WO2021247916A1

WO2021247916A1 - Azetidine and spiroazetidine compounds and uses thereof

Info

Publication number: WO2021247916A1
Application number: PCT/US2021/035771
Authority: WO
Inventors: Iwona WRONA; Kerem OZBOYA; Bertand LE BOURDONNEC; Madeline MACDONNELL; Vanessa KURIA; Matthew Lucas; Mitch RIVERS; Byron Delabarre
Original assignee: Yumanity Therapeutics, Inc.
Priority date: 2020-06-03
Filing date: 2021-06-03
Publication date: 2021-12-09
Also published as: WO2021247916A8

Abstract

The present invention features compounds useful in the treatment of neurological disorders. The compounds of the invention, alone or in combination with other pharmaceutically active agents, can be used for treating or preventing neurological disorders.

Description

AZETIDINE AND SPIROAZETIDINE COMPOUNDS AND USES THEREOF

Background

An incomplete understanding of the molecular perturbations that cause disease, as well as a limited arsenal of robust model systems, has contributed to a failure to generate successful disease-modifying therapies against common and progressive neurological disorders, such as ALS and FTD. Progress is being made on many fronts to find agents that can arrest the progress of these disorders. However, the present therapies for most, if not all, of these diseases provide very little relief. Accordingly, a need exists to develop therapies that can alter the course of neurodegenerative diseases. More generally, a need exists for better methods and compositions for the treatment of neurodegenerative diseases in order to improve the quality of the lives of those afflicted by such diseases.

Summary

TDP-43 is a nuclear DNA/RNA binding protein involved in RNA splicing. Under pathological cell stress, TDP-43 translocates to the cytoplasm and aggregates into stress granules. These phenotypes are hallmarks of degenerating motor neurons and are found in 97% of all ALS cases. The highly penetrant nature of this pathology indicates that TDP-43 is broadly involved in both familial and sporadic ALS. Additionally, TDP-43 mutations that promote aggregation are linked to higher risk of developing ALS, suggesting protein misfolding and aggregation act as drivers of toxicity. TDP-43 toxicity can be recapitulated in yeast models, where the protein induces a viability deficit and localizes to stress granules. The present inventors have discovered that the CYP51 A1 inhibitors described herein are capable of reversing TDP-43 induced toxicity. Accordingly, the present invention describes such CYP51A1 inhibitors and methods of using these compounds for the treatment of disorders related to TDP-43 toxicity such as ALS.

In an aspect, the invention features a compound, or a pharmaceutically acceptable salt thereof, having the structure:

Formula I wherein R¹ has the structure:

Formula II m is 0, 1 , 2, or 3;

X is CH, CR⁵, or N; each R⁵ is, independently, hydrogen, halo, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 alkoxy;

R² is hydrogen, halo, optionally substituted amino, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, hydroxy, -CH2OH, or morpholino;

R³ is hydrogen or optionally substituted C1-C6 alkyl; L¹ is absent, -0-, -SO2-, or optionally substituted C1-C6 alkyl; L² has the structure:

Formula III Formula IV Formula V n, 0, p, q, r, and s are, independently, 0 or 1 ;

R⁶ is hydrogen, hydroxy, or optionally substituted C1-C6 alkyl;

L³ is absent, -O-, or optionally substituted C1-C6 alkyl; and

R⁴ is optionally substituted C6-C10 aryl, optionally substituted C1-C6 alkyl C6-C10 aryl, or optionally

In some embodiments, X is CR⁵ (e.g., CH). In some embodiments, R¹ has the structure:

some embodiments, X is

N. In some embodiments, R¹ has the structure:

In some embodiments, R² is hydroxy or -CH2OH (e.g., hydroxy). In some embodiments, R³ is hydrogen or methyl (e.g., hydrogen). In some embodiments, L³ is absent, -CH2-, or -O- (e.g., absent). In some embodiments, L² has the structure:

Formula IV p, q, r, and s are, independently, 0 or 1 ; and R⁶ is hydrogen or optionally substituted C1-C6 alkyl. in some embodiments, L² has the structure: OO s

, or

In some embodiments, L² has the structure:

Formula V p, q, r, and s are, independently, 0 or 1 ; and R⁶ is hydrogen or optionally substituted C1-C6 alkyl. In some embodiments, L² has the structure: In some embodiments, L² has the structure:

Formula III n and o are, independently, 0 or 1 ; and

R⁶ is hydrogen or optionally substituted C1-C6 alkyl.

In some embodiments, L² has the structure:

In some embodiments, R⁶ is hydrogen, methyl, or ethyl. In some embodiments, R⁶ is hydrogen. In some embodiments, L¹ is absent, -CH2-, or-SC>2-. In some embodiments, L¹ is absent.

In some embodiments, R⁴ is optionally substituted C6-C10 aryl (e.g., phenyl, 3-chloro-phenyl, 4- chloro-phenyl, 3,4-chloro-phenyl, 3-chloro-4-fluoro-phenyl, 3,5-chloro-phenyl, 2-fluoro-3-chloro-phenyl, 3- fluoro-4-chloro-phenyl, 3,4-fluoro-phenyl, 3-chloro-4-cyano-phenyl, 3-fluoro-4-trifluoromethoxy-phenyl, 2- fluoro-4-chloro-phenyl, 2-fluoro-4-trifluormethyl-phenyl, 2,4-fluoro-phenyl, 3-fluoro-4-cyano-phenyl, 2- chloro-4-fluoro-phenyl, 2, 3-chloro-phenyl, 2-cyano-5-iodo-phenyl, 2-trifluoromethoxy-5-bromo-phenyl, 2- bromo-trifluoromethyl-phenyl, or 2-cyano-5-fluoro-phenyl).

In an aspect, the invention features a compound, or a pharmaceutically acceptable salt thereof, having the structure of any one of compounds 1-123 in Table 1. Table 1 . Compounds of the Invention

In an aspect, the invention features a pharmaceutical composition comprising any of the foregoing compounds and a pharmaceutically acceptable excipient.

In an aspect, the invention features a method of treating a neurological disorder (e.g., frontotemporal dementia (FTLD-TDP), chronic traumatic encephalopathy, ALS, Alzheimer’s disease, limbic-predominant age-related TDP-42 encephalopathy (LATE), or frontotemporal lobar degeneration) in a subject in need thereof. This method includes administering an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In an aspect, the invention features a method of inhibiting toxicity in a cell (e.g., mammalian neural cell) related to a protein (e.g., TDP-43). This method includes administering an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In an aspect, the invention features a method of treating a CYP51A1 -associated disorder (e.g., FTLD-TDP, chronic traumatic encephalopathy, ALS, Alzheimer’s disease, LATE, or frontotemporal lobar degeneration) in a subject in need thereof. This method includes administering an effective amount of any of the foregoing compounds pharmaceutical compositions. In an aspect, the invention features a method of inhibiting CYP51 A1. This method includes contacting a cell with an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method of treating a neurological disorder in a patient, such as a human patient, identified as likely to benefit from treatment with a CYP51 A1 inhibitor on the basis of TDP-43 aggregation. In this aspect, the method may include (i) determining that the patient exhibits, or is prone to develop, TDP-43 aggregation, and (ii) providing to the patient a therapeutically effective amount of a CYP51A1 inhibitor. In some embodiments, the patient has previously been determined to exhibit, or to be prone to developing, TDP-43 aggregation, and the method includes providing to the patient a therapeutically effective amount of a CYP51A1 inhibitor. The susceptibility of the patient to developing TDP-43 aggregation may be determined, e.g., by determining whether the patient expresses a mutant isoform of TDP-43 containing a mutation that is associated with TDP-43 aggregation and toxicity, such as a mutation selected from Q331K, M337V, Q343R, N345K, R361S, and N390D. This may be performed, for example, by determining the amino acid sequence of a TDP-43 isoform isolated from a sample obtained from the patient or by determining the nucleic acid sequence of a TDP-43 gene isolated from a sample obtained from the patient. In some embodiments, the method includes the step of obtaining the sample from the patient.

In an additional aspect, the invention features a method of treating a neurological disorder in a patient, such as a human patient, identified as likely to benefit from treatment with a CYP51 A1 inhibitor on the basis of TDP-43 expression. In this aspect, the method includes (i) determining that the patient expresses a mutant form of TDP-43 having a mutation associated with TDP-43 aggregation (e.g., a mutation selected from Q331K, M337V, Q343R, N345K, R361S, and N390D), and (ii) providing to the patient a therapeutically effective amount of a CYP51A1 inhibitor. In some embodiments, the patient has previously been determined to express a mutant form of TDP-43 having a mutation associated with TDP- 43 aggregation, such as a Q331K, M337V, Q343R, N345K, R361S, or N390D mutation, and the method includes providing to the patient a therapeutically effective amount of a CYP51A1 inhibitor.

In another aspect, the invention features a method of determining whether a patient (e.g., a human patient) having a neurological disorder is likely to benefit from treatment with a CYP51 A1 inhibitor by (i) determining whether the patient exhibits, or is prone to develop, TDP-43 aggregation and (ii) identifying the patient as likely to benefit from treatment with a CYP51A1 inhibitor if the patient exhibits, or is prone to develop, TDP-43 aggregation. In some embodiments, the method further includes the step of (iii) informing the patient whether he or she is likely to benefit from treatment with a CYP51 A1 inhibitor. The susceptibility of the patient to developing TDP-43 aggregation may be determined, e.g., by determining whether the patient expresses a mutant isoform of TDP-43 containing a mutation that is associated with TDP-43 aggregation and toxicity, such as a mutation selected from Q331K, M337V, Q343R, N345K, R361 S, and N390D. This may be performed, for example, by determining the amino acid sequence of a TDP-43 isoform isolated from a sample obtained from the patient or by determining the nucleic acid sequence of a TDP-43 gene isolated from a sample obtained from the patient. In some embodiments, the method includes the step of obtaining the sample from the patient.

In another aspect, the invention features a method of determining whether a patient (e.g., a human patient) having a neurological disorder is likely to benefit from treatment with a CYP51 A1 inhibitor by (i) determining whether the patient expresses a TDP-43 mutant having a mutation associated with TDP-43 aggregation (e.g., a mutation selected from Q331K, M337V, Q343R, N345K, R361S, and N390D) and (ii) identifying the patient as likely to benefit from treatment with a CYP51 A1 inhibitor if the patient expresses a TDP-43 mutant. In some embodiments, the method further includes the step of (iii) informing the patient whether he or she is likely to benefit from treatment with a CYP51 A1 inhibitor. The TDP-43 isoform expressed by the patient may be assessed, for example, by isolated TDP-43 protein from a sample obtained from the patient and sequencing the protein using molecular biology techniques described herein or known in the art. In some embodiments, the TDP-43 isoform expressed by the patient is determined by analyzing the patient’s genotype at the TDP-43 locus, for example, by sequencing the TDP-43 gene in a sample obtained from the patient. In some embodiments, the method includes the step of obtaining the sample from the patient.

In some embodiments of any of the above aspects, the CYP51A1 inhibitor is provided to the patient by administration of the CYP51A1 inhibitor to the patient. In some embodiments, the CYP51A1 inhibitor is provided to the patient by administration of a prodrug that is converted in vivo to the CYP51A1 inhibitor.

In some embodiments of any of the above aspects, the neurological disorder is a neuromuscular disorder, such as a neuromuscular disorder selected from amyotrophic lateral sclerosis, congenital myasthenic syndrome, congenital myopathy, cramp fasciculation syndrome, Duchenne muscular dystrophy, glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis,

Isaac's Syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal and bulbar muscular atrophy, spinal muscular atrophy, Stiff person syndrome, Troyer syndrome, and Guillain- Barre syndrome. In some embodiments, the neurological disorder is amyotrophic lateral sclerosis.

In some embodiments of any of the above aspects, the neurological disorder is selected from frontotemporal degeneration (also referred to as frontotemporal lobar degeneration and frontotemporal dementia), Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy.

In some embodiments, the neurological disorder is amyotrophic lateral sclerosis, and following administration of the CYP51A1 inhibitor to the patient, the patient exhibits one or more, or all, of the following responses:

(i) an improvement in condition as assessed using the amyotrophic lateral sclerosis functional rating scale (ALSFRS) or the revised ALSFRS (ALSFRS-R), such as an improvement in the patient’s ALSFRS or ALSFRS-R score within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement in the patient’s ALSFRS or ALSFRS-R score within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks,

9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the patient);

(ii) an increase in slow vital capacity, such as an increase in the patient’s slow vital capacity within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an increase in the patient’s slow vital capacity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the patient);

(iii) a reduction in decremental responses exhibited by the patient upon repetitive nerve stimulation, such as a reduction that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., a reduction that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks,

(iv) an improvement in muscle strength, as assessed, for example, by way of the Medical

Research Council muscle testing scale (as described, e.g., in Jagtap et at, Ann. Indian. Acad. Neurol. 17:336-339 (2014), the disclosure of which is incorporated herein by reference as it pertains to measuring patient response to neurological disease treatment), such as an improvement that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks,

25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks,

44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the

CYP51A1 inhibitorto the patient); (v) an improvement in quality of life, as assessed, for example, using the amyotrophic lateral sclerosis-specific quality of life (ALS-specific QOL) questionnaire, such as an improvement in the patient’s quality of life that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement in the subject’s quality of life that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the patient);

(vi) a decrease in the frequency and/or severity of muscle cramps, such as a decrease in cramp frequency and/or severity within one or more days, weeks, or months following administration of the CYP51 A1 inhibitor (e.g., a decrease in cramp frequency and/or severity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the patient); and/or

(vii) a decrease in TDP-43 aggregation, such as a decrease in TDP-43 aggregation within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., a decrease in TDP-

43 aggregation within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks,

44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the patient.

Chemical Terms

It is to be understood that the terminology employed herein is for the purpose of describing particular embodiments and is not intended to be limiting. Those skilled in the art will appreciate that certain compounds described herein can exist in one or more different isomeric (e.g., stereoisomers, geometric isomers, tautomers) and/or isotopic (e.g., in which one or more atoms has been substituted with a different isotope of the atom, such as hydrogen substituted for deuterium) forms. Unless otherwise indicated or clear from context, a depicted structure can be understood to represent any such isomeric or isotopic form, individually or in combination.

In some embodiments, one or more compounds depicted herein may exist in different tautomeric forms. As will be clear from context, unless explicitly excluded, references to such compounds encompass all such tautomeric forms. In some embodiments, tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. In certain embodiments, a tautomeric form may be a prototropic tautomer, which is an isomeric protonation states having the same empirical formula and total charge as a reference form. Examples of moieties with prototropic tautomeric forms are ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1 H- and 3H-imidazole, 1 H-, 2H- and 4H- 1 ,2,4-triazole, 1 H- and 2H- isoindole, and 1 H- and 2H-pyrazole. In some embodiments, tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. In certain embodiments, tautomeric forms result from acetal interconversion, e.g., the interconversion illustrated in the scheme below:

Those skilled in the art will appreciate that, in some embodiments, isotopes of compounds described herein may be prepared and/or utilized in accordance with the present invention. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium. In some embodiments, an isotopic substitution (e.g., substitution of hydrogen with deuterium) may alter the physiciochemical properties of the molecules, such as metabolism and/or the rate of racemization of a chiral center.

As is known in the art, many chemical entities (in particular many organic molecules and/or many small molecules) can adopt a variety of different solid forms such as, for example, amorphous forms and/or crystalline forms (e.g., polymorphs, hydrates, solvates, etc). In some embodiments, such entities may be utilized in any form, including in any solid form. In some embodiments, such entities are utilized in a particular form, e.g., in a particular solid form.

In some embodiments, compounds described and/or depicted herein may be provided and/or utilized in salt form.

In certain embodiments, compounds described and/or depicted herein may be provided and/or utilized in hydrate or solvate form.

At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term “C1-C6 alkyl” is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, Cs alkyl, and C6 alkyl. Furthermore, where a compound includes a plurality of positions at which substitutes are disclosed in groups or in ranges, unless otherwise indicated, the present disclosure is intended to cover individual compounds and groups of compounds (e.g., genera and subgenera) containing each and every individual subcombination of members at each position.

Herein a phrase of the form “optionally substituted X” (e.g., optionally substituted alkyl) is intended to be equivalent to “X, wherein X is optionally substituted” (e.g., “alkyl, wherein said alkyl is optionally substituted”). It is not intended to mean that the feature “X” (e.g. alkyl) perse is optional.

The term “acyl,” as used herein, represents a hydrogen or an alkyl group, as defined herein that is attached to a parent molecular group through a carbonyl group, as defined herein, and is exemplified by formyl (i.e., a carboxyaldehyde group), acetyl, trifluoroacetyl, propionyl, and butanoyl. Exemplary unsubstituted acyl groups include from 1 to 6, from 1 to 11 , or from 1 to 21 carbons.

The term “alkyl,” as used herein, refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of 1 to 20 carbon atoms (e.g., 1 to 16 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms). An alkylene is a divalent alkyl group.

The term “alkenyl,” as used herein, alone or in combination with other groups, refers to a straight-chain or branched hydrocarbon residue having a carbon-carbon double bond and having 2 to 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 to 6, or 2 carbon atoms).

The term “alkynyl,” as used herein, alone or in combination with other groups, refers to a straight-chain or branched hydrocarbon residue having a carbon-carbon triple bond and having 2 to 20 carbon atoms (e.g., 2 to 16 carbon atoms, 2 to 10 carbon atoms, 2 to 6, or 2 carbon atoms).

The term “amino,” as used herein, represents -N(R^N1)2, wherein each R^N1 is, independently, H, OH, NO2, N(R^N2)2, S0₂0R^N2, S0₂R^N2, SOR^N2, an /V-protecting group, alkyl, alkoxy, aryl, arylalkyl, cycloalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), wherein each of these recited R^N1 groups can be optionally substituted; or two R^N1 combine to form an alkylene or heteroalkylene, and wherein each R^N2 is, independently, H, alkyl, or aryl. The amino groups of the invention can be an unsubstituted amino (i.e., -NH2) or a substituted amino (i.e., -N(R^N1)2).

The term “aryl,” as used herein, refers to an aromatic mono- or polycarbocyclic radical of 6 to 12 carbon atoms having at least one aromatic ring. Examples of such groups include, but are not limited to, phenyl, naphthyl, 1 ,2,3,4-tetrahydronaphthyl, 1 ,2-dihydronaphthyl, indanyl, and 7/-/-indenyl.

The term “arylalkyl,” as used herein, represents an alkyl group substituted with an aryl group. Exemplary unsubstituted arylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C1-C6 alkyl Ce-io aryl, C1-C10 alkyl Ce-io aryl, or C1-C20 alkyl Ce-io aryl), such as, benzyl and phenethyl. In some embodiments, the akyl and the aryl each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective groups.

The term “azido,” as used herein, represents a -N3 group.

The term “cyano,” as used herein, represents a CN group.

The terms “carbocyclyl,” as used herein, refer to a non-aromatic C3-C12 monocyclic, bicyclic, or tricyclic structure in which the rings are formed by carbon atoms. Carbocyclyl structures include cycloalkyl groups and unsaturated carbocyclyl radicals.

The term “cycloalkyl,” as used herein, refers to a saturated, non-aromatic, monovalent mono- or polycarbocyclic radical of three to ten, preferably three to six carbon atoms. This term is further exemplified by radicals such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and adamantyl.

The term “halo,” as used herein, means a fluorine (fluoro), chlorine (chloro), bromine (bromo), or iodine (iodo) radical.

The term “heteroalkyl,” as used herein, refers to an alkyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for alkyl groups. Examples of heteroalkyl groups are an “alkoxy” which, as used herein, refers alkyl-O- (e.g., methoxy and ethoxy). A heteroalkylene is a divalent heteroalkyl group.

The term “heteroalkenyl,” as used herein, refers to an alkenyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkenyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for alkenyl groups. Examples of heteroalkenyl groups are an “alkenoxy” which, as used herein, refers alkenyl-O-. A heteroalkenylene is a divalent heteroalkenyl group.

The term “heteroalkynyl,” as used herein, refers to an alkynyl group, as defined herein, in which one or more of the constituent carbon atoms have been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkynyl group can be further substituted with 1 , 2, 3, or 4 substituent groups as described herein for alkynyl groups. Examples of heteroalkynyl groups are an “alkynoxy” which, as used herein, refers alkynyl-O-. A heteroalkynylene is a divalent heteroalkynyl group.

The term “heteroaryl,” as used herein, refers to an aromatic mono- or polycyclic radical of 5 to 12 atoms having at least one aromatic ring containing one, two, or three ring heteroatoms selected from N,

O, and S, with the remaining ring atoms being C. One or two ring carbon atoms of the heteroaryl group may be replaced with a carbonyl group. Examples of heteroaryl groups are pyridyl, pyrazoyl, benzooxazolyl, benzoimidazolyl, benzothiazolyl, imidazolyl, oxaxolyl, and thiazolyl.

The term “heteroarylalkyl,” as used herein, represents an alkyl group substituted with a heteroaryl group. Exemplary unsubstituted heteroarylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C1-C6 alkyl C2-C9 heteroaryl, C1-C10 alkyl C2-C9 heteroaryl, or C1-C20 alkyl C2-C9 heteroaryl). In some embodiments, the akyl and the heteroaryl each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective groups.

The term “heterocyclyl,” as used herein, denotes a mono- or polycyclic radical having 3 to 12 atoms having at least one ring containing one, two, three, or four ring heteroatoms selected from N, O or S and no aromatic ring containing any N, O, or S atoms. Examples of heterocyclyl groups include, but are not limited to, morpholinyl, thiomorpholinyl, furyl, piperazinyl, piperidinyl, pyranyl, pyrrolidinyl, tetrahydropyranyl, tetrahydrofuranyl, and 1 ,3-dioxanyl.

The term “heterocyclylalkyl,” as used herein, represents an alkyl group substituted with a heterocyclyl group. Exemplary unsubstituted heterocyclylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C1-C6 alkyl C2-C9 heterocyclyl, C1-C10 alkyl C2-C9 heterocyclyl, or C1-C20 alkyl C2-C9 heterocyclyl). In some embodiments, the akyl and the heterocyclyl each can be further substituted with 1 , 2, 3, or 4 substituent groups as defined herein for the respective groups.

The term “hydroxyl,” as used herein, represents an -OH group. The term “/V-protecting group,” as used herein, represents those groups intended to protect an amino group against undesirable reactions during synthetic procedures. Commonly used /V-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3^rd Edition (John Wiley &

Sons, New York, 1999). /V-protecting groups include acyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, a-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl,

4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, and phenylalanine; sulfonyl-containing groups such as benzenesulfonyl, and p-toluenesulfonyl; carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1 -(p-biphenylyl)-l -methylethoxycarbonyl, a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, and phenylthiocarbonyl, arylalkyl groups such as benzyl, triphenylmethyl, and benzyloxymethyl, and silyl groups, such as trimethylsilyl. Preferred /V-protecting groups are alloc, formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

The term “nitro,” as used herein, represents an NO2 group.

The term “thiol,” as used herein, represents an -SH group.

The alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl (e.g., cycloalkyl), aryl, heteroaryl, and heterocyclyl groups may be substituted or unsubstituted. When substituted, there will generally be 1 to 4 substituents present, unless otherwise specified. Substituents include, for example, aryl (e.g., substituted and unsubstituted phenyl), carbocyclyl (e.g., substituted and unsubstituted cycloalkyl), halo (e.g., fluoro), hydroxyl, oxo, heteroalkyl (e.g., substituted and unsubstituted methoxy, ethoxy, orthioalkoxy), heteroaryl, heterocyclyl, amino (e.g., NH2 or mono- or dialkyl amino), azido, cyano, nitro, or thiol. Aryl, carbocyclyl (e.g., cycloalkyl), heteroaryl, and heterocyclyl groups may also be substituted with alkyl (unsubstituted and substituted such as arylalkyl (e.g., substituted and unsubstituted benzyl)).

Compounds of the invention can have one or more asymmetric carbon atoms and can exist in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates. The optically active forms can be obtained, for example, by resolution of the racemates, by asymmetric synthesis or asymmetric chromatography (chromatography with a chiral adsorbent or eluant). That is, certain of the disclosed compounds may exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center. "Enantiomer" means one of a pair of molecules that are mirror images of each other and are not superimposable. Diastereomers are stereoisomers that are not related as mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms and represent the configuration of substituents around one or more chiral carbon atoms. Enantiomers of a compound can be prepared, for example, by separating an enantiomer from a racemate using one or more well-known techniques and methods, such as, for example, chiral chromatography and separation methods based thereon. The appropriate technique and/or method for separating an enantiomer of a compound described herein from a racemic mixture can be readily determined by those of skill in the art. "Racemate" or "racemic mixture" means a compound containing two enantiomers, wherein such mixtures exhibit no optical activity; i.e., they do not rotate the plane of polarized light. “Geometric isomer" means isomers that differ in the orientation of substituent atoms in relationship to a carbon-carbon double bond, to a cycloalkyl ring, or to a bridged bicyclic system. Atoms (other than H) on each side of a carbon- carbon double bond may be in an E (substituents are on opposite sides of the carbon- carbon double bond) or Z (substituents are oriented on the same side) configuration. "R," "S," "S*," "R*," "E," "Z," "cis," and "trans," indicate configurations relative to the core molecule. Certain of the disclosed compounds may exist in atropisomeric forms. Atropisomers are stereoisomers resulting from hindered rotation about single bonds where the steric strain barrier to rotation is high enough to allow for the isolation of the conformers. The compounds of the invention may be prepared as individual isomers by either isomer-specific synthesis or resolved from an isomeric mixture. Conventional resolution techniques include forming the salt of a free base of each isomer of an isomeric pair using an optically active acid (followed by fractional crystallization and regeneration of the free base), forming the salt of the acid form of each isomer of an isomeric pair using an optically active amine (followed by fractional crystallization and regeneration of the free acid), forming an ester or amide of each of the isomers of an isomeric pair using an optically pure acid, amine or alcohol (followed by chromatographic separation and removal of the chiral auxiliary), or resolving an isomeric mixture of either a starting material or a final product using various well known chromatographic methods. When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99% or 99.9%) by weight relative to the other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight optically pure. When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure. Percent optical purity is the ratio of the weight of the enantiomer or over the weight of the enantiomer plus the weight of its optical isomer. Diastereomeric purity by weight is the ratio of the weight of one diastereomer or over the weight of all the diastereomers. When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by mole fraction pure relative to the other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by mole fraction pure. When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least 60%, 70%, 80%, 90%, 99% or 99.9% by mole fraction pure. Percent purity by mole fraction is the ratio of the moles of the enantiomer or over the moles of the enantiomer plus the moles of its optical isomer.

Similarly, percent purity by moles fraction is the ratio of the moles of the diastereomer or over the moles of the diastereomer plus the moles of its isomer. When a disclosed compound is named or depicted by structure without indicating the stereochemistry, and the compound has at least one chiral center, it is to be understood that the name or structure encompasses either enantiomer of the compound free from the corresponding optical isomer, a racemic mixture of the compound or mixtures enriched in one enantiomer relative to its corresponding optical isomer. When a disclosed compound is named or depicted by structure without indicating the stereochemistry and has two or more chiral centers, it is to be understood that the name or structure encompasses a diastereomer free of other diastereomers, a number of diastereomers free from other diastereomeric pairs, mixtures of diastereomers, mixtures of diastereomeric pairs, mixtures of diastereomers in which one diastereomer is enriched relative to the other diastereomer(s) or mixtures of diastereomers in which one or more diastereomer is enriched relative to the other diastereomers. The invention embraces all of these forms.

Definitions

In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

As used herein, the term “administration” refers to the administration of a composition (e.g., a compound, a complex or a preparation that includes a compound or complex as described herein) to a subject or system. Administration to an animal subject (e.g., to a human) may be by any appropriate route. For example, in some embodiments, administration may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and vitreal.

As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In some embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

As used herein, the terms “approximately” and “about” are each intended to encompass normal statistical variation as would be understood by those of ordinary skill in the art as appropriate to the relevant context. In certain embodiments, the terms “approximately” or “about” each refer to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated value, unless otherwise stated or otherwise evident from the context (e.g., where such number would exceed 100% of a possible value).

Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility of the disease, disorder, or condition (e.g., across a relevant population).

As used herein, the terms “benefit” and “response” are used interchangeably in the context of a subject, such as a human subject undergoing therapy for the treatment of a neurological disorder, for example, amyotrophic lateral sclerosis, frontotemporal degeneration (also referred to as frontotemporal lobar degeneration and frontotemporal dementia), Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy. The terms “benefit” and “response” refer to any clinical improvement in the subject’s condition. Exemplary benefits in the context of a subject undergoing treatment for a neurological disorder using the compositions and methods described herein (e.g., in the context of a human subject undergoing treatment for a neurological disorder described herein, such as amyotrophic lateral sclerosis, with a cytochrome P450 isoform 51 A1 (CYP51A1) inhibitor described herein, such as an inhibitory small molecule, antibody, antigen-binding fragment thereof, or interfering RNA molecule) include the slowing and halting of disease progression, as well as suppression of one or more symptoms associated with the disease. Particularly, in the context of a patient (e.g., a human patient) undergoing treatment for amyotrophic lateral sclerosis with a CYP51A1 inhibitor described herein, examples of clinical “benefits” and “responses” are (i) an improvement in the subject’s condition as assessed using the amyotrophic lateral sclerosis functional rating scale (ALSFRS) or the revised ALSFRS (ALSFRS-R) following administration of the CYP51A1 inhibitor, such as an improvement in the subject’s ALSFRS or ALSFRS-R score within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement in the subject’s ALSFRS or ALSFRS-R score within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the subject, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the subject); (ii) an increase in the subject’s slow vital capacity following administration of the CYP51A1 inhibitor, such as an increase in the subject’s slow vital capacity within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an increase in the subject’s slow vital capacity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51 A1 inhibitor to the subject, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks,

18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the subject); (iii) a reduction in decremental responses exhibited by the subject upon repetitive nerve stimulation, such as a reduction that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., a reduction that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51 A1 inhibitor to the subject, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks,

9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the subject); (iv) an improvement in the subject’s muscle strength, as assessed, for example, by way of the Medical Research Council muscle testing scale (as described, e.g., in Jagtap et at, Ann. Indian. Acad. Neurol. 17:336-339 (2014), the disclosure of which is incorporated herein by reference as it pertains to measuring patient response to neurological disease treatment), such as an improvement that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the subject, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the subject); (v) an improvement in the subject’s quality of life, as assessed, for example, using the amyotrophic lateral sclerosis-specific quality of life (ALS-specific QOL) questionnaire, such as an improvement in the subject’s quality of life that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement in the subject’s quality of life that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the subject, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the subject); and (vi) a decrease in the frequency and/or severity of muscle cramps exhibited by the subject, such as a decrease in cramp frequency and/or severity within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., a decrease in cramp frequency and/or severity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the subject, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitor to the subject).

In the practice of the methods of the present invention, an “effective amount” of any one of the compounds of the invention or a combination of any of the compounds of the invention or a pharmaceutically acceptable salt thereof, is administered via any of the usual and acceptable methods known in the art, either singly or in combination.

As used herein, the terms “cytochrome P450 isoform 51 A1 ,” “CYP51 A1 ,” and “lanosterol 14- alpha demethylase” are used interchangeably and refer to the enzyme that catalyzes the conversion of lanosterol to 4,4-dimethylcholesta-8(9),14,24-trien-3p-ol, for example, in human subjects. The terms “cytochrome P450 isoform 51 A1 ,” “CYP51 A1 ,” and “lanosterol 14-alpha demethylase” refer not only to wild-type forms of CYP51 A1 , but also to variants of wild-type CYP51A1 proteins and nucleic acids encoding the same. The amino acid sequence and corresponding mRNA sequence of a wild-type form of human CYP51A1 are provided herein as SEQ ID NOs: 1 and 2, which correspond to GenBank Accession No. AAC50951.1 and NCBI Reference Sequence NO. NM_000786.3, respectively. These sequences are shown in Table 2, below.

Table 2. Amino acid and nucleic acid sequences of wild-type human CYP5A1

The terms “cytochrome P450 isoform 51 A1 “CYP51 A1 and “lanosterol 14-alpha demethylase” as used herein include, for example, forms of the human CYP51A1 protein that have an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the amino acid sequence of SEQ ID NO: 1) and/or forms of the human CYP51 A1 protein that contain one or more substitutions, insertions, and/or deletions (e.g., one or more conservative and/or nonconservative amino acid substitutions, such as up to 5, 10, 15, 20, 25, or more, conservative or nonconservative amino acid substitutions) relative to a wild-type CYP51A1 protein. Similarly, the terms “cytochrome P450 isoform 51A1 ,” “CYP51A1 ,” and “lanosterol 14-alpha demethylase” as used herein include, for example, forms of the human CYP51 A1 gene that encode an mRNA transcript having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the amino acid sequence of SEQ ID NO: 2). As used herein, the terms “cytochrome P450 isoform 51A1 inhibitor,” “CYP51A1 inhibitor,” and “lanosterol 14-alpha demethylase inhibitor” are used interchangeably and refer to substances, such as compounds of Formula I. Inhibitors of this type may, for example, competitively inhibit CYP51 A1 activity by specifically binding the CYP51A1 enzyme (e.g., by virtue of the affinity of the inhibitor for the CYP51A1 active site), thereby precluding, hindering, or halting the entry of one or more endogenous substrates of CYP51A1 into the enzyme’s active site. Additional examples of CYP51A1 inhibitors that suppress the activity of the CYP51A1 enzyme include substances that may bind CYP51 A1 at a site distal from the active site and attenuate the binding of endogenous substrates to the CYP51A1 active site by way of a change in the enzyme’s spatial conformation upon binding of the inhibitor. In addition to encompassing substances that modulate CYP51A1 activity, the terms “cytochrome P450 isoform 51 A1 inhibitor,” “CYP51A1 inhibitor,” and “lanosterol 14-alpha demethylase inhibitor” refer to substances that reduce the concentration and/or stability of CYP51A1 mRNA transcripts in vivo, as well as those that suppress the translation of functional CYP51A1 enzyme.

As used herein, the term “CYP51A1 -associated disorder” refers to an undesired physiological condition, disorder, or disease that is associated with and/or mediated at least in part by CYP51 A1. In some instances, CYP51A1 -associated disorders are associated with excess CYP51A1 levels and/or activity. Exemplary CYP51A1 -associated disorders include CYP51A1 -associated disorders include but are not limited to central nervous system (CNS) disorders, dementia, Alzheimer's Disease, chronic traumatic encephalopathy, FTLD-TDP, LATE, or frontotemporal lobar degeneration.

As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic agents. In some embodiments, two or more compounds may be administered simultaneously; in some embodiments, such compounds may be administered sequentially; in some embodiments, such compounds are administered in overlapping dosing regimens.

As used herein, the term “dosage form” refers to a physically discrete unit of an active compound (e.g., a therapeutic or diagnostic agent) for administration to a subject. Each unit contains a predetermined quantity of active agent. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or compound administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic compound has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e. , is a therapeutic dosing regimen).

As used herein, the term “neuromuscular disorder” refers to a disease impairing the ability of one or more neurons to control the activity of an associated muscle. Examples of neuromuscular disorders are amyotrophic lateral sclerosis, congenital myasthenic syndrome, congenital myopathy, cramp fasciculation syndrome, Duchenne muscular dystrophy, glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis, Isaac's Syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal and bulbar muscular atrophy, spinal muscular atrophy, Stiff person syndrome, Troyer syndrome, and Guillain-Barre syndrome, among others.

The term “pharmaceutical composition,” as used herein, represents a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other pharmaceutically acceptable formulation.

A “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients may include, for example, antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration.

As used herein, the term “pharmaceutically acceptable salt” means any pharmaceutically acceptable salt of the compound of formula (I). For example, pharmaceutically acceptable salts of any of the compounds described herein include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et at, J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable organic acid.

The compounds of the invention may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds of the invention be prepared from inorganic or organic bases. Frequently, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases and methods for preparation of the appropriate salts are well-known in the art. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases.

The term “pure” means substantially pure or free of unwanted components (e.g., other compounds and/or other components of a cell lysate), material defilement, admixture or imperfection.

A variety of clinical indicators can be used to identify a patient as “at risk” of developing a particular neurological disease. Examples of patients (e.g., human patients) that are “at risk” of developing a neurological disease, such as amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy, include (i) subjects exhibiting or prone to exhibit aggregation of TAR-DNA binding protein (TDP)-43, and (ii) subjects expressing a mutant form of TDP-43 containing a mutation associated with TDP-43 aggregation and toxicity, such as a mutation selected from Q331 K, M337V, Q343R, N345K, R361 S, and N390D. Subjects that are “at risk” of developing amyotrophic lateral sclerosis may exhibit one or both of these characteristics, for example, prior to the first administration of a CYP51A1 inhibitor in accordance with the compositions and methods described herein.

As used herein, the terms “TAR-DNA binding protein-43” and “TDP-43” are used interchangeably and refer to the transcription repressor protein involved in modulating HIV-1 transcription and alternative splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) pre-mRNA transcript, for example, in human subjects. The terms “TAR-DNA binding protein-43” and “TDP-43” refer not only to wild-type forms of TDP-43, but also to variants of wild-type TDP-43 proteins and nucleic acids encoding the same. The amino acid sequence and corresponding mRNA sequence of a wild-type form of human TDP-43 are provided herein as SEQ ID NOs: 3 and 4, which correspond to NCBI Reference Sequence NOs. NM_007375.3 and NP_031401.1 , respectively. These sequences are shown in Table 3, below.

Table 3. Amino acid and nucleic acid sequences of wild-type human TDP-43

The terms “TAR-DNA binding protein-43” and “TDP-43” as used herein include, for example, forms of the human TDP-43 protein that have an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the amino acid sequence of SEQ ID NO: 3) and/or forms of the human TDP-43 protein that contain one or more substitutions, insertions, and/or deletions (e.g., one or more conservative and/or nonconservative amino acid substitutions, such as up to 5, 10, 15, 20, 25, or more, conservative or nonconservative amino acid substitutions) relative to a wild- type TDP-43 protein. For instance, patients that may be treated for a neurological disorder as described herein, such as amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer’s disease,

Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy, include human patients that express a form of TDP-43 having a mutation associated with elevated TDP-43 aggregation and toxicity, such as a mutation selected from Q331 K, M337V, Q343R, N345K, R361S, and N390D. Similarly, the terms “TAR-DNA binding protein-43” and “TDP-43” as used herein include, for example, forms of the human TDP-43 gene that encode an mRNA transcript having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 4 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to the amino acid sequence of SEQ ID NO: 4).

As used herein, the term “subject” refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject may seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be a human or animal who is under care by a trained professional for a particular disease or condition.

As used herein, the terms "treat," "treated," or "treating" mean both therapeutic treatment and prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e. , not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

A “therapeutic regimen” refers to a dosing regimen whose administration across a relevant population is correlated with a desired or beneficial therapeutic outcome.

The term “therapeutically effective amount” means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. It is specifically understood that particular subjects may, in fact, be “refractory” to a “therapeutically effective amount.” To give but one example, a refractory subject may have a low bioavailability such that clinical efficacy is not obtainable. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.

Brief Description of the Drawings

FIGS. 1A - 1C demonstrate that the viability of a yeast TDP-43 model is restored by the Erg11 inhibitor, fluconazole. (FIG. 1A) Structure of the Erg 11 inhibitor and anti-fungal, fluconazole. (FIG. 1 B) Fluconazole rescues viability of TDP-43-expressing yeast using a resazurin-reduction endpoint. A 2-fold serial dilution of fluconazole was applied to TDP-43-expressing yeast for 24 hours prior to analysis. (FIG. 1 C) Wild-type yeast cultures were treated with fluconazole for eight hours prior to HPLC analysis for lanosterol and ergosterol. Data are expressed as the area under the curve (AUC) normalized to cell mass based on optical density of cultures at 600 nm. Fluconazole treatment reduces ergosterol, while simultaneously leading to an increase in the Erg 11 substrate, lanosterol.

FIG. 2 shows the structures of compounds used in primary rat cortical neuron TDP-43 wild type and Q331K mutant survival studies.

FIGS. 3A and 3B demonstrate that compound A promotes survival in primary rat cortical neurons transfected with wild-type TDP-43. Rat primary cortical neurons were co-transfected with a red fluorescent protein (RFP) as a morphological marker and either control (empty vector) or wild-type TDP- 43 expression plasmids and treated with vehicle (DMSO) or a titration of compound A. (FIG. 3A) Risk of neuron death plots. The lifetime of each neuron was determined by either loss of RFP signal or morphological indicators of death such as loss of neurites and cell blebbing and used to generate cumulative hazard plots of risk of death overtime (hrs) post-transfection. (FIG. 3B) Forest plots. Hazard ratios for each treatment group (relative to TDP-43 DMSO group) were determined by cox regression analysis and used to generate forest plots. Hazard ratios (HR) < 1 in which the confidence interval (Cl) does not encompass 1 represent treatments that significantly reduce probability of neuron death relative to the TDP-43 DMSO control. P, p-value.

FIGS. 4A and 4B demonstrate that compound A promotes survival in primary rat cortical neurons transfected with Q331 K Mutant TDP-43. Rat primary cortical neurons were co-transfected with a red fluorescent protein (RFP) as a morphological marker and either control (empty vector) or Q331 K mutant TDP-43 expression plasmids and treated with vehicle (DMSO) or a titration of compound A. (FIG. 4A) Risk of neuron death plots. The lifetime of each neuron was determined by either loss of RFP signal or morphological indicators of death such as loss of neurites and cell blebbing and used to generate cumulative hazard plots of risk of death overtime (hrs) post-transfection. (FIG. 4B) Forest plots. Hazard ratios for each treatment group (relative to TDP-43 DMSO group) were determined by cox regression analysis and used to generate forest plots. Hazard ratios (HR) < 1 in which the confidence interval (Cl) does not encompass 1 represent treatments that significantly reduce probability of neuron death relative to the TDP-43 DMSO control. P, p-value.

FIGS. 5A and 5B demonstrate that compound B promotes survival in primary rat cortical neurons transfected with wild-type TDP-43. Rat primary cortical neurons were co-transfected with a red fluorescent protein (RFP) as a morphological marker and either control (empty vector) or wild type TDP- 43 expression plasmids and treated with vehicle (DMSO) or a titration of compound B. (FIG. 5A) Risk of neuron death plots. The lifetime of each neuron was determined by either loss of RFP signal or morphological indicators of death such as loss of neurites and cell blebbing and used to generate cumulative hazard plots of risk of death overtime (hrs) post-transfection. (FIG. 5B) Forest plots. Hazard ratios for each treatment group (relative to TDP-43 DMSO group) were determined by cox regression analysis and used to generate forest plots. Hazard ratios (HR) < 1 in which the confidence interval (Cl) does not encompass 1 represent treatments that significantly reduce probability of neuron death relative to the TDP-43 DMSO control. P, p-value. Detailed Description

The present invention features compositions and methods for treating neurological disorders, such as amyotrophic lateral sclerosis and other neuromuscular disorders, as well as frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy among others. Particularly, the invention provides inhibitors of cytochrome P450 isoform 51 A1 (CYP51A1), also referred to herein as lanosterol 14-alpha demethylase, that may be administered to a patient (e.g., a human patient) so as to treat or prevent a neurological disorder, such as one or more of the foregoing conditions. In the context of therapeutic treatment, the CYP51 A1 inhibitor may be administered to the patient to alleviate one or more symptoms of the disorder and/or to remedy an underlying molecular pathology associated with the disease, such as to suppress or prevent aggregation of TAR-DNA binding protein (TDP)-43.

The disclosure herein is based, in part, on the discovery that CYP51 A1 inhibition modulates TDP- 43 aggregation in vivo. Suppression of TDP-43 aggregation exerts beneficial effects in patients suffering from a neurological disorder. Many pathological conditions have been correlated with TDP-43-promoted aggregation and toxicity, such as amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, IBMPFD, sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy. Without being limited by mechanism, by administering an inhibitor of CYP51 A1 , patients suffering from diseases associated with TDP-43 aggregation and toxicity may be treated, for example, due to the suppression of TDP-43 aggregation induced by the CYP51A1 inhibitor.

Patients that are likely to respond to CYP51 A1 inhibition as described herein include those that have or are at risk of developing TDP-43 aggregation, such as those that express a mutant form of TDP- 43 associated with TDP-43 aggregation and toxicity in vivo. Examples of such mutations in TDP-43 that have been correlated with elevated TDP-43 aggregation and toxicity include Q331K, M337V, Q343R, N345K, R361S, and N390D, among others. The compositions and methods described herein thus provide the additional clinical benefit of enabling the identification of patients that are likely to respond to CYP51A1 inhibitor therapy, as well as processes for treating these patients accordingly.

The sections that follow provide a description of exemplary CYP51A1 inhibitors that may be used in conjunction with the compositions and methods disclosed herein. The sections below additionally provide a description of various exemplary routes of administration and pharmaceutical compositions that may be used for delivery of these substances for the treatment of a neurological disorder.

CYP51A1 Inhibitors

Exemplary CYP51A1 inhibitors described herein include compounds having a structure according to Formula I:

Formula I wherein R¹ has the structure: V

(R⁵)_m

Formula II m is 0, 1 , 2, or 3;

X is CH, CR⁵, or N; each R⁵ is, independently, halo, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 alkoxy;

R² is hydrogen, halo, optionally substituted amino, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy hydroxy, -CH2OH, or morpholino;

R³ is hydrogen or optionally substituted C1-C6 alkyl;

L¹ is absent, -O-, -SO2-, or optionally substituted C1-C6 alkyl;

L² has the structure:

Formula III Formula IV Formula V n, o, p, q, r, and s are, independently, 0 or 1 ;

R⁶ is hydrogen, hydroxy, or optionally substituted C1-C6 alkyl;

L³ is absent, -O-, or optionally substituted C1-C6 alkyl; and

R⁴ is optionally substituted C6-C10 aryl, optionally substituted C1-C6 alkyl C6-C10 aryl, or optionally substituted C2-C9 heteroaryl.

In some embodiments, the compound has the structure of any one of compounds 1-123 in Table

1.

Other embodiments, as well as exemplary methods for the synthesis or production of these compounds, are described herein.

Methods of Treatment

Suppression of CYP51A1 Activity and TDP-43 Aggregation to Treat Neurological Disorders Using the compositions and methods described herein, a patient suffering from a neurological disorder may be administered a CYP51A1 inhibitor, such as a small molecule, antibody, antigen-binding fragment thereof, or interfering RNA molecule described herein, so as to treat the disorder and/or to suppress one or more symptoms associated with the disorder. Exemplary neurological disorders that may be treated using the compositions and methods described herein are, without limitation, amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington’s disease, IBMPFD, sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy, as well as neuromuscular diseases such as congenital myasthenic syndrome, congenital myopathy, cramp fasciculation syndrome, Duchenne muscular dystrophy, glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis, Isaac's Syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal and bulbar muscular atrophy, spinal muscular atrophy, Stiff person syndrome, Troyer syndrome, and Guillain-Barre syndrome.

The present disclosure is based, in part, on the discovery that CYP51 A1 inhibitors, such as the agents described herein, are capable of attenuating TDP-43 aggregation in vivo. TDP-43-promoted aggregation and toxicity have been associated with various neurological diseases. The discovery that CYP51A1 inhibitors modulate TDP-43 aggregation provides an important therapeutic benefit. Using a CYP51A1 inhibitor, such as a CYP51A1 inhibitor described herein, a patient suffering from a neurological disorder or at risk of developing such a condition may be treated in a manner that remedies an underlying molecular etiology of the disease. Without being limited by mechanism, the compositions and methods described herein can be used to treat or prevent such neurological conditions, for example, by suppressing the TDP-43 aggregation that promotes pathology.

Additionally, the compositions and methods described herein provide the beneficial feature of enabling the identification and treatment of patients that are likely to respond to CYP51 A1 inhibitor therapy. For example, in some embodiments, a patient (e.g., a human patient suffering from or at risk of developing a neurological disease described herein, such as amyotrophic lateral sclerosis) is administered a CYP51A1 inhibitor if the patient is identified as likely to respond to this form of treatment. Patients may be identified as such on the basis, for example, of susceptibility to TDP-43 aggregation. In some embodiments, the patient is identified is likely to respond to CYP51 A1 inhibitor treatment based on the isoform of TDP-43 expressed by the patient. For example, patients expressing TDP-43 isoforms having a mutation selected from Q331K, M337V, Q343R, N345K, R361S, and N390D, among others, are more likely to develop TDP-43-promoted aggregation and toxicity relative to patients that do not express such isoforms of TDP-43. Using the compositions and methods described herein, a patient may be identified as likely to respond to CYP51A1 inhibitor therapy on the basis of expressing such an isoform of TDP-43, and may subsequently be administered a CYP51A1 inhibitor so as to treat or prevent one or more neurological disorders, such as one or more of the neurological disorders described herein.

Assessing Patient Response

A variety of methods known in the art and described herein can be used to determine whether a patient having a neurological disorder (e.g., a patient at risk of developing TDP-43 aggregation, such as a patient expressing a mutant form of TDP-43 having a mutation associated with elevated TDP-43 aggregation and toxicity, for example, a mutation selected from Q331 K, M337V, Q343R, N345K, R361S, and N390D) is responding favorably to CYP51 A1 inhibition. For example, successful treatment of a patient having a neurological disease, such as amyotrophic lateral sclerosis, with a CYP51A1 inhibitor described herein may be signaled by:

(i) an improvement in condition as assessed using the amyotrophic lateral sclerosis functional rating scale (ALSFRS) or the revised ALSFRS (ALSFRS-R), such as an improvement in the patient’s ALSFRS or ALSFRS-R score within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement in the patient’s ALSFRS or ALSFRS-R score within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the patient);

(ii) an increase in slow vital capacity, such as an increase in the patient’s slow vital capacity within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an increase in the patient’s slow vital capacity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51 A1 inhibitor to the patient);

(iii) a reduction in decremental responses exhibited by the patient upon repetitive nerve stimulation, such as a reduction that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., a reduction that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks,

28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks,

47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the patient);

(iv) an improvement in muscle strength, as assessed, for example, by way of the Medical Research Council muscle testing scale (as described, e.g., in Jagtap et at, Ann. Indian. Acad. Neurol. 17:336-339 (2014), the disclosure of which is incorporated herein by reference as it pertains to measuring patient response to neurological disease treatment), such as an improvement that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the patient);

(v) an improvement in quality of life, as assessed, for example, using the amyotrophic lateral sclerosis-specific quality of life (ALS-specific QOL) questionnaire, such as an improvement in the patient’s quality of life that is observed within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., an improvement in the subject’s quality of life that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the patient);

(vii) a decrease in TDP-43 aggregation, such as a decrease in TDP-43 aggregation within one or more days, weeks, or months following administration of the CYP51A1 inhibitor (e.g., a decrease in TDP- 43 aggregation within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the CYP51A1 inhibitorto the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the CYP51A1 inhibitorto the patient.

Combination Formulations and Uses Thereof

The compounds of the invention can be combined with one or more therapeutic agents. In particular, the therapeutic agent can be one that treats or prophylactically treats any neurological disorder described herein.

Combination Therapies

A compound of the invention can be used alone or in combination with other agents that treat neurological disorders or symptoms associated therewith, or in combination with other types of treatment to treat, prevent, and/or reduce the risk of any neurological disorders. In combination treatments, the dosages of one or more of the therapeutic compounds may be reduced from standard dosages when administered alone. For example, doses may be determined empirically from drug combinations and permutations or may be deduced by isobolographic analysis (e.g., Black et at, Neurology 65:S3-S6, 2005). In this case, dosages of the compounds when combined should provide a therapeutic effect.

Pharmaceutical Compositions

The compounds of the invention are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo. Accordingly, in another aspect, the present invention provides a pharmaceutical composition comprising a compound of the invention in admixture with a suitable diluent, carrier, or excipient.

The compounds of the invention may be used in the form of the free base, in the form of salts, solvates, and as prodrugs. All forms are within the scope of the invention. In accordance with the methods of the invention, the described compounds or salts, solvates, or prodrugs thereof may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, ortransdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

A compound of the invention may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, a compound of the invention may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers.

A compound of the invention may also be administered parenterally. Solutions of a compound of the invention can be prepared in water suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington’s Pharmaceutical Sciences (2003, 20^th ed.) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19), published in 1999.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that may be easily administered via syringe.

Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels, and powders. Aerosol formulations typically include a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device, such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant, which can be a compressed gas, such as compressed air or an organic propellant, such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier, such as sugar, acacia, tragacanth, gelatin, and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter.

The compounds of the invention may be administered to an animal, e.g., a human, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration, and standard pharmaceutical practice.

Dosages

The dosage of the compounds of the invention, and/or compositions comprising a compound of the invention, can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the animal to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. The compounds of the invention may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In general, satisfactory results may be obtained when the compounds of the invention are administered to a human at a daily dosage of, for example, between 0.05 mg and 3000 mg (measured as the solid form). Dose ranges include, for example, between 10-1000 mg.

Alternatively, the dosage amount can be calculated using the body weight of the patient. For example, the dose of a compound, or pharmaceutical composition thereof, administered to a patient may range from 0.1-50 mg/kg.

EXAMPLES

Abbreviations:

BINAP: (2,2'-bis(diphenylphosphino)-1 ,1 '-binaphthyl)

BAST: Bis(2-methoxyethyl)aminosulfur trifluoride

CDI: 1 ,T-Carbonyldiimidazole

CMBP: cyanomethylene)tributylphosphorane

DBU: 1 ,8-Diazabicyclo[5.4.0]undec-7-ene

DCE: 1 ,2-dichloroethane

DCM: Dichloromethane

DEAD: Diethyl azodicarboxylate

DIAD: Diisopropyl azodicarboxylate

DIBAL-H: Diosobutylaluminum hydride

DIPEA: Diisopropyl ethylamine

DMAP: 4-Dimethylaminopyridine

DMF: N,N-dimethylformamide

DMP: Des-Martin periodinane

DPPA: Diphenylphosphoryl azide

EDTA: Ethylenediaminetetraacetic acid

GDH: Glucose dehydrogenase

HATU: Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium)

HgO: Mercury (II) oxide

LDA: Lithium diisopropylamide

LAH: Lithium aluminium hydride

LiHMDS: Lithium hexamethyldisilazide

MsCI: Methanesulfonyl chloride

NADP: Nicotinamide adenine dinucleotide phosphate

NaHMDS: Sodium hexamethyldisilazide

NaOAc: Sodium acetate

NBS: N-Bromosuccinimide

NBS: N-bromosuccinimide

NMM: N-Methylmorpholine

NMO: N-methylmorpholine oxide

P(Cy)3: Tricyclohexylphosphine

Pd₂(dba)3 : Tris(dibenzylideneacetone)dipalladium(0)

RuPhos: 2-Dicyclohexylphosphino-2',6'-diisopropoxybiphenyl SPhos: 2-Dicyclohexylphosphino-2',6'-dimethoxybiphenyl

SFC: Supercritical fluid chromatography

TEA: Triethylamine

TFA: Trifluoroacetic acid

THF: Tetrahydrofuran

TMP: 2,2,6,6-tetramethylpiperidine

TsCI: Toluenesulfonyl chloride

Example 1. General Schemes

General Scheme 1.

An intramolecular SN2 reaction of appropriately substituted chiral alcohol I under basic conditions affords epoxide II. Opening of epoxide II with appropriately substituted amine III affords B-amino alcohol

IV. General Scheme 2.

Cross coupling of an appropriately substituted ketone I and an appropriately substituted aryl halide II under Buchwald-Hartwig conditions affords ketone intermediate III. Reduction of the ketone to the alcohol IV is realized with a reducing reagent. Alternatively, racemic alcohol IV can be purified using SFC to afford S- and R- enantiomers IV. General Scheme 3.

A palladium catalyzed coupling of an appropriately substituted aryl halide I and a cyclic amine II yields ester intermediate III. The intermediate ester III is reduced to aldehyde intermediate IV using an appropriate reducing agent (e.g. DIBAL-H). The aldehyde IV is then coupled with a pyridine halide V under Grignard conditions to yield alcohol product VI. General Scheme 4.

X=l, Br

An aldehyde amine with an appropriate protecting group (e.g. PG = Boc) I is coupled with a pyridine halide II under Grignard conditions to afford alcohol intermediate III. Removal of the protecting group under acidic conditions (e.g. HCI) affords the free amine intermediate IV. Alkylation of amine IV with an appropriately substituted halide V under basic conditions affords product VI. General Scheme 5

The spirocyclic ketone I with an appropriate protecting group (e.g. Boc) is homologated to alkene ether II under Wittig conditions. The intermediate II is treated with an acid (e.g. HCI) to reveal spirocyclic aldehyde III. Grignard reaction between aldehyde III and pyridine halide IV under Grignard affords the desired alcohol intermediate V. Removal of the protecting group (e.g. HCI) gives the secondary amine VI. This intermediate VI is coupled with an appropriately substituted aryl halide VII under Buchwald-Hartwig conditions to afford intermediate VIII. This intermediate CXI is subjected to a reducing agent (e.g. sodium borohydride) to produce the appropriately substituted alcohol IX.

General Scheme 6

A pyridine sulfonyl halide II is coupled with spirocyclic halide I under basic conditions to afford protected intermediate III. Removal of the protecting group under acid conditions (e.g. HCI) affords secondary amine intermediate IV. Palladium catalyzed coupling of amine IV with appropriately substituted aromatic halide V affords product VI.

General Scheme 7

An appropriately substituted spirocyclic amine I is coupled with a appropriately substituted pyridine sulfonyl halide II under basic conditions to afford sulfonamide III.

General Scheme 8

A nucleophilic substitution between an appropriately substituted mesylate intermediate I with an amine II affords the spirocyclic product III.

Example 2: Preparation of [1-(3,4-dichlorophenyl)-4-piperidyl]-(3-pyridyl)methanol (Compound 1)

Step 1 : Preparation of fe/f-Butyl 4-[methoxy(methyl)carbamoyl]piperidine-1-carboxylate.

To a solution of 1-fe/f-butoxycarbonylpiperidine-4-carboxylic acid (10 g, 43.62 mmol) in dichloromethane (250 ml_) was added 1 ,T-carbonyldiimidazole (7.07 g, 43.62 mmol) at 25 °C. The mixture was stirred at 25 °C for 1 hour. Then, /V-methoxymethanamine hydrochloride (7.45 g, 76.33 mmol) was added to the mixture solution at 25 °C. The reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was treated with saturate sodium bicarbonate (aq) (200 ml_) and the combined organic layers were washed with water (100 ml_), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by ISCO column chromatography (25 g silica, 0- 100 % ethyl acetate in petroleum ether, gradient over 20 minutes) to afford fe/f-butyl 3- [methoxy(methyl)carbamoyl]pyrrolidine-1-carboxylate (10 g, 36.72 mmol, 84%) as a yellow oil. ¹H NMR (400 MHz, Chloroform-d) d 4.15 - 4.14 (m, 1 H), 3.71 (s, 3H), 3.18 (s, 3H), 2.92 - 2.77 (m, 4H), 1 .70 - 1.64 (m, 4H), 1.45 (s, 9H).

Step 2: Preparation of bromo(3-pyridyl)magnesium.

To a solution of 3-bromopyridine (1.2 g, 7.60 mmol, 731.71 pL) in tetrahydrofuran (10 ml_) was added isopropylmagnesium chloride (1 .3 M, 7.01 ml_). The mixture was stirred at 25 °C for 1 hour and the resultantsolution containing bromo(3-pyridyl)magnesium (1.01 g, crude) (yellow liquid)) was taken to the next step.

Step 3: Preparation of fe/f-butyl 3-(pyridine-3-carbonyl)pyrrolidine-1-carboxylate.

To a solution of fe/f-butyl 3-[methoxy(methyl)carbamoyl]pyrrolidine-1-carboxylate (1 g, 3.67 mmol) in tetrahydrofuran (20 ml_) was added chloro(3-pyridyl)magnesium (1.01 g, 7.34 mmol) at 0 °C under nitrogen. The mixture was stirred at 25 °C for 20 hours The reaction mixture was treated with saturate ammonium chloride (aq) (20 ml_) and extracted with Ethyl acetate (30 ml_). The combined organic layers were washed with brine (20 ml_), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by ISCO column chromatography (25 g silica, 0- 100 % ethyl acetate in petroleum ether, gradient over 20 minutes) to obtain fe/f-butyl 4-(pyridine-3- carbonyl)piperidine-1-carboxylate (0.5 g, 47%) as a yellow liquid.

Step 4: Preparation of 4-piperidyl(3-pyridyl)methanone.

A solution of hydrochloric acid in ethyl acetate (10 ml_) was added to fe/f-butyl 4-(pyridine-3- carbonyl)piperidine-1-carboxylate (0.5 g, 1 .72 mmol) and the resultant mixture was stirred at 25 °C for 30 minutes. LCMS showed starting material was consumed completely and desired mass was detected. The mixture was then concentrated under reduced pressure to obtain 4-piperidyl(3-pyridyl)methanone.HCI (0.38 g, crude) as a light yellow solid.

Step 5: Preparation of [1-(3,4-dichlorophenyl)-4-piperidyl]-(3-pyridyl)methanone.

To a solution of 4-bromo-1 ,2-dichloro-benzene (127 mg, 564 pmol) and 4-piperidyl(3- pyridyl)methanone.HCI (128 mg, 564 mol) in dioxane (5 ml_) were added 4,5-Bis(diphenylphosphino)-9,9- dimethylxanthene (6 mg, 11 pmol), tris(dibenzylideneacetone)dipalladium(0) (10 mg, 11 pmol) and potassium fe/f-butoxide (158 mg, 1 .41 mmol) at 25 °C under nitrogen. The mixture was stirred at 90 °C for 6 hours. LCMS showed starting material was consumed completely and desired mass was detected. The mixture was filtered, and the filtrate was concentrated. The crude product was purified by ISCO column chromatography (25 g silica, 0-50 % ethyl acetate in petroleum ether, gradient over 20 minutes), The product [1-(3,4-dichlorophenyl)-4-piperidyl]-(3-pyridyl)methanone (150 mg, 71 %) was obtained as a light yellow solid. Step 6: Preparation of [1-(3,4-dichlorophenyl)-4-piperidyl]-(3-pyridyl)methanol.

To a solution of [1-(3, 4-dichlorophenyl)-4-piperidyl]-(3-pyridyl)methanone (70 mg, 209 pmol) in methanol (5 ml_) was added sodium borohydride (16 mg, 418 pmol) at 25 °C. The mixture was stirred at 25 °C for 30 minutes. LCMS and HPLC showed starting material was consumed completely and desired mass was detected. Concentration under reduced pressure followed by prep-HPLC (Boston Green ODS 150*30 5p column; 10-40 % acetonitrile in an a 0.04% hydrochloric acid solution in water, 15 minute gradient) afforded [1-(3, 4-dichlorophenyl)-4-piperidyl]-(3-pyridyl)methanol (55 mg, 165 pmol, 79%) as a white solid. ¹H NMR (400 MHz, Methanol-d6) d 8.92 (s, 1H), 8.82 (d, J = 6.0 Hz, 1 H), 8.70 (d, J = 8.0 Hz, 1 H), 8.16 - 8.12 (q, 1 H), 7.79 (d, J = 2.4 Hz, 1 H), 7.66 (d, J = 8.8 Hz, 1 H), 7.52 (t, J = 2.8 Hz, 1 H), 4.91 (d, J = 5.2 Hz, 1 H ), 3.76 - 3.73 (m, 2H), 3.46 - 3.41 (m, 2H), 2.15 - 1 .86 (m, 5H); LCMS (ESI) m/z: 337.1 [M+H]⁺.

Example 3: Synthesis of 1-(3,4-dichlorophenyl)pyrrolidin-3-yl]-(3-pyridyl)methanone (Compound 6) and [1-(3,4-dichlorophenyl)pyrrolidin-3-yl]-(3-pyridyl)methanol (Compound 2).

Step 1 : Preparation of fe/f-butyl 3-[methoxy(methyl)carbamoyl]pyrrolidine-1-carboxylate.

To a solution of 1-fe/f-butoxycarbonylpyrrolidine-3-carboxylic acid (900 mg, 4.18 mmol) in dichloromethane (23 ml_) was added 1 ,1'-carbonyldiimidazole (678 mg, 4.18 mmol) at 25 °C. The mixture was stirred at 25 °C for 1 hour. Then /V-methoxymethanamine hydrochloride (713 mg, 7.32 mmol) was added to the mixture at 25 °C and stirred for 16 hours. The reaction mixture was treated with saturated sodium bicarbonate (aq) (20 ml_) and the organic layer was washed with water (10 ml_), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by ISCO column chromatogrpahy (25 g silica, 0-100 % ethyl acetate in petroleum ether, gradient over 20 minutes) to obtain fe/f-butyl 3-[methoxy(methyl)carbamoyl]pyrrolidine-1-carboxylate (900 mg, 3.66 mmol, 87%) as a yellow oil. ¹H NMR (400 MHz, Chloroform-d) d 3.72 (s, 3H), 3.59 - 3.51 (m, 2H), 3.44 - 3.42 (m, 1 H), 3.38 - 3.34 (m, 2H), 3.20 (s, 3H), 2.17 - 2.08 (m, 2H), 1.46 (s, 3H).

Step 2: Preparation of fe/f-butyl 3-(pyridine-3-carbonyl)pyrrolidine-1-carboxylate.

To a solution of fe/f-butyl 3-[methoxy(methyl)carbamoyl]pyrrolidine-1-carboxylate (600 mg, 2.32 mmol) in tetrahydrofuran (15 mL) was added bromo(3-pyridyl)magnesium (847 mg, 4.65 mmol) at 0 ^°C under nitrogen. The mixture was stirred at 25 °C for 1 hour. The reaction mixture was treated with saturated ammonium chloride (aq) (15 mL) and extracted with ethyl acetate (20 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by ISCO column chromatography (25 g silica, 0-20 % ethyl acetate in petroleum ether, gradient over 20 minutes) to afford fe/f-butyl 3-(pyridine-3-carbonyl)pyrrolidine-1-carboxylate (0.3 g, 47%) as a yellow oil. ¹H NMR (400 MHz, Chloroform-d) d 9.18 (s, 1H), 8.81 (s, 1 H), 8.25 (d, J = 6.0 Hz, 1 H), 7.45 (d, J = 2.0 Hz, 1 H) 4.02 -3.96 (m, 1 H), 3.68 - 3.62 (m, 2H), 3.56 - 3.48 (m, 2H), 2.30 -2.17 (m, 2H), 1 .47 (s, 9H).

Step 3: Preparation of 3-pyridyl(pyrrolidin-3-yl)methanone.

To a solution of hydrochloric acid in ethyl acetate (5 ml_) was added to fe/f-butyl 3-(pyridine-3- carbonyl)pyrrolidine-1-carboxylate (0.3 g, 1 .09 mmol) and the mixture was stirred at 25°C for 30 minutes. The reaction mixture was concentrated under reduced pressure to afford 3-pyridyl(pyrrolidin-3- yl)methanone (0.26 g, crude) as a white solid.

Step 4: Preparation of 1-(3,4-dichlorophenyl)pyrrolidin-3-yl]-(3-pyridyl)methanone.

To a solution of 4-bromo-1 ,2-dichloro-benzene (205 mg, 908 pmol) and 3-pyridyl(pyrrolidin-3- yl)methanone (160 mg, 908 pmol) in dioxane (7 ml_) was added 4,5-bis(diphenylphosphino)-9,9- dimethylxanthene (11 mg, 18 pmol), tris(dibenzylideneacetone)dipalladium(0) (17 mg, 18 pmol), and potassium fe/f-butoxide (255 mg, 2.27 mmol) at 25 ^°C under nitrogen. The mixture was stirred at 90 °C for 16 hours. The mixture was filtered, and the filtrate was concentrated. The crude product was purified by ISCO column chromatography (25 g silica, 0-50 % ethyl acetate in petroleum ether, gradient over 20 minutes) and the product [1-(3,4-dichlorophenyl)pyrrolidin-3-yl]-(3-pyridyl)methanone (0.13 g, 44%) was obtained as a light yellow solid.

¹H NMR (400 MHz, Chloroform-d) d 9.22 (d, J = 1 .6 Hz, 1 H), 8.84 (t, J = 3.2 Hz, 1 H), 8.30 - 8.27 (m,

1 H), 7.50 - 7.47 (m, 1 H),7.24, (d, J = 8.8, Hz, 1 H), 6.64 (d, J = 2.8 Hz, 1 H), 6.42, 6.40 (d,d, J = 8.8, 2.8 Hz, 1 H), 4.19 - 4.12 (m, 1 H), 3.63 (d, d = 7.2 Hz, 2H), 3.43 (t, J = 6.8 Hz, 2H), 2.44 - 2.39 (m, 2H); LCMS (ESI) m/z: 321.0 [M+H]⁺.

Step 5: Preparation of [1-(3,4-dichlorophenyl)pyrrolidin-3-yl]-(3-pyridyl)methanol .

To a solution of [1-(3,4-dichlorophenyl)pyrrolidin-3-yl]-(3-pyridyl)methanone (50 mg, 156 pmol) in methanol (5 ml_) was added sodium borohydride (12 mg, 311 pmol) at 25 °C. The mixture was stirred at 25 °C for 30 minutes and the mixture was concentrated. The crude residue was purified by prep-HPLC (Boston Green ODS 150*30 5p column; 15-40 % acetonitrile in an a 0.04% hydrochloric acid solution in water, 11 minute gradient) to obtain [1-(3,4-dichlorophenyl)pyrrolidin-3-yl]-(3-pyridyl)methanol (72 mg, 225 pmol, 48%) as a pink solid. ¹H NMR (400 MHz, Methanol-d6) d 8.93 (t, J = 4.0 Hz, 1 H), 8.80 (d, J = 5.2 Hz, 1 H), 8.71 (d,d, d = 8.0, 2.0 Hz, 1 H), 8.12 (m, 1 H), 7.21 (d,d, d = 9.2, 5.2 Hz, 1 H), 6.64 - 6.61 (m,

1 H), 6.49 - 6.44 (m, 1 H), 4.99 - 4.94 (m, 1 H), 3.34 - 3.22 (m, 4H), 2.80 - 2.78 (m, 1 H), 2.12 - 1.95 (m, 2H); LCMS (ESI) m/z: 323.0 [M+H]⁺. Example 4: Preparation of [1-(2-phenylethyl)-4-piperidyl]-(3-pyridyl)methanol (Compound 9).

Step 1 : Preparation of fe/f-butyl 4-[hydroxy(3-pyridyl)methyl]piperidine-1-carboxylate.

To a solution of 3-bromopyridine (1 g, 4.88 mmol, 452 pL) in tetrahydrofuran (20 ml_) was added isopropylmagnesium chloride (2 M, 2.68 ml_) at -20 °C, and the mixture was stirred at -20 °C for 0.5 hours. A solution of tert-butyl 4-formylpiperidine-1-carboxylate (1.04 g, 4.88 mmol) in tetrahydrofuran was then added at -20 °C. Then the mixture was warmed up to 20 °C and stirred for 16 hours. Saturated ammonium chloride solution (20 ml_) was added to the reaction, and the reaction mixture was extracted with ethyl acetate (30 ml_ x 2). The combined organic layers were washed with brine (15 ml_) and dried over sodium sulfate, filtered, and concentrated to dryness to obtain the crude product. It was further purified by ISCO column chromatography (20 g silica, 20-100 % ethyl acetate in petroleum ether, gradient over 30 minutes) to obtain fe/f-butyl 4-[hydroxy(3-pyridyl)methyl]piperidine

-1-carboxylate (0.9 g, 3.08 mmol, 63%) as a yellow gum. ¹H NMR (400 MHz, Chloroform-d) d 8.58 - 8.50 (m, 2H), 7.67 (br. d, J = 7.9 Hz, 1 H), 7.31 (dd, J = 5.0, 7.8 Hz, 1 H), 4.47 (d, J = 7.1 Hz, 1 H), 4.25 - 4.01 (m, 2H), 2.72 - 2.49 (m, 2H), 2.18 (br. s, 1H), 1.93 (br. d, J = 13.2 Hz, 1H), 1.77 (dtd, J = 3.7, 7.7, 15.4 Hz, 1 H), 1.45 (s, 9H), 1.36 - 1.12 (m, 3H).

Step 2: Preparation of 4-piperidyl(3-pyridyl)methanol.

To a solution of fe/f-butyl 4-[hydroxy(3-pyridyl)methyl]piperidine-1-carboxylate (0.3 g, 1 .03 mmol) in ethyl acetate (5 ml_) was added 4M hydrochloric acid in ethyl acetate (10 ml_). Then the mixture was stirred at 20 °C for 1 hour and concentrated to obtain 4-piperidyl(3-pyridyl)methanol.HCI (240 mg, crude) as a white solid. LCMS (ESI) m/z: 193.1 [M+H]⁺. The crude product was used further without purification.

Step 3: Preparation of [1-(2-phenylethyl)-4-piperidyl]-(3-pyridyl)methanol.

To a solution of 4-piperidyl(3-pyridyl)methanol.HCI (200 mg, 874 pmol) in dimethylformamide (3 ml_) was added 2-bromoethylbenzene (178 mg, 962 pmol) and sodium bicarbonate (220 mg, 2.62 mmol). The resultant mixture was stirred at 80 °C for 1 hour. LCMS showed the reaction was complete. The resultant crude product was purified directly by prep-HPLC (Waters Xbridge Prep OBD C18 150*40 5p column; 20-50 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to obtain [1-(2-phenylethyl)-4-piperidyl]-(3-pyridyl)methanol (55 mg, 185 pmol, 21%) as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.63 - 8.53 (m, 2H), 7.74 (br. d, J = 7.9 Hz, 1H), 7.38 - 7.30 (m, 3H), 7.28 - 7.21 (m, 3H), 4.51 (d, J = 7.3 Hz, 1 H), 3.13 (br. d, J = 12.3 Hz, 1 H), 3.02 (br. d, J = 11.5 Hz, 2H), 2.89 - 2.80 (m, 2H), 2.65 - 2.57 (m, 2H), 2.10 - 1.93 (m, 3H), 1.76 - 1.66 (m, 1 H), 1.56 - 1.48 (m, 1 H), 1 .42 - 1 .34 (m, 2H); LCMS (ESI) m/z: 297.1 [M+H] ⁺.

Example 5: Preparation of [1-(4-chlorophenyl)-4-piperidyl]-(3-pyridyl)methanol (Compound 10)

1.) BINAP, Pd(OAc)₂, Cs₂C0₃, dioxane, 20 -0, 1 h DIBAL-H, toluene

Xr°

2.) dioxane, 105 °C,

16 h

Step 1 : Preparation of methyl 1-(4-chlorophenyl)piperidine-4-carboxylate.

2,2’-Bis(diphenylphosphino)-1 ,1 ’-binapthalene (976 mg, 1.57 mmol), palladium (II) acetate (352 mg, 1.57 mmol) and cesium carbonate (6.81 g, 20.89 mmol) were suspended in dioxane (50 ml_) and stirred at 20 °C for 1 hour. Then, 1-bromo-4-chloro-benzene (2 g, 10.45 mmol, 2.00 ml_) and methyl piperidine-4-carboxylate (1.50 g, 10.45 mmol) were added as a solution in dioxane (50 ml_). The mixture was then heated to 105 °C for 16 hours and cooled. The reaction mixture was filtered, and to the filtrate 20ml_ of water was added.. The aqueous phase was extracted with ethyl acetate (20 ml_ x 3). The combined organic phases were dried with anhydrous sodium sulfate, filtered, and concentrated in vacuum. The residue was purified by ISCO column chromatography (40 g silica, 0-10% ethyl acetate in petroleum ether, gradient over 20 minutes) to obtain methyl 1-(4-chlorophenyl)piperidine-4-carboxylate (2.2 g, 8.67 mmol, 83%) as yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 7.22 - 7.16 (m, 2H), 6.88 - 6.81 (m, 2H), 3.71 (s, 3H), 3.64 - 3.52 (m, 2H), 2.84 - 2.72 (m, 2H), 2.52 - 2.39 (m, 1 H), 2.09 - 1 .98 (m, 2H), 1 .94 - 1 .79 (m, 2H); LCMS (ESI) m/z: 254.0 [M+H]⁺.

Step 2: Preparation of 1-(4-chlorophenyl)piperidine-4-carbaldehyde.

To a stirred solution of methyl 1-(4-chlorophenyl)piperidine-4-carboxylate (0.5 g, 1.97 mmol) in toluene (10 mL) was added diisobutylalumminum hydride (1 M, 1.97 mL) dropwise at -60 °C under nitrogen, and the mixture was stirred at -60 °C for 1 h. TLC (Petroleum ether : Ethyl acetate=5:1 , R_f = 0.37) showed starting material was consumed completely and new spot was formed. The reaction mixture was carefully quenched with methanol (10mL) and brine (50mL) and warmed up to room temperature. The mixture was filtered, and the filtrate was extracted with ethyl acetate (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated undervacuum. The residue was purified by ISCO column chromatography (20 g silica, 0-10% Ethyl acetate in Petroleum ether, gradient over 15 minutes) to obtain 1-(4-chlorophenyl)piperidine-4- carbaldehyde (300 mg, 1.34 mmol, 68%) as a yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 9.70 (s,

1 H), 7.25 - 7.15 (m, 2H), 6.90 - 6.81 (m, 2H), 3.61 - 3.49 (m, 2H), 2.94 - 2.76 (m, 2H), 2.49 - 2.33 (m, 1 H), 2.05 (s, 2H), 1.87 - 1.70 (m, 2H).

Step 3: Preparation of [1-(4-chlorophenyl)-4-piperidyl]-(3-pyridyl)methanol.

To a stirred solution of 3-bromopyridine (420 mg, 2.66 mmol, 256 pL) in tetrahydrofuran (2 ml_) was added isopropylmagnesium chloride (2 M, 1 .33 ml_) at 0 °C under nitrogen, and the mixture was warmed up to 20 °C and stirred for 1 hour. To the resultant mixture, a solution of 1 -(4- chlorophenyl)piperidine-4-carbaldehyde (297 mg, 1.33 mmol) was added and the mixture was stirred at 20 °C for 1 hour. LCMS showed the starting material was consumed completely and desired product was detected. The reaction mixture was cooled to 0 °C and quenched with saturated ammonium chloride solution followed by the addition of water (5 ml_). The aqueous phase was extracted with ethyl acetate (5 ml_ x 2), the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude residue was purified by prep-HPLC (Nano-micro Kromasil C18 80*25 3mM column; 35-55% acetonitrile in a 10Mm ammonium bicarbonate solution in water, 10 minute gradient) to obain [1-(4-chlorophenyl)-4-piperidyl]-(3-pyridyl)methanol (107 mg, 343 pmol, 26%) as a white solid. ¹H NMR (400 MHz, Chloroform-d) d 8.54 (s, 2H), 7.77 - 7.64 (m, 1 H), 7.34 - 7.28 (m, 1 H),

7.23 - 7.14 (m, 2H), 6.90 - 6.77 (m, 2H), 4.56 - 4.43 (m, 1 H), 3.74 - 3.64 (m, 1 H), 3.59 (br dd, J = 1.5, 12.1 Hz, 1 H), 2.75 - 2.54 (m, 2H), 2.40 (s, 1 H), 2.16 - 2.04 (m, 1 H), 1 .88 - 1 .70 (m, 1 H), 1 .60 - 1 .47 (m, 1 H),

1 .45 - 1 .35 (m, 2H); LCMS (ESI) m/z: 303.0 [M+H] ⁺. The following compounds were synthesized according to the protocol described for the Compound 10:

Example 6: Synthesis of 1-(3,4-dichlorophenyl)-4-(3-pyridylmethyl)piperidin-4-ol (Compound 11):

Step 1 : tert-butyl 4-hydroxy-4-(3-pyridylmethyl)piperidine-1-carboxylate.

To a solution of 3-methylpyridine (1.40 g, 15.06 mmol) in THF (50 ml_) at -70 °C was added LDA (2 M, 11.29 ml_) dropwise under N2 atmosphere and stirred for 0.5 h at same temperature. The reaction mixture was then stirred at 0 °C for 0.5 h before cooling it again to -70 °C when tert-butyl 4-oxopiperidine- 1-carboxylate (2.5 g, 12.55 mmol) in THF (25 ml_) was added. The resulting mixture was stirred at 15 °C for 14 h. The resultant mixture was quenched with NH₄CI solution (10 ml_) and was concentrated. To the resultant mixture was added H₂O (20 ml_), then the aqueous phase was extracted with EtOAc (50 ml_ *2). The combined organic layers were washed with H₂O (10 mL * 1), dried over Na₂SC>₄, filtered and concentrated under reduced pressure. The resultant crude product was purified by flash column (ISCO 40 g silica, 0-100 % ethyl acetate in petroleum ether, gradient over 20 min) to obtain tert-butyl 4- hydroxy-4- (3- pyridylmethyl)piperidine-1-carboxylate (0.9 g, 2.62 mmol, 21%) as a pale yellow gum.

¹H NMR (400 MHz, CHLOROFORM-d) d = 8.53 - 8.45 (m, 2H), 7.56 (br d, J = 7.6 Hz, 1 H), 7.26 - 7.22 (m, 1 H), 3.87 (br s, 2H), 3.11 (br t, J = 10.8 Hz, 2H), 2.77 (s, 2H), 1 .68 - 1.55 (m, 2H), 1.50 (br s, 2H), 1.46 (s, 9H). LCMS (ESI) m/z: 293.2 [M+H]⁺.

Step 2: 4-(3-pyridylmethyl)piperidin-4-ol.

To a solution of tert-butyl 4-hydroxy-4-(3-pyridylmethyl)piperidine-1-carboxylate (850 mg, 2.91 mmol) in DCM (10 mL) at 0 °C was added TFA (3.31 g, 29.07 mmol). The mixture was stirred at 15 °C for 3 h and concentrated. The crude product was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40mm*10um column; 1-10% acetonitrile in a 10mM ammonium bicarbonate solution in water, 8 min gradient) (neutral). The product 4-(3-pyridylmethyl)piperidin-4-ol (300 mg, 1.40 mmol, 48 %) was used into the next step as a white solid. LCMS (ESI) m/z: 193.1 [M+H]⁺.

Step 3: 1-(3,4-dichlorophenyl)-4-(3-pyridylmethyl)piperidin-4-ol.

To a solution of 1 ,2-dichloro-4-iodo-benzene (213 mg, 780 umol) and 4-(3-pyridylmethyl)piperidin- 4-ol (150 mg, 780 umol) in dioxane (3 mL) were added t-BuONa (150 mg, 1.56 mmol), Pd₂(dba)3 (36 mg, 39 umol, 0.05 eq) and Sphos (32 mg, 78 umol). The suspension was degassed and purged with nitrogen 3 times and stirred at 100 °C for 12 h and concentrated. The resultant crude product was purified by prep- HPLC (Waters Xbridge Prep OBD C18 150*40mm*10um column; 35-55 % acetonitrile in a 10mM ammonium bicarbonate solution in water, 8 min gradient) (neutral). The compound 1-(3,4-dichlorophenyl)- 4-(3-pyridylmethyl)piperidin-4-ol (50 mg, 147 umol, 19%) was obtained as a pale yellow solid.

¹H NMR (400 MHz, CHLOROFORM-d) d = 8.57 - 8.47 (m, 2H), 7.62 - 7.55 (m, 1 H), 7.30 - 7.27 (m, 1 H), 7.26 - 7.24 (m, 1 H), 7.00 - 6.96 (m, 1 H), 6.80 - 6.73 (m, 1 H), 3.47 - 3.37 (m, 2H), 3.16 - 3.05 (m, 2H), 2.81 (s, 2H), 1 .88 - 1 .76 (m, 2H), 1 .69 - 1 .58 (m, 2H), 1.35 - 1 .22 (m, 1 H). LCMS (ESI) for (C17H18CI2N20) [M+H]⁺: 337.0.

Example 7: Preparation of 1-(4-chloro-3-fluoro-phenyl)-4-piperidyl]-(3-pyridyl)methanol (Compound 12) and its chiral separation into enantiomer 1 (Compound 75) and enantiomer 2 (Compound 16).

Enantiomer 1 Enantiomer 2

Compounds 12, 16, and 75 were synthesized according to the synthetic procedure reported for the preparation of compound 17 and 77. The compound 12, [1-(4-chloro-3-fluoro-phenyl)-4-piperidyl]-(3- pyridyl)methanol (12 mg, 36 pmol, 5%) was obtained as a white solid. Both enantiomers were obtained as white solids in 17% and 13% yields respectively.

¹H NMR (400 MHz, Dimethylsulfoxide-de) for compound 12: d 8.50 (d, J = 1.6 Hz, 1 H), 8.45 (dd, J = 1.5,

4.7 Hz, 1 H), 7.70 (br. d, J = 7.8 Hz, 1 H), 7.35 (dd, J = 4.8, 7.7 Hz, 1 H), 7.27 (t, J = 9.0 Hz, 1 H), 6.89 (dd, J

= 2.7, 13.4 Hz, 1 H), 6.73 (dd, J = 2.4, 9.0 Hz, 1 H), 5.39 (br. s, 1 H), 4.36 (br. d, J = 5.5 Hz, 1 H), 3.82 - 3.63

(m, 2H), 2.68 - 2.54 (m, 2H), 1.83 (br. d, J = 12.8 Hz, 1 H), 1.75 - 1.63 (m, 1 H), 1.36 - 1.20 (m, 3H); LCMS (ESI) m/z: 321.0 [M+H]⁺.

¹H NMR (400 MHz, Dimethylsulfoxide-de) for compound 75: d 8.49 (d, J = 1 .6 Hz, 1 H), 8.45 (dd, J = 1 .5,

= 2.7, 13.4 Hz, 1 H), 6.73 (dd, J = 2.4, 8.9 Hz, 1 H), 5.40 (br. d, J = 3.2 Hz, 1 H), 4.36 (br. d, J = 3.2 Hz,

1 H), 3.84 - 3.65 (m, 2H), 2.71 - 2.54 (m, 2H), 1.83 (br. d, J = 11.1 Hz, 1 H), 1.75 - 1.61 (m, 1 H), 1.37 - 1.19 (m, 3H); LCMS (ESI) m/z: 319.0 [M-H]-. (Rt: 3.71 min).

¹H NMR (400 MHz, Dimethylsulfoxide-de) for compound 16: d 8.49 (d, J = 1.6 Hz, 1 H), 8.45 (dd, J = 1.5,

4.7 Hz, 1 H), 7.70 (br. d, J = 7.8 Hz, 1 H), 7.35 (dd, J = 4.8, 7.7 Hz, 1 H), 7.28 (t, J = 9.0 Hz, 1 H), 6.89 (dd, J = 2.7, 13.4 Hz, 1 H), 6.73 (dd, J = 2.4, 9.0 Hz, 1 H), 5.40 (s, 1 H), 4.36 (br. d, J = 6.4 Hz, 1 H), 3.84 - 3.65 (m, 2H), 2.69 - 2.54 (m, 2H), 1.82 (br. d, J = 12.7 Hz, 1 H), 1.68 (br. d, J = 8.3 Hz, 1 H), 1.35 - 1.23 (m, 3H); LCMS (ESI) m/z: 319.0 [M-H]-. (Rt: 4.28min). Example 8: Preparation of 1-[4-[hydroxy(3-pyridyl)methyl]-1-piperidyl]-2-phenyl-ethanone (Compound 14).

To a solution of 4-piperidyl(3-pyridyl)methanol.2HCI (150 mg, 780 pmol) in dimethylformamide (2 ml_) was added 2-phenylacetic acid (106 mg, 780 pmol, 98 pL), 1-hydroxybenzotriazole (127 mg, 936 pmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (179 mg, 936 pmol), 4-methylmorpholine (237 mg, 2.34 mmol, 257 pL) andthe mixture stirred at 20 °C for 2 hours. LCMS showed the starting material was consumed completely and desired compound was detected. The resultant crude product was purified directly by prep-HPLC (Waters Xbridge BEH C18 100*25mm*5pm column; 10-50 % acetonitrile in a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to obtain1-[4-[hydroxy(3- pyridyl)methyl]-1-piperidyl]-2-phenyl-ethanone (107 mg, 344 pmol, 44%) as a white solid.

¹H NMR (400 MHz, Dimethylsulfoxide-de) d 8.44 (dd, J = 1.7, 4.6 Hz, 2H), 7.70 - 7.62 (m, 1 H), 7.34 (dd, J = 4.8, 7.7 Hz, 1 H), 7.31 - 7.25 (m, 2H), 7.23 - 7.14 (m, 3H), 5.38 (d, J = 4.5 Hz, 1 H), 4.46 - 4.27 (m, 2H), 4.03 - 3.86 (m, 1 H), 3.69 - 3.63 (m, 2H), 2.96 - 2.79 (m, 1 H), 2.46 - 2.31 (m, 1 H), 1 .79 - 1 .65 (m, 2H), 1 .28 - 1.13 (m, 1 H), 1.09 - 0.90 (m, 2H); LCMS (ESI) m/z: 311.1 [M+H]⁺.

The following compounds were synthesized according to the protocol described for the Compound 14:

Example 9: Preparation of 2-[1-(2-phenylethyl)-4-piperidyl]-1-(3-pyridyl)ethanol (Compound 18).

Step 1 : Preparation of fe/f-butyl 4-[2-hydroxy-2-(3-pyridyl)ethyl]piperidine-1-carboxylate.

To a solution of 3-iodopyridine (1.44 g, 7.04 mmol) in tetrahydrofuran (15 ml_) was added isopropylmagnesium chloride (2 M, 3.52 ml_) in tetrahydrofuran at 0 °C. The mixture was stirred at 0 °C for 30 minutes. Then, fe/f-butyl 4-(2-oxoethyl)piperidine-1-carboxylate (0.8 g, 3.52 mmol) was added at 0 °C. Then the mixture was warmed and stirred at 25 °C for 1 .5 hours. Saturated ammonium chloride solution (20 ml_) was added to the reaction, and the reaction mixture was extracted with ethyl acetate (30 ml_ x 2). The combined organic layers were washed with brine (20 ml_), dried over sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by ISCO column chromatography (10 g silica, 50-100 % ethyl acetate in petroleum ether, gradient over 40 minutes) to obtain fe/f-butyl 4-[2- hydroxy-2-(3-pyridyl)ethyl]piperidine-1-carboxylate (0.94 g, 3.07 mmol, 87%) as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.45 (d, J = 1 .6 Hz, 1 H), 8.41 (dd, J = 1.5, 4.6 Hz, 1 H), 7.64 (br. d, J = 7.8 Hz, 1 H), 7.26 - 7.19 (m, 1 H), 4.76 (dd, J = 4.6, 9.0 Hz, 1 H), 4.10 - 3.92 (m, 2H), 2.70 - 2.53 (m, 2H), 1.78 - 1.66 (m, 2H), 1.64 - 1.53 (m, 2H), 1.52 - 1.42 (m, 1 H), 1.38 (s, 9H), 1.15 - 1.02 (m, 2H).

Step 2: Preparation of 2-(4-piperidyl)-1-(3-pyridyl)ethanol.

To a solution of fe/f-butyl 4-[2-hydroxy-2-(3-pyridyl)ethyl]piperidine-1-carboxylate (0.89 g, 2.90 mmol) in ethyl acetate (10 ml_) was added 4M hydrochloric acid in ethyl acetate (30 ml_). Then the mixture was stirred at 20 °C for 30 minutes. LCMS showed the reaction was complete. The reaction mixture was concentrated to dryness to obtain the crude product2-(4-piperidyl)-1-(3-pyridyl)ethanol.2HCI (840 mg, crude) as a yellow solid. It was further used without purification.

Step 3: Preparation of 2-[1-(2-phenylethyl)-4-piperidyl]-1-(3-pyridyl)ethanol.

To a solution of 2-bromoethylbenzene (199 mg, 1.07 mmol, 145 pL) in dimethylformamide (4 ml_) was added 2-(4-piperidyl)-1-(3-pyridyl)ethanol.2HCI (0.25 g, 895 pmol) and sodium bicarbonate (226 mg, 2.69 mmol). The resultant mixture was stirred at 80 °C for 1 hour. LCMS showed the reaction was complete. The resultant crude product was purified directly by prep-HPLC (Welch Xtimate C18 250*50 10m column; 10-50 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 minute gradient) to obtain 2-[1-(2-phenylethyl)-4-piperidyl]-1-(3-pyridyl)ethanol (32 mg, 104 pmol, 12%) as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.57 (d, J = 2.0 Hz, 1 H), 8.53 (dd, J = 1 .6, 4.8 Hz, 1 H), 7.77 - 7.66 (m, 1 H), 7.32 - 7.27 (m, 3H), 7.23 - 7.15 (m, 3H), 4.85 (br dd, J = 5.0, 8.5 Hz, 1 H), 3.06 - 2.94 (m, 2H), 2.87 - 2.75 (m, 2H), 2.62 - 2.52 (m, 2H), 2.32 (br. s, 1 H), 2.06 - 1 .94 (m, 2H), 1 .86 - 1 .72 (m, 3H), 1 .64 - 1 .54 (m, 1 H), 1 .53 - 1 .26 (m, 3H); LCMS (ESI) m/z: 311.1 [M+H]⁺.

Example 10: Preparation of stereoisomer 1 (Compound 20) and stereoisomer 2 (Compound 79) of (3,4-dichlorophenyl)-[3-[hydroxy(3-pyridyl)methyl]pyrrolidin-1-yl]methanone.

Step 1 : Preparation of fe/f-butyl 3-[hydroxy(3-pyridyl)methyl]pyrrolidine-1-carboxylate.

To a solution of 3-bromopyridine (1.59 g, 10.04 mmol in tetrahydrofuran (10 mL) was added isopropylmagnesium chloride (2 M, 5.02 mL) at 0 °C under nitrogen. The mixture was stirred at 20 °C for 1 hour, then fe/f-butyl 3-formylpyrrolidine-1-carboxylate (1 g, 5.02 mmol) was added. The mixture was stirred at 20 °C for 2 hours. The reaction mixture was quenched with saturated ammonium chloride (10 ml_) at 0 °C, water was added and the aqueous phase was extracted with ethyl acetate (10 ml_ x 2). The combined organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated in vacuum to give the crude productfe/f-butyl 3-[hydroxy(3-pyridyl)methyl]pyrrolidine-1-carboxylate (1.0 g, crude) as a red gum; LCMS (ESI) m/z: 279.1 [M+H]⁺.

Step 2: Preparation of 3-pyridyl(pyrrolidin-3-yl)methanol.

To a solution of fe/f-butyl 3-[hydroxy(3-pyridyl)methyl]pyrrolidine-1-carboxylate (1 g, 3.59 mmol) in ethyl acetate (15 ml_) was added hydrochloric acid/ethyl acetate (15 ml_, 4 M) at 0 °C. The mixture was warmed and stirred at 20 °C for 2 hours The reaction mixture was concentrated under reduced pressure to obtain the crude product which was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10pm; 1-10% acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient). The product 3-pyridyl(pyrrolidin-3-yl)methanol (200 mg, 1.12 mmol, 31% ) was obtained as a white solid. ¹H NMR (400 MHz, Methanol-d4) d 8.59 - 8.53 (m, 1 H), 8.46 (br. d, J = 4.9 Hz, 1 H), 7.89 (br. d, J = 7.7 Hz, 1 H), 7.49 - 7.40 (m, 1 H), 4.77 - 4.52 (m, 1 H), 3.28 - 2.91 (m, 4H), 2.72 - 2.57 (m, 1 H), 1 .97 - 1.64 (m, 2H); LCMS (ESI) m/z: 179.1 [M+H]⁺.

Step 3: Preparation of (3,4-dichlorophenyl)-[3-[hydroxy(3-pyridyl)methyl]pyrrolidin-1-yl]methanone.

To a solution of 3,4-dichlorobenzoic acid (106 mg, 555 pmol) in dimethylformamide (1 mL) were added 4-methylmorpholine (153 mg, 1.51 mmol, 167 pL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (117 mg, 606 pmol), 1-hydroxybenzotriazole (82 mg, 606 pmol), and 3-pyridyl(pyrrolidin-3-yl)methanol (90 mg, 505 pmol). The mixture was stirred at 20 °C for 3 hours and filtered. The crude product was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*30mm*10pm; 20-45% acetonitrile in a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to obtain stereoisomer 1 and stereoisomer 2 of (3,4-dichlorophenyl)-[3-[hydroxy(3-pyridyl)methyl]pyrrolidin-1-yl]methanone as pale yellow solids. Compound 20: ¹H NMR (400 MHz, Methanol-d4) d 8.71 - 8.43 (m, 2H), 8.02 - 7.83 (m, 1 H), 7.81 - 7.59 (m, 2H), 7.58 - 7.40 (m, 2H), 4.77 - 4.50 (m, 1 H), 3.92 - 3.47 (m, 4H), 2.77 - 2.55 (m, 1 H), 1 .89 - 1 .63 (m, 2H); LCMS (ESI) m/z: 351 .0 [M+H]⁺.

Compound 79: ¹H NMR (400 MHz, Chloroform-d) d 8.65 - 8.45 (m, 2H), 7.65 (br. d, J = 7.7 Hz, 1 H), 7.60 - 7.55 (m, 1 H), 7.51 - 7.43 (m, 1 H), 7.36 - 7.27 (m, 2H), 4.75 - 4.63 (m, 1 H), 3.83 - 3.23 (m, 4H), 2.74 - 2.52 (m, 2H), 2.19 - 1.87 (m, 2H); LCMS (ESI) m/z: 351.1 [M+H]⁺.

Example 11: Preparation of [2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanol (Compound 23)

100 °C, 20 m

Step 1 : Preparation of [2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanone. To a solution of 2-azaspiro[3.3]heptan-6-yl(3-pyridyl)methanol (0.6 g, 2.94 mmol) in dioxane (6 ml_) were added 1 ,2-dichloro-4-iodo-benzene (802 mg, 2.94 mmol), sodium fe/f-butoxide (847 mg, 8.81 mmol), tris(dibenzylideneacetone)dipalladium(0) (135 mg, 147 pmol), and 2-dicyclohexylphosphino-2',6'- diisopropoxybiphenyl (27 mg, 59 pmol) under a nitrogen atmosphere. The mixture was stirred at 100 °C for 20 minutes and concentrated. The resultant crude product was purified by ISCO column chromatography (40 g silica, 0-20 % ethyl acetate in petroleum ether, gradient over 20 minutes) to obtain[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanone (0.38 g, 1.09 mmol, 37%) as a pale yellow solid. LCMS (ESI) m/z: 347.1 [M+H]⁺.

Step 2: Preparation of [2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanol.

To a solution of [2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanone (0.36 g, 829 pmol) in methanol (7 ml_) was added sodium borohydride (62.75 mg, 1 .66 mmol) at 0 °C. The mixture was warmed up and stirred at 25 °C for 2 hours. The reaction mixture was quenched by the addition of water (2 ml_), and concentrated under vacuum. The residue was purified by prep-HPLC ( Kromasil C18 (250*50mm*10 pm) column; 35-65 % acetonitrile in a 10mM ammonium bicarbonate solution in water, 10 minute gradient ) to obtain [2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanol (113 mg, 323 pmol, 39%) as a white solid.

¹H NMR (400 MHz, Chloroform-d) d 8.69 - 8.49 (m, 2H), 7.74 - 7.63 (m, 1 H), 7.30 (dd, J = 4.8, 7.8 Hz,

1 H), 7.23 - 7.18 (m, 1 H), 6.45 (d, J = 2.6 Hz, 1 H), 6.23 (dd, J = 2.6, 8.6 Hz, 1 H), 4.70 - 4.60 (m, 1 H), 3.92 - 3.71 (m, 4H), 2.63 - 2.48 (m, 1 H), 2.37 - 2.24 (m, 2H), 2.20 - 2.05 (m, 3H); LCMS (ESI) m/z: 349.0 [M+H]⁺.

Example 12: Preparation of stereoisomer 1 (Compound 24) and stereoisomer 2 (Compound 22) of [1 -[(3,4-dichlorophenyl)methyl]pyrrolidin-3-yl]-(3-pyridyl)methanol.

stereoisomer 1 stereoisomer 2

To a solution of 3-pyridyl(pyrrolidin-3-yl)methanol (90 mg, 505 pmol) and 4-(bromomethyl)-1 ,2- dichloro-benzene (133 mg, 555 pmol) in dimethylformamide (1 mL) was added triethylamine (255 mg,

2.52 mmol, 351 pL). The mixture was stirred at 20 °C for 3 hours. The reaction mixture was filtered to give a clear liquid which was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*30mm*10pm; 25- 55% acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 minute gradient) to obtain [1 - [(3,4-dichlorophenyl)methyl]pyrrolidin-3-yl]-(3-pyridyl)methanol (45 mg, 123 pmol, 24%, hydrochloric acid) as a white solid.

Compound 24: ¹H NMR (400 MHz, Methanol-d4) d 8.95 (br. d, J = 8.9 Hz, 1 H), 8.82 (d, J = 5.7 Hz, 1 H), 8.72 (br t, J = 7.3 Hz, 1H), 8.17 - 8.08 (m, 1 H), 7.87 - 7.77 (m, 1 H), 7.70 - 7.61 (m, 1 H), 7.58 - 7.48 (m,

1 H), 5.16 - 4.99 (m, 1 H), 4.51 - 4.33 (m, 2H), 3.77 - 3.42 (m, 2H), 3.41 - 3.33 (m, 1 H), 3.41 - 3.33 (m, 1 H), 3.28 - 3.18 (m, 1 H), 3.16 - 2.88 (m, 1 H), 2.28 - 1.79 (m, 1 H); LCMS (ESI) m/z: 337.0 [M+H]⁺. Compound 22: ¹H NMR (400 MHz, Methanok/4) d 8.51 (d, J = 2.0 Hz, 1 H), 8.46 - 8.39 (m, 1 H), 7.88 - 7.80 (m, 1 H), 7.51 - 7.36 (m, 3H), 7.26 - 7.18 (m, 1 H), 4.57 (d, J = 7.7 Hz, 1 H), 3.64 - 3.48 (m, 2H), 2.70 - 2.54 (m, 3H), 2.48 - 2.40 (m, 1 H), 2.28 - 2.19 (m, 1 H), 2.04 - 1 .85 (m, 2H); LCMS (ESI) m/z: 337.1 [M+H]⁺.

Example 13: Preparation of 2-[1-(3,4-dichlorophenyl)-4-piperidyl]-1-(3-pyridyl)ethanol (Compound 27).

Step 1 : Preparation of ethyl 2-[1-(3,4-dichlorophenyl)-4-piperidyl]acetate.

To a solution of 2,2’-bis(diphenylphosphino)-1 ,T-binapthalene (331 mg, 531 pmol) in dioxane (30 ml_) was added palladium (II) acetate (119 mg, 531 pmol) and cesium carbonate (3.46 g, 10.62 mmol). The reaction mixture was degassed with nitrogen three times and stirred at 20 °C for 1 hour. Then, 4- bromo-1 ,2-dichloro-benzene (1.2 g, 5.31 mmol) and ethyl 2-(4-piperidyl)acetate (910 mg, 5.31 mmol) were added to the solution. The mixture was heated to 105°C and stirred for 15h. TLC (Petroleum ether : Ethyl acetate = 5:1 , R_f = 0.62) showed the reaction was complete. Water (40 mL) was added to the reaction, and the reaction mixture was extracted with ethyl acetate (50 mL x 2). The combined organic layers were washed with brine (30 mL), dried over sodium sulfate, filtered, and concentrated.. The crude product was purified by ISCO column chromatography (20 g silica, 0-10 % ethyl acetate in petroleum ether, gradient over 30 minutes)to obtain ethyl 2-[1-(3,4-dichlorophenyl)-4-piperidyl]acetate (1.08 g, 3.42 mmol, 64%) as a pale yellow oil.

¹H NMR (400 MHz, Chloroform-d) d 7.25 (d, J = 9.0 Hz, 1 H), 6.96 (d, J = 2.8 Hz, 1 H), 6.75 (dd, J = 2.8, 8.9 Hz, 1 H), 4.16 (q, J = 7.2 Hz, 2H), 3.62 (br. d, J = 12.5 Hz, 2H), 2.75 (dt, J = 2.1 , 12.3 Hz, 2H), 2.28 (d, J = 7.1 Hz, 2H), 1.96 (ttd, J = 3.7, 7.5, 11.1 Hz, 1 H), 1.83 (br. d, J = 13.0 Hz, 2H), 1.39 (dq, J = 3.9, 12.3 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H).

Step 2: Preparation of 2-[1-(3,4-dichlorophenyl)-4-piperidyl]acetaldehyde.

To a solution of ethyl 2-[1-(3,4-dichlorophenyl)-4-piperidyl]acetate (1 g, 3.16 mmol) in tetrahydrofuran (15 ml_) was added diisobutylalumminum hydride (1 M, 6.32 ml_) in toluene at -50 °C and the mixture was stirred for 1 hour. TLC (Petroleum ether : Ethyl acetate = 5:1) showed the reaction was complete. Hydrochloric acid (15 ml_, 1 M) was added to the reaction, and the reaction mixture was extracted with ethyl acetate (30 ml_ x 2). The combined organic layers were washed with brine (15 ml_), dried over sodium sulfate, filtered ,and concentrated to dryness to obtain crude product. The crude product was purified by ISCO column chromatography (10 g silica, 0-10 % ethyl acetate in petroleum ether, gradient over 20 minutes) to obtain 2-[1-(3,4-dichlorophenyl)-4-piperidyl]acetaldehyde (490 mg,

1 .80 mmol, 57%) as a pale yellow oil.

¹H NMR (400 MHz, Chloroform-d) d 9.82 (s, 1 H), 7.28 - 7.23 (m, 1 H), 6.96 (d, J = 2.4 Hz, 1 H), 6.75 (dd, J = 2.6, 8.8 Hz, 1 H), 3.62 (br. d, J = 12.6 Hz, 2H), 2.77 (brt, J = 12.2 Hz, 2H), 2.44 (br. d, J = 6.8 Hz, 2H),

2.16 - 2.01 (m, 1 H), 1.83 (br. d, J = 12.8 Hz, 2H), 1.51 - 1.32 (m, 2H).

Step 3: Preparation of 2-[1 -(3,4-dichlorophenyl)-4-piperidyl]-1 -(3-pyridyl) ethanol.

To a solution of 3-iodopyridine (331 mg, 1.62 mmol) in tetrahydrofuran (3 ml_) was added isopropylmagnesium chloride (2 M, 808 pL) in tetrahydrofuran at 0 °C. The mixture was stirred at 0 °C for 0.5 h. Then, 2-[1-(3,4-dichlorophenyl)-4-piperidyl]acetaldehyde (220 mg, 808 pmol) was added at 0 °C. The mixture was warmed and stirred at 25 °C for 1 .5 hours. LCMS showed the reaction was complete. Saturated ammonium chloride solution (10 ml_) was added to the reaction, and the reaction mixture was extracted with ethyl acetate (30 ml_ x 2). The combined organic layers were washed with brine (20 ml_), dried over sodium sulfate, filtered, and concentrated to dryness to obtain crude product. The crude was purified by prep-HPLC (Kromasil C18 250*50 5m column; 30-70 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 minute gradient) to obtain 2-[1-(3,4-dichlorophenyl)-4-piperidyl]-1-(3- pyridyl)ethanol (143 mg, 401 pmol, 50%) as a pale yellow solid.

¹H NMR (400 MHz, Chloroform-d) d 8.58 (d, J = 1 .6 Hz, 1 H), 8.54 (dd, J = 1 .5, 4.8 Hz, 1 H), 7.73 (br. d, J = 7.8 Hz, 1 H), 7.31 (dd, J = 5.0, 7.6 Hz, 1 H), 7.25 (d, J = 9.0 Hz, 1 H), 6.96 (d, J = 2.8 Hz, 1 H), 6.75 (dd, J

= 2.8, 8.9 Hz, 1 H), 4.98 - 4.78 (m, 1 H), 3.72 - 3.51 (m, 2H), 2.72 (brt, J = 11 .9 Hz, 2H), 2.11 (br. s, 1 H),

1 .96 - 1 .79 (m, 3H), 1.71 - 1 .63 (m, 1 H), 1 .62 - 1 .55 (m, 1 H), 1 .49 - 1 .32 (m, 2H); LCMS (ESI) m/z: 351 .1 [M+H]⁺.

Example 14: Preparation of stereoisomer 1 (Compound 36), stereoisomer 2 (Compound 89), stereoisomer 3 (Compound 37) and stereoisomer 4 (Compound 90) of [1 -(3,4- dichlorophenyl)pyrrolidin-3-yl]-(3-pyridyl)methanol.

Stereoisomer 3 Stereoisomer 4 Step 1 : Preparation of fe/f-butyl 3-[hydroxy(3-pyridyl)methyl]pyrrolidine-1-carboxylate.

To a solution of 3-iodopyridine (5.14 g, 25.09 mmol) in tetrahydrofuran (30 ml_) was added isopropylmagnesium chloride (2 M, 12.55 ml_) dropwise in 0 °C. The mixture was stirred at 25 °C for 1 hour. Then, fe/f-butyl 3-formylpyrrolidine-1-carboxylate (2.5 g, 12.55 mmol) in tetrahydrofuran (20 ml_) was added dropwise the mixture at 0 °C. The mixture was warmed up and stirred at 25 °C for 3 hours. To the mixture was added water (12 ml_), and the aqueous phase was extracted with ethyl acetate (25 ml_ x 3). The combined organic phase was washed with brine (10 ml_*3), dried with anhydrous sodium sulfate, filtered, and concentrated in vacuum. The product fe/f-butyl 3-[hydroxy(3-pyridyl)methyl]pyrrolidine-1- carboxylate (3.4 g, crude) was obtained as a yellow gum. LCMS (ESI) m/z: 279.1 [M+H]⁺. Step 2: Preparation of 3-pyridyl(pyrrolidin-3-yl)methanol.

A mixture of fe/f-butyl 3-[hydroxy(3-pyridyl)methyl]pyrrolidine-1-carboxylate (3.4 g, 12.22 mmol) in hydrochloric acid/ethyl acetate (4 M, 30 ml_) was stirred at 25 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to give the crude product 3-pyridyl(pyrrolidin-3-yl)methanol (3.2 g, crude, hydrochloric acid) as a yellow gum. Step 3: Synthesis of [1-(3,4-dichlorophenyl)pyrrolidin-3-yl]-(3-pyridyl)methanol and chiral separation into enantiomer 1 (compound 36), enantiomer 2 (compound 89), enantiomer 3 (compound 37) and enantiomer 4 (compound 90). To a solution of 3-pyridyl(pyrrolidin-3-yl)methanol.HCI (999 mg, 4.65 mmol) in dimethylformamide

(30 ml_) weres added 1 ,2-dichloro-4-iodo-benzene (900 mg, 3.30 mmol), cesium carbonate (2.15 g, 6.60 mmol), bis(dibenzylideneacetone)palladium(0) (190 mg, 330 pmol), and 4,5-bis(diphenylphosphino)-9,9- dimethylxanthene (191 mg, 330 pmol). The mixture was stirred at 120 °C for 16 hours under nitrogen. To the mixture was added water (10 ml_), and the aqueous solution was extracted with ethyl acetate (20 ml_ x 3). The combined organic phase was washed with brine (25 ml_ x 3), dried with anhydrous sodium sulfate, filtered, and concentrated in vacuum. The crude product was purified by prep-HPLC (Kromasil 250*50mm*10pmcolumn; 40%-60% acetonitrile in a 0.04% ammonium hydroxide andl OmM ammonium bicarbonate solution, 10 minute gradient) to obtain the racemic product. 100 mg of the racemic compound was subjected to preparative SFC (DAICEL CHIRALPAK AD(250mm*30mm,10pm); column, 40 °C, eluting with 60% ethanol containing 0.1% ammonium hydroxide in a flow of 70 g/min carbon dioxide at 100 bar) to obtain enantiomerically pure compounds.

Example 15: Preparation of (5-fluoro-3-pyridyl)-[2-[6-(trifluoromethyl)-3-pyridyl]-2- azaspiro[3.3]heptan-6-yl]methanol (Compound 38).

100 °C, 20 m To a solution of 2-azaspiro[3.3]heptan-6-yl-(5-fluoro-3-pyridyl)methanol (54 mg, 245 pmol) and 5- bromo-2-(trifluoromethyl)pyridine (55 mg, 245 pmol) in dioxane (1.5 ml_) was added sodium fe/f-butoxide (71 mg, 734 pmol), tris(dibenzylideneacetone)dipalladium(0) (11 mg, 12 pmol), and 2- dicyclohexylphosphino^'.O'-diisopropoxybiphenyl (2 mg, 5 pmol). The suspension was degassed and purged with nitrgoen 3 times, and then stirred at 100 °C for 20 minutes. LCMS showed starting material was consumed completely and one main peak with desired mass was detected. The mixture was filtered, and the filtrate was dried in vacuo to afford a crude residue. The residue was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40mm*10pm column; 35-55 % acetonitrile in a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to obtain (5-fluoro-3-pyridyl)-[2-[6-(trifluoromethyl)-3- pyridyl]-2-azaspiro[3.3]heptan-6-yl]methanol (22 mg, 59 pmol, 24%) as a pale yellow gum. ¹H NMR (400 MHz, Chloroform-d) d 8.41 - 8.38 (m, 1 H), 8.36 (s, 1 H), 7.83 - 7.80 (m, 1 H), 7.47 - 7.41 (m, 2H), 6.69 (dd,

J = 2.6, 8.6 Hz, 1 H), 4.76 - 4.67 (m, 1 H), 4.02 - 3.97 (m, 2H), 3.96 - 3.89 (m, 2H), 2.61 - 2.48 (m, 1 H),

2.38 - 2.28 (m, 3H), 2.25 - 2.15 (m, 2H); LCMS (ESI) m/z: 368.0 [M+H]⁺.

The following compounds were synthesized according to the procedure described for Compound 38:

Example 16: Preparation of (S)-[2-(1,3-benzothiazol-6-yl)-2-azaspiro[3.3]heptan-6-yl]-(3- pyridyl)methanol (Compound 42)

To a solution of (S)-2-azaspiro[3.3]heptan-6-yl(3-pyridyl)methanol (120 mg, 587 pmol) in dichloromethane (3 ml_) was added 1 ,3-benzothiazol-6-ylboronic acid (105 mg, 587 pmol), copper(ll) acetate (128 mg, 705 pmol), and A/,A/-diisopropylethylamine (304 mg, 2.35 mmol, 409 pL). The mixture was stirred at 40 °C for 16 hours under oxygen (15 Psi. The reaction solution was filtered, and the filtrate was purified directly by prep-HPLC (Phenomenex Gemini-NX 150*30 5pm column; 15-35 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient). The product (S)-[2-(1 ,3- benzothiazol-6-yl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanol (12 mg, 35 pmol, 6%) was obtained as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.67 (s, 1H), 8.60 - 8.52 (m, 2H), 7.91 (d, J = 8.8 Hz, 1 H), 7.68 (br. d, J = 7.8 Hz, 1 H), 7.30 (dd, J = 4.8, 7.9 Hz, 1 H), 6.85 (d, J = 2.2 Hz, 1 H), 6.63 (dd, J = 2.2, 8.8 Hz, 1 H), 4.66 (br. d, J = 7.0 Hz, 1 H), 3.98 - 3.90 (m, 2H), 3.89 - 3.82 (m, 2H), 2.63 - 2.52 (m, 1 H), 2.39 - 2.27 (m, 2H), 2.22 - 2.09 (m, 2H), 1 .96 (br. s, 1 H); LCMS (ESI) m/z: 338.2 [M+H]⁺.

Example 17: Preparation of [2-(3-chloro-4-fluoro-phenyl)-2-azaspiro[3.3]heptan-6-yl]-pyridazin-3-yl- methanol (Compound 44).

Compound 44 was synthesized according to the synthetic procedure reported for the Preparation of compound 95. It was obtained as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 9.16 (dd, J = 2.1 , 4.3 Hz, 1 H), 7.57 - 7.42 (m, 2H), 6.97 (t, J = 8.9 Hz, 1 H), 6.41 (dd, J = 2.8, 6.1 Hz, 1 H), 6.24 (td, J = 3.3, 8.9 Hz, 1 H), 4.92 (d, J = 5.4 Hz, 1 H), 4.01 - 3.72 (m, 5H), 2.73 - 2.62 (m, 1 H), 2.43 (dd, J = 8.3, 11 .8 Hz, 1 H), 2.33 - 2.22 (m, 2H), 2.15 - 2.03 (m, 1 H); LCMS (ESI) m/z: 334.1 [M+H]⁺. Example 18: Synthesis of N-[(R)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3- pyridyl)methyl]methanesulfonamide (Compound 45):

Step 1 : (S)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanol.

To a solution of 1 ,2-dichloro-4-iodo-benzene (668 mg, 2.45 mmol) in DMSO (10 ml_) were added K2CO3 (1.35 g, 9.79 mmol), Cul (93 mg, 490 mol), pyrrolidine-2-carboxylic (113 mg, 979 umol) and (S)-2- azaspiro[3.3]heptan-6-yl(3-pyridyl)methanol (500 mg, 2.45 mmol) under N2. The mixture was stirred at 90 °C for 12 h, filtered, and the filtrate was purified directly by prep-HPLC (Kromasil C18 (250*50mm*10 um)column; 40-70 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 min gradient). The compound (S)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanol (122 mg, 349 umol, 14%) was obtained as a pale yellow solid. LCMS (ESI) m/z: 349.1 [M+H]⁺.

Step 2: 6-[(R)-azido(3-pyridyl)methyl]-2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptane.

To a solution of (S)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanol (120 mg, 344 umol) in THF (1.5 ml_) were added DPPA (104 mg, 378 umol), DIAD (76 mg, 378 umol) and PPhi3 (99 mg, 378 umol, 1.1 eq). The mixture was stirred at 20 °C for 12 h and concentrated in vacuum. The crude product was purity by prep-TLC (Petroleum ether: Ethyl acetate = 2:1 and Petroleum ether: Ethyl acetate = 0:1 , R_f =0.43) to obtain 6-[(R)-azido(3-pyridyl)methyl]-2-(3,4-dichlorophenyl)-2- azaspiro[3.3]heptane (130 mg, 327 umol, 953%) was obtained as a colorless gum. LCMS (ESI) m/z: 374.1 [M+H]⁺.

Step 3: (R)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanamine.

To a solution of 6-[(R)-azido(3-pyridyl)methyl]-2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptane (120 mg, 321 umol) in THF (2 mL) and H2O (0.2 mL) was added PPhi3 (126 mg, 481 umol). The mixture was stirred at 50 °C for 12 h and concentrated. The crude product was purified by Prep-TLC (Dichloromethane: Methanol = 5:1 , R_f =0.5) to obtain (R)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanamine (80 mg, 224 umol, 70%) as a colorless gum. LCMS (ESI) m/z: 348.1 [M+H]⁺.

Step 4: N-[(R)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methyl]methanesulfonamide.

To a solution of (R)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanamine (40 mg, 115 umol) in DCM (1 mL) was added Et3N (23 mg, 230 umol) and MsCI (26 mg, 230 umol) at 0 °C. The mixture was stirred at 20 °C for 2 h and was quenched by addition H2O (0.5 mL) and the resultant mixture was concentrated in vacuum. The residue was purified by prep-HPLC (Waters Xbridge 150*25 5u column; 41-71 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 min gradient) to obtain N-[(R)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methyl]methanesulfonamide (15 mg, 36 umol, 31%) was obtained as a white solid.

¹H NMR (400 MHz, CHLOROFORM-d) d 8.65 - 8.55 (m, 2H), 7.66 - 7.58 (m, 1 H), 7.39 - 7.30 (m, 1 H), 7.20 (d, J = 8.6 Hz, 1 H), 6.44 (d, J = 2.6 Hz, 1 H), 6.26 - 6.18 (m, 1 H), 5.01 - 4.92 (m, 1 H), 4.44 - 4.32 (m, 1 H), 3.90 - 3.69 (m, 4H), 2.69 - 2.62 (s, 3H), 2.61 - 2.40 (m, 2H), 2.29 - 2.18 (m, 1 H), 2.16 - 2.04 (m, 1 H), 2.01 -

1.92 (m, 1 H). LCMS (ESI) for (C19H21CI2N302S) [M+H]+: 426.1.

Example 19: Preparation of 6-(3-pyridylmethyl)-2-[6-(trifluoromethyl)-3-pyridyl]-2- azaspiro[3.3]heptane (Compound 47)

To a solution of 3-pyridyl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]methanol (300 mg, 859 pmol) in acetic acid (1 .5 ml_) was added palladium on activated charcoal (101 mg, 86 pmol, 10% purity) and perchloric acid (388 mg, 3.86 mmol) under hydrogen. The mixture was stirred at 20 °C for 18 hours. The mixture was then filtered, and the filtrate was dried over in vacuo to afford the crude product. It then was purified by prep-HPLC (Waters Xbridge BEH C18 100*30mm*10pm column; 25-55 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 6 minute gradient) to afford 6-(3- pyridylmethyl)-2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptane (16 mg, 46 pmol, 5%) as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.50 - 8.45 (m, 1 H), 8.42 (d, J = 1 .6, 1 H), 7.82 (d, J =

2.6 Hz, 1 H), 7.48 - 7.39 (m, 2H), 7.22 (ddd, J = 0.8, 4.8, 7.8 Hz, 1 H), 6.71 - 6.65 (m, 1 H), 3.99 (s, 2H), 3.89 (s, 2H), 2.75 - 2.68 (m, 2H), 2.57 - 2.43 (m, 1 H), 2.42 - 2.32 (m, 2H), 2.05 - 1 .95 (m, 2H). LCMS (ESI) m/z: 334.1 [M+H]⁺.

Example 20: Preparation of pyridin-3-yl(7-(6-(trifluoromethyl)pyridin-3-yl)-7-azaspiro[3.5]nonan-2- yl)methanol (Compound 49).

Step 1 : Preparation of fe/f-butyl 2-(hydroxy(pyridin-3-yl)methyl)-7-azaspiro[3.5]nonane-7-carboxylate.

To a solution of 3-iodopyridine (728 mg, 3.55 mmol) in tetrahydrofuran (6 ml_) was added isopropylmagnesium chloride (2 M, 1.78 ml_) in tetrahydrofuran dropwise by syringe at 0 °C. The mixture was stirred at 0 °C for 1 hour. Then, fe/f-butyl 2-formyl-7-azaspiro[3.5]nonane-7-carboxylate (600 mg,

2.37 mmol) was added to the solution at 0 °C under nitrogen. The reaction mixture was warmed up and stirred at 20 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to give crude product which was purified by ISCO column chromatography (10 g silica, 20-50% ethyl acetate in petroleum ether, gradient over 20 minutes). The product fe/f-butyl 2-[hydroxy(3-pyridyl)methyl]-7- azaspiro[3.5]nonane-7-carboxylate (480 mg, crude) was obtained as a yellow oil. LCMS (ESI) m/z: 333.3 [M+H]⁺.

Step 2: Preparation of pyridin-3-yl(7-azaspiro[3.5]nonan-2-yl)methanol.

A solution of fe/f-butyl 2-[hydroxy(3-pyridyl)methyl]-7-azaspiro[3.5]nonane-7-carboxylate (480 mg, 1.44 mmol) in hydrochloric acid/ethyl acetate (4 M, 4.80 ml_) was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give 7-azaspiro[3.5]nonan-2-yl(3-pyridyl)methanol (360 mg, crude, Hydrochloric acid) as a white solid. LCMS (ESI) m/z: 233.1 [M+H]⁺. The crude product was used further without purification.

Step 3: Preparation of pyridin-3-yl(7-(6-(trifluoromethyl)pyridin-3-yl)-7-azaspiro[3.5]nonan-2-yl)methanol.

To a solution of 1 ,8-iazabicyclo[5.4.0]undec-7-ene (170 mg, 1.12 mmol, 168 pL) in dimethylsulfoxide (2 mL) was added 5-fluoro-2-(trifluoromethyl)pyridine (92 mg, 558 pmol) and 7- azaspiro[3.5]nonan-2-yl(3-pyridyl)methanol (150 mg, 558 pmol, hydrochloric acid). The mixture was stirred at 80 °C for 2 hours. The reaction mixture was filtered, and the filtrate was concentrated under vacuum. The crude residue was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40mm*10pm column; 25-60% acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 minute gradient). The product 3-pyridyl-[7-[6-(trifluoromethyl)-3-pyridyl]-7-azaspiro[3.5]nonan-2- yl]methanol (68 mg, 180 pmol, 32% ) was obtained as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.59 - 8.49 (m, 2H), 8.30 (d, J = 2.8 Hz, 1 H), 7.68 (br. d, J = 7.8 Hz, 1 H), 7.47 (d, J = 8.8 Hz, 1 H), 7.30 - 7.27 (m, 1 H), 7.16 (dd, J = 2.6, 8.7 Hz, 1 H), 4.65 (br. d, J = 5.9 Hz, 1 H), 3.33 - 3.15 (m, 4H), 2.62 (sxt, J = 8.4 Hz, 1 H), 2.24 (br. d, J = 2.3 Hz, 1 H), 2.03 - 1 .93 (m, 1 H), 1 .90 - 1 .82 (m, 1 H), 1 .81 - 1 .72 (m, 3H), 1 .69 - 1 .61 (m, 3H); LCMS (ESI) m/z: 378.2 [M+H]⁺.

The following Compound 105 was synthesized according to the protocol described for the Compound 49.

Example 21: Preparation of 2-[1-[(3,4-dichlorophenyl)methyl]azetidin-3-yl]-1-(3-pyridyl)ethanol (Compound 50)

To a solution of 2-(azetidin-3-yl)-1-(3-pyridyl)ethanol (200 mg, 1.12 mmol) in dichloromethane (4 ml_) was added 3,4-dichlorobenzaldehyde (196 mg, 1 .12 mmol). The mixture was stirred at 20 °C for 2 hours. Then, sodium triacetoxyborohydride (476 mg, 2.24 mmol) was added and the mixture was stirred at 20 °C for 12 hours. The reaction mixture was concentrated to dryness to give the crude product. The crude product was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40 10p column; 35-65 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to obtain2-[1-[(3,4- dichlorophenyl)methyl]azetidin-3-yl]-1-(3-pyridyl)ethanol (116 mg, 343 pmol, 31%) as a pale yellow gum.

1 H NMR (400 MHz, Chloroform-d) d 8.59 - 8.49 (m, 2H), 7.70 (br. d, J = 7.9 Hz, 1 H), 7.40 - 7.33 (m, 2H), 7.31 - 7.27 (m, 1 H), 7.09 (dd, J = 1 .8, 8.2 Hz, 1 H), 4.81 (dd, J = 5.1 , 8.2 Hz, 1 H), 3.51 (s, 2H), 3.43 - 3.30 (m, 2H), 2.99-2.96(t, 1 H), 2.86-2.83 (t, 1 H), 2.66-2.60 (m, 1 H), 2.09 - 1.90 (m, 2H); LCMS (ESI) m/z: 337.1 [M+H]⁺. Example 22: Preparation of 1-(3-pyridyl)-2-[1-[5-(trifluoromethoxy)-2-pyridyl]azetidin-3-yl]ethanol (Compound 54) and its chiral separation into enantiomer 1 (Compound 26) and enantiomer 2 (Compound 29).

To a solution of 2-(azetidin-3-yl)-1-(3-pyridyl)ethanol (200 mg, 1.12 mmol) in dimethylsulfoxide (3 ml_) weres added potassium carbonate (620 mg, 4.49 mmol), copper(l) iodide (36 mg, 191 pmol), pyrrolidine-2-carboxylic acid (39 mg, 337 pmol), and 2-bromo-5-(trifluoromethoxy)pyridine (272 mg, 1.12 mmol). The mixture was stirred at 90 °C for 12 hours under nitrogen. Water (5 ml_) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (10 mL x 4). The combined organic layer was concentrated to dryness to give the crude product which was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40 10pm column; 25-55 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to afford the racemic compound 1 -(3-pyridyl)-2-[1-[5- (trifluoromethoxy)-2-pyridyl]azetidin-3-yl]ethanol (167 mg) as a pale yellow gum. The racemic product was subjected to preparative SFC (DAICEL CHIRALPAK AD (250mm*30mm,10pm) column, 40 °C, eluting with 40% methanol containing 0.1% ammonium hydroxide in a flow of 70 g/min carbon dioxide at 100 bar) to obtain enantiomer 1 and 2 in pure form.

¹H NMR (400 MHz, Chloroform-d) for compound 54: for d 8.61 - 8.50 (m, 2H), 8.03 (d, J = 2.6 Hz, 1 H), 7.75 - 7.67 (m, 1 H), 7.31 (dd, J = 4.6, 7.7 Hz, 2H), 6.20 (d, J = 9.0 Hz, 1 H), 4.84 - 4.76 (m, 1 H), 4.17 - 4.05 (m, 2H), 3.74 - 3.58 (m, 2H), 2.99 - 2.85 (m, 1 H), 2.27 (d, J = 2.6 Hz, 1 H), 2.25 - 2.15 (m, 1 H), 2.08 (ddd, J = 5.3, 7.9, 13.7 Hz, 1 H); LCMS (ESI) m/z: 340.2 [M+H]⁺.

¹H NMR (400 MHz, Chloroform-d) for compound 26: d 8.60 - 8.50 (m, 2H), 8.02 (d, J = 2.7 Hz, 1 H), 7.71 (td, J = 1.8, 7.8 Hz, 1H), 7.35 - 7.27 (m, 2H), 6.19 (d, J = 9.0 Hz, 1 H), 4.80 (dd, J = 5.2, 7.8 Hz, 1 H), 4.17 - 4.04 (m, 2H), 3.74 - 3.59 (m, 2H), 2.99 - 2.85 (m, 1 H), 2.44 (br. s, 1 H), 2.26 - 2.02 (m, 2H); LCMS (ESI) m/z: 340.2 [M+H]⁺. HPLC retention time: 2.47min

¹H NMR (400 MHz, Chloroform-d) for compound 29: d 8.63 - 8.48 (m, 2H), 8.02 (d, J = 2.4 Hz, 1 H), 7.76 - 7.66 (m, 1 H), 7.35 - 7.27 (m, 2H), 6.19 (d, J = 9.0 Hz, 1 H), 4.79 (dd, J = 5.2, 7.6 Hz, 1 H), 4.18 - 4.02 (m, 2H), 3.75 - 3.58 (m, 2H), 3.00 - 2.84 (m, 1 H), 2.53 (br. s, 1 H), 2.29 - 1 .98 (m, 2H); LCMS (ESI) m/z:

340.2 [M+H]⁺. HPLC retention time: 2.91 min Example 23: Preparation of 1-(2-(3-chloro-4-fluorophenyl)-2-azaspiro[3.3]heptan-6-yl)-1-(pyridin-3- yl)ethanol (Compound 57).

Step 1 : Preparation of (2-(3-chloro-4-fluorophenyl)-2-azaspiro[3.3]heptan-6-yl)(pyridin-3-yl)methanone.

To a solution of 2-azaspiro[3.3]heptan-6-yl(3-pyridyl)methanol (800 mg, 3.92 mmol) in dioxane (10 ml_), was added 2-chloro-1-fluoro-4-iodo-benzene (913 mg, 3.56 mmol) sodium fe/f-butoxide (1.03 g, 10.68 mmol), 2-dicyclohexylphosphino-2^,,6'-diisopropoxybiphenyl (33 mg, 71 pmol), and tris(dibenzylideneacetone)dipalladium(0) (163 mg, 178 pmol). Tthe mixture was stirred at 100 °C for 20 min under nitrogen. LCMS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was concentrated under reduced pressure to give obtain crude product. The crude product was purified by ISCO column chromatography (40 g silica, 60-80 % ethyl acetate in petroleum ether, gradient over 20 minutes) to obtain[2-(3-chloro-4-fluoro-phenyl)-2-azaspiro[3.3]heptan-6- yl]-(3-pyridyl)methanone (130 mg, 393 pmol, 11 %) as a yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 9.08 (s, 1 H), 8.78 (br. d, J = 4.3 Hz, 1 H), 8.20 (br. d, J = 7.6 Hz, 1 H), 7.49 - 7.38 (m, 1 H), 7.02 - 6.84 (m,

1 H), 6.46 - 6.32 (m, 1 H), 6.27 - 6.17 (m, 1 H), 3.97 - 3.87 (m, 3H), 3.77 (s, 2H), 2.69 - 2.55 (m, 4H); LCMS (ESI) m/z: 331.2 [M+H]⁺.

Step 2: Preparation of 1-(2-(3-chloro-4-fluorophenyl)-2-azaspiro[3.3]heptan-6-yl)-1-(pyridin-3-yl)ethanol.

To a solution of [2-(3-chloro-4-fluoro-phenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanone (120 mg, 363 pmol) in tetrahydrofuran (1.5 mL), was added methylmagnesium bromide solution (3 M, 157 pL) at 0 °C. The mixture was warmed up to 20 °C and stirred for 1 hour. Concentration under reduced pressure and purification by prep-HPLC (Waters Xbridge BEH C18 100*25mm*5pm column; 40%-70% acetonitrile in an a 10mM ammonium bicarbonate solution, 10 minute gradient) afforded product 1-[2-(3- chloro-4-fluoro-phenyl)-2-azaspiro[3.3]heptan-6-yl]-1-(3-pyridyl)ethanol (56 mg, 161 pmol, 44%) as a colorless gum. ¹H NMR (400 MHz, Chloroform-d) d 8.65 (d, J = 1 .9 Hz, 1 H), 8.47 (dd, J = 1 .5, 4.8 Hz,

1 H), 7.76 (td, J = 1.9, 8.0 Hz, 1 H), 7.26 (s, 1H), 6.94 (t, J = 8.9 Hz, 1 H), 6.37 (dd, J = 2.8, 6.1 Hz, 1 H), 6.20 (td, J = 3.3, 8.8 Hz, 1 H), 3.84 - 3.81 (m, 1 H), 3.78 - 3.75 (m, 1 H), 3.71 - 3.64 (m, 2H), 2.58 (quin, J = 8.6 Hz, 1 H), 2.34 - 2.20 (m, 2H), 2.16 - 2.07 (m, 2H), 1 .87 (ddd, J = 3.8, 8.2, 11.8 Hz, 1 H), 1.50 (s, 3H); LCMS (ESI) m/z: 347.1 [M+H]⁺. Example 24: Preparation of 2-[1-(4-chloro-2-fluoro-phenyl)azetidin-3-yl]-1-(3-pyridyl)ethanol (Compound 65) and its chiral resolution into enantiomer 1 (compound 101) and enantiomer 2 (compound 52).

Enantiomer 1 Enantiomer 2

To a solution of 2-(azetidin-3-yl)-1-(3-pyridyl)ethanol (200 mg, 1.12 mmol) in dioxane (3 ml_) were added 4-chloro-2-fluoro-1-iodo-benzene (96 mg, 374 pmol), 4,5-Bis(diphenylphosphino)-9,9- dimethylxanthene (43 mg, 75 pmol), cesium carbonate (487 mg, 1.50 mmol), and Tris(dibenzylideneacetone)dipalladium(0) (51 mg, 56 pmol). The resultant mixture was stirred at 100 °C for 4 hours under nitrogen. Water (10 ml_) was added to the reaction, and the mixture was extracted with ethyl acetate (15 mL x 3). The combined organic layers were washed with brine (10 ml_), dried over sodium sulfate, filtered, and concentrated. The crude product was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40 10pm column; 35-55 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to afford2-[1-(4-chloro-2-fluoro-phenyl)azetidin-3-yl]-1-(3-pyridyl)ethanol (57 mg) as a yellow gum. The racemic mixture was purified by preparative SFC DAICEL CHIRALPAK IG (250mm*30mm,10pm) column, 40 °C, eluting with 50% methanol containing 0.1% ammonium hydroxide in a flow of 70 g/min carbon dioxide at 100 bar) to afford enantiomerically pure compounds 101 and 52.

¹H NMR (400 MHz, Chloroform-d) for compound 65: d 8.62 - 8.52 (m, 2H), 7.71 (td, J = 1.8, 7.8 Hz, 1 H), 7.31 (dd, J = 4.8, 7.7 Hz, 1 H), 7.01 - 6.89 (m, 2H), 6.38 - 6.28 (m, 1 H), 4.80 (dd, J = 5.3, 7.6 Hz, 1 H), 4.12 - 3.98 (m, 2H), 3.66 - 3.48 (m, 2H), 2.95 - 2.81 (m, 1 H), 2.23 - 1 .99 (m, 3H); LCMS (ESI) m/z: 307.1 [M+H]⁺.

¹H NMR (400 MHz, Chloroform-d) for compound 101 : d 8.60 - 8.51 (m, 2H), 7.75 - 7.68 (m, 1 H), 7.30 (dd, J = 4.9, 7.9 Hz, 1 H), 6.99 - 6.90 (m, 2H), 6.33 (t, J = 8.9 Hz, 1 H), 4.79 (dd, J = 5.2, 7.8 Hz, 1 H), 4.11 - 3.97 (m, 2H), 3.63 - 3.48 (m, 2H), 2.88 (quind, J = 7.1 , 14.1 Hz, 1 H), 2.21 - 2.01 (m, 3H); LCMS (ESI) m/z: 307.1 [M+H]⁺.

¹H NMR (400 MHz, Chloroform-d) for compound 52: d 8.62 - 8.49 (m, 2H), 7.77 - 7.66 (m, 1 H), 7.30 (dd, J = 4.9, 7.7 Hz, 1 H), 6.97 - 6.90 (m, 2H), 6.33 (t, J = 8.9 Hz, 1 H), 4.79 (dd, J = 5.3, 7.7 Hz, 1 H), 4.10 -3.98 (m, 2H), 3.64 - 3.49 (m, 2H), 2.88 (quind, J = 7.1 , 14.2 Hz, 1 H), 2.27 - 2.01 (m, 3H); LCMS (ESI) m/z: 307.1 [M+H]⁺. The following compounds were synthesized according to the protocol described for the Compound 65.

The following compounds were chirally separated using the conditions described for the Compounds 101 and 52.

Example 25: Synthesis of (1S)-2-[3-(3,4-dichlorophenyl)azetidin-1-yl]-1-(3-pyridyl)ethanol (Compound 67)

(1 R,2R)-2-aminocyclohexanol

Step 1 : Preparation of fe/f-butyl 3-(3,4-dichlorophenyl)azetidine-1-carboxylate.

To a solution of (3,4-dichlorophenyl)boronic acid (809 mg, 4.24 mmol) in isopropyl alcohol (10 ml_) was added diiodonickel (66 mg, 212 pmol), (1R,2R)-2-aminocyclohexanol;hydrochloride (32 mg, 212 pmol), sodium bis(trimethylsilyl)amide (1 M, 7.06 ml_) and fe/f-butyl 3-iodoazetidine-1-carboxylate (1 g, 3.53 mmol). The mixture was stirred at 80 °C for 2 hours. TLC (Petroleum ether : Ethyl acetate = 5:1 , R_f = 0.43) showed the reaction was complete. The reaction mixture was concentrated to dryness to give the crude product. The crude product was purified by ISCO column chromatography (40 g silica, 0-20 % ethyl acetate in petroleum ether, gradient over 30 minutes) to obtain fe/f-butyl 3-(3,4-dichlorophenyl)azetidine- 1-carboxylate (680 mg) as a pale yellow oil. ¹H NMR (400 MHz, Chloroform-d) d 7.44 - 7.41 (m, 2H), 7.16 (dd, J = 2.1, 8.3 Hz, 1 H), 4.33 (t, J = 8.7 Hz, 2H), 3.95 - 3.91 (m, 2H), 3.68 (tt, J = 5.8, 8.7 Hz, 1 H), 1.47 (s, 9H).

Step 2: Preparation of 3-(3,4-dichlorophenyl)azetidine.

Tert-butyl 3-(3,4-dichlorophenyl)azetidine-1-carboxylate (650 mg, 2.15 mmol) was dissolved in 4M hydrochloric acid in ethyl acetate (20 ml_). The mixture was stirred at 25 °C for 1 hour. TLC (Petroleum ether : Ethyl acetate = 3:1 , R_f = 0.00) showed the reaction was complete. The reaction mixture was concentrated to dryness to obtain 3-(3,4-dichlorophenyl)azetidine (490 mg, 2.05 mmol, 95%) as hydrochloride salt. It was used further without purification.

Step 3: Preparation of (1S)-2-[3-(3,4-dichlorophenyl)azetidin-1-yl]-1-(3-pyridyl)ethanol.

To a solution of 3-(3,4-dichlorophenyl)azetidine.HCI (150 mg, 629 pmol,) in ethanol (2 mL) was added (1S)-2-bromo-1-(3-pyridyl)ethanol (127 mg, 629 pmol) and triethylamine(191 mg, 1.89 mmol, 263 pL). The mixture was stirred at 80 °C for 16 hours. LCMS showed the reaction was complete. The mixture was concentrated and purified by prep-HPLC (Xtimate C18 150*40 5p column; 35-55 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 minute gradient) to obtain (1S)-2-[3-(3,4- dichlorophenyl)azetidin-1-yl]-1-(3-pyridyl)ethanol (41 mg, 124 pmol, 20%) as a yellow gum. ¹H NMR (400 MHz, Chloroform-d) d 8.60 (d, J = 1.98 Hz, 1 H), 8.54 (dd, J = 1.65, 4.74 Hz, 1 H), 7.70-7.77 (m, 1 H), 7.40 (dd, J = 2.98, 5.18 Hz, 2H), 7.30 (dd, J = 4.96, 7.83 Hz, 1H), 7.15 (dd, J = 1.98, 8.16 Hz, 1 H), 4.63 (dd, J = 5.18, 7.83 Hz, 1H), 3.82-3.90 (m, 1 H), 3.62-3.78 (m, 3H), 3.32 (t, J = 6.17 Hz, 1 H), 3.25 (t, J = 6.73 Hz, 1 H), 2.67-2.77 (m, 2H); LCMS (ESI) m/z: 323.0 [M+H] ⁺.

The following compound was synthesized according to the protocol described for the compound 67:

Example 26: Preparation of 4-[(R)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3- pyridyl)methyl]morpholine (Compound 69)

Step 1 : Preparation of [(S)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methyl] methanesulfonate.

To a solution of (S)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanol (300 mg, 859 pmol) and triethylamine (261 mg, 2.58 mmol, 359 pL) in dichloromethane (3 ml_) was added methanesulfonyl chloride (118 mg, 1 .03 mmol, 80 pl_) dropwise at 0 °C. The mixture was stirred at 15 °C for 1 hour. The reaction mixture was concentrated in vacuo and the crude product was dissolved in ethyl acetate (10 ml_) and water (5ml_), organic layer separated, and aqueous layer was extracted with ethyl acetate (10 ml_ x 2). The organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The compound [(S)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methyl] methanesulfonate (500 mg, crude) was obtained as a yellow gum. Step 2: Preparation of 4-[(R)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3- pyridyl)methyl]morpholine.

To a solution of [(S)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methyl] methanesulfonate (120 mg, 281 pmol) in dimethylsulfoxide (1 ml_) was added morpholine (147 mg, 1.68 mmol). The mixture was stirred at 60 °C for 3 hours. The mixture was filtered, and the filtrate was purified by prep-HPLC (Phenomenex Luna C18 100*30mm*5pm column; 20-50 % acetonitrile in a 0.225% formic acid solution in water, 9 minute gradient) to obtain4-[(R)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6- yl]-(3-pyridyl)methyl]morpholine (7 mg, 15% formic acid) as a white solid. ¹H NMR (400 MHz, Methanol- d4) d 8.44 (br. d, J = 2.6 Hz, 2H), 7.80 - 7.75 (m, 1 H), 7.42 (dd, J = 4.9, 7.8 Hz, 1 H), 7.21 (d, J = 8.7 Hz,

1 H), 6.49 (d, J = 2.6 Hz, 1 H), 6.32 (dd, J = 2.7, 8.7 Hz, 1 H), 3.91 - 3.85 (m, 1 H), 3.83 - 3.78 (m, 1 H), 3.71 - 3.66 (m, 1 H), 3.64 - 3.60 (m, 5H), 3.28 (s, 1 H), 2.78 (br. d, J = 8.8 Hz, 1 H), 2.56 - 2.41 (m, 3H), 2.40 - 2.32 (m, 2H), 2.23 (dd, J = 8.7, 11 .7 Hz, 1 H), 1 .81 (br. s, 1 H), 1 .72 - 1 .62 (m, 1 H); LCMS (ESI) m/z: 418.2 [M+H]⁺.

Example 27: Preparation of [1-(3-chloro-4-fluoro-phenyl)-4-piperidyl]-(3-pyridyl)methanol (Compound 71) and its chiral separation into enantiomer 1 (compound 17) and enantiomer 2 (compound 77).

Enantiomer 1 Enantiomer 2

Step 1 : Preparation of [1-(3-chloro-4-fluoro-phenyl)-4-piperidyl]-(3-pyridyl)methanone.

To a solution of 4-piperidyl(3-pyridyl)methanone.2HCI (0.5 g, 1.90 mmol) in dioxane (8 ml_) was added 2-chloro-1-fluoro-4-iodo-benzene (585 mg, 2.28 mmol), sodium fe/f-butoxide (548 mg, 5.70 mmol), tris(dibenzylideneacetone)dipalladium (0) (87 mg, 95 pmol), and 2-dicyclohexylphosphino-2’,6’- dimethoxybiphenyl (78 mg, 190 pmol). The reaction mixture was bubbled with nitrogen for 10 seconds then stirred at 100 °C for 12 hours. TLC (Petroleum ether : Ethyl acetate = 0:1 , R_f = 0.43) showed the reaction was complete. The reaction mixture was cooled to 20 °C followed by addition of water (15 ml_) and extracted with ethyl acetate (30 ml_ x 2). The combined organic layers were washed with brine (15 ml_), dried over sodium sulfate, filtered, and concentrated to dryness to obtain crude product. The crude product was purified by ISCO column chromatography (10 g silica, 10-60 % ethyl acetate in petroleum ether, gradient over 30 minutes) to obtain [1-(3-chloro-4-fluoro-phenyl)-4-piperidyl]-(3-pyridyl)methanone (470 mg, 1 .47 mmol, 78%) as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 9.19 (d, J = 1 .8 Hz, 1 H), 8.81 (dd, J = 1.5, 4.9 Hz, 1 H), 8.25 (td, J = 2.0, 8.0 Hz, 1 H), 7.46 (dd, J = 4.9, 7.9 Hz, 1 H), 7.03 (t, J = 8.8 Hz, 1 H), 6.95 (dd, J = 3.0, 6.3 Hz, 1 H), 6.80 (td, J = 3.4, 9.0 Hz, 1 H), 3.64 (td, J = 3.1 , 12.4 Hz, 2H), 3.43 - 3.30 (m, 1 H), 2.87 (dt, J = 3.3, 11 .7 Hz, 2H), 2.07 - 1 .90 (m, 4H).

Step 2: Preparation of [1-(3-chloro-4-fluoro-phenyl)-4-piperidyl]-(3-pyridyl)methanol, and chiral separation to enantiomer 1 and enantiomer 2.

To a solution of [1-(3-chloro-4-fluoro-phenyl)-4-piperidyl]-(3-pyridyl)methanone (250 mg, 784 pmol) in methanol (4 ml_) was added sodium borohydride (59 mg, 1 .57 mmol). The mixture was stirred at 20 °C for 1 hour. LCMS showed the reaction was complete. The reaction mixture was concentrated to dryness to give the crude product. The crude was purified by prep-HPLC (Welch Xtimate C18 250*50 10p column; 30-70 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 minute gradient) to obtain [1-(3-chloro-4-fluoro-phenyl)-4-piperidyl]-(3-pyridyl)methanol (140 mg) as a white solid. For the chiral resolution an amount 120 mg of racemic product was purified by preparative SFC [DAICEL CHIRALPAK AD (250mm*30mm,10pm) column, 40°C, eluting with 45% methanol containing 0.1% ammonium hydroxide in a flow of 70 g/min carbon dioxide at 100 bar]. Enantiomer 1 and enantiomer 2 were obtained as white solids in 24% and 17% yields.

¹H NMR (400 MHz, Dimethylsulfoxide-de) for compound 71 : d 8.50 (d, J = 1 .6 Hz, 1 H), 8.45 (dd, J = 1 .4, 4.7 Hz, 1 H), 7.71 (br. d, J = 7.8 Hz, 1 H), 7.36 (dd, J = 4.8, 7.6 Hz, 1 H), 7.19 (t, J = 9.2 Hz, 1 H), 7.01 (dd, J = 2.9, 6.3 Hz, 1 H), 6.88 (td, J = 3.5, 9.0 Hz, 1 H), 5.40 (d, J = 4.5 Hz, 1 H), 4.45 - 4.30 (m, 1 H), 3.74 - 3.53 (m, 2H), 2.62 - 2.52 (m, 2H), 1 .85 (br. d, J = 11 .5 Hz, 1 H), 1 .64 (br. d, J = 7.2 Hz, 1 H), 1 .40 - 1 .21 (m,

3H); LCMS (ESI) m/z: 321 .0 [M-H]⁺.

¹H NMR (400 MHz, Dimethylsulfoxide-de) for compound 17: d 8.50 (d, J = 1.6 Hz, 1 H), 8.45 (dd, J = 1.5,

4.7 Hz, 1 H), 7.71 (br. d, J = 7.8 Hz, 1 H), 7.36 (dd, J = 4.7, 7.6 Hz, 1 H), 7.19 (t, J = 9.1 Hz, 1 H), 7.03 - 6.99 (m, 1 H), 6.88 (td, J = 3.5, 9.1 Hz, 1 H), 5.39 (d, J = 4.5 Hz, 1 H), 4.37 (dd, J = 4.9, 6.5 Hz, 1 H), 3.73 - 3.54 (m, 2H), 2.61 - 2.51 (m, 2H), 1 .85 (br. d, J = 11 .2 Hz, 1 H), 1 .72 - 1 .56 (m, 1 H), 1 .39 - 1 .21 (m, 3H); LCMS (ESI) m/z: 321 .0 [M+H]⁺. (Rt: 3.26min).

¹H NMR (400 MHz, Dimethylsulfoxide-de) for compound 77: d 8.50 (d, J = 1 .6 Hz, 1 H), 8.45 (dd, J = 1 .5,

4.8 Hz, 1 H), 7.71 (br. d, J = 7.8 Hz, 1 H), 7.36 (dd, J = 4.8, 7.7 Hz, 1 H), 7.19 (t, J = 9.2 Hz, 1 H), 7.01 (dd, J = 2.9, 6.4 Hz, 1 H), 6.93 - 6.81 (m, 1 H), 5.39 (d, J = 4.4 Hz, 1 H), 4.42 - 4.32 (m, 1 H), 3.73 - 3.55 (m, 2H), 2.62 - 2.51 (m, 2H), 1 .89 - 1 .80 (m, 1 H), 1 .72 - 1 .57 (m, 1 H), 1 .40 - 1 .22 (m, 3H); LCMS (ESI) m/z: 321 .0 [M-H]⁺. (Rt: 3.77min).

Example 28: Preparation of 1-(3,4-dichlorophenyl)-4-[hydroxy(3-pyridyl)methyl]piperidin-2-one (Compound 72)

To a solution of [1-(3,4-dichlorophenyl)-4-piperidyl]-(3-pyridyl)methanol (60 mg, 178 pmol) in water (3 mL) was added mercury (II) oxide (39 mg, 178 pmol) and 2-[2-[bis(carboxymethyl)amino]ethyl- (carboxymethyl)amino]acetic acid (52 mg, 178 pmol). The resultant mixture was stirred at 100 °C for 1 hour. LCMS showed the reaction was complete. The reaction mixture was filtered, and the filtrate was extracted with ethyl acetate (10 mL x 2). The combined organic layers were washed with brine (5 mL), dried over sodium sulfate, filtered, and concentrated to dryness to afford the crude product. The crude product was further purified by prep-HPLC (Welch Xtimate C18 150*25 5p column; 30-60 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 minute gradient) To obtiain 1 -(3,4- dichlorophenyl)-4-[hydroxy(3-pyridyl)methyl]piperidin-2-one (10 mg, 27 pmol, 15%) as a pale yellow solid. ¹H NMR (400 MHz, Dimethylsulfoxide-de) d 8.56 (s, 1 H), 8.48 (dd, J = 1 .5, 4.8 Hz, 1 H), 7.81 - 7.71 (m,

1 H), 7.67 - 7.56 (m, 2H), 7.39 (dd, J = 4.8, 7.8 Hz, 1 H), 7.30 (dd, J = 2.3, 8.7 Hz, 1 H), 5.62 (br. s, 1 H), 4.60 - 4.46 (m, 1 H), 3.71 - 3.51 (m, 2H), 2.35 - 2.24 (m, 2H), 2.10 - 1 .99 (m, 1 H), 1 .74 - 1 .57 (m, 2H); LCMS (ESI) m/z: 350.9 [M+H]⁺.

Example 29: Preparation of enantiomer 1 (Compound 76) and enantiomer 2 (Compound 15) of [1- (3,4-dichlorophenyl)-4-piperidyl]-(3-pyridyl)methanol.

Enantiomer 1 Enantiomer 2

Compound 15 and compound 76 were synthesized according to the synthetic procedure reported for the preparation of compounds 17 and 77. The enantiomer 1 product [1-(3,4-dichlorophenyl)-4- piperidyl]-(3-pyridyl)methanol (27 mg, 80 pmol, 11%) was obtained as a white solid. The enantiomer 2 product [1-(3,4-dichlorophenyl)-4-piperidyl]-(3-pyridyl)methanol (42 mg, 122 pmol, 16%) was obtained as a white solid.

¹H NMR (400 MHz, Dimethylsulfoxide-de) for compound 76 d 8.50 (d, J = 1 .7 Hz, 1 H), 8.45 (dd, J = 1 .5, 4.7 Hz, 1 H), 7.70 (br. d, J = 7.8 Hz, 1 H), 7.43 - 7.26 (m, 2H), 7.06 (d, J = 2.8 Hz, 1 H), 6.88 (dd, J = 2.9, 9.0 Hz, 1 H), 5.39 (d, J = 4.5 Hz, 1 H), 4.37 (dd, J = 4.8, 6.5 Hz, 1 H), 3.83 - 3.60 (m, 2H), 2.73 - 2.55 (m, 2H), 1.90 - 1.77 (m, 1 H), 1.75 - 1.60 (m, 1 H), 1.37 - 1.17 (m, 3H); LCMS (ESI) m/z: 335.0 [M-H]⁺. (Rt: 3.44min)

¹H NMR (400 MHz, Dimethylsulfoxide-de) for compound 15: d 8.50 (d, J = 1.7 Hz, 1 H), 8.45 (dd, J = 1.5, 4.6 Hz, 1 H), 7.70 (br. d, J = 7.8 Hz, 1 H), 7.42 - 7.28 (m, 2H), 7.06 (d, J = 2.7 Hz, 1 H), 6.88 (dd, J = 2.8, 9.0 Hz, 1 H), 5.38 (d, J = 4.6 Hz, 1 H), 4.45 - 4.28 (m, 1 H), 3.84 - 3.65 (m, 2H), 2.70 - 2.53 (m, 2H), 1 .88 - 1 .77 (m, 1 H), 1 .75 - 1 .61 (m, 1 H), 1 .37 - 1 .17 (m, 3H); LCMS (ESI) m/z: 335.0 [M-H]⁺. (Rt: 3.79min). Example 30: Preparation of [1-[(3,4-dichlorophenyl)methyl]-4-piperidyl]-(3-pyridyl)methanol (Compound 78).

To a stirred solution of 4-piperidyl(3-pyridyl)methanol (150 mg, 780 pmol) in tetrahydrofuran (2 ml_) was added 3,4-dichlorobenzaldehyde (137 mg, 780 pmol), acetic acid (94 mg, 1 .56 mmol, 89 mI_), and sodium triacetoxyborohydride (331 mg, 1 .56 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 hour and warmed up to 20 °C and stirred further for 1 h. LCMS showed the starting material was consumed completely and desired compound was detected. The resultant crude product was purified directly by prep-HPLC (Waters Xbridge BEH C18 100*30mm*10pm column; 27-57 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 minute gradient) to obtain [1-[(3,4-dichlorophenyl)methyl]-4- piperidyl]-(3-pyridyl)methanol (53 mg, 150 pmol, 19%) as a white solid.

¹H NMR (400 MHz, Chloroform-d) d 8.54 (br. d, J = 2.6 Hz, 2H), 7.66 (br. d, J = 7.9 Hz, 1 H), 7.42 (d, J =

1 .3 Hz, 1 H), 7.37 (d, J = 8.2 Hz, 1 H), 7.32 - 7.28 (m, 1 H), 7.14 (br. d, J = 7.8 Hz, 1 H), 4.48 (br. d, J = 7.2 Hz, 1 H), 3.42 (s, 2H), 2.91 (br. d, J = 10.9 Hz, 1 H), 2.82 (br. d, J = 10.6 Hz, 1 H), 2.07 - 1.83 (m, 4H), 1.60 (br. s, 1 H), 1 .48 - 1 .38 (m, 1 H), 1 .37 - 1 .26 (m, 2H); LCMS (ESI) m/z: 351 .0 [M+H]⁺.

Example 31: Preparation of [1-[(2,3-dichlorophenyl)methyl]-4-piperidyl]-(3-pyridyl)methanol (Compound 80).

To a solution of 4-piperidyl(3-pyridyl)methanol.HCI (100 mg, 437 pmol) and 1-(bromomethyl)-2,3- dichloro-benzene (115 mg, 481 pmol) in dimethylformamide (1 mL) was added triethylamine (221 mg, 2.19 mmol, 304 pL). The mixture was stirred at 20 °C for 2 hours. The reaction mixture was filtered and the resultant product was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10pm; 35-65% acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient). Product [1-[(2,3-dichlorophenyl)methyl]-4-piperidyl]-(3-pyridyl)methanol (90 mg, 252 pmol,

57%) was obtained as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.61 - 8.51 (m, 2H), 7.73 - 7.66 (m, 1 H), 7.45 - 7.29 (m, 3H), 7.23 - 7.14 (m, 1 H), 4.50 (d, J = 7.2 Hz, 1 H), 3.61 (s, 2H), 2.98 (br. d, J = 11 .2 Hz, 1 H), 2.93 - 2.83 (m, 1 H), 2.19 - 1 .90 (m, 4H), 1 .74 - 1 .62 (m, 1 H), 1 .53 - 1 .39 (m, 1 H), 1 .39 - 1.28 (m, 2H); LCMS (ESI) m/z: 351.1 [M+H]⁺. Example 32: Preparation of [1-(4,5-dichlorothiazol-2-yl)-4-piperidyl]-(3-pyridyl)methanol (Compound 81).

Step 1 : Preparation of 2,4,5-trichlorothiazole.

To a solution of 2,4-dichlorothiazole (0.5 g, 3.25 mmol) in chloroform (7 ml_) was added sulfuryl chloride (876 mg, 6.49 mmol, 649 pL) and the resultant mixture was stirred at 70 °C for 3 hours. The reaction mixture was concentrated to dryness to obtain 2,4,5-trichlorothiazole (540 mg, 2.87 mmol, 88%) as a colorless oil. The crude product was used further without purification.

Step 2: Preparation of [1-(4,5-dichlorothiazol-2-yl)-4-piperidyl]-(3-pyridyl)methanol.

To a solution of 2,4,5-trichlorothiazole (0.2 g, 1.06 mmol) in acetonitrile (5 ml_) was added 4- piperidyl(3-pyridyl)methanol (243 mg, 1.06 mmol, hydrochloric acid) and potassium carbonate (587 mg, 4.24 mmol). The mixture was stirred at 20 °C for 16 hours. The crude was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40 10m column; 40-60 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to obtain [1-(4,5-dichlorothiazol-2-yl)-4-piperidyl]-(3-pyridyl)methanol (121 mg, 351 pmol, 33%) as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.59 - 8.48 (m, 2H), 7.68 (br. d, J = 7.9 Hz, 1 H), 7.32 (dd, J = 4.9, 7.7 Hz, 1 H), 4.50 (br. d, J = 7.1 Hz, 1 H), 3.98 - 3.78 (m,

2H), 3.02 - 2.82 (m, 2H), 2.48 (br. s, 1 H), 2.11 - 2.01 (m, 1 H), 1 .86 (dtd, J = 3.7, 7.6, 11 .3 Hz, 1 H), 1 .52 - 1 .30 (m, 3H); LCMS (ESI) m/z: 344.0 [M+H]⁺.

Example 33: Synthesis of (1S)-2-[4-(3,4-dichlorophenoxy)-1-piperidyl]-1-(3-pyridyl)ethanol (

Step 1 : tert-butyl 4-(3,4-dichlorophenoxy)piperidine-1-carboxylate.

To a solution of 3,4-dichlorophenol (1 g, 6.13 mmol) and tert-butyl 4-hydroxypiperidine-1- carboxylate (1.36 g, 6.75 mmol) in THF (30 ml_) was added PPhi3 (3.22 g, 12.27 mmol). Then the mixture was cooled to 0 °C and DIAD (2.48 g, 12.27 mmol) was added drop wise to the above solution and the resultant mixture was stirred at 25 °C for 48 h. The reaction mixture was concentrated to give crude product which was purified by flash column (ISCO 40 g silica, 0-20 % ethyl acetate in petroleum ether, gradient over 20 min) to afford tert-butyl 4-(3,4-dichlorophenoxy)piperidine-1-carboxylate (1.4g) as a colorless oil. Step 2: 4-(3,4-dichlorophenoxy)piperidine.

To a solution of tert-butyl 4-(3,4-dichlorophenoxy)piperidine-1-carboxylate (1.3 g, 3.75 mmol) in EtOAc (10 ml_) was added HCI/EtOAc (4 M, 10 ml_). The mixture was stirred at 25 °C for 2h, filtered and the solids were dried over in vacuo to afford 4-(3,4-dichlorophenoxy)piperidine (0.8 g, 3.25 mmol, 87%) as a white solid.

Step 3: (1 S)-2-[4-(3,4-dichlorophenoxy)-1 -piperidyl]-1 -(3-pyridyl)ethanol.

To a solution of 4-(3,4-dichlorophenoxy)piperidine (0.25 g, 1.02 mmol) in n-BuOH (5 ml_) was added Et3N (123 mg, 1.22 mmol) and then (1S)-2-bromo-1-(3-pyridyl)ethanol (205 mg, 1.02 mmol) was added to the mixture. The resultant mixture was stirred at 120 °C for 12h and concentrated. The crude product was purified by prep-HPLC (Welch Xtimate C18 250*50mm*10um column; 30-55 % acetonitrile in a 10mM ammonium bicarbonate solution in water, 10 min gradient) (neutral) to obtain (1 S)-2-[4-(3,4- dichlorophenoxy)-1-piperidyl]-1-(3-pyridyl)ethanol (79 mg, 216 umol, 11%) as a brown solid. An additional regioisomer was also isolated during this step.

¹H NMR (400 MHz, CHLOROFORM-d) d 8.63 - 8.58 (m, 1 H), 8.57 - 8.51 (m, 1 H), 7.78 - 7.72 (m, 1 H),

7.35 - 7.28 (m, 2H), 7.02 (d, J = 2.8 Hz, 1 H), 6.77 (dd, J = 2.8, 8.8 Hz, 1 H), 4.83 - 4.74 (m, 1 H), 4.35 (tt, J = 3.4, 7.0 Hz, 1 H), 4.08 (s, 1 H), 3.07 - 2.95 (m, 1 H), 2.77 - 2.64 (m, 2H), 2.63 - 2.56 (m, 1 H), 2.52 - 2.44 (m, 1 H), 2.39 (ddd, J = 3.3, 8.1 , 11 .4 Hz, 1 H), 2.10 - 1 .96 (m, 2H), 1 .95 - 1 .81 (m, 2H). LCMS (ESI) for (C18H20CI2N2O) [M+H]+: 367.0.

Example 34: Preparation of pyridin-3-yl(1-(6-(trifluoromethyl)pyridin-3-yl)pyrrolidin-3-yl)methanol (Compound 84) and separation of stereoisomer 1 (Compound 124) and stereoisomer 2 (Compound 28).

stereoisomer 1 stereoisomer 2

To a solution of 2,2’-bis(diphenylphosphino)-1 ,T-binapthalene (140 mg, 224.43 pmol) in dioxane (10 ml_) was added palladium (II) acetate (50 mg, 24 pmol) and cesium carbonate (1.46 g, 4.49 mmol). The reaction mixture was degassed with nitrogen three times, and the mixture was stirred at 20 °C for 1 hour. Then, 3-pyridyl(pyrrolidin-3-yl)methanol (400 mg, 2.24 mmol) and 5-bromo-2 - trifluoromethyl)pyridine (507 mg, 2.24 mmol) were added to the reaction, the mixture was heated to 105 °C and stirred for 15 hours The reaction mixture was concentrated in vacuum to obtain the compound 84. This was further subjected to prep-HPLC ( Waters Xbridge BEH C18 100*25mm*5pm column; 28- 40% acetonitrile in a 10mM ammonium bicarbonate solution in water, 8 minute gradient)(neutral) to obtain stereoisomer 1 of pyridin-3-yl(1-(6-(trifluoromethyl)pyridin-3-yl)pyrrolidin-3-yl)methanol (65 mg, 199 pmol, 9%) was obtained as a pale yellow gum and the stereoisomer 2 (52 mg, 160 pmol, 7%) was obtained as a white solid.

¹H NMR (400 MHz, Chloroform-d) for compound 124: d 8.62 - 8.50 (m, 2H), 7.93 - 7.87 (m, 1 H), 7.76 (td, J = 1 .8, 7.8 Hz, 1 H), 7.47 - 7.40 (m, 1 H), 7.32 (dd, J = 4.8, 7.8 Hz, 1 H), 6.73 (dd, J = 2.4, 8.6 Hz, 1 H),

4.74 (d, J = 7.2 Hz, 1H), 3.55 - 3.31 (m, 2H), 3.26 - 3.08 (m, 2H), 2.78 (s, 1 H), 2.34 - 2.22 (m, 1 H), 2.16 (br. d, J = 8.8 Hz, 1 H); LCMS (ESI) m/z: 324.0 [M+H]⁺.

¹H NMR (400 MHz, Chloroform-d) for compound 28: d 8.63 - 8.52 (m, 2H), 8.02 - 7.96 (m, 1 H), 7.82 -

7.75 (m, 1 H), 7.52 - 7.45 (m, 1 H), 7.39 - 7.32 (m, 1 H), 6.87 - 6.80 (m, 1 H), 4.67 (d, J = 8.6 Hz, 1 H), 3.64 (dd, J = 7.6, 9.6 Hz, 1 H), 3.53 - 3.41 (m, 2H), 3.33 (d, J = 8.2 Hz, 1 H), 3.08 - 2.91 (m, 1 H), 2.75 (br. d, J = 7.6 Hz, 1 H), 1.91 - 1.73 (m, 2H); LCMS (ESI) m/z: 324.0 [M+H]⁺.

Example 35: Preparation of (1-benzyl-4-piperidyl)-(3-pyridyl)methanol (Compound 85).

To a solution of 4-piperidyl(3-pyridyl)methanol.HCI (300 mg, 1.13 mmol) in dimethylformamide (3 ml_) was added bromomethylbenzene (213 mg, 1 .24 mmol) and triethylamine (572 mg, 5.66 mmol, 787 mI_). The mixture was stirred at 25 °C for 3 hours. The reaction mixture was filtered, concentrated and was purified by prep-HPLC (column: Welch Xtimate C18 150*30mm*5pm; 25-50% acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to afford (1-benzyl-4-piperidyl)-(3- pyridyl)methanol (121 mg, 420 pmol, 37%) as a pink solid. ¹H NMR (400 MHz, Methanol-cM) d 8.59 - 8.34 (m, 2H), 7.80 (br. d, J = 7.8 Hz, 1 H), 7.53 - 7.16 (m, 6H), 4.42 (br. d, J = 7.2 Hz, 1 H), 3.49 (s, 2H), 3.07 - 2.77 (m, 2H), 2.14 - 1 .83 (m, 3H), 1 .72 - 1 .54 (m, 1 H), 1.51 - 1.18 (m, 3H); LCMS (ESI) m/z: 283.2 [M+H]⁺.

Example 36: Preparation of 2-[1-(3,4-dichlorophenyl)azetidin-3-yl]-1-(3-pyridyl)ethanol (Compound 86) and chiral separation into enantiomer 1 (compound 31) and enantiomer 2 (compound 32).

Step 1 : Preparation of methyl 2-[1-(3,4-dichlorophenyl)azetidin-3-yl]acetate.

To a solution of 1 ,2-dichloro-4-iodo-benzene (1 .68 g, 6.17 mmol) and methyl 2-(azetidin-3- yl)acetate.TFA (1.8 g, 7.40 mmol) in dimethylformamide (20 ml_) were added cesium carbonate (6.03 g, 18.50 mmol), bis(dibenzylideneacetone)palladium(0) (142 mg, 247 pmol), and 4,5- bis(diphenylphosphino)-9,9-dimethylxanthene (357 mg, 617 pmol). The mixture was stirred at 120 °C for 2 hours.. The resultant reaction mixture was partitioned between ethyl acetate (100 ml_) and water (100 ml_), then extracted with ethyl acetate (50 ml_ * 2). The organic phase was separated, washed with brine (100 ml_), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by ISCO column chromatography (10 g silica, 0-40% ethyl acetate in petroleum ether, gradient over 20 minutes) to afford methyl 2-[1 -(3,4- dichlorophenyl)azetidin-3-yl]acetate (1.2 g, 4.38 mmol, 71%) as a red oil. ¹H NMR (400 MHz, Chloroforme d 7.22 (d, J = 8.6 Hz, 1 H), 6.47 (d, J = 2.3 Hz, 1 H), 6.30 - 6.18 (m, 1 H), 4.10 - 3.98 (m, 2H), 3.71 (s, 3H), 3.55 (br t, J = 6.3 Hz, 2H), 3.17 - 3.02 (m, 1H), 2.71 (br. d, J = 7.7 Hz, 2H); LCMS (ESI) m/z: 274.0 [M+H]⁺.

Step 2: Preparation of 2-[1-(3,4-dichlorophenyl)azetidin-3-yl]acetaldehyde.

To a solution of methyl 2-[1-(3,4-dichlorophenyl)azetidin-3-yl]acetate (1.1 g, 4.01 mmol) in dichloromethane (50 ml_) was added diisobutylalumminum hydride (1 M, 12 ml_). The mixture was stirred at -78 °C for 1 hour And reaction mixture was quenched by addition water (20 ml_) at 0 °C, and then extracted with ethyl acetate (20 ml_ x 2). The combined organic layers were washed with brine (10 ml_), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product was purified by ISCO column chromatography (40 g silica, 0-20% ethyl acetate in petroleum ether, gradient over 20 minutes). The product 2-[1 -(3, 4-dichlorophenyl)azetidin-3-yl]acetaldehyde (0.8 g, 3.28 mmol, 82%) was obtained as a yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 9.82 (s, 1 H), 7.22 (d, J = 8.8 Hz, 1 H), 6.46 (d, J = 2.6 Hz, 1 H), 6.27 - 6.20 (m, 1 H), 4.15 - 4.03 (m, 2H), 3.56 - 3.46 (m, 2H), 3.21 - 3.06 (m, 1 H), 2.95 - 2.87 (m, 2H).

Step 3: Preparation of 2-[1-(3,4-dichlorophenyl)azetidin-3-yl]-1-(3-pyridyl)ethanol and chiral separation into enantiomer 1 and enantiomer 2.

To a solution of 3-iodopyridine (1 g, 4.92 mmol) in tetrahydrofuran (2 ml_) was added a solution of isopropylmagnesium chloride (2 M, 2.46 ml_) dropwise at 0 °C. The mixture was stirred at 25 °C for 1 hour. Then the mixture was cooled to 0 °C and in was added 2-[1-(3,4-dichlorophenyl)azetidin-3- yljacetaldehyde (600 mg, 2.46 mmol). The mixture was stirred at 25 °C for 2 hours and was quenched by addition water (20 ml_) at 0 °C, and then extracted with ethyl acetate (20 ml_ x 2). The combined organic layers were washed with brine (10 ml_), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude residue was purified by prep-HPLC (column: Kromasil C18 (250*50mm*10 pm); 40-65% acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 minute gradient). The product 2-[1 -(3,4-dichlorophenyl)azetidin-3-yl]-1-(3- pyridyl)ethanol (130 mg, 402 pmol, 16%) was obtained as a pale yellow gum. A portion of the compound 2-[1-(3,4-dichlorophenyl)azetidin-3-yl]-1-(3-pyridyl)ethanol was subjected to chiral purification using SFC (column: DAICEL CHIRALPAK IC(250mm*30mm,10pm); mobile phase: [0.1 % ammonium:isopropyl alcohol]; B%: 45%-45%, 6 minutes) to afford enantiomers 1 and 2.

¹H NMR (400 MHz, Chloroform-d) for compound 86 d 8.59 - 8.50 (m, 2H), 7.76 - 7.68 (m, 1 H), 7.35 - 7.29 (m, 1 H), 7.20 (d, J = 8.8 Hz, 1 H), 6.44 (d, J = 2.6 Hz, 1 H), 6.25 - 6.18 (m, 1 H), 4.86 - 4.77 (m, 1 H), 4.03 - 3.89 (m, 2H), 3.56 - 3.47 (m, 1 H), 3.47 - 3.39 (m, 1 H), 2.98 - 2.84 (m, 1 H), 2.32 - 2.12 (m, 2H), 2.12 - 2.00 (m, 1 H); LCMS (ESI) m/z: 323.0 [M+H]⁺.

¹H NMR (400 MHz, Chloroform-d) for compound 31 d 8.68 - 8.45 (m, 2H), 7.82 - 7.63 (m, 1 H), 7.37 - 7.29 (m, 1 H), 7.24 - 7.16 (m, 1 H), 6.44 (d, J = 2.6 Hz, 1 H), 6.28 - 6.17 (m, 1 H), 4.92 - 4.73 (m, 1 H), 4.09 - 3.86 (m, 2H), 3.62 - 3.37 (m, 2H), 3.00 - 2.82 (m, 1 H), 2.29 - 2.12 (m, 2H), 2.1 1 - 1 .97 (m, 1 H); LCMS (ESI) m/z: 323.0 [M+H]⁺.

¹H NMR (400 MHz, Chloroform-d) for compound 32 d 8.66 - 8.44 (m, 2H), 7.82 - 7.63 (m, 1 H), 7.36 - 7.29 (m, 1 H), 7.20 (d, J = 8.6 Hz, 1 H), 6.48 - 6.40 (m, 1 H), 6.27 - 6.17 (m, 1 H), 4.88 - 4.73 (m, 1 H), 4.09 - 3.86 (m, 2H), 3.59 - 3.37 (m, 2H), 2.99 - 2.83 (m, 1 H), 2.51 - 2.26 (m, 1 H), 2.25 - 2.12 (m, 1 H), 2.11 - 1 .98 (m, 1 H); LCMS (ESI) m/z: 323.0 [M+H]⁺.

Example 37: Preparation of [1-(3,4-dichlorophenyl)pyrrolidin-3-yl]-pyridazin-3-yl-methanol (Compound 87).

s_{2 3}, oxane 120 °C, 15 h

Step 1 : Preparation of fe/f-butyl 3-[hydroxy(pyridazin-3-yl)methyl]pyrrolidine-1-carboxylate.

To a solution of 2,2,6, 6-tetramethylpiperidine (1 .06 g, 7.53 mmol, 1.28 ml_) in tetrahydrofuran (20 ml_) was added n-butyllithium (2.5 M, 3.01 ml_) dropwise at -30 °C. The mixture was stirred at 0 °C for 30 minutes. Then, fe/f-butyl 3-formylpyrrolidine-1-carboxylate (1 g, 5.02 mmol) in tetrahydrofuran (2 ml_) and pyridazine (442 mg, 5.52 mmol, 398 pL) in tetrahydrofuran (2 ml_) were added simultaneously to a cold solution of lithium tetramethylpiperidide at -70 °C. Then the mixture was stirred at -70 °C for 4 hours. To the mixture was added water (10 ml_), and the mixture was extracted with ethyl acetate (20 ml_ x 6). The organic layer was washed with brine (20 ml_), dried over sodium sulfate, filtered, and concentrated to give crude product. The crude product was purified by ISCO column chromatography (20 g silica, 80-100 % ethyl acetate in petroleum ether, gradient over 20 minutes). Product fe/f-butyl 3-[hydroxy(pyridazin-3- yl)methyl]pyrrolidine-1-carboxylate (600 mg, 2.15 mmol, 43%) was obtained as a brown oil. LCMS (ESI) m/z: 280.2 [M+H]⁺.

Step 2: Preparation of pyridazin-3-yl(pyrrolidin-3-yl)methanol.

A mixture of fe/f-butyl 3-[hydroxy(pyridazin-3-yl)methyl]pyrrolidine-1-carboxylate (600 mg, 2.15 mmol) in ethyl acetate (5 ml_) was added hydrochloric acid/ethyl acetate (2 ml_). The mixture was stirred at 25 °C for 30 minutes. The mixture was concentrated to give crude product which was carried onto the next step without purification.

Step 3: Preparation of [1-(3,4-dichlorophenyl)pyrrolidin-3-yl]-pyridazin-3-yl-methanol.

To a solution of pyridazin-3-yl(pyrrolidin-3-yl)methanol (330 mg, 1.53 mmol, hydrochloric acid) in dimethylformamide (6 ml_) was added 1 ,2-dichloro-4-iodo-benzene (459 mg, 1.68 mmol), cesium carbonate (997 mg, 3.06 mmol), bis(dibenzylideneacetone)palladium(0) (88 mg, 153 pmol), and 4,5- bis(diphenylphosphino)-9,9-dimethylxanthene (89 mg, 153 pmol). The mixture was stirred at 120 °C for 15 hours under nitrogen. Then to the mixture was added water (10 ml_), and the mixture was extracted with ethyl acetate (20 ml_ x 4). The organic layer was washed with brine (10 ml_), dried over sodium sulfate, filtered, and concentrated to give crude product. The crude product was purified by prep-HPLC (Kromasil C18 (250*50mm*10 pm) column; 35-60 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 minute gradient) to afford[1-(3,4-dichlorophenyl)pyrrolidin-3-yl]-pyridazin-3-yl- methanol (56 mg, 173 pmol, 11%) as a yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 9.20 (dt, J = 1.8, 4.4 Hz, 1 H), 7.63 - 7.51 (m, 2H), 7.22 (d, J = 8.8 Hz, 1 H), 6.59 (t, J = 2.4 Hz, 1 H), 6.39 - 6.33 (m, 1 H),

5.11 - 4.96 (m, 1 H), 3.91 - 3.67 (m, 1 H), 3.45 - 3.20 (m, 4H), 2.93 - 2.77 (m, 1 H), 2.19 - 1.96 (m, 2H) LCMS (ESI) m/z: 324.0 [M+H]⁺.

Example 38: Preparation of [6-[hydroxy(3-pyridyl)methyl]-2-azaspiro[3.3]heptan-2-yl]-[6- (trifluoromethyl)-3-pyridyl]methanone (Compound 91)

To a solution of 6-(trifluoromethyl)pyridine-3-carboxylic acid (66 mg, 343 pmol) in dimethylformamide (2 mL) was added 2-(1 H-benzotriazole-1-yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate (156 mg, 411 pmol), A/,A/-diisopropylethylamine (133 mg, 1.03 mmol, 179 pL), and 2-azaspiro[3.3]heptan-6-yl(3-pyridyl)methanol (70 mg, 343 pmol). The mixture was stirred at 25 °C for 2 hours. The reaction mixture was concentrated in vacuum and the residue was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40mm*10pm column; 15-45 % acetonitrile in a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to obtain [6-[hydroxy(3-pyridyl)methyl]-2- azaspiro[3.3]heptan-2-yl]-[6-(trifluoromethyl)-3-pyridyl]methanone (11 mg, 29 pmol, 8%) as a yellow gum. ¹H NMR (400 MHz, Methanok/4) d 8.94 (s, 1 H), 8.50 (br. s, 1H), 8.45 - 8.39 (m, 1 H), 8.26 (br. d, J = 8.2 Hz, 1 H), 7.95 - 7.87 (m, 1 H), 7.85 - 7.78 (m, 1 H), 7.46 - 7.37 (m, 1 H), 4.58 (t, J = 7.4 Hz, 1 H), 4.46 - 4.36 (m, 1 H), 4.35 - 4.27 (m, 1 H), 4.25 - 4.17 (m, 1 H), 4.16 - 4.07 (m, 1 H), 2.59 - 2.40 (m, 1 H), 2.35 - 2.24 (m, 2H), 2.21 - 2.09 (m, 2H); LCMS (ESI) m/z: 378.1 [M+H]⁺.

Example 39: Preparation of 6-(3,4-dichlorophenyl)-2-(3-pyridylsulfonyl)-2,6-diazaspiro[3.3]heptane (Compound 94)

Step 1 : Preparation of fe/f-butyl 6-(3,4-dichlorophenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate.

To a solution of fe/f-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (500 mg, 2.52 mmol) in dioxane (5 ml_) was added 1 ,2-dichloro-4-iodo-benzene (688 mg, 2.52 mmol), sodium fe/f-butoxide (727 mg, 7.57 mmol) , tris(dibenzylideneacetone)dipalladium(0) (115 mg, 126 pmol), and 2- dicyclohexylphosphino^'.e'-diisopropoxybiphenyl (24 mg, 50 pmol). The mixture was stirred at 100 °C for 20 minutes under nitrogen. Water (10 ml_) was added to the reaction, and the reaction mixture was extracted with ethyl acetate (20 ml_ x 2). The combined organic layers were washed with brine (10 ml_), dried over sodium sulfate, filtered, and concentrated to dryness to give the crude product which was purified by ISCO column chromatography (10 g silica, 0-10 % ethyl acetate in petroleum ether, gradient over 20 minutes). The product fe/f-butyl 6-(3,4-dichlorophenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (0.36 g, 1.05 mmol, 42%) was obtained as a white solid. ¹H NMR (400 MHz, Chloroform-d) d 7.23 (d, J = 8.6 Hz, 1 H), 6.49 (d, J = 2.6 Hz, 1 H), 6.27 (dd, J = 2.8, 8.7 Hz, 1 H), 4.10 (s, 4H), 3.96 (s, 4H), 1.46 (s,

9H).

Step 2: Preparation of 2-(3,4-dichlorophenyl)-2,6-diazaspiro[3.3]heptane.

To a solution of fe/f-butyl 6-(3,4-dichlorophenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (200 mg, 583 pmol) in dichloromethane (4 ml_) was added trifluoroacetic acid (930 mg, 8.16 mmol, 604 pl_). The mixture was stirred at 20 °C for 1 hour. The reaction mixture was concentrated to dryness to give the crude product. Product 2-(3,4-dichlorophenyl)-2,6-diazaspiro[3.3]heptane (0.42 g, crude, trifluoroacetic acid) was obtained as a brown solid. LCMS (ESI) m/z: 242.9 [M+H]⁺.

Step 3: Preparation of 6-(3,4-dichlorophenyl)-2-(3-pyridylsulfonyl)-2,6-diazaspiro[3.3]heptane.

To a solution of 2-(3,4-dichlorophenyl)-2,6-diazaspiro[3.3]heptane (200 mg, 560 pmol, trifluoroacetic acid) in dichloromethane (5 ml_) was added pyridine-3-sulfonyl chloride (199 mg, 1.12 mmol) and triethylamine(227 mg, 2.24 mmol). The mixture was stirred at 20 °C for 1 hour andconcentrated to dryness to give the crude product. The crude product was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40 10mM column; 40-70 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient). The product 6-(3,4-dichlorophenyl)-2-(3-pyridylsulfonyl)- 2,6-diazaspiro[3.3]heptane (56 mg, 147 pmol, 26%) was obtained as a gray solid. ¹H NMR (400 MHz, Chloroform-d) d 9.09 (s, 1 H), 8.91 (d, J = 4.8 Hz, 1 H), 8.15 (br. d, J = 7.9 Hz, 1 H), 7.56 (dd, J = 4.9, 7.9 Hz, 1 H), 7.22 (d, J = 8.7 Hz, 1 H), 6.43 (d, J = 2.3 Hz, 1 H), 6.21 (dd, J = 2.4, 8.7 Hz, 1 H), 4.02 (s, 4H),

3.86 (s, 4H); LCMS (ESI) m/z: 384.1 [M+H]⁺.

Example 40: Preparation of pyridazin-3-yl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan- 6-yl]methanol (Compound 95).

Step 1 : Preparation of fe/f-butyl 6-[hydroxy(pyridazin-3-yl)methyl]-2-azaspiro[3.3]heptane-2-carboxylate.

To a solution of 2,2,6, 6-tetramethylpiperidine (941 mg, 6.66 mmol, 1 .13 ml_) in tetrahydrofuran (15 ml_) was added dropwise n-butyllithium (2.5 M, 2.66 ml_) at -30 °C. Then the mixture was warmed up to 0 °C and stirred for 30 minutes. The mixture was then cooled to -70 °C followed by the addition of pyridazine (391 mg, 4.88 mmol, 352 pL) in tetrahydrofuran (1 ml_), fe/f-butyl 6-formyl-2- azaspiro[3.3]heptane-2-carboxylate (1 g, 4.44 mmol), and 2,2,6,6-tetramethylpiperidine (941 mg, 6.66 mmol, 1.13 ml_) in tetrahydrofuran (3 ml_). The mixture was stirred at -70 °C for 2 hours. The mixture was then quenched with water (10 ml_) and extracted with ethyl acetate (20 ml_ x 5). The organic layer was washed with brine (10 ml_), dried over sodium sulfate, filtered, and concentrated to give crude product. The crude product was purified by ISCO column chromatography (20 g silica, 90-100 % ethyl acetate in petroleum ether, gradient over 10 minutes) to affordfe/f-butyl 6-[hydroxy(pyridazin-3-yl)methyl]-2- azaspiro[3.3]heptane-2-carboxylate (0.6 g, 1.96 mmol, 44%) as a yellow oil. LCMS (ESI) m/z: 306.2 [M+H]⁺.

Step 2: Preparation of 2-azaspiro[3.3]heptan-6-yl(pyridazin-3-yl)methanol.

To a solution of fe/f-butyl 6-[hydroxy(pyridazin-3-yl)methyl]-2-azaspiro[3.3]heptane-2-carboxylate (0.6 g, 1 .96 mmol) in dichloromethane (8 ml_) was added trifluoroacetic acid (1.57 g, 13.75 mmol, 1.02 ml_). The mixture was stirred at 20 °C for 1 hour. LCMS showed the reaction was complete. The reaction mixture was concentrated to dryness to give the crude product 2-azaspiro[3.3]heptan-6-yl(pyridazin-3- yl)methanol (0.8 g, crude, trifluoroacetic acid salt) as a brown oil which was used further without purification. LCMS (ESI) m/z: 206.2 [M+H]⁺.

Step 3: Preparation of pyridazin-3-yl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6- yl]methanone.

To a solution of 2-azaspiro[3.3]heptan-6-yl(pyridazin-3-yl)methanol. (0.2 g, 626 pmol, trifluoroacetic acid salt) in dioxane (2 mL) were added 5-bromo-2-(trifluoromethyl)pyridine (142 mg, 626 pmol), sodium fe/f-butoxide (181 mg, 1.88 mmol), tris(dibenzylideneacetone)dipalladium(0) (29 mg, 31 pmol), and 2-dicyclohexylphosphino-2^,,6'-diisopropoxybiphenyl (6 mg, 13 pmol). The mixture was stirred at 100 °C for 20 minutes. LCMS showed the reaction was complete. Water (15 mL) was added to the reaction, and the reaction mixture was extracted with ethyl acetate (30 mL x2). The combined organic layers were washed with brine (15 mL), dried over sodium sulfate, filtered, and concentrated to give crude product. The crude product was purified by prep-HPLC (Phenomenex Gemini-NX 150*30 5p column; 16- 46 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to afford pyridazin-3-yl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]methanone (10 mg, 29 pmol,

5%) as a pale yellow solid. LCMS (ESI) m/z: 349.0 [M+H]⁺.

Step 4: Preparation of pyridazin-3-yl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]methanol.

To a solution of pyridazin-3-yl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6- yl]methanone (10 mg, 29 pmol) in methanol (2 mL) was added sodium borohydride (2 mg, 57 pmol). The mixture was stirred at 20 °C for 30 minutes. LCMS showed the reaction was complete. The reaction mixture was concentrated to dryness to give the crude product. The crude was purified by prep-HPLC (Phenomenex Gemini-NX 150*30 5p column; 15-45 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 11 minute gradient) to affordpyridazin-3-yl-[2-[6-(trifluoromethyl)-3-pyridyl]-2- azaspiro[3.3]heptan-6-yl]methanol (2 mg, 7 pmol, 23%) as a white solid. ¹H NMR (400 MHz, Chloroformed) d 9.20 - 9.12 (m, 1H), 7.83 (d, J = 2.7 Hz, 1 H), 7.59 - 7.39 (m, 3H), 6.69 (dd, J = 2.5, 8.5 Hz, 1H), 4.93 (t, J = 4.8 Hz, 1 H), 4.03 - 3.89 (m, 5H), 2.74 - 2.63 (m, 1 H), 2.48 (dd, J = 7.9, 11.6 Hz, 1 H), 2.39 - 2.25 (m, 2H), 2.16 - 2.06 (m, 1 H); LCMS (ESI) m/z: 351 .1 [M+H]⁺. Example 41: Preparation of 3-pyridyl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.5]nonan-7- yl]methanol (Compound 97).

Step 1 : Preparation of fe/f-butyl 7-[hydroxy(3-pyridyl)methyl]-2-azaspiro[3.5]nonane-2-carboxylate.

To a solution of 3-iodopyridine (947 mg, 4.62 mmol) in tetrahydrofuran (15 ml_) was added isopropylmagnesium chloride (2 M, 2.31 ml_) in tetrahydrofuran dropwise by syringe at 0 °C. The mixture was stirred at 0°C for 30 minutes. To the reaction was added fe/f-butyl 7-formyl-2-azaspiro[3.5]nonane-2- carboxylate (780 mg, 3.08 mmol) 0 °C. The solution was stirred at 20 °C for 1.5 hours. Saturated ammonium chloride solution (5 ml_) was added to the reaction, and the reaction mixture was extracted with Ethyl acetate (30 ml_ x 2). The combined organic layers were washed with brine (20 ml_), dried over sodium sulfate, filtered, and concentrated to to obtin the crude product which was purified by ISCO column chromatography (10 g silica, 0-5 % methanol in dichloromethane, gradient over 20 min). The product fe/f-butyl 7-[hydroxy(3-pyridyl)methyl]-2-azaspiro[3.5]nonane-2-carboxylate (0.8 g, 2.41 mmol, 78%) was obtained as a white solid. ¹H NMR (400 MHz, Chloroform-d) d 8.53 - 8.47 (m, 2H), 7.66 (br. d, J = 7.7 Hz, 1 H), 7.31 - 7.28 (m, 1 H), 4.42 (d, J = 7.1 Hz, 1 H), 3.60 - 3.49 (m, 4H), 2.55-2.47 (m, 1 H), 1 .98 - 1.82 (m, 3H), 1.63 - 1.52 (m, 1 H), 1.43 (s, 9H), 1 .41 - 1.30 (m, 3H), 1.15 - 0.93 (m, 2H).

Step 2: Preparation of 2-azaspiro[3.5]nonan-7-yl(3-pyridyl)methanol.

A mixture of fe/f-butyl 7-[hydroxy(3-pyridyl)methyl]-2-azaspiro[3.5]nonane-2-carboxylate (0.4 g,

1.20 mmol) in hydrochloric acid/methanol (4M, 4 ml_) was stirred at 20 °C for 1 hour The reaction mixture was concentrated to dryness to give the crude product. Product 2-azaspiro[3.5]nonan-7-yl(3- pyridyl)methanol (380 mg, crude, hydrochloric acid) was obtained as a pale yellow gum. LCMS (ESI) m/z: 233.1 [M+H]⁺.

Step 3: Preparation of 3-pyridyl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.5]nonan-7-yl]methanol.

To a solution of 1 ,8-diazabicyclo[5.4.0]undec-7-ene (157 mg, 1.03 mmol, 156 pL) in dimethylsulfoxide (1.5 ml_) was added 2-azaspiro[3.5]nonan-7-yl(3-pyridyl)methanol (120 mg, 517 pmol) and 5-fluoro-2-(trifluoromethyl)pyridine (128 mg, 775 pmol). The mixture was stirred at 80 °C for 2 hours. The reaction solution was filtered, and the filtrate was purified directly using prep-HPLC (Phenomenex Gemini-NX 150*30 5pM column; 20%-50% acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient).to afford 3-pyridyl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.5]nonan-7- yl]methanol (77 mg, 203 pmol, 39%) as a white solid. ¹H NMR (400 MHz, Dimethylsulfoxide-cf6) d 8.48 (d, J = 1 .8 Hz, 1 H), 8.44 (dd, J = 1.5, 4.8 Hz, 1 H), 7.86 (d, J = 2.6 Hz, 1 H), 7.68 (br. d, J = 7.9 Hz, 1 H), 7.56 (d, J = 8.7 Hz, 1H), 7.34 (dd, J = 4.8, 7.8 Hz, 1 H), 6.86 (dd, J = 2.5, 8.5 Hz, 1 H), 5.32 (d, J = 4.5 Hz, 1 H), 4.37 - 4.30 (m, 1 H), 3.71 - 3.55 (m, 4H), 1.98 - 1.71 (m, 3H), 1.57 - 1.25 (m, 4H), 1.14 - 0.98 (m, 2H);

LCMS (ESI) m/z: 378.2 [M+H]⁺.

The following compound was synthesized according to the protocol described for the Compound

97.

Example 42: Synthesis of N-(pyridin-3-yl)-2-(6-(trifluoromethyl)pyridin-3-yl)-2-azaspiro[3.3]heptan- 6-amine (Compound 98):

Step 1 : preparation of tert-butyl 6-(pyridin-3-ylamino)-2-azaspiro[3.3]heptane-2-carboxylate.

To a solution of 3-iodopyridine (386 mg, 1.88 mmol) in dioxane (4 ml_) were added tert-butyl 6- amino-2-azaspiro[3.3]heptane-2-carboxylate (400 mg, 1.88 mmol), t-BuONa (543 mg, 5.65 mmol, 3 eq), Pd₂(dba)3 (86 mg, 94 umol, 0.05 eq) and RuPhos (18 mg, 38 umol), then the mixture was stirred at 100 °C for 20 min under N2. The reaction mixture was diluted with 2 ml_ H2O and extracted with EtOAc(5 ml_ *3). The combined organic phase was dried over anhydrous Na₂SC>₄ and concentrated under reduced pressure. The crude product was purified by flash column (ISCO 40 g silica, 67-80% ethyl acetate in petroleum ether, gradient over 20 min) to obtain tert-butyl 6-(3-pyridylamino)-2-azaspiro[3.3]heptane-2-carboxylate (630 mg, crude) was obtained as a red solid.

¹H NMR (400MHz, CHLOROFORM-d) d 8.32 (dd, J=1 .2, 4.6 Hz, 1 H), 8.05 (d, J=2.3 Hz, 1 H), 7.90 (d, J=4.4 Hz, 1H), 7.24 - 7.12 (m, 1 H), 6.99 - 6.91 (m, 1 H), 4.11 (q, J=7.2 Hz, 1 H), 3.93 - 3.78 (m, 2H), 2.75 - 2.52 (m, 2H), 2.16 - 1 .97 (m, 3H), 1 .46 - 1 .35 (m, 9H), 1 .25 (t, J=7.2 Hz, 1 H). LCMS (ESI) m/z: 290.2

[M+H]⁺.

Step 2: preparation of N-(pyridin-3-yl)-2-azaspiro[3.3]heptan-6-amine.

To a solution of tert-butyl 6-(3-pyridylamino)-2-azaspiro[3.3]heptane-2-carboxylate (600 mg, 2.07 mmol) in DCM (6 ml_), was added TFA (9.46 g, 82.94 mmol) and the resultant mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure to obtain N-(3-pyridyl)-2- azaspiro[3.3]heptan-6-amine (830 mg, crude, TFA) as a brown oil. LCMS (ESI) m/z: 190.3 [M+H]⁺.

Step 3: preparation of N-(pyridin-3-yl)-2-(6-(trifluoromethyl)pyridin-3-yl)-2-azaspiro[3.3]heptan-6-amine.

To a solution of DBU (322 mg, 2.11 mmol) in DMSO (3 mL), were added 5-fluoro-2-(trifluoromethyl)pyridine (174 mg, 1.06 mmol) and N-(3-pyridyl)-2-azaspiro[3.3]heptan-6-amine (200 mg, 1.06 mmol) and then the mixture was stirred at 80 °C for 2 h. The reaction mixture was filtered and the filtrate was concentrated under vacuum. The crude product was purified by prep-HPLC ( Phenomenex Luna C18 200*40mm*10um column; 20-50% acetonitrile in an a 0.2% formic acid solution in water, 8 min gradient) to give N-(3-pyridyl)- 2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-amine (29 mg, 77 umol, 7% FA) as a pale yellow solid.

¹H NMR (400MHz, METHANOL-d4) d 7.94 - 7.72 (m, 3H), 7.54 (d, J=8.6 Hz, 1 H), 7.18 (dd, J=4.8, 8.3 Hz, 1 H), 7.01 (br d, J=8.6 Hz, 1 H), 6.91 (br d, J=8.4 Hz, 1 H), 4.11 (s, 2H), 3.99 (s, 2H), 3.88 (quin, J=7.4 Hz, 1 H), 2.78 (ddd, J=2.5, 7.4, 10.1 Hz, 2H), 2.23 - 2.09 (m, 2H). LCMS (ESI for C17H17F3N4 [M+H]⁺: 335.1.

Example 43: Preparation of 2-(3,4-dichlorophenyl)-6-(3-pyridylsulfonyl)-2-azaspiro[3.3]heptane (Compound 99).

1.) Na₂S0 NaHC0 H₂0

, 100 °C, 30 m

Step 1 : Preparation of fe/f-butyl 6-iodo-2-azaspiro[3.3]heptane-2-carboxylate.

A solution of sodium sulfite (936 mg, 7.43 mmol) and sodium bicarbonate (624 mg, 7.43 mmol) in water (5 mL) was heated to 75 °C, and pyridine-3-sulfonyl chloride (594 mg, 3.34 mmol) was added dropwise. The reaction mixture was stirred at 75 °C for 1 h. The mixture was concentrated in vacuo.

Dimethylformamide (5 ml_), sodium bicarbonate (624 mg, 7.43 mmol, 289 pL), and fe/f-butyl 6-iodo-2- azaspiro[3.3]heptane-2-carboxylate (1.20 g, 3.71 mmol) were added to the residue. The reaction mixture was stirred at 75 °C for 2 hours. The mixture was filtered and washed with dimethylformamide (1 ml_) to give filtrate. The crude product was purified by prep-HPLC (Kromasil C18 (250*50mm*10 pm column; 20%-50% acetonitrile in 10mM ammonium acetate bicarbonate in water, 10 minute gradient). Product 6- (3-pyridylsulfonyl)-2-azaspiro[3.3]heptane-2-carboxylate (310 mg, 916 pmol, 25%) was obtained as a white solid. LCMS (ESI) m/z: 339.2 [M+H]⁺.

Step 2: Preparation of 6-(3-pyridylsulfonyl)-2-azaspiro[3.3]heptane.

A solution of fe/f-butyl 6-(3-pyridylsulfonyl)-2-azaspiro[3.3]heptane-2-carboxylate (280 mg, 827 pmol) in trifluoroacetic acid (0.5 ml_) and dichloromethane (0.5 ml_) was stirred at 15 °C for 1 hour. The mixture was concentrated in vacuo and dissolved in methanol (4 ml_). The solution was basified by ion exchange resin. Product 6-(3-pyridylsulfonyl)-2-azaspiro[3.3]heptane (220 mg, crude) was obtained as a yellow gum. LCMS (ESI) m/z: 239.1 [M+H]⁺.

Step 3: Preparation of 2-(3,4-dichlorophenyl)-6-(3-pyridylsulfonyl)-2-azaspiro[3.3]heptane.

To a solution of 6-(3-pyridylsulfonyl)-2-azaspiro[3.3]heptane (200 mg, 839 pmol), 1 ,2-dichloro-4- iodo-benzene (229 mg, 839 pmol), and sodium fe/f-butoxide (323 mg, 3.36 mmol) in dioxane (3 mL) was added tris(dibenzylideneacetone)dipalladium(0) (38 mg, 42 pmol) and 2-dicyclohexylphosphino-2',6'- diisopropoxybiphenyl (8 mg, 17 pmol). The suspension was stirred at 100°C for 30 minutes under nitrogen.. The mixture was filtered and filtrate was concentrated. The crude product was purified by prep- HPLC (Waters Xbridge BEH C18 100*25mm*5pm column; 45%-75% acetonitrile in an10mM ammonium acetate bicarbonate in water, 10 minute gradient). The product 2-(3,4-dichlorophenyl)-6-(3- pyridylsulfonyl)-2-azaspiro[3.3]heptane (50 mg, 129 pmol, 15%) was obtained as a white solid. ¹H NMR (400 MHz, Chloroform-d-d) d 9.01-9.09 (s, 1H), 8.92 - 8.90 (d, 1H), 8.20 - 8.17 (m, 1H), 7.56 - 7.53 (m,

1 H), 7.23 - 7.21 (s, 1 H), 6.46 - 6.45 (d, 1 H), 6.25 - 6.23 (m, 1 H), 3.90 - 3.84 (m, 2H), 3.80 - 3.76 (m, 2H), 3.75 - 3.71 (m, 1 H), 2.85 - 2.80 (m, 2H), 2.57 - 2.51 (m, 2H); LCMS (ESI) m/z: 383.1 [M+H]⁺.

Example 44: Preparation of (S)-(2-(3-chloro-2-fluorophenyl)-2-azaspiro[3.3]heptan-6-yl)(pyridin-3- yl)methanol (Compound 107)

To a solution of (S)-2-azaspiro[3.3]heptan-6-yl(3-pyridyl)methanol (150 mg, 734 pmol) in dimethylsulfoxide (4 mL), was added 1-chloro-2-fluoro-3-iodo-benzene (188 mg, 734 pmol), potassium carbonate (477 mg, 3.45 mmol), pyrrolidine-2-carboxylic acid (34 mg, 294 pmol), and copper(l) iodide (28 mg, 147 pmol). The mixture was stirred at 90 °C for 3 hours. LCMS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered, and the filtrate was concentrated under vacuum. Purification by prep-HPLC ( Waters Xbridge Prep OBD C18 150*40mm*10pm column; 30%-70% acetonitrile in an a 0.04% ammonium hydroxide and 10mM ammonium bicarbonate solution, 8 minute gradient) afforded(S)-[2-(3-chloro-2-fluoro-phenyl)-2- azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanol (25 mg, 74 pmol, 10% as a pale yellow gum. ¹H NMR (400 MHz, Chloroform-d) d 8.53 (br. s, 2H), 7.67 (br. d, J = 7.9 Hz, 1 H), 7.28 (br. d, J = 4.9 Hz, 1 H), 6.92 - 6.83 (m, 1 H), 6.76 - 6.67 (m, 1 H), 6.29 (dt, J = 1 .3, 8.1 Hz, 1 H), 4.62 (d, J = 7.1 Hz, 1 H), 4.01 - 3.93 (m, 2H), 3.92 - 3.84 (m, 2H), 2.60 - 2.47 (m, 1 H), 2.38 - 2.22 (m, 3H), 2.19 - 2.04 (m, 2H); LCMS (ESI) m/z: 333.1 [M+H]⁺.

The following compounds were synthesized according to the protocol described for the

Compound 107.

Example 45: Preparation of (S)-[2-(6-bromopyridazin-3-yl)-2-azaspiro[3.3]heptan-6-yl]-(3- pyridyl)methanol (Compound 112) and (S)-3-pyridyl-[2-[6-(trifluoromethoxy)pyridazin-3-yl]-2- azaspiro[3.3]heptan-6-yl]methanol (Compound 7).

Step 1 : Preparation of 3-bromo-6-(trifluoromethoxy)pyridazine.

To a solution of 6-bromopyridazin-3-ol (1 g, 5.71 mmol) in nitromethane (15 ml_) was added 1- (trifluoromethyl)-l ,2-benziodoxol-3-one (903 mg, 2.86 mmol). The mixture was stirred at 100 °C for 5 hours. The reaction mixture was concentrated in vacuum and the crude product was purified by ISCO column chromatography (20 g silica, 0-18 % ethyl acetate in petroleum ether, gradient over 20 minutes) to obtain 3-bromo-6-(trifluoromethoxy)pyridazine (150 mg, 432 pmol, 15%) as a white solid. LCMS (ESI) m/z: 243.1 [M+H]⁺. Step 2: Preparation of (S)-3-pyridyl-[2-[6-(trifluoromethoxy)pyridazin-3-yl]-2-azaspiro[3.3]heptan-6- yljmethanol, (S)-[2-(6-bromopyridazin-3-yl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanol.

To a solution of (S)-2-azaspiro[3.3]heptan-6-yl(3-pyridyl)methanol (120 mg, 588 pmol) in dimethylsulfoxide (3 ml_) were added copper(l) iodide (22 mg, 118 pmol), 3-bromo-6- (trifluoromethoxy)pyridazine (143 mg, 588 pmol), potassium carbonate (325 mg, 2.35 mmol), and pyrrolidine-2-carboxylic acid (27 mg, 235 pmol) under nitrogen. The mixture was stirred at 90 °C for 3 hours and concentrated. The resultant crude product was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40mm*10pm column; 1-60 % acetonitrile in an a 0.05% ammonia solution in water, 8 minute gradient). The product (S)-3-pyridyl-[2-[6-(trifluoromethoxy)pyridazin-3-yl]-2-azaspiro[3.3]heptan-6- yljmethanol (7 mg, 18 pmol, 3%) was obtained as a pale yellow solid and the product (S)-[2-(6- bromopyridazin-3-yl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanol (16 mg, 42 pmol, 7%) was obtained as a pale yellow solid.

¹H NMR (400 MHz, Chloroform-d) for compound 7 d 8.59 - 8.48 (m, 2H), 7.72 - 7.65 (m, 1 H), 7.32 - 7.27 (m, 1 H), 7.03 - 6.95 (m, 1 H), 6.65 - 6.58 (m, 1 H), 4.71 - 4.62 (m, 1 H), 4.17 - 4.09 (m, 2H), 4.09 - 4.02 (m, 2H), 2.67 - 2.50 (m, 2H), 2.37 - 2.30 (m, 2H), 2.24 - 2.13 (m, 2H). LCMS (ESI) m/z: 367.2 [M+H]⁺. ¹H NMR (400 MHz, Chloroform-d) for compound 112 d 8.55 - 8.51 (m, 2H), 7.72 - 7.66 (m, 1 H), 7.32 - 7.27 (m, 1 H), 7.25 (s, 1 H), 6.37 (d, J = 9.2, 1 H), 4.66 (d, J = 6.8 Hz, 1 H), 4.15 - 4.07 (m, 2H), 4.07 - 3.99 (m, 2H), 2.67 - 2.50 (m, 2H), 2.38 - 2.29 (m, 2H), 2.24 - 2.11 (m, 2H); LCMS (ESI) m/z: 361 .0 [M+H]⁺.

Example 46: Preparation of 2-[3-ethyl-1-[6-(trifluoromethyl)-3-pyridyl]azetidin-3-yl]-1-(3- pyridyl)ethanol (Compound 113) and its chiral separation into enantiomer 1 (compound 116) and enantiomer 2 (compound 46).

Step 1 : Preparation of fe/f-butyl 3-ethyl-3-(hydroxymethyl)azetidine-1-carboxylate.

To a solution of 1-fe/f-butyl-3-methyl 3-ethylazetidine-1 ,3-dicarboxylate (8 g, 32.88 mmol) in tetrahydrofuran (100 ml_) was added lithium borohydride (5.01 g, 230.17 mmol). The mixture was stirred at 20 °C for 3 hours. The mixture was quenched by ice-water (50 ml_), and the aqueous solution was extracted with ethyl acetate (30 ml_ x 3). The combined organic phase was washed with brine (10 mL x 3), dried with anhydrous sodium sulfate, filtered, and concentrated in vacuum. The crude product was purified by ISCO column chromatography (40 g silica, 0-40 % ethyl acetate in petroleum ether, gradient over 30 minutes) to obtain fe/f-butyl 3-ethyl-3-(hydroxymethyl)azetidine-1-carboxylate (6.5 g, 30.19 mmol, 92%) was obtained as a colorless oil. ¹H NMR (400 MHz, Chloroform-d) d 3.71 - 3.67 (m, 2H), 3.66 - 3.62 (m, 2H), 3.57 (d, J = 8.6 Hz, 2H), 1 .73 - 1 .56 (m, 2H), 1 .48 - 1 .37 (m, 9H), 0.93 - 0.83 (m, 3H).

Step 2: Preparation of fe/f-butyl 3-ethyl-3-(methylsulfonyloxymethyl)azetidine-1-carboxylate.

To a solution of fe/f-butyl 3-ethyl-3-(hydroxymethyl)azetidine-1-carboxylate (6.2 g, 28.80 mmol) in dichloromethane (100 mL) were added triethylamine (5.83 g, 57.60 mmol, 8.02 mL), and methanesulfonyl chloride (3.96 g, 34.56 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 hour and the mixture was quenched by water (30 mL) at 0°C, and the aqueous solution was extracted with ethyl acetate (10 mL x 3). The combined organic phase was washed with brine (10 mL x 3), dried with anhydrous sodium sulfate, filtered, and concentrated in vacuum. The crude product was purified by ISCO column chromatography (40 g silica, 0-40 % ethyl acetate in petroleum ether, gradient over 20 minutes) to obtain fe/f-butyl 3-ethyl-3-(methylsulfonyloxymethyl)azetidine-1-carboxylate (8 g, 27.27 mmol, 95%) was obtained as a pale yellow oil. ¹H NMR (400 MHz, Chloroform-d) d 4.24 (s, 2H), 3.74 - 3.68 (m, 2H), 3.67 - 3.61 (m, 2H), 3.04 (s, 3H), 1.79 - 1.61 (m, 2H), 1.43 (s, 9H), 0.99 - 0.82 (m, 3H).

Step 3: Preparation of fe/f-butyl 3-(cyanomethyl)-3-ethyl-azetidine-1-carboxylate.

To a solution of fe/f-butyl 3-ethyl-3-(methylsulfonyloxymethyl)azetidine-1-carboxylate (4 g, 13.63 mmol) in dimethylsulfoxide (8 ml_) was added sodium cyanide (935 mg, 19.09 mmol). The mixture was stirred at 80 °C for 16 h. To the mixture was added water (10 ml_), and the mixture was extracted with ethyl acetate (10 ml_ x 3). The combined organic phase was washed with brine (10 ml_ x 3), dried with anhydrous sodium sulfate, filtered, and concentrated in vacuum. The crude product was purified by ISCO column chromatography (40 g silica, 0-80 % ethyl acetate in petroleum ether, gradient over 20 minutes) to obtainfe/f-butyl 3-(cyanomethyl)-3-ethyl-azetidine-1-carboxylate (2.6 g, 11.59 mmol, 85%) as a colorless oil. ¹H NMR (400 MHz, Chloroform-d) d 3.80 - 3.62 (m, 4H), 2.61 (s, 2H), 1.78 (q, J = 7.5 Hz, 2H), 1.44 (s, 9H), 0.94 (t, J = 7.5 Hz, 3H).

Step 4: Preparation of fe/f-butyl 3-ethyl-3-(2-oxoethyl)azetidine-1-carboxylate.

To a solution of fe/f-butyl 3-(cyanomethyl)-3-ethyl-azetidine-1-carboxylate (1 g, 4.46 mmol) in dichloromethane (12 ml_) was added diisobutylalumminum hydride (1 M, 11.15 ml_) at -70 °C. The mixture was warmed to 20 °C and stirred at 20 °C for 12 hours. The mixture was then quenched with saturated ammonium chloride (4 ml_) and extracted with dichloromethane (10 mL x 3). The organic layer was washed with brine (5 mL), dried over sodium sulfate, filtered, and concentrated to give crude product. The crude product was purified by ISCO column chromatography (4 g silica, 20-50 % ethyl acetate in petroleum ether, gradient over 20 minutes) to obtain fe/f-butyl 3-ethyl-3-(2-oxoethyl)azetidine-1- carboxylate (0.13 g, 572 pmol, 13%) as a yellow oil. ¹H NMR (400 MHz, Chloroform-d) d 9.72 (t, J = 1.6 Hz, 1 H), 3.70 - 3.62 (m, 4H), 2.67 (d, J = 1.6 Hz, 2H), 1.70 - 1.61 (m, 2H), 1.37 (s, 9H), 0.81 (t, J = 7.4 Hz, 3H).

Step 5: Preparation of fe/f-butyl 3-ethyl-3-[2-hydroxy-2-(3-pyridyl)ethyl]azetidine-1-carboxylate.

To a solution of 3-iodopyridine (152 mg, 744 pmol) in tetrahydrofuran (5 mL) was added isopropylmagnesium chloride (2 M, 372 pL) at 0 °C. The mixture was stirred at 0 °C for 30 minutes. Then, fe/f-butyl 3-ethyl-3-(2-oxoethyl)azetidine-1-carboxylate (130 mg, 572 pmol) in tetrahydrofuran (0.5 mL) was added dropwise to the reaction. The mixture was stirred at 20 °C for 3 hours and was quenched with water (5 mL) and extracted with ethyl acetate (15 mL x 4). The organic layer was washed with brine (10 mL), dried over sodium sulfate, filtered, and concentrated to give crude product. The crude product was purified by ISCO column chromatography (10 g silica, 50-70 % ethyl acetate in petroleum ether, gradient over 20 minutes) to obtain fe/f-butyl 3-ethyl-3-[2-hydroxy-2-(3-pyridyl)ethyl]azetidine-1-carboxylate (240 mg, crude) as a yellow oil. ¹H NMR (400 MHz, Chloroform-d) d 8.52 - 8.42 (m, 2H), 7.66 (br. d, J = 7.9 Hz, 1 H), 7.23 (dd, J = 4.8, 7.8 Hz, 1 H), 4.78 (dd, J = 3.4, 9.7 Hz, 1 H), 3.83 (d, J = 8.6 Hz, 1 H), 3.56 (d, J = 8.8 Hz, 1 H), 3.53 - 3.43 (m, 2H), 2.11 - 1.99 (m, 1 H), 1.81 - 1.68 (m, 3H), 1.36 (s, 9H), 0.86 (t, J = 7.4 Hz, 3H). Step 6: Preparation of 2-(3-ethylazetidin-3-yl)-1-(3-pyridyl)ethanol.

A mixture of fe/f-butyl 3-ethyl-3-[2-hydroxy-2-(3-pyridyl)ethyl]azetidine-1-carboxylate (240 mg,

783 pmol) in dichloromethane (2 ml_) and trifluoroacetic aid (1 ml_) was stirred at 20 °C for 1 .5 hours.

The mixture was concentrated to obtain the crude product. Then the crude product was dissolved in methanol (10 ml), basified by ion exchange resin, and the turbid liquid was filtered to remove the insoluble solids and the filtrate was concentrated in vacuo. The product 2-(3-ethylazetidin-3-yl)-1-(3-pyridyl)ethanol (130 mg, 630 pmol, 80%) was obtained as yellow oil. LCMS (ESI) m/z: 207.2 [M+H]⁺.

Step 7: Preparation of 2-[3-ethyl-1-[6-(trifluoromethyl)-3-pyridyl]azetidin-3-yl]-1-(3-pyridyl)ethanol and its chiral separation into pure enantiomers.

To a solution of 2-(3-ethylazetidin-3-yl)-1-(3-pyridyl)ethanol (130 mg, 630 pmol) in dimethylformamide (2 ml_) were added 5-fluoro-2-(trifluoromethyl)pyridine (114 mg, 693 pmol) and triethylamine (128 mg, 1 .26 mmol). The mixture was stirred at 70 °C for 12 hours and concentrated. The resultant crude product purified directly by prep-HPLC Phenomenex Gemini-NX 150*30mm*5pm column; 21-51 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to obtain the racemic compound 2-[3-ethyl-1-[6-(trifluoromethyl)-3-pyridyl]azetidin-3-yl]-1-(3-pyridyl)ethanol. Then the racemic compound was subjected to preparative SFC (DAICEL CHIRALPAK AD(250mm*30mm,10pm) column, 40°C, eluting with 40% ethanol containing 0.1% ammonium hydroxide in a flow of 65 g/min carbon dioxide at 100 bar) to obtain enantiomer 1 (Rt=1 .210min) and enantiomer 2 (Rt=1 .344min).

The product 2-[3-ethyl-1-[6-(trifluoromethyl)-3-pyridyl]azetidin-3-yl]-1-(3-pyridyl)ethanol (67 mg, 190 pmol, 30%) was obtained as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.58 (d, J = 1 .8 Hz, 1 H), 8.54 (d, J = 3.6 Hz, 1 H), 7.83 (d, J = 2.5 Hz, 1 H), 7.76 (br. d, J = 7.9 Hz, 1 H), 7.46 (d, J = 8.6 Hz, 1 H), 7.32 (dd, J = 4.8, 7.8 Hz, 1 H), 6.70 (dd, J = 2.6, 8.5 Hz, 1 H), 4.94 (br. d, J = 8.4 Hz, 1 H), 4.03 (d, J =

7.8 Hz, 1 H), 3.77 (d, J = 7.6 Hz, 1 H), 3.72 (d, J = 7.4 Hz, 1 H), 3.63 (d, J = 7.4 Hz, 1 H), 2.50 (br. s, 1 H), 2.22 (dd, J = 9.9, 14.6 Hz, 1 H), 2.02 - 1.88 (m, 3H), 1.03 - 0.94 (t, 3H) LCMS (ESI) m/z: 352.2 [M+H]⁺. Enantiomer 1 (20 mg, 57 pmol) was obtained as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.59 (d, J = 1 .9 Hz, 1 H), 8.55 (dd, J = 1 .4, 4.7 Hz, 1 H), 7.83 (d, J = 2.6 Hz, 1 H), 7.75 (br. d, J = 7.9 Hz,

1 H), 7.45 (d, J = 8.5 Hz, 1 H), 7.31 (dd, J = 4.8, 7.8 Hz, 1 H), 6.69 (dd, J = 2.6, 8.6 Hz, 1 H), 4.94 (br. d, J =

9.9 Hz, 1 H), 4.02 (d, J = 7.8 Hz, 1 H), 3.77 (d, J = 7.6 Hz, 1 H), 3.72 (d, J = 7.4 Hz, 1 H), 3.61 (d, J = 7.4 Hz, 1 H), 2.27 - 2.11 (m, 2H), 2.01 - 1 .83 (m, 3H), 1 .01 (t, J = 7.4 Hz, 3H) LCMS (ESI) m/z: 352.2 [M+H]⁺. Enantiomer 2 (22 mg, 61 pmol) was obtained as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.59 (d, J = 1 .9 Hz, 1 H), 8.55 (dd, J = 1 .5, 4.8 Hz, 1 H), 7.83 (d, J = 2.6 Hz, 1 H), 7.75 (br. d, J = 7.9 Hz,

1 H), 7.45 (d, J = 8.7 Hz, 1 H), 7.32 (dd, J = 4.8, 7.8 Hz, 1 H), 6.69 (dd, J = 2.6, 8.5 Hz, 1 H), 4.94 (br. d, J = 9.8 Hz, 1 H), 4.02 (d, J = 7.7 Hz, 1 H), 3.77 (d, J = 7.6 Hz, 1 H), 3.72 (d, J = 7.4 Hz, 1 H), 3.61 (d, J = 7.3 Hz, 1 H), 2.26 - 2.13 (m, 2H), 2.03 - 1.84 (m, 3H), 1.01 (t, J = 7.4 Hz, 3H)LCMS (ESI) m/z: 352.2 [M+H]⁺. Example 47: Preparation of (4-methoxypyridin-3-yl)(2-(6-(trifluoromethyl)pyridin-3-yl)-2- azaspiro[3.3]heptan-6-yl)methanol (Compound 114) and its chiral separation into enantiomer 1 (Compound 100) and enantiomer 2 (Compound 51).

Step 1 : Preparation of fe/f-butyl 6-(hydroxy(4-methoxypyridin-3-yl)methyl)-2-azaspiro[3.3]heptane-2- carboxylate.

To a solution of 3-iodo-4-methoxy-pyridine (390 mg, 1.66 mmol) in tetrahydrofuran (5 ml_) was added isopropylmagnesium chloride (2 M, 1.24 ml_) in tetrahydrofuran dropwise by syringe at 0 °C. The mixture was stirred at 0 °C for 1 hour. Then, fe/f-butyl 6-formyl-2-azaspiro[3.3]heptane-2-carboxylate (374 mg, 1.66 mmol) was added to the solution at 0 °C under nitrogen. The solution was stirred at 20 °C for 1 hour and was diluted with ammonium chloride (2 ml_) and extracted with ethyl acetate (10ml_ x 3). The combined organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by I SCO column chromatography (10 g silica, 70-100% ethyl acetate in petroleum ether, gradient over 20 minutes) to obtain fe/f-butyl 6-[hydroxy-(4-methoxy-3-pyridyl)methyl]-2-azaspiro[3.3]heptane-2-carboxylate (550 mg,

1.64 mmol, 99%) as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.49 - 8.29 (m, 2H), 6.76 (d, J = 5.6 Hz, 1 H), 4.76 (br. d, J = 7.0 Hz, 1H), 3.92 - 3.85 (m, 5H), 3.84 - 3.77 (m, 2H), 2.97 - 2.84 (m, 1H), 2.57 (qd, J = 7.8, 15.6 Hz, 1H), 2.19 (br. d, J = 7.9 Hz, 2H), 2.10 - 1.96 (m, 2H), 1.41 (s, 9H); LCMS (ESI) m/z: 335.2 [M+H]⁺.

Step 2: Preparation of (4-methoxypyridin-3-yl)(2-azaspiro[3.3]heptan-6-yl)methanol.

To a solution of fe/f-butyl 6-[hydroxy-(4-methoxy-3-pyridyl)methyl]-2-azaspiro[3.3]heptane-2- carboxylate (500 mg, 1.50 mmol) in dichloromethane (3 ml_) was added trifluoroacetic acid (2.39 g, 20.93 mmol). The mixture was stirred at 20 °C for 2 hours. The mixture was basified by ion exchange resin to pH ~9 at 0 °C and concentrated to obtain 2-azaspiro[3.3]heptan-6-yl-(4-methoxy-3-pyridyl)methanol (380 mg, 1.30 mmol, 87%, crude) as a white solid. LCMS (ESI) m/z: 235.1 [M+H]⁺. It was taken to the next step without purification.

Step 3: Preparation of (4-methoxypyridin-3-yl)(2-(6-(trifluoromethyl)pyridin-3-yl)-2-azaspiro[3.3]heptan-6- yl)methanol.

To a solution of 1 ,8-Diazabicyclo[5.4.0]undec-7-ene (390 mg, 2.56 mmol, 386 pL) in dimethylsulfoxide (5 mL) were added 5-fluoro-2-(trifluoromethyl)pyridine (211 mg, 1.28 mmol) and 2- azaspiro[3.3]heptan-6-yl-(4-methoxy-3-pyridyl)methanol (300 mg, 1.28 mmol). The mixture was stirred at 60 °C for 12 hours. The reaction was filtered, and the filtrate was concentrated under vacuum. The crude residue was purified by prep-HPLC (Kromasil C18 (250*50mm*10 pm column; 20-50% acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 minute gradient) to obtain (4-methoxy-3-pyridyl)-[2- [6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]methanol (100% purity) ( total 471 mg ) as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.45 (s, 1H), 8.40 (s, 1 H), 7.82 (d, J = 2.6 Hz, 1 H), 7.43 (d, J = 8.6 Hz, 1 H), 6.81 (d, J = 5.7 Hz, 1 H), 6.68 (dd, J = 2.6, 8.6 Hz, 1 H), 4.81 (br. d, J = 5.5 Hz, 1 H), 4.03 - 3.96 (m, 2H), 3.96 - 3.88 (m, 5H), 2.69 (sxt, J = 7.8 Hz, 1 H), 2.43 - 2.38 (m, 1 H), 2.35 - 2.30 (m,

1 H), 2.27 - 2.09 (m, 2H); LCMS (ESI) m/z: 380.2 [M+H]⁺.

Step 4: Chiral separation of (4-methoxy-3-pyridyl)-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan- 6-yl]methanol to enantiomer 1 and enantiomer 2.

The racemic (4-methoxy-3-pyridyl)-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6- yljmethanol (454 mg, 1.20 mmol) was subjected to chiral chromatography: SFC (column: Lux Cellulose-2 (50x4.6 mm,I.D.,3um);mobile phase: [0.05%ammonium:ethanol];B%: 5%-50%, minutes). The enantiomer 1 was obtained as white solid in 46% yield and enantiomer 2 was obtained in 48% yield as white solid.

¹H NMR (400 MHz, Chloroform-d) for compound 100: d 8.42 -8.39 (m, 2H), 7.82 (d, J = 2.6 Hz, 1 H), 7.43 (d, J = 8.6 Hz, 1 H), 6.81 (d, J = 5.7 Hz, 1 H), 6.68 (dd, J = 2.6, 8.6 Hz, 1 H), 4.81 (br. d, J = 5.5 Hz, 1 H), 4.03 - 3.96 (m, 2H), 3.96 - 3.88 (m, 5H), 2.69 (sxt, J = 7.8 Hz, 1 H), 2.43 - 2.30 (m, 2H), 2.27 - 2.09 (m, 2H); LCMS (ESI) m/z: 380.2 [M+H]⁺; (Rt: 1.430min).

¹H NMR (400 MHz, Chloroform-d) for compound 51 : d 8.45 - 8.40 (m, 2H), 7.82 (d, J = 2.6 Hz, 1 H), 7.43 (d, J = 8.6 Hz, 1 H), 6.81 (d, J = 5.7 Hz, 1 H), 6.68 (dd, J = 2.6, 8.6 Hz, 1 H), 4.81 (br. d, J = 5.5 Hz, 1 H), 4.03 - 3.96 (m, 2H), 3.96 - 3.88 (m, 5H), 2.69 (sxt, J = 7.8 Hz, 1 H), 2.43 - 2.30 (m, 2H), 2.27 - 2.09 (m, 2H); LCMS (ESI) m/z: 380.2 [M+H]⁺; (Rt: 1.509min).

Example 48: Preparation of 1-(3-pyridyl)-1-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan- 6-yl]ethanol (Compound 117)

Step 1 : Preparation of 3-pyridyl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]methanone.

To a solution of 3-pyridyl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]methanol (0.9 g, 2.58 mmol) in ethylene dichloride (18 mL) was added manganese(IV) oxide (2.24 g, 25.76 mmol) The mixture was stirred at 60 °C for 40 hours. The mixture was filtered, and the filtrate was dried over in vacuo to afford the crude product 3-pyridyl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6- yl]methanone (0.55 g, 1 .58 mmol, 62%, crude) which was used into the next step without further purification.

Step 2: Preparation of 1-(3-pyridyl)-1-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]ethanol.

To a solution of 3-pyridyl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]methanone (60 mg, 173 pmol) in tetrahydrofuran (0.5 ml_) was added methylmagnesium bromide (3 M, 115 pL, in tetrahydrofuran) at 0 °C. The mixture was stirred at 20 °C for 2 hours. The reaction mixture was quenched by addition of ammonium chloride (1 ml_) and the reaction mixture was concentrated in vacuum. The residue was purified by prep-HPLC (Waters Xbridge 150*25 5pm column; 40-60 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 10 minute gradient) to obtain 1 -(3- pyridyl)-1-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]ethanol (31 mg, 86 pmol, 25%) was obtained as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.67 (d, 1 H), 8.51 (d, J = 4, 1 H),

7.81 (d, J = 2.4, 1 H), 7.80 - 7.73 (m, 1 H), 7.47 - 7.40 (m, 1 H), 7.29 (br. d, J = 4.8 Hz, 1 H), 6.70 - 6.64 (m,

1 H), 4.03 - 3.92 (m, 2H), 3.90 - 3.83 (m, 2H), 2.66 - 2.53 (m, 1 H), 2.42 - 2.26 (m, 2H), 2.22 - 2.12 (m, 1 H), 1 .98 - 1 .83 (m, 2H), 1.52 (s, 3H). LCMS (ESI) m/z: 364.1 [M+H]⁺.

Example 49: Preparation of 6-[fluoro(3-pyridyl)methyl]-2-[6-(trifluoromethyl)-3-pyridyl]-2- azaspiro[3.3]heptane (Compound 118)

To a solution of 3-pyridyl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]methanol (200 mg, 573 pmol) in dichloromethane (4 ml_) was added bis(2-methoxyethyl)aminosulfur trifluoride (253 mg, 1.14 mmol) under nitrogen. The mixture was stirred at -70 °C for 1 .5 hours, then was stirred at -10 °C for 30 minutes The reaction mixture was concentrated in vacuum and the resultant crude product was purified by prep-HPLC (Phenomenex Gemini-NX 150*30mm*5pm column; 35-55 % acetonitrile in a 10mM ammonium bicarbonate solution in water, 8 minute gradient). Then the product was purified again by prep-HPLC (Phenomenex Luna C18 100*30mm*5pm column; 15-45 % acetonitrile in an a 0.225% formic acid solution in water, 9 minute gradient). The product 6-[fluoro(3-pyridyl)methyl]-2-[6- (trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptane (12 mg, 34 pmol, 6%) was obtained as a pale yellow gum. ¹H NMR (400 MHz, Chloroform-d) d 8.63 - 8.58 (m, 1 H), 8.57 - 8.53 (m, 1 H), 7.86 - 7.81 (m, 1 H), 7.67 - 7.62 (m, 1 H), 7.47 - 7.42 (m, 1 H), 7.36 - 7.30 (m, 1 H), 6.73 - 6.65 (m, 1 H), 5.50 - 5.36 (m, 1 H), 4.04 - 3.98 (m, 2H), 3.97 - 3.90 (m, 2H), 2.82 - 2.64 (m, 1 H), 2.43 - 2.26 (m, 4H). LCMS (ESI) m/z: 352.1 [M+H]⁺. Example 50: Synthesis of (3aR,6aS)-2-(3,4-dichlorophenyl)-5-(3-pyridylsulfonyl)-1,3,3a,4,6,6a- hexahydropyrrolo[3,4-c]pyrrole (Compound 121):

Step 1 : tert-butyl (3aR,6aS)-2-(3,4-dichlorophenyl)-1 ,3,3a,4,6,6a-hexahydropyrrolo[3,4-c]pyrrole-5- carboxylate.

To a solution of tert-butyl (3aR,6aS)-2,3,3a,4,6,6a-hexahydro-1 H-pyrrolo[3,4-c]pyrrole-5- carboxylate (400 mg, 1.88 mmol) in dioxane (4 ml_) were added t-BuONa (543 mg, 5.65 mmol), Pd₂(dba)3 (86 mg, 94 umol, 0.05 eq), RuPhos (18 mg, 38 umol) and 1 ,2-dichloro-4-iodo-benzene (514 mg, 1 .88 mmol) and the resultant mixture was stirred at 100 °C for 20 min under N2. 10 mL of water was added to the reaction mixture and the reaction mixture was extracted with Ethyl acetate (20 ml_*3). The combined organic layers were washed with brine (10 mL), dried over Na₂SC>₄ and concentrated. The crude product was purified by flash column (ISCO 10 g silica, 0-15 % ethyl acetate in petroleum ether, gradient over 20 min) tert-butyl (3aR,6aS)-2-(3,4-dichlorophenyl)-1 ,3,3a,4,6,6a-hexahydropyrrolo[3,4-c]pyrrole-5-carboxylate (360 mg, 1 .01 mmol, 53%) was obtained as a pale yellow solid.

1 H NMR (400MHz, CHLOROFORM-d) d = 7.23 (d, J=8.9 Hz, 1 H), 6.59 (d, J=2.8 Hz, 1H), 6.37 (dd, J=2.8, 8.8 Hz, 1 H), 3.65 (br s, 2H), 3.50 (br s, 2H), 3.36 (br d, J=9.4 Hz, 1 H), 3.26 (br s, 1 H), 3.18 (dd, J=3.9, 9.6 Hz, 2H), 3.01 (br s, 2H), 1.46 (s, 9H)

Step 2: (3aR,6aS)-5-(3,4-dichlorophenyl)-2,3,3a,4,6,6a-hexahydro-1 H-pyrrolo[3,4-c]pyrrole.

To a solution of tert-butyl (3aR,6aS)-2-(3,4-dichlorophenyl)-1 ,3,3a,4,6,6a-hexahydropyrrolo[3,4- c]pyrrole-5-carboxylate (340 mg, 952 umol) in DCM (4 mL) was added TFA (1.52 g, 13.32 mmol) at 0 °C, then the mixture was stirred at 20 °C for 2 h and concentrated. The crude product (3aR,6aS)-5-(3,4- dichlorophenyl)-2,3,3a,4,6,6a-hexahydro-1 H-pyrrolo[3,4-c]pyrrole (760 mg, crude, TFA) was obtained as a brown gum.

Step 3: (3aR,6aS)-2-(3,4-dichlorophenyl)-5-(3-pyridylsulfonyl)-1 ,3,3a,4,6,6a-hexahydropyrrolo[3,4- cjpyrrole.

To a solution of (3aR,6aS)-5-(3,4-dichlorophenyl)-2,3,3a,4,6,6a-hexahydro-1 H-pyrrolo[3,4- cjpyrrole (300 mg, 1.17 mmol) in DCM (4 mL) was added pyridine-3-sulfonyl chloride (414 mg, 2.33 mmol) and Et3N (472 mg, 4.67 mmol), then the mixture was stirred at 20 °C for 1 h and concentrated. The resultant crude product was purified by prep-HPLC (Phenomenex Gemini-NX C18 75*30 3u column; 30-60 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 6 min gradient) to obtain (3aR,6aS)-2- (3,4-dichlorophenyl)-5-(3-pyridylsulfonyl)-1 ,3,3a,4,6,6a-hexahydropyrrolo[3,4-c]pyrrole (32 mg, 76 umol, 7%) was obtained as a white solid.

1 H NMR (400MHz, DMSO-d6) d = 8.97 (d, J=1 .9 Hz, 1 H), 8.87 (dd, J=1 .4, 4.8 Hz, 1 H), 8.22 (td, J=1 .8, 8.1 Hz, 1 H), 7.66 (dd, J=4.8, 7.8 Hz, 1 H), 7.32 (d, J=8.9 Hz, 1 H), 6.62 (d, J=2.8 Hz, 1 H), 6.44 (dd, J=2.8, 8.9 Hz, 1 H), 3.42 (br dd, J=6.8, 10.1 Hz, 2H), 3.31 - 3.27 (m, 2H), 3.11 (dd, J=3.2, 10.2 Hz, 2H), 3.00 - 2.90 (m, 4H). LCMS (ESI) for C17H17CI2N302S [M+H]+: 398.1

Example 51: Preparation of 2,2,2-trifluoro-1-(3-pyridyl)-1-[2-[6-(trifluoromethyl)-3-pyridyl]-2- azaspiro[3.3]heptan-6-yl]ethanol (Compound 122)

To a solution of 3-pyridyl-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]methanone (150 mg, 432 pmol) in tetrahydrofuran (3 ml_) were added trimethyl(trifluoromethyl)silane (246 mg, 1.73 mmol) and cesium fluoride (72 mg, 475 pmol, 18 pL). The mixture was stirred at 60 °C for 12 hours. The reaction mixture was concentrated in vacuum and the crude product was purified by prep-HPLC (Phenomenex Gemini-NX C18 75*30mm*3pm column; 40-60 % acetonitrile in an a 10mM ammonium bicarbonate solution in water, 8 minute gradient) to afford2,2,2-trifluoro-1-(3-pyridyl)-1-[2-[6- (trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]ethanol (30 mg, 72 pmol, 17%) as a pale yellow solid. ¹H NMR (400 MHz, Chloroform-d) d 8.78 - 8.73 (m, 1 H), 8.61 (d, J = 3.8 Hz, 1 H), 7.92 - 7.85 (m,

1 H), 7.84 - 7.77 (m, 1 H), 7.49 - 7.41 (m, 1 H), 7.40 - 7.32 (m, 1 H), 6.68 (dd, J = 2.6, 8.6 Hz, 1 H), 4.15 - 3.96 (m, 2H), 3.94 - 3.78 (m, 2H), 3.75 - 3.48 (m, 1 H), 3.23 - 3.06 (m, 1 H), 2.62 - 2.48 (m, 1 H), 2.47 - 2.34 (m, 1 H), 2.20 - 2.03 (m, 1 H), 2.02 - 1.87 (m, 1 H). LCMS (ESI) m/z: 418.1 [M+H]⁺.

Example 52: Preparation of 2-(3-pyridylsulfonyl)-6-[5-(trifluoromethoxy)-2-pyridyl]-2,6- diazaspiro[3.3]heptane (Compound 48)

Compound 48 was synthesized according to the synthetic procedure reported for the Preparation of compound 121 . Product 2-(3-pyridylsulfonyl)-6-[5-(trifluoromethoxy)-2-pyridyl]-2,6- diazaspiro[3.3]heptane (54 mg, 135 pmol, 34%) was obtained as a white solid. ¹H NMR (400 MHz, Chloroform-d) d 9.09 (s, 1 H), 8.91 (d, J = 4.8 Hz, 1 H), 8.16 (br. d, J = 8.1 Hz, 1 H), 8.06 (s, 1 H), 7.56 (dd, J = 4.9, 7.9 Hz, 1 H), 7.35 (br. d, J = 8.9 Hz, 1 H), 6.24 (d, J = 9.0 Hz, 1 H), 4.03 (d, J = 4.9 Hz, 8H) LCMS (ESI) m/z: 401.1 [M+H]⁺.

Example 53: Preparation of 2-(3-pyridyl)-2-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan- 6-yl]ethanol (Compound 60)

Step 1 : Preparation of 6-[(E)-2-methoxy-1-(3-pyridyl)vinyl]-2-[6-(trifluoromethyl)-3-pyridyl]-2- azaspiro[3.3]heptane

A solution of methoxymethyl(triphenyl)phosphonium;chloride (1.07 g, 3.11 mmol) in tetrahydrofuran (1 ml_) was added drop wise lithium diisopropylamide (2 M, 1 .55 ml_) at 0°C . The mixture was stirred at 0 °C for 1 h. Then, a solution of 3-pyridyl-[2-[6-(trifluoromethyl)-3-pyridyl]-2- azaspiro[3.3]heptan-6-yl]methanone (360 mg, 1.04 mmol) in tetrahydrofuran (1 ml_) was added dropwise at 0 °C and the mixture was allowed to warm to 15 °C and stirred for 12 hours. LCMS showed the reaction was complete. The reaction mixture was quenched by addition ice water (5 mL) at 0°C, and the mixture was extracted with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine (1 OmL x 3), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The crude product was purified by ISCO column chromatography (40 g silica, 0-50 % ethyl acetate in petroleum ether, gradient over 30 minutes). Product 6-[(E)-2-methoxy-1-(3-pyridyl)vinyl]-2-[6- (trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptane (740 mg, crude) was obtained as yellow solid . LCMS (ESI) m/z: 376.1 [M+H]⁺.

Step 2: Preparation of 2-(3-pyridyl)-2-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6- yljacetaldehyde

A solution of 6-[(Z)-2-methoxy-1 -(3-pyridyl)vinyl]-2-[6-(trifluoromethyl)-3-pyridyl]-2- azaspiro[3.3]heptane (500 mg, 1 .33 mmol) in dichloromethane (1 ml_) was added trifluoroacetic acid (759 mg, 6.66 mmol, 493 pL). The mixture was stirred at 15 °C for 3 hours. LCMS showed the reaction was complete. The mixture was cooled to 0°C and in was added an ion exchange resin to turn pH = 8~9. The mixture was stirred for 1 hour. The mixture was filtered and the filtrate was concentrated to dryness to give the crude product. Product 2-(3-pyridyl)-2-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6- yljacetaldehyde (480 mg, crude) was obtained as a yellow oil. LCMS (ESI) m/z: 362.1 [M+H]⁺.

Step 3: Preparation of 2-(3-pyridyl)-2-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6-yl]ethanol

To a solution of 2-(3-pyridyl)-2-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6- yljacetaldehyde (480 mg, 1.33 mmol) in methanol (0.5 mL) was added sodium borohydride (151 mg, 3.98 mmol) at 0°C. The solution was stirred at 15 °C for 1 hour. LCMS showed the reaction was complete. To the mixture was added water (1 mL) and the mixture was filtered. The filtrate was purified by prep-HPLC (Kromasil 150*25mm*10pm column; 15-45 % acetonitrile in an a 0.05% hydrogen chloride solution in water, 10 minutes gradient). Product 2-(3-pyridyl)-2-[2-[6-(trifluoromethyl)-3-pyridyl]-2- azaspiro[3.3]heptan-6-yl]ethanol was obtained (120 mg) as pale yellow solid. Note: The ring opened when it was separated by prep- HPLC (hydrochloric acid system).

Step 4: Preparation of 2-(3-pyridyl)-2-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan-6- yljacetaldehyde

To a solution of 2-[3-(chloromethyl)-3-[[[6-(trifluoromethyl)-3-pyridyl]amino]methyl]cyclobutyl]-2-(3- pyridyl)ethanol (100 mg, 250 pmol) in dimethylformamide (2 mL) was added sodium hydride (45 mg, 1.13 mmol, 60% purity) at 0°C. The mixture was stirred at 20°C for 2 hours. LCMS showed the reaction was complete. The mixture was filtered, and the filtrate was purified directly by prep-HPLC (Waters Xbridge BEH C18 100*25mm*5pm column; 30-55 % acetonitrile in an a 10Mm ammonium bicarbonate solution in water, 10 minute gradient). Product 2-(3-pyridyl)-2-[2-[6-(trifluoromethyl)-3-pyridyl]-2-azaspiro[3.3]heptan- 6-yl]ethanol (21 mg, 57 pmol, 23%) was obtained as a pale yellow solid. ¹H NMR (400 MHz, Chloroformed) d 8.55 - 8.48 (m, 2H), 7.83 (d, J = 2.7 Hz, 1 H), 7.58 (br. d, J = 7.9 Hz, 1 H), 7.44 (d, J = 8.4 Hz, 1 H),

7.31 (dd, J = 5.0, 7.8 Hz, 1 H), 6.68 (dd, J = 2.4, 8.4 Hz, 1 H), 4.07 - 3.98 (m, 2H), 3.91 - 3.87 (m, 1 H), 3.87 - 3.81 (m, 2H), 3.80 - 3.73 (m, 1 H), 2.81 - 2.72 (m, 1 H), 2.67 - 2.50 (m, 2H), 2.20 - 2.08 (m, 2H), 1 .83 (dd, J = 8.3, 11 .9 Hz, 1 H) LCMS (ESI) m/z: 364.2 [M+H]⁺.

Example 54: Stereoisomer Separation

The following stereoisomers were separated similar to the conditions described for the compounds 100 and 51 using the one of the conditions mentioned below. General chiral prep HPLC conditions (applies to all chiral separations described in the patent).

Condition A: SFC (DAICEL CHIRALPAK IC(250mm*30mm,5um column, 40°C, eluting with organic solvent containing 0.1% ammonium hydroxide in a flow of 65 g/min CO2 at 100 bar).

Condition B: SFC (Phenomenex-Cellulose-2 (250mm*30mm,10um) column, 40°C, eluting with organic solvent containing 0.1% ammonium hydroxide in a flow of 65 g/min CO2 at 100 bar).

Organic solvent: 25-60% methanol, ethanol or isopropanol.

The retention times mentioned for the possible stereoisomers are based on the order they are eluted from the column under the same condition.

In this table, * on the structure denotes chiral center which is enantiomerically pure (either R or S). Example 55: Confirmation of structure of Compound 34

Step 1 : tert-butyl 6-[hydroxy(3-pyridyl)methyl]-2-azaspiro[3.3]heptane-2-carboxylate.

To a solution of 3-iodopyridine (32.76 g, 159.80 mmol) in THF (350 ml_) was added i-PrMgCI (2 M, 79.90 ml_) at 0 °C and the mixture was stirred at 20 °C for 1 h, then tert-butyl 6-formyl-2- azaspiro[3.3]heptane-2-carboxylate (18 g, 79.90 mmol) in THF (50 ml_) was added to the mixture at 0 °C. The resultant mixture was stirred at 20 °C for 1h and quenched by the addition NH4CI (300 ml_), extracted with ethyl acetate (500 ml_ *4). The combined organic layers were dried over Na₂SC>₄, filtered and concentrated under reduced pressure to give the crude product which was purified by flash column (ISCO 100 g silica, 70-100% ethyl acetate in petroleum ether, gradient over 20 min). The product tert-butyl 6- [hydroxy(3-pyridyl)methyl]-2-azaspiro[3.3]heptane-2-carboxylate (16 g, 52.57 mmol, 66%) was obtained as a pale yellow solid.

Step 2: tert-butyl 6-(pyridine-3-carbonyl)-2-azaspiro[3.3]heptane-2-carboxylate.

To a solution of tert-butyl 6-[hydroxy(3-pyridyl)methyl]-2-azaspiro[3.3]heptane-2-carboxylate (7 g, 23.00 mmol) in DCE (120 ml_) was added MnC>2 (19.99 g, 229.97 mmol). The mixture was stirred at 60 °C for 36 h, filtered and the filtrate was concentrated in vacuum. The crude product was purified by flash column (ISCO 40 g silica, 56-80% ethyl acetate in petroleum ether, gradient over 20 min) to afford tert- butyl 6-(pyridine-3-carbonyl)-2-azaspiro[3.3]heptane-2-carboxylate (6.2 g, 19.89 mmol, 87%) as a pale yellow gum. LCMS (ESI) m/z: 247.1 [M-56+H]⁺.

Step 3: tert-butyl 6-[(S)-hydroxy(3-pyridyl)methyl]-2-azaspiro[3.3]heptane-2-carboxylate.

To a solution of glucose dehydrogenase (GDH) (313 mg, 20.50 mmol),NADP (150 mg, 20.50 mmol), glucose (11.18 g, 20.50 mmol) and keto reductase (1.25 g, 20.50 mmol) in buffer (190 ml_) was added drop wise tert-butyl 6-(pyridine-3-carbonyl)-2-azaspiro[3.3]heptane-2-carboxylate (6.2 g, 20.50 mmol, 1 eq) in DMSO (19 ml_). Then the mixture was stirred at 30 °C for 12h (pH was maintained around 7 using 4M NaOH). The mixture was filtered and the filtrate was extracted with ethyl acetate (80 ml_*4). The combined organic layers were dried over anhydrous sodium sulfate and concentrated to afford the crude product. The compound tert-butyl 6-[(S)-hydroxy(3-pyridyl)methyl]-2-azaspiro[3.3]heptane-2- carboxylate (6 g, 19.71 mmol, 96%) was obtained as a pale yellow solid.

Note: Buffer: A mixture of NaH₂P04.2H₂0 (3.96 g) and Na₂HPC>₄.12H₂0 (11.1 g) were dissolved in H2O (500 ml_) to make 0.1 M (pH = 7) aqueous solution. ¹H NMR (400 MHz, CHLOROFORM-d) d 8.51 - 8.41 (m, 2H), 7.7 - 7.6 (m, 1 H), 7.26 - 7.18 (m, 1 H), 4.59 - 4.50 (m, 1 H), 3.94 - 3.71 (m, 4H), 2.5 - 2.35 (m, 1 H), 2.24 - 2.12 (m, 2H), 2.09 - 1 .95 (m, 2H), 1 .48 - 1.35 (s, 9H). LCMS (ESI) m/z: 305.2 [M+H]⁺.

Step 4: (S)-2-azaspiro[3.3]heptan-6-yl(3-pyridyl)methanol.

To a solution of tert-butyl 6-[(S)-hydroxy(3-pyridyl)methyl]-2-azaspiro[3.3]heptane-2-carboxylate (5 g, 16.43 mmol) in DCM (34 ml_) was added TFA (26.22 g, 229.97 mmol). The mixture was stirred at 15 °C for 1 h and concentrated. MeOH (10 ml_) was added and the mixture was basified by resin AMBERSEP(R)9000H until pH > 7. Then the mixture was filtered and the filtrate was concentrated to obtain (S)-2-azaspiro[3.3]heptan-6-yl(3-pyridyl)methanol (4.5 g, crude) as a yellow gum. The crude product was used in the next steps without further purification.

¹H NMR (400 MHz, CHLOROFORM-d) d 8.53 (s, 1 H), 8.43 - 8.42 (m, 1 H), 7.9 - 7.76 (m, 1 H), 7.45 - 7.36 (m, 1 H), 4.64 - 4.56 (m, 1 H), 4.02 - 3.97 (d, 4H), 2.57 - 2.43 (m, 1 H), 2.34 - 2.12 (m, 4H). LCMS (ESI) m/z: 205.2 [M+H]⁺. SFC (Rt =2.282) method: AD_IPA_DEA_5_40_34_35_4min.

Step 5: (S)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3-pyridyl)methanol.

To a solution of (S)-2-azaspiro[3.3]heptan-6-yl(3-pyridyl)methanol (150 mg, 734 umol) in DMSO (2 mL) was added K2CO3 (406 mg, 2.94 mmol), 1 ,2-dichloro-4-iodo-benzene (200 mg, 734 umol), pyrrolidine-2-carboxylic acid (34 mg, 294 umol) and Cul (28 mg, 147 umol) under N2. The mixture was stirred at 90 °C for 3 h and concentrated in vacuum. The residue was purified by prep-HPLC (Waters Xbridge Prep OBD C18 150*40mm*10um column;35-65% acetonitrile in an a 0.04% ammonia solution in water, 8 min gradient) to obtain (S)-[2-(3,4-dichlorophenyl)-2-azaspiro[3.3]heptan-6-yl]-(3- pyridyl)methanol (75 mg, 215 umol, 29%) as a pale yellow solid. LCMS (ESI) m/z: 349.2 [M+H]⁺, SFC (Rt =2.120) method: AD_MeOH_DEA_40_4_35. The chiral purity and enantiomeric form was confirmed by HPLC and X-ray crystallography respectively.

Example 56: Inhibition of CYP51A1 by Compounds of the Invention

Method: Recombinant human CYP51A1 (lanosterol-14a-demethylase) enzyme was coexpressed with CYP reductase in bacterial membranes and the fluorescent substrate BOMCC (a nonnatural substrate that causes increases in fluorescence upon CYP51A1 -dependent demethylation) was used to obtain 8-point dose concentration-response curves for each compound.

Results: As shown in Table 4, the compounds of the invention inhibit CYP51A1.

Table 4.

“+++” = <0.1 mM; “++” = >0.1 mM to <1 mM; “+” = >1 mM

Example 57. Inhibition of CYP51A1 modulates TDP-43 aggregation Introduction

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is an aggressive, debilitating disease in which affected patients succumb within two to five years after diagnosis. ALS presents with heterogeneous clinical features but has a common underlying pathology of motor neuron loss that limits the central nervous system’s ability to effectively regulate voluntary and involuntary muscle activity. Additionally, without neuronal trophic support muscles being to atrophy, further exacerbating motor deterioration. Cellular and tissue degeneration results in motor impairment such as fasciculations and weakening in the arms, legs and neck, difficulty swallowing, slurred speech and ultimately failure of the diaphragm muscles that control breathing.

At the cellular level, 97% of all ALS cases have the common pathological feature of misfolded and aggregated TAR-DNA binding protein (TDP)-43 in spinal motor neuron inclusions. TDP-43 is a DNA/RNA binding protein involved in RNA splicing and is typically localized to the nucleus but can be translocated to the cytoplasm under conditions of cell stress. Nuclear clearing and cytoplasmic accumulation of misfolded and aggregated TDP-43 are hallmarks of degenerating motor neurons in ALS, but it remains unclear if mechanism of toxicity is due to aggregation-dependent loss of TDP-43 function or if the aggregates acquire toxic gain of function. Aggregates of TDP-43 accumulate in discrete cellular domains known as stress granules, which are also enriched with translationally inactive mRNAs. Stress granules are observed in multiple cellular types and are thought to be directly related to TDP-43- dependent toxicity in ALS and FTD. Dysfunction in DNA/RNA binding protein activity plays a crucial role in susceptible motor neurons in ALS, as familial cases have also been traced to mutations in the protein Fused in Sarcoma (FUS), a DNA/RNA binding protein that recently has been shown to be involved in gene silencing. Preclinical studies suggest that FUS mutations promote a toxic gain of function that may be causative in motor neuron degeneration.

Mutations in the TDP-43 gene (TARDBP) have also been causally linked to familial forms of ALS. A common TDP-43 mutation is known as Q331K, in which glutamine (Q) 331 has been mutated to a lysine (K). This mutation results in a TDP-43 protein that is more aggregation prone and exhibits enhanced toxicity. A recent study has also demonstrated that the Q331 K mutation can confer a toxic gain of function in a TDP-43 knock-in mouse, which exhibits cognitive deficits and histological abnormalities similar to that which occurs in frontotemporal dementia (FTD). FTD refers to a group of degenerative disorders that are characterized by atrophy in the frontal and temporal cortices due to progressive neuron loss. Due to the functional nature of the brain regions impacted in FTD, the most common symptoms involve noticeable alterations in personality, behavior and linguistic ability and can also present with loss of speech. The pathological basis of FTD appears to be multifactorial involving mutations in genes such as C9orf72, progranulin (GRN) and MAPT, but intracellular inclusions of aggregated TDP-43, FUS and tau have been observed. Although ALS and FTD may have different genetic and molecular triggers and occur in different cell types, similar protein misfolding and degenerative mechanisms may operate in multiple diseases.

The toxic gain of function features of TDP-43 can be faithfully recapitulated in the simple model organism, budding yeast, where the protein also localizes to stress granules. Human disease mutations in TDP-43 enhance toxicity and yeast genetic screens have revealed key connections that are conserved to humans. The yeast model thus provides a robust cell-based screening platform for small molecules capable of ameliorating toxicity. To validate compounds from such phenotypic screens, it is imperative to test compounds in a mammalian neuronal context. In an effort to develop TDP-43-related mammalian models of neuron loss that occurs in ALS and FTD, primary cultures of rat cortical neurons were transfected with human wild type or Q331 K mutant TDP-43. These cells were compared to cells which received an empty expression vector control. Validation studies have demonstrated that cells expressing either wild type or Q331 K TDP-43 have are more susceptible to dying over time in culture. In the experiments described in this example, this model system is used to interrogate new therapeutic approaches to ameliorate TDP-43 toxicity.

Results

From the TDP-43 yeast model, a compound with known mode of action was identified that restored viability to TDP-43-expressing yeast (FIG. 1A). Fluconazole is an antifungal known to inhibit Erg11 , the yeast lanosterol 14-alpha demethylase (FIG. 1B). Inhibition of Erg 11 reduces ergosterol synthesis (yeast equivalent of cholesterol), while increasing lanosterol levels, the substrate of Erg 11 (FIG. 1C). The human homolog of Erg11 is Cyp51 A1 , a member of the cytochrome P450 superfamily of enzymes but does not appear to have a role in detoxification of xenobiotics. CYP51A1 has also been known as lanosterol 14-alpha demethylase, which describes its function in removing the 14-alpha-methyl group from lanosterol to generate 4,4-dimethylcholesta-8(9),14,24-trien-3p-ol, which is a critical step in the cholesterol biosynthetic pathway.

To evaluate the potential role of CYP51 A1 in TDP-43 pathology, the aforementioned primary rat cortical neuron TDP-43 models were utilized to test the efficacy of published inhibitors (FIG. 2). Rat cortical neurons transfected with wild type human TDP-43 exhibited a significant reduction in survival compared to neurons transfected with empty vector control, and this reduction in survival was partially alleviated by treatment with compound A (FIGS. 3A and 3B). Compound A has the structure:

A similar survival befit was conferred by compound A when applied to cells transfected with Q331 K mutant TDP-43 (FIGS. 4A and 4B). A similar effect in rescuing a survival deficit was observed for a structurally differentiated compound, compound B, when applied to cells transfected with wild-type TDP-43 (FIGS. 5A and 5B). Compound B has the structure:

These studies demonstrate that inhibition of Erg11 in yeast and inhibition of Cyp51 A1 has a beneficial effect of rescuing cells from wild type and mutant TDP-43 toxicity and promotes cell survival. This is the first demonstration that inhibition of CYP51A1 is beneficial in treating and preventing TDP-43 pathological processes and represents a novel therapeutic approach for the treatment of ALS.

Other Embodiments

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

Other embodiments are in the claims.

Claims

1 . A compound, or a pharmaceutically acceptable salt thereof, having the structure:

Formula I wherein R¹ has the structure:

Formula II m is 0, 1 , 2, or 3;

X is CH, CR⁵, or N;

R⁵ is halo, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 alkoxy;

R³ is hydrogen or optionally substituted C1-C6 alkyl;

L¹ is absent, -0-, -SO2-, or optionally substituted C1-C6 alkyl;

L² has the structure:

R⁶ is hydrogen, hydroxy, or optionally substituted C1-C6 alkyl;

L³ is absent, -0-, or optionally substituted C1-C6 alkyl; and

2. The compound of claim 1 , wherein X is CR⁵.

3. The compound of claim 2, wherein X is CH.

4. The compound of any one of claims 1 to 3, wherein R¹ has the structure:

5. The compound of claim 4, wherein R¹ has the structure:

6. The compound of claim 1 , wherein X is N.

7. The compound of claim 6, wherein R¹ has the structure:

8. The compound of any one of claims 1 to 7, wherein R² is hydroxy or-ChhOH.

9. The compound of claim 8, wherein R² is hydroxy.

10. The compound of any one of claims 1 to 9, wherein R³ is hydrogen or methyl.

11. The compound of claim 10, wherein R³ is hydrogen.

12. The compound of any one of claims 1 to 11 , wherein L³ is absent, -CH2-, or -0-.

13. The compound of claim 12, wherein L³ is absent.

14. The compound of any one of claims 1 to 13, wherein L² has the structure:

Formula IV p, q, r, and s are, independently, 0 or 1 ; and R⁶ is hydrogen or optionally substituted C1-C6 alkyl.

15. The compound of claim 14, wherein L² has the structure:

16. The compound of claim 15, wherein L² has the structure:

17. The compound of any one of claims 1 to 13, wherein L² has the structure:

Formula V p, q, r, and s are, independently, 0 or 1 ; and R⁶ is hydrogen or optionally substituted C1-C6 alkyl.

18. The compound of claim 17, wherein L² has the structure:

19. The compound of any one of claims 1 to 13, wherein L² has the structure:

Formula III n and 0 are, independently, 0 or 1 ; and

R⁶ is hydrogen or optionally substituted C1-C6 alkyl.

20. The compound of claim 19, wherein L² has the structure:

21 . The compound of claim 20, wherein L² has the structure:

22. The compound of claim 21 , wherein R⁶ is hydrogen, methyl, or ethyl.

23. The compound of claim 22, wherein R⁶ is hydrogen.

24. The compound of any one of claims 1 to 23, wherein L¹ is absent, -CH2-, or -SC>2-.

25. The compound of claim 24, wherein L¹ is absent.

26. The compound of any one of claims 1 to 25, wherein R⁴ is optionally substituted C6-C10 aryl.

27. The compound of claim 26, wherein R⁴ is phenyl, 3-chloro-phenyl, 4-chloro-phenyl, 3,4- chloro-phenyl, 3-chloro-4-fluoro-phenyl, 3,5-chloro-phenyl, 2-fluoro-3-chloro-phenyl, 3-fluoro-4-chloro- phenyl, 3,4-fluoro-phenyl, 3-chloro-4-cyano-phenyl, 3-fluoro-4-trifluoromethoxy-phenyl, 2-fluoro-4-chloro- phenyl, 2-fluoro-4-trifluormethyl-phenyl, 2,4-fluoro-phenyl, 3-fluoro-4-cyano-phenyl, 2-chloro-4-fluoro- phenyl, 2, 3-chloro-phenyl, 2-cyano-5-iodo-phenyl, 2-trifluoromethoxy-5-bromo-phenyl, 2-bromo- trifluoromethyl-phenyl, or 2-cyano-5-fluoro-phenyl.

28. The compound of claim 27, wherein R⁴ is 3, 4-chloro-phenyl, 3-chloro-4-fluoro-phenyl, or 3,5- chloro-phenyl.

29. The compound of any one of claims 1 to 25, wherein R⁴ is optionally substituted C2-C9 heteroaryl.

30. The compound of claim 29, wherein

32. A compound, or pharmaceutically acceptable salt thereof, having the structure of any one of compounds 1-123 in Table 1.

33. A pharmaceutical composition comprising a compound of any one of claims 1 to 32 and a pharmaceutically acceptable excipient.

34. A method of treating a neurological disorder in a subject in need thereof, the method comprising administering an effective amount of a compound, or pharmaceutically acceptable salt thereof, of any one of claims 1 to 32 or a pharmaceutical composition of claim 33.

35. A method of inhibiting toxicity in a cell related to a protein, the method comprising administering an effective amount of a compound of any one of claims 1 to 32 or a pharmaceutical composition of claim 33.

36. The method of claim 35, wherein the toxicity is TDP-43-related toxicity.

37. The method of any one of claims 35 to 36, wherein the cell is a mammalian neural cell.

38. A method of treating a CYP51A1 -associated disorder in a subject in need thereof, the method comprising administering an effective amount of a compound, or pharmaceutically acceptable salt thereof, of any one of claims 1 to 32 or a pharmaceutical composition of claim 33.

39. The method of claim 38, wherein the CYP51A1 -associated disorder is ALS.

40. A method of inhibiting CYP51 A1 , the method comprising contacting a cell with an effective amount of a compound of any one of claims 1 to 32 or a pharmaceutical composition of claim 33.