RU2824599C2 - Methods of treating symptoms and disorders associated with lysosomal storage diseases - Google Patents

Abstract

FIELD: chemistry; pharmaceutics.

SUBSTANCE: invention relates to chemistry and pharmaceutical industry, namely to a method of treating or preventing cognitive dysfunction associated with lysosomal storage disease in a human subject in need thereof. Disclosed method involves administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) has the structure , wherein: R¹ is hydrogen; R² and R³ are independently selected from C_1-3-alkyl; R⁴, R⁵ and R⁶ are independently selected from hydrogen and halogen; A is thiazolyl.

EFFECT: invention provides a method which is effective for improving cognitive abilities or reducing cognitive deficits, as measured by a reduction in the time required to complete the route building test (TMT), TMT-A and/or TMT-B, reducing the difference between time TMT-A and time TMT-B (TMT-A - TMT-B).

16 cl, 2 dwg, 20 tbl, 7 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is an international application that claims priority from U.S. Provisional Applications No. 62/800,996, filed February 4, 2019, No. 62/851,433, filed May 22, 2019, No. 62/894,167, filed August 30, 2019, No. 62/937,618, filed November 19, 2019, and No. 62/962,647, filed January 17, 2020, the contents of each of which are incorporated herein by reference in their entireties.

AREA OF TECHNOLOGY

The present invention relates to methods for treating or preventing specific symptoms and disorders that are associated with lysosomal storage diseases, using quinuclidine compounds of formula (I), optionally in combination with enzyme replacement therapy. These include supranuclear gaze palsies, including horizontal and vertical paresis of saccadic eye movements, as well as cognitive impairment or gait disturbances, for example in patients with Gaucher disease or Niemann-Pick disease type C.

LEVEL OF TECHNOLOGY

Lysosomal storage diseases

Lysosomal storage diseases (LSDs) are a group of about 50 rare inherited metabolic disorders caused by defects in lysosomal function. Typically, patients with LSDs accumulate harmful levels of substrate (i.e., stored material) in lysosomes due to a deficiency or defect in the enzyme responsible for metabolizing the substrate, or a deficiency in an enzyme activator needed for proper enzymatic function. Most LSDs are caused by a single enzymatic defect or deficiency, usually an enzyme involved in the metabolism of fats or glycoproteins. Some of the more common LSDs include Gaucher disease, Fabry disease, and Niemann-Pick disease (type C). Gaucher, Fabry, and Niemann-Pick are examples of sphingolipidoses. Each of these diseases is associated with a set of symptoms that are directly or indirectly caused by underlying genetic defects. As a result, it is often difficult to predict which symptoms or disorders associated with them can be effectively treated with different treatments. Symptoms common to several LSDs include changes in saccadic eye movements, cognitive dysfunction, and gait disturbances such as ataxia. These symptoms are particularly common in Gaucher disease (e.g., type 3) and Neumann-Pick disease (type C).

Saccadic eye movement defects in LSD

There are several functional classes of eye movements, including rapid saccade, smooth pursuit, optokinetic nystagmus (OKN), vestibular reflexes, and vergence, each of which is controlled by distinct cortical, brainstem, and supranuclear networks in the cerebellum. Failure of the supranuclear brainstem saccadic centers results in supranuclear gaze palsy, also called saccadic eye palsy. "Supranuclear" refers to the location of the defect above the corresponding cranial nerve nuclei in the midbrain of the brainstem (oculomotor nerve, trochlear nerve) or pons of the brainstem (abducens nerve). The oculomotor, trochlear, and abducens nerves are the only cranial nerves that control the small muscles that move the eye, and damage to the nerves themselves does not result in gaze palsy.

Fast saccades are rapid simultaneous movements of both eyes between two or more fixations in the same direction. Fast saccades contrast with smooth pursuit, in which the eyes move smoothly without saccades, typically tracking objects in the visual field. Fast saccades serve as visual fixation, rapid eye movement, and the fast phase of optokinetic nystagmus. Fast saccades are controlled cortically via the frontal oculomotor field region of the frontal lobe, or subcortically via the superior colliculus (a midbrain region). Fast saccades are particularly important during reading and when looking at the immediate surroundings. Because the high-resolution area of the retina, the fovea, is very small (approximately 1–2 degrees of visual field), saccadic eye movements are critical for resolving small objects in the visual field. Experienced readers move their eyes on average every 250 milliseconds while reading, and during each 20-40 millisecond saccade, the gaze target moves an average of 7-9 characters (range 1-20 characters).

The peak angular velocity of the eye during a saccade can reach up to 900 degrees per second in humans. Fast saccades in response to an unexpected stimulus typically begin within 200 milliseconds and last between 20 milliseconds and 200 milliseconds, depending on their amplitude. The amplitude of a fast saccade is the angular distance the eye traverses during an eye movement. Fast saccades with the head fixed can have an amplitude of up to 90 degrees, but in most cases any shift in gaze of more than 20 degrees is accompanied by head movement. During these fast saccades, the eye first makes a rapid saccade to shift gaze to the target, and the head follows more slowly while the eyes maintain focus on the target. The latter is called the vestibulo-ocular reflex (VOR), and its function is to slowly shift the eyes in the direction opposite to the head movement in order to maintain focus of vision on the retina. Because the head is almost always in slight motion, the VOR is essential for stabilizing vision under almost all circumstances, but especially during reading.

Saccadic eye movement palsy may result in slowing of rapid saccadic movements in horizontal, vertical, or both directions, and may or may not be limited in range. Whether horizontal or vertical saccadic eye movement palsy exists depends on the specific brain region involved.

Gaucher disease (GD) is a rare autosomal recessive lysosomal storage disorder. Patients with GD have a mutation in the GBA1 gene, which encodes glucosylceramidase (GC), also known as beta-glucocerebrosidase. This enzyme is responsible for breaking down glycosphingolipids into their components, such as the breakdown of glucosylceramide (GLC; also known as glucocerebroside) into glucose and ceramide. Monocytes and macrophages have particularly high levels of GLC-containing lysosomes, and in patients with GD, these cells become enlarged and accumulate toxic concentrations of GLC. These so-called “Gaucher cells” accumulate in several organs, including bone, bone marrow, spleen, liver, lungs, and brain. Systemically, this leads to splenomegaly, hepatomegaly, anemia, thrombocytopenia, leukopenia, osteopenia, osteonecrosis and other pathological disorders.

There are three subtypes of Gaucher disease, which differ in age of onset, severity, and presence of neurologic manifestations. Gaucher disease type 1 (GD-1), non-neuronopathic GD, is the most common form with a median age at diagnosis of 28 years and a moderately shortened life expectancy. In GD-1, the GC enzyme retains some functionality and there is no neurologic involvement. GD type 2 is an acute neuronopathic GD that is diagnosed in infancy, causes severe neurologic involvement, and causes death usually within the first two years of life. The GC enzyme in a type 2 patient has more severe dysfunction compared to GD-1. GD type 3 is a chronic neuronopathic GD that is diagnosed in childhood, is characterized by gradually worsening neurologic involvement, and a life expectancy of typically no more than 30 years. Symptoms of GD-3 include spleen and liver abnormalities, fatigue, bleeding, seizures, and supranuclear gaze palsy. Neurologic manifestations in patients with GD-3 gradually develop as the disease progresses. One of the most debilitating features is gaze paresis, which is a defect in the neural pathways that control rapid saccade movements of the eyes. In the early stages of the disease, there is a slowing of horizontal rapid saccade movements. The disease progresses to complete horizontal paresis of saccadic eye movements with varying degrees of vertical paresis of saccadic eye movements. The VOR may also be impaired in patients with GD-3. These features of the disease have a profound impact on the quality of life of patients with GD-3 and may interfere with educational attainment and employment prospects.

Current treatment for GD-1 and GD-3 is limited to recombinant enzyme replacement therapy (ERT) with imiglucerase, velaglucerase, or taliglucerase, and substrate reduction therapy (SRT) with miglustat or eliglustat. See, e.g., Lunawati L. Bennett & Chris Fellner, Pharmacotherapy of Gaucher Disease: Current and Future Options, P&T 43(5): 274–280, 309 (2018). Imiglucerase, the leading regimen, is a recombinant version of human GC made in Chinese hamster ovary cells and given by slow intravenous injection (usually over 1–2 hours) every 1–2 weeks. It has been available in the US since 1998. Velaglucerase is another recombinant human GC analogue that is made in a fibrosarcoma cell line and was approved by the FDA in 2010. Taliglucerase is a similar one made using genetically modified carrot root cells and approved since 2012. All of these regimens require IV administration in a hospital or other healthcare facility, and the recombinant enzymes do not cross the blood-brain barrier and are therefore unable to treat the neurological symptoms of GD. Thus, while these ERT regimens have been shown to be effective in treating patients with GD-1, they are only effective in treating the non-neurological symptoms of the disease in patients with GD-3.

Substrate reduction therapy is an alternative approach to the treatment of GD. The goal of this therapy is to reduce the accumulation of GLC by inhibiting the enzyme responsible for GLC synthesis. Glucosylceramide synthase (GCS), also known as UDP-glucosaccharide synthase, is the enzyme that catalyzes the initial glycosylation step of ceramide to produce glucosylceramide.

GCS inhibitors have been proposed for the treatment of a variety of diseases, including glycolipid storage diseases and lysosomal storage diseases, including Gaucher disease. See, for example, WO 2005/068426 (Actelion Pharm. Ltd.). Miglustat (Zavesca) is an iminoglycoside inhibitor of GCS. It is an N-alkylated iminosugar and acts as a reversible, competitive inhibitor of GCS by binding to the active site of the enzyme. Although it is designed to treat the neuronopathic forms of GD, GD-2 and GD-3, the FDA has only approved it for the treatment of patients with mild-moderate GD-1, and only as second-line therapy (patients must be unable to receive ERT treatment). Although miglustat crosses the blood-brain barrier, it has been found to be ineffective in clinical trials for the treatment of the neurologic manifestations of GD-3. Eliglustat is also a GCS inhibitor and is an analog of ceremide. It has been approved by the FDA only for the treatment of systemic symptoms in patients with GD-1.

Niemann-Pick disease type C (NPC) is also a lysosomal storage disorder, and although its cause is quite different from Gaucher disease, in some ways the end result is similar. NPC is caused by mutations in the NPC1 or NPC2 genes. NPC1 is a membrane protein that mediates the intracellular transport of cholesterol to post-lysosomal destinations. Specifically, NPC1 acts in concert with NPC2 to promote the efflux of cholesterol from the endosomal/lysosomal compartment. Unesterified cholesterol that has been released from low-density lipoproteins in the lumen of late endosomes/lysosomes is transported by NPC2 into the cholesterol-binding pocket of NPC1. Approximately 95% of patients with NPC have mutations in NPC1, while most of the remainder have mutations in NPC2. One consequence of this impaired cholesterol migration is the accumulation of cholesterol and glycosphingolipids (including GLC) in liver, spleen, and brain cells. One of the hallmarks of NPC, like GD3, is the progressive development of supranuclear gaze palsy, including horizontal and vertical paresis of saccadic eye movements.

Another group of diseases and disorders commonly associated with saccadic eye movement paresis include the GM2 gangliosidoses (such as Tay-Sachs disease, Sandhoff disease, and AB variant GM2 gangliosidosis).

GM2 gangliosidoses, like Gaucher disease, are lysosomal storage disorders marked by genetic defects in glycosphingolipid metabolism. GM2 gangliosidoses are marked by defects in the enzyme hexosaminidase A and/or its cofactor GM2 activator protein, which are responsible for the degradation of GM2 to GM3. GM2 and GM3 are related gangliosides that are part of the same metabolic pathway that degrades glucosylceramide to ceramide. Thus, GM3 is produced in a stepwise manner that begins with the conversion of ceramide to glucosylceramide (by GLC), followed by conversion to galactosyl-glucosylceramide, followed by conversion to GM3 (N-acetyl-a-neuraminidyl-galactosyl-glucosylceramide), followed by conversion to GM2 (N-acetylgalactosyl N-acetyl-a-neuraminidyl-galactosyl-glucosylceramide). Thus, the pathological accumulation of GM2, which is a hallmark of GM2 gangliosidoses, can be reduced by a GCS inhibitor that inhibits an earlier step in glucosylceramide synthesis.

The quinuclidine compounds described herein have activity as inhibitors of the enzyme glucosylceramide synthase (GCS). These compounds have been described as commonly used in the treatment of lysosomal storage diseases such as Fabry disease, Gaucher disease, and Niemann-Pick disease. See, e.g., WO 2012/129084 and U.S. 2016/0361301.

There is a real need in the field to develop therapeutic agents effective in alleviating or managing the neurological symptoms associated with type 3 Gaucher disease, particularly saccadic eye movement deficits.

ESSENCE OF THE INVENTION

The present invention relates to a quinuclidine compound (compound 1) according to formula (I),

(I)

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

R ¹ is selected from hydrogen, halogen (e.g. fluorine), cyano, nitro, hydroxy, thio, amino, C _1-6 alkyl (e.g. methyl or ethyl), C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkyloxy, C _2-6 alkenyloxy and C _2-6 alkynyloxy, wherein said alkyl, alkenyl, alkynyl, alkyloxy, alkenyloxy or alkynyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen, cyano, nitro, hydroxy, thio or amino;

R ² and R ³ are independently selected from C _1-3 -alkyl optionally substituted with one or more (e.g. 1, 2 or 3) halogens, or R ² and R ³ together form a cyclopropyl or cyclobutyl group optionally substituted with one or more (e.g. 1 or 2) halogens;

R ⁴ , R ⁵ and R ⁶ are each independently selected from hydrogen, halogen, nitro, hydroxy, thio, amino, C _1-6 -alkyl and C _1-6 -alkyloxy, wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen, hydroxy, cyano and C _1-6 -alkyloxy; and

A is a 5- or 6-membered aryl or heteroaryl group optionally substituted with 1, 2 or 3 groups independently selected from halogen, hydroxy, thio, amino, nitro, C _1-6 alkoxy or C _1-6 alkyl.

In a first aspect, the present application provides a method for treating or preventing supranuclear gaze palsies, including horizontal and vertical saccadic eye palsies, in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a quinuclidine compound as described herein, such as a compound of formula I. In other aspects, the present application further provides the use of the quinuclidine compounds described herein for treating or preventing supranuclear gaze palsies, including horizontal and vertical saccadic eye palsies, and/or for the manufacture of a medicament for treating or preventing supranuclear gaze palsies, including horizontal and vertical saccadic eye palsies.

Additional features and advantages of the compounds, compositions and methods described herein will be apparent from the following detailed description.

BRIEF DESCRIPTION OF DRAWINGS

Figures 1 and 2 show horizontal saccadic eye movements measured in five patients as described in Example 5 (Figure 1 shows patients 1-3 and Figure 2 shows patients 4-5). The amplitude of the fast saccadic movements and the peak velocity are measured when the target moves horizontally either 15° (gray dots) or 30° (black dots) from the central position to either the left or the right. Eye movements to the right are represented by positive peak velocities, and movements to the left are represented by negative peak velocities. The gray shaded area on each graph represents the normal range of peak velocities at any given amplitude.

DETAILED DESCRIPTION

Although specific embodiments of the present disclosure will now be described with reference to production examples and circuits, it should be understood that such embodiments are provided by way of example only and merely illustrate only a small number of the many possible specific embodiments that may represent applications of the principles of the present disclosure. Various changes and modifications will be apparent to those skilled in the art, given the benefit of the present disclosure, and are considered to be within the spirit and scope of the present disclosure, as further defined in the appended claims.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods, devices, and materials are now described. All technical and patent publications cited herein are hereby incorporated by reference in their entirety. Nothing herein shall be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art.

All numerical designations, such as pH, temperature, time, concentration, molecular weight, including ranges, are approximate, varying (+) or (-) in increments of 0.1 or 1.0, where appropriate. It should be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about". It should also be understood, although not always explicitly stated, that the reagents described herein are merely typical, and that their equivalents are known in the art.

As used herein, the term “optionally substituted” is equivalent to the phrase “unsubstituted or substituted.”

As used herein, the phrase "in a method of treating or preventing" (e.g., in the phrase "in a method of treating or preventing supranuclear gaze palsies") is equivalent to the phrase "in the treatment or prevention" (e.g., in the phrase "in the treatment or prevention of supranuclear gaze palsies").

As used in the specification and claims, the singular forms include the plural unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells, including mixtures thereof. Unless specifically stated or obvious from the context, as used herein, the term "or" is to be construed as inclusive. The term "including" is used herein to mean and is used interchangeably with the phrase "including, but not limited to."

As used herein, the term "comprising" or "comprises" is intended to mean that the compositions and methods include the listed elements but do not exclude others. "Consisting essentially of," when used to define compositions and methods, means to the exclusion of other elements that are essential to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein does not exclude trace impurities from the isolation and purification process and pharmaceutically acceptable carriers such as phosphate buffered saline, preservatives, and the like. "Consisting of" means to the exclusion of more than trace elements of other ingredients and essential steps in the method of administering the compositions of the present invention or steps in the method of preparing the composition or achieving the desired result. Embodiments defined by each of these transition terms are within the scope of the present invention. The use of the term "comprising" herein is intended to encompass "consisting essentially of" and "consisting of."

The terms "subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, mice, rats, rabbits, monkeys, cattle, sheep, pigs, dogs, cats, farm animals, sport animals, pets, horses, primates, and humans. In one embodiment, mammals include horses, dogs, and cats. In some embodiments, the mammal is a human, such as a human suffering from a particular disease or disorder, such as Gaucher disease (e.g., GD-3) or Niemann-Pick disease type C.

"Administration" is defined herein as the means of providing an agent or a composition containing the agent to a subject such that the agent is within the subject's body. Such administration may be by any route, including, without limitation, oral, transdermal (e.g., vaginal, rectal, oral-mucosal), injection (e.g., subcutaneous, intravenous, parenteral, intraperitoneal, intra-CNS), or inhalation (e.g., oral or nasal). Of course, pharmaceutical preparations are available in forms suitable for each route of administration.

"Treat" or "treating" a disease generally includes: (1) inhibiting the disease, i.e. stopping or reducing the progression of the disease or its clinical symptoms; and/or (2) alleviating the disease, i.e. causing regression of the disease or its clinical symptoms.

As used in this document, the terms "treat" and "treatment" also refer to either reversal of supranuclear gaze palsy or stabilization of supranuclear gaze palsy. This is because the diseases and disorders described herein are progressive disorders - if left untreated, supranuclear gaze palsy will continue to worsen until complete paresis (i.e. paralysis) occurs. For example, a patient may have slow or inhibited rapid saccade movements of the eyes at the onset of the disease, but as the disease progresses, patients may develop a complete absence of rapid saccade movements. Thus, treatment involves both slowing this progressive deterioration (i.e., stabilization) and reversing this progressive deterioration (i.e., improvement).

"Prophylaxis" or "prevention" of a disease generally involves preventing the development of clinical symptoms of a disease in a patient who may be predisposed to the disease but does not yet experience or show symptoms of the disease.

As used herein, the terms "prevention" or "prophylaxis" also include preventing the development of supranuclear gaze palsy in a patient suspected of having or diagnosed with a disease or disorder described herein. Because the diseases and disorders described herein are progressive disorders, various signs and symptoms may appear progressively as the disease progresses. For example, a patient may be diagnosed with GD-3 or NPC before the development of supranuclear gaze palsy. In such a patient, the treatments described herein may be effective in preventing the development of supranuclear gaze palsy.

The term "paresis" is synonymous with "paralysis" and includes any degree of loss of motor function of one or more skeletal muscles. Thus, as used herein, the term "paresis" includes both complete paresis, i.e., complete paralysis, and partial paresis. Complete paresis means that a muscle or group of muscles, such as extraocular muscles, have lost the ability to contract. Thus, the affected eye or eyes may be unable to move. Partial paresis may manifest as retardation of movements, slowness of movements, or other movement defects. These may include loss of range of motion. With respect to rapid saccades, this may include inhibition of initiation of rapid saccades (e.g., in response to stimuli), changes in frequency of rapid saccades, changes in peak velocity of rapid saccades, changes in amplitude of rapid saccades, changes in latency between rapid saccades, and/or loss of the ability to maintain gaze or shift gaze. As used herein, in some embodiments, paresis includes ophthalmoparesis and/or ophthalmoplegia. Thus, the term covers both weakness and paralysis of the extraocular muscles. Extraocular muscles include any one or more of the superior rectus, inferior, middle, lateral, inferior oblique, and superior oblique muscles of the eye. Weakness and/or paralysis may involve one or more of the horizontal movements, vertical movements, or rotational movements.

The term "affected," when used in the context of "treatment," refers to a patient or person who has been diagnosed with a disease. The term "affected," when used in the context of "prevention," refers to a patient or person who is predisposed to a disease. A patient may also be considered "at risk" of having a disease because of a family history of the disease or because of the presence of genetic mutations associated with the disease. A patient at risk of having a disease has not yet developed some or all of the characteristic pathologies of the disease.

"An effective amount" or "therapeutically effective amount" is an amount sufficient to achieve the desired or beneficial results. An effective amount may be administered in one or more administrations, applications, or dosages. Such delivery depends on a number of variables, including the period of time over which the individual dosage form is to be used, the bioavailability of the therapeutic agent, and the route of administration. However, it is understood that specific dosage levels of the therapeutic agents of the present invention for any particular subject will depend on a variety of factors, including, for example, the activity of the particular compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of elimination, the drug combination, and the severity of the particular disorder being treated, and the route of administration. Therapeutic dosages can typically be titrated to optimize safety and efficacy. In general, dose-effect relationships obtained from in vitro and/or in vivo testing can initially provide useful guidance on the proper dosages to administer to a patient. It is generally desirable to administer an amount of the compound that is effective to achieve serum levels commensurate with concentrations that have proven effective in vitro. Determination of these parameters is within the skill of the art. These considerations, as well as effective formulations and administration procedures, are well known in the art and described in standard textbooks. In accordance with this definition, as used herein, the term "therapeutically effective amount" is an amount sufficient to treat (e.g., ameliorate) one or more symptoms associated with a disease or disorder described herein (e.g., in any of Method 1 et seq. or Method 4 et seq.) ex vivo, in vitro, or in vivo.

As used herein, the term "pharmaceutically acceptable excipient" encompasses any standard pharmaceutical excipients, including carriers such as phosphate buffered saline, water, and emulsions such as oil/water or water/oil emulsions and various types of wetting agents. The pharmaceutical compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Remington's Pharmaceutical Sciences ( ^20th ed., Mack Publishing Co., 2000).

As used herein, the term "prodrug" means a pharmacological derivative of a parent drug molecule that requires biotransformation, either spontaneous or enzymatic, in the body to release the active drug. For example, prodrugs are variations or derivatives of the quinuclidine compound described herein that have groups that are cleavable under certain metabolic conditions that, when cleaved, yield a quinuclidine compound described herein, such as a compound of formula I. Such prodrugs are then pharmaceutically active in vivo when they undergo solvolysis under physiological conditions or undergo enzymatic degradation. Prodrug compounds may be referred to herein as single, double, triple, etc., depending on the number of biotransformation steps required to release the active drug in the body and the number of functional groups present in the precursor form. Prodrug forms often have advantages of solubility, histocompatibility, or delayed release in mammals.

Prodrugs, which are widely known in the art, include well-known acid derivatives such as, for example, esters prepared by reacting acidic compounds with a suitable alcohol, amides prepared by reacting acidic compounds with an amine, and basic groups that react to form an acylated base derivative. Other prodrug derivatives can be combined with other features described herein to enhance bioavailability. Thus, those skilled in the art will understand that some of the compounds described herein, having, for example, free amino or hydroxy groups, can be converted into prodrugs. Prodrugs include compounds having an amino acid residue, or a polypeptide chain of two or more (e.g., two, three, or four) amino acid residues, which are covalently linked through peptide bonds to free amino, hydroxy, or carboxylic acid groups of the compounds described herein. Amino acid residues include 20 naturally occurring amino acids, commonly designated by three-letter symbols, and also include 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvaline, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine, and methionine sulfone. Prodrugs also include compounds having a carbonate, carbamate, amide, or alkyl ester group covalently bonded to any of the above substituents described herein.

As used herein, the term "pharmaceutically acceptable salt" means a pharmaceutically acceptable acid addition salt or a pharmaceutically acceptable base addition salt of a compound as described herein that can be administered without any resulting significant undesirable biological effects or any resulting deleterious interaction with any other component of the pharmaceutical composition in which it may be contained.

As used herein, the term "C _1-6 alkyl" means a saturated or branched free radical consisting essentially of 1-6 carbon atoms and the appropriate number of hydrogen atoms. Exemplary C _1-6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl. Other C _1-6 alkyl groups will be apparent to those skilled in the art, given the benefit of this disclosure. The terms "C _1-3 alkyl", "C _1-4 alkyl", etc. have equivalent meanings, i.e., a saturated linear or branched free radical consisting essentially of 1-3 (or 4) carbon atoms and the appropriate number of hydrogen atoms.

As used herein, the term " _C2-6 -alkenyl" means an unsaturated linear or branched free radical consisting essentially of 2-6 carbon atoms and a corresponding number of hydrogen atoms, the free radical containing at least one carbon-carbon double bond. Exemplary C2-6-alkenyl groups include ethenyl, prop-1-enyl, prop _{-2-enyl, isopropenyl, but-1-enyl, 2-methylprop-1-enyl, and 2-methyl-prop-2-enyl. Other C2-6 -} alkenyl groups will be apparent to those skilled in the art, given the benefit of this disclosure.

As used herein, the term " _C2-6 -alkynyl" means an unsaturated linear or branched free radical consisting essentially of 2-6 carbon atoms and a corresponding number of hydrogen atoms, the free radical containing at least one carbon-carbon triple bond. Exemplary _C2-6 -alkynyl groups include ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, and 3-methylbut-1-ynyl. Other _C2-6 -alkynyl groups will be apparent to those skilled in the art, given the benefit of this disclosure.

As used herein, the term "C _1-6 -alkyloxy" means a saturated linear or branched free radical consisting essentially of 1-6 carbon atoms (and the appropriate number of hydrogen atoms) and an oxygen atom. A C _1-6 -alkyloxy group is attached through an oxygen atom. Representative C _1-6 -alkyloxy groups include methyloxy, ethyloxy, n-propyloxy, isopropyloxy, n-butyloxy and isobutyloxy. Other C _1-6 -alkyloxy groups will be apparent to those skilled in the art having the benefit of this disclosure. The terms "C _1-3 -alkyloxy", "C _1-4 -alkyloxy" and the like have equivalent meanings, i.e., a saturated linear or branched free radical consisting essentially of 1-3 (or 4) carbon atoms (and the appropriate number of hydrogen atoms) and an oxygen atom, where the group is attached through an oxygen atom.

As used herein, the term "C _2-6 -alkenyloxy" means an unsaturated linear or branched free radical consisting essentially of 2-6 carbon atoms (and an appropriate number of hydrogen atoms) and an oxygen atom, the free radical containing at least one carbon-carbon double bond. The C _2-6 -alkenyloxy group is attached through an oxygen atom. An exemplary C _2-6 -alkenyloxy group is ethenyloxy; others will be apparent to those skilled in the art, given the benefit of this disclosure.

As used herein, the term "C _2-6 -alkynyloxy" means an unsaturated linear or branched free radical consisting essentially of 2-6 carbon atoms (and an appropriate number of hydrogen atoms) and an oxygen atom, the free radical containing at least one carbon-carbon triple bond. A C _2-6 -alkenyloxy group is attached through an oxygen atom. An exemplary C _2-6 -alkenyloxy group is ethynyloxy; others will be apparent to those skilled in the art, given the benefit of this disclosure.

As used herein, the term "heteroaryl" means an aromatic free radical containing 5 or 6 atoms (i.e., ring atoms) that form a ring, wherein 1 to 5 ring atoms are carbon and the remaining 1 to 5 ring atoms (i.e., ring heteroatoms) are independently selected from the group consisting of nitrogen, sulfur, and oxygen. Exemplary 5-membered heteroaryl groups include furyl, thienyl, thiazolyl (e.g., thiazol-2-yl), pyrazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrrolyl, triazolyl, imidazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 6-membered heteroaryl groups include pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, 1,2,4-triazinyl, benzoxazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, and benzimidazolyl. Other heteroaryl groups will be apparent to those skilled in the art having the benefit of this disclosure. Typically, the heteroaryl group is attached to the parent structure through a carbon atom. However, those skilled in the art will appreciate that certain other atoms, such as ring heteroatoms, may be attached to the parent structure.

As used herein, the term "aryl" means an aromatic free radical containing 5 or 6 atoms (i.e., ring atoms) that form a ring, where all the ring atoms are carbon. A typical aryl group is the phenyl group.

As used herein, the term "aliphatic" means a non-aromatic compound containing carbon and hydrogen atoms, such as containing from 1 to 9 carbon atoms. Aliphatic compounds may be linear or branched, may contain one or more ring structures, and may contain one or more carbon-carbon double bonds (provided that the compound does not contain an unsaturated ring structure having aromatic character). Examples of aliphatic compounds include ethane, propylene, cyclobutane, and cyclohexadiene.

As used herein, the terms "halo" and "halogen" mean fluorine, chlorine, bromine or iodine. These terms are used interchangeably and may refer to a free radical halogen group or to a halogen atom per se. Those skilled in the art will readily be able to establish the identification given the context in which the term is used in the present description.

As used herein, the term "cyano" means a free radical having a carbon atom bonded to a nitrogen atom by a triple bond. The cyano radical is bonded through a carbon atom.

In this document, the term "nitro" means the _-NO2 radical that is attached through the nitrogen atom.

As used herein, the terms "hydroxy" and "hydroxyl" mean the -OH radical that is attached through an oxygen atom. The term "thio" means the -SH radical that is attached through a sulfur atom.

As used herein, the term "amino" means a free radical containing a nitrogen atom and 1 or 2 hydrogen atoms. Thus, the term "amino" generally refers to primary and secondary amines. In this regard, as used herein, a tertiary amine is represented by the general formula RR'N-, where R and R' are carbon radicals that may or may not be identical. However, the term "amino" may generally be used herein to describe a primary, secondary or tertiary amine, and those skilled in the art will readily be able to establish the identification given the context in which the term is used herein.

As used herein, the term "oxo" means an oxygen radical that is attached via a double bond. If the atom attached to this oxygen is a carbon atom, the bond is a carbon-oxygen double bond, which may be represented as -(C=O)- and may be referred to as a ketone.

The recitation of a list of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of the recited groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein may be combined with one or more other compositions and methods provided herein.

The following abbreviations are used in this document:

sh wide signal KDI carbonyldiimidazole CNS central nervous system d doublet DAFI 4',6-diamidino-2-phenioindole dd doublet of doublets DME dimethoxyethane DMEM Dulbecco's modified Eagle's medium DMSO-d6 dimethyl sulfoxide-d6 DMF dimethylformamide DNA deoxyribonucleic acid DTBZ dihydrotetrabenazine Carbon-11 EDTA ethylenediaminetetraacetic acid ELISA enzyme immunoassay _Et2O diethyl ether EtMgBr ethyl magnesium bromide EtOAc ethyl acetate GL1 glucosylceramide (GlcCer) GM1 monosialotetrahexosylganglioside GM3 monosialodihexosylganglioside GSL glycosphingolipid H&E hematoxylin and eosin staining HPLC high pressure/high performance liquid chromatography HSA human serum albumin IPS isopropyl alcohol J combination constant LCMS liquid chromatography mass spectrometry m multiplet ppm parts per million rHA recombinant human albumin With singlet TBME tert-butyl methyl ether THF tetrahydrofuran Tris tris(hydroxymethyl)aminomethane TWEEN20 Polysorbate 20 TWEEN80 Polysorbate 80 WT wild type UZHMS ultra performance liquid chromatography mass spectrometry

Connections

The present disclosure relates to quinuclidine compounds for use in therapeutic methods relating to the treatment or prevention of the diseases and disorders discussed herein. In all of its various aspects, the invention relates to a quinuclidine compound (Compound 1) according to formula (I),

(I)

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

R ¹ is selected from hydrogen, halogen (e.g. fluorine), cyano, nitro, hydroxy, thio, amino, C _1-6 alkyl (e.g. methyl or ethyl), C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkyloxy, C _2-6 alkenyloxy and C _2-6 alkynyloxy, wherein said alkyl, alkenyl, alkynyl, alkyloxy, alkenyloxy or alkynyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen, cyano, nitro, hydroxy, thio or amino;

R ² and R ³ are independently selected from C _1-3 -alkyl optionally substituted with one or more (e.g. 1, 2 or 3) halogens, or R ² and R ³ together form a cyclopropyl or cyclobutyl group optionally substituted with one or more (e.g. 1 or 2) halogens;

R ⁴ , R ⁵ and R ⁶ are each independently selected from hydrogen, halogen, nitro, hydroxy, thio, amino, C _1-6 -alkyl and C _1-6 -alkyloxy, wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen, hydroxy, cyano and C _1-6 -alkyloxy; and

A is a 5- or 6-membered aryl or heteroaryl group (e.g. phenyl or thiazolyl) optionally substituted with 1, 2 or 3 groups independently selected from halogen, hydroxy, thio, amino, nitro, C _1-6 alkoxy or C _1-6 alkyl.

In other embodiments of any aspects of the present disclosure, the present disclosure further relates to compounds as follows:

1.1 Compound 1, wherein R ¹ is selected from hydrogen, halogen, cyano, nitro, hydroxy, thio, amino, C _1-6 -alkyl, C _1-6 -alkyloxy, wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen, cyano, nitro, hydroxy, thio or amino;

1.2 Compound 1, wherein R ¹ is selected from hydrogen, halogen, C _1-6 -alkyl, C _1-6 -alkyloxy, wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen, cyano, nitro, hydroxy, thio or amino;

1.3 Compound 1, wherein R ¹ is selected from hydrogen, halogen, C _1-4 -alkyl, C _1-4 -alkyloxy, wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen, cyano, nitro, hydroxy, thio or amino;

1.4 Compound 1, wherein R ¹ is selected from hydrogen, halogen, C _1-4 -alkyl, C _1-4 -alkyloxy, wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3, or 1 or 2) groups selected from cyano, nitro, hydroxy, thio or amino;

1.5 Compound 1, wherein R ¹ is selected from hydrogen, halogen and C _1-4 -alkyl, wherein said alkyl is optionally substituted with one or more (e.g. 1 or 2) groups selected from halogen, hydroxy, thio or amino;

1.6 Compound 1, wherein R ¹ is selected from hydrogen, fluoro, methyl and ethyl, wherein said methyl or ethyl is optionally substituted with 1 or 2 groups selected from halogen, hydroxy, thio or amino;

1.7 Compound 1, wherein R ¹ is selected from hydrogen and methyl, wherein said methyl is optionally substituted with 1 or 2 halogens;

1.8 Compound 1, where R ¹ is hydrogen;

1.9 Compound 1, or any of 1.1-1.8, wherein R ¹ is not attached to the nitrogen atom of the quinuclidine group;

1.10 Compound 1, or any of 1.1-1.9, wherein R ² and R ³ are each independently C _1-3 -alkyl optionally substituted with one or more (e.g. 1, 2 or 3) halogens;

1.11 Compound 1.11, wherein R ² and R ³ are each independently methyl or ethyl, optionally substituted with 1 or 2 halogens;

1.12 Compound 1.11, wherein R ² and R ³ are each independently selected from methyl and ethyl, optionally substituted with one or more fluorines, for example 1, 2 or 4 fluorines;

1.13 Compound 1.11, wherein R ² and R ³ are each independently methyl substituted with 0, 1, 2, or 3 fluorines;

1.14 Compound 1.11, where R ² and R ³ are each methyl or trifluoromethyl;

1.15 Compound 1.11, R ² and R ³ are each methyl;

1.16 Compound 1, or any of 1.1-1.9, wherein R ² and R ³ together form a cyclopropyl or cyclobutyl group optionally substituted with one or more (e.g. 1 or 2) halogens;

1.17 Compound 1.16, where R ² and R ³ together form a cyclopropyl group;

1.18 Compound 1 or any of 1.1-1.9, wherein R ² and R ³ are each methyl, or R ² and R ³ together form a cyclopropyl group;

1.19 Compound 1, or any of 1.1-1.9, wherein R ⁴ , R ⁵ and R ⁶ are each independently selected from hydrogen, halogen, C _1-6 alkyl and C _1-6 alkyloxy, wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen, hydroxy, cyano and C _1-6 alkyloxy;

1.20 Compound 1, or any of 1.1-1.9, wherein R ⁴ , R ⁵ and R ⁶ are each independently selected from hydrogen, halogen, C _1-3 -alkyl and C _1-3 -alkyloxy, wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen, hydroxy, cyano and C _1-3 -alkyloxy;

1.21 Compound 1.19, wherein R ⁴ , R ⁵ and R ⁶ are each independently selected from hydrogen, halogen, C _1-3 -alkyl and C _1-3 -alkyloxy, wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen, cyano and C _1-3 -alkyloxy;

1.22 Compound 1.19, wherein R ⁴ , R ⁵ and R ⁶ are each independently selected from hydrogen, halogen, C _1-3 -alkyl and C _1-3 -alkyloxy, wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen and C _1-3 -alkyloxy;

1.23 Compound 1.19, wherein R ⁴ , R ⁵ and R ⁶ are each independently selected from halogen, C _1-3 -alkyl and C _1-3 -alkyloxy, wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen and C _1-3 -alkyloxy;

1.24 Compound 1, or any of 1.19-1.23, R ⁴ is selected from hydrogen, halogen, C _1-3 -alkyl and C _1-3 -alkyloxy, wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen and C _1-3 -alkyloxy;

1.25 Compound 1.24, R ⁴ is selected from halogen (e.g. fluorine), C _1-3 -alkyl (e.g. methyl) and C _1-3 -alkyloxy (e.g. methoxy or ethoxy), wherein said alkyl or alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen and C _1-3 -alkyloxy (e.g. methoxy or ethoxy);

1.26 Compound 1.26, R ⁴ is selected from halogen (e.g. fluorine) and C _1-3 -alkyloxy (e.g. methoxy or ethoxy), wherein said alkyloxy is optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen and C _1-3 -alkyloxy (e.g. methoxy or ethoxy);

1.27 Compound 1.26, R ⁴ is fluoro or C _1-3 -alkyloxy (e.g. ethoxy), optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen and C _1-3 -alkyloxy (e.g. methoxy);

1.28 Compound 1.26, wherein R ⁴ is fluoro or ethoxy optionally substituted with one or more (e.g. 1, 2 or 3) C _1-3 -alkyloxy (e.g. methoxy);

1.29 Compound 1, or any of 1.19-1.28, where R ⁶ is hydrogen;

1.30 Compound 1, or any of 1.19-1.28, wherein R ⁵ and R ⁶ are each hydrogen;

1.31 Compound 1, or any of 1.19-1.28, R ⁵ and R ⁶ are each hydrogen and R ⁴ is fluoro or C _1-3 -alkyloxy (e.g. ethoxy) optionally substituted with one or more (e.g. 1, 2 or 3) groups selected from halogen and C _1-3 -alkyloxy (e.g. methoxy);

1.32 Compound 1.31, wherein R ⁵ and R ⁶ are each hydrogen, and R ⁴ is fluoro or ethoxy optionally substituted with one or more (e.g. 1, 2 or 3) C _1-3 -alkyloxy (e.g. methoxy);

1.33 Compound 1.32, where R ⁵ and R ⁶ are each hydrogen, and R ⁴ is fluorine or ethoxy substituted by methoxy (e.g. 2-methoxyethoxy);

1.34 Compound 1.32, where R ⁴ is fluorine or 2-methoxyethoxy;

1.35 Compound 1, or any of 1.1-1.34, wherein at least one of R ⁴ , R ⁵ and R ⁶ is not hydrogen;

1.36 Compound 1, or any of 1.1-1.35, wherein R ⁶ is hydrogen, and R ⁴ and R ⁵ are located at the 2, 4, or 6 positions of the phenyl ring to which they are attached (i.e., ortho or para to substituent A);

1.37 Compound 1, or any of 1.1-1.35, wherein R ⁶ is hydrogen and R ⁴ and R ⁵ are independently located at the 2 and 3 (i.e., adjacent ortho and meta), 3 and 4 (i.e., adjacent meta and para), or 3 and 5 positions (i.e., meta) of the phenyl ring to which they are attached (with respect to substituent A);

1.38 Compound 1, or any of 1.1-1.35, wherein R ⁶ is hydrogen and R ⁴ and R ⁵ are located at the 3 and 5 positions (i.e., meta) of the phenyl ring to which they are attached (with respect to substituent A);

1.39 Compound 1, or any of 1.1-1.35, wherein R ⁵ and R ⁶ are hydrogen and R ⁴ is located at the 2, 3, or 4 position of the phenyl ring to which it is attached (e.g., ortho, meta, or para to substituent A);

1.40 Compound 1, or any of 1.1-1.35, wherein R ⁵ and R ⁶ are hydrogen and R ⁴ is located at the 2 or 4 position of the phenyl ring to which it is attached (e.g., ortho or para to substituent A);

1.41 Compound 1, or any of 1.1-1.35, wherein R ⁵ and R ⁶ are hydrogen and R ⁴ is located at the 4-position of the phenyl ring to which it is attached (e.g., para to substituent A);

1.42 Compound 1, or any of 1.1-1.35, wherein none of R ⁴ , R ⁵ , and R ⁶ is hydrogen, and each of R ⁴ , R ⁵ , and R ⁶ is independently in the 2, 4, or 6 positions of the phenyl ring to which they are attached (i.e., ortho or para to substituent A);

1.43 Compound 1, or any of 1.1-1.42, wherein R ⁴ is located at the 4-position of the phenyl ring to which it is attached (i.e., para to substituent A);

1.44 Compound 1, or any of 1.1-1.43, wherein A is a 6-membered aryl group, a 5-membered heteroaryl group (e.g. containing 1, 2, or 3 heteroatoms in the heteroaryl ring selected from N, O, and S), or a 6-membered heteroaryl group (e.g. containing 1, 2, or 3 nitrogen atoms in the heteroaryl ring);

1.45 Compound 1.44, wherein A is a 6-membered aryl group or a 5-membered heteroaryl group (e.g. containing 1, 2 or 3 heteroatoms in the heteroaryl ring selected from N, O and S), optionally wherein the 5-membered heteroaryl group contains 1 or 2 heteroatoms selected from N and S (e.g. one N and/or one S);

1.46 Compound 1.44 or 1.45, wherein A is selected from the group consisting of phenyl, furyl, thienyl, thiazolyl, pyrazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrrolyl, triazolyl, imidazolyl, oxadiazolyl and thiadiazolyl;

1.47 Compound 1.46, wherein A is selected from the group consisting of phenyl, thienyl, thiazolyl, pyrrolyl and imidazolyl;

1.48 Compound 1.46, wherein A is selected from the group consisting of phenyl and thiazolyl, for example 2-thiazol-4-yl or 4-thiazol-2-yl;

1.49 Compound 1, or any of 1.1-1.48, wherein A is unsubstituted;

1.50 Compound 1, or any of 1.1-1.48, wherein A is substituted with one or more (e.g. 1, 2 or 3) groups independently selected from halogen, hydroxy, thio, amino, nitro, C _1-6 alkoxy and C _1-6 alkyl (e.g. methyl);

1.51 Compound 1.50, wherein A is thiazolyl substituted with one halogen (e.g. fluorine) or C _1-6 alkyl (e.g. methyl);

1.52 Compound 1.50, wherein A is phenyl substituted with 1, 2, or 3 groups independently selected from halogen (e.g., fluorine) and C _1-6 alkyl (e.g., methyl);

1.53 Compound 1.52, where A is phenyl substituted with 1 or 2 fluorine or methyl groups;

1.54 Compound 1, or any of 1.1-1.53 wherein the two groups attached to substituent A (i.e., the phenyl ring (-(C ₆ H ₂ R ⁴ R ⁵ R ⁶ )) and the -C(R ² R ³ )- group) are in a 1,2-, 1,3-, or 1,4-relationship to each other (i.e., ortho, meta, or para);

1.55 Compound 1.54, where the two groups attached to substituent A are in a 1,3-relationship to each other (i.e., meta);

1.56 Compound 1.54, where the two groups attached to substituent A are in a 1,4-relationship to each other (i.e., a pair);

1.57 Any of the compounds 1.54-1.56, wherein the A substituent is ^a 5-membered heteroaryl group and at least one of the two groups attached to the A substituent (i.e., the _phenyl ring (-( _C6H2R4R5R6 )) ^{and the} -C( ^R2R3 ) group) is attached to a carbon atom of the ^heteroaryl ring, optionally wherein both such groups are attached to carbon atoms of the heteroaryl ring;

1.58 Compound 1, or any of 1.1-1.57, wherein the compound of formula I may be represented by one or more of the following substructures:

1.59 Compound 1, or any of 1.1-1.58, wherein the compound of formula I or any of formulas II-XII has the (S) configuration;

1.60 Compound 1, or any of 1.1-1.58, wherein the compound of formula I or any of formulas II-XII has the (R) configuration;

1.61 Compound 1, or any of 1.1-1.60, wherein the compound of formula I or any of formulas II-XII has an enantiomeric excess (e.g., (S) configuration) of at least 90%, such as at least 92%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%;

1.62 Compound 1, or any of 1.1-1.58, wherein the compound of formula I or any of formulas II-XII is racemic (i.e., approximately 50:50 enantiomers), or is a mixture of enantiomers in some other ratio (e.g., less than 50:50 or more than 50:50);

1.63 Compound 1, or any of 1.1-1.62, wherein the compound of formula I is selected from the group consisting of:

Connection No. Compound 1 Quinuclidin-3-yl (2-(4'-fluoro[1,1'-biphenyl]-3-yl)propan-2-yl)carbamate 2 (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate 3 (S)-quinuclidin-3-yl (2-(4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate 4 1-azabicyclo[2.2.2]oct-3-yl [2-(biphenyl-3-yl)propan-2-yl]carbamate 5 (S)-quinuclidin-3-yl 2-(biphenyl-4-yl)propan-2-ylcarbamate 6 Quinuclidin-3-yl 1-(biphenyl-4-yl)cyclopropylcarbamate 7 (S)-quinuclidin-3-yl 1-(4'-fluorobiphenyl-4-yl)cyclopropylcarbamate 8 (S)-1-azabicyclo[2.2.2]oct-3-yl [1-(2',4'-difluorobiphenyl-4-yl)cyclopropyl]carbamate 9 1-azabicyclo[2.2.2]oct-3-yl [1-(4'-methoxybiphenyl-4-yl)cyclopropyl]carbamate 10 Quinuclidin-3-yl 2-(5-(4-fluorophenyl)thiophen-3-yl)propan-2-ylcarbamate 11 (S)-quinuclidin-3-yl 2-(3-(4-fluorophenyl)isothiazol-5-yl)propan-2-ylcarbamate 12 (S)-quinuclidin-3-yl 2-(4-(4-fluorophenyl)thiazol-2-yl)propan-2-ylcarbamate 13 Quinuclidin-3-yl (2-(4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate 14 (S)-quinuclidin-3-yl (2-(3'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate 15 Quinuclidin-3-yl (2-(4'-(2-methoxyethoxy)-[1,1'-biphenyl]-3-yl)propan-2-yl)carbamate 16 Quinuclidin-3-yl (2-(4'-(3-methoxypropoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate 17 Quinuclidin-3-yl (2-(4'-(hydroxymethyl)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate 18 Quinuclidin-3-yl (2-(4'-(2-hydroxyethyl)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate 19 Quinuclidin-3-yl (2-(2-(4-(3-methoxypropoxy)phenyl)thiazol-4-yl)propan-2-yl)carbamate 20 Quinuclidin-3-yl (2-(2-(4-(2-methoxyethoxy)phenyl)thiazol-4-yl)propan-2-yl)carbamate 21 Quinuclidin-3-yl 2-(5-(4-(2-methoxyethoxy)phenyl)pyridin-2-yl)propan-2-ylcarbamate 22 Quinuclidin-3-yl (2-(4'-(3-cyanopropoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate 23 Quinuclidin-3-yl (2-(4'-(cyanomethoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate

1.64 Compound 1, or any of 1.1-1.63, wherein the compound is selected from quinuclidin-3-yl (2-(4'-fluoro-[1,1'-biphenyl]-3-yl)propan-2-yl)carbamate, (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate, and (S)-quinuclidin-3-yl (2-(4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate;

1.65 Compound 1, or any of 1.1-1.63, wherein the compound is quinuclidin-3-yl (2-(4'-fluoro-[1,1'-biphenyl]-3-yl)propan-2-yl)carbamate;

1.66 Compound 1 or any of 1.1-1.63, wherein the compound is quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate, such as (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate;

1.67 Compound 1, or any of 1.1-1.66, wherein the compound of formula I, or any of II-XII, is in the free base form;

1.68 Compound 1, or any of 1.1-1.66, wherein the compound of formula I, or any of II-XII, is in the form of a pharmaceutically acceptable salt;

1.69 Compound 1.68, wherein said salt form is an acid addition salt form;

1.70 Compound 1.69, wherein said acid addition salt form is a salt selected from hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, hydrogen phosphate, acetate, lactate, citrate, hydrogen citrate, tartrate, bitartrate, succinate, hydroxysuccinate, malate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate and pamoate;

1.71 Compound 1.70, wherein the acid addition salt form is selected from hydrochloride, hydroxysuccinate (e.g. 2-hydroxysuccinate) and malate;

1.72 Compound 1.68, wherein the indicated salt form is the base addition form of the salt;

1.73 Compound 1, or any of 1.1-1.72, wherein the compound is (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate in the malate form;

1.74 Compound 1, or any of 1.1-1.73, wherein the compound of formula I, or any of II-XII, is in the form of a prodrug as described herein;

1.75 Compound 1, or any of 1.1-1.74, wherein the compound of formula I, or any of II-XII, is in the form of a hydrate, solvate and/or polymorph.

Salts

The disclosed compounds, for example any of Compounds 1 or 1.1-1.75, which are basic in nature, are generally capable of forming a large number of different salts with various inorganic and/or organic acids. Although such salts are generally pharmaceutically acceptable for administration to animals and humans, in practice it is often desirable to first isolate the compound from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base form of the compound by treatment with an alkaline reagent, followed by conversion of the free base to a pharmaceutically acceptable acid addition salt. Acid addition salts of basic compounds can be readily prepared using conventional techniques, for example by treating the basic compound with a substantially equivalent amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent such as, for example, methanol or ethanol. After careful evaporation of the solvent, the desired solid salt is obtained. The described compounds, which are positively charged, for example, containing quaternary ammonium, can also form salts with the anionic component of various inorganic and/or organic acids.

Acids that can be used to prepare pharmaceutically acceptable salts of the quinuclidine compound include those that can form non-toxic acid addition salts, for example, salts containing pharmacologically acceptable anions such as chloride, bromide, iodide, nitrate, sulfate or bisulfate, phosphate or acid phosphate, acetate, lactate, citrate or acid citrate, tartrate or bitartrate, succinate, malate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate and pamoate [i.e., 1,1'-methylenebis(2-hydroxy-3-naphthoate)].

The compounds described which are acidic in nature, for example those containing a thiol group, are generally capable of forming a large number of different salts with various inorganic and/or organic bases. Although such salts are generally pharmaceutically acceptable for administration to animals and humans, in practice it is often desirable to first isolate the compound from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free acid compound by treatment with an acidic reagent and then convert the free acid into a pharmaceutically acceptable base addition salt. These base addition salts can be readily prepared using conventional techniques, for example by treating the corresponding acidic compounds with an aqueous solution containing the desired pharmacologically acceptable cations and then evaporating the resulting solution to dryness, for example under reduced pressure. Alternatively, they can also be prepared by mixing lower alkanol solutions of the acidic compounds and the desired alkali metal alkoxide together and then evaporating the resulting solution to dryness in the same manner as before. In any case, stoichiometric amounts of reagents can be used to ensure completeness of the reaction and maximum yield of the desired solid salt product.

Bases that can be used to prepare pharmaceutically acceptable base addition salts of the quinuclidine compound include those that can form non-toxic base addition salts, for example, salts containing pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium), alkaline earth metal cations (e.g., calcium and magnesium), ammonium or other water-soluble amino addition salts such as N-methylglucamine (meglumine), lower alkanolammonium and other similar organic amine bases.

In one embodiment, the pharmaceutically acceptable salt is succinate. In another embodiment, the pharmaceutically acceptable salt is 2-hydroxysuccinate, such as (S)-2-hydroxysuccinate. In another embodiment, the pharmaceutically acceptable salt is hydrochloride (i.e., salt with HCl). In another embodiment, the pharmaceutically acceptable salt is malate.

Prodrugs

The present disclosure further encompasses prodrugs of compounds 1 and 1.1-1.75. The pharmaceutically acceptable prodrugs described herein are derivatives of quinuclidine compounds that can be converted in vivo to the quinuclidine compounds described herein. The prodrugs, which may themselves have some activity, become pharmaceutically active in vivo when they undergo, for example, solvolysis under physiological conditions or enzymatic degradation. Methods for preparing prodrugs of the compounds as described herein will be apparent to one skilled in the art based on the present disclosure.

In one embodiment, the carbamate group of the quinuclidine compound is modified. For example, the carbamate group of the quinuclidine compound can be modified by the addition of water and/or one or two aliphatic alcohols. In this case, the carbon-oxygen double bond of the carbamate group adopts what could be considered a hemiacetal or acetal functionality. In one embodiment, the carbamate group of the quinuclidine compound can be modified by the addition of an aliphatic diol, such as 1,2-ethanediol.

In one embodiment, one or more hydroxy, thio, or amino groups on the quinuclidine compound are modified. For example, one or more hydroxy, thio, and/or amino groups on the quinuclidine compound can be modified to form acidic derivatives, such as esters, thioesters (or thiol esters), and/or amides. Acid derivatives can be formed, for example, by reacting a quinuclidine compound that contains one or more hydroxy, thio, or amino groups with an acetylating agent. Examples of acetylating agents include anhydrides such as acetic anhydride, acid chlorides such as benzyl chloride, and dicarbonates such as di-tert-butyl dicarbonate.

Stereochemistry

The present description further covers stereoisomers and mixtures of stereoisomers of compounds 1 and 1.1-1.75. Stereoisomers (e.g., cis and trans isomers) and all optical isomers of the described compound (e.g., R and S enantiomers), as well as racemic, diastereomeric and other mixtures of such isomers, are within the scope of the present description.

In one embodiment, the quinuclidin-3-yl group of the quinuclidine compound as defined herein has the R-configuration. Accordingly, the quinuclidine compound can be selected from the group consisting of compounds of formulae (Ia) - (XIIa):

and their pharmaceutically acceptable salts and prodrugs.

In another embodiment, the quinuclidin-3-yl group of the quinuclidine compound as defined herein has an S-configuration. Accordingly, the quinuclidine compound may be selected from the group consisting of compounds of formulae (Ib) to (XIIb):

and their pharmaceutically acceptable salts and prodrugs.

In one embodiment, the quinuclidine compound is a compound of formula (Xb), or a pharmaceutically acceptable salt or prodrug thereof. In another embodiment, the quinuclidine compound is a compound of formula (XIIb), or a pharmaceutically acceptable salt or prodrug thereof.

In one embodiment, the quinuclidin-3-yl group of a quinuclidine compound, as defined herein, exists in a mixture of isomers having R- and S-configurations. For example, the quinuclidine compound may be a mixture of compounds selected from the group consisting of compounds of formulas (Ia) and (Ib), (IIa) and (IIb), (IIIa) and (IIIb), (IVa) and (IVb), ( Va) and (Vb), (VIa) and (VIb), (VIIa) and (VIIb), (VIIIa) and (VIIIb), (IXa) and (IXb), (Xa) and (Xb), (XIa) and (XIb) and (XIIa) and (XIIb) and pharmaceutically acceptable salts and prodrugs thereof. In one embodiment, the quinuclidine compound is present as a racemic mixture, e.g., the R- and S-isomers of the quinuclidin-3-yl group are present in approximately equal amounts. In another embodiment, the quinuclidine compound is present as isomers having R- and S -configurations wherein the R- and S-isomers are present in different amounts. In one embodiment, the S-isomer is present in an enantiomeric excess of at least about 5%, 10%, 25%, 40%, 70%, 80% , 90%, 95%, 97%, 98% or 99%, for example, approximately 100%. In another embodiment, the R-isomer is present in an enantiomeric excess of at least about 5%, 10%, 25%, 40%, 70%, 80%, 90%, 95%, 97%, 98%, or 99%. , for example, approximately 100%.

Methods for preparing enantioenriched and/or enantiosensitive quinuclidine compounds will be apparent to those skilled in the art based on the present disclosure.

The compounds described may exist in several tautomeric forms, including enol and imine forms, keto and enamine forms, geometric isomers, and mixtures thereof. Tautomers exist as mixtures of tautomers in solution. In the solid form, one tautomer usually predominates. Although one tautomer may be described, all tautomers are within the scope of the present disclosure.

Atropisomers are also included within the scope of the present description. Atropisomers refer to compounds that can be separated into isomers with limited rotation.

Other forms

The present disclosure also includes hydrates, solvates, and polymorphs of compound 1 and 1.1-1.75. Pharmaceutically acceptable hydrates, solvates, and polymorphs of the quinuclidine compounds described herein are within the scope of the present disclosure. The quinuclidine compounds described herein may be in amorphous form and/or in one or more crystalline forms.

Isotopically labeled compounds are also included within the scope of the present disclosure. As used herein, the term "isotopically labeled compound" refers to a disclosed compound, including pharmaceutical salts and prodrugs thereof, each of which is disclosed herein, in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature. Examples of isotopes that may be included in the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as ^2H , ^3H , ^13C , ^14C , ^15N , ^18O , ^17O , ^31P , ^32P , ^35S , ^18F , and ^36Cl , respectively.

Medical indications

The quinuclidine compounds and pharmaceutical compositions containing them described herein are used in therapy, in particular in the therapeutic treatment of supranuclear gaze palsies, including horizontal and vertical paresis of saccadic eye movements, neurological disorders, including dementia and gait disturbances in a patient having a disease such as Gaucher disease. Subjects to be treated in accordance with the methods described herein include vertebrates, such as mammals. In particular embodiments, the mammal is a human patient.

In a first aspect, the present invention provides a method (Method 1) for the treatment or prevention of supranuclear gaze palsies, including horizontal and vertical saccadic eye palsies, in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a quinuclidine compound as described herein, for example a compound of formula I or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or 1.1-1.75. Also provided is a quinuclidine compound as described herein, e.g. a compound of formula I or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or 1.1-1.75, for use in a method of treating or preventing supranuclear gaze palsies, including horizontal and vertical saccadic eye palsies, in a subject in need thereof, e.g. for use in method 1, or any of 1.1-1.62. Also provided is the use of a quinuclidine compound as described herein, e.g. a compound of formula I or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or 1.1-1.75, in the manufacture of a medicament for use in a method for the treatment or prevention of supranuclear gaze palsies, including horizontal and vertical saccadic eye palsies, in a subject in need thereof, e.g. in the manufacture of a medicament for use in method 1, or any of 1.1-1.62.

In other specific embodiments of method 1, the present description provides:

1.1 Method 1, wherein the method comprises administering to a subject an effective amount of a compound of formula I or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or any of 1.1-1.75;

1.2 Method 1, wherein the method comprises administering to a subject an effective amount of compound 1 or any one or more of compounds 1.1-1.75;

1.3 Method 1 or any of 1.1-1.2, wherein the method comprises administering to the subject an effective amount of a pharmaceutical composition comprising a compound according to formula I or any of II-XII, Ia-XIIa or Ib-XIIb, or any of Compounds 1 or any of 1.1-1.75;

1.4 Method 1 or any of 1.1-1.2, wherein the method comprises administering to the subject an effective amount of a pharmaceutical composition comprising compound 1 or any one or more of compounds 1.1-1.75;

1.5 Method 1.3 or 1.4, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient as described herein;

1.6 Method 1 or any of 1.1-1.5, wherein the method comprises administering a pharmaceutical dosage form containing an effective amount of the compound or an effective amount of the pharmaceutical composition;

1.7 Method 1.6, wherein the dosage form is an oral dosage form (e.g., a pill, capsule, caplet, tablet, dragee, powder, granule, film, lozenge, or liquid);

1.8 Method 1.7, where the dosage form is a chewable tablet;

1.9 Method 1.6, wherein the dosage form is a parenteral dosage form (e.g. where the pharmaceutical composition is formulated for injection);

1.10 Method 1.9, wherein the injection is intravenous, intramuscular, intrathecal or subcutaneous, optionally by sterile injection;

1.11 Method 1.6, wherein the dosage form is a topical or rectal dosage form;

1.12 Method 1.6, where the dosage form is an intranasal dosage form (e.g. an aerosol);

1.13 Method 1 or any of 1.1-1.12, wherein the method further comprises co-administering a second active agent, such as a second compound, capable of treating or preventing supranuclear gaze palsies in a patient in need thereof, as described herein;

1.14 Method 1.13, wherein the second active agent is administered in the same pharmaceutical composition or dosage form as the quinuclidine compound;

1.15 Method 1.13 or 1.14, wherein the second active agent is a GCS inhibitor (eg, miglustat or eliglustat);

1.16 Method 1, or any of 1.1-1.15, where the subject is a mammal;

1.17 Method 1.16, where the subject is a primate;

1.18 Method 1.17, where the subject is a human being;

1.19 Method 1 or any of 1.1-1.18, where the supranuclear gaze palsy is gaze paresis;

1.20 Method 1 or any of 1.1-1.19, wherein the supranuclear gaze palsy includes horizontal paresis of saccadic eye movements, vertical paresis of saccadic eye movements, impairment of the vestibulo-ocular reflex, impairment of gaze fixation, impairment of horizontal smooth pursuit and/or impairment of vertical smooth pursuit;

1.21 Method 1, or any of 1.1-1.20, where the supranuclear gaze palsy includes horizontal paresis of saccadic eye movements;

1.22 Method 1, or any of 1.1-1.21, where the supranuclear gaze palsy includes vertical paresis of saccadic eye movements;

1.23 Method 1, or any of 1.1-1.22, where the supranuclear gaze palsy is a complete paresis (e.g., paralysis);

1.24 Method 1, or any of 1.1-1.23, wherein the subject has type 3 Gaucher disease;

1.25 Method 1, or any of 1.1-1.24, wherein the subject has Niemann-Pick disease type C;

1.26 Method 1, or any of 1.1-1.24, wherein the subject has GM2 gangliosidosis (e.g., Tay-Sachs disease, Sandhoff disease, or AB variant GM2 gangliosidosis);

1.27 Method 1, or any of 1.1-1.24, wherein the subject is diagnosed with a mutation in the GBA1 gene;

1.28 Method 1, or any of 1.1-1.24, wherein the subject is diagnosed with a mutation in the NPC1 and/or NPC2 genes;

1.29 Method 1, or any of 1.1-1.24, wherein the subject is diagnosed with a mutation in the HEXA gene (encoding hexosaminidase A) and/or a mutation in the HEXB gene (encoding hexosaminidase B) and/or a mutation in the GM2A gene (encoding GM2 ganglioside activator protein);

1.30 Method 1, or any of 1.1-1.29, wherein the subject is diagnosed with Parkinson's disease;

1.31 Method 1, or any of 1.1-1.30, wherein the subject is concomitantly treated with enzyme replacement therapy (ERT), such as with glucocerebrosidase (e.g. imiglucerase, velaglucerase or taliglucerase), optionally wherein each of such enzymes is a recombinant enzyme;

1.32 Method 1.31, wherein the subject is concomitantly treated with one or more of imiglucerase, velaglucerase (e.g., velaglucerase alfa), and taliglucerase (e.g., taliglucerase alfa);

1.33 Method 1.32, wherein the subject is concomitantly treated with imiglucerase;

1.34 Method 1.33, wherein the subject is concomitantly treated with imiglucerase at a dose of 2.5 units/kg body weight to 80 units/kg body weight every 1 to 3 weeks, such as 40 to 60 units/kg body weight every 2 weeks (1 unit of imiglucerase is equal to the amount of enzyme that catalyzes the hydrolysis of 1 micromole of the synthetic substrate p-nitrophenyl-β-D-glucopyranoside per minute at 37°C);

1.35 Method 1.34, wherein the dosage of imiglucerase to the subject at each administration (e.g., every 1-3 weeks, e.g., every 2 weeks) is administered as an intravenous (IV) infusion over a period of 1-3 hours (e.g., 1-2 hours);

1.36 Method 1 or any of 1.1-1.35, wherein the subject is administered an enzyme replacement therapy (e.g., imiglucerase, velaglucerase, and/or taliglucerase) prior to treatment with a compound of formula I (or any of II-XII, Ia-XIIa, or Ib-XIIb, or any of compounds 1 or 1.1-1.75);

1.37 Method 1.36, wherein the subject is administered imiglucerase therapy for at least 6 months from the start of therapy with a compound of Formula I (or any of II-XII, Ia-XIIa, or Ib-XIIb, or any of compounds 1 or 1.1-1.75), such as at least 12 months (1 year), or at least 18 months, or at least 2 years, or at least 3 years.

1.38 Method 1.36 or 1.37, wherein the subject is administered imiglucerase therapy for at least 6 months at a stable dose prior to initiation of therapy with a compound of formula I (or any of II-XII, Ia-XIIa, or Ib-XIIb, or any of compounds 1 or 1.1-1.75);

1.39 Method 1 or any of 1.1-1.38, wherein the method further comprises the step of switching the subject from ERT therapy (e.g., imiglucerase, velaglucerase, or taliglucerase) to treatment with a compound of formula I (or any of II-XII, Ia-XIIa, or Ib-XIIb, or any of compounds 1 or 1.1-1.75);

1.40 Method 1 or any of 1.1-1.39, wherein the subject has a hemoglobin level of at least 11 g/dL for females and at least 12 g/dL for males;

1.41 Method 1, or any of 1.1-1.40, wherein the subject has a platelet count of at least 100,000/cubic millimeter;

1.42 Method 1, or any of 1.1-1.41, wherein the subject has a spleen volume less than 10 times normal (MN) and/or a liver volume less than ±1.5 MN;

1.43 Method 1, or any of 1.1-1.42, where the subject is diagnosed with an oculomotor disorder, such as an oculomotor disorder characterized by a horizontal abnormality of saccadic eye movements;

1.44 Method 1, or any of 1.1-1.43, wherein the subject is at least 18 years of age (e.g. 18-30 years of age) at the start of treatment with a compound of formula I (or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or 1.1-1.75);

1.45 Method 1, or any of 1.1-1.44, wherein the subject has a glucosylceramide (GL1) concentration of 4.4-11.1 ng/mL in cerebrospinal fluid (CSF) and 4.9-8.3 mcg/mL in plasma;

1.46 Method 1, or any of 1.1-1.45, wherein the subject has a glucosylsphingosine (lyso-GL1) concentration of 20.1-67.6 pg/mL in CSF and 8.8-159.0 ng/mL in plasma;

1.47 Method 1, or any of 1.1-1.46, wherein the subject is administered a daily dose of from about 1 mg to about 150 mg of a compound of Formula I (or any of II-XII, Ia-XIIa, or Ib-XIIb, or any of Compounds 1 or 1.1-1.75), such as from 5 to 50 mg, or from 10 to 40 mg, or from 10 to 30 mg, or from 10 to 20 mg, or from 20 to 30 mg, or from 30 to 40 mg, or from 40 to 50 mg, or from 5 to 25 mg, or from 20 to 50 mg, or from 5 to 15 mg, or from 15 to 30 mg, or about 15 mg, or selected from 2, 5, 15, 25, 50, 100, or 150 mg;

1.48 Method 1, or any of 1.1-1.47, wherein the subject is an adult human patient, such as between 18 and 80 years of age, such as between 18 and 60 years of age, or between 18 and 40 years of age, or between 18 and 30 years of age, or between 18 and 25 years of age;

1.49 Method 1, or any of 1.1-1.47, wherein the subject is a human patient of pediatric age, such as between the ages of 0 and 18, such as between 1 and 15, or between 1 and 5, or between 5 and 10, or between 10 and 15, or between 10 and 18;

1.50 Method 1, or any of 1.1-1.49, wherein the method is effective to stabilize the progression of supranuclear gaze palsy, such as for at least 6 months, or at least 9 months, or at least 12 months;

1.51 Method 1, or any of 1.1-1.49, wherein the method is effective to reverse the progression of supranuclear gaze palsy, such as for at least 6 months, or at least 9 months, or at least 12 months;

1.52 Method 1, or any of 1.1-1.51, wherein the method results in a reduction in the concentration of glucosylceramide in the CSF and/or plasma by at least 30% after 6 months of treatment, such as at least 40%, at least 50%, at least 60%, or at least 70%;

1.53 Method 1, or any of 1.1-1.52, wherein the method results in an increase in the concentration of glucosylsphingosine in the CSF and/or plasma of at least 30% after 6 months of treatment, such as at least 40%, at least 50%, at least 60%, or at least 70%;

1.54 Method 1, or any of 1.1-1.53, where the method yields a statistically or clinically unchanged modified Severity Assessment Tool (mSST) value for neurological disorders after 6 months of treatment;

1.55 Method 1, or any of 1.1-1.54, wherein the compound of formula I (or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or 1.1-1.75) or a pharmaceutically acceptable salt or prodrug thereof is administered by systemic administration, for example, parenteral or non-parenteral;

1.56 Method 1.55, where the route of administration is oral (enteral);

1.57 Method 1.55, wherein the route of administration is parenteral, such as by injection, such as by intravenous injection.

1.58 Method 1, or any of 1.1-1.57, wherein the compound of formula I (or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or 1.1-1.75), or a pharmaceutically acceptable salt or prodrug thereof, is administered by topical administration, such as topical administration;

1.59 Method 1, or any of 1.1-1.58, wherein the compound is (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate or quinuclidin-3-yl (2-(4'-fluoro-[1,1'-biphenyl]-3-yl)propan-2-yl)carbamate;

1.60 Method 1.59, wherein the dose of the compound is 15 mg/day when administered orally;

1.61 Method 1.60, wherein the dose of the compound is 15 mg/day as a single oral dose;

1.62 Method 1, or any of 1.1-1.61, wherein the subject is administered a single daily dose of 5 mg, 10 mg, 15 mg or 20 mg of a compound, such as (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate, optionally in the form of an acid addition malate.

Diseases and disorders, such as those causing supranuclear gaze palsy, are often associated with one or more genetic mutations. In some embodiments of the present disclosure, a subject or entity is diagnosed with a particular disease or disorder, and is also diagnosed with a particular genetic mutation, such as one that is known to cause the disease or disorder in question, although it is often impossible to prove that a particular disease or disorder of a patient is caused by a particular mutation that has been diagnosed in the person. In this case, the term "diagnosed with a particular genetic mutation" means that the subject or patient has been tested, such as by DNA or RNA sequencing, protein profiling, or other suitable means, and has been found to have the mutation in question. However, as discussed below, many genetic diseases and disorders may have multiple genetic causes (e.g., mutations), and patients may have multiple mutations, each of which may, under some circumstances, be sufficient to cause the disease or disorder without being proof that a particular mutation causes a particular disease or disorder in a particular patient.

The methods of method 1 et seq. may be beneficial for subjects who have been diagnosed with a lysosomal storage disease, such as Gaucher type 3 or Niemann-Pick type C, but who do not yet have ocular symptoms associated with the disease state. The methods of method 1 et seq. may also be beneficial for subjects who are at risk of developing a lysosomal storage disease, such as Gaucher type 3 or Niemann-Pick type C, due to, for example, a mutation in the subject or a family history that is known to cause such a disease. Therefore, in some embodiments of the methods described herein, the subject is diagnosed as being at risk of developing such a disease or disorder, and the method prevents or delays the onset and/or development of ocular symptoms of the disease or disorder (e.g., supranuclear gaze palsy) in the subject. In some embodiments, the subject is diagnosed as being at risk of developing said disease or disorder through the presence of a mutation in a gene as described herein.

In a second aspect, the present invention provides a method (Method 4) for the treatment or prevention of cognitive dysfunction and/or gait disorders, including ataxia, associated with a lysosomal storage disease, in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a quinuclidine compound as described herein, for example a compound of formula I or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or 1.1-1.75. Also provided is a quinuclidine compound as described herein, e.g. a compound of formula I or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or 1.1-1.75, for use in a method of treating or preventing cognitive dysfunction and/or gait disorders, including ataxia, associated with a lysosomal storage disease in a subject in need thereof, e.g. for use in method 4 or any of 4.1-4.62. Also provided is the use of a quinuclidine compound as described herein, e.g. a compound of formula I or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or 1.1-1.75, in the manufacture of a medicament for use in a method for the treatment or prevention of cognitive dysfunction and/or gait disorders, including ataxia, associated with a lysosomal storage disease, in a subject in need thereof, e.g. the manufacture of a medicament for use in Method 4 or any of 4.1-4.62.

In specific additional embodiments of method 4, the present description provides:

4.1 Method 4, wherein the method comprises administering to the subject an effective amount of a compound of formula I or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or any of 1.1-1.75;

4.2 Method 4, wherein the method comprises administering to the subject an effective amount of compound 1 or any one or more of compounds 1.1-1.75;

4.3 Method 4 or any of 4.1-4.2, wherein the method comprises administering to the subject an effective amount of a pharmaceutical composition comprising a compound of formula I or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or any of 1.1-1.75;

4.4. Method 4 or any of 4.1-4.2, wherein the method comprises administering to the subject an effective amount of a pharmaceutical composition comprising compound 1 or any one or more of compounds 1.1-1.75;

4.5 Method 4.3 or 4.4, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient as described herein;

4.6 Method 4 or any of 4.1-4.5, wherein the method comprises administering a pharmaceutical dosage form containing an effective amount of the compound or an effective amount of the pharmaceutical composition;

4.7 Method 4.6, wherein the dosage form is an oral dosage form (e.g., a pill, capsule, caplet, tablet, dragee, powder, granule, film, lozenge, or liquid);

4.8 Method 4.7, where the dosage form is a chewable tablet;

4.9 Method 4.6, wherein the dosage form is a parenteral dosage form (e.g. where the pharmaceutical composition is formulated for injection);

4.10 Method 4.9, wherein the injection is intravenous, intramuscular, intrathecal or subcutaneous, optionally by sterile injection;

4.11 Method 4.6, wherein the dosage form is a topical or rectal dosage form;

4.12 Method 4.6, wherein the dosage form is an intranasal dosage form (e.g. an aerosol);

4.13 Method 4 or any of 4.1-4.12, wherein the method further comprises co-administering a second active agent, such as a second compound, capable of treating or preventing supranuclear gaze palsies in a patient in need thereof, as described herein;

4.14 Method 4.13, wherein the second active agent is administered in the same pharmaceutical composition or dosage form as the quinuclidine compound;

4.15 Method 4.13 or 4.14, wherein the second active agent is a GCS inhibitor (eg, miglustat or eliglustat);

4.16 Method 1, or any of 4.1-4.15, where the subject is a mammal;

4.17 Method 4.16, where the subject is a primate;

4.18 Method 4.17, where the subject is a human being;

4.19 Method 4 or any of 4.1-4.18, where the ataxia is cerebellar ataxia;

4.20 Method 4 or any of 4.1-4.19, where the ataxia exhibits symptoms selected from unstable gait, asthenia, asynergia, slow reaction time, dyschronometry, dysarthria, dysphagia, hypotonia, dysmetria, hypometria, hypermetria, dysdiadochokinesia, slurred speech, vocal tremor, ataxic breathing, postural instability, and combinations thereof, for example, where the primary ataxic deficit is gait instability;

4.21 Method 4, or any of 4.1-4.20, wherein the subject has a baseline ataxia of at least 0.5 on the SARA scale at the start of therapy according to the method, e.g., a baseline SARA score of at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 10, or at least 20;

4.22 Method 4, or any of 4.1-4.21, where the cognitive dysfunction is dementia;

4.23 Method 4, or any of 4.1-4.22, where dementia shows evidence of impairment in visual search speed, reading processing speed, mental flexibility and/or executive functioning, such as evidenced by a TMT-A of more than 30 seconds, or more than 45 seconds, or more than 60 seconds and/or a TMT-B of more than 70 seconds, or more than 90 seconds, or more than 120 seconds, or more than 150 seconds, or more than 180 seconds, and/or where TMT-B minus TMT-A is more than 40 seconds, or more than 60 seconds, or more than 90 seconds, or more than 120 seconds;

4.24 Method 4, or any of 4.1-4.23, wherein the subject has type 3 Gaucher disease;

4.25 Method 4, or any of 4.1-4.24, wherein the subject has Niemann-Pick disease type C;

4.26 Method 4, or any of 4.1-4.24, wherein the subject has GM2 gangliosidosis (e.g., Tay-Sachs disease, Sandhoff disease, or AB variant GM2 gangliosidosis);

4.27 Method 4, or any of 4.1-4.24, wherein the subject is diagnosed with a mutation in the GBA1 gene;

4.28 Method 4, or any of 4.1-4.24, wherein the subject is diagnosed with a mutation in the NPC1 and/or NPC2 genes;

4.29 Method 4, or any of 4.1-4.24, wherein the subject is diagnosed with a mutation in the HEXA gene (encoding hexosaminidase A) and/or a mutation in the HEXB gene (encoding hexosaminidase B) and/or a mutation in the GM2A gene (encoding GM2 ganglioside activator protein);

4.30 Method 4, or any of 4.1-4.29, wherein the subject is diagnosed with Parkinson's disease;

4.31 Method 4, or any of 4.1-4.30, wherein the subject is concomitantly treated with enzyme replacement therapy (ERT), such as with glucocerebrosidase (e.g. imiglucerase, velaglucerase or taliglucerase), optionally wherein each of such enzymes is a recombinant enzyme;

4.32 Method 4.31, wherein the subject is concomitantly treated with one or more of imiglucerase, velaglucerase (e.g., velaglucerase alfa), and taliglucerase (e.g., taliglucerase alfa);

4.33 Method 4.32, wherein the subject is concomitantly treated with imiglucerase;

4.34 Method 4.33, wherein the subject is concomitantly treated with imiglucerase at a dose of 2.5 units/kg body weight to 80 units/kg body weight every 1 to 3 weeks, such as 40 to 60 units/kg body weight every 2 weeks (1 unit of imiglucerase is equal to the amount of enzyme that catalyzes the hydrolysis of 1 micromole of the synthetic substrate p-nitrophenyl-β-D-glucopyranoside per minute at 37°C);

4.35 Method 4.34, wherein the dosage of imiglucerase to the subject at each administration (e.g., every 1-3 weeks, such as every 2 weeks) is administered as an intravenous (IV) infusion over a period of 1-3 hours (e.g., 1-2 hours);

4.36 Method 4 or any of 4.1-4.35, wherein the subject is administered an enzyme replacement therapy (e.g., imiglucerase, velaglucerase, and/or taliglucerase) prior to treatment with a compound of formula I (or any of II-XII, Ia-XIIa, or Ib-XIIb, or any of compounds 1 or 1.1-1.75);

4.37 Method 4.36, wherein the subject is administered imiglucerase therapy for at least 6 months from the start of therapy with a compound of Formula I (or any of II-XII, Ia-XIIa, or Ib-XIIb, or any of compounds 1 or 1.1-1.75), such as at least 12 months (1 year), or at least 18 months, or at least 2 years, or at least 3 years.

4.38 Method 4.36 or 4.37, wherein the subject is administered imiglucerase therapy for at least 6 months at a stable dose prior to initiation of therapy with a compound of formula I (or any of II-XII, Ia-XIIa, or Ib-XIIb, or any of compounds 1 or 1.1-1.75);

4.39 Method 4 or any of 4.1-4.38, wherein the method further comprises the step of switching the subject from ERT therapy (e.g., imiglucerase, velaglucerase, or taliglucerase) to treatment with a compound of formula I (or any of II-XII, Ia-XIIa, or Ib-XIIb, or any of compounds 1 or 1.1-1.75);

4.40 Method 4 or any of 4.1-4.39, wherein the subject has a hemoglobin level of at least 11 g/dL for females and at least 12 g/dL for males;

4.41 Method 4, or any of 4.1-4.40, wherein the subject has a platelet count of at least 100,000/cubic millimeter;

4.42 Method 4, or any of 4.1-4.41, wherein the subject has a spleen volume less than 10 times normal (MN) and/or a liver volume less than ±1.5 MN;

4.43 Method 4, or any of 4.1-4.42, where the subject is diagnosed with a co-existing dementia, such as Alzheimer's disease or Parkinson's disease;

4.44 Method 4, or any of 4.1-4.43, wherein the subject is at least 18 years of age (e.g. 18-30 years of age) at the start of treatment with a compound of formula I (or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or 1.1-1.75);

4.45 Method 4, or any of 4.1-4.44, wherein the subject has a glucosylceramide (GL1) concentration of 4.4-14.1 ng/mL in cerebrospinal fluid (CSF) and 4.9-8.3 mcg/mL in plasma;

4.46 Method 4, or any of 4.1-4.45, wherein the subject has a glucosylsphingosine (lyso-GL1) concentration of 20.1-67.6 pg/mL in CSF and 8.8-159.0 ng/mL in plasma;

4.47 Method 4, or any of 4.1-4.46, wherein the subject is administered a daily dose of from about 1 mg to about 150 mg of a compound of Formula I (or any of II-XII, Ia-XIIa, or Ib-XIIb, or any of Compounds 1 or 1.1-1.75), such as from 5 to 50 mg, or from 10 to 40 mg, or from 10 to 30 mg, or from 10 to 20 mg, or from 20 to 30 mg, or from 30 to 40 mg, or from 40 to 50 mg, or from 5 to 25 mg, or from 20 to 50 mg, or from 5 to 15 mg, or from 15 to 30 mg, or about 15 mg, or selected from 2, 5, 15, 25, 50, 100, or 150 mg;

4.48 Method 4, or any of 4.1-4.47, wherein the subject is an adult human patient, such as between 18 and 80 years of age, such as between 18 and 60 years of age, or between 18 and 40 years of age, or between 18 and 30 years of age, or between 18 and 25 years of age;

4.49 Method 4, or any of 4.1-4.47, wherein the subject is a human patient of pediatric age, such as between the ages of 0 and 18, such as between the ages of 1 and 15, or between the ages of 1 and 5, or between the ages of 5 and 10, or between the ages of 10 and 15, or between the ages of 10 and 18;

4.50 Method 4, or any of 4.1-4.49, wherein the method is effective to reduce the SARA ataxia scale by at least 0.5, such as reducing the SARA score by at least 1, or at least 2, or at least 3, or at least 5, or at least 10; or wherein the method is effective to reduce the SARA score to a value of from 0.00 to 3.00, or from 0.00 to 2.00, or from 0.00 to 1.50, or from 0.00 to 1.00, or from 0.00 to 0.50;

4.51 Method 4, or any of 4.1-4.49, wherein the method is effective in improving cognitive performance or reducing cognitive deficit, such as as measured by a reduction in the time required to complete the Tracking Test (TMT), TMT-A and/or TMT-B, a reduction in the difference between TMT-A time and TMT-B time (TMT-A - TMT-B), such as a reduction of at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% (e.g. where TMT-A is reduced by 5-20%, and/or TMT-B is reduced by 25-30%, and/or [TMT-A - TMT-B] is reduced by 25-30%);

4.52 Method 4, or any of 4.1-4.51, wherein the method results in a reduction in the concentration of glucosylceramide in the CSF and/or plasma by at least 30% after 6 months of treatment, such as at least 40%, at least 50%, at least 60%, or at least 70%;

4.53 Method 4, or any of 4.1-4.52, wherein the method results in an increase in the concentration of glucosylsphingosine in the CSF and/or plasma of at least 30% after 6 months of treatment, such as at least 40%, at least 50%, at least 60%, or at least 70%;

4.54 Method 4, or any of 4.1-4.53, where the method yields a statistically or clinically unchanged modified Severity Assessment Tool (mSST) value for neurological disorders after 6 months of treatment;

4.55 Method 4, or any of 4.1-4.54, wherein the compound of formula I (or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or 1.1-1.75) or a pharmaceutically acceptable salt or prodrug thereof is administered by systemic administration, for example, parenteral or non-parenteral;

4.56 Method 4.55, where the route of administration is oral (enteral);

4.57 Method 4.55, wherein the route of administration is parenteral, such as by injection, such as by intravenous injection.

4.58 Method 4, or any of 4.1-4.57, wherein the compound of formula I (or any of II-XII, Ia-XIIa or Ib-XIIb, or any of compounds 1 or 1.1-1.75), or a pharmaceutically acceptable salt or prodrug thereof, is administered by topical administration, such as topical administration;

4.59 Method 4, or any of 4.1-4.58, wherein the compound is (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate or quinuclidin-3-yl (2-(4'-fluoro-[1,1'-biphenyl]-3-yl)propan-2-yl)carbamate;

4.60 Method 4.59, wherein the dose of the compound is 15 mg/day when administered orally;

4.61 Method 4.60, wherein the dose of the compound is 15 mg/day as a single oral dose;

4.62 Method 4, or any of 4.1-4.61, wherein the subject is administered a single daily dose of 5 mg, 10 mg, 15 mg or 20 mg of a compound, such as (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate, optionally in the form of the acid addition malate.

In some embodiments of the present disclosure, a subject or entity is diagnosed with a particular disease or disorder, and is also diagnosed with a particular genetic mutation, such as one that is known to cause the disease or disorder in question, although it is often impossible to prove that the particular disease or disorder of the patient is caused by the particular mutation that has been diagnosed in the person. As used herein, the term "diagnosed with a particular genetic mutation" means that the subject or patient has been tested, such as by DNA or RNA sequencing, protein profiling, or other suitable means, and has been found to have the mutation in question. However, as discussed below, many genetic diseases and disorders may have multiple genetic causes (e.g., mutations), and patients may have multiple mutations, each of which may, under some circumstances, be sufficient to cause the disease or disorder without being proof that the particular mutation causes the particular disease or disorder in the particular patient.

The methods of method 4 et seq. may be beneficial for subjects who have been diagnosed with a lysosomal storage disease, such as Gaucher type 3 or Niemann-Pick type C, but who do not yet have the cognitive and/or ataxic symptoms associated with the disease state. The methods of method 4 et seq. may also be beneficial for subjects at risk for developing a lysosomal storage disease, such as Gaucher type 3 or Niemann-Pick type C, due to, for example, a mutation in the subject or a family history that is known to cause such a disease. Therefore, in some embodiments of the methods described herein, the subject is diagnosed as being at risk for developing such a disease or disorder, and the method prevents or delays the onset and/or development of cognitive and/or ataxic symptoms of the disease or disorder (e.g., supranuclear gaze palsy) in the subject. In some embodiments, the subject is diagnosed as being at risk of developing said disease or disorder through the presence of a mutation in a gene as described herein.

Pharmaceutical compositions

Also provided herein are pharmaceutical compositions comprising at least one quinuclidine compound as described herein and at least one pharmaceutically acceptable excipient, e.g., for use in accordance with the methods described herein. The pharmaceutically acceptable excipient can be any such excipient known in the art, including those described in, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro ed. 1985). Pharmaceutical compositions of the compounds described herein can be prepared by conventional methods known in the art, e.g., by mixing at least one compound described herein with a pharmaceutically acceptable excipient.

Thus, in one aspect, the present disclosure provides a pharmaceutical dosage form comprising a quinuclidine compound as described herein and a pharmaceutically acceptable excipient, wherein the dosage form is formulated to provide upon administration (e.g., upon oral administration) an amount of said compound sufficient to treat a disease or disorder described herein (e.g., in any of Method 1 et seq. or Method 4 et seq.).

A pharmaceutical composition or dosage form according to the invention may comprise an agent and another carrier, for example, a compound or composition, inert or active, such as a detectable agent, a label, an adjuvant, a diluent, a binder, a stabilizer, buffers, salts, lipophilic solvents, a preservative, an adjuvant or the like. Carriers also include pharmaceutical excipients and additives, for example, proteins, peptides, amino acids, fats and carbohydrates (for example, sugars, including monosaccharides, di-, tri-, tetra- and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which may be present alone or in combination, including alone or in combination from 1 to 99.99% by weight or by volume. Examples of protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein and the like. Exemplary amino acid/antibody components that can also function as a buffering agent include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame and the like. Carbohydrate excipients are also within the scope of the present invention, examples thereof include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose and the like; polysaccharides such as raffinose, melezitose, maltodextrins, dextrans, starches and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol) and myoinositol.

Carriers that can be used include a buffer or pH adjusting agent; typically, the buffer is a salt derived from an organic acid or base. Typical buffers include salts of organic acids such as citric acid, ascorbic acid, gluconic acid, carboxylic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Additional carriers include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavorings, antimicrobial agents, sweeteners, antioxidants, antistatic agents. surfactants (e.g. polysorbates such as TWEEN 20 and TWEEN 80), fats (e.g. phospholipids, fatty acids), steroids (e.g. cholesterol) and chelating agents (e.g. EDTA).

The present disclosure also provides pharmaceutical compositions and kits comprising the compositions, which comprise at least one quinuclidine compound disclosed herein and at least one other pharmaceutically active agent. These pharmaceutical compositions and kits can be adapted for simultaneous, sequential, and/or separate administration of the quinuclidine compound and the additional active agent. For example, the quinuclidine compound and the additional active agent can be formulated as separate dosage forms, such as in separate tablets, capsules, lyophilisates, or liquids, or they can be formulated in the same dosage form, such as in the same tablet, capsule, lyophilisate, or liquid. If the quinuclidine compound and the additional active agent are formulated in the same dosage form, the quinuclidine compound and the additional active agent may be present substantially in admixture, such as in a tablet core, or they may be present substantially in separate regions of the dosage form, such as in different layers of the same tablet. In one embodiment, the pharmaceutical dosage form comprises an additional agent that is capable of treating or preventing supranuclear gaze palsy, such as in a patient having, diagnosed with, or predisposed to a lysosomal storage disease, such as Gaucher type 3 or Niemann-Pick type C, or pain, such as in a patient diagnosed with, or predisposed to, a lysosomal storage disease, such as Fabry disease, as described herein.

In a further aspect, provided herein is a pharmaceutical composition comprising: (i) a quinuclidine compound as described herein; (ii) an additional active agent; and (iii) a pharmaceutically acceptable excipient. In one embodiment, the additional active agent is an agent that is capable of treating or preventing supranuclear gaze palsy, gait disorder, or cognitive dysfunction (e.g., dementia), for example, in a patient having, diagnosed with, or predisposed to a lysosomal storage disease such as Gaucher type 3 or Niemann-Pick type C, as described herein.

The disclosed quinuclidine compounds and pharmaceutical compositions can be used in animals or humans. Thus, the disclosed compound can be formulated as a pharmaceutical composition for oral, buccal, parenteral (e.g., intravenous, intramuscular, or subcutaneous), topical, rectal, or intranasal administration, or in a form suitable for administration by inhalation or insufflation. In particular embodiments, the quinuclidine compound or pharmaceutical composition is formulated for systemic administration, e.g., by a non-parenteral route. In one embodiment, the quinuclidine compound or pharmaceutical composition is formulated for oral administration, e.g., in solid form. Such routes of administration and methods for preparing corresponding pharmaceutical compositions are described, for example, in Gibaldi's Drug Delivery Systems in Pharmaceutical Care (1 ^st ed., American Society of Health-System Pharmacists 2007).

The pharmaceutical compositions can be formulated to provide slow, sustained or controlled release of the active ingredient using, for example, hydroxypropyl methylcellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. The pharmaceutical compositions can also optionally contain opacifying agents, and can be of a composition that releases the active ingredients only or preferentially in a certain part of the gastrointestinal tract, optionally in a delayed manner, for example, by an enteric coating. Examples of sealing compositions include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if desired, with one or more pharmaceutically acceptable carriers, excipients or diluents well known in the art (see, for example, Remington's). The disclosed compounds can be formulated for continuous delivery by methods well known to those skilled in the art. Examples of such compositions can be found in U.S. Patents 3,119,742; 3,492,397; 3,538,214; 4,060,598; and 4,173,626.

In solid dosage forms for oral administration (e.g. capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients or diluents, such as sodium citrate or dicalcium phosphate and/or any of the following: (1) fillers or diluents, such as starches, lactose, sucrose, glucose, mannitol, microcrystalline cellulose, calcium phosphate and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, pregelatinized corn starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, sodium starch glycolate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) dissolution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, sodium lauryl sulfate, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricating agents, such as talc, silicon dioxide, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions can also contain buffering agents. Solid compositions of a similar type can also be prepared using fillers in soft and hard gelatin capsules and excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more additional ingredients. Compressed tablets may be prepared using binding agents (e.g., gelatin or hydroxypropyl methylcellulose), lubricating agents, inert diluents, preservatives, disintegrating agents (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants and/or dispersing agents. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. Tablets and other solid dosage forms such as dragees, capsules, pills and granules may optionally be scored or prepared with coatings and shells such as enteric coatings and other coatings well known in the art.

In embodiments, the pharmaceutical compositions are administered orally in liquid form. Liquid dosage forms for oral administration of the active ingredient include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid preparations for oral administration may be presented as a dry product for reconstitution with water or other suitable vehicle before use. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (e.g., cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and mixtures thereof. In addition to inert diluents, liquid pharmaceutical compositions may include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, coloring agents, perfumes and preservatives and the like. Suspensions, in addition to the active ingredient, may contain suspending agents such as, but not limited to, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Suitable liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as a suspending agent (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); an emulsifier (e.g., lecithin or acacia); a non-aqueous vehicle (e.g., almond oil, oily esters or ethyl alcohol); and/or a preservative (e.g., methyl or propyl parahydroxybenzoates or sorbic acid). The active ingredients may also be administered as a bolus, electuary or paste.

For buccal administration, the composition may take the form of tablets or lozenges prepared in the usual manner.

In embodiments, the pharmaceutical compositions are administered by non-oral means, such as topical application, transdermal application, injection, and the like. In related embodiments, the pharmaceutical compositions are administered parenterally by injection, infusion, or implantation (e.g., intravenously, intramuscularly, intraarterially, subcutaneously, etc.).

The disclosed compounds may be formulated for parenteral administration by injection, including using conventional catheterization or infusion techniques. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain a formulatory agent such as a suspending, stabilizing and/or dispersing agent recognized by those skilled in the art. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, for example, sterile, pyrogen-free water, before use.

The pharmaceutical compositions can be administered directly into the central nervous system. Accordingly, in some embodiments, the compositions are administered directly into the central nervous system to avoid the blood-brain barrier. In some embodiments, the composition can be administered via direct injection into the spinal cord. In embodiments of the invention, the composition is administered by intrathecal injection. In some embodiments, the composition is administered by intracerebroventricular injection. In embodiments, the composition is administered into the cerebral lateral ventricle. In embodiments, the composition is administered into both cerebral lateral ventricles. In further embodiments, the composition is administered by intrahippocampal injection. The compositions can be administered by a single injection or by multiple injections. In other embodiments, the composition is administered to more than one site (e.g., two sites in the central nervous system).

The pharmaceutical compositions may be in the form of sterile injections. The pharmaceutical compositions may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water or some other sterile injection medium immediately before use. To prepare such a composition, the active ingredient is dissolved or suspended in a parenterally acceptable liquid carrier. Typical carriers and solvents include, but are not limited to, water, water adjusted to a suitable pH by adding an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution. The pharmaceutical composition may also contain one or more preservatives, for example, methyl, ethyl or n-propyl para-hydroxybenzoate. To improve solubility, a dissolution enhancing or solubilizing agent may be added, or the solvent may contain 10-60% by weight of propylene glycol or the like.

The pharmaceutical compositions may contain one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders which can be reconstituted into sterile injection solutions or dispersions immediately before use. Such pharmaceutical compositions may contain antioxidants; buffers; bacteriostats; solutes which render the composition isotonic to the blood of the intended recipient; suspending agents; thickening agents; preservatives; and the like.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants. In some embodiments, in order to prolong the action of the active ingredient, it is desirable to slow the absorption of the compound upon subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material having poor solubility in water. The rate of absorption of the active ingredient then depends on the rate of its dissolution, which in turn can depend on the size of the crystals and the crystalline form. Alternatively, delayed absorption of the parenterally administered active ingredient is achieved by dissolving or suspending the compound in an oil vehicle. Additionally, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

The parenteral controlled release compositions may be in the form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, emulsions, or the active ingredient may be incorporated into biocompatible carriers, liposomes, nanoparticles, implants or infusion devices. Materials for use in the manufacture of microspheres and/or microcapsules include, but are not limited to, biodegradable/biodegradable polymers such as polyglactin, poly(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamine) and poly(lactic acid). Biocompatible carriers that can be used in the preparation of a parenteral controlled release formulation include carbohydrates such as dextrans, proteins such as albumin, lipoproteins or antibodies. Materials for use in implants may be non-biodegradable, such as polydimethylsiloxane, or biodegradable, such as poly(caprolactone), poly(lactic acid), poly(glycolic acid), or poly(orthoesters).

For topical application, the described compound may be formulated as an ointment or cream. The described compounds may also be formulated in rectal compositions such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides.

For intranasal or inhalation administration, the disclosed compounds may conveniently be delivered in the form of a solution or suspension from a pump-action nebulizer that is squeezed or pumped by the patient, or as an aerosol spray from a pressurized container or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the unit dose can be determined by providing a valve for delivering a metered amount. The pressurized container or nebulizer may contain a solution or suspension of the disclosed compound. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the disclosed compound and a suitable powder base, such as lactose or starch.

Typically, the agents and compositions described herein are administered in an effective amount or an amount sufficient to treat or prevent supranuclear gaze palsy in a subject in need thereof. Typically, the dose can be adjusted within this range based on, for example, age, physical condition, body weight, sex, diet, time of administration, and other clinical factors. Determination of an effective amount is within the skill of the art.

While having been generally described herein, the following non-limiting examples are presented to further illustrate the invention.

EXAMPLES

General methods of chemical synthesis

General Procedure A: Formation of Carbamate with Triphosgene

Triphosgene (0.35 equivalent) was added to a suspension of amine hydrochloride (1 equivalent) and triethylamine (3-4 equivalents) in THF (~0.2 M concentration) at room temperature. The reaction mixture was stirred for 10 min and a small amount of ether (1-2 ml) was added. The triethylammonium salt was filtered off to give a clear solution of the isocyanate in THF/ether.

To a solution of the alcohol (1.5 equivalents) in THF (concentration ~0.2 M) at room temperature was added NaH [60%, oil] (1.5 equivalents). The reaction mixture was stirred for 15 min and the above solution (isocyanate in THF/ether) was added dropwise. During standard workup, the reaction was quenched with brine. The solution was extracted with EtOAc and the organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified on combiflash (SiO ₂ cartridge, CHCl ₃ and 2N NH ₃ in MeOH) to afford the corresponding carbamate.

General Procedure B: Alkylation with an Organocerium Compound

A suspension of _CeCl3 (4 equivalents) in THF (~0.2 M concentration) was stirred at room temperature for 1 h. The suspension was cooled to -78 °C and MeLi/ether [1.6 M] (4 equivalents) was added dropwise. The organocerium complex formed within 1 h and a solution of the nitrile (1 equivalent) in THF (2.0 M concentration) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 18 h. The solution was cooled to 0 °C and quenched with water (~1 mL), then 50% aqueous ammonium hydroxide solution (~3 mL) was added until a precipitate formed and settled to the bottom of the flask. The mixture was filtered through a pad of celite and concentrated. The crude product was triturated with HCl/dioxane solution [4.0 M]. The intermediate arylpropan-2-amine hydrochloride is triturated with ether and used as is in the next step. Alternatively, the crude amine free base is purified on a combiflash ( _SiO2 cartridge, _CHCl3 and 2N _NH3 in MeOH) to give the corresponding arylpropylamine.

General Method C: Suzuki Combination

To a solution of the aryl halide (1 equivalent) in DME/water [4:1] (~0.2 M concentration) were added boronic acid (2 equivalents), palladium catalyst (0.1-0.25 equivalents), and sodium carbonate (2 equivalents). The reaction mixture was microwaved for 25 min at 150°C. After filtration through a pad of celite and concentration, the crude product was purified on a combiflash ( _SiO2 cartridge, _CHCl3 , and 2N _NH3 in MeOH) to afford the corresponding coupling product.

Alternatively, to a solution of the aryl halide (1 equivalent) in toluene/water [20:1] (~0.2 M concentration) are added boronic acid (1.3-2.5 equivalents), palladium catalyst (0.05-0.15 equivalents), tricyclohexylphosphine (0.15-0.45 equivalents), and potassium phosphate (5 equivalents). The reaction mixture is microwaved for 25 min at 150°C. After filtration through a pad of celite and concentration, the crude product is purified on a combiflash ( _SiO2 cartridge, _CHCl3 , and 2N _NH3 in MeOH) to afford the corresponding coupling product.

General Procedure D: Cyclopropanation

To a mixture of aryl nitrile (1 equivalent) and Ti(Oi-Pr) ₄ (1.7 equivalents) stirred at -70 °C was added dropwise EtMgBr [3.0 M in ether] (1.1 equivalents). The reaction mixture was warmed to 25 °C and stirred for 1 h. To the above mixture was added BF ₃ Et ₂ O (3 equivalents) dropwise at 25 °C. After addition, the mixture was stirred for another 2 h and then quenched with aqueous HCl [2 M]. The resulting solution was then made basic by adding aqueous NaOH [2 M]. The organic product was extracted with ethyl ether. The organic layers were combined, dried over Na ₂ SO ₄ , filtered and concentrated. The crude product is purified by column chromatography on silica gel (eluting with petroleum ether/EtOAc: 10/1 to 1/1) to give the corresponding 1-arylcyclopropanamine.

General Procedure E: Biaryl Coupling Using Suzuki Conditions

To a stirred solution of the aryl halide component (1 equivalent) in 5:1 (v/v) dioxane/water (~0.15 M) or 5:1 (v/v) were added N,N-dimethylformamide (~0.15 M), aryl boronate or aryl boronic acid component (1-1.5 equivalents), sodium carbonate (2-3 equivalents), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.05 equivalents). The mixture was heated (90°C) overnight and then filtered through a pad of celite. The celite was washed with ethyl acetate and the combined filtrate was washed with brine, dried (Na ₂ SO ₄ ), and concentrated. The residue was purified by flash chromatography over silica.

General Procedure F: Preparation of a Carbamate Using an Isocyanate Created via the Mixed Anhydride/Curtius Rearrangement Route

To a stirred solution of the carboxylic acid component (1 equivalent) in tetrahydrofuran (~0.1 M) was added triethylamine (2 equivalents). The reaction mixture was cooled (0 °C) and treated with isobutyl chloroformate (1.5 equivalents). After 1 h at 0 °C, a solution of sodium azide (2 equivalents) in water (~1 M) was added and the reaction mixture was warmed to room temperature. After stirring overnight, the reaction mixture was diluted with water and extracted with ethyl acetate. The combined extracts were washed with aqueous sodium bicarbonate and brine, dried (Na ₂ SO ₄ ), and concentrated. The crude acyl azide was further dried by coevaporation with toluene and then taken up in toluene (~0.1 M). The stirred solution is heated at reflux for 2-2.5 h, cooled, and treated with the alcohol component (1.25-2 equivalents). The reaction mixture is heated at reflux overnight and then concentrated. The residue is taken up in either ethyl acetate or chloroform and washed with aqueous sodium carbonate (Na ₂ SO ₄ ) and concentrated. The crude product is purified by flash chromatography over silica using solvent gradients of chloroform/methanol (less polar carbamates) or chloroform/methanol/ammonia (more polar carbamates).

Example 1: Synthesis of quinuclidine compounds

1-azabicyclo[2,2,2]oct-3-yl [2-(4'-fluorobiphenyl-3-yl)propan-2-yl]carbamate (Compound 1)

Using General Procedure C, 1-azabicyclo[2,2,2]oct-3-yl [2-(3-bromophenyl)propan-2-yl]carbamate (600 mg, 1.63 mmol), 4-fluorophenylboronic acid (457 mg, 3.27 mmol) and palladium(II) acetate gave the title compound as a white solid (373 mg; 60%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.56 (s, 1H), 7.52 (dd, J=5.4, 8.4 Hz, 2H), 7.42-7.38 (m, 3H), 7.12 (m, 2H), 5.18 (5, 1H), 4.62 (s, 1H), 2.66 (m, 6H), 1.72 (s, 6H), 2.01-0.83 (m, 5H) ppm. ¹³ C NMR (100 MHz, CDCl ₃ ) δ 125.0, 124.0, 123.8, 116.0, 116.0, 71.3, 55.9, 55.5, 47.6, 46.7, 29.6, 25.6, 24.8, 19.8 ppm. Purity: 98.0% ULCMS (210 nm); retention time 0.95 min; (M+1) 382.9. Anal. Calc. for C ₂₃ H ₂₇ FN ₂ O ₂ 0.37 (CHCl ₃ ): C, 65.86; H, 6.47; N, 6.57. Found: C, 65.85; H, 6.69; N, 6.49.

(S)-quinuclidin-3-yl 2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-ylcarbamate (Compound 2)

To a stirred solution of 4-fluorothiobenzamide (8.94 g, 57.6 mmol) in ethanol (70 mL) was added ethyl 4-chloroacetoacetate (7.8 mL, 58 mmol). The reaction mixture was heated at reflux for 4 h, quenched with an aliquot of ethyl 4-chloroacetoacetate (1.0 mL, 7.4 mmol) and heated at reflux for an additional 3.5 h. The reaction mixture was then concentrated and the residue was partitioned between ethyl acetate (200 mL) and aqueous NaHCO ₃ (200 mL). The organic layer was combined with the back extract of the aqueous layer (ethyl acetate, 1 x 75 mL), dried (Na ₂ SO ₄ ) and concentrated. The resulting amber oil was purified by flash chromatography using a hexane/ethyl acetate gradient to give ethyl 2-(2-(4-fluorophenyl)thiazol-4-yl)acetate as a low melting point, nearly colorless solid (13.58 g, 89%).

To a stirred solution of ethyl 2-(2-(4-fluorophenyl)thiazol-4-yl)acetate (6.28 g, 23.7 mmol) in DMF (50 mL) was added sodium hydride [60% dispersion in mineral oil] (2.84 g, 71.0 mmol). The foamy mixture was stirred for 15 min, then cooled in an ice bath and iodomethane (4.4 mL, 71 mmol) was added. The reaction mixture was stirred overnight, allowing the cooling bath to warm slowly to room temperature. The mixture was then concentrated and the residue was partitioned between ethyl acetate (80 mL) and water (200 mL). The organic layer was washed with a second portion of water (1 x 200 mL), dried (Na ₂ SO ₄ ) and concentrated. The resulting amber oil was purified by flash chromatography using a hexane/ethyl acetate gradient to give ethyl 2-(2-(4-fluorophenyl)thiazol-4-yl)-2-methylpropanoate as a colorless oil (4.57 g, 66%).

To a stirred solution of ethyl 2-(2-(4-fluorophenyl)thiazol-4-yl)-2-methylpropanoate (4.56 g, 15.5 mmol) in 1:1:1 THF/ethanol/water (45 mL) was added lithium hydroxide monohydrate (2.93 g, 69.8 mmol). The reaction mixture was stirred overnight, concentrated and redissolved in water (175 mL). The solution was washed with ether (1 x 100 mL), acidified by adding 1.0 N HCl (80 mL) and extracted with ethyl acetate (2 x 70 mL). The combined extracts were dried (Na ₂ SO ₄ ) and concentrated to give 2-(2-(4-fluorophenyl)thiazol-4-yl)-2-methylpropanoic acid as a white solid (4.04 g, 98%). This product is used in the next step without purification.

To a stirred and cooled (0°C) solution of 2-(2-(4-fluorophenyl)thiazol-4-yl)-2-methylpropanoic acid (4.02 g, 15.2 mmol) in THF (100 mL) was added trimethylamine (4.2 mL, 30 mmol) followed by isobutyl chloroformate (3.0 mL, 23 mmol). The reaction mixture was stirred cold for an additional 1 h then a solution of sodium azide (1.98 g, 30.5 mmol) in water (20 mL) was added. The reaction mixture was stirred overnight allowing the cooling bath to warm slowly to room temperature. The mixture was then diluted with water (100 mL) and extracted with ethyl acetate (2 x 60 mL). The combined extracts were washed with aqueous NaHCO ₃ (1 x 150 mL) and brine (1 x 100 mL), dried (Na ₂ SO ₄ ) and concentrated. After co-evaporation with toluene (2 x 50 mL), the resulting white solid was taken up in toluene (100 mL) and heated at reflux for 4 h. (S)-3-quinuclidinol (3.87 g, 30.4 mmol) was then added and reflux was continued overnight. The reaction mixture was concentrated and the residue was partitioned between ethyl acetate (100 mL) and aqueous NaHCO ₃ (150 mL). The organic layer was washed with water (1 x 150 mL), dried (Na ₂ SO ₄ ) and concentrated. The resulting off-white solid was purified by flash chromatography using a chloroform/methanol/ammonia gradient to afford the title compound as a white solid (4.34 g, 73%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.96-7.88 (m, 2H), 7.16-7.04 (m, 3H), 5.55 (br s, 1H), 4.69-4.62 (m, 1H), 3.24-3.11 (m, 1H), 3.00-2.50 (m, 5H), 2.01-1.26 (m, 11H) ppm. ¹³ C NMR (400 MHz, CDCl ₃ ) δ 166.4, 165.1, 163.8 (d, J=250.3 Hz), 162.9, 155.0, 130.1 (d, J=3.3 Hz), 128.4 (d, J= 8.5 Hz), 115.9 (d, J= 22.3 G c), 112.5, 71.2, 55.7, 54.2, 47.5, 46.5, 28.0, 25.5, 24.7, 19.6 ppm. Purity: 100% ULCMS (210 nm and 254 nm); retention time 0.83 min; (M+1) 390.

(S)-quinuclidin-3-yl (2-(4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 3)

Using General Procedure E and introducing ethyl 2-(4-bromophenyl)-2-methylpropanoate and 4-(2-methoxyethoxy)phenylboronic acid into the reaction mixture, ethyl 2-(4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)-2-methylpropanoate was obtained as an off-white solid. To a stirred solution of this compound (3.01 g, 8.78 mmol) in 1:1:1 (v/v/v) tetrahydrofuran/ethanol/water (45 mL) was added lithium hydroxide monohydrate (1.47 g, 61.4 mmol). The mixture was heated at reflux overnight and then concentrated. The residue was dissolved in water, treated with 1N hydrochloric acid (65 mL), and extracted with ethyl acetate. The combined organic layers were washed with _brine , dried ( _Na2SO4 ) and concentrated to give 2-(4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)-2-methylpropanoic acid as a white solid (2.75 g, 100%). This intermediate and (S)-quinuclidin-3-ol were reacted according to General Procedure F to give the title compound as a colourless glassy solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.62-7.29 (m, 7H), 7.01 (d, J=8.9 Hz, 2H), 4.47-4.37 (m, 1H), 4.17-4.08 (m, 2H), 3.72-3.62 (m, 2H), 3.32 (s 3H), 3.09-2.25 (m, 6H), 2.05-1.18 (m, 11H) ppm. ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 157.9, 154.5, 146.7, 137.4, 132.5, 127.5, 125.7, 125.2, 114.8, 70.4, 70.0, 66.9, 58.2, 55.4, 54 ,2, 46.9, 45.9, 29.4, 25.3, 24.2, 19.2 ppm. Purity: 100%, 100% (210 and 254 nm) ULCMS; retention time: 0.87 min; (M+H ⁺ ) 439.5.

1-azabicyclo[2,2,2]oct-3-yl [2-(biphenyl-3-yl)propan-2-yl]carbamate (Compound 4)

Using General Procedure C, 1-azabicyclo[2,2,2]oct-3-yl [2-(3-bromophenyl)propan-2-yl]carbamate (600 mg, 1.63 mmol), phenylboronic acid (398 mg, 3.27 mmol) and palladium(II) acetate gave the title compound as a white solid (379 mg, 64%). ^1H NMR (400 MHz, CDCl ₃ ) δ 7.61 (s, 1H), 7.56 (d, J= 7.4 Hz, 2H), 7.50-7.38 (m, 4H), 7.34 (m, 2H), 5.16 (s, 1H), 4.63 (s, 1H), 3.39-2.09 (m, 6H), 1.72 (s, 6H), 2.02-0.73 (m, 5H) ppm. ¹³ C NMR (100 MHz, CDCl ₃ ) δ 154.8, 147.8, 141.6, 129.0, 129.0, 128.6, 127.5, 125.8, 125.0, 124.0, 71.6, 71.3, 55.9, 55.5, 47.6, 46.8, 31.5, 30.2, 30.0, 29.5, 25.6, 24.8, 19.8 ppm. Purity: 99% LCMS (210 nm); retention time 0.84 min; (M+1) 365.0. Anal. calc. for C ₂₃ H ₂₈ N ₂ O ₂ 0.29 (CHCl ₃ ): C, 70.02; H, 7.14; N, 7.01. Found: C, 70.02; H, 7.37; N, 6.84.

(S)-quinuclidin-3-yl 2-(biphenyl-4-yl)propan-2-ylcarbamate (Compound 5)

Using General Procedure B, bromobenzonitrile (2.00 g, 11.0 mmol) was converted to the corresponding 2-(4-bromophenyl)propan-2-amine (1.20 g, 51%) as a brown oil.

Using General Procedure A, 2-(4-bromophenyl)propan-2-amine (1.0 g, 4.7 mmol) and (S)-quinuclidin-3-ol afforded (S)-quinuclidin-3-yl 2-(4-bromophenyl)propan-2-ylcarbamate (1.0 g, 58%) as a brown oil.

Using General Procedure C, the above bromide (200 mg, 0.540 mmol), phenylboronic acid (133 mg, 1.10 mmol) and [PdCl ₂ (pddf)]CH ₂ Cl ₂ gave the title compound as a white solid (70 mg, 35%). ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.60-7.53 (m, 4H), 7.47 (d, J=8.5 Hz, 2H), 7.42 (t, J=7.5 Hz, 2H), 7.33 (t, J=7.5 Hz, 1H), 5.26 (shs, 1H), 4.64 (m, 1 H), 3.33-3.15 (m, 1H), 3.10-2.45 (m, 5H), 2.40-1.80 (m, 2H), 1.78-1.58 (m, 7H), 1.55-1.33 (m, 2H) ppm. ¹³ C NMR (125 MHz, CDCl ₃ ) δ 154.5, 146.1, 140.8, 139.5, 128.7, 127.2, 127.1, 127.1, 125.2, 70.9, 55.5, 55.1, 47.4, 46.4, 31.1, 29.5, 25.3, 24.5, 19.5 ppm. Purity: 100% LCMS (214 nm and 254 nm); retention time 1.56 min; (M+1) 365.

Quinuclidin-3-yl 1-(biphenyl-4-yl)cyclopropylcarbamate (Compound 6)

Using General Procedure D, bromobenzonitrile (3.00 g, 16.5 mmol) was converted to the corresponding 1-(4-bromophenyl)cyclopropanamine (1.80 g, 51%) as a yellow solid.

Using General Procedure A, 1-(4-bromophenyl)cyclopropanamine (1.0 g, 4.7 mmol) and quinuclidin-3-ol afforded quinuclidin-3-yl 1-(4-bromophenyl)cyclopropylcarbamate (1.3 g, 75%) as a white semi-solid.

Using General Procedure C, the above carbamate (400 mg, 1.12 mmol), phenylboronic acid (267 mg, 2.22 mmol) and [PdCl ₂ (pddf)]CH ₂ Cl ₂ gave the title compound as a viscous oil (100 mg, 25%). ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.47 (d, J = 7.5 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 7.26-7.15 (m, 3H), 5.93 (br s, 0.6H), 5.89 (br s, 0.4H), 4.67 (m, 1H), 3.20-3.06 (m, 1H), 2.88-2.42 (m, 5H), 1.98-1.08 (m, 9H) ppm. ¹³ C NMR (125 MHz, CDCl ₃ ) δ 155.0, 141.0, 139.7, 138.2, 127.7, 126.1, 126.0, 124.8, 124.1, 70.0, 54.5, 46.3, 45.4, 34.1, 24.3, 23.2, 18.3, 17.0 ppm. Purity: 100% LCMS (214 nm and 254 nm); retention time 1.52 min; (M+1) 363.

(S)-quinuclidin-3-yl 1-(4'-fluorobiphenyl-4-yl)cyclopropylcarbamate (Compound 7)

Using the General Method C, (S)-quinuclidin-3-yl 1-(4-bromophenyl)cyclopropyl carbamate, 4-F-phenylboronic acid and [PdCl ₂ (pddf)]CH ₂ Cl ₂ gave the title compound as a white solid (45%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.06-7.83 (d, 1H), 7.69-7.66 (m, 2H), 7.59-7.55 (m, 2H), 7.29-7.22 (m, 4H), 4.56-4.54 (m, 1H), 3.13-2.32 (m, 6H), 1.91-1.19 (m, 9H) ppm. ¹³ C NMR (125 MHz, DMSO-d ₆ ) δ 163.2, 161.2, 156.4, 143.7, 136.9, 128.9, 128.8, 126.8, 125.6, 116.2, 116.0, 70.7, 55.8, 47.4, 46.4, 34.8, 25.7, 24.6, 19.6, 18.7, 18.6 ppm. Purity: > 97% LCMS (214 nm and 254 nm); retention time 1.96 min; (M+1) 381.2.

(S)-1-azabicyclo[2,2,2]oct-3-yl [1-(2',4'-difluorobiphenyl-4-yl)cyclopropyl]carbamate (Compound 8)

Using the general procedure C, (S)-quinuclidin-3-yl 1-(4-bromophenyl)cyclopropylcarbamate (0.446 g, 1.22 mmol), 2,4-difluorophenylboronic acid (0.386 g, 2.44 mmol) and Pd(OAc) ₂ (0.015 g, 0.067 mmol) gave the title compound as a tan solid (0.111 g, 23%). ¹ H NMR (CDCl ₃ ) δ 7.43 (dd, J=8.4, 1.6 Hz, 2H), 7.40-7.33 (m, 1H), 7.31 (d, J=7.7 Hz, 2H), 6.99-6.81 (m, 2H), 5.54 (d, J=48.0 Hz, 1H), 4.82- 4.65 (m, 1H), 3.30-3.07 (m, 1H), 2.98-2.44 (m, 5H), 1.97 (d, J=32.7 Hz, 1H), 1.83 (d, J=10.3 Hz, 1H), 1.64 (s, 1H), 1.52 (s, 1H), 1.39 (s, 1H), 1.31 (d, J=6.8 Hz, 4H) ppm. ¹³ C NMR main rotomer (CDCl ₃ ) δ 162.2 (dd, J=12.8, 249.1 Hz), 159.8 (dd, J=11.8, 251.0 Hz), 156.9, 156.0, 142.6, 133.1, 131.3 (m), 128.9, , 124.9, 111.5 (dd, J=3.9, 21.2 Hz) 104.4 (dd, J=25.2, 29.4 Hz), 72.1, 71.6, 55.7, 47.4, 46.5, 35.7, 35.3, 25.5, 24.6, 24.4, 19.5, 18.1 ppm. Purity: LCMS >99.3% (214 nm and 254 nm); retention time 0.90 min; (M+1) 399.0.

1-azabicyclo[2,2,2]oct-3-yl [1-(4'-methoxybiphenyl-4-yl)cyclopropyl]carbamate (Compound 9)

Using General Procedure C, quinuclidin-3-yl 1-(4-bromophenyl)cyclopropylcarbamate (0.485 g, 1.33 mmol), 4-methoxyphenylboronic acid (0.404 g, 2.66 mmol) and Pd(OAc) ₂ (0.016 g, 0.071 mmol) gave the title compound as a gray solid (0.337 mg, 65%). ¹ H NMR (CDCl ₃ ) δ 7.48 (dd, J=8.6, 5.5 Hz, 4H), 7.29 (d, J=7.6 Hz, 2H), 6.96 (d, J=8.8 Hz, 2H), 5.58 (d, J=48.7 Hz, 1H), 4.83-4.63 (m, 1H), 3.84 (s, 3H), 3.20 (dd, J=24.0, 15.5 Hz, 1H), 2.97-2.42 (m, 5H), 1.97 (d, J=30.9 Hz, 1H), 1.81 (s, 1H), 1.75-1.33 (m, 3H), 1.28 (d, J=6.8 Hz, 4H) ppm. ¹³ C NMR major rotomers (CDCl ₃ ) δ 159.1, 156.0, 141.4, 139.0, 133.4, 128.0, 126.7, 125.9, 114.2, 71.5, 55.7, 55.3, 47.4, 46.5, 35.3, 25.5, 24.6, 19.6, 17.8 ppm. Purity: LCMS >97.1% (214 nm and 254 nm); retention time 0.88 min; (M+1) 393.4.

Quinuclidin-3-yl 2-(5-(4-fluorophenyl)thiophen-3-yl)propan-2-ylcarbamate (Compound 10)

To a stirred and cooled (0°C) solution of ethyl 5-bromothiophene-3-carboxylate (13.30 g, 56.57 mmol) in THF (100 mL) was added a solution of methylmagnesium bromide in diethyl ether [3.0 M] (55.0 mL, 165 mmol) dropwise over 20 min. After 2 h, the reaction solution was concentrated. The residue was taken up in aqueous NH ₄ Cl (200 mL) and extracted with ethyl acetate (2×100 mL). The combined extracts were dried (Na ₂ SO ₄ ) and concentrated. The resulting amber oil was purified by flash chromatography using a hexane/ethyl acetate gradient to give 2-(5-bromothiophen-3-yl)propan-2-ol as a pale amber oil (8.05 g, 64%).

To a stirred solution of 2-(5-bromothiophen-3-yl)propan-2-ol (8.03 g, 36.3 mmol) in methylene chloride (80 mL) was added sodium azide (7.08 g, 109 mmol) followed by trifluoroacetic acid (8.0 mL; dropwise over 5–6 min). The thickening suspension was stirred for 1.5 h, then diluted with water (350 mL) and extracted with ethyl acetate (1 x 200 mL). The organic layer was washed with aqueous NaHCO ₃ (1 x 250 mL), dried (Na ₂ SO ₄ ), and concentrated to give the crude azide product. To a stirred solution of this product in THF (160 mL) was added water (11 mL) followed by triphenylphosphine (23.8 g, 90.7 mmol). The reaction mixture was stirred for 2 days and then concentrated. The resulting residue was dissolved in ethyl acetate (250 mL) and extracted with 1 N aqueous HCl (4 x 75 mL). The combined extracts were basified with concentrated NH ₄ OH and extracted with ethyl acetate (2 x 100 mL). These extracts were in turn dried (Na ₂ SO ₄ ) and concentrated. The resulting amber oil was purified by flash chromatography using a methylene chloride/methanol/ammonia gradient to give a mixture of 2-(5-bromothiophen-3-yl)propan-2-amine and triphenylphosphine oxide (~70/30 ratio) as a viscous amber oil (1.32 g, 17%).

To a stirred solution of 3-quinuclidinol (3.00 g, 23.6 mmol) in THF (100 mL) was added 4-nitrophenyl chloroformate (5.94 g, 29.5). After stirring for 4 h, the precipitate was filtered, washed with THF, and air-dried on a frit under laboratory vacuum. The filter cake was dissolved in ethyl acetate (150 mL) and washed with aqueous NaHCO ₃ (1 x 150 mL) and water (2 x 150 mL). The organic layer was dried (Na ₂ SO ₄ ) and concentrated to give crude 4-nitrophenylquinuclidin-3-yl carbonate, which was used in the next step without purification.

To a stirred solution of 2-(5-bromothiophen-3-yl)propan-2-amine (0.366 g, 1.66 mmol) in THF (10 mL) were added 4-nitrophenylquinuclidin-3-yl carbonate (0.571 g, 1.95 mmol) and a few pellets of 4-(dimethylamino)pyridine. The mixture was refluxed overnight, concentrated, and partitioned between ethyl acetate (50 mL) and aqueous NaHCO ₃ (50 mL). The organic layer was washed again with aqueous NaHCO ₃ (1 x 50 mL), dried (Na ₂ SO ₄ ), and concentrated. The resulting dirty yellow gum was purified by flash chromatography using a chloroform/methanol/ammonia gradient to give quinuclidin-3-yl (1-(5-bromothiophen-3-yl)cyclopropyl)carbamate as an off-white solid (0.305 g, 49%).

Using General Procedure C, quinuclidin-3-yl (1-(5-bromothiophen-3-yl)cyclopropyl)carbamate (0.227 g, 0.742 mmol), 4-fluorophenylboronic acid (0.208 g, 1.49 mmol), tricyclohexylphosphine (0.021 g, 0.075 mmol), potassium phosphate (0.866, 4.08 mmol), and palladium acetate (8.0 mg, 36 μmol) gave the title compound as a gray solid (0.142 g, 49%). ^1H NMR (400 MHz, CDCl ₃ ) δ 7.60-7.45 (m, 2H), 7.24-7.19 (m, 1H), 7.10-6.97 (m, 3H), 5.23 (bs, 1H), 4.72-4.61 (m, 1H), 3.30-3.04 (m, 1H), 3 .03-2.25 (m, 5H), 2.09-1.02 (m, 11H) ppm. ¹³ C NMR (400 MHz, CDCl ₃ ) δ 162.3 (d, J=247.1 Hz), 154.5, 149.8, 143.6, 130.7, 127.4 (d, J=8.1 Hz), 121.8, 118.9, 115.8 (d, J=21.6 Hz), 70 ,8, 55.5, 53.4, 47.3, 46.4, 29.0, 25.4, 24.4, 19.4 ppm. Purity: 95.8% ULCMS (210 nm and 254 nm); retention time 0.90 min; (M+1) 389.

(S)-quinuclidin-3-yl 2-(3-(4-fluorophenyl)isothiazol-5-yl)propan-2-ylcarbamate (Compound 11)

To a stirred solution of 2-(3-(4-fluorophenyl)isothiazol-5-yl)propan-2-amine (1.21 g, 5.12 mmol) in toluene was added a solution of phosgene in toluene [~1.9 M] (10.8 mL, 20.5 mmol). The reaction mixture was heated at reflux for 2 h and then concentrated. The residue was coevaporated with toluene (2 x 15 mL) to give the crude intermediate isocyanate as a golden oil. This product was taken up in toluene (10 mL) and treated with (S)-3-quinuclidinol (0.749 g, 5.89 mmol). The reaction mixture was heated at reflux overnight and concentrated. The residue was purified by flash chromatography using a chloroform/methanol/ammonia gradient to give the title compound as a white solid (0.971 g, 49%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.09-8.00 (m, 2H), 7.87 (shs, 1H), 7.75 (s, 1H), 7.35-7.25 (m, 2H), 4.54-4.45 (m, 1H), 3.14-2.92 (m, 1H), 2.8 7-2.17 (m, 5H), 1.98-0.98 (m, 11H) ppm. ¹³ C NMR (400 MHz, DMSO-d ₆ ) δ 180.1, 165.6, 162.6 (d, J=246.4 Hz), 154.7, 131.2 (d, J=3.0 Hz), 128.7 (d, J=8.4 Hz), 118.2, 115.7 (d, J=21.8 Hz), 70.6, 55.3, 52.8, 46.9, 45.9, 29.9, 25.2, 24.2, 19.2 ppm. Purity: 100% ULCMS (210 nm and 254 nm); retention time 0.82 min; (M+1) 390.

(S)-quinuclidin-3-yl 2-(4-(4-fluorophenyl)thiazol-2-yl)propan-2-ylcarbamate (Compound 12)

To a stirred solution of ethyl 3-amino-3-thiopropanoate (20.00 g, 135.9 mmol) in ethanol (120 mL) was added 2-bromo-4'-fluoroacetophenone (29.49 g, 135.9 mmol). The mixture was heated under reflux for 1 h, concentrated, and partitioned between ethyl acetate (300 mL) and aqueous NaHCO ₃ (400 mL). The organic layer was combined with the back extract of the aqueous layer (ethyl acetate, 1×100 mL), dried (Na ₂ SO ₄ ), and concentrated. The resulting light brown solid was purified by flash chromatography using a hexane/ethyl acetate gradient to give ethyl 2-(4-(4-fluorophenyl)thiazol-2-yl)acetate as an off-white solid (29.92 g, 83%).

To a stirred and cooled (-78°C) solution of ethyl 2-(4-(4-fluorophenyl)thiazol-2-yl)acetate (10.00 g, 37.69 mmol) in THF (250 mL) was added a solution of potassium tert-butoxide in THF [1.0 M] (136 mL, 136 mmol) dropwise over 15 min, then 18-crown-6 (1.6 mL, 7.5 mmol). After another 30 min at -78°C, iodomethane (8.5 mL) was added dropwise over 5 min. The reaction mixture was stirred cold for another 2 h, then poured into water (450 mL) and extracted with ethyl acetate (2×150 mL). The combined extracts were washed with brine (1 x 200 mL), dried (Na ₂ SO ₄ ) and concentrated. The resulting brown oil was purified by flash chromatography using a hexane/ethyl acetate gradient to give ethyl 2-(4-(4-fluorophenyl)thiazol-2-yl)-2-methylpropanoate as a pale amber oil (8.64 g, 78%).

To a stirred solution of ethyl 2-(4-(4-fluorophenyl)thiazol-2-yl)-2-methylpropanoate (0.900 g, 3.07 mmol) in 1:1:1 THF/ethanol/water (15 mL) was added lithium hydroxide monohydrate (0.451 g, 10.7 mmol). After stirring overnight, the reaction mixture was concentrated and redissolved in water (80 mL). The solution was washed with ether (1 x 50 mL), acidified by adding 1N HCl (15 mL), and extracted with ethyl acetate (2 x 50 mL). The combined extracts _were dried ( _Na2SO4 ) and concentrated to give 2-(4-(4-fluorophenyl)thiazol-2-yl)-2-methylpropanoic acid as a pale golden solid (0.808 g, 99%).

To a stirred and cooled (0°C) solution of 2-(4-(4-fluorophenyl)thiazol-2-yl)-2-methylpropanoic acid (0.784 g, 2.96 mmol) in THF (25 mL) was added triethylamine (0.82 mL, 5.9 mmol) followed by isobutyl chloroformate (0.58 mL, 4.4 mmol). The reaction mixture was stirred cold for a further 1 h then a solution of sodium azide (0.385 g, 5.92 mmol) in water (7 mL) was added. The reaction mixture was stirred overnight allowing the cooling bath to warm slowly to room temperature. The mixture was then diluted with water (100 mL) and extracted with ethyl acetate (2 x 60 mL). The combined extracts were washed with aqueous _NaHCO3 (1 x 150 mL) and brine (1 x 100 mL), dried ( _Na2SO4 ) and concentrated. After co-evaporation with toluene (2 x 30 mL), the resulting off-white solid was taken up in toluene (25 mL) and heated at reflux for 4 h. (S)-3-quinuclidinol ( _0.753 g, 5.92 mmol) was then added and reflux was continued for 3 h. The reaction mixture was concentrated and the residue was purified by flash chromatography using a chloroform/methanol/ammonia gradient to give the title compound as a white solid (0.793 g, 69%). ^1H NMR (400 MHz, CDCl ₃ ) δ 7.90-7.81 (m, 2H), 7.32 (s, 1H), 7.14-7.05 (m, 2H), 5.76 (shs, 1H), 4.72-4.65 (m, 1H), 3.26-3.10 (m, 1H), 3.03-2 .37 (m, 5H), 2.05-1.23 (m, 11H) ppm. ¹³ C NMR (400 MHz, CDCl ₃ ) δ 177.6, 162.6 (d, J=248.4 Hz), 154.8, 153.6, 130.8 (d, J=3.2 Hz), 128.1 (d, J=8.1 Hz), 115.9 (d, J=21.7 Hz), 112.2, 71.6, 55.7, 47.4, 46.5, 29.1, 25.4, 24.7, 19.6 ppm. Purity: 100% ULCMS (210 nm and 254 nm); retention time 0.82 min; (M+1) 390.

Quinuclidin-3-yl (2-(4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 13)

Using General Procedure F and introducing 2-(4'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)-2-methylpropanoic acid (prepared as described in Example 3) and quinuclidin-3-ol into the reaction mixture, the title compound was obtained as a colourless glassy solid (23%). NMR consistent with Example 3. Purity: 100%, 99.1% (210 & 254 nm) UPLCMS; retention time: 0.87 min; (M+H ⁺ ) 439.0.

(S)-quinuclidin-3-yl (2-(3'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 14)

Substituting 3-(2-methoxyethoxy)phenylboronic acid for 4-(2-methoxyethoxy)phenylboronic acid, the reaction sequence described in Example 3 is used to prepare 2-(3'-(2-methoxyethoxy)-[1,1'-biphenyl]-4-yl)-2-methylpropanoic acid. This intermediate and quinuclidin-3-ol are reacted according to General Procedure F to give the title compound as a glassy, colourless solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.63-7.31 (m, 6H), 7.24-7.10 (m, 2H), 6.92 (dd, J=8.2, 1.9 Hz, 1H), 4.51-4.34 (m, 1H), 4.21-4.08 (m, 2H), 3, 72-3.64 (m, 2H), 3.32 (s, 3H), 3.09-2.26 (m, 5H), 2.04-1.22 (m, 9H) ppm. ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 158.9, 154.6, 147.6, 141.5, 137.6, 129.9, 126.3, 125.2, 118.9, 113.2, 112.5, 70.4, 70.0, 66.9, 58.2, 55.4, 54.2, 46.9, 45.9, 29.4, 25.3, 24.2, 19.2 ppm. Purity: 100%, 100% (210 and 254 nm) ULCMS; retention time: 0.91 min; 15 (M+H ⁺ ) 439.4.

Quinuclidin-3-yl (2-(4'-(2-methoxyethoxy)-[1,1'-biphenyl]-3-yl)propan-2-yl)carbamate (Compound 15)

Substituting ethyl 2-(3-bromophenyl)-2-methylpropanoate for ethyl 2-(4-bromophenyl)-2-methylpropanoate, the reaction sequence described in Example 3 was used to prepare 2-(4'-(2-methoxyethoxy)-[1,1'-biphenyl]-3-yl)-2-methylpropanoic acid. This intermediate and quinuclidin-3-ol were reacted according to General Procedure F to give the title compound as a yellow solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.62-7.20 (m, 7H), 7.03 (d, J=8.7 Hz, 2H), 4.48-4.35 (m, 2H), 4.18-4.08 (m, 2H), 3.72-3.62 (m, 2H), 3.32 (s, 3H), 3.10-2.19 (m, 6H), 2.10-1.10 (m, 11H) ppm. ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 158.0, 154.6, 148.8, 139.5, 133.1, 128.5, 127.7, 123.8, 123.2, 122.7, 114.8, 70.4, 69.9, 67.0, 58.2, 55.3, 54.5, 47.0, 45.9, 29.4, 25.3, 24.2, 19.2 ppm. Purity: 97.4%, 94.6% (210 and 254 nm) ULCMS; retention time: 0.88 min; (M+H ⁺ ) 439.3.

Quinuclidin-3-yl (2-(4'-(3-methoxypropoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 16)

To a stirred solution of 4-iodophenol (10.05 g, 45.68 mmol) in acetonitrile (100 mL) were added potassium carbonate (6.95 g, 50.2 mmol) and 1-chloro-3-methoxypropane (6.4 mL, 57.1 mmol). The mixture was heated at reflux overnight and then concentrated. The residue was taken up in water and extracted with ethyl acetate. The combined extracts were washed with aqueous sodium bicarbonate, dried (Na ₂ SO ₄ ) and concentrated. The crude product was purified by flash chromatography over silica using hexane/ethyl acetate as eluent to give 1-iodo-4-(3-methoxypropoxy)benzene as a colourless oil (4.39 g, 33%). This intermediate and ethyl 2-methyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate were reacted according to General Procedure E to give ethyl 2-(4'-(3-methoxypropoxy)-[1,1'-biphenyl]-4-yl)-2-methylpropanoate. To a stirred solution of this compound (0.693 g, 1.94 mmol) in 1:1:1 (v/v/v) tetrahydrofuran/ethanol/water (10 mL) was added lithium hydroxide monohydrate (0.326 g, 7.77 mmol). The mixture was heated at reflux overnight and then concentrated. The residue was dissolved in water, treated with 1N hydrochloric acid (10 mL), and extracted with ethyl acetate. The combined organic layers were washed with brine, dried ( _Na2SO4 ) and concentrated to give 2-(4'-(3-methoxypropoxy)-[1,1'-biphenyl]-4-yl)-2-methylpropanoic acid as a waxy off-white solid ( _0.630 g, 99%). This intermediate and quinuclidin-3-ol were reacted according to General Procedure F to give the title compound as a glassy, colourless solid (62%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.61-7.29 (m, 7H), 7.00 (d, J= 8.8 Hz, 2H), 4.47-4.36 (m, 1H), 4.05 (t, J= 6.4 Hz, 2H), 3.48 (t, J=6.3 Hz, 2H), 3.26 (s, 3H), 3.10-2.25 (m, 6H), 2.04-1.74 (m, 4H), 1.65-1.23 (m, 9H) ppm, 1 ³ C NMR (100 MHz, DMSO-d6) δ 158.0, 154.5, 146.7, 137.4, 132.4, 127.5, 125.7, 125.2, 114.8, 69.9, 68.5, 64.6, 57.9, 55.4, 54.2, 46.9, 46.0, 29.4, 29.0, 25.2, 24.1, 19.2 ppm. Purity: 97.7%, 98.2% (210 and 254 nm) UPLCMS; retention time: 0.96 min; (M+H ⁺ ) 453.5.

Quinuclidin-3-yl (2-(4'-(hydroxymethyl)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 17)

Using General Procedure E and introducing ethyl 2-(4-bromophenyl)-2-methylpropanoate and 4-formylphenylboronic acid into the reaction mixture, ethyl 2-(4'-formyl-[1,1'-biphenyl]-4-yl)-2-methylpropanoate was obtained as a pale amber solid. This intermediate and quinuclidin-3-ol were reacted according to General Procedure F to give quinuclidin-3-yl (2-(4'-formyl-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate as a foamy yellow solid. To a stirred solution of this product (0.755 g, 1.92 mmol) in 2:1 (v/v) tetrahydrofuran/ethanol (15 mL) was added sodium borohydride (0.073 g, 1.93 mmol). After 45 min, the reaction mixture was diluted with water and extracted with chloroform. The combined extracts were dried (Na ₂ SO ₄ ) and concentrated onto silica. Flash chromatography on silica using chloroform/methanol/ammonia as eluent gave the title compound as a white solid (0.323 g, 43%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.66-7.29 (m, 9H), 5.18 (t, J= 5.7 Hz, 1H), 4.53 (d, J= 5.7 Hz, 2H), 4.46-4.37 (m, 1H), 3.11-2.19 (m, 6H), 2.11-1.10 (m, 11H) ppm. ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 154.7, 147.3, 141.5, 138.4, 137.7, 127.0, 126.2, 126.1, 125.3, 70.0, 62.6, 55.4, 54.2, 46.9, .9, 29.4, 25.3, 24.2, 19.2 ppm. Purity: 97.5%, 99.1% (210 and 254 nm) ULCMS; retention time: 0.73 min; (M+H ⁺ ) 395.

Quinuclidin-3-yl (2-(4'-(2-hydroxyethyl)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 18)

By employing General Procedure E and introducing 1-(2-(benzyloxy)ethyl)-4-bromobenzene and ethyl 2-methyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate into the reaction mixture, ethyl 2-(4'-(2-(benzyloxy)ethyl)-[1,1'-biphenyl]-4-yl)-2-methylpropanoate was obtained as a colorless gum. To a stirred solution of this compound (1.34 g, 3.33 mmol) in 1:1:1 (v/v/v) tetrahydrofuran/ethanol/water (18 mL) was added lithium hydroxide monohydrate (0.698 g, 16.6 mmol). After heating at reflux overnight, the reaction mixture was concentrated and partitioned between water and diethyl ether. The resulting emulsion was re-extracted with 0.2 N aqueous sodium hydroxide solution (5 x 50 mL). The clear portion of the aqueous layer was removed each time. The combined aqueous layers were then treated with 1.0 N hydrochloric acid (80 mL) and the resulting suspension of white solid was extracted with ethyl acetate. The combined organic layers were dried (Na ₂ SO ₄ ) and concentrated to give 2-(4'-(2-(benzyloxy)ethyl)-[1,1'-biphenyl]-4-yl)-2-methylpropanoic acid as a white solid (1.20 g, 96%). This compound and quinuclidin-3-ol were reacted according to General Procedure F to give quinuclidin-3-yl (2-(4'-(2-benzyloxyethyl)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate. To a stirred solution of this product (0.435 g, 0.806 mmol) in methanol was added 1.0 N hydrochloric acid (1 mL) and 10% palladium on carbon (50% water; 0.087 g). The mixture was cycled between vacuum and nitrogen flush several times, backfilling with hydrogen after the last vacuum. After 1.25 h, the reaction mixture was filtered through Celite and concentrated. The residue was taken up in aqueous sodium carbonate and extracted with 4:1 (v/v) chloroform/isopropanol. The combined extracts were dried ( _Na2SO4 ) and concentrated into _silica . Flash chromatography over silica using a chloroform/methanol/ammonia gradient afforded the purified title compound as a colourless solid. ¹ H NMR (400 MHz, DMSO- _d6 ) δ 7.85-7.63 (m,1H), 7.63-7.19 (m,8H), 4.78-4.62 (m,2H), 3.71-2.78 (m,8H), 2.76 (t, J= 6.8 Hz, 2H), 2.26-1.96 (m,2H), 1.96-1.40 (m,9H) ppm. ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 153.8, 146.8, 138.7, 137.9, 137.6, 129.4, 126.3, 126.1, 125.3, 66.2, 62.1, 54.4, 52.8, 45.4, .5, 38.6, 29.5, 29.2, 24.0, 19.9, 16.6 ppm. Purity: 100%, 100% (210 and 254 nm) ULCMS; retention time: 0.75 min; (M+H ⁺ ) 409.

Quinuclidin-3-yl (2-(2-(4-(3-methoxypropoxy)phenyl)thiazol-4-yl)propan-2-yl)carbamate (Compound 19)

To a stirred suspension of 4-methoxythiobenzamide (9.99 g, 59.7 mmol) in ethanol (75 mL) was added ethyl 4-chloroacetoacetate (8.1 mL, 60 mmol). The mixture was heated at reflux for 4 h, then cooled, more ethyl 4-chloroacetoacetate (0.81 mL, 6.0 mmol) was added, and the mixture was returned to reflux. After another 4 h of heating, the reaction mixture was concentrated and partitioned between ethyl acetate and aqueous sodium bicarbonate. The organic layer was combined with additional ethyl acetate extracts, dried (Na ₂ SO ₄ ), and concentrated. The crude product was purified by flash chromatography over silica using a hexane/ethyl acetate gradient to give ethyl 2-(2-(4-methoxyphenyl)thiazol-4-yl)acetate as a pale amber oil (14.51 g, 87%). To a stirred solution of this compound (14.48 g, 52.2 mmol) in N,N-dimethylformamide (125 mL) was added sodium hydride (60% dispersion in mineral oil; 6.27 g, 157 mmol) portionwise over 15 min. The resulting red suspension was cooled (0 °C) and treated dropwise with iodomethane (9.80 mL, 157 mmol) over 10 min. The cooling bath was removed and the reaction mixture was stirred for 4 h, then concentrated and the residue partitioned between ethyl acetate and water. The organic layer was washed twice with water, dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash chromatography over silica using a hexane/ethyl acetate gradient to give ethyl 2-(2-(4-methoxyphenyl)thiazol-4-yl)-2-methylpropanoate as a pale amber oil (14.12 g, 89%). To a stirred solution of this intermediate (14.12 g, 46.24 mmol) in methylene chloride (250 mL) was added boron tribromide (11.0 mL, 116 mmol) dropwise over 5 min. After stirring overnight, the reaction was quenched by the slow addition of methanol (~20 mL) and then concentrated. The residue was taken up in methanol (250 mL) and concentrated sulfuric acid (7.0 mL). The stirred solution was heated at reflux for 2 h, concentrated, and partitioned between ethyl acetate and aqueous sodium bicarbonate. The organic layer was combined with a second ethyl acetate extract of the aqueous layer, dried (Na ₂ SO ₄ ), and concentrated to give methyl 2-(2-(4-hydroxyphenyl)thiazol-4-yl)-2-methylpropanoate as a white solid (12.56 g, 98%). To a stirred solution of 1-bromo-3-methoxypropane (1.66 g, 10.8 mmol) in acetone (30 mL) were added the intermediate phenol (2.00 g, 7.21 mmol) and potassium carbonate (1.25 g, 9.04 mmol). The mixture was heated overnight at reflux, filtered, and concentrated. The residue was purified by flash chromatography over silica using a hexane/ethyl acetate gradient to give methyl 2-(2-(4-(3-methoxypropoxy)phenyl)thiazol-4-yl)-2-methylpropanoate as a pale amber gum (2.47 g, 98%). To a stirred solution of this compound (2.45 g, 7.01 mmol) in 1:1:1 (v/v/v) tetrahydrofuran/ethanol/water (45 mL) was added lithium hydroxide monohydrate (1.47 g, 35.0 mmol). After stirring overnight, the reaction mixture was concentrated and partitioned between water and diethyl ether. The aqueous layer was treated with 1.0 N hydrochloric acid (40 mL) and extracted with ethyl acetate. The combined extracts were dried (Na ₂ SO ₄ ) and concentrated to give 2-(2-(4-(3-methoxypropoxy)phenyl)thiazol-4-yl)-2-methylpropanoic acid as a white solid (2.19 g, 40.93%). This compound and quinuclidin-3-ol were reacted according to General Procedure F to give the title compound as a soft, pale amber solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.82 (d, J=8.9 Hz, 2H), 7.36 (br s, 1H), 7.24 (br s, 1H), 7.03 (d, J=8.9 Hz, 2H), 4.49-4.41 (m, 1H), 4.07 (t, J=6.4 Hz, 2H), 3.48 (t, J=6.4 Hz, 2H), 3.26 (s, 3H), 3.09-2.26 (m, 6H), 2.02-1.91 (m, 2H), 1.91-1.03 (m, 11H) ppm. ¹³ C NMR (100 MHz, DMSO-d6) δ 165.8, 162.4, 160.0, 154.6, 127.5, 126.1, 114.9, 112.1, 70.1, 68.4, 64.8, 57.9, 55.4, 53.5, 46.9, 45.9, 28.9, 28.3, 25.2, 24.2, 19.2 ppm. Purity: 100%, 100% (210 and 254 nm) LCMS; retention time: 0.87 min; (M+H ⁺ ) 460.

Quinuclidin-3-yl (2-(2-(4-(2-methoxyethoxy)phenyl)thiazol-4-yl)propan-2-yl)carbamate (Compound 20)

To a stirred solution of 2-bromoethyl methyl ether (1.88 g, 13.5 mmol) in acetone was added methyl 2-(2-(4-hydroxyphenyl)thiazol-4-yl)-2-methylpropanoate (prepared as described in Example 19, 2.00 g, 7.21 mmol) and potassium carbonate (1.56 g, 11.3 mmol). After heating at reflux overnight, the mixture was treated with additional 2-bromoethyl methyl ether (1.88 g, 13.5 mmol) and potassium carbonate (1.56 g, 11.3 mmol). The reaction mixture was heated at reflux for a second night, filtered, and concentrated. The residue was purified by flash chromatography over silica using a hexane/ethyl acetate gradient to give methyl 2-(2-(4-(2-methoxyethoxy)phenyl)thiazol-4-yl)-2-methylpropanoate as a white solid (2.71 g, 90%). To a stirred solution of this compound (2.71 g, 8.08 mmol) in 1:1:1 (v/v/v) tetrahydrofuran/ethanol/water (50 mL) was added lithium hydroxide monohydrate (1.70 g, 40.5 mmol). After stirring overnight, the reaction mixture was concentrated and partitioned between water and diethyl ether. The aqueous layer was treated with 1.0 N hydrochloric acid (41 mL) and extracted with ethyl acetate. The combined extracts were dried ( _Na2SO4 ) and concentrated to give 2-(2-(4-(2-methoxyethoxy)phenyl)thiazol-4-yl)-2-methylpropanoic acid as a white solid (2.57 g, 99%). This compound and _quinuclidin -3-ol were reacted according to General Procedure F to give the title compound as a pale amber solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.82 (d, J=8.8 Hz, 2H), 7.36 (brs, 1H), 7.24 (brs, 1H), 7.04 (d, J=8.8 Hz, 2H), 4.49-4.41 (m, 1H), 4.19-4.12 (m, 2H ), 3.71-3.65 (m,2H), 3.32 (s, 3H), 3.11-2.87 (m,1H), 2.86-2.19 (m,5H), 1.92-1.16 (m,11H) ppm. ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 165.7, 162.9, 159.9, 154.6, 127.5, 126.2, 114.9, 112.2, 70.3, 70.1, 67.1, 58.2, 55.4, 53.5, 46, 9, 45.9, 28.3, 25.2, 24.3, 19.2 ppm Purity: 100%, 100% (210 and 254 nm) ULCMS; holding time: 0.85 min; (M+H ⁺ ) 446.

Quinuclidin-3-yl 2-(5-(4-(2-methoxyethoxy)phenyl)pyridin-2-yl)propan-2-ylcarbamate (Compound 21)

By using General Procedure E and introducing 5-bromopicolinonitrile and 2-(4-(2-methoxyethoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane into the reaction mixture, 5-(4-(2-methoxyethoxy)phenyl)picolinonitrile was obtained. Cerium trichloride (8.05 g, 21.6 mmol) was charged to the flask and dried by heating (170 °C) under vacuum for 3 h. The solid was taken up in tetrahydrofuran (20 mL) and stirred vigorously for 30 min. The suspension was cooled to -78 °C and treated dropwise with a 3.0 M solution of methyllithium in diethyl ether (7.2 mL, 21.6 mmol). After the addition, the reaction mixture was stirred at -78 °C for 1 h, then a solution of the above aryl borate (1.83 g, 7.20 mmol) in tetrahydrofuran (20 mL) was added. The mixture was kept at -78 °C for 2 h and then warmed to room temperature. At this time, the reaction was quenched by the addition of aqueous ammonium hydroxide (10 mL) and filtered through a pad of celite. The filtrate was extracted with ethyl acetate, and the combined extracts were washed with brine, dried (Na ₂ SO ₄ ), and concentrated. The residue was purified by flash chromatography over silica using ethyl acetate as eluent to give 2-(5-(4-(2-methoxyethoxy)phenyl)pyridin-2-yl)propan-2-amine as a yellow solid (0.800 g, 39%). To a stirred suspension of this intermediate (0.500 g, 1.75 mmol) in water (10 ml) and concentrated hydrochloric acid (0.44 ml) was added toluene (10 ml). The mixture was cooled (0°C) and treated simultaneously for 1 h with solutions of triphosgene (0.776 g, 2.62 mmol) in toluene (10 ml) and sodium bicarbonate (2.2 g, 26 mmol) in water (20 ml). After the additions, the reaction mixture was stirred for a further 30 min, then the upper toluene layer was removed and dried (Na ₂ SO ₄ ). At this time, a stirred solution of quinuclidin-3-ol (0.445 g, 3.64 mmol) in tetrahydrofuran (10 mL) was treated with sodium hydride (60% dispersion in mineral oil; 0.154 g, 3.85 mmol). This mixture was stirred for 5 min and then added to a solution of the crude isocyanate in toluene. The reaction mixture was stirred for 10 min, quenched by addition of brine (5 mL), and extracted with ethyl acetate. The combined extracts were dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash chromatography over reverse-phase silica to give the title compound as a light yellow solid (0.100 g, 13%). ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.70-8.70 (d, J=2.0 Hz, 1H), 7.83-7.81 (m, 1H), 7.49-7.47 (d, J=9.0 Hz, 2H), 7.45-7.43 (d, J=8.0 Hz, 1H), 7.03-7.01 (d, J=8.5 Hz, 2H), 6.63 (br s, 1H), 4.68-4.66 (m, 1H), 4.16 (t, J=5.0 Hz, 2H), 3.77 (t, J=5.0 Hz, 2H), 3.45 (s, 3H), 3.19-2.70 (m, 6H), 2.15-1.89 (m,2H), 1.76 (s, 6H), 1.73-1.36 (m,3H) ppm. ¹³ C NMR (125 MHz, CDCl ₃ ) δ 162.7, 158.9, 154.9, 145.9, 134.8, 134.3, 130.1, 128.1, 119.2, 115.2, 71.0, 70.8, 67.4, 59.2, , 55.7, 47.4, 46.5, 46.4, 27.9, 25.4, 24.6, 19.5 ppm. Purity: >99% (214 and 254 nm) LCMS; retention time: 1.32 min; (M+H ⁺ ) 440.2.

Quinuclidin-3-yl (2-(4'-(3-cyanopropoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 22)

To a stirred solution of 4-bromophenol (17.1 g, 98.8 mmol) in acetonitrile (150 mL) were added 1-bromobutylnitrile (12.3 mL, 124 mmol) and potassium carbonate (15.0 g, 109 mmol). The mixture was heated to reflux overnight, cooled and concentrated. The residue was taken up in water and extracted with ethyl acetate. The combined extracts were dried (Na ₂ SO ₄ ) and concentrated, and the crude product was purified by flash chromatography over silica using hexane/ethyl acetate as eluent to give 4-(4-bromophenoxy)butanenitrile as a white solid (20.8 g, 88%). To a stirred solution of this product in N,N-dimethylformamide (100 mL) were added bis(pinacolato)diborone (4.60 g, 18.1 mmol), potassium acetate (7.41 g, 75.5 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (0.616 g, 1.04 mmol). The mixture was heated to reflux overnight and then concentrated. The residue was taken up in ethyl acetate and washed with water and brine. The organic layer was dried ( _Na2SO4 ) and concentrated and the crude product was purified by flash chromatography over silica using hexane/ethyl acetate as eluent to give 4-(4-( _4,4,5,5 -tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butanenitrile as a white solid (3.43 g, 79%). This product and quinuclidin-3-yl (2-(4-bromophenyl)propan-2-yl)carbamate (prepared by reacting quinuclidin-3-ol and 2-(4-bromophenyl)propan-2-amine using General Procedure F) were reacted according to General Procedure E to give the title compound as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.67-7.26 (m, 7H), 7.02 (d, J=8.8 Hz, 2H), 4.50-4.33 (m, 1H), 4.08 (t, J=6.0 Hz, 2H), 3.14-2.18 (m, 8H), 2.04 (quin, J=6.7 Hz, 2H), 1.94-1.70 (m, 11H) ppm. ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 157.7, 154.5, 146.8, 137.4, 132.7, 127.6, 125.7, 125.2, 120.2, 114.9, 70.0, 65.8, 55.4, 54.2, 4 6.9, 45.9, 29.4, 25.3, 24.7, 24.2, 19.2, 13.4 ppm. Purity: 100%, 98.9% (210 and 254 nm) ULCMS; retention time: 0.88 min; (M+H ⁺ ) 448.6.

Quinuclidin-3-yl (2-(4'-(cyanomethoxy)-[1,1'-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 23)

Using General Procedure E and introducing into the reaction mixture quinuclidin-3-yl (2-(4-bromophenyl)propan-2-yl)carbamate (prepared by reacting quinuclidin-3-ol and 2-(4-bromophenyl)propan-2-amine using General Procedure F) and 4-(cyanomethoxy)phenylboronic acid, the title compound is obtained as a pale amber solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.65 (d, J= 8.2 Hz, 2H), 7.60-7.31 (m, 5H), 7.15 (d, J=8.9 Hz, 2H), 5.21 (s, 2H), 4.53-4.30 (m, 1H), 3.18-2.19 (m, 6H), 2.05-1.18 (m, 11H) ppm. ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 155.8, 154.6, 147.2, 137.2, 134.4, 127.8, 126.0, 125.3, 116.7, 115.3, 70.0, 55.4, 54.2, 53.5, 4 6.9, 45.9, 29.4, 25.2, 24.2, 19.2 ppm Purity: 100%, 100% (210 and 254 nm) ULCMS; holding time: 0.85 min; (M+H ⁺ ) 420.3.

Example 2: Preparation of free base (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate

Step 1: Dimethylation with methyl iodide

Chemical formula: C ₁₃ H ₁₂ FNO ₂ S Chemical formula: C ₁₅ H ₁₆ FNO ₂ S Exact weight: 265.06 Exact mass: 293.09 Molecular weight: 265.30 Molecular weight: 293.36

A 3N KD flask was fitted with a thermometer, dropping funnel and nitrogen inlet. The flask was flushed with nitrogen and potassium tert-butoxide (MW 112.21, 75.4 mmol, 8.46 g, 4.0 equiv, white powder) was weighed and added to the flask via the powder funnel, followed by THF (60 mL). Most of the potassium tert-butoxide dissolved to give a cloudy solution. This mixture was cooled in an ice-water bath to 0-2 °C (internal temperature). In a separate flask, the starting ester (MW 265.3, 18.85 mmol, 5.0 g, 1.0 equiv) was dissolved in THF (18 mL + 2 mL as a wash) and transferred to the dropping funnel. This solution was added dropwise to the cooled mixture over 25–30 min, maintaining the internal temperature below 5 °C during the addition. The reaction mixture was cooled back to 0–2 °C. In a separate flask, a solution of methyl iodide (MW 141.94, 47.13 mmol, 6.7 g, 2.5 equiv) in THF (6 mL) was prepared and transferred to the addition funnel. The flask containing the methyl iodide solution was then washed with THF (1.5 mL), then transferred to the addition funnel now containing a clear, colorless solution of methyl iodide in THF. This solution was carefully added dropwise to the dark brown reaction mixture over 30–40 min, maintaining the internal temperature below 10 °C at all times during the addition. After the addition was complete, the slightly cloudy mixture was stirred for an additional 1 h, during which time the internal temperature dropped to 0–5 °C. After stirring for 1 h at 0-5 °C, the reaction mixture was quenched by slowly adding 5.0 M aqueous HCl (8 mL) dropwise over 5-7 min. The internal temperature was kept below 20 °C during this addition. After the addition, water (14 mL) was added and the mixture was stirred for 2-3 min. Stirring was stopped and the two layers were separated. The two layers were then transferred to a 250 mL 1N CD flask and the THF was evaporated in vacuo as much as possible to obtain a biphasic layer of THF/product and water. The two layers were separated. The THF solution of the product from step 1 was used in the next reaction.

Step 2: Hydrolysis of ethyl ester with LiOH monohydrate

Chemical formula: C ₁₅ H ₁₆ FNO ₂ S Chemical formula: C ₁₃ H ₁₂ FNO ₂ S Exact mass: 293.09 Exact weight: 265.06 Molecular weight: 293.36 Molecular weight: 265.30

The crude ester in THF was added to the reaction flask. Separately, LiOH.H ₂ O (MW 41.96, 75.0 mmol, 3.15 grams, 2.2 equiv.) was weighed into a 100 mL beaker and a stir bar was added. Water (40 mL) was added and the mixture was stirred until all solids had dissolved to give a clear, colorless solution. This aqueous solution was then added to a 250 mL CD flask containing a solution of the ester in tetrahydrofuran (THF). A condenser was attached to the neck of the flask and a nitrogen inlet was attached to the top of the condenser. The mixture was heated at reflux for 16 h. After 16 h, heating was stopped and the mixture was cooled to room temperature. The THF was evaporated in vacuo to give a brown solution. An aliquot of the brown aqueous solution is analyzed by HPLC and LC/MS to ensure complete hydrolysis of the ethyl ester. Water (15 mL) is added and this aqueous alkaline solution is extracted with TBME (2 x 40 mL) to remove the tert-butyl ester. The aqueous alkaline layer is cooled in an ice-water bath to 0-10 °C and acidified by dropwise addition of concentrated HCl to pH ~ 1 with stirring. To this sticky solid in aqueous acid solution is added TBME (60 mL) and the mixture is shaken and then vigorously stirred to dissolve all the acid in the TBME layer. The two layers are transferred to a separatory funnel and the TBME layer is separated. The pale yellow aqueous acidic solution is re-extracted with TBME (40 mL) and the TBME layer is separated and combined with the previous TBME layer. The aqueous acidic layer was discarded. The combined TBME layers were dried over _{anhydrous Na2SO4} , filtered and evaporated in vacuo to remove TBME and give the crude acid as an orange/dark yellow oil which solidified under high vacuum to a dirty yellow solid. The crude acid was weighed and crystallized by heating in heptane/TBME (3:1, 5 ml/g crude product) to give the acid as a yellow solid.

Step 3: Formation of hydroxamic acid with NH ₂ OH.HCl

Chemical formula: C ₁₃ H ₁₂ FNO ₂ S Chemical formula: C ₁₃ H ₁₃ FN ₂ O ₂ S Exact weight: 265.06 Exact weight: 280.07 Molecular weight: 265.30 Molecular weight: 280.32

The carboxylic acid (MW 265.3, 18.85 mmol, 5.0 g, 1.0 equiv) was weighed and transferred to a 25 mL 1N KD flask under nitrogen. THF (5.0 mL) was added and the acid dissolved readily to give a clear, dark yellow/brown solution. The solution was cooled to 0-2 °C (bath temperature) in an ice bath and N,N'-carbonyldiimidazole (CDI; MW 162.15, 20.74 mmol, 3.36 g, 1.1 equiv) was slowly added in small portions over 10-15 min. The ice bath was removed and the solution was stirred at room temperature for 1 h. After 1 h of stirring, the solution was again cooled in an ice-water bath to 0-2 °C (bath temperature). Hydroxylamine hydrochloride (NH ₂ OH.HCl; MW 69.49, 37.7 mmol, 2.62 g, 2.0 equiv) was added slowly in small portions as a solid over 3-5 min, as the addition was exothermic. After complete addition, water (1.0 mL) was added to the heterogeneous mixture dropwise over 2 min, and the reaction mixture was stirred at 0-10 °C in an ice-water bath for 5 min. The cooling bath was removed, and the reaction mixture was stirred under nitrogen at room temperature overnight for 20-22 h. The solution became clear as all of the NH ₂ OH.HCl dissolved. After 20-22 h, an aliquot of the reaction mixture was analyzed by high-pressure liquid chromatography (HPLC). The THF was then evaporated in vacuo and the residue taken up in dichloromethane (120 ml) and water (60 ml). The mixture was transferred to a separatory funnel where it was shaken and the two layers were separated. The aqueous layer was discarded and the dichloromethane layer was washed with 1N hydrochloride (HCl; 60 ml). The acidic layer was discarded. The dichloromethane layer was dried over _{anhydrous Na2SO4} , filtered and the solvent evaporated in vacuo to give the crude hydroxamic acid as a pale yellow solid which was dried under high vacuum overnight.

Step 3 (continued): Conversion of hydroxamic acid to a cyclic intermediate (not isolated)

Chemical formula: C ₁₃ H ₁₃ FN ₂ O ₂ S Chemical formula: C ₁₄ H ₁₁ FN ₂ O ₃ S Exact weight: 280.07 Exact weight: 306.05 Molecular weight: 280.32 Molecular weight: 306.31

The crude hydroxamic acid (MW 280.32, 5.1 g) was transferred to a 250 mL 1N CD flask with a nitrogen inlet. A stir bar was added, then acetonitrile (50 mL) was added. The solid was insoluble in acetonitrile. The yellow heterogeneous mixture was stirred for 2–3 min under nitrogen, and CDI (MW 162.15, 20.74 mmol, 3.36 g, 1.1 equiv) was added in one portion at room temperature. No exotherm was observed. The solid dissolved immediately, and the clear yellow solution was stirred at room temperature for 2–2.5 h. After 2–2.5 h, an aliquot was analyzed by HPLC and LC/MS, which showed conversion of the hydroxamic acid to the desired cyclic intermediate.

The acetonitrile is then evaporated in vacuo to give the crude cyclic intermediate as a reddish thick oil. The oil is taken up in toluene (60 ml) and the reddish mixture is heated to reflux for 2 h, during which time the cyclic intermediate evolves CO ₂ and rearranges to the isocyanate (see below).

Chemical formula: C ₁₄ H ₁₁ FN ₂ O ₃ S Chemical formula: C ₁₃ H ₁₁ FN ₂ OS Exact weight: 306.05 Exact weight: 262.06 Molecular weight: 306.31 Molecular weight: 262.30

Step 3 (continued): Conversion of isocyanate to free base

Chemical formula: C ₁₃ H ₁₁ FN ₂ OS _{Chemical formula : C20H24FN2O2S} Exact weight: 262.06 Exact mass: 389.16 Molecular weight: 262.30 Molecular weight: 389.49

The reaction mixture was cooled to 50-60 °C and (S)-(+)-quinuclidinol (MW 127.18, 28.28 mmol, 3.6 g, 1.5 equiv) was added to the mixture as a solid in one portion. The mixture was reheated to reflux for 18 h. After 18 h, an aliquot was analyzed by HPLC and LC/MS, which showed complete conversion of the isocyanate to the desired product. The reaction mixture was transferred to a separatory funnel and toluene (25 mL) was added. The mixture was washed with water (2 x 40 mL) and the aqueous layers were separated. The combined aqueous layers were re-extracted with toluene (30 mL) and the aqueous layer was discarded. The combined toluene layers were extracted with 1 N HCl (2 x 60 mL) and the toluene layer (containing the O-acyl impurity) was discarded. The combined HCl layers were transferred to a 500 mL Erlenmeyer flask equipped with a stirrer. The stirred yellow/reddish-orange solution was basified to pH 10-12 by the dropwise addition of 50% w/w aqueous NaOH. The desired free base precipitated as an off-yellow sticky solid that supported the stirrer paddle. Isopropyl acetate (100 mL) was added to this mixture and the mixture was stirred vigorously for 5 min when the sticky solid converted to isopropyl acetate. Stirring was stopped and the two layers were separated. The yellow isopropyl acetate layer was separated and the basic layer was re-extracted with isopropyl acetate (30 mL). The basic aqueous layer is discarded and the combined isopropyl _acetate layers are dried over anhydrous _Na2SO4 , filtered into a pre-weighed CD flask and the solvent is evaporated in vacuo to give the crude free base as a beige/tan solid which is dried under high vacuum overnight.

Step 3 (continued): Recrystallization of the crude free base

The beige/tan solid was weighed and recrystallized from heptane/isopropyl acetate (3:1, 9.0 mL solvent/g crude free base). An appropriate amount of heptane/isopropyl acetate was added to the crude free base with a stirrer and the mixture was heated to reflux for 10 min (the free base was initially partially soluble but dissolved to give a clear reddish-orange solution upon heating at reflux). The heat source was removed and the mixture was cooled to room temperature with stirring when a white precipitate formed. After stirring at room temperature for 3-4 h, the precipitate was filtered through a vacuum hose using a Buchner funnel, washed with heptane (20 mL) and dried under a vacuum hose on a Buchner funnel overnight. The precipitate was transferred to a crystallizer and dried at 55°C overnight in a vacuum oven. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.04-7.83 (m, 2H), 7.20-6.99 (m, 3H), 5.53 (s, 1H), 4.73-4.55 (m, 1H), 3.18 (dd, J=14.5, 8.4 Hz, 1H), 3.05-2.19 (m, 5H), 2.0-1.76 (m, 11H) ppm. ¹³ C NMR (100 MHz, CDCl ₃ ) δ 166.38, 165.02, 162.54, 162.8-155.0 (d, CF), 130.06, 128.43, 128.34, 116.01, 115.79, 112.46, 71.18, 5 5.70, 54.13, 47.42, 46.52, 27.94, 25.41, 24.67, 19.58 ppm.

Example 3: Preparation of crystalline forms of salts of (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate

Crystalline salts of (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate can be formed from the free base prepared as described in Example 23.

For example, (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate free base (ca. 50 mmol) is dissolved in IPA (140 mL) at room temperature and filtered. The filtrate is added to a 1 L c.d. flask equipped with an overhead stirrer and a nitrogen inlet/outlet. L-malic acid (ca. 50 mmol) is dissolved in IPA (100+30 mL) at room temperature and filtered. The filtrate is added to the above 1 L flask. The resulting solution is stirred at room temperature (with or without seeding) under nitrogen for 4-24 h. During this time, crystals form. The product is collected by filtration and washed with a small amount of IPA (30 mL). The crystalline solid is dried in a vacuum oven at 55°C for 72 hours to give the desired malate.

Crystalline forms of other salts, such as acid addition salts with succinic acid or HCl, can be obtained by a similar method.

Example 4: In vitro GCS inhibition (Compound 2 and analogues)

Inhibition of glucosylceramide synthase activity can be measured using one or more assays. The first assay is a microsomal assay that directly measures the conversion of ceramide to glucosylceramide using HPLC. Microsomes are the source of glucosylceramide synthase activity in the microsomal assay. The second assay is a cellular, phenotypic assay that monitors cell surface expression of descending fat GM3 using antibody-mediated immunofluorescence. Specific protocols are provided below.

Microsomal analysis of glucosylceramide synthase activity:

Enzyme assay using microsomes as a source of glucosylceramide synthase activity. A fluorescent ceramide substrate is delivered to the membrane-bound enzyme as a complex with albumin. Following the reaction, ceramide and glucosylceramide are separated and quantified by fluorescence-mediated reversed-phase HPLC. Enzyme activity is assessed using the fluorescently labeled substrate and microsomes as a source of glucosylceramide synthase. C ₆ -NBD-ceramide is complexed with albumin for delivery to microsomes, which are isolated as described below. The final concentration of C ₆ -NBD-ceramide in the stock solution is 0.5 mM; the final density of ABS is 0.5 mM. Separation and quantification of substrate and product (glucosylceramide) is performed using reversed-phase HPLC with fluorescence detection.

Preparation of microsomes from A375 human melanoma cells;

Microsomes were isolated from A375 human melanoma cells. Eight to ten million cells were harvested by trypsinization and washed with ice-cold PBS. The cells were resuspended in ice-cold lysis buffer containing protease inhibitors. The cell lysate was sonicated on ice using a probe sonicator. Following sonication, the cell lysate was separated from debris by centrifugation at 10,000 g for 10 minutes at 4°C. The supernatant was removed and clarified by additional centrifugation at 100,000 g for 1 hour at 4°C. The pellet in the tube was then resuspended in lysis buffer, aliquoted, and stored at -80°C until use.

Glucosylceramide synthase assay

To determine glucosylceramide synthase inhibition, substrates at 2x their Km (fluorescent ceramide and UDP-glucose, 3 μM and 4 μM, respectively) and microsomes (1:50 dilution) were combined 1:1 and incubated at room temperature for 1 h in the dark on a plate shaker. The reaction mixture was stopped by adding 150 μl of 100 μM C ₈ -ceramide in 50% aqueous isopropanol; 10 μl of the final mixture was analyzed by HPLC (with a fluorescence detector). The mobile phase was 1% formic acid added to 81% methanol/19% water at a flow rate of 0.5 ml/min. Fluorescence is determined with λ _ex = 470 nm and λ _em = 530 nm. Under these conditions, NBD-C ₆ -GluCer has a retention time of approximately 1.7 min, and NBD-C ₆ -Cer elutes from the column after approximately 2.1 min. The two peaks are separated from each other and by baseline and integrated automatically using the HPLC software. The fraction of substrate to product conversion is used as data for inhibitor testing.

GM3 Fluorescently linked enzyme assay (FLISA):

This is a phenotypic assay that measures GM3 expression in B16 mouse melanoma or C32 human melanoma cells following treatment with test compounds. Cell surface GM3 expression is detected by antibody-mediated fluorescence.

Compounds were diluted in medium and plated in 384-well plates in DMSO. B16 and C32 cells were assayed at a density of 20,000 cells/mL and 62,500 cells/mL, respectively, per well. Each filtration curve contains 10 points, which were assayed in duplicate for each assay run. Plates were incubated for 48 h at 37°C, 5% CO2 and then washed once with TBS. Anti-GM3 antibody was added to each well and the plates were then incubated for another hour at room temperature. Plates were then washed twice and incubated for another hour with labeled secondary antibody. After the final incubation, plates were washed twice and fluorescence at λ _ex = D640/20 nm and λ _em = 657 nm was determined on a fluorescence reader.

Results of the analysis

The results of individual assays for some representative compounds in these assays are presented in the table below. The results of the microsomal assays are reported as the "GCS IC ₅₀ ", which is the concentration of the compound that causes 50% inhibition of glucosylceramide synthase activity. The results of the cellular assays are reported as the "GM3 B16 IC ₅₀ " or "GM3 C32 IC ₅₀ " for the B16 assay and the C32 assay, respectively. These values represent the concentration of the compound that causes 50% inhibition of GM3 expression on the cell surface.

Connection No. GCS IC ₅₀ (nM) GM3 B16 IC ₅₀ (nM) GM3 C32 IC ₅₀ (nM) 1 0.0019 0,0156 0.0021 2 0,0601 0.1068 0.0096 3 0,00414 0,0437 0,00131 4 0,0015 0,0116 0,0008 5 0,0012 0,0193 0,0003 6 0.0028 0,0181 0,0006 7 0,0014 0,0081 0,0004 8 0,0010 0.0075 0,0004 9 0,0014 0,0168 0,0004 10 0.0064 0,0213 0.0022 11 0,0149 0,0819 0.0018 12 0,0203 0.0878 0.0037 13 0.0035 0.0386 0,0007 14 0,0104 0.1096 0.0053 15 0.0267 0.0295 0.0049 16 0.0024 0,0666 0,0016 17 0.4544 0.8786 0,0216 18 0,1480 0.6555 0.0223 19 0,1701 0,1972 0.0426 20 0.3601 0.1065 0.0198 21 0,0506 0.2658 0,0111 22 0.0096 0.0865 0.0032 23 0.0026 0,0477 0,0008

These comparative results demonstrate that the compounds according to the present description have comparable in vitro activity as GCS inhibitors and, as a result, are expected to demonstrate similar benefits in vivo.

Example 5: Clinical Study of Compound 2 in Patients with GD-3

A 156-week, open-label, multinational, multi-arm study is being initiated to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and exploratory efficacy endpoints of Compound 2 in combination with imiglucerase in adult patients with imiglucerase-stabilized type 3 Gaucher disease.

Patients aged 18 years or older with a clinical diagnosis of GD3 and documented deficiency of acid beta-glucosidase activity, treated with ERT for at least 3 years and imiglucerase (Cerezyme) at a stable monthly dose for at least 6 months prior to inclusion, were included in the study. Patients must achieve the following GD therapeutic goals: hemoglobin level ≥11.0 g/dL for women and ≥12.0 g/dL for men; platelet count ≥100,000/ ^mm3 ; spleen volume <10 times normal (MN) or total splenectomy (provided that splenectomy occurred >3 years before randomization); liver volume <1.5 MN; and absence of bone crisis and absence of symptomatic bone diseases such as bone pain related to osteonecrosis and/or pathological fractures within the last year. Patients must have GD3 with oculomotor impairment (supranuclear gaze palsy) characterized by horizontal saltatory abnormality.

(A) Interim analysis at week 26

An interim analysis was conducted when 5 patients completed 26 weeks of concomitant treatment with (1) imiglucerase (Cerezyme by Sanofi Genzyme) as prescribed for each patient and (2) compound 2 administered orally at 15 mg/day as a single dose. During the study, patients will be assessed for safety and tolerability, CSF and plasma biomarkers (glucosylceramide, GL-1; glucosylsphingosine, lyso-GL1), pharmacokinetics, markers of systemic disease (spleen and liver volume measured by magnetic resonance imaging (MRI), platelet count, hemoglobin level), evidence of interstitial lung disease (high-resolution lung computed tomography (CT)) and horizontal eye movement.

At baseline, four patients had mild neurological impairment and one had moderate neurological impairment as measured by the modified severity scoring tool (mSST; Davies, et al., 2011). All patients had evidence of interstitial lung disease based on chest CT. One patient had anemia as evidenced by a plasma hemoglobin level of 10.6 g/dL.

All patients reported no serious or long-term treatment-emergent adverse events. The most common events were headache and back pain, which were considered mild to moderate and transient in duration (likely related to the lumbar puncture performed for CSF testing).

Compound 2 was found to effectively cross the blood-brain barrier in all patients, as shown in the table below:

Compound 2 in plasma Day 1 (N=5) AUC _0-24 , ng⋅h/ml (mean ± SD) 715 ± 225 C _max , ng/ml (mean ± SD) 48.2 ± 19.2 t _max , h (median) 2.00 Compound 2 in CSF Week 4 (N=5) Week 26 (N=4) Concentration 4 hours after dose, ng/ml (mean ± SD) 4.17 ± 0.83 4.71 ± 2.50

The higher variability in CSF concentrations of compound 2 observed at week 26 is explained by a decrease in exposure in one patient (Patient 5) between weeks 12 and 26 for unknown reasons. Patient 1 was excluded from CSF determination at week 26 due to a sample collection error.

Significant improvements in plasma and CSF biomarkers were found for GD-3 after 26 weeks. At baseline, the mean (±SD) CSF GL-1 concentration was 7.1 ± 2.8 ng/mL (range 4.4-11.1 ng/mL), while the mean (±SD) CSF lyso-GL-1 concentration was 39.3 ± 22.9 pg/mL (range 20.1-67.6 pg/mL). For comparison, the CSF GL-1 concentration in healthy individuals is 4.5-5.9 ng/mL, and the CSF lyso-GL-1 concentration is less than 5.0 pg/mL. After 4 weeks and 26 weeks, the following individual reductions in CSF biomarkers were found (shown as a proportion of the reduction from baseline CSF concentration):

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Average (n=4)* GL-1 in 4 weeks -86% -55% -53% -62% -36% -64% GL-1 at 26 weeks -86% -78% -58% -78% +4% -75% Lyso-GL1 after 4 weeks -38% -16% -34% -39% -11% -32% Lyso-GL1 at 26 weeks -52% -42% -41% -57% +94% -48% *Except for Patient 5, who showed reduced exposure in the PK analysis at weeks 12-26.

The severity of interstitial lung disease is characterized by the proportion of lung volume affected by ILD, as determined by high-resolution CT in four lung regions (aortic arch, carina, L3 inferior, L4 inferior). Patients are graded as having severe ILD (51-100% of lung volume affected), moderate ILD (26-50% of lung volume affected), mild ILD (1-25% of lung volume affected), or normal (0% of lung volume showing ILD). All patients had baseline ILD, and 4 of 5 patients showed regression of ILD after 26 weeks of treatment (patient 5 showed minor progression of ILD):

Aortic arch Keel Zone L3 Zone L4 Left Right Left Right Left Right Left Right Patient 1 Original Easy. Easy. Easy. Easy. Norm. Norm. Easy. Easy. Week 26 Norm. Norm. Norm. Norm. Norm. Norm. Norm. Norm. Patient 2 Original Easy. Easy. Easy. Easy. Easy. Norm. Moderate. Moderate. Week 26 Norm. Norm. Norm. Easy. Norm. Norm. Norm. Norm. Patient 3 Original Easy. Easy. Moderate Easy. Heavy. Moderate Heavy. Heavy. Week 26 Norm. Norm. Norm. Norm. Easy. Easy. Moderate. Moderate. Patient 4 Original Norm. Easy. Norm. Norm. Norm. Norm. Norm. Norm. Week 26 Norm. Norm. Norm. Norm. Norm. Norm. Norm. Norm. Patient 5 Original Easy. Easy. Easy. Easy. Easy. Easy. Easy. Easy. Week 26 Easy. Easy. Easy. Moderate Easy. Easy. Moderate. Moderate.

No systemic deterioration was observed in any patient. Two patients showed a decrease in spleen volume of 10% or more. There were no clinically significant changes in hemoglobin levels. On average, platelet counts increased by 17% from baseline to 26 weeks, and the three patients with the lowest baseline platelet counts showed individual increases of 23-42% at 26 weeks. Individual patient data for platelet counts are shown in the table below (shown as 10 ⁹ platelets/L):

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Screening 192 207 259* 313 149 Day 1 186* 192* n/a 307* 127* Week 4 203 243 247 239 154 Week 12 214 294 264 314 180 Week 26 264 236 248 267 173 % change from baseline to 26 weeks + 42% + 23% -4.3% -13% +36% *indicates the number used as the initial one.

Quantitative assessment of horizontal and vertical saccade eye movements (HSEM and VSEM, respectively) was performed for all 5 patients. In the five patients, the mean peak velocity (PV) of horizontal 15° fast saccades to the right was 50.8°/sec (±8.1°/sec) at baseline and 47.5°/min (±12.6°/sec) at week 26; and the mean PV of horizontal 15° fast saccades to the left was 44.7°/sec (±17.9°/sec) at baseline and 32.3°/sec (±15.9°/sec) at week 26. Slower velocity implies greater neurologic impairment. The mean PV of horizontal 30° rapid saccades to the right was 77.7°/s (±16.4°/s) at baseline and 68.1°/min (±24.7°/s) at week 26; and the mean PV of horizontal 30° rapid saccades to the left was 58.7°/s (±21.5°/s) at baseline and 49.9°/s (±8.5°/s) at week 26. The normal range for 15° and 30° for horizontal rapid saccades has previously been described as >200°/s and >400°/s (Bremova-Ertl et al, 2018). HSEM measurements for each of the five patients are shown in Figures 1 and 2. Briefly, no clinically significant changes in HSEM were observed over the 26-week treatment period. As with HSEM, VSEM measurements were stable between baseline and 26 weeks.

Four exploratory biomarkers were quantified in plasma, serum, and/or CSF of GD3 patients at week 4 and week 26: chitotriosidase (CHITO; an enzyme known to be elevated in GD patients) was measured in CSF and serum; GM3 (a glycosphingolipid marker known to be elevated in GD patients) was measured in CSF and plasma; and glycoprotein non-metastatic melanoma protein B (GPNMB; a reported biomarker of neuropathic GD3) was measured in CSF. The results are shown in the table below as the proportion of change from baseline at week 4 and week 26 for each parameter:

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 CHITO CSF- Week 4 -5% +11% +9% -6% -6% CSF- Week 26 -10% -11% +20% -1% +68% Serum - Week 4 -11% +8% -7% -12% -1% Serum - Week 26 -43% -55% -1% +98% -11% GM3 CSF - Week 4 -51% 0% -59% -59% 0% CSF - Week 26 -51% 0% -59% -59% 0% Plasma - Week 4 -70% -63% -65% -62% -47% Plasma - Week 26 -86% -69% -74% -72% +10% GPNMB CSF - Week 4 -63% +30% +9% -6% -2% CSF - Week 26 -14% 0% +45% +10% -8%

(B) Interim analysis at week 52

The second interim analysis is performed when the first 6 patients reach 52 weeks of treatment as described above in section (A). This analysis includes patients 1-5 as described in section (A) as well as a new patient, patient 6. All six patients had the homozygous L444P (1448T/C) Gaucher phenotype.

At week 52, all patients remained enrolled in the study. A total of 30 treatment-emergent adverse events were reported in six patients, all of which were mild or moderate in severity, and none were considered related to treatment with Compound 2 or imiglucerase. These were primarily headache and back pain, probably related to the lumbar puncture.

Analysis of plasma and CSF concentrations of compound 2 shows largely comparable values to those obtained at week 26. However, patient 5 has approximately 50% lower plasma and CSF concentrations of compound 2 at week 26 and undetectable concentrations at week 52. This is thought to be due to either non-compliance or dosing errors, so the analysis is repeated without patient 5's data for weeks 26 and 52. These data support the conclusion that steady-state concentrations of compound 2 are achieved in plasma and CSF at or before week 4:

Compound 2 in plasma Day 1 (N=6) AUC _0-24 , ng⋅h/ml (mean ± SD) 729 ± 205 C _max , ng/ml (mean ± SD) 49.1 ± 17.3 t _max , h (median) 2.00 Compound 2 in plasma Day 1 (N=6) Week 4 (N=6) Week 26 (N=6) Week 52 (N=6) Concentration 2-4 hours after dose, ng/ml (mean ± SD) 39.7 ± 12.6 92.3 ± 36.4 102.0 ± 49.5 69.8 ± 58.3 Excluding patient 5 112 84 Compound 2 in CSF Day 1 (N=6) Week 4 (N=6) Week 26 (N=6) Week 52 (N=6) Concentration 2-4 hours after dose, ng/ml (mean ± SD) < LLOQ 4.56 ± 1.20 5.26 ± 2.49 4.43 ± 3.23 Excluding patient 5 6.13 5.32

At week 52, data also showed sustained significant improvements in plasma and CSF biomarkers for GD-3. Results were similar to those seen at week 26. For all six GD3 patients, plasma and CSF GL-1 and lyso-GL-1 concentrations were as follows:

Lizo-GL-1 GL-1 Initial level 52 weeks Initial level 52 weeks Plasma 29.3 ng/ml (6.3-159.0) 15.2 ng/ml (2.5-46.8) 6.21 mcg/ml (4.2-8.3) 1.59 mcg/ml (0.9-2.7) CSF 34.0 pg/ml (20.1-67.6) 17.3 pg/ml (5.8-37.4) 6.36 ng/ml (4.4-11.1) 2.48 ng/ml (1.0-6.1)

Thus, at week 52, compared with baseline, plasma and CSF concentrations changed as follows:

Lyso-GL-1 (% change) GL-1 (% change) Plasma concentration -56.7% -71.6% Concentration in CSF -55.9% -55.4%

Additionally, exploratory biomarkers quantified in the CSF of GD3 patients included ceramide (GL-1 precursor), chitotriosidase (CHITO), GM3, and GPNMB. After 52 weeks of treatment, no significant changes were observed in CSF concentrations of ceramide, CHITO, or GPNMB. Four of the six patients had measurable CSF GM3 concentrations at baseline, and each of these patients had undetectable CSF GM3 at 4 weeks, 26 weeks, and 52 weeks.

At 52 weeks, horizontal and vertical saccadic eye movements were quantified in all six patients in a similar manner as described in section (A). However, it was determined that the methodology used to account for noise (e.g., caused by blinking or head movements) may have biased the results. Therefore, the method for accounting for noise was modified and a set of control criteria was developed to assess the reliability of the eye movement device data sets. When the 26-week saccadic eye movement data were re-evaluated and when the 52-week data were assessed, it was found that the noise level was too high to allow any conclusions to be drawn from the data.

In addition, after 52 weeks, 5 of the 6 patients showed improvement in ataxia. The degree of ataxia at baseline and throughout the study was assessed using the Score of Ataxia Rating and Assessment (SARA; Schmitz-Hübsch et al. [2006]), which assesses eight individual features of cerebellar ataxia on a scale of 0 to 40. The eight features are: gait, stance phase, sitting, speech impairment, finger tracking, nose-toe test, rapid alternating arm movement, and heel-to-knee test. The SARA ataxia scores for all six patients are presented in the table below:

SARA Overall Score During screening Week 26 Week 52 Patient 1 3.0 1.0 0,0 Patient 2 3.0 2.0 1.5 Patient 3 3.5 0,0 0,0 Patient 4 3.0 5.0 7.5 Patient 5 0.5 0,0 0,0 Patient 6 4.0 2.0 3.0 Average rating 2.83 1.67 2.00 Average rating excluding patient 4 2.80 1.00 0.90

As shown in the table, five of the six patients have mild ataxia at baseline, with a mean SARA total score of 2.8 (SD = 1.2). The most common deficit at baseline is gait abnormalities. Excluding patient 5 due to the low level of compound 2 exposure in this patient and the patient's essentially normal baseline ataxia score (only 0.5), 4 of the 5 patients demonstrate improvement in ataxia at week 52 (mean improvement = -0.9; SD = 3.2). Patient 4 demonstrates an increase in ataxia score from a baseline score of 3 to a week 52 score of 7.5. It should be noted that this apparent worsening is almost entirely due to a change in the stance phase score parameter (stance phase score at baseline and week 26 = 1; score at week 52 = 5) and that the patient complained of left knee pain during the study. In addition, the subject injured his left big toe prior to the study; 11 days after the study, this injury had resolved. With the exception of this outlier effect in Patient 4, Compound 2 treatment resulted in a significant reduction in the mean SARA score at week 26, which was further slightly improved at week 52.

The Trail Making Test (TMT) is used to assess cognitive function in patients. The TMT is one of the most widely used neuropsychological tests and is included in most test panels. The TMT is a diagnostic tool for assessing general intelligence and cognitive dysfunction (Tombaugh et al. [2004]; Cavaco et al. [2013]). In Part A of the TMT, subjects are asked to connect a group of numbers in ascending order. This task is a combination of visual search and general visual and motor processing speed. Part B is a sequence in which digits and letters alternate. Subjects must actively switch between both categories, connecting them in an ascending but alternating order. Therefore, this task is considered to involve a component of executive function, since the subject must actively switch between categories while connecting the symbols (MacPherson et al. [2017]).

TMT-A primarily assesses perceptual speed and psychomotor speed. TMT-B more specifically assesses mental flexibility and shifting ability. The TMT B minus TMT A score is used to remove variability related to the graphomotor and visual scanning components of TMT A. The resulting score reflects the unique demands of the TMT B task.

In a study of normative data for TMT A and TMT B in community-dwelling older adults aged 18–89 years (n=911), the mean (SD) values in the 18–24 year old group (n=155) were 22.9 s (6.9) for TMT A and 49 s (12.7) for TMT B (Tombauch et al. [2004]). In contrast, the mean times taken to complete Trial A and Trial B for the patients in this study were 67.8 s (SD=60.3 s) and 193.8 s (SD=197.0), respectively. At baseline, the mean difference in the time taken to complete Trial B minus Trial A was 126.0 s (SD=142.9 s). This indicates that the GD-3 patients in this study exhibit some degree of cognitive dysfunction at baseline.

At week 52, the mean time to complete Trial A was 56.5 s (SD=55.2 s) and for Trial B it was 122.7 s (SD=91.8 s). Four of six patients demonstrated a reduction in the time taken to complete Trial A and six of six demonstrated a reduction in the time taken to complete Trial B. With the exception of Patient 5, due to the low level of Compound 2 exposure in this patient, four of five patients demonstrated a reduction in TMT A and five of five patients demonstrated a reduction in TMT-B.

At 52 weeks, 5 of 6 patients showed a reduction in time (TMT B - TMT A). Individual results are shown in the table below.

TMT-A (c) - TMT-B (c) During screening Week 26 Week 52 Change (%) from baseline to week 52 Patient 1 13 21 16 + 23% Patient 2 71 37 57 - 20% Patient 3 72 56 20 - 72% Patient 4 116 (no data) 66 - 43% Patient 5 74 60 72 - 3% Patient 6 410 440 166 - 60% Average 126 (n=6) 123 (n=5) 66 (n=6) - 29%

At week 52, the mean difference in time taken to complete Trial B minus Trial A was 66.2 s (SD=54.3). Except for patient 5, four of the five patients demonstrated improvement in Trial B minus Trial A at week 52, with a mean improvement of -71.4 s (-31.6%) (SD 99.3 s (37.6%)).

Neurological function is further assessed using functional magnetic resonance imaging (fMRI). Patient 2 was excluded because fMRI data were not collected at the week 52 session. Resting-state fMRI sessions are performed at baseline, screening, week 26, and week 52 visits. Connectivity scores from four subjects (patients 1, 3, 4, and 5) are entered into second-level analyses as the “matched” group. Patient 5 was isolated due to probable noncompliance with study medication as described above. Analyses are performed as described elsewhere (Smith et al. [2009]).

Matching subjects were found to exhibit increased connectivity between a more widely distributed set of brain regions than nonmatching subjects, with increased strength between posterior and anterior aspects as the most prominent feature. At the anatomical level, matching patients showed widespread and robust increases in connectivity between occipito-parietal structures and frontal, temporal, and limbic targets. Connectivity changes in patient 5 were more modest and limited to spatially proximal structures. At the functional level, improved connectivity between default mode and medial frontal networks was observed in all subjects except patient 5, suggesting that the signal in these disparate networks is becoming more coherent, such that brain activity can be more efficiently transmitted between cognitive reserve (posterior) and higher-order executive functions (anterior). Also evident is the consistent reciprocal mapping of resting-state networks (RSNs) 2 and 3 (cognition-language-orthography and cognition-space) to RSNs 8 and 9 (executive and left frontoparietal). The spatial distribution of connectivity changes is much more important for patient 5, primarily reflecting the overlap of medial frontal and frontoparietal networks. Both views suggest that a patient who is fully compliant with the treatment protocol has developed greater coherence between the back and front of the brain, such that the whole brain has become amenable to efficient information transfer. Where this is evident, the altered connectivity for patient 5 is confined to a narrower set of frontal brain regions and represents less holistic evidence of a therapeutic effect.

The results are presented in the table below. Spatial connectivity analysis between different anatomical brain regions is performed to determine the correlation coefficient for the regressed mean voxel intensity in a single pass. The results show that connectivity between the default mode network (resting state) and the executive function network increased in patients 1, 3, 4, and 6, but decreased in patient 5.

Patient 1 Patient 3 Patient 4 Patient 5 Patient 6 Change in correlation coefficient + 0.20 +0.20 +0.20 -0.13 +0.70

Additionally, two patients were found to have a decrease in spleen volume at week 52, and mean platelet concentration increased by 9.3% on average (range -8.2% to +45.3%), all patients maintaining the therapeutic goal of more than 120 x 10 ⁹ /L platelet count. The increase in mean platelet concentration was primarily driven by increases in 3 of 6 patients. There were no clinically significant changes in hemoglobin levels.

Example 6: Pharmacokinetics of Compound 2 in healthy human volunteers

Two phase 1 clinical studies were conducted to evaluate the pharmacokinetics, pharmacodynamics, safety, and tolerability of Compound 2 in healthy human volunteers in the presence and absence of food. Compound 2 is also known as venglustat.

Study 1

Study 1 is a 2-part, single-center study in healthy adult male volunteers. Part 1 is a double-blind, randomized, placebo-controlled, sequential, ascending, single-dose study of Compound 2 to evaluate safety, tolerability, and PK. Part 2 is an open-label, single-cohort, randomized, crossover study with 2 sequences, 2 periods, and 2 treatments of Compound 2 to determine PK with and without a high-fat meal.

Part 1 of the study included 55 healthy men and randomized them (placebo, n=14; doses of 2, 5, 15, 25, 50, and 100 mg, n=6 each; dose of 150 mg, n=5). Part 2 included eight healthy men.

In Part 1, subjects were randomly assigned to receive 2, 5, 15, 25, 50, 100, or 150 mg of Compound 2 (L-apple salt form) or matching placebo on the morning of Day 1 after at least a 10-hour fast. In Part 2, subjects were randomly assigned to receive a single oral dose of 5 mg of Compound 2 either during the fast (at least 10 hours before and 4 hours after dosing) or 30 minutes after a standardized high-fat breakfast (~815 kcal). After a 7-day washout period, subjects were crossed over to the other condition.

In Study 1, Part 1, blood will be collected for determination of plasma Compound 2 concentrations at the time of study drug administration (0 hour) and at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 48, 72, and 96 hours post-dose. Urine samples will be collected for analysis of Compound 2 concentrations beginning 2 hours before and 48 hours after study drug administration.

In Study 1, Part 2, blood was collected to determine plasma Compound 2 concentrations at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, and 48 hours post-dose.

In Part 1, it was found that following single oral dosing of 2-150 mg doses of Compound 2, peak plasma concentration (C _max ) occurred at a median time of 3-5.5 hours, then plasma concentrations began to decline exponentially, with a geometric mean t _1/2 of 28.9 hours. Exposure increased proportionally with dose across the dose range, with a 75-fold increase in dose yielding 97.3-, 89.2-, and 85.9-fold increases in geometric mean C _max , AUC _last , and AUC _inf , respectively. The PK results are shown in the following table (AUC=area under the time-concentration curve, either for the last measurable concentration or extrapolated to infinity; t _1/2= terminal half-life; CL/F=apparent total plasma clearance; CV=coefficient of variation; SD=standard deviation; t _max =time to C _max ; Vss/F=apparent volume of distribution at steady state):

Parameter 2 mg (N=6) 5 mg (N=6) 15 mg (N=6) 25 mg (N=6) 50 mg (N=6) 100 mg (N=6) 150 mg (N=5) C _max , ng/ml Average (SD) 5.7 (1.2) 14.7 (1.61) 53.0 (16.7) 84.4 (31.8) 181 (56) 374 (38) 529 (109) Geometric mean (CV) 5.6 (21.4) 14.6 (10.9) 50.7 (31.5) 79.9 (37.7) 173 (31) 372 (10.3) 520 (21) t _max , median, h (range) 3.50 (3.00-8.00) 5.50 (4.00-8.00) 3.50 (2.00-5.00) 5.00 (4.00-8.00) 4.00 (3.00-6.00) 3.00 (2.00-4.00) 4.00 (1.00-8.00) PPK _last , ng⋅h/ml Average (SD) 214 (52) 560 (71) 1,830 (520) 3,380 (1100) 6,310 (1880) 13,000 (2330) 18,600 (5480) Geometric mean (CV) 209 (24.3) 556 (12.7) 1,760 (29) 3,240 (33) 6,070 (30) 12,800 (18) 18,000 (30) PPK _inf , ng⋅h/ml Average (SD) 243 (61) 652 (122) 2,070 (600) 3,810 (1,080) 7,130 (2,320) 14,400 (3,010) 20,600 (6,640) Geometric mean (CV) 237 (25) 643 (19) 1,990 (29) 3,690 (28) 6,800 (33) 14,100 (21) 19,900 (32) t _1/2 , h Average (SD) 29.2 (43) 33.3 (8.1) 29.7 (7.1) 30.2 (5.5) 28.9 (5.3) 27.8 (3.6) 26.9 (5.7) Geometric mean (CV) 28.9 (14.8) 32.5 (24.4) 29.0 (24.0) 29.8 (18.1) 28.5 (18.4) 27.6 (12.8) 26.4 (21.3) CL/F, l/h Average (SD) 6.43 (1.41) 5.86 (1.01) 5.85 (1.89) 5.18 (1.31) 5.75 (2.01) 5.38 (1.25) 5.80 (1.55) Geometric mean (CV) 6.3 (22.0) 5.8 (17.3) 5.6 (32.2) 5.0 (25.3) 5.5 (34.9) 5.3 (23.4) 5.6 (26.7) V _ss /F, l Average (SD) 275 (54) 274 (30) 245 (81) 240 (78) 239 (62) 213 (22) 228 (50) Geometric mean (CV) 270 (20) 273 (11) 233 (33) 228 (33) 232 (26) 212 (10) 223 (22)

From Part 2, it was found that administration of the 5 mg dose in the high-fat meal had no effect on the effects of Compound 2 compared to the fasted state. The median t _max was 6.00 hours regardless of the fed or fasted state. The fed/fasted geometric mean ratios were 0.92 and 0.91 for C _max and AUC _last , respectively. Within-subject variability (i.e., fed/fasted) was less than half of the total subject variability.

Study 2

Study 2 was a single-center, double-blind, randomized, placebo-controlled, sequential, ascending, repeat-dose study of the safety, tolerability, PK, and pharmacodynamics of Compound 2 in healthy adult male and female volunteers.

The study enrolled and randomly assigned 36 healthy adults (19 men and 17 women) (n=9 in each group). Subjects were randomly assigned to receive once daily Compound 2 at a dose of 5, 10, or 20 mg (as 5 mg capsules of L-malt salt) or placebo for 14 days after at least 10 hours of fasting.

Blood is collected for determination of plasma concentrations of Compound 2 as follows: Day 1 at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 16 hours post-dosing; Days 2-5, 8, 11, and 13, at 0 h; Day 14 at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12 hours post-dosing; Days 15-17 at 24, 48, and 72 hours, respectively, post-dosing on Day 14. Urine samples are collected for analysis of Compound 2 concentrations on Day 1 (0 hours post-dosing) and again on Day 14 at 0-24 hours post-dosing. Pharmacodynamic endpoints (plasma GL-1, GL-3, and GM3 concentrations) will be assessed on days 1-5, 8, 11, 13, and 14, at 0 hours post-dose; and on day 15 through 24 hours post-dose on day 14.

In subjects receiving 5, 10, or 20 mg of Compound 2 once daily for 14 days, plasma C _max was found to occur at a median time of 2-5 hours after dosing on days 1 and 14. C _trough values reached a plateau at day 5. 5. Compound 2 exposure increased closely in proportion to dose over the 5-20 mg dosing range: this 4-fold increase in dose resulted in 3.76- and 3.69-fold increases in geometric mean C _max and AUC _0-24 values on day 14, respectively. The PK results from Study 2 are summarized in the following table:

Day 1 Parameter 5 mg (N=9) 10 mg (N=9) 20 mg (N=9) C _max , ng/ml Average (SD) 18.5 (3.2) 38.5 (7.4) 68.0 (15.7) Geometric mean (CV) 18.2 (17.3) 37.8 (19.3) 66.5 (23.1) t _max , median, h (range) 5.00 (2.00-8.17) 3.00 (2.00-5.00) 3.07 (2.00-6.00) AUC _0-24 , ng⋅h/ml Average (SD) 296 (54) 635 (132) 1,100 (211) Geometric mean (CV) 292 (18) 623 (21) 1,080 (19) Day 14 C _max , ng/ml Average (SD) 37.0 (6.4) 89.7 (29.1) 142 (40) Geometric mean (CV) 36.5 (17.2) 86.0 (32.5) 137 (28.3) t _max , median, h (range) 3.00 (2.00-6.00) 2.00 (2.00-6.00) 3.00 (2.00-8.00) AUC _0-24 , ng⋅h/ml Average (SD) 642 (121) 1,550 (464) 2,420 (705) Geometric mean (CV) 632 (19) 1,490 (30) 2,340 (29) C _trough , ng/ml Average (SD) 19.4 (4.0) 49.9 (19.3) 73.3 (24.4) Geometric mean (CV) 19.0 (20.5) 47.5 (38.7) 69.9 (33.2) t _1/2 , h Average (SD) 29.3 (4.6) 31.3 (3.3) 35.0 (6.3) Geometric mean (CV) 29.0 (15.8) 31.2 (10.5) 34.5 (18.0) CL _ss /F, l/h Average (SD) 5.98 (1.17) 5.13 (1.25) 6.58 (1.70) Geometric mean (CV) 5.9 (19.5) 5.0 (24.4) 6.4 (25.8) CL _R(0-24) , l/h Average (SD) 1.55 (0.68) 1.49 (0.41) 2.07 (0.58) Geometric mean (CV) ND ^a (44.0) 1.4 (27.7) 2.0 (28.0)

Following 14 once daily doses of Compound 2, the 24-hour unchanged urinary fractional excretion (mean fe _0-24 ) ranged between 26.3% and 33.1%, with no apparent dose relationship. Mean CL _R(0-24) ranged from 1.49 L/h to 2.07 L/h, approximately 3.18- to 3.86-fold lower than the observed plasma CL/F.

Plasma GL-1, GL-3, and GM3 in placebo recipients remain similar to baseline, while plasma GL-1 and GM3 levels decrease from baseline in a time- and dose-dependent manner in the Compound 2 dose group as shown in the following table (point estimate of glucosylceramide (GL-1), globotriaosylceramide (GL-3), and GM3 ganglioside (GM3) treatment ratios at day 15 in a repeated dose escalation study):

Parameter Comparison Grade 90% confidence interval GL-1 5 mg to placebo 0.39 0.29-0.50 10 mg to placebo 0.32 0.25-0.42 20 mg to placebo 0.23 0.17-0.30 GL-3 5 mg to placebo 0.61 0.47-0.79 10 mg to placebo 0.69 0.53-0.89 20 mg to placebo 0.67 0.51-0.89 GM3 5 mg to placebo 0.56 0.45-0.70 10 mg to placebo 0.49 0.39-0.60 20 mg to placebo 0.40 0.32-0.50

The maximum sustained effect on GL-1 was observed at day 11 in the 5 and 10 mg groups and at day 8 in the 20 mg group. The mean calculated reductions in GL-1 from baseline at day 15 were 41.9%, 69.6%, and 74.6% in the respective 5, 10, and 20 mg groups. GL-1 values were below the lower limit of quantification (LLOQ) at baseline in 1 recipient of 5 mg Compound 2 and at day 15 in 3, 5, and 9 subjects in the 5, 10, and 20 mg dose groups, respectively.

Maximal sustained reductions in GM3 were observed across all Compound 2 dose groups beginning on day 13. Mean plasma GM3 levels on day 15 were 42.7%, 49.4%, and 57.8% of baseline for the 5, 10, and 20 mg dose groups, respectively. GM3 was below the LLOQ on day 15 in 1 and 2 subjects in the 10 and 20 mg dose groups, respectively.

Plasma GL-3 also decreased over time in all Compound 2 dose groups, but variable and low baseline GL-3 values compared with LLOQ were limited by mean estimated decreases in GL-3. In the placebo, 5, 10, and 20 mg dose groups, GL-3 values were below LLOQ in 1, 3, 1, and 6 subjects, respectively, at baseline and in 4, 9, 7, and 9 subjects, respectively, at Day 15.

The mean estimated decreases in plasma GL-1 from baseline (90% CI) associated with Compound 2C _trough in the 5, 10, and 20 mg dose groups (19.0, 47.5, and 69.9 ng/mL, respectively) were 67.0% (54.4-79.7%), 74.4% (63.7-85.2%), and 76.3% (64.8-87.8%), respectively.

Conclusions

In these studies, the effects of Compound 2 in healthy subjects (C _max and AUC) were nearly dose-dependent when administered as single doses ranging from 2 to 150 mg or as repeated, single doses ranging from 5 to 20 mg for 14 days. Compared with fasting, a high-fat meal had no effect on effects in subjects receiving a single 5 mg dose. With repeated doses of 5 to 20 mg once daily, steady state was achieved within 5 days; accumulation was not affected by age or gender. Pharmacodynamically, repeated daily doses of Compound 2 decreased plasma GL-1 and GM3 in a time- and dose-dependent manner, consistent with Compound 2-mediated inhibition of GCS, although baseline GL-3 levels were too low to be useful as a pharmacodynamic biomarker. The dose-dependent reduction in GL-1 confirmed the proposed mechanism of action of Compound 2: inhibition of GL-1 formation from ceramide via GCS.

In all studies, the safety profile is assessed by monitoring treatment-emergent adverse events (TEAEs) for 10 days after the last dose of study drug, including serious adverse events [SAEs]), ECG monitoring, laboratory parameters, and physical examinations.

There were no deaths, SAEs, severe TEAEs, or TEAEs leading to study discontinuation in any of the studies.

No clinically significant hematological or biochemical abnormalities were reported in any of the studies. Vital signs did not show significant changes from baseline in any of the studies. ECG parameters did not show significant changes in the single ascending dose and food studies; in the multiple ascending dose study, ECG parameters did not change statistically significantly from mean baseline compared to placebo in Compound 2 recipients at any dose. It should be understood that while the invention has been described in connection with the above embodiments, the above description and examples are intended to illustrate and not to limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

Example 7: Clinical study of Compound 2 in patients with Fabry disease

Way

A 3-year, open-label study of Compound 2 in young patients with classical Fabry disease was conducted to evaluate the long-term safety, pharmacodynamics, and exploratory efficacy endpoints of Compound 2 in adult male patients with Fabry disease. Eleven subjects were enrolled in the study, and seven subjects completed all aspects of the study. All subjects were male with diagnosed classical Fabry disease, confirmed by genotype, and residual alpha-galactosidase activity below detection levels (9 of 11 had a nonsense mutation in the GLA gene). All subjects had plasma lyso-GL3 levels of at least 65 ng/mL and had not received prior specific treatment for Fabry disease. The median age of subjects was 24 years (range 19-37 years).

Patients are administered a daily oral dose of 15 mg Compound 2. Clearance of GL-3 deposits in skin is monitored by taking biopsies at 12, 26, 52, and 156 weeks and assessing them semiquantitatively by light microscopy (focusing on skin capillary endothelial cells). Each specimen is independently assessed by 3 pathologists for the presence of GL-3 deposits on a 4-point scale and graded as 0 (none/trace), 1 (mild), 2 (moderate), or 3 (severe) according to Eng et al., N. Engl. J. Med. 345:9-16 (2001). A single score for each patient at time point is obtained by taking the scores of a majority of the 3 pathologists. If a majority score cannot be obtained, the mean score is used (resulting in some fractional scores). Plasma samples were also analyzed for GL-3, lyso-GL-3, GL-1, and GM3 at baseline and weeks 12, 26, 52, and 156. Pain and abdominal symptom scores were analyzed at baseline and weeks 12, 26, 52, 104, and 156 using the SF-36 scoring protocol.

Patients are assessed using the Short Form-36 (SF-36) questionnaire at multiple visits from baseline to week 156. This is a 36-item questionnaire used to measure eight different aspects of health (vitality, physical functioning, bodily pain, general perception of health, physical role functioning, emotional role functioning, social role functioning, and mental health). The score for each of the eight aspects ranges from 0 (maximum disability) to 100 (no disability), with higher scores indicating good health. In addition, gastrointestinal symptoms, including abdominal pain, bloating, and bowel movements, are assessed using a modified version of the Inflammatory Bowel Severity Scoring System. Specific questions asked as part of these assessments include: (1) whether the patient has had abdominal pain in the past ten days, (2) what was the severity of the abdominal pain in the past ten days, using a rating scale from 0 (no pain) to 100 (very severe pain), and (3) how many days in the past ten days did the patient experience abdominal pain.

Results

At week 156, five patients had a 1-point decrease in skin GL-3 score, two patients had complete removal of GL-3 inclusions, one patient had no change, and one patient had no samples. Over the 156-week study, mean plasma GL-1 decreased by 69%, mean plasma GM3 decreased by 60%, mean plasma GL-3 decreased by 77%, and mean plasma lyso-GL3 decreased by 52%. Plasma GL-1 and GM3 showed very rapid decreases during the first 2-4 weeks of treatment. All four parameters showed a steady maintenance of reduced plasma loads, which had largely stabilized by week 52.

Plasma and urine data are summarized in the table below:

Measured parameter: Change from original measurement to: 26 weeks (n=9) 52 weeks (n=7) 104 weeks (n=7) 156 weeks (n=7) Plasma GL-3 (µg/ml) -3.62 mcg/ml -5.06 mcg/ml -6.32 mcg/ml -6.97 mcg/ml lyso-GL-3 in plasma (ng/ml) -30.99 ng/ml -37.10 ng/ml -39.84 ng/ml -48.13 ng/ml Plasma GL-1 (µg/ml) -3.26 mcg/ml -3.58 mcg/ml -3.70 mcg/ml -3.23 mcg/ml M3 in plasma G (µg/ml) -10.77 mcg/ml -8.84 mcg/ml -9.92 mcg/ml -8.12 mcg/ml GL-3 in urine (mg per mmol urine creatinine) -0.25 mg/mmol
(n=8) -0.20 mg/mmol
(n=7) -0.18 mg/mmol
(n=7) -0.18 mg/mmol
(n=7)

These results demonstrate that Compound 2, administered at a dose of 15 mcg/day, consistently reduces whole body GL-1, lyso-GL-1, and GM3 levels in a progressive manner.

In addition, data from a previously completed, placebo-controlled phase 3 study of agalsidase beta (Fabrazyme) were analyzed for comparison (see Eng et al., N. Eng. J. Med., 345:9 (2001). For comparison with agalsidase beta, the historical control group includes patients treated with agalsidase beta in the phase 3 trial, and the change in plasma GL-3 is compared at multiple time points for up to three years. Inclusion criteria and baseline characteristics were similar between the two studies. To strengthen the comparison, patients treated with Compound 2 are matched to patients in the phase 3 study based on propensity scores using the baseline variables of age, plasma GL-3, sex, UPCR (<500 mg/g to 500–1000 mg/g to >1000 mg/g), and eGFR (<80 to ≥80 ml/min/1.73 ^m2 ). Eleven patients treated with Compound 2 were compared with 19 patients for the placebo comparison and with 28 patients for the agalsidase beta comparison. All patients in all three groups were male and had elevated plasma GL-3, UPCR <500 mg/g, and eGFR ≥80 ml/min/1.73 ^m2 . The mean age was similar in all three groups. The comparison shows that treatment with Compound 2 for 26 weeks resulted in a significant decrease in plasma GL-3 compared with placebo, -3.62 μg/mL versus -1.06 μg/mL (P < 0.0001). Although Compound 2 treatment produced similar reductions in plasma GL-3 at week 52 compared with agalsidase beta, at week 104 and at week 156, the reduction in plasma GL-3 with Compound treatment was significantly greater (p=0.0351 at week 104; p=0.0081 at week 156). Plasma GL-3 levels at week 156 were 1.90 mcg/mL for Compound 2-treated patients compared with 4.44 mcg/mL for agalsidase beta-treated patients.

Detailed results of GL-3 incorporation assessment in skin are shown in the tables below (a score of 0 means no GL-3 incorporation):

Endothelial cells, superficial vessels Assessment of GL-3 changes in skin Number of patients in each change category (change from baseline skin score) by: 12 weeks 26 weeks 52 weeks 156 weeks Rating 1 is reduced to 0 2/6 (33%) Rating 1 does not change 4/9 (44%) 4/9 (44%) 3/6 (50%) 1/6 (17%) Rating 1 increases to 2 1/9 (11%) 1/9 (11%) Rating 2 is reduced to 1 3/9 (33%) 3/9 (33%) 2/6 (33%) 3/6 (50%) Rating 2 does not change 1/9 (11%) 1/9 (11%) 1/6 (17%) Endothelial cells, deep vessels Assessment of GL-3 changes in skin Number of patients in each change category (change from baseline skin score) by: 12 weeks 26 weeks 52 weeks 156 weeks Rating 1 does not change 1/9 (11%) 1/9 (11%) 1/6 (17%) Rating 2 is reduced to 0.5 1/6 (17%) Rating 2 is reduced to 1 2/9 (22%) 2/9 (22%) 3/6 (50%) 3/6 (50%) Rating 2 is reduced to 1.5 1/6 (17%) Rating 2 does not change 6/9 (67%) 6/9 (67%) 2/6 (33%) 1/6 (17%) Smooth muscle cells, deep vessels Assessment of GL-3 changes in skin Number of patients in each change category (change from baseline skin score) by: 12 weeks 26 weeks 52 weeks 156 weeks Rating 1.5 does not change 2/9 (22%) Rating 1.5 increases to 2 2/9 (22%) 1/5 (20%) 1/6 (17%) Rating 2 is reduced to 0.5 Rating 2 is reduced to 1 1/6 (17%) Rating 2 is reduced to 1.5 1/6 (17%) Rating 2 does not change 7/9 (78%) 7/9 (78%) 4/5 (80%) 3/6 (50%) Perineurium cells Assessment of GL-3 changes in skin Number of patients in each change category (change from baseline skin score) by: 12 weeks 26 weeks 52 weeks 156 weeks Rating 1 does not change 1/6 (17%) Rating 1 increases to 2 1/9 (11%) 1/9 (11%) 1/6 (17%) Rating 2 is reduced to 1 1/6 (17%) Rating 2 is reduced to 1.5 1/6 (17%) 1/6 (17%) Rating 2 does not change 8/9 (89%) 8/9 (89%) 3/6 (50%) 4/6 (67%)

In addition to counting GL-3 inclusions in the skin using light microscopy, the proportion of endothelial cell cytoplasmic volume occupied by GL-3 inclusions was estimated using point counting of electron microscopic images with a masked reader. Images of at least 50 capillaries of superficial endothelial cells were obtained using electron microscopy at 7500x magnification. Two-way t-tests were used to evaluate differences between baseline and post-treatment values at each time point. The results are presented in the table below:

Baseline (N=9) 3 months (N=9) 6 months (N=8) 3 years (N=6) Volume fraction (volume of inclusions/total volume of cytoplasm) 0.29 ± 0.03 0.29 ± 0.06 0.23 ± 0.04 0.18 ± 0.03 Changes from baseline -1.9% -21.1% -38.7% P value to baseline 0.74 0,001 0,001

These results demonstrate that Compound 2, administered at 15 mg/day, consistently reduces GL-3 inclusion levels in the skin in a generally progressive manner. The results were generally more pronounced in superficial vascular endothelium compared to deep vascular endothelial cells and other skin tissues.

Seven of nine patients improved their overall body pain scores at week 26 (SF-36), while three of six patients improved their overall body pain scores at week 156 (SF-36). Among patients with gastrointestinal pain at baseline, pain severity (abdominal pain) decreased in four of five at week 26 and in four of four at week 156. The number of days with gastrointestinal pain decreased in five of five patients at week 26 and in three of four patients at week 156.

Detailed abdominal pain measurement results are shown in the tables below.

Abdominal pain Number of patients who responded “Yes” to abdominal pain 10 days prior to visit Initial level 4 weeks 12 weeks 26 weeks 52 weeks 104 weeks 156 weeks 6/11 (55%) 4/11 (36%) 2/10 (20%) 3/9 (33%) 2/7 (28%) 2/7 (28%) 2/7 (28%) Assessment of abdominal pain severity 10 days before the visit (mean, scale 0-100) Baseline (n=6) 4 weeks (n=4) 12 weeks (n=2) 26 weeks (n=3) 52 weeks (n=1) 104 weeks (n=1) 156 weeks (n=2) 52.50 29.75 40,00 31,33 21.00 21.00 15.00 Number of days in the previous 10 days with abdominal pain (mean) Baseline (n=6) 4 weeks (n=4) 12 weeks (n=2) 26 weeks (n=3) 52 weeks (n=3) 104 weeks (n=2) 156 weeks (n=2) 3.83 2.50 3.50 3.00 0.70 0.50 0.20

These results demonstrate that Compound 2, administered at a dose of 15 mg/day, consistently reduces abdominal pain and overall body discomfort in a progressive manner.

Furthermore, when features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is similarly described in terms of any individual member or subgroup of members of the Markush group.

All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, shall control.

Claims (26)

1. A method for treating or preventing cognitive dysfunction associated with a lysosomal storage disease in a human subject in need thereof, wherein the method is effective for improving cognitive performance or reducing cognitive deficits as measured by a reduction in the time required to complete the trail making test (TMT), TMT-A and/or TMT-B or a reduction in the difference between TMT-A time and TMT-B time (TMT-A - TMT-B); comprising administering to the subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, where: R ¹ is hydrogen; R ² and R ³ are independently selected from C _1-3 -alkyl; R ⁴ , R ⁵ and R ⁶ are each independently selected from hydrogen and halogen; and A is thiazolyl. 2. The method according to claim 1, wherein R ² and R ³ are each independently selected from methyl and ethyl groups. 3. The method according to claim 1 or 2, wherein R ⁴ is fluorine and R ⁵ and R ⁶ are hydrogen; and/or wherein R ⁴ is located in the 4 position of the phenyl ring to which it is attached. 4. The method according to any one of claims 1 to 3, wherein said compound is represented by the following structural formulas (III), (IV), (V), (VIII), or (XI): or a pharmaceutically acceptable salt thereof. 5. The method according to claim 4, wherein said compound is represented by the structural formula (XI) and wherein R ⁴ is fluorine. 6. The method according to claim 1, wherein said compound is ( S )-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate or a pharmaceutically acceptable salt thereof. 7. The method according to claim 1, wherein said compound is ( S )-quinuclidin-3-yl(2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate in the form of a malate salt. 8. The method according to any one of claims 1 to 7, wherein the cognitive dysfunction is a dementia, such as Alzheimer's disease or Parkinson's disease, optionally wherein the dementia exhibits signs of impairment in visual search speed, reading processing speed, mental flexibility and/or executive functioning. 9. The method according to any one of claims 1 to 8, wherein the reduction in the time required to complete the route construction test (TMT), TMT-A and/or TMT-B or the reduction in the difference between the TMT-A time and the TMT-B time (TMT-A - TMT-B) is at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%. 10. The method according to any one of claims 1 to 9, wherein the method is a method for treating or preventing cognitive dysfunction and gait disorders, including ataxia associated with lysosomal storage disease. 11. The method of claim 10, wherein the ataxia is cerebellar ataxia, wherein the ataxia is optionally manifested by symptoms selected from gait instability, asthenia, asynergia, reaction delay, dyschronometry, dysarthria, dysphagia, hypotonia, dysmetria, hypometria, hypermetria, dysdiadochokinesia, slurred speech, vocal tremor, ataxic breathing, postural instability, and a combination thereof, for example, wherein the primary ataxic deficit is gait instability. 12. The method according to claim 10 or 11, wherein the subject has a baseline ataxia of at least 0.5 on the scale of ataxia rating and ranking (SARA) at the beginning of therapy according to the method, for example, the baseline SARA score is at least 1 or at least 2 or at least 3 or at least 4 or at least 5 or at least 10 or at least 20. 13. The method of any one of claims 10 to 12, wherein the method is effective to reduce the SARA ataxia scale by at least 0.5, such as reducing the SARA score by at least 1, or at least 2, or at least 3, or at least 5, or at least 10; or wherein the method is effective to reduce the SARA score to values from 0.00 to 3.00, or from 0.00 to 2.00, or from 0.00 to 1.50, or from 0.00 to 1.00, or from 0.00 to 0.50. 14. The method according to any one of claims 1 to 13, wherein the subject has Gaucher disease type 3 or Niemann-Pick disease type C or GM2 gangliosidosis. 15. The method according to any one of claims 1 to 14, wherein the subject is undergoing concomitant treatment with enzyme replacement therapy (ERT), for example using glucocerebrosidase, optionally wherein the subject is undergoing treatment with enzyme replacement therapy prior to treatment with said compound, and/or wherein the method further comprises the step of switching the subject from ERT therapy to treatment with said compound. 16. The method according to any one of claims 1 to 15, wherein said compound or a pharmaceutically acceptable salt thereof is administered systemically, for example, non-parenterally or orally, and/or wherein the subject is administered a daily dose of from about 1 mg to about 150 mg of said compound, for example, from 5 to 50 mg, or from 10 to 40 mg, or from 10 to 30 mg, or from 10 to 20 mg, or from 20 to 30 mg, or from 30 to 40 mg, or from 40 to 50 mg, or from 5 to 25 mg, or from 20 to 50 mg, or from 5 to 15 mg, or from 15 to 30 mg, or about 15 mg.