in which NNR plays a role. Examples include autoimmune disorders such as lupus, disorders associated with cytokine release, cachexia secondary to infection (e.g., as found in AIDS, AIDS related syndrome and neoplasias), metabolic disorders including type I diabetes, type II diabetes, metabolic syndrome, obesity or hyperglycemia, pemphitis, urinary incontinence, retinal diseases, infectious diseases, myasthenia gravis, easy myasthenia gravis (Eaton-Lambert) syndrome, hypertension, osteoporosis, vasoconstriction, vasodilation, arrhythmia, bulimia, anorexia and those indications described in published PCT application WO98/25619, which is incorporated herein by reference for these disorders. The compounds of the invention may also be administered to treat convulsions, such as those that are symptoms of epilepsy, and for the treatment of conditions such as syphilis and creutzfeldt-jakob disease.

Diagnostic applications

The compounds are useful in diagnostic compositions, such as probes, particularly when they are modified to include suitable labels. Probes can be used, for example, to determine the relative number and/or function of specific receptors, particularly the α 7 receptor subtype. For this purpose, the compounds of the invention are most preferably selected from radioisotope moieties such as¹¹C、¹⁸F、⁷⁶Br、¹²³I or¹²⁵And I, marking.

The administered compound can be detected using known detection methods appropriate to the label used. Examples of detection methods include Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT). The radioactive labels described above can be used in PET (e.g.,¹¹C，¹⁸f or⁷⁶Br) and SPECT (for example,¹²³I) in the imaging process, the imaging device is used,¹¹the half-life of C is about 20.4 minutes,¹⁸the half-life of F is about 109 minutes,¹²³the half-life of I is about 13 hours, an⁷⁶The half-life of Br was about 16 hours. High specific radioactivity is desirable to allow imaging of the selected receptor subtype at non-saturating concentrations. The dose administered is typically lower than the toxic dose and provides a strong contrast image. The compounds are expected to be able to be administered at non-toxic levels. The dose is determined in a manner known to those skilled in the art of radiolabeling imaging. See, for example, U.S. patent No. 5,969,144 to London et al, which is incorporated herein by reference for the administration of such compounds.

The compounds may be administered using known techniques. See, for example, U.S. patent No. 5,969,144 to London et al, which is incorporated herein by reference for such administration. The compounds can be administered in a formulation composition that incorporates other ingredients, such as those types of ingredients useful in formulating diagnostic compositions. The compounds which can be used according to the invention are most preferably used in highly pure form. See U.S. patent No. 5,853,696 to Elmalch et al, which is incorporated by reference herein for this analysis.

After a compound is administered to a subject (e.g., a human subject), the compound present in the subject can be imaged and quantified by suitable techniques to indicate the presence, amount, and function of a selected NNR subtype. In addition to humans, the compounds can also be administered to animals, such as mice, rats, dogs, and monkeys. SPECT and PET imaging can be performed using any suitable techniques and apparatus. See Villemagne et al, Arneric et al, (eds.) neuronal Receptives: Pharmacology and Therapeutic Opportunities, 235-250(1998) and Elmalch et al, U.S. Pat. No. 5,853,696, each of which is incorporated herein by reference for representative imaging techniques.

The radiolabeled compounds bind with high affinity to selective NNR subtypes (e.g., α 7) and preferably exhibit little non-specific binding to other nicotinic cholinergic receptor subtypes (e.g., those associated with muscle and ganglia). As such, the compounds are useful as agents for the non-invasive imaging of nicotinic cholinergic receptor subtypes within a subject, particularly within the brain for diagnosis associated with various CNS diseases and disorders.

In one aspect, the diagnostic composition may be used in a method of diagnosing a disease in a subject, such as a human patient. The methods involve administering to a patient a detectably labeled compound described herein and detecting binding of the compound to a selected NNR subtype (e.g., the α 7 receptor subtype). Those skilled in the art using diagnostic tools such as PET and SPECT can use the radiolabeled compounds described herein to diagnose various conditions and disorders, including conditions and disorders associated with dysfunction of the central and autonomic nervous systems. These disorders include a variety of CNS diseases and disorders, including alzheimer's disease, parkinson's disease and schizophrenia. These and other representative diseases and disorders that may be evaluated include those described in U.S. patent 5,952,339 to Bencherif et al, which is hereby incorporated by reference.

In another aspect, the diagnostic compositions can be used in methods of monitoring a selective nicotinic receptor subtype of a subject, such as a human patient. The methods involve administering to a patient a detectably labeled compound described herein and detecting binding of the compound to a selected nicotinic receptor subtype, the α 7 receptor subtype.

Receptor binding

The compounds of the invention are useful as reference ligands in binding assays for compounds that bind to NNR subtypes, particularly the α 7 receptor subtype. For this purpose, the compounds of the invention are preferably substituted with a radioisotope moiety such as³H or¹⁴And C, marking.

Synthesis example

The following synthetic examples are provided to illustrate the present invention and are not to be construed as limiting the scope of the invention. In these examples, all parts and percentages are by weight unless otherwise indicated. All solutions are aqueous unless otherwise specified.

Example 1: Small-Scale Synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octan-3-yl) benzofuran-2-carboxamide (Compound A) and its enantiomer (2R,3S) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octan-3-yl) benzofuran-2-carboxamide

2- ((3-pyridinyl) methylene) -1-azabicyclo [2.2.2] octan-3-one

Potassium hydroxide (56g, 0.54 moles) was dissolved in methanol (420 mL). 3-quinuclidinone hydrochloride (75g, 0.49 mole) was added and the mixture was stirred at ambient temperature for 30 minutes. 3-pyridinecarboxaldehyde (58g, 0.54 mole) was added and the mixture was stirred at ambient temperature for 16 hours. The reaction mixture turned yellow during this time with solids adhering to the flask walls. The solids are scraped from the wall and broken up. Water (390mL) was added with rapid stirring. When the solid dissolved, the mixture was cooled at 4 ℃ overnight. The crystals were collected by filtration, washed with water, and air-dried to give 80g of a yellow solid. A second crop (8g) of product was obtained by concentrating the filtrate to about 10% of its original volume and cooling overnight at 4 ℃. Both batches had sufficient purity for further conversion (88g, 82% yield).

2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octan-3-one

2- ((3-pyridinyl) methylene) -1-azabicyclo [2.2.2] octan-3-one (20g, 93mmol) was suspended in methanol (200mL) and treated with 46mL of 6M hydrochloric acid. 10% palladium on charcoal (1.6g) was added and the mixture shaken under 25psi of hydrogen for 16 h. The mixture was filtered through celite and the solvent was removed from the filtrate by rotary evaporation. Crude 2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octan-3-one hydrochloride was obtained as a white gum (20g), which was subsequently treated with 2M sodium hydroxide (50mL) and chloroform (50mL) and stirred for 1 hour. The chloroform layer was separated and the aqueous phase was treated with 2M sodium hydroxide (. about.5 mL, sufficient to raise the pH to 10) and saturated aqueous sodium chloride (25 mL). The aqueous mixture was extracted with chloroform (3 × 10mL), and the combined chloroform extracts were dried (anhydrous magnesium sulfate) and concentrated by rotary evaporation. The residue (18g) was dissolved in warm ether (320mL) and cooled to 4 ℃. The white solid was filtered off, washed with a small amount of cold ether and air dried. The filtrate was concentrated to about 10% of its original volume and cooled at 4 ℃ to give a second crop. The combined yield was 16g (79%).

3-amino-2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octane

To a stirred solution of 2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octan-3-one (3.00g, 13.9mmol) in dry methanol (20mL) under nitrogen was added a 1M solution of zinc chloride in ether (2.78mL, 2.78 mmol). After stirring for 30 minutes at ambient temperature, the mixture was treated with solid ammonium formate (10.4g, 167 mmol). After stirring at ambient temperature for a further 1h, solid sodium cyanoborohydride (1.75g, 27.8mmol) was added in portions. The reaction was then stirred at ambient temperature overnight and quenched by the addition of water (. about.5 mL). The quenched reaction was partitioned between 5M sodium hydroxide (10mL) and chloroform (20 mL). The aqueous layer was extracted with chloroform (20mL) and the combined organic layers were dried (sodium sulfate), filtered and concentrated. 2.97g of a yellow gum are obtained. GCMS analysis showed that the product was a 1:9 mixture of cis-and trans-amines with traces of the corresponding alcohol present together (98% overall mass recovery).

(2R,3S) and (2S,3R) -3-amino-2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octane

di-p-toluoyl-D-tartaric acid (5.33g, 13.8mmol) was added to a solution of crude 3-amino-2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octane (6.00g, 27.6mmol of a 1:9 cis/trans mixture) in methanol (20 mL). After complete dissolution, the clear solution was concentrated by rotary evaporation to form a solid material. The solid was dissolved in a minimum amount of boiling methanol (-5 mL). The solution was cooled slowly, first to ambient temperature (1 hour), then at 5 ℃ for about 4 hours, and finally at-5 ℃ overnight. The precipitated salt was collected by suction filtration and recrystallized from 5mL of methanol. Air-dried to give 1.4g of a white solid, which was partitioned between chloroform (5mL) and 2M sodium hydroxide (5 mL). The chloroform layers and 5mL chloroform extracts of the aqueous layer were combined, dried (anhydrous sodium sulfate) and concentrated to give a colorless oil (0.434 g). The enantiomeric purity of the free base was determined by converting a portion to its N- (tert-butoxycarbonyl) -L-prolinamide, followed by analysis of diastereomeric purity (98%) using LCMS.

The mother liquor from the initial crystallization was made basic (. about.pH 11) with 2M sodium hydroxide and extracted twice with chloroform (10 mL). The chloroform extract was dried (anhydrous sodium sulfate) and concentrated to give an oil. The amine (3.00g, 13.8mmol) was dissolved in methanol (10mL) and treated with di-p-toluoyl-L-tartaric acid (2.76g, 6.90 mmol). The mixture was warmed to aid dissolution, then slowly cooled to-5 ℃ and held at-5 ℃ overnight. The precipitate was collected by suction filtration, recrystallized from methanol and dried. 1.05g of a white solid was obtained. The salt was converted to the free base (yield =0.364g) and enantiomeric purity (97%) was determined using the prolinamide method as described above for the other enantiomer.

Trans-enantiomer A of N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octan-3-yl) benzofuran-2-carboxamide

Diphenyl chlorophosphate (0.35mL, 0.46g, 1.7mmol) was added dropwise to a solution of benzofuran-2-carboxylic acid (0.28g, 1.7mmol) and triethylamine (0.24mL, 0.17g, 1.7mmol) in dry dichloromethane (5 mL). After stirring for 30 minutes at ambient temperature, (2S,3R) -3-amino-2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] is added]A solution of octane (0.337g, 1.55mmol), which is derived from di-p-toluoyl-D-tartrate, and triethylamine (0.24mL, 0.17g, 1.7mmol) in dry dichloromethane (5 mL). The reaction mixture was stirred at ambient temperature overnight and then treated with 10% sodium hydroxide (1 mL). The biphasic mixture was separated and the organic layer was concentrated on a Genevac centrifugal evaporator. The residue was dissolved in methanol (6mL) and purified by HPLC on a C18 silica gel column using an acetonitrile/water gradient containing 0.05% trifluoroacetic acid as eluent. The selected fractions were concentrated, the resulting residue partitioned between chloroform and saturated aqueous sodium bicarbonate and the chloroform evaporated to give 0.310g (42% yield) of a white powder (95% GCMS purity).¹H NMR(300MHz，CDCl₃)δ8.51(d，1H)，8.34(dd，1H)，7.66(d，1H)，7.58(dt，1H)，7.49(d，1H)，7.44(s，1H)，7.40(dd，1H)，7.29(t，1H)，7.13(dd，1H)，6.63(d，1H)，3.95(t，1H)，3.08(m，1H)，2.95(m，4H)，2.78(m，2H)，2.03(m，1H)，1.72(m，3H)，1.52(m，1H)。

This material (trans enantiomer a) was then identified by chiral chromatographic analysis to be the same as the material whose absolute configuration was 2S,3R (identified by X-ray crystallography).

Trans-enantiomer B of N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octan-3-yl) benzofuran-2-carboxamide

Diphenyl chlorophosphate (96. mu.L, 124mg, 0.46mmol) was added dropwise to a solution of benzofuran-2-carboxylic acid (75mg, 0.46mmol) and triethylamine (64. mu.L, 46mg, 0.46mmol) in anhydrous dichloromethane (1 mL). After stirring for 45 minutes at ambient temperature, a solution of (2R,3S) -3-amino-2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octane (0.10g, 0.46mmol), which is derived from di-p-toluoyl-L-tartrate, and triethylamine (64 μ L, 46mg, 0.46mmol) in anhydrous dichloromethane (1mL) was added. The reaction mixture was stirred at ambient temperature overnight and then treated with 10% sodium hydroxide (1 mL). The biphasic mixture was separated and the chloroform extracts (2mL) of the organic and aqueous layers were concentrated by rotary evaporation. The residue was dissolved in methanol and purified by HPLC on a C18 silica gel column using an acetonitrile/water gradient containing 0.05% trifluoroacetic acid as eluent. The selected fractions were concentrated, the resulting residue partitioned between chloroform and saturated aqueous sodium bicarbonate, and the chloroform evaporated to give 82.5mg (50% yield) of a white powder. The NMR spectrum was the same as that of the 2S,3R isomer. Since the direct precursor of this substance (trans enantiomer B) is in enantiomeric relationship with the direct precursor of the 2S,3R compound (trans enantiomer a), the absolute configuration of trans enantiomer B is assumed to be 2R, 3S.

Example 2: large Scale Synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide and (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) -1-benzofuran-2-carboxamide p-toluenesulfonate

2- ((3-pyridinyl) methylene) -1-azabicyclo [2.2.2]Octane-3-ones

3-quinuclidinone hydrochloride (8.25kg, 51.0mol) and methanol (49.5L) were charged under a nitrogen atmosphere to a 100L glass reaction flask equipped with a mechanical stirrer, temperature probe and condenser. Potassium hydroxide (5.55kg, 99.0mol) was added over about 30 minutes via an addition funnel, resulting in an increase in the reaction temperature from 50 ℃ to 56 ℃. 3-pyridinecarboxaldehyde (4.80kg, 44.9mol) was added to the reaction mixture over about 2 hours. The resulting mixture was stirred at 20 ℃. + -. 5 ℃ for at least 12 hours and the reaction was monitored by Thin Layer Chromatography (TLC). After completion of the reaction, the reaction mixture was filtered through a sintered glass funnel and the filter cake was washed with methanol (74.2L). The filtrate was concentrated, transferred to a reaction flask, and water (66.0L) was added. The suspension was stirred for at least 30 minutes, filtered, and the filter cake was washed with water (90.0L) until the pH of the wash was 7-9. The solid was dried under vacuum at 50 ℃. + -. 5 ℃ for at least 12 hours to give 8.58kg (89.3%) of 2- ((3-pyridinyl) methylene) -1-azabicyclo [2.2.2] octan-3-one.

(2S) -2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Octane-3-keto di-p-toluoyl base-D-tartrate salt

Under an inert atmosphere, reacting 2- ((3-pyridyl) methylene) -1-azabicyclo [2.2.2]Octane-3-one (5.40kg, 25.2mol) and methanol (40.5L) were charged to a 72L reaction vessel equipped with a mechanical stirrer, a temperature probe, a low pressure gas regulation system and a pressure gauge. The headspace was purged with nitrogen and the mixture was stirred to obtain a clear yellow solution. To the flask was added 10% palladium on charcoal (50% humidity) (270 g). The atmosphere of the reactor was evacuated using a vacuum pump and the headspace was replaced with hydrogen to a pressure of 10-20 inches of water. The venting and pressurization with hydrogen were repeated 2 more times so that after the third pressurization the reactor was in hydrogen at a pressure of 20 inches of water, the reaction mixture was stirred at 20 ℃ ± 5 ℃ for at least 12 hours, and the reaction was monitored by TLC. After completion of the reaction, the suspension was filtered through a sintered glass funnel545 pad (1.9kg) and the filter cake was washed with methanol (10.1L). The filtrate was concentrated to a semi-solid which was transferred under nitrogen to a 200L reaction flask equipped with a mechanical stirrer, condenser and temperature probe. The semi-solid was dissolved in ethanol (57.2L) and di-p-toluoyl-D-tartaric acid (DTTA) (9.74kg, 25.2mol) was added. The stirred reaction mixture was heated at reflux for at least 1 hour, the reaction was held for an additional at least 12 hours while cooling the reaction to 15 ℃ to 30 ℃. The suspension was filtered using a tabletop filter and the filter cake was washed with ethanol (11.4L). The product was dried under vacuum at ambient temperature to give 11.6kg (76.2% yield, 59.5% purity factor) of (2S) -2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]]Octane-3-keto di-p-toluoyl-D-tartrate.

(2S,3R) -3-amino-2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Octane di-p-methyl benzoyl-D-tartrate salts

Water (46.25L) and sodium bicarbonate (4.35kg, 51.8mol) were added to a 200L flask. After complete dissolution, dichloromethane (69.4L) was added. (2S) -2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octan-3-one di-p-toluoyl-D-tartrate (11.56kg, 19.19mol) was added and the reaction mixture was stirred for 2-10 minutes. The layers were allowed to separate for at least 2 minutes (additional water (20L) was added to separate the layers when necessary). The organic phase was removed and dried over anhydrous sodium sulfate. To the remaining aqueous phase was added dichloromethane (34.7L) and the suspension was stirred for 2-10 minutes. The layers were allowed to separate for 2-10 minutes. Again, the organic phase was removed and dried over anhydrous sodium sulfate. The aqueous phase was again extracted with dichloromethane (34.7L) again as described above. Samples from each extraction were used for chiral HPLC analysis. The sodium sulfate was removed by filtration and the filtrate was concentrated to give (2S) -2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octan-3-one (4.0kg) as a solid.

Under a nitrogen atmosphere, (2S) -2- ((3-pyridyl) methyl) -1-azabicyclo [2.2.2]Octane-3-one (3.8kg) was transferred to a clean 100LGlass reaction flask, equipped with mechanical stirrer and temperature probe. Anhydrous tetrahydrofuran (7.24L) and (+) - (R) - α -methylbenzylamine (2.55L, 20.1mol) were added. Titanium (IV) isopropoxide (6.47L, 21.8mol) was added to the stirred reaction mixture over 1 hour. The reaction was stirred under nitrogen atmosphere for at least 12 hours. Ethanol (36.17L) was added to the reaction mixture. The reaction mixture was cooled to below-5 ℃ and sodium borohydride (1.53kg, 40.5mol) was added in portions, maintaining the reaction temperature below 15 ℃ (this addition takes several hours). The reaction mixture was then stirred at 15 ℃. + -. 10 ℃ for at least 1 hour. The reaction was monitored by HPLC and was complete (from less than 0.5% remaining (2S) -2- ((3-pyridinyl) methyl) -1-azabicyclo [ 2.2.2%]Octane-3-one indicated), 2M sodium hydroxide (15.99L) was added and the mixture was stirred for at least 10 minutes. Filtering the reaction mixture through a tabletop funnel545 of the pad. The filter cake was washed with ethanol (15.23L) and the filtrate was concentrated to give an oil.

The concentrate was transferred under an inert atmosphere to a clean 100L glass reaction flask equipped with a mechanical stirrer and a temperature probe. Water (1L) was added and the mixture was cooled to 0 ℃. + -. 5 ℃.2M hydrochloric acid (24L) was added to the mixture to adjust the pH of the mixture to 1. The mixture was then stirred for at least 10 minutes and 2M sodium hydroxide (24L) was added slowly to adjust the pH of the mixture to 14. The mixture was stirred for at least 10 minutes and the aqueous phase was extracted with dichloromethane (3X 15.23L). The organic phase was dried over anhydrous sodium sulfate (2.0kg), filtered and concentrated to give (2S,3R) -N- ((1R) -phenylethyl) -3-amino-2- ((3-pyridinyl) methyl)) -1-azabicyclo [2.2.2] octane (4.80kg, 84.7% yield).

Under an inert atmosphere, (2S,3R) -N- ((1R) -phenylethyl) -3-amino-2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]The octane was transferred to a 22L glass flask equipped with a mechanical stirrer and a temperature probe. Water (4.8L) was added and the stirred mixture was cooled to 5 ℃. + -. 5 ℃. Adding strong saltAcid (2.97L) was added slowly to the reaction flask, maintaining the temperature of the mixture below 25 ℃. The resulting solution was transferred under an inert atmosphere to a 72L reaction flask containing ethanol (18L) and equipped with a mechanical stirrer, temperature probe and condenser. To the flask was added 10% palladium on charcoal (50% humidity) (311.1g) and cyclohexene (14.36L). The reaction mixture was heated at a temperature near reflux for at least 12 hours and the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled to below 45 ℃ and filtered through a sintered glass funnel545 pad (1.2 kg). The filter cake was rinsed with ethanol (3L) and the filtrate was concentrated to give an aqueous phase. Water (500mL) was added to the concentrated filtrate and the combined aqueous layers were washed with methyl tert-butyl ether (MTBE) (2X 4.79L). 2M sodium hydroxide (19.5L) was added to the aqueous phase to adjust the pH of the mixture to 14. The mixture was then stirred for at least 10 minutes. The aqueous phase was extracted with chloroform (4X 11.96L) and the combined organic phases were dried over anhydrous sodium sulfate (2.34 kg). Filtering the filtrate and concentrating to obtain (2S,3R) -3-amino-2- ((3-pyridyl) methyl) -1-azabicyclo [2.2.2]Octane (3.49kg, greater than quantitative yield) was an oil.

Under an inert atmosphere, (2S,3R) -3-amino-2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octane is transferred to a clean 100L reaction flask equipped with a mechanical stirrer, condenser, and temperature probe. Ethanol (38.4L) and di-p-toluoyl-D-tartaric acid (3.58kg, 9.27mol) were added. The reaction mixture was heated under gentle reflux for at least 1 hour. The reaction mixture was then stirred for at least 12 hours while it was cooled to 15 ℃ to 30 ℃. The resulting suspension was filtered and the filter cake was washed with ethanol (5.76L). The filter cake was transferred under an inert atmosphere to a clean 100L glass reaction flask equipped with a mechanical stirrer, temperature probe and condenser. A9: 1 ethanol/water solution (30.7L) was added and the resulting slurry was heated under gentle reflux for at least 1 hour. The reaction mixture was then stirred for at least 12 hours while it was cooled to 15 ℃ to 30 ℃. The mixture was filtered and the filter cake was washed with ethanol (5.76L). The product was collected and dried under vacuum at 50 ℃. + -. 5 ℃ for at least 12 hours to give 5.63kg (58.1% yield) of (2S,3R) -3-amino-2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octane di-p-toluoyl-D-tartrate.

(2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Octane-3-yl) benzo Furan-2-carboxamides

(2S,3R) -3-amino-2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octane di-p-toluoyl-D-tartrate (3.64kg, 5.96mol) and 10% aqueous sodium chloride (14.4L, 46.4mol) were added to a 72L glass reaction flask equipped with a mechanical stirrer under an inert atmosphere. 5M sodium hydroxide (5.09L) was added to the stirred mixture to adjust the pH of the mixture to 14. The mixture was then stirred for at least 10 minutes. The aqueous solution was extracted with chloroform (4X12.0L), and the combined organic layers were dried over anhydrous sodium sulfate (1.72 kg). The combined organic layers were filtered and the filtrate was concentrated to give (2S,3R) -3-amino-2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octane (1.27kg) as an oil.

(2S,3R) -3-amino-2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octane is transferred under an inert atmosphere to a 50L glass reaction flask equipped with a mechanical stirrer. Methylene chloride (16.5L), triethylamine (847mL, 6.08mol), benzofuran-2-carboxylic acid (948g, 5.85mol) and O- (benzotriazol-1-yl) -N, N, N, 1-tetramethylammonium Hexafluorophosphate (HBTU) (2.17kg, 5.85mol) were added to the reaction mixture. The mixture was stirred at ambient temperature for at least 4 hours and the reaction was monitored by HPLC. After completion of the reaction, 10% aqueous potassium carbonate solution (12.7L, 17.1mol) was added to the reaction mixture and the mixture was stirred for at least 5 minutes. The layers were separated and the organic phase was washed with 10% brine (12.7L), the layers were separated and the organic phase was cooled to 15 ℃. + -. 10 ℃.3M hydrochloric acid (8.0L) was slowly added to the reaction mixture to adjust the pH of the mixture to 1. The mixture was then stirred for at least 5 minutes. The layers were allowed to separate for at least 5 minutes. The solids were filtered using a tabletop filter. The filtrates were separated into layers and the aqueous phase and the solid from the funnel were transferred to a reaction flask. 3M sodium hydroxide (9.0L) was slowly added in portions to the flask to adjust the pH of the mixture to 14. The aqueous phase was extracted with dichloromethane (2X 16.5L). The combined organic phases were dried over anhydrous sodium sulfate (1.71 kg). The mixture was filtered and the filtrate was concentrated to give (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octan-3-yl) benzofuran-2-carboxamide (1.63kg, 77.0% yield) as a yellow solid.

(2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Oct-3-yl]Benzofuran derivatives Pyran-2-carboxamide p-toluenesulfonate salt

Reacting (2S,3R) -N- (2- ((3-pyridyl) methyl) -1-azabicyclo [2.2.2]Octane-3-yl) benzofuran-2-carboxamide (1.62kg, 4.48mol) and methylene chloride (8.60kg) were added to a small glass vial. Weight/weight% of material in solution was determined by HPLC analysis. The solution was concentrated to an oil, acetone (4L) was added and the mixture was concentrated to an oily solid. Additional acetone (12L) was added to the oily solid in the rotary evaporator evaporation bulb and the resulting slurry was transferred under an inert atmosphere to a 50L glass reaction flask equipped with a mechanical stirrer, condenser, temperature probe and condenser. The reaction mixture was heated to 50 ℃. + -. 5 ℃. Water (80.7g) was added to the solution and it was stirred for at least 10 minutes. P-toluenesulfonic acid (853g, 4.44mol) was added in portions to the reaction mixture over about 15 minutes. The reaction mixture was heated to reflux and held at this temperature for at least 30 minutes to obtain a solution. The reaction was cooled to 40 ℃. + -. 5 ℃ over about 2 hours. Isopropyl acetate (14.1L) was added over about 1.5 hours. The reaction mixture was slowly cooled to ambient temperature over at least 10 hours. The mixture was filtered and the filter cake was washed with isopropyl acetate (3.5L). The isolated product was dried under vacuum at 105 ℃. + -. 5 ℃ for 2-9 hours to give 2.19kg (88.5% yield) of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Oct-3-yl) benzofuran-2-carboxamide p-toluenesulfonate, mp226-228 ℃.¹H NMR(500MHz，D₂O)δ8.29(s，1H)，7.78(m，J＝5.1，1H)，7.63(d，J＝7.9，1H)，7.54(d，J＝7.8，1H)，7.49(d，J＝8.1，2H)，7.37(m，J＝8.3，1H)，7.33(m，J＝8.3，6.9，1.0，1H)，7.18(m，J＝7.8，6.9，1.0，1H)，7.14(d，J＝8.1，2H)，7.09(s，1H)，6.99(dd，J＝7.9，5.1，1H)，4.05(m，J＝7.7，1H)，3.74(m，1H)，3.47(m，2H)，3.28(m，1H)，3.22(m，1H)，3.15(dd，J＝13.2，4.7，1H)，3.02(dd，J＝13.2，11.5，1H)，2.19(s，3H)，2.02(m，2H)，1.93(m，2H)，1.79(m，1H).¹³C NMR(126MHz，D₂O)δ157.2，154.1，150.1，148.2，146.4，145.2，138.0，137.0，130.9，128.2(2)，126.9，126.8，125.5(2)，123.7，123.3，122.7，111.7，100.7，61.3，50.2，48.0，40.9，33.1，26.9，21.5，20.8，17.0。

A sample of this material was converted to compound a free base (for salt selection studies) by treatment with aqueous sodium hydroxide and extraction with chloroform. The chloroform was evaporated thoroughly to give an off-white powder, mp167-170 ℃, with the following spectral characteristics: positive ion electrospray MS [ M + H [ ]]⁺Ion m/z = 362.¹H NMR(500MHz，DMSO-d₆)δ8.53(d，J＝7.6Hz，1H)，8.43(d，J＝1.7Hz，1H)，8.28(dd，J＝1.6，4.7Hz，1H)，7.77(d，J＝7.7Hz，1H)，7.66(d，J＝8.5Hz，1H)，7.63(dt，J＝1.7，7.7Hz，1H)，7.52(s，1H)，7.46(m，J＝8.5，7.5Hz，1H)，7.33(m，J＝7.7，7.5Hz，1H)，7.21(dd，J＝4.7，7.7Hz，1H)，3.71(m，J＝7.6Hz，1H)，3.11(m，1H)，3.02(m，1H)，2.80(m，2H)，2.69(m，2H)，2.55(m，1H)，1.80(m，1H)，1.77(m，1H)，1.62(m，1H)，1.56(m，1H)，1.26(m，1H).¹³C NMR(126MHz，DMSO-d₆)δ158.1，154.1，150.1，149.1，146.8，136.4，135.4，127.1，126.7，123.6，122.9，122.6，111.8，109.3，61.9，53.4，49.9，40.3，35.0，28.1，26.1，19.6。

The monohydrochloride salt of compound a (see example 5) was used for X-ray crystallography. The resulting crystal structures (shown in fig. 10A and 10B, respectively) confirm the absolute 2S,3R configurations of compound a.

Example 3: synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide phosphate

(2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] in round bottom flask]Oct-3-yl) benzofuran-2-carboxamide (8.18g, 22.6mmol) and 2-propanol (180 mL). The mixture was stirred and heated at 65-70 ℃ until all solids were dissolved. The solution was stirred vigorously at 65-70 ℃ and phosphoric acid (1.65mL, 24.3mmol) was added dropwise by pipette. A white granular solid formed immediately. The mixture was stirred at 65-70 ℃ for 30 minutes, cooled to ambient temperature (23 ℃) and stirred for another 24 hours. The white solid was collected by suction filtration, the filter cake was washed with 2-propanol (20mL) and the solid was air dried for at least 1 hour. The solid was further dried in a vacuum oven at 75 deg.C overnight (16 hours) to give 10.7g of the product (greater than quantitative yield), melting point 265 ℃ and 273 deg.C (decomposition), with a change in crystallinity observed at about 180 deg.C.¹H-NMR(DMSO-d₆) The presence of 2-propanol (a strong solvate) was shown, which could explain why the yield was greater than quantitative. Chiral LC analysis gave a purity of 97.1% (270 nm).

Example 4: synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide maleate

Maleic acid (0.067g, 0.630mmol) was added to (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]]In a hot slurry of oct-3-yl) benzofuran-2-carboxamide (0.203g, 0.560mmol) in isopropyl acetate (2mL) a fine white solid precipitated with a gummy residue. Additional isopropyl acetate (3mL) and maleic acid (0.006g) were added, the mixture heated to reflux and isopropanol (5mL) was added under reflux. The resulting white solid mixture was cooled to ambient temperature, filtered, and the solid was washed with isopropyl acetate (2 mL). The product was dried under vacuum at 60 ℃ for 18 hours to give 0.228g off-white flaky solid (84.7% yield), mp180-182 ℃.¹H NMR(DMSO-d₆) Shown is the single salt stoichiometry. C₂₂H₂₃N₃O₂C₄H₄O₄The calculated value of (a): c, 65.40, H, 5.70, N, 8.80; measured value: c, 65.35, 65.29, H, 5.86, 5.68, N, 8.69, 8.78.

Example 5: synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide hydrochloride

A monohydrochloride salt: a hydrochloric acid/THF solution was prepared by adding concentrated hydrochloric acid (1.93mL, 12M, 23.2mmol) dropwise to 8.5mL of cold THF. The solution was warmed to ambient temperature. (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] in round bottom flask]Oct-3-yl) benzofuran-2-carboxamide (8.49g, 23.5mmol) and acetone (85 mL). The mixture was stirred and heated at 45-50 ℃ until complete dissolution was achieved. The hydrochloric acid/THF solution prepared above was added dropwise over 5 minutes, using additional THF (1.5mL) for transfer. A granular white solid began to form during the addition of the acidic solution. The mixture was cooled to ambient temperature and stirred overnight (16 hours). The solid was collected by suction filtration, the filter cake was washed with acetone (10mL), and the solid was air dried under suction for 30 minutes. The solid was dried in a vacuum oven at 75 ℃ for an additional 2 hours to give 8.79g of fine white crystals (94% yield), mp255-262 ℃. Chiral LC analysis gave a purity of 98.8% (270 nm).¹H-NMR(DMSO-d₆) Showing no residual solvent and confirming single stoichiometry.¹H NMR(300MHz，DMSO-d₆) Δ 10.7 (broad s, 1H-quat), 8.80 (broad s, 1H-amide H), 8.54(s, 1H), 8.23(d, 1H), 7.78(d, 1H), 7.74(d, 1H), 7.60(d, 1H), 7.47(m, 2H), 7.33(m, 1H), 7.19(m, 1H), 4.19(m, 1H), 4.08(m, 1H), 3.05-3.55(m, 6H), 2.00-2.10(m, 3H), 1.90(m, 1H), 1.70(m, 1H). X-ray crystallographic analysis of the salt confirmed stereochemical assignment and stoichiometry (see fig. 10A and 10B).

Dihydrochloride salt: slowly bubbling hydrogen chloride gas through (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Oct-3-yl) benzofuran-2-carboxamide (1.9g, 5.3mmol) in ice-cooled solution in anhydrous ether (25 mL). Volatiles were removed first in a nitrogen gas stream and then using a high vacuum (sodium hydroxide scrubber in a high vacuum tube). The residue was triturated with a small volume of anhydrous ether (discarded) and the remaining solid was dried under high vacuum. 2.17g (94% yield) of an off-white powder are obtained, mp210-212 deg.C (hygroscopic). Chiral LC analysis gave a purity of 93.7% (270 nm). Positive ion electrospray MS [ M + H [ ]]⁺Ion m/z = 362.¹H NMR(300MHz，CD₃OD)δ9.15(s，1H)，8.84(d，1H)，8.63(d，1H)，7.97(t，1H)，7.75(d，1H)，7.61(d，1H)，7.52(m，2H)，7.35(t，1H)，4.50(m，1H)，4.32(m，1H)，3.40-3.85(m，6H)，1.95-2.40(m，5H)。

Example 6: synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide hemi-galactaric acid salt

Galactaric acid (mucic acid) (36.3mg, 0.173mmol) was added to (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Oct-3-yl) benzofuran-2-carboxamide (125mg, 0.346mmol) in hot ethanol (1 mL). The mixture was refluxed while water (8 drops) was added; the hot mixture was then filtered through a cotton plug, which was subsequently rinsed with ethanol (1 mL). Cooling did not yield a precipitate. Volatiles were removed by rotary evaporation, the residue (white foam) was triturated with isopropanol (discarded), and the remaining solid was dissolved in refluxing acetone/water (4mL, 7: 1). Cooling slowly to 5 ℃ produced a white solid which was filtered off, washed with isopropanol (3X 1mL) and dried under high vacuum. This gave 118mg (73% yield) of a fine white flake, mp134-139 ℃.¹H NMR(300MHz，D₂O) Δ 8.29(s, 1H), 7.78(d, 1H), 7.62(d, 1H), 7.54(d, 1H), 7.35(m, 2H), 7.18(t, 1H), 7.10(s, 1H), 6.98(m, 1H), 4.08(s, 1H, galactaric acid), 3.98 (m,1H)(d, 1H), 3.77(s, 1H, galactaric acid), 3.66(m, 1H), 3.35(m, 1H), 2.95-3.30(m, 4H), 1.65-2.05(m, 5H).

Example 7: synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide D-tartrate

Tartaric acid (25.6mg, 0.173mmol) was added to (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]]Oct-3-yl) benzofuran-2-carboxamide (125mg, 0.346mmol) in hot ethanol (1 mL). The resulting solution was slowly cooled to ambient temperature. No solid precipitated out, so the solution was concentrated to give a white foam. Attempts to crystallize in isopropanol ended up in failure. The foam was dissolved in methanol and the other half equivalent of tartaric acid (25.6mg, 0.173mmol) was added. The mixture was concentrated to give a white foam which did not crystallize from a mixture of methanol and isopropanol. The concentrated material (mixture of solid and gummy liquid) was then slurried in ethyl acetate (1mL) to give a white solid. It was isolated by filtration (washing with ethyl acetate) and dried in a vacuum oven (18 h at 40 ℃ C.) to give 141mg (79.7% yield) of the mono-stoichiometric salt (NMR), mp136-140 ℃. Chiral LC analysis gave a purity of 98.1% (270 nm).¹H NMR(300MHz，D₂O) δ 8.50(s, 1H), 8.01(d, 1H), 7.86(d, 1H), 7.75(d, 1H), 7.56(m, 2H), 7.38(t, 1H), 7.32(s, 1H), 7.21(t, 1H), 4.34(s, 2H, tartaric acid), 4.26(d, 1H), 3.95(m, 1H), 3.64(m, 2H), 3.15-3.55(m, 4H), 1.90-2.30(m, 5H).

Example 8: synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide mesylate

Methanesulfonic acid (33.2mg, 0.346mmol) was added to (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]]Octin-3-yl) benzofuran-2-carboxamide (125mg, 0.346mmol) in hot ethanol (1 mL). Cooling did not produce a precipitate. The mixture was refluxed and the hot mixture was filtered through a cotton plug, which was then rinsed with methanol (1 mL). Volatiles were removed by rotary evaporation and the residue (pale yellow foam) was dissolved in hot isopropanol (1 mL). Again, cooling did not produce a precipitate. The isopropanol was evaporated and the residue was slurried in acetone (1 mL). Filtered and dried in a vacuum oven (18 hours at 50 ℃) to give 146mg (92.5% yield) of a pale beige solid, mp240-243 ℃.¹H NMR(300MHz，D₂O) delta 8.32(s, 1H), 7.82(d, 1H), 7.66(d, 1H), 7.57(d, 1H), 7.38(m, 2H), 7.20(m, 1H), 7.12(s, 1H), 7.01(m, 1H), 4.09(d, 1H), 3.75(m, 1H), 3.47(m, 2H), 3.00-3.40(m, 4H), 2.67(s, 3H, methanesulfonic acid), 1.75-2.15(m, 5H).

Example 9: synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide D-mandelate salt

D-mandelic acid (52.6mg, 0.346mmol) was added to (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [ 2.2.2)]Oct-3-yl) benzofuran-2-carboxamide (125mg, 0.346mmol) in hot ethanol (1 mL). Dilution with ethyl acetate (4ml) and cooling did not produce a precipitate. Volatiles were removed by rotary evaporation and the residue (white foam) was dissolved in hot isopropanol (0.5 mL). Cooling to 5 ℃ produced white crystals which were collected by suction filtration. Drying in a vacuum oven (18 h at 45 ℃) gives 111mg (62.4% yield) of a pale beige solid, mp188.5-193 ℃.¹H NMR(300MHz，D₂O) delta 8.33(s, 1H), 7.83(s, 1H), 7.67(d, 1H), 7.60(d, 1H), 7.27(m, 8H, including mandelic acid), 7.12(s, 1H), 7.01(m, 1H), 4.85(s, 1H, mandelic acid), 4.10(d, 1H), 3.75(m, 1H), 3.48(m, 2H), 3.00-3.40(m, 4H), 1.75-2.15(m, 5H).

Example 10: synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide R-camphorsulfonate

R-10-Camphorsulfonic acid (80.3mg, 0.346mmol) was added to (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Oct-3-yl) benzofuran-2-carboxamide (125mg, 0.346mmol) in hot ethanol (1 mL). Cooling did not deposit any precipitate. Volatiles were removed by rotary evaporation and the residue (white foam) was dissolved in hot isopropanol (0.5 mL). Cooling to 5 ℃ produced little white crystals and a milky suspension. The flask was scraped at its side walls with a spatula to finally convert the mixture into a thick mass of fine white crystals. An additional 0.5mL of isopropanol was added and the crystals were collected by suction filtration. Drying in a vacuum oven (at 70 ℃ for 5 hours, then at 110 ℃ for 2 hours) gave 193mg (93.8% yield) of a white solid. mp149.5-156 ℃.¹H NMR(300MHz，D₂O) δ 8.30(s, 1H), 7.79(d, 1H), 7.64(d, 1H), 7.55(d, 1H), 7.36(m, 2H), 7.18(m, 1H), 7.11(s, 1H), 6.99(m, 1H), 4.07(d, 1H), 3.73(m, 1H), 3.45(m, 2H), 3.95-3.35(m, 5H, including camphorsulfonic acid), 2.64(d, 1H, camphorsulfonic acid), 2.22(m, 2H), 1.70-2.10(m, 8H, including camphorsulfonic acid), 1.45(m, 1H, camphorsulfonic acid), 1.25(m, 1H, camphorsulfonic acid), 0.85(s, 3H, camphorsulfonic acid), 0.68(s, 3H, sulfonic acid).

Example 11: synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide S-camphorsulfonate

S-10-Camphorsulfonic acid (80.3mg, 0.346mmol) was added to (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Octyl-3-yl) benzofuran-2-carboxamide (125mg, 0.346mmol) in hot ethanol (1 mL). Dilution with ethyl acetate (4mL) did not precipitate any precipitate on cooling. Volatiles were removed by rotary evaporation and the residue (white foam) was dissolved in hot isopropanol (1.5 mL). Cooling to 5 ℃ produced white crystals. The mixture was concentrated to about 0.5mL and cooled again to 5 ℃. Then is filtered and collected by suction filtrationThe solids were collected and dried initially under vacuum at 45 ℃ for 18 hours, then under vacuum at a sustained higher temperature (eventually 110 ℃) to remove residual isopropanol. This gives 143mg (69.7% yield) of a white solid, mp153.5-157 ℃.¹H NMR(300MHz，D₂O) δ 8.29(s, 1H), 7.79(d, 1H), 7.63(d, 1H), 7.54(d, 1H), 7.34(m, 2H), 7.18(m, 1H), 7.10(s, 1H), 6.99(m, 1H), 4.05(d, 1H), 3.73(m, 1H), 3.44(m, 2H), 3.95-3.35(m, 5H, including camphorsulfonic acid), 2.67(d, 1H, camphorsulfonic acid), 2.23(m, 2H), 1.70-2.10(m, 8H, including camphorsulfonic acid), 1.46(m, 1H, camphorsulfonic acid), 1.25(m, 1H, camphorsulfonic acid), 0.84(s, 3H, camphorsulfonic acid), 0.64(s, 3H, sulfonic acid).

Several other salt forms were characterized using procedures similar to those described above (examples 3-11). The results of these preparations are described in examples 12-14.

Example 12: synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide sulfate

The sulfate salt precipitates out of the mixture of isopropyl acetate and water. MP278 ℃.¹HNMR(400MHz，DMSO-d₆) Δ 9.28 (broad s, 1H, amide), 8.56(dd, 1H), 8.24(t, 1H), 7.77(d, 1H), 7.74(d, 1H), 7.60(s, 1H), 7.40(m, 2H), 7.35(s, 1H), 7.21(m, 1H), 4.21(m, 1H), 3.93(m, 2H), 3.10-3.60(m, 5H), 2.05(m, 3H), 1.92(m, 1H), 1.73(m, 1H).

Example 13: synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide ketoglutarate

Alpha-ketoglutarate is precipitated from isopropyl acetate. MP177 ℃.¹H NMR(400MHz，DMSO-d₆) Δ 8.64(s, 1H, amide), 8.50(d, 1H), 8.20(d, 1H), 7.74(d, 1H)) 7.70(d, 1H), 7.60(m, 1H), 7.45(m, 1H), 7.32(m, 2H), 7.18(m, 1H), 4.10(m, 1H), 3.78(m, 2H), 3.00-3.45(m, 5H), 2.81(m, 2H, ketoglutaric acid), 2.41(m, 2H, ketoglutaric acid), 1.96(m, 3H), 1.83(m, 1H), 1.60(m, 1H).

Example 14: synthesis of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide hippurate

Hippurate precipitates out of acetone (hygroscopic too strongly to obtain a melting point).¹H NMR(400MHz，DMSO-d₆) δ 8.79(s, 1H, amide), 8.56(d, 1H), 8.44(s, 1H, hippuric acid), 8.29(m, 1H), 7.87(m, 2H, hippuric acid), 7.76(d, 1H), 7.65(m, 1H), 7.54(m, 1H), 7.49(m, 4H, including hippuric acid), 7.34(m, 2H), 7.21(m, 1H), 3.91(m, 1H), 3.74(m, 2H), 3.00-3.50(m, 5H), 2.80(m, 2H, hippuric acid), 1.79(m, 2H), 1.60(m, 2H), 1.30(m, 1H).

Example 15: isolation and conversion of (2R,3R) -and (2S,3S) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide to galactaric acid salt

(2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] by rotary evaporation]Oct-3-yl]A sample of the supernatant obtained from benzofuran-2-carboxamide tosylate (example 2) was concentrated, adjusted to pH10 with 10% aqueous sodium hydroxide and extracted with dichloromethane. The dichloromethane extract was evaporated and the residue (1.8g) was dissolved in absolute ethanol (55mL) containing 0.5% di-n-butylamine. The solution was injected at 0.25mL per serving to 25 cm. times.2.1 cmAD chiral HPLC column, eluting with 60:40:0.2 hexane/ethanol/di-n-butylamine (flow =30mL/min), monitoring at 270 nm. Isolate the eluate eluted at-7.5 minutesAnd the eluate eluted at-13.5 minutes, to give, after evaporation of the solvent, 0.48g (98% chiral purity) and 0.47g (99% chiral purity), respectively, of a colorless oil. Both NMR spectra were identical.¹H NMR(300MHz，CDCl₃)δ8.49(s，1H)，8.45(d，1H)，7.74(d，1H)，7.52(m，4H)，7.35(t，1H)，7.20(dd，1H)，7.05(d，1H)，4.55(dt，1H)，3.43(m，1H)，3.22(m，1H)，2.90(m，5H)，2.09(m，1H)，1.88(m，4H)。

Each warm solution of the free base sample in absolute ethanol (10mL) was treated with one equivalent of galactaric acid. The resulting mixture was heated at 75 ℃ for 5 minutes with stirring and cooled to ambient temperature. The resulting solid was collected by suction filtration and dried in vacuo to give 0.65g (87% yield) and 0.62g (85% yield) of a white granular solid (mp 200-205 ℃ C. in each case).¹H NMR(300MHz，D₂O)δ8.38(s，1H)，8.28(d，1H)，7.94(d，1H)，7.70(d，1H)，7.59(d，1H)，7.48(t，1H)，7.40(m，1H)，7.32(m，2H)，4.42(m，1H)，4.21(s，2H)，3.87(s，2H)，3.68(m，1H)，3.35(m，6H)，2.25(m，2H)，2.02(m，3H)。

Example 16: synthesis of (2R,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide p-chlorobenzoate

Solid p-chlorobenzoic acid (46.8mg, 0.299mmol) was added in one portion to the previously eluted N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] from example 15]Oct-3-yl]Benzofuran-2-carboxamide isomer (108mg, 0.299mmol) in acetone (10 mL). The mixture was warmed to near reflux for 30 minutes and cooled to ambient temperature. No precipitate formed, so the solution was concentrated to about 20% of its original volume (hot plate), at which point crystallization began to form. The mixture was cooled and diluted with isopropanol (2 mL). The mixture was concentrated by slowly evaporating the solvent at ambient temperature, and the resulting solid was collected and dried. This gave 145mg (94% yield) of pale yellow crystals, mp150-152 ℃.¹H NMR(300MHz，CDCl₃) Δ 8.49(s, 1H), 8.38(d, 1H), 7.93(d, 2H, p-chlorobenzoic acid), 7.67(m, 2H), 7.57(d, 1H), 7.45(m, 1H), 7.36(d, 2H, p-chlorobenzoic acid), 7.30(m, 1H), 7.27(s, 1H), 7.16(m,1H), 7.00(d, 1H, amide), 6.90 (broad s, quaternary ammonium), 4.62(m, 1H), 3.85(dd, 1H), 3.36(m, 1H), 2.95-3.25(m, 5H), 2.16(s, 1H), 1.70-2.10(m, 4H).

X-ray crystallography analysis of this sample revealed that its absolute stereochemistry was 2R,3R (see fig. 11A and 11B). The latter eluted isomer in example 15 thus has the absolute configuration 2S,3S by exclusion.

Example 17: chiral chromatographic method for stereoisomer analysis

Obtaining a chiral chromatographic method for separating the four stereoisomers from each other has proven to be very challenging. Initial attempts (using hexane/isopropanol/triethylamine mobile phase) resulted in peaks overlapping and sub-optimal peak shapes. Changing isopropanol to ethanol and triethylamine to di-n-butylamine improves resolution and peak shape and shortens run time. The method is described in detail as follows:

and (3) analyzing the column:AD(250×4.6mm，5μm)

mobile phase: 60:40:0.2 Hexane/ethanol/di-n-butylamine

Injection volume: 10 μ l

Flow rate: 1.0 ml/min

Temperature: 20 deg.C

And (3) detection: UV at 270nm

Total run time: 25 minutes

Elution order (RT): 2S,3R (5.3 min); 2R,3S (7.3 min); 2R,3R (8.3 min); 2S,3S (12.1 min)

Representative chromatograms of the stereoisomeric analogs are shown in figure 12.

Example 18: XRPD

XRPD analysis was performed on several salt samples described in this specification. Diffraction patterns for the hydrochloride (fig. 13) and tosylate (fig. 14) salts are provided.

X-ray powder diffraction (XRPD)

X-ray powder diffraction patterns were collected from one or both instruments. Some patterns were collected on a Siemens D5000 diffractometer using CuK α radiation (40kV, 40mA), a θ - θ goniometer, V20 divergence and acceptance slit, a graphite secondary monochromator and scintillation counter. A performance check of the instrument was performed using a certified emery standard (NIST 1976). The run samples were made into flat plate samples using the as received powder under ambient conditions. Approximately 35mg of the sample was lightly packed into a chamber cut into ground zero-background (510) silicon wafers. During analysis the sample was rotated in its own plane, scanning in 0.05 ° steps ranging from 2 ° to 42 °, 4 seconds each, using CuK α 1

Some X-ray powder diffraction patterns were collected on a Bruker AXS C2GADDS diffractometer using CuK alpha radiation (40kV, 40mA), an automated XYZ stage, a laser video microscope for sample auto-positioning and a HiStar 2-dimensional area detector. The X-ray optical system comprises a single pinhole collimator connected with a 0.3mm pinhole collimatorA multilayer mirror. The beam divergence (i.e. the effective size of the X-ray beam on the sample) is about 4 mm. Using a theta-theta continuous scanning mode, adopting a sample-detector distance of 20cm,this results in an effective 2 theta range of 3.2 deg. -30.0 deg.. Typically, the sample will be exposed to the X-ray beam for 120 seconds. The run samples were made into flat plate samples using as-received powder without milling under ambient conditions. Approximately 1-2mg of the sample was gently pressed onto the silicon wafer to obtain a flat surface. The run samples were placed on a silicon wafer with a thermally conductive compound under non-ambient conditions. The sample was then heated to the appropriate temperature at approximately 10 deg.c/minute and then held isothermally for about 5 minutes before data collection was initiated.

Differential Scanning Calorimetry (DSC)

DSC data were collected on a TA instrument Q1000 equipped with a 50-bit autosampler. The instrument was energy and temperature calibrated using certified indium. Typically, 0.5-1.5mg of each sample in a pinhole aluminum pot was heated at 10 deg.C/min from 25 deg.C to 175-200 deg.C. A30 mL/min nitrogen purge was maintained over the sample.

Thermogravimetric analysis (TGA)

TGA data were collected on a TA instrument Q500TGA equipped with a 16-position autosampler. The instrument was temperature calibrated using certified Alumel. Typically, 5-10mg of each sample was loaded into a pre-tared platinum crucible and aluminum DSC pan and heated from ambient temperature to 350 ℃ at 10 ℃/min. A nitrogen purge of 60mL/min was maintained over the sample.

Polarized Light Microscopy (PLM)

The samples were studied on a Leica LM/DM polarized light microscope with a digital camera for capturing images. A small amount of each sample was placed on a slide, placed in oil immersion and covered with a coverslip to separate as well as possible the individual particles. The sample was observed using appropriate magnification and partially polarized light coupled to a lambda false color filter.

Hot Stage Microscopy (HSM)

Hot stage microscopy was performed using a Leica LM/DM polarized light microscope combined with a Mettler-Toledo MTFP82HT hot stage and a digital camera for capturing images. A small amount of each sample was placed on the slide so that the individual particles were as well separated as possible. The sample was observed using appropriate magnification and partially polarized light connected to a lambda pseudo-optical filter while heating from ambient temperature at typically 10 deg.c/min.

Gravimetric vapor adsorption (GVS)

Adsorption isotherms were determined on one or two instruments. Some samples were tested using a VTI Corporation SGA-100 moisture sorption analyzer under the control of VTIFlowSystem4 software. The sample temperature was maintained at 25 ℃ by means of a Polyscience thermostatic bath. Humidity is controlled by mixing dry and wet nitrogen streams. Changes in weight as a function of% RH were monitored using Cahn digital recording Balance D-200 with an accuracy of +/-0.0001 g.

Typically, 10-20mg of the sample is placed in a tared balance pan under ambient conditions. The sample was dried at 50 ℃ for 1 hour. The standard adsorption isotherms were plotted at 25 ℃ in the range of 5-95% RH at 5% RH intervals and similarly the desorption isotherms were plotted at 25 ℃ in the range of 95-5% RH at 5% RH intervals. For each% RH data point, the sample equilibration standard comprises 0.0100wt% within a maximum equilibration time of 5 minutes or 180 minutes.

Several adsorption isotherms were obtained using a Hiden IGASorp moisture adsorption analyzer under the control of CFRSorp software. The sample temperature was maintained at 25 ℃ by means of a Huber recirculating water bath. Humidity was controlled by mixing dry and wet nitrogen streams, using a total flow rate of 250 mL/min. The relative humidity was measured by a calibrated Vaisala RH probe (dynamic range 0-95% RH) located near the sample. The weight change (mass loss) of the samples relative to RH% was monitored uninterruptedly by means of a microbalance (accuracy ± 0.001 mg). Typically, 10-20mg of the sample is placed in a tared grid stainless steel basket at ambient conditions. The loading and unloading of the samples was performed at 40% RH and 25 ℃ (typical ambient conditions). Moisture sorption isotherms were plotted as described below (2 scans gave 1 complete cycle). Standard isotherms were plotted at 25 ℃ at 10% RH intervals over a range of 0-90% RH.

GVS general method parameters

Parameter(s)	Value of
		Adsorption-scanning 1	40-90
Desorption/adsorption-Scan 2	85-Dry, Dry-40
		Interval (% RH)	10
Number of scans	2
		Flow rate (mL/min)	250
Temperature (. degree.C.)	25
		Stability (. degree. C./min)	0.05
Minimum adsorption time (hours)	1
		Maximum adsorption time (hours)	4
Mode(s)	AF2
		Precision (%)	98

The software uses a least squares minimization process and a mass mitigation model to predict the asymptotic value. The measured mass loss value must be within 5% of the predicted value for the software and then the next RH% value is selected. The minimum equilibration time was set to 1 hour and the maximum equilibration time was set to 4 hours. Typically, the sample is recovered after completion of the isotherm and analyzed again by XRPD.

Water measurement by Karl Fischer method (KF)

The water content of each sample was measured on a Mettler ToledoDL39 coulometer using Hydranal Coulomat AG reagent and argon purge. The weighed solid sample was introduced onto a platinum TGA tray inside the container, which was attached to the subaeal to avoid water ingress. Approximately 10mg of sample was used for each titration and duplicate determinations were performed.

Determination of thermodynamic aqueous solubility by HPLC

Aqueous solubility was determined by suspending enough compound in 0.25mL of water to obtain the maximum final concentration of the parent-free form of the compound ≧ 10 mg/mL. The suspension was equilibrated at 25 ℃ for 24 hours, and then the pH was measured. The suspension was then filtered through a glass fiber C filter into a 96-well plate. The filtrate was then diluted 101-fold. HPLC quantification was performed relative to a standard solution of about 0.1mg/mL in DMSO. Different volumes of standards, diluted and undiluted sample solutions were injected. The solubility was calculated using the peak area determined by integrating the peaks found at the same retention time as the main peak in the standard injection. XRPD was collected if there was sufficient solids in the filter plate.

Detailed description of the general methods for thermodynamic water solubility methods

Determination of chemical purity by HPLC

Purity analysis was performed on an Agilent HP1100 tandem system equipped with a diode array detector and using ChemStation software v 9. One of two methods detailed below was used.

Method 1

Method 2

Ion chromatography

Data were collected on a Metrohm861Advanced Compact IC using IC Net software v 2.3. The samples were prepared as 1000ppm mother liquor in water. When the solubility of the sample is low, a suitable co-solvent, such as DMSO, is used. Prior to testing, the samples were diluted to 50ppm or 100ppm using a suitable solvent. Quantitative determinations are made by comparison to standard solutions of known concentrations of the ions being analyzed.

Ion chromatography of anions

Cation ion chromatography

About 50mg of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide hydrochloride are weighed in a glass vial and heated to 50 ℃. To the solid was added a 100 μ l aliquot of 1-butanol/water (5 vol% water) until a clear solution (500 μ l total) was formed. The sample was stirred at 50 ℃ for 1 hour and observed. After heating at 50 ℃ for 1 hour, the sample was still a clear solution and was cooled from 50 ℃ to 25 ℃ at a rate of 1.4 ℃/hour. The sample remained a clear solution when cooled and was covered with a pinhole parafilm, which was allowed to evaporate at ambient temperature. After 2 weeks, large crystals were visible in the partially evaporated samples. FIG. 13 is an XRPD of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide monohydrochloride showing the observed pattern (lighter) and the calculated pattern (darker).

Experimental patterns were obtained for samples of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide monohydrochloride, while the calculated examples were obtained from the single crystal X-ray structure described in this specification and are depicted in fig. 10A and 10B. Both graphs are consistent in terms of 2 θ values, and slight differences in intensity and peak width are attributable to the effects of instrument resolution and preferred orientation. In addition, minor differences can be attributed to temperature variations due to observed data collected at room temperature and calculated data collected for structures at 120K.

The diffraction patterns of the tosylate salt, particularly the crystalline monosalt, confirmed and obtained using CuK α radiation (40kV, 40mA), θ - θ goniometer, V20 divergence and acceptance slit, graphite secondary monochromator and scintillation counter are shown in fig. 14. The XRPD diffractogram of the tosylate at 40 ℃/75% RH over 1 week revealed a change, but the sample was still form 1. This change is likely due to a greater degree of hydration of the form.

Biological assay

The ability of compound a and its stereoisomers to bind and modulate the function of various NNR subtypes is assessed as described in us patent 6,953,855 to Mazurov et al, the contents of which are incorporated herein by reference. Receptor-selective characterization of Compound A (including 5 HT)₃Receptors and muscarinic receptors) fromBiosciences Corporation.

Electrophysiological measurements of α 7NNR responses were performed within two expression systems: rat α 7NNR in mammalian GH4C1 cells and human α 7NNR in Xenopus (Xenopus) oocytes.

GH4C1 cells expressing rat α 7NNR were prepared as described in Placzek et al, mol.Pharm.68(6):1863-1876(2005), which is incorporated herein by reference. Electrophysiological measurements of agonist activity were achieved using this GH4C1 cell expression system using a hydrodynamic rapid perfusion system and patch clamp. Both acetylcholine and nicotine produce concentration-dependent activation of the current mediated by α 7. Literature-derived agonists EC₅₀Values and those EC obtained using this method₅₀The values are comparable (see Dunlop et al biochem pharmacol mercaptal (2007) and Dynaflow online materials (www.cellectricon.com), each incorporated by reference for this method).

The whole cell current recorded with the Axopatch700A amplifier was filtered at 1kHz and sampled at 5kHz using a PCI card (National Instrument). The saline solution was adjusted as described to increase current stability compared to previous studies. Cells were recorded at room temperature in the following extracellular medium: 130mM NaCl, 5mM KCl, 2mM CaCl₂，2mM MgCl₂10mM HEPES, adjusted to pH7.4 with aqueous NaOH. Borosilicate electrodes (3-5 M.OMEGA.) were filled with the following medium: 130mM TRIS phosphate, 5mM NaCl, 2mM MgCl₂10mM HEPES, 10mM EGTA, adjusted to pH7.4 with aqueous KOH (see, Wu et al, J. physiol.576:103-118(2006), which is incorporated by reference for this teaching). Under these conditions, the high current activity obtained using NNR whole cell recordings lasted up to 60 minutes when primed with 1000 μ M acetylcholine (ACh) concentration.

Annotated cell processing programs were applied using Cellecricon from Dynaflow. Briefly, after the cells are removed from the incubator, the cells are washed three times thoroughly with recording media and placed on the platform of an inverted Zeiss microscope. An average of 5 minutes is necessary before establishing the whole-cell record structure. To avoid changing cell conditions, single cells loaded per single cell were recorded into Dynaflow silicon wafers. No differences between the fractions of responsive cells could be detected under the experimental conditions. Greater than 95% of cells respond to ACh and all cells that provide a measurable current are considered. Cells were maintained at-60 mV throughout the experiment. All test article solutions were prepared daily from stock solutions. Fresh stock solutions of acetylcholine (ACh) were prepared daily in ringer's solution and diluted. Dose response curves were plotted by a single Hill equation using prism5.0 software.

Xenopus oocytes expressing human α 7NNR were prepared as described by Papke and Papke Brit.J.Pharmacol.137:49-61(2002), which are incorporated herein by reference. Mature (>9cm) female Xenopus laevis (Xenopus laevis) Xenopus laevis (Nasco, ft. atkinson, WI) was used as the source of oocytes. Before surgery, the animals were anesthetized by placing them in a solution of 1.5g/L ethyl-3-aminobenzoate for 30 minutes. Oocytes were removed from incisions made in the abdomen.

To remove the follicular cell layer, at room temperature, in Barth's solution (88mM NaCl, 1mM KCl, 15mM HEPES pH7.6, 0.81mM Mg) without calciumSO₄，2.38mMNaHCO₃0.1mg/mL gentamicin sulfate), the harvested oocytes were treated with 1.25mg/mL collagenase from Worthington Biochemical Corporation (Freehold, NJ) for 2 hours. Then, stage 5 (stage) oocytes were isolated and injected with human α 7cRNA at 50nL (5-20ng) each. Recordings were made on days 2-7 post-injection. Stock solutions of fresh acetylcholine (ACh) were prepared daily in ringer's solution.

Experiments were performed using an OpusXpress6000A (Axon Instruments, Union City CA). OpusXpress is an integrated system providing automated piercing and voltage clamping of up to eight oocytes in parallel. Both voltage and current electrodes are filled with 3M KCl. The cells were subjected to a voltage clamp test at a holding potential of-60 mV. Data were collected at 50Hz and filtered at 20 Hz. Cells were bath-perfused with ringer's solution and agonist solution was released from the 96-well plate with the aid of a disposable tip, which eliminated the possibility of any cross-contamination. The flow rate was set to 2 ml/min. Drug administration was alternated between ACh control and experimental agonist. Application is of a duration of 12 seconds followed by a rest period of 181 seconds.

The response of the α 7 receptor was calculated as net charge (see, Papke and Papke, brit.j. pharmacol.137:49-61(2002) as described above). Each oocyte received an initial control application of ACh, followed by an experimental drug application, followed by a subsequent control application of ACh (300 μ M). The response to the experimental drug application was calculated relative to the last ACh control response to normalize the data to compensate for varying channel expression levels between oocytes. Note that 300 μ M ACh stimulated the maximum net charge response from the α 7 receptor, so that normalization to the ACh control effectively normalized the data to ACh maximum response. Mean and Standard Error (SEM) were calculated from the normalized responses of at least four oocytes at the concentration of each experiment. For the concentration-response relationship, data from the net charge analysis was plotted using Kaleidagraph3.0.2(Abelbeck software; Reading, Pa.), and curves were generated from the Hill equation.

Behavioral characterization of compound a was performed according to the following protocol. The object identification (OR) task is performed according to the instructions of Behav. brain Res.100:85-92(1988) by Ennaceur and Delaour, which are incorporated herein by reference. The Radial Arm Maze (RAM) model was developed in accordance with the teachings of Levin et al, Behav.Pharm.10:675-680(1999), which is incorporated herein by reference. The prepulse inhibition (PPI) assay was performed according to the specification of Brit.J.Pharmacol.142(5):843-850(2004) of Suemaru et al. Reversal of apomorphine-induced locomotor activity (APO LOCO) was performed according to the instructions of Curr.

Summary of in vitro biological Activity

Compound a competitively inhibited the binding of radiolabeled MLA to rat brain hippocampal α 7NNR with an equilibrium constant (Ki) of-1 nM, showing that it has very high affinity for the α 7NNR subtype. The stereoisomer of compound a had the following Ki values at rat α 7 NNR: 2R,3S (42nM) [ previously reported as 28nM ]; 2R,3R (1 nM); 2S,3S (25. mu.M) (see FIG. 1A). As shown in fig. 1a2, the 2S,3R enantiomer of compound a showed opposite activity at the α 7 subtype compared to the other three enantiomeric analogs, which were provided as overlapping spots with weak activity. Compound a binds to α 4 β 2NNR without any significant affinity (Ki value >2 μ M).

The patch-clamp electrophysiological technique using rat α 7NNR stably expressed in GH4C1 (mammalian) cells examined (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Functional activity of oct-3-yl) benzofuran-2-carboxamide or a pharmaceutically acceptable salt thereof (compound a) and stereoisomers thereof. In these experiments, compound a gave significantly different functional properties compared to the other individual isomers and the racemic mixture of all four isomers. As can be seen from FIGS. 1A and 1B, Compound A is responsible for eliciting a functional response (E)_max=93% relative to acetylcholine (ACh); EC (EC)₅₀=14nM) has greater efficacy and is more effective than either of the other isomers or the mixture of the four isomers. In fact, compound A (2S,3R isomer)) Is N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Oct-3-yl) benzofuran-2-carboxamide is the only isomer that provides potent agonism over the entire concentration range of 1-50nM, using 10nM associated with in vivo activity described in this specification, as shown in fig. 1B.

The functional activity of compound a was also assessed electrophysiologically in xenopus oocytes transiently expressing human α 7 NNR. In this system, Compound A has an EC of 33nM₅₀E with 100% ACh response_max. At an administration concentration of greater than 100nM (IC)₅₀(2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [ 2.2.2) of =200nM]Oct-3-yl) benzofuran-2-carboxamide followed by a subsequent increase in control response to ACh. In contrast to the previously described alpha 7 full agonists (see Astles et al, Current Drug Targets CNS neurological disorders1(4):337-348(2002), which is incorporated herein by reference for this report), (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [ 2.2.2)]EC of oct-3-yl) benzofuran-2-carboxamide₅₀And IC₅₀The difference between indicates that the concentration that produces half the maximal functional response of α 7 results in minimal, but not complete, residual inhibition. When (2S,3R) -N- (2- ((3-pyridyl) methyl) -1-azabicyclo [2.2.2]Oct-3-yl) benzofuran-2-carboxamide has no detectable activation when administered to oocytes expressing human α 4 β 2 subtype and no significant decrease in subsequent control response to ACh, indicating (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2]Oct-3-yl) benzofuran-2-carboxamide is neither an agonist nor an antagonist at α 4 β 2.

The compounds show little or no agonist activity in functional models carrying muscle-type receptors (the α 1 β 1 γ δ subtype in human TE671/RD clone cells), or ganglion-type receptors (the α 3 β 4 subtype in rat pheochromocytoma PC12 cells and in human SHSY-5Y clone cells), producing nicotinic responses at these subtypes of 10% (human muscle), 20% (rat ganglion), and 10% (human ganglion). These data show that selectivity for CNS subtypes is higher than for PNS subtypes.

Due to the presence of alpha 7 and 5-hydroxytryptamine (5 HT)₃) The close sequence and structural homology between the receptors, as well as the cross-reactivity observed with other nicotinic ligands to the two receptors, examined compound a for 5HT₃The affinity of the receptor. Compound A (10. mu.M) was shown to be 5HT in mice₃59% inhibition of radioligand binding at the receptor and 25% inhibition of radioligand binding at the human receptor. For human 5HT₃Examination of functional activation at the receptor suggests a minimum to no activation (i.e., a maximum response of 15% activation at 100 μ M).

Muscarinic receptors are another area of interest due to the interactions that have been observed with other nicotinic ligands. When examined in competitive binding inhibition assays for M1, M2, non-selective central muscarinic receptors and non-selective peripheral muscarinic receptors, compound a showed minimal interaction with no interaction.

The data show that compound a is selective for the α 7NNR ligand. Compound A is at these subtypes of nicotinic receptors characteristic of the peripheral nervous system or at muscarinic receptors or 5HT₃Good binding does not occur at the 5-hydroxytryptamine-capable receptor. Thus, compound a has therapeutic potential in the treatment of central nervous system disorders, and does not produce side effects associated with interactions with the peripheral nervous system.

Summary of in vivo biological Activity

Compound a (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide or a pharmaceutically acceptable salt thereof shows significant efficacy in two models of cognitive behavior. Compound a showed potent activity after i.p. (intraperitoneal administration, fig. 3) and p.o. (oral administration, fig. 4) in an object recognition model in rats, and also showed activity over a wide dose range after oral administration (fig. 4). Compound a administered intraperitoneally at the same low dose (0.3 and 1mg/kg) tended to reverse the defect induced by MK-801 in the OR task (fig. 5), and compound a administered orally at 0.3mg/kg had a cognitive effect lasting at least 18 hours (fig. 6). In the Radial Arm Maze (RAM) (fig. 7) model, which examined working memory, compound a significantly increased the number of correct selections before the wrong selection. These results show the potential of (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide in the treatment of cognitive deficits and dysfunctions associated with schizophrenia, including dysfunctions associated with working memory.

For compounds suitable for the treatment of cognitive dysfunction in schizophrenia, the effect of typical or atypical antipsychotics against the positive symptoms of schizophrenia is not impaired. Thus, in addition to the cognitive enhancing properties of compound a, it is striking that compound a is also effective in reversing apomorphine-induced locomotor activity (APO LOCO) (fig. 8) and is effective in a prepulse inhibition (PPI) (fig. 9) model of the positive symptoms of schizophrenia. Thus, (2S,3R) -N- (2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide would be expected to provide additional benefits in combating the positive symptoms associated with schizophrenia as well as cognitive symptoms.

The particular pharmacological responses observed may vary according to or depending upon the particular active compound selected, or whether a pharmaceutical carrier is present, as well as the type of formulation and mode of administration employed, and such expected variations or differences in the results are contemplated in accordance with the practice of the present invention.

While specific embodiments of the present invention have been illustrated and described in detail, the present invention is not limited to these specific embodiments. The above detailed description is provided as an example of the present invention and should not be construed as limiting the present invention in any way. Modifications will be apparent to those skilled in the art and such modifications do not depart from the spirit of the invention and are intended to be included within the scope of the appended claims.

Claims (11)

1. The compound (2S,3R) N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, or a pharmaceutically acceptable salt thereof, that is substantially free of (2S,3S) N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, or a pharmaceutically acceptable salt thereof, (2R,3S) N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, or a pharmaceutically acceptable salt thereof, or (2R,3R) N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide, or a pharmaceutically acceptable salt thereof -yl) benzofuran-2-carboxamide or a pharmaceutically acceptable salt thereof.

2. The use of a compound of claim 1 in the manufacture of a medicament for the treatment or prevention of a central nervous system disorder, inflammation, pain, or neovascularization.

3. The use of claim 2, wherein the central nervous system disorder is selected from mild cognitive impairment, age-related memory impairment, pre-senile dementia, early onset alzheimer's disease, senile dementia, dementia of the alzheimer's type, alzheimer's disease, lewy body dementia, micro-infarct dementia, AIDS-related dementia, HIV-dementia, multiple cerebral infarcts, parkinsonism, parkinson's disease, pick's disease, progressive supranuclear palsy, huntington's chorea, tardive dyskinesia, hyperkinesia, mania, attention deficit disorder, attention deficit hyperactivity disorder, anxiety, depression, dyslexia, schizophrenia, cognitive dysfunction in schizophrenia, depression, obsessive-compulsive disorders, or tourette's syndrome.

4. The use of claim 2, wherein the central nervous system disorder is selected from alzheimer's disease, mania, attention deficit disorder, attention deficit hyperactivity disorder, anxiety, dyslexia, schizophrenia, cognitive dysfunction in schizophrenia, depression, obsessive-compulsive disorders, or tourette's syndrome.

5. The use according to claim 2, wherein the central nervous system disorder is schizophrenia or cognitive dysfunction in schizophrenia.

6. The use of claim 2, wherein the central nervous system disorder is attention deficit disorder or attention deficit hyperactivity disorder.

7. A pharmaceutical composition comprising a compound of claim 1 and one or more pharmaceutically acceptable diluents, excipients or carriers.

8. A process for the preparation of (2S,3R) -N- (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide or a pharmaceutically acceptable salt thereof comprising sequential kinetic resolution and stereoselective reductive amination of (2- ((3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-one.

9. A process for preparing (2S,3R) -N- (2- (3-pyridinyl) methyl-1-azabicyclo [2.2.2] oct-3-yl) benzofuran-2-carboxamide or a pharmaceutically acceptable salt thereof comprising an intermediate of (2S,3R) - (2- ((3-pyridinyl) methyl) -3-amino-1-azabicyclo [2.2.2] octane.

10. Stereoisomer-enriched (2S,3R) -2- ((3-pyridinyl) methyl) -3-amino-1-azabicyclo [2.2.2] octane.

11. Stereoisomer-enriched (2S) -2- ((3-pyridinyl) methyl) -1-azabicyclo [2.2.2] octan-3-one di-p-toluoyl-D-tartrate.